Assessment of therapeutic drug efficacy in the epithelial to mesenchymal transition of human liver cancer

Das hepatozelluläre Karzinom ist die sechst häufigste Krebserkrankung weltweit, dessen Inzidenz nach wie vor steigend ist. Die epitheliale zur mesenchymalen Transition (EMT), ein Prozess bei dem epitheliale Zellen ihre typische Organisation verlieren und dadurch in das umliegende Gewebe invadieren können, wurde als eine Hauptursache der fortschreitenden Leberkrebserkrankung und deren Metastasierung identifiziert. Ziel dieser experimentellen Studie war, ein humanes zelluläres Modell der EMT zu entwickeln, um einerseits molekulare Mechanismen der EMT zu identifizieren und andererseits die Effizienz antitumoraler Medikamente in diesem Prozess zu untersuchen. Dafür wurden zwei Leber-Zelllinien, welche einen epithelialen beziehungsweise einen mesenchymalen Phänotyp aufweisen, aus dem Tumor eines HCC Patienten isoliert. Die Immunfluoreszenzanalyse bestätigte die epithelialen und mesenchymalen Charakteristika dieser HCC Zelllinien. Weiters konnten wir zeigen, dass die mesenchymalen HCC Zellen, im Vergleich zu den epithelialen, eine höhere Migration und Invasion in vitro aufweisen. Die chromosomale Analyse mittels vergleichender genomischen Hybridisierung und eine ‘Short Tandem Repeat‘ Analyse bestätigten, dass beide Zelltypen ursprünglich aus einer Zelle entstanden sind. Dies zeigt damit eindeutig, dass sich die mesenchymalen Zellen durch eine EMT in vivo aus den epithelialen Zellen entwickelt haben. Durch die Expressionsanalyse des gesamten Transkriptoms konnte der Verlust von epithelialen und der Erwerb von mesenchymalen Markern nachgewiesen werden, wodurch eine EMT bestätigt wurde. Eine methylierungsspezifische Polymerase- Kettenreaktion (PCR) ergab weiters, dass das Protein ‘Secreted Protein, Acidic, Rich in Cystein‘ (SPARC), das in den mesenchymalen HCC Zellen stark überexprimiert ist, durch Demethylierung seines Promotors epigenetisch reguliert wird. Ebenso konnten wir zeigen, dass mesenchymale HCC Zellen im Vergleich zu den epithelialen HCC Zellen eine höhere Resistenz gegen zielgerichte Therapien wie Sorafenib und Erlotinib aufweisen, wohingegen epitheliale HCC Zellen stärker resistent gegen zytostatische Medikamente sind. Die Kombination von Doxorubicin und Sorafenib verursacht eine erhöhte Empfindlichkeit von beiden Zelllinien, was dazu führt, dass sich die maximalen mittleren inhibitorischen Konzentrationen (IC50) einander angleichen. Diese Kombination ist folglich eine effiziente Behandlung um beide, eptiheliale als auch mesenchymale Hepatomzellen erfolgreich zu eliminieren. Zusammenfassend konnten wir ein einzigartiges EMT Modell des humanen HCCs entwickeln, das in präklinischen Studien eingesetzt werden kann, um (i) die cytostatische Effizienz einer Substanz in vitro zu testen, (ii) ein Profil des Wirkstoffs mit dessen Auswirkung auf zelluläre Mechanismen zu erstellen und (iii) Synergismen mit weiteren Antikrebsmitteln zu identifizieren.Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the incidence is still increasing. The epithelial to mesenchymal transition (EMT) of malignant hepatocytes in HCC is considered as a major cause of liver cancer progression and metastasis. In this study we aimed to establish a human cellular model of HCC to investigate the molecular mechanisms of EMT and to examine the efficacy of anti-cancer agents in this process.Two liver cell lines were established from one HCC patient showing either an epithelial or mesenchymal phenotype. Immunofluorescence analysis confirmed the distinct epithelial and mesenchymal characteristics of HCC cells. Most remarkably, mesenchymal HCC cells showed enhanced migration and invasion in vitro. Comparative genomic hybridization and short tandem repeat analysis indicated a unique cellular origin of both cell types, suggesting that mesenchymal cells derived from epithelial hepatocytes via EMT in vivo. Loss of epithelial as well as gain of mesenchymal markers analyzed by whole genome expression profiling verified EMT. Interestingly, methylation-specific PCR analysis revealed epigenetic upregulation of secreted protein, acidic, rich in cystein (SPARC) in mesenchymal cells via promoter demethylation. Upon drug exposure, mesenchymal HCC cells showed a higher resistance to the targeted therapeutic agents sorafenib and erlotinib as compared to epithelial HCC cells, which were slightly more resistant to cytostatic drugs. Combination of doxorubicin and sorafenib caused an increased susceptibility of both cell lines to cytostatic effects resulting in an equalization of IC50 values, thus showing an effective treatment to target both, epithelial and mesenchymal cells. In conclusion, we established a unique EMT model of human HCC for pre-clinical studies which allows studying drug efficacy during HCC progression including the assessment of synergistic anti-cancer drug profiles

Proteome analysis of human substantia nigra in Parkinson's disease

<p>Abstract</p> <p>Background</p> <p>Parkinson's disease (PD) is the most common neurodegenerative disorder involving the motor system. Although not being the only region involved in PD, affection of the substantia nigra and its projections is responsible for some of the most debilitating features of the disease. To further advance a comprehensive understanding of nigral pathology, we conducted a tissue based comparative proteome study of healthy and diseased human substantia nigra.</p> <p>Results</p> <p>The gross number of differentially regulated proteins in PD was 221. In total, we identified 37 proteins, of which 16 were differentially expressed. Identified differential proteins comprised elements of iron metabolism (H-ferritin) and glutathione-related redox metabolism (GST M3, GST P1, GST O1), including novel redox proteins (SH3BGRL). Additionally, many glial or related proteins were found to be differentially regulated in PD (GFAP, GMFB, galectin-1, sorcin), as well as proteins belonging to metabolic pathways sparsely described in PD, such as adenosyl homocysteinase (methylation), aldehyde dehydrogenase 1 and cellular retinol-binding protein 1 (aldehyde metabolism). Further differentially regulated proteins included annexin V, beta-tubulin cofactor A, coactosin-like protein and V-type ATPase subunit 1. Proteins that were similarly expressed in healthy or diseased substantia nigra comprised housekeeping proteins such as COX5A, Rho GDI alpha, actin gamma 1, creatin-kinase B, lactate dehydrogenase B, disulfide isomerase ER-60, Rab GDI beta, methyl glyoxalase 1 (AGE metabolism) and glutamine synthetase. Interestingly, also DJ-1 and UCH-L1 were expressed similarly. Furthermore, proteins believed to serve as internal standards were found to be expressed in a constant manner, such as 14-3-3 epsilon and hCRMP-2, thus lending further validity to our results.</p> <p>Conclusion</p> <p>Using an approach encompassing high sensitivity and high resolution, we show that alterations of SN in PD include many more proteins than previously thought. The results point towards a heterogeneous aetiopathogenesis of the disease, including alterations of GSH-related proteins as well as alterations of proteins involved in retinoid metabolism, and they indicate that proteins involved in familial PD may not be differentially regulated in idiopathic Parkinson's disease.</p

Anything goes: Wissenschaft wider den Methodenzwang

Empörung oder Ernüchterung, Belustigung oder Mitleid, Langeweile oder Ohnmacht? – Man weiß gar nicht, mit welcher Regung man sich zu den jüngsten Vorkommnissen wissenschaftlichen Fehlgehens (oder: fehlenden wissenschaftlichen Vorgehens) in der Rechtswissenschaft verhalten soll. Die Vorgänge sind so musterhaft bekannt und vertraut, die Indizien dabei so eindeutig, dass sich in jedem Fall eine gewisse Ermüdung einstellt

Effectiveness of a community-based positive prevention intervention for people living with HIV who are not receiving antiretroviral treatment: a prospective cohort study

Background: We report effectiveness of an HIV-prevention intervention delivered by community health workers (CHWs) in Mombasa, Kenya, to PLHIV who have not initiated or who have discontinued ART-an often difficult-to-reach population because they fall outside the ambit of health care and prevention services. Methods: A 2-arm cohort study assessed a structured risk-reduction intervention involving at least 4 one-to-one counseling sessions and personalized support. The control group received standard prevention services. CHWs recruited treatment-naïve people living with HIV (PLHIV) or those who had previously taken antiretroviral drugs. Data were analyzed using a Propensity Score Matched (PSM)-sample to control for baseline differences between the groups. Results: 634 PLHIV were recruited and followed for 6 months. Median age was 35 years, and 74.3% were female. Participants in the intervention group reported reduced risky sexual behaviors both at endline compared with baseline and compared with the control group. At endline, in the PSM analysis, participants in the intervention arm were less likely than participants in the control group to report unprotected sex with a spouse (Odds Ratio [OR] = 0.08, 95% confidence interval [CI] = 0.03-0.24), and they reported fewer unprotected sex acts (12.3% versus 46.0%, respectively; OR = 0.16, 95% CI = 0.09-0.29; P\u3c0.001). Further, 92.4% of participants in the intervention group reported zero unsafe sex acts (with partners of negative or unknown HIV status) compared with 70.8% in the control group (P\u3c0.001), and more participants in the intervention arm were receiving ART (34.3% versus 12.7%, respectively; P\u3c0.001). Conclusion: CHWs effectively reached PLHIV who had never received or who had discontinued ART, and they delivered a risk-reduction intervention that led to declines in reported sexual risk behaviors, as well as to increases in ART uptake. A scaled-up intervention warrants consideration

Effects on Clinical Outcomes of a 5-Year Surgical Safety Checklist Implementation Experience: A Large-scale Population-Based Difference-in-Differences Study

The adoption of a surgical checklist is strongly recommended worldwide as an effective practice to improve patient safety; however, several studies have reported mixed results and a number of issues are still unresolved. The main objective of this study was to explore the impact of the first 5-year period of a surgical checklist-based intervention in a large regional health care system in Italy (4 500 000 inhabitants). We conducted a retrospective longitudinal study on 1 166 424 patients who underwent surgery in 48 public hospitals between 2006 and 2014. The adherence to the checklist was measured between 2011 and 2013 through a computerized database. The effects of the intervention were explored through multivariable logistic regression and difference-in-differences (DID) approaches, based on current administrative data sources. In-hospital and 30-days mortality, 30-days readmissions and length-of-stay (LOS) \u2a7e8 days were the observed outcomes. Adherence to the checklist showed marked variations across hospitals (0%-93.3%). A pre/post analysis detected statistically significant differences between surgical interventions performed in hospitals with higher adherence to the checklist (\u2a7e75% of the surgeries) and those performed in other hospitals, as for the 30-days readmissions rate (odds ratio [OR]: 0.96; 95% confidence interval [CI]: 0.94-0.98) and LOS \u2a7e 8 days rate (OR: 0.88; 95% CI: 0.87-0.89). These findings were confirmed after risk adjustment and DID analysis. No association was observed with mortality outcomes. On the whole, our study attained mixed results. Although a protective effect of the surgical checklist use could not be proved over the first 5 years of this regional implementation experience, our research offers some methodological insights for practical use in the evaluation process of large-scale implementation projects

Elastase activity on sputum neutrophils correlates with severity of lung disease in cystic fibrosis

Neutrophil elastase (NE) is a key risk factor for severity of cystic fibrosis (CF) lung disease. Recent studies identified increased NE activity on the surface of airway neutrophils from CF-like mice and patients with CF. However, the role of surface-bound NE in CF lung disease remains unknown. We determined the relationship between surface-bound NE activity and severity of lung disease in CF.Surface-bound NE activity was measured on sputum neutrophils from 35 CF patients and eight healthy controls using novel lipidated Förster resonance energy transfer reporters and correlated with free NE activity, neutrophil counts, interleukin-8, myeloperoxidase and antiproteases in sputum supernatant, and with lung function parameters.Surface-bound NE activity was increased in CF compared to healthy controls (p&lt;0.01) and correlated with free NE activity (p&lt;0.05) and other inflammation markers (p&lt;0.001). Surface-bound and free NE activity correlated with forced expiratory volume in 1 s % predicted (p&lt;0.01 and p&lt;0.05), but only surface-bound NE activity correlated with plethysmographic functional residual capacity % pred (p&lt;0.01) in patients with CF.We demonstrate that surface-bound NE activity on airway neutrophils correlates with severity of lung disease in patients with CF. Our results suggest that surface-bound NE activity may play an important role in the pathogenesis and serve as novel biomarker in CF lung disease.</jats:p

Isolation of human MHC class II-restricted T cell receptors from the autologous T-cell repertoire with potent anti-leukaemic reactivity

Summary Adoptive transfer of T cells genetically modified with tumour-specific T-cell receptors (TCR) is a promising novel approach in the treatment of cancer. We have previously isolated an allorestricted MHC class I-restricted TCR with specificity for Formin-like protein 1 (FMNL1) with potent activity against chronic lymphocytic leukaemia cells. CD4 + T cells have been described to be highly important for tumour elimination although TCR derived from CD4 + T cells with anti-tumour reactivity have been only rarely described. In this study we aimed to isolate MHC class-II-restricted CD4 + T cells and TCR with specificity for leukaemia antigens. We used professional antigen-presenting cells pulsed with the leukaemiaassociated and tumour-associated antigen FMNL1 for stimulation of autologous T cells in vitro. We isolated two CD4 + HLA-DR-restricted T-cell clones and T-cell-derived TCR with so far unknown specificity but high reactivity against lymphoma cells and native malignant cells derived from HLA-matched patients with diverse leukaemias. Moreover, characterization of the TCR after TCR gene transfer revealed that specific characteristics of isolated TCR as reactivity in response to Toll-like receptors were transferable on effector cells. Our results have a major impact on the development of novel immunotherapies. They demonstrate that TCR with potent HLA-DR-restricted anti-leukaemic reactivity against so far undefined self-restricted antigens can be isolated from the healthy autorestricted CD4 + T-cell repertoire and these TCR are highly interesting candidate tools for novel immunotherapies

Exploring Clinical and Epidemiological Characteristics of Interstitial Lung Diseases: Rationale, Aims, and Design of a Nationwide Prospective Registry-The EXCITING-ILD Registry

Despite a number of prospective registries conducted in past years, the current epidemiology of interstitial lung diseases (ILD) is still not well defined, particularly regarding the prevalence and incidence, their management, healthcare utilisation needs, and healthcare-associated costs. To address these issues in Germany, a new prospective ILD registry, &quot;Exploring Clinical and Epidemiological Characteristics of Interstitial Lung Diseases&quot; (EXCITING-ILD), is being conducted by the German Centre for Lung Research in association with ambulatory, inpatient, scientific pulmonology organisations and patient support groups. This multicentre, noninterventional, prospective, and observational ILD registry aims to collect comprehensive and validated data from all healthcare institutions on the incidence, prevalence, characteristics, management, and outcomes regarding all ILD presentations in the real-world setting. Specifically, this registry will collect demographic data, disease-related data such as ILD subtype, treatments, diagnostic procedures (e.g., HRCT, surgical lung biopsy), risk factors (e.g., familial ILD), significant comorbidities, ILD managements, and disease outcomes as well as healthcare resource consumption. The EXCITING-ILD registry will include in-patient and out-patient ILD healthcare facilities in more than 100 sites. In summary, this registry will document comprehensive and current epidemiological data as well as important health economic data for ILDs in Germany