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

Survival-Adjusted FEV1 and BMI Percentiles for Patients with Cystic Fibrosis before the Era of Triple CFTR Modulator Therapy in Germany

Background: Pulmonary disease is the major cause for morbidity and mortality in cystic fibrosis (CF). In CF, forced expiratory volume in 1 s (FEV1) referenced against a healthy population (FEV1%predicted) and body mass index (BMI) do not allow for the comparison of disease severity across age and gender. Objectives: We aimed to determine updated FEV1 and BMI percentiles for patients with CF and to study their dependence on mortality attrition. Methods: Age- and height-adjusted FEV1 and BMI percentiles for CF patients aged 6-50 years were calculated from 4,947 patients of the German CF Registry for the period 2016-2019 utilizing quantile regression and a Generalized Additive Model for Location, Scale and Shape (GAMLSS). Further, survival-adjusted percentiles were estimated. Results: In patients with CF, FEV1 increased throughout childhood until maximal median values at 16 years in females (2.46 L) and 18 years in males (3.27 L). During adulthood, FEV1 decreased substantially. At 17 years of age, the 25th BMI percentile of patients with CF (females 18.50 and males 18.15 kg/m(2)) was below the 10th BMI percentile of the German reference cohort. From the age of 20 years, survival (96.3%) decreased tremendously. At 50 years of age (survival 15.0%), the 50th CF-specific FEV1 or BMI percentile among the survivors corresponded to the 92.5th percentile among the total CF birth cohort. Conclusions: Continuously updated disease-specific FEV1 and BMI percentiles with correction for survival may serve as age-independent measure of disease severity in CF (accessible via https://cfpercentiles.statup.solutions)

Specifically Increased Rate of Infections in Children Post Measles in a High Resource Setting

Objectives: Post-measles increased susceptibility to subsequent infections seems particularly relevant in low-resource settings. We tested the hypothesis that measles causes a specifically increased rate of infections in children, also in a high-resource setting. Methods: We conducted a retrospective cohort study on a large measles outbreak in Berlin, Germany. All children with measles who presented to hospitals in Berlin were included as cases, children with non-infectious and children with non-measles infectious diseases as controls. Repeat visits within 3 years after the outbreak were recorded. Results: We included 250 cases, 502 non-infectious, and 498 infectious disease controls. The relative risk for cases for the diagnosis of an infectious disease upon a repeat visit was 1.6 (95% CI 1.4–2.0, p < 0.001) vs. non-infectious and 1.3 (95% CI 1.1–1.6, p = 0.002) vs. infectious disease controls. 33 cases (27%), 35 non-infectious (12%) and 57 (18%) infectious disease controls presented more than three times due to an infectious disease (p = 0.01, and p = 0.02, respectively). This results in a relative risk of more than three repeat visits due to an infection for measles cases of 1.8 (95% CI 1.3–2.4, p = 0.01), and 1.4 (95% CI 1.0–1.9, p = 0.04), respectively. Conclusion: Our study demonstrates for the first time in a high-resource setting, that increased post-measles susceptibility to subsequent infections in children is measles-specific—even compared to controls with previous non-measles infections.Peer Reviewe

Corrigendum: Specifically increased rate of infections in children post measles in a high resource setting

Peer Reviewe