Evaluation of the biomarker candidate MFAP4 for non-invasive assessment of hepatic fibrosis in hepatitis C patients

$\textbf {Background:}$ The human microfibrillar-associated protein 4 (MFAP4) is located to extracellular matrix fibers and plays a role in disease-related tissue remodeling. Previously, we identified MFAP4 as a serum biomarker candidate for hepatic fibrosis and cirrhosis in hepatitis C patients. The aim of the present study was to elucidate the potential of MFAP4 as biomarker for hepatic fibrosis with a focus on the differentiation of no to moderate (F0–F2) and severe fibrosis stages and cirrhosis (F3 and F4, Desmet-Scheuer scoring system). $\textbf {Methods:}$ MFAP4 levels were measured using an AlphaLISA immunoassay in a retrospective study including $\it n$ = 542 hepatitis C patients. We applied a univariate logistic regression model based on MFAP4 serum levels and furthermore derived a multivariate model including also age and gender. Youden-optimal cutoffs for binary classification were determined for both models without restrictions and considering a lower limit of 80% sensitivity (correct classification of F3 and F4), respectively. To assess the generalization error, leave-one-out cross validation (LOOCV ) was performed. $\textbf {Results:}$ MFAP4 levels were shown to differ between no to moderate fibrosis stages F0–F2 and severe stages (F3 and F4) with high statistical significance ($\it t$ test on log scale, $\it p$ value $<2.2·10^{-16}$). In the LOOCV, the univariate classification resulted in 85.8% sensitivity and 54.9% specificity while the multivariate model yielded 81.3% sensitivity and 61.5% specificity (restricted approaches). $\textbf {Conclusions:}$ We confirmed the applicability of MFAP4 as a novel serum biomarker for assessment of hepatic fibrosis and identification of high-risk patients with severe fibrosis stages in hepatitis C. The combination of MFAP4 with existing tests might lead to a more accurate non-invasive diagnosis of hepatic fibrosis and allow a cost-effective disease management in the era of new direct acting antivirals

Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC

Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets. We identified 34 differentially abundant proteins in phosphoprotein enriched lysates. Expression and distribution of the candidate protein eEF2 and its phosphorylated isoform was validated immunohistochemically on 78 hepatocellular carcinoma and non-tumorous tissue samples. Validation showed that total eEF2 and phosphorylated eEF2 at threonine 56 are prognostic markers for overall survival of HCC-patients. The activity of the regulating eEF2 kinase, compared between tumor and non-tumorous tissue lysates by in vitro kinase assays, is more than four times higher in tumor tissues. Functional analyzes regarding eEF2 kinase were performed in JHH5 cells with CRISPR/Cas9 mediated eEF2 kinase knock out. Proliferation and growth is decreased in eEF2 kinase knock out cells

Spatial proteomics revealed a CX3CL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder

The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CX3CL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CX3CL1 signaling in Cx3cr1gfp/gfp mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CX3CL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder. © 2020, The Author(s)

Comparative transcriptomic and proteomic signature of lung alveolar macrophages reveals the integrin CD11b as a regulatory hub during pneumococcal pneumonia infection

IntroductionStreptococcus pneumoniae is one of the main causes of community-acquired infections in the lung alveoli in children and the elderly. Alveolar macrophages (AM) patrol alveoli in homeostasis and under infectious conditions. However, the molecular adaptations of AM upon infections with Streptococcus pneumoniae are incompletely resolved.MethodsWe used a comparative transcriptomic and proteomic approach to provide novel insights into the cellular mechanism that changes the molecular signature of AM during lung infections. Using a tandem mass spectrometry approach to murine cell-sorted AM, we revealed significant proteomic changes upon lung infection with Streptococcus pneumoniae.ResultsAM showed a strong neutrophil-associated proteomic signature, such as expression of CD11b, MPO, neutrophil gelatinases, and elastases, which was associated with phagocytosis of recruited neutrophils. Transcriptomic analysis indicated intrinsic expression of CD11b by AM. Moreover, comparative transcriptomic and proteomic profiling identified CD11b as the central molecular hub in AM, which influenced neutrophil recruitment, activation, and migration.DiscussionIn conclusion, our study provides novel insights into the intrinsic molecular adaptations of AM upon lung infection with Streptococcus pneumoniae and reveals profound alterations critical for effective antimicrobial immunity

Extracorporeal life support in COVID-19-related acute respiratory distress syndrome: A EuroELSO international survey

Extracorporeal life support (ECLS) is a means to support patients with acute respiratory failure. Initially, recommendations to treat severe cases of pandemic coronavirus disease 2019 (COVID-19) with ECLS have been restrained. In the meantime, ECLS has been shown to produce similar outcomes in patients with severe COVID-19 compared to existing data on ARDS mortality. We performed an international email survey to assess how ECLS providers worldwide have previously used ECLS during the treatment of critically ill patients with COVID-19. A questionnaire with 45 questions (covering, e.g., indication, technical aspects, benefit, and reasons for treatment discontinuation), mostly multiple choice, was distributed by email to ECLS centers. The survey was approved by the European branch of the Extracorporeal Life Support Organization (ELSO); 276 ECMO professionals from 98 centers in 30 different countries on four continents reported that they employed ECMO for very severe COVID-19 cases, mostly in veno-venous configuration (87%). The most common reason to establish ECLS was isolated hypoxemic respiratory failure (50%), followed by a combination of hypoxemia and hypercapnia (39%). Only a small fraction of patients required veno-arterial cannulation due to heart failure (3%). Time on ECLS varied between less than 2 and more than 4 weeks. The main reason to discontinue ECLS treatment prior to patient’s recovery was lack of clinical improvement (53%), followed by major bleeding, mostly intracranially (13%). Only 4% of respondents reported that triage situations, lack of staff or lack of oxygenators, were responsible for discontinuation of ECLS support. Most ECLS physicians (51%, IQR 30%) agreed that patients with COVID-19-induced ARDS (CARDS) benefitted from ECLS. Overall mortality of COVID-19 patients on ECLS was estimated to be about 55%. ECLS has been utilized successfully during the COVID-19 pandemic to stabilize CARDS patients in hypoxemic or hypercapnic lung failure. Age and multimorbidity limited the use of ECLS. Triage situations were rarely a concern. ECLS providers stated that patients with severe COVID-19 benefitted from ECLS

Proteome profiling of enzalutamide-resistant cell lines and serum analysis identified ALCAM as marker of resistance in castration-resistant prostate cancer

Enzalutamide (ENZA) is a frequently used therapy in metastatic castration‐resistant prostate cancer (mCRPC). Baseline or acquired resistance to ENZA have been observed, but the molecular mechanisms of resistance are poorly understood. We aimed to identify proteins involved in ENZA resistance and to find therapy‐predictive serum markers. We performed comparative proteome analyses on ENZA‐sensitive parental (LAPC4, DuCaP) and ‐resistant prostate cancer cell lines (LAPC4‐ENZA, DuCaP‐ENZA) using liquid chromatography tandem mass spectrometry (LC‐MS/MS). The top four most promising candidate markers were selected using bioinformatic approaches. Serum concentrations of selected markers (ALCAM, AGR2, NDRG1, IDH1) were measured in pretreatment samples of 72 ENZA‐treated mCRPC patients using ELISA. In addition, ALCAM serum levels were measured in 101 Abiraterone (ABI) and 100 Docetaxel (DOC)‐treated mCRPC patients' baseline samples. Results were correlated with clinical and follow‐up data. The functional role of ALCAM in ENZA resistance was assessed in vitro using siRNA. Our proteome analyses revealed 731 significantly differentially abundant proteins between ENZA‐sensitive and ‐resistant cells and our filtering methods identified four biomarker candidates. Serum analyses of these proteins revealed only ALCAM to be associated with poor patient survival. Furthermore, higher baseline ALCAM levels were associated with poor survival in ABI‐ but not in DOC‐treated patients. In LAPC4‐ENZA resistant cells, ALCAM silencing by siRNA knockdown resulted in significantly enhanced ENZA sensitivity. Our analyses revealed that ALCAM serum levels may help to identify ENZA‐ and ABI‐resistant patients and may thereby help to optimize future clinical decision‐making. Our functional analyses suggest the possible involvement of ALCAM in ENZA resistance

Comparative proteome analysis identified CD44 as a possible serum marker for docetaxel resistance in castration-resistant prostate cancer

Baseline or acquired resistance to docetaxel (DOC) represents a significant risk for patients with metastatic prostate cancer (PC). In the last years, novel therapy regimens have been approved providing reasonable alternatives for DOC‐resistant patients making prediction of DOC resistance of great clinical importance. We aimed to identify serum biomarkers, which are able to select patients who will not benefit from DOC treatment. DOC‐resistant PC3‐DR and DU145‐DR sublines and their sensitive parental cell lines (DU145, PC3) were comparatively analyzed using liquid chromatography‐coupled tandem mass spectrometry (LC‐MS/MS). Results were filtered using bioinformatics approaches to identify promising serum biomarkers. Serum levels of five proteins were determined in serum samples of 66 DOC‐treated metastatic castration‐resistant PC patients (mCRPC) using ELISA. Results were correlated with clinicopathological and survival data. CD44 was subjected to further functional cell culture analyses. We found at least 177 two‐fold significantly overexpressed proteins in DOC‐resistant cell lines. Our bioinformatics method suggested 11/177 proteins to be secreted into the serum. We determined serum levels of five (CD44, MET, GSN, IL13RA2 and LNPEP) proteins in serum samples of DOC‐treated patients and found high CD44 serum levels to be independently associated with poor overall survival (p = 0.001). In accordance, silencing of CD44 in DU145‐DR cells resulted in re‐sensitization to DOC. In conclusion, high serum CD44 levels may help identify DOC‐resistant patients and may thereby help optimize clinical decision‐making regarding type and timing of therapy for mCRPC patients. In addition, our in vitro results imply the possible functional involvement of CD44 in DOC resistance

Herstellung monoklonaler Antikörper gegen den Integrin-α2β1-Antagonisten Rhodocetin zur Analyse dessen Eigenschaften in vitro und in vivo

Integrin a2ß1 ist ein Adhäsionsrezeptor, der verschiedene Kollagentypen bindet. Als solcher spielt es eine bedeutende Rolle für Zellfunktionen wie Migration und die Regulation des Zytoskeletts, Zellteilung, Apoptose und Differenzierung. Integrin a2ß1 übernimmt deshalb eine tragende Funktion in Prozessen wie Wundheilung, Angiogenese und der Progression und Metastasierung von Krebs. Rhodocetin ist ein Antagonist des Integrins a2ß1. Es ist ein C-Typ-Lektin-artiges Protein das im Schlangengift der Malaiischen Grubenotter (Calloselasma rhodostoma) entdeckt wurde. Rhodocetin besteht aus vier Untereinheiten a, ß, ? und d, die zwei Dimere formen (aß und ?d), welche wiederum eine kreuzförmige heterotetramere Quartiärstruktur bilden, die bislang nur für Rhodocetin beschrieben wurde. Rhodocetin bindet an die Integrin-a2A-Domäne der a-Untereinheit des Integrins a2ß1. Rhodocetin unterbindet damit die Bindung von Kollagen und die Aktivierung des Integrins. Der genaue Interaktionsmechanismus von Rhodocetin mit der Integrin-a2A-Domäne ist noch unbekannt. In der vorliegenden Arbeit wurden mittels Hybridomtechnik monoklonale Antikörper gegen Rhodocetin generiert. Es wurden sechs Fusionen von Lymphozyten mit Myelomazellen durchgeführt, für die sowohl Mäuse als auch Ratten mit Rhodocetin immunisiert wurden. Die erzeugten Hybridomaklone wurden zunächst im ELISA auf ihre Fähigkeit getestet, Rhodocetin zu binden. Positiv getestete Klone wurden selektiert und die monoklonalen Antikörper aus den Zellüberständen aufgereinigt. Es konnten 14 monoklonale Antikörper etabliert werden. Die Antikörper wurden zunächst in ihren Bindungseigenschaften charakterisiert. Hierzu wurden verschiedene ELISAs, Immunblot und Immunpräzipitation genutzt. In der Duchflusszytometrie wurde getestet, ob die Antikörper an Integrin a2ß1 gebundenes Rhodocetin detektieren. Die Antikörper lassen sich hinsichtlich ihr Bindungseigenschaften und der Lage ihrer Bindungsepitope auf den verschiedenen Rhodocetin-Heterodimeren in unterschiedliche Klassen unterteilen. Es wurde nachgewiesen, dass Rhodocetin Konformationsänderungen unterliegt, die durch die Bindung einiger der generierten Antikörper induziert werden. Um den Einfluss divalenter Kationen auf die Konformation von Rhodocetin sowie den Interaktionsmechanismus von Rhodocetin mit der Integrin-a2A-Domäne zu untersuchen, wurde die analytische Gelfiltrationschromatographie genutzt. Es konnte gezeigt werden, dass die Bindung divalenter Kationen die Konformation von Rhodocetin beeinflusst. Um die Interaktionsstudien von Rhodocetin mit der a2A-Domäne auszuwerten wurde ein Sandwich-ELISA etabliert. Dieser ermöglichte es, im Anschluss an die Gelfiltration, die beiden Rhodocetin-Heterodimere unabhängig voneinander im Eluat nachzuweisen und zu quantifizieren. Es wurde nachgewiesen, dass das Rhodocetin-Heterotetramer während der Interaktion mit der Integrin-a2A-Domäne dissoziiert und nur das ?d-Dimer einen stabilen Komplex mit der a2A-Domäne bildet, während das aß-Dimer freigesetzt und unabhängig von diesem Komplex eluiert wird. Die pharmakologischen Eigenschaften von Rhodocetin wurden in einem Tumor-Xenograft-Modell untersucht, für das immundefizienten Mäusen HT1080 Fibrosarkomzellen implantiert wurden. Rhodocetin wurde den Mäusen intravenös appliziert und die Auswirkungen auf die Tumoren sowie der Verbleib von Rhodocetin im Körper analysiert. Die Entwicklung der Serumkonzentration von Rhodocetin wurde mit dem etablierten Sandwich-ELISA untersucht, welches hierzu mit einer Standardreihe kalibriert wurde. Die Elimination von Rhodocetin folgt einer Kinetik erster Ordnung. Die Exkretion von Rhodocetin erfolgt über die Niere. Es zeigte sich, dass etwa zwei Drittel des applizierten Rhodocetins unmittelbar nach Injektion von Integrin a2ß1-exprimierenden Zellen des Blutes gebunden werden. Die immunhistologische Auswertung von Tumoren und Kontrollgeweben ergab, dass Rhodocetin an Endothelzellen innerhalb der Nierenglomeruli sowie der Leber bindet. Rhodocetin konnte auch auf Endothelzellen der Tumorvaskulatur nachgewiesen werden. Innerhalb der Tumoren wurde Rhodocetin auch außerhalb von Blutgefäßen gefunden, wo es an HT1080 Tumorzellen bindet. Die Behandlung mit Rhodocetin beeinträchtigte die Integrität der Blutgefäße innerhalb der Tumoren und führte zu Hämorrhagien. Um die Auswirkungen von Rhodocetin auf die Tumoren genauer zu untersuchen und zu quantifizieren, wurde dynamische Magnetresonanztomographie genutzt. Es zeigte sich, dass Rhodocetin die Durchlässigkeit der Blutgefäße in Tumoren signifikant erhöht