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Abstract Introduction In 2013, rituximab was approved in France for the treatment of ANCA-associated vasculitis (AAV). The aim of the study was to compare the treatment and health events of adult incident patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), included before rituximab approval (over 2010–2012, Group 1) and those included after rituximab approval (over 2014–2017, Group 2). Method Data were extracted from the French National Health Insurance database (SNDS) including outpatient health care consumption and hospital discharge forms. Comparisons between inclusion periods were performed using Wilcoxon and χ² tests. Kaplan-Meier method was used to model the duration of treatment induction, maintenance, and off-drug periods. Fine and Gray tests were used to compare treatment phase durations. Results A total of 694 GPA and 283 MPA patients were included in Group 1, while 668 GPA and 463 MPA patients were included in Group 2. Between the two inclusion periods, the proportions of patients treated with rituximab increased in the induction and maintenance phases whereas treatment with azathioprine declined. These proportions remained stable in the case of methotrexate, cyclophosphamide, and glucocorticoid-treated patients. Frequency of first-time hospitalized infections, diabetes and renal failure during the first year after inclusion increased for both groups. Limitations of the study This is a retrospective study based on claims data including only 76% of people covered by health insurance in France. The period studied includes the learning phase of using rituximab. This study lacks biological data and precise quantitative analysis for the use of steroids, therefore the criteria for establishing diagnosis and therapeutic choice were unknown. Conclusions Introduction of rituximab reduced the use of azathioprine without affecting the use of glucocorticoids or cyclophosphamide.

Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH)-induced cirrhosis, are characterized by an increasing accumulation of stressed, damaged, or dying hepatocytes. Hepatocyte damage triggers the activation of resident immune cells, such as Kupffer cells (KC), as well as the recruitment of immune cells from the circulation toward areas of inflammation. After infiltration, monocytes differentiate into monocyte-derived macrophages (MoMF) which are functionally distinct from resident KC. We herein aim to compare the in vitro signatures of polarized macrophages and activated hepatic stellate cells (HSC) with ex vivo-derived disease signatures from human NASH. Furthermore, to shed more light on HSC activation and liver fibrosis progression, we investigate the effects of the secretome from primary human monocytes, macrophages, and NK cells on HSC activation. Interleukin (IL)-4 and IL-13 treatment induced transforming growth factor beta 1 (TGF-β1) secretion by macrophages. However, the supernatant transfer did not induce HSC activation. Interestingly, PMA-activated macrophages showed strong induction of the fibrosis response genes COL10A1 and CTGF, while the supernatant of IL-4/IL-13-treated monocytes induced the upregulation of COL3A1 in HSC. The supernatant of PMA-activated NK cells had the strongest effect on COL10A1 induction in HSC, while IL-15-stimulated NK cells reduced the expression of COL1A1 and CTGF. These data indicate that other factors, aside from the well-known cytokines and chemokines, might potentially be stronger contributors to the activation of HSCs and induction of a fibrotic response, indicating a more diverse and complex role of monocytes, macrophages, and NK cells in liver fibrosis progression.

Summary: Persistent liver injury triggers a fibrogenic program that causes pathologic remodeling of the hepatic microenvironment (i.e., liver fibrosis) and portal hypertension. The dynamics of gene regulation during liver disease progression and early regression remain understudied. Here, we generated hepatic transcriptome profiles in two well-established liver disease models at peak fibrosis and during spontaneous regression after the removal of the inducing agents. We linked the dynamics of key disease readouts, such as portal pressure, collagen area, and transaminase levels, to differentially expressed genes, enabling the identification of transcriptomic signatures of progressive vs. regressive liver fibrosis and portal hypertension. These candidate biomarkers (e.g., Tcf4, Mmp7, Trem2, Spp1, Scube1, Islr) were validated in RNA sequencing datasets of patients with cirrhosis and portal hypertension, and those cured from hepatitis C infection. Finally, deconvolution identified major cell types and suggested an association of macrophage and portal hepatocyte signatures with portal hypertension and fibrosis area.

Abstract The current obesity epidemic and high prevalence of metabolic diseases necessitate efficacious and safe treatments. Brown adipose tissue in this context is a promising target with the potential to increase energy expenditure, however no pharmacological treatments activating brown adipose tissue are currently available. Here, we identify AXL receptor tyrosine kinase as a regulator of adipose function. Pharmacological and genetic inhibition of AXL enhance thermogenic capacity of brown and white adipocytes, in vitro and in vivo. Mechanistically, these effects are mediated through inhibition of PI3K/AKT/PDE signaling pathway, resulting in induction of nuclear FOXO1 localization and increased intracellular cAMP levels via PDE3/4 inhibition and subsequent stimulation of the PKA-ATF2 pathway. In line with this, both constitutive Axl deletion as well as inducible adipocyte-specific Axl deletion protect animals from diet-induced obesity concomitant with increases in energy expenditure. Based on these data, we propose AXL receptor as a target for the treatment of obesity.

Summary: Immune dysregulation and inflammation by hepatic-resident leukocytes is considered a key step in disease progression of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis toward cirrhosis and hepatocellular carcinoma. Here, we provide a protocol for isolation and characterization of liver-resident immune cells from fine-needle biopsies obtained from a rodent model and humans. We describe steps for isolating leukocytes, cell sorting, and RNA extraction and sequencing. We then detail procedures for low-input mRNA sequencing analyses. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract There is a high need for predictive human ex vivo models for non-alcoholic fatty liver disease (NAFLD). About a decade ago, precision-cut liver slices (PCLSs) have been established as an ex vivo assay for humans and other organisms. In the present study, we use transcriptomics by RNASeq to profile a new human and mouse PCLSs based assay for steatosis in NAFLD. Steatosis as quantified by an increase of triglycerides after 48 h in culture, is induced by incremental supplementation of sugars (glucose and fructose), insulin, and fatty acids (palmitate, oleate). We mirrored the experimental design for human vs. mouse liver organ derived PCLSs and profiled each organ at eight different nutrient conditions after 24 h and 48 h time in culture. Thus, the provided data allows a comprehensive analysis of the donor, species, time, and nutrient factor specific regulation of gene expression in steatosis, despite the heterogeneity of the human tissue samples. Exemplified this is demonstrated by ranking homologous gene pairs by convergent or divergent expression pattern across nutrient conditions.

In diesem Beitrag wird eine Methode zur Eruierung der Perspektiven von Kindern auf digitale Medien in der Kindertageseinrichtung sowie auf ihre Mediennutzung in der Familie vorgestellt. Hierzu wurde auf ein dialoggestütztes Gruppeninterview zurückgegriffen, das durch einen Kitarundgang und spielerische Elemente in Form eines Memo-Spiels ergänzt wurde. Der Fokus des Beitrags liegt auf der Beschreibung der Methode. Diese soll für den Einsatz in der Praxis zur Verfügung gestellt werden mit dem Ziel, dass Fachkräfte mit den Kindern zum Thema Medien ins Gespräch kommen und auf dieser Basis deren Perspektiven in ihre pädagogische Planung einbeziehen, indem sie ihr eigenes medienpädagogisches Handeln reflektieren und ggf. anpassen. Erste Erprobungen bestätigten die Praxistauglichkeit, es wurde aber auch deutlich, dass je nach Situation flexible Anpassungen sinnvoll sein können und darauf zu achten ist, dass die Auswertung im Rahmen der zeitlichen Ressourcen der Fachkräfte leistbar ist.

Abstract Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology.

Abstract Non‐alcoholic steatohepatitis (NASH) has emerged as a major challenge for public health because of high global prevalence and lack of evidence‐based therapies. Most animal models of NASH lack sufficient validation regarding disease progression and pharmacological treatment. The Gubra‐Amylin NASH (GAN) diet‐induced obese (DIO) mouse demonstrate clinical translatability with respect to disease etiology and hallmarks of NASH. This study aimed to evaluate disease progression and responsiveness to clinically effective interventions in GAN DIO‐NASH mice. Disease phenotyping was performed in male C57BL/6J mice fed the GAN diet high in fat, fructose, and cholesterol for 28–88 weeks. GAN DIO‐NASH mice with biopsy‐confirmed NASH and fibrosis received low‐caloric dietary intervention, semaglutide (30 nmol/kg/day, s.c.) or lanifibranor (30 mg/kg/day, p.o.) for 8 and 12 weeks, respectively. Within‐subject change in nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) and fibrosis stage was evaluated using automated deep learning‐based image analysis. GAN DIO‐NASH mice showed clear and reproducible progression in NASH, fibrosis stage, and tumor burden with high incidence of hepatocellular carcinoma. Consistent with clinical trial outcomes, semaglutide and lanifibranor improved NAS, whereas only lanifibranor induced regression in the fibrosis stage. Dietary intervention also demonstrated substantial benefits on metabolic outcomes and liver histology. Differential therapeutic efficacy of semaglutide, lanifibranor, and dietary intervention was supported by quantitative histology, RNA sequencing, and blood/liver biochemistry. In conclusion, the GAN DIO‐NASH mouse model recapitulates various histological stages of NASH and faithfully reproduces histological efficacy profiles of compounds in advanced clinical development for NASH. Collectively, these features highlight the utility of GAN DIO‐NASH mice in preclinical drug development.

Objective: Obesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. Methods: BI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. Results: BI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906’s superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. Conclusions: BI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide. In adults with NAFLD, fibrosis can develop and progress to liver cirrhosis and liver failure. However, the underlying molecular mechanisms of fibrosis progression are not fully understood. Using total RNA-Seq, we investigated the molecular mechanisms of NAFLD and fibrosis. We sequenced liver tissue from 143 adults across the full spectrum of fibrosis stage including those with stage 4 fibrosis (cirrhosis). We identified gene expression clusters that strongly correlate with fibrosis stage including four genes that have been found consistently across previously published transcriptomic studies on NASH i.e. COL1A2, EFEMP2, FBLN5 and THBS2. Using cell type deconvolution, we estimated the loss of hepatocytes versus gain of hepatic stellate cells, macrophages and cholangiocytes with advancing fibrosis stage. Hepatocyte-specific functional analysis indicated increase of pro-apoptotic pathways and markers of bipotent hepatocyte/cholangiocyte precursors. Regression modelling was used to derive predictors of fibrosis stage. This study elucidated molecular and cell composition changes associated with increasing fibrosis stage in NAFLD and defined informative gene signatures for the disease.

Schumann et al. demonstrate that the loss of a lactate transporter Slc16a13 increases mitochondrial respiration in the liver, which reduces hepatic lipid accumulation while increasing hepatic insulin sensitivity in mice fed a high-fat diet. This study suggests SLC16A13 as a potential target for the treatment of type 2 diabetes and non-alcoholic fatty liver disease.

Abstract Diabetic Retinopathy (DR) is among the major global causes for vision loss. With the rise in diabetes prevalence, an increase in DR incidence is expected. Current understanding of both the molecular etiology and pathways involved in the initiation and progression of DR is limited. Via RNA-Sequencing, we analyzed mRNA and miRNA expression profiles of 80 human post-mortem retinal samples from 43 patients diagnosed with various stages of DR. We found differentially expressed transcripts to be predominantly associated with late stage DR and pathways such as hippo and gap junction signaling. A multivariate regression model identified transcripts with progressive changes throughout disease stages, which in turn displayed significant overlap with sphingolipid and cGMP–PKG signaling. Combined analysis of miRNA and mRNA expression further uncovered disease-relevant miRNA/mRNA associations as potential mechanisms of post-transcriptional regulation. Finally, integrating human retinal single cell RNA-Sequencing data revealed a continuous loss of retinal ganglion cells, and Müller cell mediated changes in histidine and β-alanine signaling. While previously considered primarily a vascular disease, attention in DR has shifted to additional mechanisms and cell-types. Our findings offer an unprecedented and unbiased insight into molecular pathways and cell-specific changes in the development of DR, and provide potential avenues for future therapeutic intervention.

Abstract Exploration of genetic variant-to-gene relationships by quantitative trait loci such as expression QTLs is a frequently used tool in genome-wide association studies. However, the wide range of public QTL databases and the lack of batch annotation features complicate a comprehensive annotation of GWAS results. In this work, we introduce the tool “Qtlizer” for annotating lists of variants in human with associated changes in gene expression and protein abundance using an integrated database of published QTLs. Features include incorporation of variants in linkage disequilibrium and reverse search by gene names. Analyzing the database for base pair distances between best significant eQTLs and their affected genes suggests that the commonly used cis-distance limit of 1,000,000 base pairs might be too restrictive, implicating a substantial amount of wrongly and yet undetected eQTLs. We also ranked genes with respect to the maximum number of tissue-specific eQTL studies in which a most significant eQTL signal was consistent. For the top 100 genes we observed the strongest enrichment with housekeeping genes (P = 2 × 10–6) and with the 10% highest expressed genes (P = 0.005) after grouping eQTLs by r2 > 0.95, underlining the relevance of LD information in eQTL analyses. Qtlizer can be accessed via https://genehopper.de/qtlizer or by using the respective Bioconductor R-package ( https://doi.org/10.18129/B9.bioc.Qtlizer ).

Abstract Background The ability to generate recombinant drug target proteins is important for drug discovery research as it facilitates the investigation of drug-target-interactions in vitro. To accomplish this, the target’s exact protein sequence is required. Public databases, such as Ensembl, UniProt and RefSeq, are extensive protein and nucleotide sequence repositories. However, many sequences for non-human organisms are predicted by computational pipelines and may thus be incomplete or incorrect. This could lead to misinterpreted experimental outcomes due to gaps or errors in orthologous drug target sequences. Transcriptome analysis by RNA-Seq has been established as a standard method for gene expression analysis. Apart from this common application, paired-end RNA-Seq data can also be used to obtain full coverage cDNA sequences via de novo transcriptome assembly. Methods To assess whether de novo transcriptome assemblies can be used to determine a protein’s sequence by searching the assembly for a known orthologous sequence, we generated 3 × 6 = 18 tissue specific assemblies (three organs: brain, kidney and liver; six species: human, mouse, rat, dog, pig and cynomolgus monkey). These assemblies and the manually curated human protein sequences from UniProtKB/Swiss-Prot were used in a reciprocal BLAST search to identify best matching hits. We automated and generalised our approach and present the a&o-tool, a workflow which exploits de novo assemblies of paired-end RNA-Seq data and orthology information for target sequence validation and refinement across related species. Furthermore, the a&o-tool extracts best hits’ sequences from a reciprocal BLAST search, translates them into protein sequences, computes a multiple sequence alignment and quantifies the refinement. Results For the three human assemblies we observed a hit rate greater than 60% with 100% sequence coverage and identity. For assemblies from the other species we observed similar hit rates and coverage with highest identities for cynomolgus monkey. Conclusions In summary, we show how to refine protein sequences using RNA-Seq data and sequence information from closely related species. With the a&o-tool we provide a fully automated pipeline to perform refinement including cDNA translation and multiple sequence alignment for visual inspection. The major prerequisite for applying the a&o-tool is high quality sequencing data.

This study employed GIS tools to help optimise faecal sludge (FS) management in the Greater Accra Metropolitan Area (GAMA) and its environs in Ghana. First, the rates of excreta generation, FS generation and FS collection were quantified based on literature, census and FS discharge data obtained from treatment plants in the study area. Next, we mapped the FS collection to the administrative areas in GAMA based on discharge records obtained from Lavender Hill, the main faecal treatment (FTP) and estimated the travel distance and travel time from the various FS desludging neighbourhoods to the plant. The results of the study show that the excreta and FS generation rates in GAMA are 604 L/cap/yr and 4,137 L/cap/yr, respectively. About 1 million m3 of FS was collected and treated in the study area in 2018, with a collection rate of 244 L/cap/yr. The private sector dominates this collection, haulage and treatment of FS in GAMA. The GIS analysis has provided fundamental data that will be useful in rationalising the FS emptying and transport cost in the study area. Moreover, it revealed that about 20–40% of the localities were outside the 15–25 km sustainable maximum transport distance recommended by some scholars. Finally, the findings highlight the importance of looking beyond administrative boundaries when planning for FS management logistics and infrastructure and also show that the most impoverished communities in the Accra metropolis may not necessarily be the least served when it comes to FS collection and haulage.

While Bmp4 has a well-established role in the commitment of mesenchymal stem cells into the adipogenic lineage, its role in brown adipocyte formation and activity is not well defined. Here, we show that Bmp4 has a dual function in adipogenesis by inducing adipocyte commitment while inhibiting the acquisition of a brown phenotype during terminal differentiation. Selective brown adipose tissue overexpression of Bmp4 in mice induces a shift from a brown to a white-like adipocyte phenotype. This effect is mediated by Smad signaling and might be in part due to suppression of lipolysis, via regulation of hormone sensitive lipase expression linked to reduced Ppar activity. Given that we observed a strong correlation between BMP4 levels and adipocyte size, as well as insulin sensitivity in humans, we propose that Bmp4 is an important factor in the context of obesity and type 2 diabetes.

BACKGROUND:Activation of the AMP-activated protein kinase (AMPK) is an attractive approach for the treatment of type 2 diabetes. AMPK activation reduces glucose levels in animal models of type 2 diabetes by increasing glucose uptake in skeletal muscles and reducing hepatic glucose production. Furthermore, AMPK activation ameliorates hepatic steatosis in animal models. For the clinical development of AMPK activators it is essential to have a reliable target engagement marker for appropriate dose finding and to support proof of clinical principle. While the activation of AMPK by quantification of the phosphorylation of AMPK at Thr172 in target tissues can be assessed pre-clinically, this is not feasible in clinical studies. Therefore, we attempted to identify and translate a peripheral target engagement biomarker downstream of AMPK activation for clinical use in blood samples. METHODS:For pharmacological activation of AMPK, two AMPK activators were synthesized (compound 1 and 2). A compound with structural similarities but no pharmacological effect on AMPK phosphorylation was synthesized as negative control (compound 3). Whole blood from healthy volunteers was incubated with an AMPK activator for up to 6 hours and mRNA sequencing was performed. Additionally, human PBMCs were isolated to evaluate Thr172-phosphorylation of AMPK in Western blots. In order to enable identification of translatable biomarker candidates, blood samples from HanWistar rats treated for two weeks with an AMPK activator were also subjected to mRNA sequencing. Furthermore, concentration-response curves for four biomarker candidates were recorded in human blood samples using Nanostring nCounter technology. Finally, ZDF rats were treated with increasing doses of compound 2 for five weeks to investigate the glucose-lowering efficacy. To investigate changes of mRNA expression of two selected biomarker candidates in this ZDF rat study, qRT-PCR was performed. RESULTS:Pharmacological activation of AMPK in human PBMCs revealed an increase in Thr172-phosphorylation of AMPK, confirming target engagement in these blood cells. RNA sequencing of human blood samples identified 608 deregulated genes after AMPK activation. Additionally, AMPK activation led to deregulation of 367 genes in whole blood from HanWistar rats which mapped to the respective human genes. 22 genes out of the intersection of genes deregulated in both species are proposed as potential translatable target engagement biomarker candidates. The most prominent genes were transmembrane glycoprotein NMB (GPNMB, osteoactivin), calcium-binding protein A9 (S100A9), peptidoglycan recognition protein (PGLYRP1) and Ras homolog gene family, member B (RHOB). Specificity for AMPK was shown by testing inactive compound 3 in HanWistar rats. The exposure-effect relationship for GPNMB was investigated in a subchronic study in diabetic ZDF rats. GPNMB showed a dose-dependent up-regulation both acutely and after subchronic dosing. GPNMB up-regulation correlated with an increased Thr172-phosphorylation of AMPK in liver and quadriceps muscle in rats. CONCLUSION:GPNMB has been identified as a translatable target engagement biomarker for use in clinical studies.

Through its history, agricultural cooperation has developed standards and values, leading specific decision making processes. The article proposes a model of decision based on "dialectical tension". By taking into account the dynamic relationship between stakeholders and organizations, this innovative model overcomes the limits of the stakeholders’ theory.