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Hintergrund: Die Interaktion zwischen β-HCG und TSH in der Schwangerschaft stellt ein differentialdiagnostisches Problem dar, weil die Wertung von supprimierten TSH-Spiegeln erschwert wird. Dies kann im schlimmsten Fall zu Fehlinterpretationen führen. Ziel der vorliegenden Arbeit war, diese Interaktion an einem großen Kollektiv in einen zeitlichen Kontext mit dem Verlauf der Schwangerschaft zu bringen, da der Zeitpunkt des Screenings entscheidenden Einfluss auf die Höhe des TSH-Spiegels hat. Zusätzlich wurden anhand der vorliegenden Daten Referenzbereiche für Schwangere berechnet und der Einfluss einer Jodmedikation untersucht.
Patienten und Methoden: Aus einem unselektionierten Pool von Patientinnen eines nuklearmedizinischen Praxisverbundes wurden die Daten von 1283 schilddrüsengesunden Schwangeren zwischen 16 und 48 Jahren ausgewertet. Neben der TSH-Bestimmung lag ein Schwerpunkt auf dem zeitlichen Verlauf, so dass die Schwangeren in Untergruppen von je 2 Wochen analysiert wurden. Untersucht wurde der Einfluss der Jodmedikation auf die TSH-Werte. Am Ende erfolgte mit Hilfe der logarithmischen Transformation unter Verwendung der 2-Sigma-Grenzen die Bestimmung neuer TSH-Referenzbereiche für Schwangere.
Ergebnisse: Es zeigt sich zu Beginn der Schwangerschaft ein Anstieg der mittleren TSH-Werte von 1,22 mU/l in der 2. SSW bis auf 1,7 mU/l um die 7. SSW mit einem konsekutiven Abfall der TSH-Werte bis auf 0,9 mU/l bis zur 16. SSW (entsprechend 52,9%). Der größte Abfall findet sich in der 12. bis 14. SSW, also zum Zeitpunkt des ersten Screenings. Die Jodmedikation hat keinen maßgeblichen Einfluss auf den TSH-Wert. Die Berechnung schwangerschaftskorrigierter Referenzbereiche zeigt im ersten Drittel TSH-Werte von 0,08 – 3,67 mU/l, im 2. Drittel 0,04 – 2,88 mU/l und im 3. Trimenon 0,17 – 3,19 mU/l.
Schlussfolgerungen: Die Arbeit zeigt, dass die niedrigsten TSH-Werte zum Zeitpunkt des ersten Screenings zu erwarten sind und deswegen möglicherweise zu Fehlentscheidungen führen können. Ein relevanter Zusammenhang der Jodmedikation mit dem TSH-Wert lässt sich nicht nachweisen. Neue Referenzbereiche für Schwangere könnten hilfreich sein, dieses diagnostische Dilemma zu vermeiden.
Mesenchymal stem/stromal cells (MSCs) feature promising potential for cellular therapies, yet significant progress in development of MSC therapeutics and assays is hampered because of remarkable MSC heterogeneity in vivo and in vitro. This heterogeneity poses challenges for standardization of MSC characterization and potency assays as well as for MSC study comparability and manufacturing. This review discusses promising marker combinations for prospective MSC subpopulation enrichment and expansion, and reflects MSC phenotype changes due to environment and age. In order to address animal modelling in MSC biology, comparison of mouse and human MSC markers highlights current common ground of MSCs between species.
Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.
Vimentin is currently used to differentiate between malignant renal carcinomas and benign oncocytomas. Recent reports showing Vimentin positive oncocytomas seriously question the validity of this present diagnostic approach. Vimentin 3 is a spliced variant and ends with a unique C-terminal ending after exon 7 which differentiates it from the full length version that has 9 exons. Therefore, the protein size is different; the full length Vimentin version has a protein size of ~57 kDa and the truncated version of ~47 kDa. We designed an antibody, called Vim3, against the unique C-terminal ending of the Vimentin 3 variant. Using immune histology, immune fluorescence, Western blot, and qRT-PCR analysis, a Vim3 overexpression was detectable exclusively in oncocytoma, making the detection of Vim3 a potential specific marker for benign kidney tumors. This antibody is the first to clearly differentiate benign oncocytoma and the mimicking eosinophilic variants of the RCCs. This differentiation between malignant and benign RCCs is essential for operative planning, follow-up therapy, and patients' survival. In the future the usage of Vimentin antibodies in routine pathology has to be applied with care. Consideration must be given to Vimentin specific binding epitopes otherwise a misdiagnosis of the patients' tumor samples may result.
Protein turnover and quality control by the proteasome is of paramount importance for cell homeostasis. Dysfunction of the proteasome is associated with aging processes and human diseases such as neurodegeneration, cardiomyopathy, and cancer. The regulation, i.e. activation and inhibition of this fundamentally important protein degradation system, is still widely unexplored. We demonstrate here that the evolutionarily highly conserved type II triple-A ATPase VCP and the proteasome inhibitor PSMF1/PI31 interact directly, and antagonistically regulate proteasomal activity. Our data provide novel insights into the regulation of proteasomal activity.
The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3’UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3’UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3’UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3’ UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.
Objectives: We aimed to investigate the contemporary usage rate and habits of the WHO Surgical Safety Checklist (SSC) in German urological departments.
Methods: We designed a 26-item questionnaire that was sent to all urological departments in Germany. The primary aim of this study was to evaluate the usage rate of the SSC. Secondary aims were to compare perioperative characteristics of users vs. non-users of the SSC and to assess circumstances of the SSC application.
Results: A total of 213 of 234 (91 %) urological departments were users of the SSC, and 21 (9 %) were non-users. SSC users had more often a standard protocol, took less time and had fewer people involved for checking perioperative patient data compared to non-users. Financial budgeting for the SSC existed in 55 (24 %) departments and for patient safety in 73 (32 %) departments.
Conclusions: The usage rate of the SSC in urological departments in Germany is high despite restricted financial budgeting. Users of the SSC profit by saving time and manpower for checking perioperative patient data.
Objective: To evaluate two ultrafast cone-beam CT (UF-CBCT) imaging protocols with different acquisition and injection parameters regarding image quality and required contrast media during image-guided hepatic transarterial chemoembolization (TACE).
Methods: In 80 patients (male: 46, female: 34; mean age: 56.8 years; range: 33–83) UF-CBCT was performed during TACE for intraprocedural guidance. Imaging was performed using two ultrafast CBCT acquisition protocols with different acquisition and injection parameters (imaging protocol 1: acquisition time 2.54 s, and contrast 6 mL with 3 s delay; imaging protocol 2: acquisition time 2.72 s, and contrast 7 mL with 6 s delay). Image evaluation was performed with both qualitative and quantitative methods. Contrast injection volume and dose parameters were compared using values from the literature.
Results: Imaging protocol 2 provided significantly better (P < 0.05) image quality than protocol 1 at the cost of slightly higher contrast load and patient dose. Imaging protocol 1 provided good contrast perfusion but it mostly failed to delineate the tumors (P < 0.05). On the contrary, imaging protocol 2 showed excellent enhancement of hepatic parenchyma, tumor, and feeding vessels.
Conclusion: Tumor delineation, visualization of hepatic parenchyma, and feeding vessels are clearly possible using imaging protocol 2 with ultrafast CBCT imaging. A reduction of required contrast volume and patient dose were achieved due to the ultrafast CBCT imaging.
Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.
The current Review article provides a narrative review about the neurobiological underpinnings and treatment of treatment resistant late-life depression (TRLLD). The manuscript focuses on therapeutic targets of late-life depression, which include pharmacological, psychological, biophysical and exercise treatment approaches. Therefore, we summarize available evidences on that kind of therapies for patients suffering from late-life depression. The search for evidences of therapeutic options of late-life depression were done using searching websites as “pubmed”, and using the searching terms “depression”, “late-life depression”, “treatment”, “biophysical therapy”, “exercise therapy”, “pharmacological therapy” and “psychological therapy”. To the end, we summarize and discuss current data, providing some directions for further research.
Treatment recommendations for elderly depressive patients favour a multimodal approach, containing psychological, pharmacological and secondary biophysical therapeutic options. Particularly, a combination of psychotherapy and antidepressant medication reflects the best therapeutic option. However, mostly accepted and used is the pharmacological treatment although evidence suggests that the drug therapy is not as effective as it is in younger depressive patients. Further studies employing larger samples and longer follow-up periods are necessary and may focus on comparability of study designs and involve novel approaches to establish the validity and reliability of multimodal treatment programs.
Background Reaching the therapeutic target of remission or low-disease activity has improved outcomes in patients with rheumatoid arthritis (RA) significantly. The treat-to-target recommendations, formulated in 2010, have provided a basis for implementation of a strategic approach towards this therapeutic goal in routine clinical practice, but these recommendations need to be re-evaluated for appropriateness and practicability in the light of new insights.
Objective To update the 2010 treat-to-target recommendations based on systematic literature reviews (SLR) and expert opinion.
Methods A task force of rheumatologists, patients and a nurse specialist assessed the SLR results and evaluated the individual items of the 2010 recommendations accordingly, reformulating many of the items. These were subsequently discussed, amended and voted upon by >40 experts, including 5 patients, from various regions of the world. Levels of evidence, strengths of recommendations and levels of agreement were derived.
Results The update resulted in 4 overarching principles and 10 recommendations. The previous recommendations were partly adapted and their order changed as deemed appropriate in terms of importance in the view of the experts. The SLR had now provided also data for the effectiveness of targeting low-disease activity or remission in established rather than only early disease. The role of comorbidities, including their potential to preclude treatment intensification, was highlighted more strongly than before. The treatment aim was again defined as remission with low-disease activity being an alternative goal especially in patients with long-standing disease. Regular follow-up (every 1–3 months during active disease) with according therapeutic adaptations to reach the desired state was recommended. Follow-up examinations ought to employ composite measures of disease activity that include joint counts. Additional items provide further details for particular aspects of the disease, especially comorbidity and shared decision-making with the patient. Levels of evidence had increased for many items compared with the 2010 recommendations, and levels of agreement were very high for most of the individual recommendations (≥9/10).
Conclusions The 4 overarching principles and 10 recommendations are based on stronger evidence than before and are supposed to inform patients, rheumatologists and other stakeholders about strategies to reach optimal outcomes of RA.
Tracheomalacia or tracheobronchomalacia (TM or TBM) is a common problem especially for elderly patients often unfit for surgical techniques. Several surgical or minimally invasive techniques have already been described. Stenting is one option but in general long-time stenting is accompanied by a high complication rate. Stent removal is more difficult in case of self-expandable nitinol stents or metallic stents in general in comparison to silicone stents. The main disadvantage of silicone stents in comparison to uncovered metallic stents is migration and plugging. We compared the operation time and in particular the duration of a sufficient Dumon stent fixation with different techniques in a patient with severe posttracheotomy TM and strongly reduced mobility of the vocal cords due to Parkinson’s disease. The combined approach with simultaneous Dumon stenting and endoluminal transtracheal externalized suture under cone-beam computer tomography guidance with the Berci needle was by far the fastest approach compared to a (not performed) surgical intervention, or even purely endoluminal suturing through the rigid bronchoscope. The duration of the endoluminal transtracheal externalized suture was between 5 minutes and 9 minutes with the Berci needle; the pure endoluminal approach needed 51 minutes. The alternative of tracheobronchoplasty was refused by the patient. In general, 180 minutes for this surgical approach is calculated. The costs of the different approaches are supposed to vary widely due to the fact that in Germany 1 minute in an operation room costs on average approximately 50–60€ inclusive of taxes. In our own hospital (tertiary level), it is nearly 30€ per minute in an operation room for a surgical approach. Calculating an additional 15 minutes for patient preparation and transfer to wake-up room, therefore a total duration inside the investigation room of 30 minutes, the cost per flexible bronchoscopy is per minute on average less than 6€. Although the Dumon stenting requires a set-up with more expensive anesthesiology accompaniment, which takes longer than a flexible investigation estimated at 1 hour in an operation room, still without calculation of the costs of the materials and specialized staff that the surgical approach would consume at least 3,000€ more than a minimally invasive approach performed with the Berci needle. This difference is due to the longer time of the surgical intervention which is calculated at approximately 180 minutes in comparison to the achieved non-surgical approach of 60 minutes in the operation suite.
Translation elongation factor Tuf of Acinetobacter baumannii is a plasminogen-binding protein
(2015)
Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system.
Traditional chinese medicine and herbal hepatotoxicity: a tabular compilation of reported cases
(2015)
Traditional Chinese Medicine (TCM) with its focus on herbal use became popular worldwide. Treatment was perceived as safe, with neglect of rare adverse reactions including liver injury. To compile worldwide cases of liver injury by herbal TCM, we undertook a selective literature search in the PubMed database and searched for the items Traditional Chinese Medicine, TCM, Traditional Asian Medicine, and Traditional Oriental Medicine, also combined with the terms herbal hepatotoxicity or herb induced liver injury. The search focused primarily on English-language case reports, case series, and clinical reviews. We identified reported hepatotoxicity cases in 77 relevant publications with 57 different herbs and herbal mixtures of TCM, which were further analyzed for causality by the Council for International Organizations of Medical Sciences (CIOMS) scale, positive reexposure test results, or both. Causality was established for 28/57 different herbs or herbal mixtures, Bai Xian Pi, Bo He, Ci Wu Jia, Chuan Lian Zi, Da Huang, Gan Cao, Ge Gen, Ho Shou Wu, Huang Qin, Hwang Geun Cho, Ji Gu Cao, Ji Xue Cao, Jin Bu Huan, Jue Ming Zi, Jiguja, Kudzu, Ling Yang Qing Fei Keli, Lu Cha, Rhen Shen, Ma Huang, Shou Wu Pian, Shan Chi, Shen Min, Syo Saiko To, Xiao Chai Hu Tang, Yin Chen Hao, Zexie, and Zhen Chu Cao. In conclusion, this compilation of liver injury cases establishes causality for 28/57 different TCM herbs and herbal mixtures, aiding diagnosis for physicians who care for patients with liver disease possibly related to herbal TCM.
Highlights
• A panel of 20 biomarkers was identified capable of differentiating BD patients from controls.
• Excellent discrimination between established BD patients and controls.
• Good to excellent discrimination between misdiagnosed BD patients and first onset MDD patients.
• Fair to good discrimination between pre-diagnostic BD patients and controls.
• Study demonstrates the potential utility of a protein biomarker panel as a diagnostic test for BD.
Abstract
Background: Bipolar disorder (BD) is a costly, devastating and life shortening mental disorder that is often misdiagnosed, especially on initial presentation. Misdiagnosis frequently results in ineffective treatment. We investigated the utility of a biomarker panel as a diagnostic test for BD.
Methods and findings: We performed a meta-analysis of eight case-control studies to define a diagnostic biomarker panel for BD. After validating the panel on established BD patients, we applied it to undiagnosed BD patients. We analysed 249 BD, 122 pre-diagnostic BD, 75 pre-diagnostic schizophrenia and 90 first onset major depression disorder (MDD) patients and 371 controls. The biomarker panel was identified using ten-fold cross-validation with lasso regression applied to the 87 analytes available across the meta-analysis studies.
We identified 20 protein analytes with excellent predictive performance [area under the curve (AUC) ⩾ 0.90]. Importantly, the panel had a good predictive performance (AUC 0.84) to differentiate 12 misdiagnosed BD patients from 90 first onset MDD patients, and a fair to good predictive performance (AUC 0.79) to differentiate between 110 pre-diagnostic BD patients and 184 controls. We also demonstrated the disease specificity of the panel.
Conclusions: An early and accurate diagnosis has the potential to delay or even prevent the onset of BD. This study demonstrates the potential utility of a biomarker panel as a diagnostic test for BD.
Chronic inflammation as an important epigenetic and environmental factor for putative tumorigenesis and tumor progression may be associated with specific activation of Toll-like receptors (TLR). Recently, carcinogenesis has been suggested to be dependent on TLR7 signaling. In the present study, we determined the role of both TLR7 and TLR8 expression and signaling in tumor cell proliferation and chemoresistance in pancreatic cancer. Expression of TLR7/TLR8 in UICC stage I-IV pancreatic cancer, chronic pancreatitis, normal pancreatic tissue and human pancreatic (PANC1) cancer cell line was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of (un-)stimulated TLR expression on tumor cell proliferation and chemoresistance. TLR expression was increased in pancreatic cancer, with stage-dependent upregulation in advanced tumors, compared to earlier stages and chronic pancreatitis. Stimulation of TLR7/TLR8 overexpressing PANC1 cells resulted in elevated NF-κB and COX-2 expression, increased cancer cell proliferation and reduced chemosensitivity. More importantly, TLR7/TLR8 expression increased tumor growth in vivo. Our data demonstrate a stage-dependent upregulation of both TLR7 and TLR8 expression in pancreatic cancer. Functional analysis in human pancreatic cancer cells point to a significant role of both TLRs in chronic inflammation-mediated TLR7/TLR8 signaling leading to tumor cell proliferation and chemoresistance.
Background. Bile leakage testing may help to detect and reduce the incidence of biliary leakage after hepatic resection. This review was performed to investigate the value of the White-test in identifying intraoperative biliary leakage and avoiding postoperative leakage.
Material and methods. A systematic review and meta-analysis was performed. Two researchers performed literature research. Primary outcome measure was the incidence of post-hepatectomy biliary leakage; secondary outcome measure was the ability of detecting intraoperative biliary leakage with the help of the White-test.
Results. A total of 4 publications (including original data from our center) were included in the analysis. Evidence levels of the included studies had medium quality of 2b (individual cohort studies including low quality randomized controlled trials). Use of the White-test led to a significant reduction of post-operative biliary leakage [OR: 0.3 (95% CI: 0.14, 0.63), p = 0.002] and led to a significant higher intraoperative detection of biliary leakages [OR: 0.03 (95%CI: 0.02, 0.07), p < 0.00001].
Conclusion. Existing evidence implicates the use of the White-test after hepatic resection to identify bile leaks intraoperatively and thus reduce incidence of post-operative biliary leakage. Nonetheless, there is a requirement for a high-quality randomized controlled trial with adequately powered sample-size to confirm findings from the above described studies and further increase evidence in this field.
Study Design: Survey of 100 worldwide spine surgeons.
Objective: To develop a spine injury score for the AOSpine Thoracolumbar Spine Injury Classification System.
Methods: Each respondent was asked to numerically grade the severity of each variable of the AOSpine Thoracolumbar Spine Injury Classification System. Using the results, as well as limited input from the AOSpine Trauma Knowledge Forum, the Thoracolumbar AOSpine Injury Score was developed.
Results: Beginning with 1 point for A1, groups A, B, and C were consecutively awarded an additional point (A1, 1 point; A2, 2 points; A3, 3 points); however, because of a significant increase in the severity between A3 and A4 and because the severity of A4 and B1 was similar, both A4 and B1 were awarded 5 points. An uneven stepwise increase in severity moving from N0 to N4, with a substantial increase in severity between N2 (nerve root injury with radicular symptoms) and N3 (incomplete spinal cord injury) injuries, was identified. Hence, each grade of neurologic injury was progressively given an additional point starting with 0 points for N0, and the substantial difference in severity between N2 and N3 injuries was recognized by elevating N3 to 4 points. Finally, 1 point was awarded to the M1 modifier (indeterminate posterolateral ligamentous complex injury).
Conclusion: The Thoracolumbar AOSpine Injury Score is an easy-to-use, data-driven metric that will allow for the development of a surgical algorithm to accompany the AOSpine Thoracolumbar Spine Injury Classification System.
Background: In the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells.
Material and methods: Colorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0–4 μM) with or without irradiation (2–8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)).
Results: BV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells.
Conclusion: The SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells.
Background: Patients with liver cirrhosis have a highly elevated risk of developing bacterial infections that significantly decrease survival rates. One of the most relevant infections is spontaneous bacterial peritonitis (SBP). Recently, NOD2 germline variants were found to be potential predictors of the development of infectious complications and mortality in patients with cirrhosis. The aim of the INCA (Impact of NOD2 genotype-guided antibiotic prevention on survival in patients with liver Cirrhosis and Ascites) trial is to investigate whether survival of this genetically defined high-risk group of patients with cirrhosis defined by the presence of NOD2 variants is improved by primary antibiotic prophylaxis of SBP.
Methods/Design: The INCA trial is a double-blind, placebo-controlled clinical trial with two parallel treatment arms (arm 1: norfloxacin 400 mg once daily; arm 2: placebo once daily; 12-month treatment and observational period). Balanced randomization of 186 eligible patients with stratification for the protein content of the ascites (<15 versus ≥15 g/L) and the study site is planned. In this multicenter national study, patients are recruited in at least 13 centers throughout Germany. The key inclusion criterion is the presence of a NOD2 risk variant in patients with decompensated liver cirrhosis. The most important exclusion criteria are current SBP or previous history of SBP and any long-term antibiotic prophylaxis. The primary endpoint is overall survival after 12 months of treatment. Secondary objectives are to evaluate whether the frequencies of SBP and other clinically relevant infections necessitating antibiotic treatment, as well as the total duration of unplanned hospitalization due to cirrhosis, differ in both study arms. Recruitment started in February 2014.
Discussion: Preventive strategies are required to avoid life-threatening infections in patients with liver cirrhosis, but unselected use of antibiotics can trigger resistant bacteria and worsen outcome. Thus, individualized approaches that direct intervention only to patients with the highest risk are urgently needed. This trial meets this need by suggesting stratified prevention based on genetic risk assessment. To our knowledge, the INCA trial is first in the field of hepatology aimed at rapidly transferring and validating information on individual genetic risk into clinical decision algorithms.
Trial registrations: German Clinical Trials Register DRKS00005616. Registered 22 January 2014. EU Clinical Trials Register EudraCT 2013-001626-26. Registered 26 January 2015.
The hallmark of Philadelphia chromosome positive (Ph+) leukemia is the BCR/ABL kinase, which is successfully targeted by selective ATP competitors. However, inhibition of BCR/ABL alone is unable to eradicate Ph+ leukemia. The t(9;22) is a reciprocal translocation which encodes not only for the der22 (Philadelphia chromosome) related BCR/ABL, but also for der9 related ABL/BCR fusion proteins, which can be detected in 65% of patients with chronic myeloid leukemia (CML) and 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. The co-expression of p96ABL/BCR enhanced the kinase activity and as a consequence, the transformation potential of p185BCR/ABL. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and Ph+ ALL patient-derived long-term cultures (PD-LTCs). Our in vitro and in vivo stem cell studies further revealed a functional hierarchy of p96ABL/BCR and p185BCR/ABL in hematopoietic stem cells. Co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph+ ALL.
The small leucine-rich proteoglycan biglycan (Bgn) is a part of the extracellular matrix providing structure and enhancing fibril stability. In its soluble form, biglycan is able to bind and signal via the innate immune receptors Toll-like receptor (TLR) 2 and 4, thereby activating MAP-kinases and the NF-κB pathway. In macrophages soluble biglycan induces the secretion of several cytokines and chemokines, including TNF-α, CCL2, CXCL5 and CXCL13. A unique feature of biglycan is its ability to stimulate the secretion of mature IL-1β. By orchestrating TLR2 and 4 with the purinergic P2X4 and P2X7 receptor signalling biglycan triggers the activation of the NLRP3/ASC inflammasome, which in turn activates caspase-1 to cleave pro-IL-1β to mature IL-1β. Furthermore, in several inflammatory diseases an upregulated biglycan expression is found. Enhanced levels of biglycan could be measured in plasma and inflamed tissue. In mouse models of sepsis, lupus nephritis and renal ischemic reperfusion injury, biglycan-deficiency improved the disease outcome. Overexpression of soluble biglycan on the other hand increased immune cell infiltration into the kidney by inducing cytokine and chemokine expression in a TLR2/4-dependent manner. These studies emphasise its importance in inflammatory processes, especially in the kidney. Furthermore, the pro-inflammatory effects on macrophages and diseases established biglycan as a danger signalling molecule, yet its role as a soluble molecule in plasma was not further investigated.
Although an increase of soluble biglycan in the circulation could be seen in several inflammatory diseases, the source is not fully unravelled. Previously it could be shown that macrophages and dendritic cells secrete soluble biglycan after stimulation with IL-6 and TGF-β1. However, since these cell are resident in organs and do not circulate in the blood stream their contribution to soluble biglycan levels in plasma is likely minor. Therefore, monocytes as precursor of both macrophages and dendritic cells were investigated as a possible source of circulating biglycan. Analysis of blood from septic patients revealed elevated soluble biglycan levels as well as an increased number of monocytes. Isolated monocytes from healthy volunteers incubated with the inflammatory cytokines IL-1β, IL-6 and TGF-β1 displayed increased biglycan mRNA expression and secretion of soluble biglycan into the supernatant, revealing monocytes as a producer of soluble biglycan in blood. Therefore this work was directed to further investigate the influence of soluble biglycan on circulating monocytes, with regard to sepsis.
Monocytes can be classified into three subtypes, while the classical monocytes express CD14 (CD14++CD16low), intermediate monocytes express both CD14 and CD16 (CD14++CD16+) and non-classical monocytes express mainly CD16 (CD14lowCD16++). The intermediate and non-classical monocytes make up about 10 % of all monocytes and are referred to as CD16-positive subtypes. The CD16-positive monocytes express higher levels of TNF-α and IL-1β upon stimulation and display different migration behaviour. In most inflammatory diseases an expansion of CD16-positive monocytes is observed, especially an increased number of intermediate monocytes frequently correlate with disease severity and mortality. Since septic patients had increased circulating biglycan levels and augmented CD16-positive monocytes, a possible correlation between these two parameters was investigated. Using FACS analysis of biglycan-stimulated monocytes from healthy donors revealed a significant shift from classical to intermediate and non-classical monocytes. This shift was mediated by increased expression of CD14 and CD16 on mRNA and protein levels upon biglycan treatment. Furthermore, biglycan induced the mRNA expression of the adhesion molecules ICAM-1, VCAM-1 and ELAM-1 in CD14-positive monocytes. Four hours after biglycan stimulation an increased ICAM-1 protein expression on the cell surface of classical and intermediate monocytes was observed. Additionally, biglycan-treated CD14-positive monocytes rolled and attached to pre-stimulated endothelial cells to a greater extent compared to untreated monocytes. This demonstrates that biglycan not only triggers the expression of CD14 and CD16 but also induces a functional shift of monocytes. ...
Background: In German breast cancer care, the S1-guidelines of the 1990s were substituted by national S3-guidelines in 2003. The application of guidelines became mandatory for certified breast cancer centers. The aim of the study was to assess guideline adherence according to time intervals and its impact on survival.
Methods: Women with primary breast cancer treated in three rural hospitals of one German geographical district were included. A cohort study design encompassed women from 1996–97 (N = 389) and from 2003–04 (N = 488). Quality indicators were defined along inpatient therapy sequences for each time interval and distinguished as guideline-adherent and guideline-divergent medical decisions. Based on all of the quality indicators, a binary overall adherence index was defined and served as a group indicator in multivariate Cox-regression models. A corrected group analysis estimated adjusted 5-year survival curves.
Results: From a total of 877 patients, 743 (85 %) and 504 (58 %) were included to assess 104 developed quality indicators and the resuming binary overall adherence index. The latter significantly increased from 13–15 % (1996–97) up to 33–35 % (2003–04). Within each time interval, no significant survival differences of guideline-adherent and -divergent treated patients were detected. Across time intervals and within the group of guideline-adherent treated patients only, survival increased but did not significantly differ between time intervals. Across time intervals and within the group of guideline-divergent treated patients only, survival increased and significantly differed between time intervals.
Conclusions: Infrastructural efforts contributed to the increase of process quality of the examined certified breast cancer center. Paradoxically, a systematic impact on 5-year survival has been observed for patients treated divergently from the guideline recommendations. This is an indicator for the appropriate application of guidelines. A maximization of guideline-based decisions instead of the ubiquitous demand of guideline adherence maximization is advocated.
Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain‐containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11‐positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi‐subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N‐terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy‐specific TRAPP subunit, forms part of a mammalian TRAPPIII‐like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.
Device-related infections in recipients of left ventricular assist devices (LVAD) have been recognized as a major source of morbidity and mortality. They require a high level of diagnostic effort as part of the overall burden resulting from infectious complications in LVAD recipients. We present a multi-allergic patient who was treated for persistent sterile intrathoracic abscess formation and pericardial empyema following minimally invasive LVAD implantation including use of a sheet of e-polytetrafluoroethylene (ePTFE) membrane to restore pericardial integrity. Sterile abscess formation and pericardial empyema recurred after surgical removal until the ePTFE membrane was removed, suggesting that in disposed patients, ePTFE may be related to sterile abscess formation or sterile empyema.
Cancer is characterized by a remarkable intertumoral, intratumoral, and cellular heterogeneity that might be explained by the cancer stem cell (CSC) and/or the clonal evolution models. CSCs have the ability to generate all different cells of a tumor and to reinitiate the disease after remission. In the clonal evolution model, a consecutive accumulation of mutations starting in a single cell results in competitive growth of subclones with divergent fitness in either a linear or a branching succession. Acute lymphoblastic leukemia (ALL) is a highly malignant cancer of the lymphoid system in the bone marrow with a dismal prognosis after relapse. However, stabile phenotypes and functional data of CSCs in ALL, the so-called leukemia-initiating cells (LICs), are highly controversial and the question remains whether there is evidence for their existence. This review discusses the concepts of CSCs and clonal evolution in respect to LICs mainly in B-ALL and sheds light onto the technical controversies in LIC isolation and evaluation. These aspects are important for the development of strategies to eradicate cells with LIC capacity. Common properties of LICs within different subclones need to be defined for future ALL diagnostics, treatment, and disease monitoring to improve the patients’ outcome in ALL.
Perioperative Blutungskomplikationen stellen, insbesondere für neurochirurgisch behandelte Patienten, eine schwerwiegende Komplikation dar und sind mit einer signifikant erhöhten Morbidität und Mortalität assoziiert [1]. Der perioperative Blutverlust ist einer der Hauptgründe für die Transfusion allogener Blutprodukte [2, 3]. Transfusionen von Erythrozytenkonzentraten (EK), Gefrorenem Frischplasma (GFP) und Thrombozytenkonzentraten (TK) sind dabei unabhängige Risikofaktoren für die Entwicklung thromboembolischer, pulmonaler, hämodynamischer oder septischer Komplikationen und selbst mit einer erhöhten perioperativen Morbidität und Mortalität assoziiert [3-10]. In der Literatur wird die Prävalenz von Blutungskomplikationen im Rahmen von intrakraniellen Eingriffen (Raten zwischen 0,8% bis 50%) sehr unterschiedlich beschrieben [1, 11-13]. Gleichwertige Voraussetzungen für die Bildung und Aufrechterhaltung eines stabilen Blutgerinnsels sind, neben einer effizienten primären Hämostase (Thrombozytenzahl und –funktion, Von-Willebrand-Faktor), auch die plasmatische Gerinnung (extrinsische und intrinsische Gerinnungsfaktoren) und der Schutz vor (Hyper)fibrinolyse [14].
Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold, EC50: 2,5% milk) without toxic effects. Surprisingly, bovine milk was identified as strong inducer of collagen 1A1 synthesis at both, the protein (4-fold, EC50: 0,09% milk) and promoter level. Regarding the underlying molecular pathways, we show functional activation of STAT6 in a p44/42 and p38-dependent manner. More upstream, we identified IGF-1 and insulin as key factors responsible for milk-induced collagen synthesis. These findings show that bovine milk contains bioactive molecules that act on human skin cells. Therefore, it is tempting to test the herein introduced concept in treatment of atrophic skin conditions induced e.g. by UV light or corticosteroids.
Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b−/− mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.
Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F)+ HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5.
The Asian bush mosquito (Aedes japonicus japonicus, Theobald 1901) is an invasive culicid species which originates in Asia but is nowadays present in northern America and Europe. It is a competent vector for several human disease pathogens. In addition to the public health threat, this invasive species may also be an ecological threat for native container-breeding mosquitoes which share a similar larval habitat. Therefore, it is of importance to gain knowledge on ecological and eco-toxicological features of the Asian bush mosquito. However, optimal laboratory feeding conditions have not yet been established. Standardized feeding methods will be needed in assessing the impact of insecticides or competitional strength of this species. To fill this gap, we performed experiments on food quality and quantity for Ae. j. japonicus larvae. We found out that the commercial fish food TetraMin (Tetra, Melle, Germany) in a dose of 10 mg per larva is the most suitable food tested. We also suggest a protocol with a feeding sequence of seven portions for all larval stages of this species.
The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is overactivated in malignant glioma and plays a key role in promoting cell survival, thereby increasing the acquired apoptosis resistance of these tumors. Here we investigated the STAT3/myeloid cell leukemia 1 (MCL1) signaling pathway as a target to overcome the resistance of glioma cells to the Bcl-2-inhibiting synthetic BH3 mimetic ABT-737. Stable lentiviral knockdown of MCL1 sensitized LN229 and U87 glioma cells to apoptotic cell death induced by single-agent treatment with ABT-737 which was associated with an early activation of DEVDase activity, cytochrome c release, and nuclear apoptosis. Similar sensitizing effects were observed when ABT-737 treatment was combined with the multikinase inhibitor sorafenib which effectively suppressed levels of phosphorylated STAT3 and MCL1 in MCL1-proficient LN229 and U87 glioma cells. In analogous fashion, these synergistic effects were observed when we combined ABT-737 with the STAT3 inhibitor WP-1066. Lentiviral knockdown of the activating transcription factor 5 combined with subsequent quantitative polymerase chain reaction analysis revealed that sorafenib-dependent suppression of MCL1 occurred at the transcriptional level but did not depend on activating transcription factor 5 which previously had been proposed to be essential for MCL1-dependent glioma cell survival. In contrast, the constitutively active STAT3 mutant STAT3-C was able to significantly enhance MCL1 levels under sorafenib treatment to retain cell survival. Collectively, these data demonstrate that sorafenib targets MCL1 in a STAT3-dependent manner, thereby sensitizing glioma cells to treatment with ABT-737. They also suggest that targeting STAT3 in combination with inducers of the intrinsic pathway of apoptosis may be a promising novel strategy for the treatment of malignant glioma.
Rationale: Early reperfusion in patients experiencing acute ischemic stroke is critical, especially for patients with large vessel occlusion who have poor prognosis without revascularization. S olitaire™ stent retriever devices have been shown to immediately restore vascular perfusion safely, rapidly, and effectively in acute ischemic stroke patients with large vessel occlusions.
Aim: The aim of the study was to demonstrate that, among patients with large vessel, anterior circulation occlusion who have received intravenous tissue plasminogen activator, treatment with S olitaire revascularization devices reduces degree of disability 3 months post stroke.
Design: The study is a global multicenter, two‐arm, prospective, randomized, open, blinded end‐point trial comparing functional outcomes in acute ischemic stroke patients who are treated with either intravenous tissue plasminogen activator alone or intravenous tissue plasminogen activator in combination with the Solitaire device. Up to 833 patients will be enrolled.
Procedures:Patients who have received intravenous tissue plasminogen activator are randomized to either continue with intravenous tissue plasminogen activator alone or additionally proceed to neurothrombectomy using the S olitaire device within six‐hours of symptom onset.
Study Outcomes: The primary end‐point is 90‐day global disability, assessed with the modified R ankin S cale (mRS). Secondary outcomes include mortality at 90 days, functional independence (mRS ≤ 2) at 90 days, change in N ational I nstitutes of H ealth S troke S cale at 27 h, reperfusion at 27 h, and thrombolysis in cerebral infarction 2b/3 flow at the end of the procedure.
Analysis:Statistical analysis will be conducted using simultaneous success criteria on the overall distribution of modified R ankin S cale (R ankin shift) and proportions of subjects achieving functional independence (mRS 0–2).
Introduction: In this article three research questions are addressed: (1) Is there an association between socioeconomic status (SES) and patient-reported outcomes in a cohort of multimorbid patients? (2) Does the association vary according to SES indicator used (income, education, occupational position)? (3) Can the association between SES and patient-reported outcomes (self-rated health, health-related quality of life and functional status) be (partly) explained by burden of disease?
Methods: Analyses are based on the MultiCare Cohort Study, a German multicentre, prospective, observational cohort study of multimorbid patients from general practice. We analysed baseline data and data from the first follow-up after 15 months (N = 2,729). To assess burden of disease we used the patients’ morbidity data from standardized general practitioner (GP) interviews based on a list of 46 groups of chronic conditions including the GP’s severity rating of each chronic condition ranging from marginal to very severe.
Results: In the cross-sectional analyses SES was significantly associated with the patient-reported outcomes at baseline. Associations with income were more consistent and stronger than with education and occupational position. Associations were partly explained (17% to 44%) by burden of disease. In the longitudinal analyses only income (but not education and occupational position) was significantly related to the patient-reported outcomes at follow-up. Associations between income and the outcomes were reduced by 18% to 27% after adjustment for burden of disease.
Conclusions: Results indicate social inequalities in self-rated health, functional status and health related quality of life among older multimorbid patients. As associations with education and occupational position were inconsistent, these inequalities were mainly due to income. Inequalities were partly explained by burden of disease. However, even among patients with a similar disease burden, those with a low income were worse off in terms of the three patient-reported outcomes under study.
Second mitochondria-derived activator of caspase (Smac) mimetics are considered as promising anticancer therapeutics that are currently under investigation in early clinical trials. They induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are frequently overexpressed in cancer. We previously reported that Smac mimetics, such as BV6, additionally exert non-apoptotic functions in glioblastoma (GBM) cells by stimulating migration and invasion in a nuclear factor kappa B (NF-κB)-dependent manner. Because NF-κB target genes mediating these effects are largely unknown, we performed whole-genome expression analyses. Here, we identify chemokine (C-C motif) ligand 2 (CCL2) as the top-listed NF-κB-regulated gene being upregulated upon BV6 treatment in GBM cells. BV6-induced upregulation and secretion of CCL2 are required for migration and invasion of GBM cells because knockdown of CCL2 in GBM cells abolishes these effects. Co-culture experiments of GBM cells with non-malignant astroglial cells reveal that BV6-stimulated secretion of CCL2 by GBM cells into the supernatant triggers migration of astroglial cells toward GBM cells because CCL2 knockdown in BV6-treated GBM cells impedes BV6-stimulated migration of astroglial cells. In conclusion, we identify CCL2 as a BV6-induced NF-κB target gene that triggers migration and invasion of GBM cells and exerts paracrine effects on the GBM's microenvironment by stimulating migration of astroglial cells. These findings provide novel insights into the biological functions of Smac mimetics with important implications for the development of Smac mimetics as cancer therapeutics.
The ability of hematopoietic stem cells (HSCs) to self-renew is a prerequisite for the establishment of definitive hematopoiesis and life-long blood regeneration. Here, we report the single-stranded DNA-binding transcriptional regulator far upstream element (FUSE)-binding protein 1 (FUBP1) as an essential factor of HSC self-renewal. Functional inactivation of FUBP1 in two different mouse models resulted in embryonic lethal anemia at around E15.5 caused by severely diminished HSCs. Fetal and adult HSCs lacking FUBP1 revealed an HSC-intrinsic defect in their maintenance, expansion, and long-term blood reconstitution, but could differentiate into all hematopoietic lineages. FUBP1-deficient adult HSCs exhibit significant transcriptional changes, including upregulation of the cell-cycle inhibitor p21 and the pro-apoptotic Noxa molecule. These changes caused an increase in generation time and death of HSCs as determined by video-microscopy-based tracking. Our data establish FUBP1 and its recognition of single-stranded genomic DNA as an important element in the transcriptional regulation of HSC self-renewal.
Simultaneous and dose dependent melanoma cytotoxic and immune stimulatory activity of betulin
(2015)
Conventional cytostatic cancer treatments rarely result in the complete eradication of tumor cells. Therefore, new therapeutic strategies focus on antagonizing the immunosuppressive activity of established tumors. In particular, recent studies of antigen-loaded dendritic cells (DCs) eliciting a specific antitumor immune response has raised the hopes of achieving the complete elimination of tumor tissue. Genistein, fingolimod and betulin have already been described as active compounds in different types of cancer. Herein, we applied an integrated screening approach to characterize both their cytostatic and their immune-modulating properties side-by-side. As will be described in detail, our data confirmed that all three compounds exerted proapoptotic and antiproliferative activity in different B16 melanoma cell lines to a given extent, as revealed by an MTT assay, CFSE and DAPI staining. However, while genistein and fingolimod also affected the survival of primary bone marrow (BM) derived DCs of C57BL/6 mice, betulin exhibited a lower cytotoxicity for BMDCs in comparison to the melanoma cells. Moreover, we could show for the first time, that only betulin caused a simultaneous, highly specific immune-stimulating activity, as measured by the IL-12p70 release of Toll-like receptor 4-stimulated BMDCs by ELISA, which was due to increased IL-12p35 mRNA expression. Interestingly, the activation of DCs resulted in enhanced T lymphocyte stimulation, indicated by increased IL-2 and IFN-γ production of cytotoxic T cells in spleen cell co-culture assays which led to a decreased viability of B16 cells in an antigen specific model system. This may overcome the immunosuppressive environment of a tumor and destroy tumor cells more effectively in vivo if the immune response is specific targeted against the tumor tissue by antigen-loaded dendritic cells. In summary, cytostatic agents, such as betulin, that simultaneously exhibit immune stimulatory activity may serve as lead compounds and hold great promise as a novel approach for an integrated cancer therapy.
Background: The peroneal muscles are the most effective lateral stabilisers whose tension braces the ankle joint complex against excessive supination. The purpose of this study was to identify the morphological and biomechanical effects of two machine-based shank muscle training methods.
Methods: Twenty-two healthy male recreationally active sports students performed ten weeks of single-set high resistance strength training with 3 training sessions per week. The subjects conducted subtalar pronator/supinator muscle training (ST) with the right leg by using a custom-made apparatus; the left foot muscles were exercised with machine-based talocrural plantar and dorsiflexor training (TT). Muscle strength (MVIC), muscle volume and foot biomechanics (rearfoot motion, ground reaction forces, muscle reaction times) during a sudden ankle supination were recorded before and after the intervention.
Results: Compared to TT, ST resulted in significantly higher pronator (14% vs. 8%, P<0.01) and supinator MVIC (25% vs. 12%, P<0.01). During sudden foot inversions, both ST and TT resulted in reduced supination velocity (-12%; P<0.01). The muscle reaction onset time was faster after the training in peroneus longus (PL) (P<0.01). Muscle volume of PL (P<0.01) and TA (P<0.01) increased significantly after both ST and TT.
Conclusion: After both ST and TT, the ankle joint complex is mechanically more stabilised against sudden supinations due to the muscle volume increase of PL and TA. As the reduced supination velocities indicate, the strength training effects are already present during free-fall. According to a sudden ankle supination in standing position, both machine-based dorsiflexor and pronator strength training is recommended for enhancing the mechanical stability of the ankle.
The aim of this study was to identify sex-dependent expression of renal transporter mRNA in lean and obese Zucker spontaneously hypertensive fatty (ZSF1) rats and to investigate the interaction of the most altered transporter, organic anion transporter 2 (Oat2), with diabetes-relevant metabolites and drugs. Higher incidence of glomerulosclerosis, tubulointerstitial fibrosis, and protein casts in Bowman’s space and tubular lumen was detected by PAS staining in obese male compared to female ZSF1 rats. Real-time PCR on RNA isolated from kidney cortex revealed that Sglt1-2, Oat1-3, and Oct1 were higher expressed in kidneys of lean females. Oct2 and Mrp2 were higher expressed in obese males. Renal mRNA levels of transporters were reduced with diabetic nephropathy in females and the expression of transcription factors Hnf1β and Hnf4α in both sexes. The highest difference between lean and obese ZSF1 rats was found for Oat2. Therefore, we have tested the interaction of human OAT2 with various substances using tritium-labeled cGMP. Human OAT2 showed no interaction with diabetes-related metabolites, diabetic drugs, and ACE-inhibitors. However, OAT2-dependent uptake of cGMP was inhibited by furosemide. The strongly decreased expression of Oat2 and other transporters in female diabetic ZSF1 rats could possibly impair renal drug excretion, for example, of furosemide.
Background: Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy.
Methods: We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry.
Results: We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival (standard beta = -0.001, p = 0.022).
Conclusions: In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies.
Secondary plant metabolites reveal numerous biological activities making them attractive as resource for drug development of human diseases. As the majority of cancer drugs clinically established during the past half century is derived from nature, cancer researchers worldwide try to identify novel natural products as lead compounds for cancer therapy. Natural products are considered as promising cancer therapeutics, either as single agents or in combination protocols, to enhance the antitumor activity of additional therapeutic modalities. Most natural compounds exert pleotrophic effects and modulate various signal transduction pathways. A better understanding of the complex mechanisms of action of natural products is expected to open new perspectives in coming years for their use alone or in combination therapies in oncology. Two major strategies to identify novel drug candidates from nature are the bioactivity-guided fractionation of medicinal plant extracts to isolate cytotoxic chemicals and the identification of small molecules inhibiting specific targets in cancer cells. In the present review, we report on our own efforts to unravel the molecular modes of action of phytochemicals in cancer cells and focus on resveratrol, betulinic acid, artesunate, dicentrine and camptothecin derivatives.
BACKGROUND: Secukinumab, a fully human anti-interleukin-17A monoclonal antibody, has shown superior efficacy to etanercept with similar safety in moderate to severe plaque psoriasis (FIXTURE study).
OBJECTIVE: We sought to directly compare efficacy and safety of secukinumab versus ustekinumab.
METHODS: In this 52-week, double-blind study (NCT02074982), 676 subjects were randomized 1:1 to subcutaneous injection of secukinumab 300 mg or ustekinumab per label. Primary end point was 90% or more improvement from baseline Psoriasis Area and Severity Index (PASI) score (PASI 90) at week 16.
RESULTS: Secukinumab (79.0%) was superior to ustekinumab (57.6%) as assessed by PASI 90 response at week 16 (P < .0001). The 100% improvement from baseline PASI score at week 16 was also significantly greater with secukinumab (44.3%) than ustekinumab (28.4%) (P < .0001). The 75% or more improvement from baseline PASI score at week 4 was superior for secukinumab (50.0%) versus ustekinumab (20.6%) (P < .0001). Percentage of subjects with the Dermatology Life Quality Index score 0/1 (week 16) was significantly higher with secukinumab (71.9%) than ustekinumab (57.4%) (P < .0001). The safety profile of secukinumab was comparable with ustekinumab and consistent with pivotal phase III secukinumab studies.
LIMITATIONS: The study was not placebo-controlled and of short-term duration.
CONCLUSIONS: Secukinumab is superior to ustekinumab in clearing skin of subjects with moderate to severe psoriasis and improving health-related quality of life with a comparable safety profile over 16 weeks.
Background: Measurement of prostate-specific antigen (PSA) advanced the diagnostic and prognostic potential for prostate cancer (PCa). However, due to PSA’s lack of specificity, novel biomarkers are needed to improve risk assessment and ensure optimal personalized therapy. A set of protein molecules as potential biomarkers was therefore evaluated in serum of PCa patients.
Methods: Serum samples from patients undergoing radical prostatectomy (RPE) for biopsy-proven PCa without neoadjuvant treatment were compared to serum samples from healthy subjects. Preliminary screening of 119 proteins in 10 PCa patients and 10 controls was carried out by the Proteome Profiler Antibody Array. Those markers showing distinct differences between patients and controls were then further evaluated by ELISA in the serum of 165 PCa patients and 19 controls. Uni- and multivariate as well as correlation analysis were performed to test the capability of these molecules to detect disease and predict pathological outcome.
Results: Screening showed that soluble (s)E-cadherin, E-selectin, MMP2, MMP9, TIMP1, TIMP2, Galectin and Clusterin warranted further evaluation. sE-Cadherin, TIMP1, Galectin and Clusterin were significantly over- and MMP9 under-expressed in PCa compared to controls. The concentration of sE-cadherin, MMP2 and Clusterin correlated negatively and that of MMP9 and TIMP1 positively with the Gleason Sum at prostatectomy. Only sE-cadherin significantly correlated with the highest Gleason pattern. Compared to serum PSA, sE-cadherin provided an independent and better matching predictive ability for discriminating PCas with an upgrade at RPE and aggressive tumors with a Gleason Sum ≥7.
Conclusions: sE-cadherin performed most favorably from a large panel of serum proteins in terms of diagnostic and predictive potential in curatively treatable PCa. sE-cadherin merits further investigation as a biomarker for PCa.
Polygenic risk scores, based on risk variants identified in genome-wide-association-studies (GWAS), explain a considerable portion of the heritability for schizophrenia (SZ) and bipolar disorder (BD). However, little is known about the combined effects of these variants, although polygenic neuroimaging has developed into a powerful tool of translational neuroscience. In this study, we used genome wide significant SZ risk variants to test the predictive capacity of the polygenic model and explored potential associations with white matter volume, a key candidate in imaging phenotype for psychotic disorders.
By calculating the combined additive schizophrenia risk of seven SNPs (significant hits from a recent schizophrenia GWAS study), we show that increased additive genetic risk for SZ was associated with reduced white matter volume in a group of participants (n = 94) consisting of healthy individuals, SZ first-degree relatives, SZ patients and BD patients. This effect was also seen in a second independent sample of healthy individuals (n = 89). We suggest that a moderate portion of variance (~4%) of white matter volume can be explained by the seven hits from the recent schizophrenia GWAS.
These results provide evidence for associations between cumulative genetic risk for schizophrenia and intermediate neuroimaging phenotypes in models of psychosis. Our work contributes to a growing body of literature suggesting that polygenic risk may help to explain white matter alterations associated with familial risk for psychosis.
RUCAM (Roussel Uclaf Causality Assessment Method) or its previous synonym CIOMS (Council for International Organizations of Medical Sciences) is a well established tool in common use to quantitatively assess causality in cases of suspected drug induced liver injury (DILI) and herb induced liver injury (HILI). Historical background and the original work confirm the use of RUCAM as single term for future cases, dismissing now the term CIOMS for reasons of simplicity and clarity. RUCAM represents a structured, standardized, validated, and hepatotoxicity specific diagnostic approach that attributes scores to individual key items, providing final quantitative gradings of causality for each suspect drug/herb in a case report. Experts from Europe and the United States had previously established in consensus meetings the first criteria of RUCAM to meet the requirements of clinicians and practitioners in care for their patients with suspected DILI and HILI. RUCAM was completed by additional criteria and validated, assisting to establish the timely diagnosis with a high degree of certainty. In many countries and for more than two decades, physicians, regulatory agencies, case report authors, and pharmaceutical companies successfully applied RUCAM for suspected DILI and HILI. Their practical experience, emerging new data on DILI and HILI characteristics, and few ambiguous questions in domains such alcohol use and exclusions of non-drug causes led to the present update of RUCAM. The aim was to reduce interobserver and intraobserver variability, to provide accurately defined, objective core elements, and to simplify the handling of the items. We now present the update of the well accepted original RUCAM scale and recommend its use for clinical, regulatory, publication, and expert purposes to validly establish causality in cases of suspected DILI and HILI, facilitating a straightforward application and an internationally harmonized approach of causality assessment as a common basic tool.
Background: While recent data show that crizotinib is highly effective in patients with ROS1 rearrangement, few data is available about the prognostic impact, the predictive value for different treatments, and the genetic heterogeneity of ROS1- positive patients.
Patients and methods: 1137 patients with adenocarcinoma of the lung were analyzed regarding their ROS1 status. In positive cases, next-generation sequencing (NGS) was performed. Clinical characteristics, treatments and outcome of these patients were assessed. Overall survival (OS) was compared with genetically defined subgroups of ROS1-negative patients.
Results: 19 patients of 1035 evaluable (1.8%) had ROS1-rearrangement. The median OS has not been reached. Stage IV patients with ROS1-rearrangement had the best OS of all subgroups (36.7 months, p < 0.001). 9 of 14 (64.2%) patients had at least one response to chemotherapy. Estimated mean OS for patients receiving chemotherapy and crizotinib was 5.3 years. Ten patients with ROS1-rearrangement (52.6%) harbored additional aberrations.
Conclusion: ROS1-rearangement is not only a predictive marker for response to crizotinib, but also seems to be the one of the best prognostic molecular markers in NSCLC reported so far. In stage IV patients, response to chemotherapy was remarkable high and overall survival was significantly better compared to other subgroups including EGFR-mutated and ALK-fusion-positive NSCLC.
Background: The presence of the recently introduced primary dengue virus vector mosquito Aedes aegypti in Nepal, in association with the likely indigenous secondary vector Aedes albopictus, raises public health concerns. Chikungunya fever cases have also been reported in Nepal, and the virus causing this disease is also transmitted by these mosquito species. Here we report the results of a study on the risk factors for the presence of chikungunya and dengue virus vectors, their elevational ceiling of distribution, and climatic determinants of their abundance in central Nepal.
Methodology/Principal findings: We collected immature stages of mosquitoes during six monthly cross-sectional surveys covering six administrative districts along an altitudinal transect in central Nepal that extended from Birgunj (80 m above sea level [asl]) to Dhunche (highest altitude sampled: 2,100 m asl). The dengue vectors Ae. aegypti and Ae. albopictus were commonly found up to 1,350 m asl in Kathmandu valley and were present but rarely found from 1,750 to 2,100 m asl in Dhunche. The lymphatic filariasis vector Culex quinquefasciatus was commonly found throughout the study transect. Physiographic region, month of collection, collection station and container type were significant predictors of the occurrence and co-occurrence of Ae. aegypti and Ae. albopictus. The climatic variables rainfall, temperature, and relative humidity were significant predictors of chikungunya and dengue virus vectors abundance.
Conclusions/Significance: We conclude that chikungunya and dengue virus vectors have already established their populations up to the High Mountain region of Nepal and that this may be attributed to the environmental and climate change that has been observed over the decades in Nepal. The rapid expansion of the distribution of these important disease vectors in the High Mountain region, previously considered to be non-endemic for dengue and chikungunya fever, calls for urgent actions to protect the health of local people and tourists travelling in the central Himalayas.
Cell death and survival programs are controlled by the cellular redox state, which is typically dysregulated during oncogenesis. A recent study reports that the inhibition of antioxidant defenses resulting from glutathione depletion can prime acute lymphoblastic leukemia cells for death induced by Smac mimetics.
Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI-null mice
(2015)
Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1-null (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.
Gallbladder cancer (GBC) is a highly malignant tumor characterized by a poor response to chemotherapy and radiotherapy. We evaluated the in vitro and in vivo antitumor efficacy of mTOR inhibitors, rapamycin and WYE-354. In vitro assays showed WYE-354 significantly reduced cell viability, migration and invasion and phospho-P70S6K expression in GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with WYE-354 or rapamycin, exhibited a significant reduction in tumor mass. A short-term treatment with a higher dose of WYE-354 decreased the tumor size by 68.6% and 52.4%, in mice harboring G-415 or TGBC-2TKB tumors, respectively, compared to the control group. By contrast, treatment with a prolonged-low-dose regime of rapamycin almost abrogated tumor growth, exhibiting 92.7% and 97.1% reduction in tumor size, respectively, compared to control mice. These results were accompanied by a greater decrease in the phosphorylation status of P70S6K and a lower cell proliferation Ki67 index, compared to WYE-354 treated mice, suggesting a more effective mTOR pathway inhibition. These findings provide a proof of concept for the use of rapamycin or WYE-354 as potentially good candidates to be studied in clinical trials in GBC patients.
Background: Clinical manifestations and outcomes of atherosclerotic disease differ between ethnic groups. In addition, the prevalence of risk factors is substantially different. Primary prevention programs are based on data derived from almost exclusively White people. We investigated how race/ethnic differences modify the associations of established risk factors with atherosclerosis and cardiovascular events.
Methods: We used data from an ongoing individual participant meta-analysis involving 17 population-based cohorts worldwide. We selected 60,211 participants without cardiovascular disease at baseline with available data on ethnicity (White, Black, Asian or Hispanic). We generated a multivariable linear regression model containing risk factors and ethnicity predicting mean common carotid intima-media thickness (CIMT) and a multivariable Cox regression model predicting myocardial infarction or stroke. For each risk factor we assessed how the association with the preclinical and clinical measures of cardiovascular atherosclerotic disease was affected by ethnicity.
Results: Ethnicity appeared to significantly modify the associations between risk factors and CIMT and cardiovascular events. The association between age and CIMT was weaker in Blacks and Hispanics. Systolic blood pressure associated more strongly with CIMT in Asians. HDL cholesterol and smoking associated less with CIMT in Blacks. Furthermore, the association of age and total cholesterol levels with the occurrence of cardiovascular events differed between Blacks and Whites.
Conclusion: The magnitude of associations between risk factors and the presence of atherosclerotic disease differs between race/ethnic groups. These subtle, yet significant differences provide insight in the etiology of cardiovascular disease among race/ethnic groups. These insights aid the race/ethnic-specific implementation of primary prevention.
In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.
The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.
Background: High reproducibility of LV mass and volume measurement from cine cardiovascular magnetic resonance (CMR) has been shown within single centers. However, the extent to which contours may vary from center to center, due to different training protocols, is unknown. We aimed to quantify sources of variation between many centers, and provide a multi-center consensus ground truth dataset for benchmarking automated processing tools and facilitating training for new readers in CMR analysis.
Methods: Seven independent expert readers, representing seven experienced CMR core laboratories, analyzed fifteen cine CMR data sets in accordance with their standard operating protocols and SCMR guidelines. Consensus contours were generated for each image according to a statistical optimization scheme that maximized contour placement agreement between readers.
Results: Reader-consensus agreement was better than inter-reader agreement (end-diastolic volume 14.7 ml vs 15.2–28.4 ml; end-systolic volume 13.2 ml vs 14.0–21.5 ml; LV mass 17.5 g vs 20.2–34.5 g; ejection fraction 4.2 % vs 4.6–7.5 %). Compared with consensus contours, readers were very consistent (small variability across cases within each reader), but bias varied between readers due to differences in contouring protocols at each center. Although larger contour differences were found at the apex and base, the main effect on volume was due to small but consistent differences in the position of the contours in all regions of the LV.
Conclusions: A multi-center consensus dataset was established for the purposes of benchmarking and training. Achieving consensus on contour drawing protocol between centers before analysis, or bias correction after analysis, is required when collating multi-center results.
Background: Cardiovascular magnetic resonance (CMR) offers quantification of phasic atrial functions based on volumetric assessment and more recently, on CMR feature tracking (CMR-FT) quantitative strain and strain rate (SR) deformation imaging. Inter-study reproducibility is a key requirement for longitudinal studies but has not been defined for CMR-based quantification of left atrial (LA) and right atrial (RA) dynamics.
Methods: Long-axis 2- and 4-chamber cine images were acquired at 9:00 (Exam A), 9:30 (Exam B) and 14:00 (Exam C) in 16 healthy volunteers. LA and RA reservoir, conduit and contractile booster pump functions were quantified by volumetric indexes as derived from fractional volume changes and by strain and SR as derived from CMR-FT. Exam A and B were compared to assess the inter-study reproducibility. Morning and afternoon scans were compared to address possible diurnal variation of atrial function.
Results: Inter-study reproducibility was within acceptable limits for all LA and RA volumetric, strain and SR parameters. Inter-study reproducibility was better for volumetric indexes and strain than for SR parameters and better for LA than for RA dynamics. For the LA, reservoir function showed the best reproducibility (intraclass correlation coefficient (ICC) 0.94–0.97, coefficient of variation (CoV) 4.5–8.2 %), followed by conduit (ICC 0.78–0.97, CoV 8.2–18.5 %) and booster pump function (ICC 0.71–0.95, CoV 18.3–22.7). Similarly, for the RA, reproducibility was best for reservoir function (ICC 0.76–0.96, CoV 7.5–24.0 %) followed by conduit (ICC 0.67–0.91, CoV 13.9–35.9) and booster pump function (ICC 0.73–0.90, CoV 19.4–32.3). Atrial dynamics were not measurably affected by diurnal variation between morning and afternoon scans.
Conclusions: Inter-study reproducibility for CMR-based derivation of LA and RA functions is acceptable using either volumetric, strain or SR parameters with LA function showing higher reproducibility than RA function assessment. Amongst the different functional components, reservoir function is most reproducibly assessed by either technique followed by conduit and booster pump function, which needs to be considered in future longitudinal research studies.
Einleitung: Der Wissenschaftsrat empfahl 2008 den Universitäten innerhalb der nächsten 5 Jahre, d. h. bis spätestens 2014, ein Qualitätsmanagementsystem (QMS), das internationalen Maßstäben entspricht, zu etablieren. Ziel der vorliegenden Studie war es, zu evaluieren, ob es derzeit ein geeignetes QMS für das elektronische Lernen (eLearning) gibt, das speziell im Fach Humanmedizin deutschlandweit eingesetzt werden kann.
Methoden: Im Rahmen einer Umfrage wurden mittels eines anonymisierten Fragebogens (8 Domänen, 50 Items) alle Universitäten (n=35) des Fachbereichs Medizin in Deutschland evaluiert.
Ergebnisse: Die Ergebnisse (46,3% Rücklaufquote) zeigen einen nur zögerlichen Einsatz von QMS für eLearning und dass vor Ort ein großes Informationsdefizit herrscht.
Schlussfolgerung: Unter Berücksichtigung der Limitationen dieser Studie kann zusammenfassend festgehalten werden, dass erheblicher Bedarf zu bestehen scheint, das existierende Informationsdefizit für QMS eLearning zu mindern, sowie zukünftig genaue Richtlinien und Standards zur Umsetzung zu definieren.
Introduction: In 2008, the German Council of Science had advised universities to establish a quality management system (QMS) that conforms to international standards. The system was to be implemented within 5 years, i.e., until 2014 at the latest. The aim of the present study was to determine whether a QMS suitable for electronic learning (eLearning) domain of medical education to be used across Germany has meanwhile been identified.
Methods: We approached all medical universities in Germany (n=35), using an anonymous questionnaire (8 domains, 50 items).
Results: Our results (response rate 46.3%) indicated very reluctant application of QMS in eLearning and a major information deficit at the various institutions.
Conclusions: Authors conclude that under the limitations of this study there seems to be a considerable need to improve the current knowledge on QMS for eLearning, and that clear guidelines and standards for their implementation should be further defined.
Introduction: ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of the craniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1) is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention.
Methods: twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2.
Results: moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2.
Conclusion: the importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents.
We provide a comprehensive classification of the proteoglycan gene families and respective protein cores. This updated nomenclature is based on three criteria: Cellular and subcellular location, overall gene/protein homology, and the utilization of specific protein modules within their respective protein cores. These three signatures were utilized to design four major classes of proteoglycans with distinct forms and functions: the intracellular, cell-surface, pericellular and extracellular proteoglycans. The proposed nomenclature encompasses forty-three distinct proteoglycan-encoding genes and many alternatively-spliced variants. The biological functions of these four proteoglycan families are critically assessed in development, cancer and angiogenesis, and in various acquired and genetic diseases where their expression is aberrant.
Sleep has been shown to subtly disrupt the spatial organization of functional connectivity networks in the brain, but in a way that largely preserves the connectivity within sensory cortices. Here we evaluated the hypothesis that sleep does impact sensory cortices, but through alteration of activity dynamics. We therefore examined the impact of sleep on hemodynamics using a method for quantifying non-random, high frequency signatures of the blood-oxygen-level dependent (BOLD) signal (amplitude variance asymmetry; AVA). We found that sleep was associated with the elimination of these dynamics in a manner that is restricted to auditory, motor and visual cortices. This elimination was concurrent with increased variance of activity in these regions. Functional connectivity between regions showing AVA during wakefulness maintained a relatively consistent hierarchical structure during wakefulness and N1 and N2 sleep, despite a gradual reduction of connectivity strength as sleep progressed. Thus, sleep is related to elimination of high frequency non-random activity signatures in sensory cortices that are robust during wakefulness. The elimination of these AVA signatures conjointly with preservation of the structure of functional connectivity patterns may be linked to the need to suppress sensory inputs during sleep while still maintaining the capacity to react quickly to complex multimodal inputs.
The α-defensins, human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed in promyelocytes and stored as mature HNPs in azurophil granules. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme that processes proHNPs nor the localization of processing has been identified. It has been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which are able to process recombinant proHNP into HNP in vitro. We investigated whether serine proteases are in fact responsible for processing of proHNP in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing in primary human bone marrow cells. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not enhance the proHNP processing capability. Finally, two patients suffering from Papillon–Lefèvre syndrome, who lack active neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s).
Background: The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry.
Methods: HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR.
Results: Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA.
Conclusions: Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.
Background: Although the risk of developing colorectal cancer (CRC) is 2-4 times higher in case of a positive family history, risk-adapted screening programs for family members related to CRC- patients do not exist in the German health care system. CRC screening recommendations for persons under 55 years of age that have a family predisposition have been published in several guidelines.
The primary aim of this study is to determine the frequency of positive family history of CRC (1st degree relatives with CRC) among 40–54 year old persons in a general practitioner (GP) setting in Germany. Secondary aims are to detect the frequency of occurrence of colorectal neoplasms (CRC and advanced adenomas) in 1st degree relatives of CRC patients and to identify the variables (e.g. demographic, genetic, epigenetic and proteomic characteristics) that are associated with it. This study also explores whether evidence-based information contributes to informed decisions and how screening participation correlates with anxiety and (anticipated) regret.
Methods/Design: Prior to the beginning of the study, the GP team (GP and one health care assistant) in around 50 practices will be trained, and about 8,750 persons that are registered with them will be asked to complete the “Network against colorectal cancer” questionnaire. The 10 % who are expected to have a positive family history will then be invited to give their informed consent to participate in the study. All individuals with positive family history will be provided with evidence-based information and prevention strategies. We plan to examine each participant’s family history of CRC in detail and to collect information on further variables (e.g. demographics) associated with increased risk. Additional stool and blood samples will be collected from study-participants who decide to undergo a colonoscopy (n ~ 350) and then analyzed at the German Cancer Research Center (DKFZ) Heidelberg to see whether further relevant variables are associated with an increased risk of CRC. One screening list and four questionnaires will be used to collect the data, and a detailed statistical analysis plan will be provided before the database is closed (expected to be June 30, 2015).
Discussion: It is anticipated that when persons with a family history of colorectal cancer have been provided with professional advice by the practice team, there will be an increase in the availability of valid information on the frequency of affected individuals and an increase in the number of persons making informed decisions. We also expect to identify further variables that are associated with colorectal cancer. This study therefore has translational relevance from lab to practice.
Trial registration: German Clinical Trials Register DRKS00006277
Polo-like kinase 1 inhibition sensitizes neuroblastoma cells for vinca alkaloid-induced apoptosis
(2015)
High polo-like kinase 1 (PLK1) expression has been linked to poor outcome in neuroblastoma (NB), indicating that it represents a relevant therapeutic target in this malignancy. Here, we identify a synergistic induction of apoptosis by the PLK1 inhibitor BI 2536 and vinca alkaloids in NB cells. Synergistic drug interaction of BI 2536 together with vincristine (VCR), vinblastine (VBL) or vinorelbine (VNR) is confirmed by calculation of combination index (CI). Also, BI 2536 and VCR act in concert to reduce long-term clonogenic survival. Importantly, BI 2536 significantly enhances the antitumor activity of VCR in an in vivo model of NB. Mechanistically, BI 2536/VCR co-treatment triggers prolonged mitotic arrest, which is necessary for BI 2536/VCR-mediated apoptosis, since pharmacological inhibition of mitotic arrest by the CDK1 inhibitor RO-3306 significantly reduces cell death. Prolonged mitotic arrest leads to phosphorylation-mediated inactivation of BCL-2 and BCL-XL as well as downregulation of MCL-1, since inhibition of mitotic arrest by RO-3306 also prevents phosphorylation of BCL-2 and BCL-XL and MCL-1 downregulation. This inactivation of antiapoptotic BCL-2 proteins promotes activation of BAX and BAK, cleavage of caspase-9 and -3 and caspase-dependent apoptosis. Engagement of the mitochondrial pathway of apoptosis is critically required for BI 2536/VCR-induced apoptosis, since ectopic expression of a non-degradable MCL-1 phospho-mutant, BCL-2 overexpression or BAK knockdown significantly reduce BI 2536/VCR-mediated apoptosis. Thus, PLK1 inhibitors may open new perspectives for chemosensitization of NB.
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(2015)
BACKGROUND: Human SAMHD1 is a triphosphohydrolase that restricts the replication of retroviruses, retroelements and DNA viruses in noncycling cells. While modes of action have been extensively described for human SAMHD1, only little is known about the regulation of SAMHD1 in the mouse. Here, we characterize the antiviral activity of murine SAMHD1 with the help of knockout mice to shed light on the regulation and the mechanism of the SAMHD1 restriction and to validate the SAMHD1 knockout mouse model for the use in future infectivity studies.
RESULTS: We found that endogenous mouse SAMHD1 restricts not only HIV-1 but also MLV reporter virus infection at the level of reverse transcription in primary myeloid cells. Similar to the human protein, the antiviral activity of murine SAMHD1 is regulated through phosphorylation at threonine 603 and is limited to nondividing cells. Comparing the susceptibility to infection with intracellular dNTP levels and SAMHD1 phosphorylation in different cell types shows that both functions are important determinants of the antiviral activity of murine SAMHD1. In contrast, we found the proposed RNase activity of SAMHD1 to be less important and could not detect any effect of mouse or human SAMHD1 on the level of incoming viral RNA.
CONCLUSION: Our findings show that SAMHD1 in the mouse blocks retroviral infection at the level of reverse transcription and is regulated through cell cycle-dependent phosphorylation. We show that the antiviral restriction mediated by murine SAMHD1 is mechanistically similar to what is known for the human protein, making the SAMHD1 knockout mouse model a valuable tool to characterize the influence of SAMHD1 on the replication of different viruses in vivo.
Background: Hypoxia is a key driver for infiltrative growth in experimental gliomas. It has remained elusive whether tumor hypoxia in glioblastoma patients contributes to distant or diffuse recurrences. We therefore investigated the influence of perioperative cerebral ischemia on patterns of progression in glioblastoma patients.
Methods: We retrospectively screened MRI scans of 245 patients with newly diagnosed glioblastoma undergoing resection for perioperative ischemia near the resection cavity. 46 showed relevant ischemia nearby the resection cavity. A control cohort without perioperative ischemia was generated by a 1:1 matching using an algorithm based on gender, age and adjuvant treatment. Both cohorts were analyzed for patterns of progression by a blinded neuroradiologist.
Results: The percentage of diffuse or distant recurrences at first relapse was significantly higher in the cohort with perioperative ischemia (61.1%) compared to the control cohort (19.4%). The results of the control cohort matched well with historical data. The change in patterns of progression was not associated with a difference in survival.
Conclusions: This study reveals an unrecognized association of perioperative cerebral ischemia with distant or diffuse recurrence in glioblastoma. It is the first clinical study supporting the concept that hypoxia is a key driver of infiltrative tumor growth in glioblastoma patients.
BACKGROUND: Plasminogen deficiency is a rare autosomal recessive disease, which is associated with aggressive periodontitis and gingival enlargement. Previously described treatments of plasminogen deficiency associated periodontitis have shown limited success. This is the first case report indicating a successful therapy approach consisting of a non-surgical supra- and subgingival debridement in combination with an adjunctive systemic antibiotic therapy and a strict supportive periodontal regimen over an observation period of 4 years.
CASE PRESENTATION: The intraoral examination of a 17-year-old Turkish female with severe plasminogen deficiency revealed generalized increased pocket probing depths ranging from 6 to 9 mm, bleeding on probing over 30%, generalized tooth mobility, and gingival hyperplasia. Alveolar bone loss ranged from 30% to 50%. Clinical attachment loss corresponded to pocket probing depths. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Prevotella nigrescens and Eikenella corrodens have been detected by realtime polymerase chain reaction. Periodontal treatment consisted of full mouth disinfection and adjunctive systemic administration of amoxicillin (500 mg tid) and metronidazole (400 mg tid). A strict supportive periodontal therapy regimen every three month in terms of supra- and subgingival debridement was rendered. The reported therapy has significantly improved periodontal health and arrested disease progression. Intraoral examination at the end of the observation period 3.5 years after non-surgical periodontal therapy showed generalized decreased pocket probing depths ranging from 1 to 6 mm, bleeding on probing lower 30%, and tooth mobility class I and II. Furthermore, microbiological analysis shows the absence of Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola after therapy.
CONCLUSION: Adjunctive antibiotic treatment may alter the oral microbiome and thus, the inflammatory response of periodontal disease associated to plasminogen deficiency and diminishes the risk of pseudomembrane formation and progressive attachment loss. This case report indicates that patients with plasminogen deficiency may benefit from non-surgical periodontal treatment in combination with an adjunctive antibiotic therapy and a strict supportive periodontal therapy regimen.
Aims: We sought to describe perfusion dyssynchrony analysis specifically to exploit the high temporal resolution of stress perfusion CMR. This novel approach detects differences in the temporal distribution of the wash-in of contrast agent across the left ventricular wall.
Methods and results: Ninety-eight patients with suspected coronary artery disease (CAD) were retrospectively identified. All patients had undergone perfusion CMR at 3T and invasive angiography with fractional flow reserve (FFR) of lesions visually judged >50% stenosis. Stress images were analysed using four different perfusion dyssynchrony indices: the variance and coefficient of variation of the time to maximum signal upslope (V-TTMU and C-TTMU) and the variance and coefficient of variation of the time to peak myocardial signal enhancement (V-TTP and C-TTP). Patients were classified according to the number of vessels with haemodynamically significant CAD indicated by FFR <0.8. All indices of perfusion dyssynchrony were capable of identifying the presence of significant CAD. C-TTP >10% identified CAD with sensitivity 0.889, specificity 0.857 (P < 0.0001). All indices correlated with the number of diseased vessels. C-TTP >12% identified multi-vessel disease with sensitivity 0.806, specificity 0.657 (P < 0.0001). C-TTP was also the dyssynchrony index with the best inter- and intra-observer reproducibility. Perfusion dyssynchrony indices showed weak correlation with other invasive and non-invasive measurements of the severity of ischaemia, including FFR, visual ischaemic burden, and MPR.
Conclusion: These findings suggest that perfusion dyssynchrony analysis is a robust novel approach to the analysis of first-pass perfusion and has the potential to add complementary information to aid assessment of CAD.
Background: SNPs near the interferon lambda (IFNL) 3 gene are predictors for sustained virological response (SVR) in patients with chronic hepatitis C genotype (GT) 1. In addition, a dinucleotide frame shift in ss469415590 was described, which generates IFNL4. In this study, we compared the role of IFNL4 variants with IFNL3-(rs12979860) and IFNL3-(rs8099917) on response to pegylated (PEG)-IFN and Ribavirin (RBV) in patients with chronic hepatitis C GT2/3.
Methods: We recruited 1006 patients with chronic hepatitis C and GT2/3 in a large German registry. A treatment with PEG-IFN and Ribavirin was started by 959 patients. We performed genotyping of IFNL3 (rs12979860, n = 726; rs8099917, n = 687) and of IFNL4 (ss469415590; n = 631).
Results: Both preferable IFNL3 genotypes were associated with RVR (both p<0.0001) rather than with SVR (rs12979860: p = 0.251; rs8099917: p = 0.447). Only RVR was linked to SVR in univariate and multivariate analyzes (both p<0.001). Concordance of genotyping in patients with available serum samples and EDTA blood samples (n = 259) was more than 96% for both IFNL3 SNPs. IFNL3-(rs12979860) correlated with IFNL4: 99.2% of patients with IFNL3-(rs12979860)-CC were IFNL4-(ss469415590)-TT/TT. IFNL3-(rs12979860)-CT was linked with IFNL4-(ss469415590)-TT/ΔG (98.0%) and IFNL3-(rs12979860)-TT was associated with IFNL4-(ss469415590)-ΔG/ΔG (97.6%).
Conclusion: IFNL3 genotyping from serum was highly efficient and can be used as an alternative if EDTA whole blood is not available. In Caucasian GT2/3 patients genotyping for INFL4-(ss469415590) does not lead to additional information for the decision-making process. Importantly, IFNL3 SNPs were not associated with SVR but with RVR. Even in the era of new direct acting antiviral (DAA) therapies, IFNL3 testing may therefore still be considered for naïve GT2/3 patients to decide if dual Peg-IFN/RBV therapy is an option in resource limited regions.
In many neural systems anatomical motifs are present repeatedly, but despite their structural similarity they can serve very different tasks. A prime example for such a motif is the canonical microcircuit of six-layered neo-cortex, which is repeated across cortical areas, and is involved in a number of different tasks (e.g. sensory, cognitive, or motor tasks). This observation has spawned interest in finding a common underlying principle, a ‘goal function’, of information processing implemented in this structure. By definition such a goal function, if universal, cannot be cast in processing-domain specific language (e.g. ‘edge filtering’, ‘working memory’). Thus, to formulate such a principle, we have to use a domain-independent framework. Information theory offers such a framework. However, while the classical framework of information theory focuses on the relation between one input and one output (Shannon’s mutual information), we argue that neural information processing crucially depends on the combination of multiple inputs to create the output of a processor. To account for this, we use a very recent extension of Shannon Information theory, called partial information decomposition (PID). PID allows to quantify the information that several inputs provide individually (unique information), redundantly (shared information) or only jointly (synergistic information) about the output. First, we review the framework of PID. Then we apply it to reevaluate and analyze several earlier proposals of information theoretic neural goal functions (predictive coding, infomax and coherent infomax, efficient coding). We find that PID allows to compare these goal functions in a common framework, and also provides a versatile approach to design new goal functions from first principles. Building on this, we design and analyze a novel goal function, called ‘coding with synergy’, which builds on combining external input and prior knowledge in a synergistic manner. We suggest that this novel goal function may be highly useful in neural information processing.
Background: From 2008–2013, the European indication for panitumumab required that patients' tumor KRAS exon 2 mutation status was known prior to starting treatment. To evaluate physician awareness of panitumumab prescribing information and how physicians prescribe panitumumab in patients with metastatic colorectal cancer (mCRC), two European multi-country, cross-sectional, observational studies were initiated in 2012: a physician survey and a medical records review. The first two out of three planned rounds for each study are reported.
Methods: The primary objective in the physician survey was to estimate the prevalence of KRAS testing, and in the medical records review, it was to evaluate the effect of test results on patterns of panitumumab use. The medical records review study also included a pathologists' survey.
Results: In the physician survey, nearly all oncologists (299/301) were aware of the correct panitumumab indication and the need to test patients' tumor KRAS status before treatment with panitumumab. Nearly all oncologists (283/301) had in the past 6 months of clinical practice administered panitumumab correctly to mCRC patients with wild-type KRAS status. In the medical records review, 97.5% of participating oncologists (77/79) conducted a KRAS test for all of their patients prior to prescribing panitumumab. Four patients (1.3%) did not have tumor KRAS mutation status tested prior to starting panitumumab treatment. Approximately one-quarter of patients (85/306) were treated with panitumumab and concurrent oxaliplatin-containing chemotherapy; of these, 83/85 had confirmed wild-type KRAS status prior to starting panitumumab treatment. All 56 referred laboratories that participated used a Conformité Européenne-marked or otherwise validated KRAS detection method, and nearly all (55/56) participated in a quality assurance scheme.
Conclusions: There was a high level of knowledge amongst oncologists around panitumumab prescribing information and the need to test and confirm patients' tumors as being wild-type KRAS prior to treatment with panitumumab, with or without concurrent oxaliplatin-containing therapy.
Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Defects in flow-induced NO formation go along with endothelial dysfunction, initiation and progression of atherosclerosis as well as with arterial hypertension. Previous work has identified several mechanotransducing signaling processes involved in fluid shear stress-induced endothelial effects. But how fluid shear stress initiates the response is poorly understood. Here, I show in human and bovine endothelial cells that the G-protein Gq/G11 and the purinergic receptor P2Y2 mediate fluid shear stress-induced endothelial responses such as Ca2+ release, nitric oxide (NO) formation and the phosphorylation of platelet-endothelial-cell-adhesion-molecule-1 (PECAM-1), vascular endothelial growth factor-2 (VEGFR-2) and Akt kinase as well as activation of the endothelial NO synthase (eNOS). P2Y2 receptor is activated by adenosine triphosphate (ATP) which is released from endothelial cells under the influence of fluid shear stress. Arteries with P2Y2 or Gαq/Gα11 deficiency have impaired flow-induced dilatation. Mice with induced endothelium-specific deficiency of P2Y2 or Gαq/Gα11 develop hypertension which is accompanied by reduced eNOS activation. My data identify P2Y2 and Gq/G11 as a critical endothelial mechano-signaling pathway located upstream of mechanotransducing processes described so far. Moreover, I demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone and blood pressure.
Background & Aims: Hepatitis C virus (HCV) cell entry is mediated by several cell surface receptors, including scavenger receptor class B type I (SR-BI). Oxidized low density lipoprotein (oxLDL) inhibits the interaction between HCV and SR-BI in a noncompetitive manner. We tested whether serum oxLDL levels correlate with sustained virologic response (SVR) rates after interferon-based treatment of chronic hepatitis C.
Methods: Baseline oxLDL was determined in 379 participants with chronic HCV genotype 1 infection from the INDIV-2 study using a commercial enzyme-linked immunosorbent assay. The mechanistic in vitro studies used full-length and subgenomic HCV genomes replicating in hepatoma cells.
Results: In the multivariate analysis, oxLDL was found to be an independent predictor of SVR. Oxidized LDL did not correlate with markers of inflammation (alanine transaminase, ferritin), nor was serum oxLDL affected by exogenous interferon administration. Also, oxLDL did not alter the sensitivity of HCV replication to interferon. However, oxLDL was found to be a potent inhibitor of cell-to-cell spread of HCV between adjacent cells in vitro. It could thus reduce the rate at which new cells are infected by HCV through either the cell-free or cell-to-cell route. Finally, serum oxLDL was significantly associated with the estimated infected cell loss rate under treatment.
Conclusions: Oxidized LDL is a novel predictor of SVR after interferon-based therapy and may explain the previously observed association of LDL with SVR. Rather than being a marker of activated antiviral defenses it may improve chances of SVR by limiting spread of infection to naive cells through the cell-to-cell route.
The extracellular matrix is rapidly emerging as a prominent contributor to various fundamental processes of tumorigenesis. In particular, decorin, a member of the small leucine-rich proteoglycan gene family, is assuming a central role as a potent soluble tumor repressor. Decorin binds and antagonizes various receptor tyrosine kinases and inhibits downstream oncogenic signaling in several solid tumors. Among other functions, decorin evokes cell cycle arrest, apoptosis, and antimetastatic, and antiangiogenic programs. Recent work has revealed a paradigmatic shift in our understanding of the molecular mechanisms underlying its tumoricidal properties. Decorin adversely compromises the genetic signature of the tumor microenvironment and induces endothelial cell autophagy downstream of VEGFR2. Moreover, decorin selectively evokes destruction of tumor cell mitochondria downstream of Met through mitophagy. Acting as a partial agonist, decorin signals via proautophagic receptors and triggers procatabolic processes that parallel the classical tumoricidal properties of this multifaceted proteoglycan.
An increasing body of evidences from preclinical as well as epidemiological and clinical studies suggest a potential beneficial role of dietary intake of omega-3 fatty acids for cognitive functioning. In this narrative review, we will summarize and discuss recent findings from epidemiological, interventional and experimental studies linking dietary consumption of omega-3 fatty acids to cognitive function in healthy adults. Furthermore, affective disorders and schizophrenia (SZ) are characterized by cognitive dysfunction encompassing several domains. Cognitive dysfunction is closely related to impaired functioning and quality of life across these conditions. Therefore, the current review focues on the potential influence of omega-3 fatty acids on cognition in SZ and affective disorders. In sum, current data predominantly from mechanistic models and animal studies suggest that adjunctive omega-3 fatty acid supplementation could lead to improved cognitive functioning in SZ and affective disorders. However, besides its translational promise, evidence for clinical benefits in humans has been mixed. Notwithstanding evidences indicate that adjunctive omega-3 fatty acids may have benefit for affective symptoms in both unipolar and bipolar depression, to date no randomized controlled trial had evaluated omega-3 as cognitive enhancer for mood disorders, while a single published controlled trial suggested no therapeutic benefit for cognitive improvement in SZ. Considering the pleiotropic mechanisms of action of omega-3 fatty acids, the design of well-designed controlled trials of omega-3 supplementation as a novel, domain-specific, target for cognitive impairment in SZ and affective disorders is warranted.
Introduction: By producing H2O2, the NADPH oxidase Nox4 is involved in differentiation of mesenchymal cells. Exercise alters the composition of slow and fast twitch fibres in skeletal. Here we hypothesized that Nox4 contributes to exercise-induced adaptation such as changes in muscle metabolism or muscle fibre specification and studied this in wildtype and Nox4-/- mice.
Results: Exercise, as induced by voluntary running in a running wheel or forced running on a treadmill induced a switch from fast twitch to intermediate fibres. However the induced muscle fibre switch was similar between Nox4-/- and wildtype mice. The same held true for exercise-induced expression of PGC1α or AMPK activation. Both are increased in response to exercise, but with no difference was observed between wildtype and Nox4-/- mice.
Conclusion: Thus, exercise-induced muscle fibre switch is Nox4-independent.
Novel insights into the regulation of cyclooxygenase-2 expression by platelet-cancer cell cross-talk
(2015)
Platelets are activated by the interaction with cancer cells and release enhanced levels of lipid mediators [such as thromboxane (TX)A2 and prostaglandin (PG)E2, generated from arachidonic acid (AA) by the activity of cyclooxygenase (COX)-1], granule content, including ADP and growth factors, chemokines, proteases and Wnt proteins. Moreover, activated platelets shed different vesicles, such as microparticles (MPs) and exosomes (rich in genetic material such as mRNAs and miRNAs). These platelet-derived products induce several phenotypic changes in cancer cells which confer high metastatic capacity. A central event involves an aberrant expression of COX-2 which influences cell-cycle progression and contribute to the acquisition of a cell migratory phenotype through the induction of epithelial mesenchymal transition genes and down-regulation of E-cadherin expression. The identification of novel molecular determinants involved in the cross-talk between platelets and cancer cells has led to identify novel targets for anti-cancer drug development.
Non-VKA oral anticoagulants (NOACs) have now widely reached the lucrative market of anticoagulation. While the marketing authorization holders claimed that no routine monitoring is required and that these compounds can be given at fixed doses, several evidences arisen from the literature tend to demonstrate the opposite. New data suggests that an assessment of the response at the individual level could improve the benefit-risk ratio of at least dabigatran. Information regarding the association of rivaroxaban and apixaban exposure and the bleeding risk is available in the drug approval package on the FDA website. These reviews suggest that accumulation of these compounds increases the risk of experiencing a bleeding complication. Therefore, in certain patient populations such as patients with acute or chronic renal impairment or with multiple drug interactions, measurement of drug exposure may be useful to ensure an optimal treatment response. More specific circumstances such as patients experiencing a haemorrhagic or thromboembolic event during the treatment duration, patients who require urgent surgery or an invasive procedure, or patient with a suspected overdose could benefit from such a measurement. This paper aims at providing guidance on how to best estimate the intensity of anticoagulation using laboratory assays in daily practice.
Disorder-relevant but task-unrelated stimuli impair cognitive performance in social anxiety disorder (SAD); however, time course and neural correlates of emotional interference are unknown. The present study investigated time course and neural basis of emotional interference in SAD using event-related functional magnetic resonance imaging (fMRI). Patients with SAD and healthy controls performed an emotional stroop task which allowed examining interference effects on the current and the succeeding trial. Reaction time data showed an emotional interference effect in the current trial, but not the succeeding trial, specifically in SAD. FMRI data showed greater activation in the left amygdala, bilateral insula, medial prefrontal cortex (mPFC), dorsal anterior cingulate cortex (ACC), and left opercular part of the inferior frontal gyrus during emotional interference of the current trial in SAD patients. Furthermore, we found a positive correlation between patients’ interference scores and activation in the mPFC, dorsal ACC and left angular/supramarginal gyrus. Taken together, results indicate a network of brain regions comprising amygdala, insula, mPFC, ACC, and areas strongly involved in language processing during the processing of task-unrelated threat in SAD. However, specifically the activation in mPFC, dorsal ACC, and left angular/supramarginal gyrus is associated with the strength of the interference effect, suggesting a cognitive network model of attentional bias in SAD. This probably comprises exceeded allocation of attentional resources to disorder-related information of the presented stimuli and increased self-referential and semantic processing of threat words in SAD.
Multiple antenatal dexamethasone treatment alters brain vessel differentiation in newborn mouse pups
(2015)
Antenatal steroid treatment decreases morbidity and mortality in premature infants through the maturation of lung tissue, which enables sufficient breathing performance. However, clinical and animal studies have shown that repeated doses of glucocorticoids such as dexamethasone and betamethasone lead to long-term adverse effects on brain development. Therefore, we established a mouse model for antenatal dexamethasone treatment to investigate the effects of dexamethasone on brain vessel differentiation towards the blood-brain barrier (BBB) phenotype, focusing on molecular marker analysis. The major findings were that in total brains on postnatal day (PN) 4 triple antenatal dexamethasone treatment significantly downregulated the tight junction protein claudin-5, the endothelial marker Pecam-1/CD31, the glucocorticoid receptor, the NR1 subunit of the N-methyl-D-aspartate receptor, and Abc transporters (Abcb1a, Abcg2 Abcc4). Less pronounced effects were found after single antenatal dexamethasone treatment and in PN10 samples. Comparisons of total brain samples with isolated brain endothelial cells together with the stainings for Pecam-1/CD31 and claudin-5 led to the assumption that the morphology of brain vessels is affected by antenatal dexamethasone treatment at PN4. On the mRNA level markers for angiogenesis, the sonic hedgehog and the Wnt pathway were downregulated in PN4 samples, suggesting fundamental changes in brain vascularization and/or differentiation. In conclusion, we provided a first comprehensive molecular basis for the adverse effects of multiple antenatal dexamethasone treatment on brain vessel differentiation.
The study of large-scale functional interactions in the human brain with functional magnetic resonance imaging (fMRI) extends almost to the first applications of this technology. Due to historical reasons and preconceptions about the limitations of this brain imaging method, most studies have focused on assessing connectivity over extended periods of time. It is now clear that fMRI can resolve the temporal dynamics of functional connectivity, like other faster imaging techniques such as electroencephalography and magnetoencephalography (albeit on a different temporal scale). However, the indirect nature of fMRI measurements can hinder the interpretability of the results. After briefly summarizing recent advances in the field, we discuss how the simultaneous combination of fMRI with electrophysiological activity measurements can contribute to a better understanding of dynamic functional connectivity in humans both during rest and task, wakefulness, and other brain states.
Background: It is assumed that different pain phenotypes are based on varying molecular pathomechanisms. Distinct ion channels seem to be associated with the perception of cold pain, in particular TRPM8 and TRPA1 have been highlighted previously. The present study analyzed the distribution of cold pain thresholds with focus at describing the multimodality based on the hypothesis that it reflects a contribution of distinct ion channels.
Methods: Cold pain thresholds (CPT) were available from 329 healthy volunteers (aged 18 - 37 years; 159 men) enrolled in previous studies. The distribution of the pooled and log-transformed threshold data was described using a kernel density estimation (Pareto Density Estimation (PDE)) and subsequently, the log data was modeled as a mixture of Gaussian distributions using the expectation maximization (EM) algorithm to optimize the fit.
Results: CPTs were clearly multi-modally distributed. Fitting a Gaussian Mixture Model (GMM) to the log-transformed threshold data revealed that the best fit is obtained when applying a three-model distribution pattern. The modes of the identified three Gaussian distributions, retransformed from the log domain to the mean stimulation temperatures at which the subjects had indicated pain thresholds, were obtained at 23.7 °C, 13.2 °C and 1.5 °C for Gaussian #1, #2 and #3, respectively.
Conclusions: The localization of the first and second Gaussians was interpreted as reflecting the contribution of two different cold sensors. From the calculated localization of the modes of the first two Gaussians, the hypothesis of an involvement of TRPM8, sensing temperatures from 25 - 24 °C, and TRPA1, sensing cold from 17 °C can be derived. In that case, subjects belonging to either Gaussian would possess a dominance of the one or the other receptor at the skin area where the cold stimuli had been applied. The findings therefore support a suitability of complex analytical approaches to detect mechanistically determined patterns from pain phenotype data.
An accurate quantification of low viremic HCV RNA plasma samples has gained importance since the approval of direct acting antivirals and since only one single measurement predicts the necessity of a prolonged or shortened therapy. As reported previously, HCV quantification assays such as Abbott RealTime HCV and Roche COBAS AmpliPrep/COBAS TaqMan HCV version 2 (CTM v2) may vary in sensitivity and precision particularly in low-level viremia. Importantly, substantial variations were previously demonstrated between some of these assays compared to the Roche High Pure System/COBAS TaqMan assay (HPS) reference assay, which was used to establish the clinical decision points in clinical studies. In this study, the reproducibility of assay performances across several laboratories was assessed by analysing quantification results generated by six independent laboratories (3× RealTime, 3× CTM v2) in comparison with one HPS reference laboratory. The 4th WHO Standard was diluted to 100, 25 and 10 IU/ml, and aliquots were tested in triplicates in 5 independent runs by each assay in the different laboratories to assess assay precision and detection rates. In a second approach, 2 clinical samples (GT 1a & GT 1b) were diluted to 100 and 25 IU/ml and tested as described above. While the result range for WHO 100 IU/ml replicates across all laboratories was similar in this analysis, the CVs of each laboratory ranged from 19.3 to 25.6 % for RealTime laboratories and were lower than CVs of CTM v2 laboratories with a range of 26.1–47.3 %, respectively, and also in comparison with the CV of the HPS reference laboratory (34.9 %). At WHO standard dilution of 25 IU/ml, 24 replicates were quantified by RealTime compared to 8 replicates with CTM v2. Results of clinical samples again revealed a higher variation of CTM v2 results as compared to RealTime values. (CVs at 100 IU/ml: RealTime: 13.1–21.0 % and CTM v2: 15.0–32.3 %; CVs at 25 IU/ml: RealTime 17.6–34.9 % and CTM v2 28.2–54.9 %). These findings confirm the superior precision of RealTime versus CTM v2 at low-level viremia even across different laboratories including the new clinical decision point at 25 IU/ml. A highly precise monitoring of HCV viral load during therapy will remain crucial for patient management with regard to futility rules, therapy efficacy and SVR.
Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1-/- PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1-/- macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.
Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.
Molecular cause and functional impact of altered synaptic lipid signaling due to a prg‐1 gene SNP
(2015)
Loss of plasticity-related gene 1 (PRG-1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg-1 (R345T/mutPRG-1) affects ~5 million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss-of-PRG-1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by this SNP. PRG-1(+/-) mice, which are animal correlates of human PRG-1(+/mut) carriers, showed an altered cortical network function and stress-related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA-synthesizing molecule autotaxin. In line, EEG recordings in a human population-based cohort revealed an E/I balance shift in monoallelic mutPRG-1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress-related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate-dependent symptoms in psychiatric diseases.
Modulation der Zytokinproduktion durch Interaktionen von IRAK4 und p47phox mit den TLR-Signalwegen
(2015)
p47phox wird als ein in mehreren Strukturen involviertes Protein beschrieben, welches in seiner Funktion einen großen Einfluss auf das Immunsystem nehmen kann und damit zu schweren Erkrankungen führen kann. Wegen der Vielfalt der Interaktionen ist es schwierig die Effekte von p47phox einer bestimmten Wechselwirkung zuzuordnen. Dennoch ist es unserer Arbeitsgruppe gelungen eine negative Rückkopplung von p47phox auf die Produktion von IL-12p70 [1] festzustellen. Ausgehend von dieser Interaktion und der Phosphorylierung von p47phox durch IRAK4 [2], wurde daher in dieser Doktorarbeit der Zusammenhang einer Modulation der IL-12p70-Produktion durch IRAK4 näher untersucht.