Publications by authors named "Robert Geffers"

226 Publications

GPR15 facilitates recruitment of regulatory T cells to promote colorectal cancer.

Cancer Res 2021 Mar 16. Epub 2021 Mar 16.

Institute of Medical Microbiology, University Hospital Essen, University of Duisburg-Essen

Colorectal cancer (CRC) is one of the most frequent malignancies worldwide. Despite considerable progress in early detection and treatment, there is still an unmet need for novel anti-tumor therapies, particularly in advanced CRC. Regulatory T cells (Tregs) are increased in the peripheral blood and tumor tissue of CRC patients. Recently, transient ablation of tumor-associated Tregs was shown to foster CD8+ T cell-mediated anti-tumoral immunity in murine CRC models. However, before considering therapies, targeting Tregs in cancer patients and detailed knowledge of the phenotype and features of tumor-associated Tregs is indispensable. Here we demonstrate in a murine model of inflammation-induced CRC that tumor-associated Tregs are mainly of thymic origin and equipped with a specific set of molecules strongly associated with enhanced migratory properties. Particularly, a dense infiltration of Tregs in mouse and human CRC lesions correlated with increased expression of the orphan chemoattractant receptor GPR15 on these cells. Comprehensive gene expression analysis revealed that tumor-associated GPR15+ Tregs have a Th17-like phenotype, thereby producing IL-17 and TNF-α. Gpr15 deficiency repressed Treg infiltration in CRC, which paved the way for enhanced anti-tumoral CD8+ T cell immunity and reduced tumorigenesis. In conclusion, GPR15 represents a promising novel target for modifying T cell-mediated anti-tumoral immunity in CRC.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2133DOI Listing
March 2021

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion.

Curr Biol 2021 Mar 6. Epub 2021 Mar 6.

Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. Electronic address:

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.
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http://dx.doi.org/10.1016/j.cub.2021.02.043DOI Listing
March 2021

Role of endothelial microRNA 155 on capillary leakage in systemic inflammation.

Crit Care 2021 02 22;25(1):76. Epub 2021 Feb 22.

Division of Nephrology and Hypertension, Hannover Medical School, Hannover, Germany.

Background: Capillary leakage is a key contributor to the pathological host response to infections. The underlying mechanisms remain incompletely understood, and the role of microRNAs (MIR) has not been investigated in detail. We hypothesized that specific MIRs might be regulated directly in the endothelium thereby contributing to vascular leakage.

Methods: SmallRNA sequencing of endotoxemic murine pulmonary endothelial cells (ECs) was done to detect regulated vascular MIRs. In vivo models: transgenic zebrafish (flk1:mCherry/l-fabp:eGFP-DPB), knockout/wildtype mouse (B6.Cg-Mir155tm1.1Rsky/J); disease models: LPS 17.5 mg/kgBW and cecal ligation and puncture (CLP); in vitro models: stimulated human umbilical vein EC (HUVECs), transendothelial electrical resistance.

Results: Endothelial MIR155 was identified as a promising candidate in endotoxemic murine pulmonary ECs (25 × upregulation). Experimental overexpression in a transgenic zebrafish line and in HUVECs was sufficient to induce spontaneous vascular leakage. To the contrary, genetic MIR155 reduction protects against permeability both in vitro and in endotoxemia in vivo in MIR155 heterozygote knockout mice thereby improving survival by 40%. A tight junction protein, Claudin-1, was down-regulated both in endotoxemia and by experimental MIR155 overexpression. Translationally, MIR155 was detectable at high levels in bronchoalveolar fluid of patients with ARDS compared to healthy human subjects.

Conclusions: We found that MIR155 is upregulated in the endothelium in mouse and men as part of a systemic inflammatory response and might contribute to the pathophysiology of vascular leakage in a Claudin-1-dependent manner. Future studies have to clarify whether MIR155 could be a potential therapeutic target.
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http://dx.doi.org/10.1186/s13054-021-03500-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901081PMC
February 2021

No impact of a short-term climatic "El Niño" fluctuation on gut microbial diversity in populations of the Galápagos marine iguana (Amblyrhynchus cristatus).

Naturwissenschaften 2021 Feb 2;108(1). Epub 2021 Feb 2.

Zoological Institute, Technische Universität Braunschweig, Braunschweig, Germany.

Gut microorganisms are crucial for many biological functions playing a pivotal role in the host's well-being. We studied gut bacterial community structure of marine iguana populations across the Galápagos archipelago. Marine iguanas depend heavily on their specialized gut microbiome for the digestion of dietary algae, a resource whose growth was strongly reduced by severe "El Niño"-related climatic fluctuations in 2015/2016. As a consequence, marine iguana populations showed signs of starvation as expressed by a poor body condition. Body condition indices (BCI) varied between island populations indicating that food resources (i.e., algae) are affected differently across the archipelago during 'El Niño' events. Though this event impacted food availability for marine iguanas, we found that reductions in body condition due to "El Niño"-related starvation did not result in differences in bacterial gut community structure. Species richness of gut microorganisms was instead correlated with levels of neutral genetic diversity in the distinct host populations. Our data suggest that marine iguana populations with a higher level of gene diversity and allelic richness may harbor a more diverse gut microbiome than those populations with lower genetic diversity. Since low values of these diversity parameters usually correlate with small census and effective population sizes, we use our results to propose a novel hypothesis according to which small and genetically less diverse host populations might be characterized by less diverse microbiomes. Whether such genetically depauperate populations may experience additional threats from reduced dietary flexibility due to a limited intestinal microbiome is currently unclear and calls for further investigation.
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http://dx.doi.org/10.1007/s00114-020-01714-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854437PMC
February 2021

p53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

Cell Mol Gastroenterol Hepatol 2021 Jan 21;11(5):1387-1404. Epub 2021 Jan 21.

Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address:

Background & Aims: A coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury.

Methods: Nitisinone was reduced or withdrawn in Fah mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2.

Results: In a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response.

Conclusions: Characteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.
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http://dx.doi.org/10.1016/j.jcmgh.2021.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024980PMC
January 2021

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

J Mol Med (Berl) 2021 Mar 23;99(3):425-438. Epub 2021 Jan 23.

Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.
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http://dx.doi.org/10.1007/s00109-020-02020-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900040PMC
March 2021

Serum Response Factor (SRF) Drives the Transcriptional Upregulation of the MDM4 Oncogene in HCC.

Cancers (Basel) 2021 Jan 8;13(2). Epub 2021 Jan 8.

Institute of Pathology, University Hospital Heidelberg, 69120 Heidelberg, Germany.

Different molecular mechanisms support the overexpression of the mouse double minute homolog 4 (MDM4), a functional p53 inhibitor, in human hepatocellular carcinoma (HCC). However, the transcription factors (TFs) leading to its transcriptional upregulation remain unknown. Following promoter and gene expression analyses, putative TFs were investigated using gene-specific siRNAs, cDNAs, luciferase reporter assays, chromatin immunoprecipitation, and XI-011 drug treatment in vitro. Additionally, MDM4 expression was investigated in transgenic mice. We observed a copy-number-independent upregulation of in human HCCs. Serum response factor (SRF), ELK1 and ELK4 were identified as TFs activating transcription. While SRF was constitutively detected in TF complexes at the promoter, presence of ELK1 and ELK4 was cell-type dependent. Furthermore, MDM4 was upregulated in SRF-VP16-driven murine liver tumors. The pharmacological inhibitor XI-011 exhibited anti- activity by downregulating the TFs driving transcription, which decreased HCC cell viability and increased apoptosis. In conclusion, SRF drives transcriptional upregulation in HCC, acting in concert with either ELK1 or ELK4. The transcriptional regulation of may be a promising target for precision oncology of human HCC, as XI-011 treatment exerts anti- activity independent from the copy number and the status.
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http://dx.doi.org/10.3390/cancers13020199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829828PMC
January 2021

Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

mSystems 2020 Dec 22;5(6). Epub 2020 Dec 22.

Center Algatech, Institute of Microbiology of the Czech Academy of Science, Třeboň, Czechia

Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation. Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in may have other functions not linked to bioenergetics.
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http://dx.doi.org/10.1128/mSystems.01044-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762795PMC
December 2020

Germline variation of Ribonuclease H2 genes in ovarian cancer patients.

J Ovarian Res 2020 Dec 22;13(1):146. Epub 2020 Dec 22.

Department of Gynaecology and Obstetrics, Gynaecology Research Unit (OE 6411), Hannover Medical School, Carl-Neuberg-Str. 1, D-30625, Hannover, Germany.

Epithelial ovarian carcinoma (EOC) is a genetically heterogeneous disease that is partly driven by molecular defects in mismatch repair (MMR) or homology-directed DNA repair (HDR). Ribonuclease H2 serves to remove mis-incorporated ribonucleotides from DNA which alleviates HDR mechanisms and guides the MMR machinery. Although Ribonuclease H2 has been implicated in cancer, the role of germline variants for ovarian cancer is unknown. In the present case-control study, we sequenced the coding and flanking untranslated regions of the RNASEH2A, RNASEH2B and RNASEH2C genes, encoding all three subunits of Ribonuclease H2, in a total of 602 German patients with EOC and of 940 healthy females from the same population. We identified one patient with a truncating variant in RNASEH2B, p.C44X, resulting in a premature stop codon. This patient had high-grade serous EOC with an 8 years survival after platinum/taxane-based therapy. Subsequent analysis of TCGA data similarly showed a significantly longer progression-free survival in ovarian cancer patients with low RNASEH2B or RNASEH2C expression levels. In conclusion, loss-of-function variants in Ribonuclease H2 genes are not common predisposing factors in ovarian cancer but the possibility that they modulate therapeutic platinum response deserves further investigation.
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http://dx.doi.org/10.1186/s13048-020-00753-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756920PMC
December 2020

Different DOACs Control Inflammation in Cardiac Ischemia-Reperfusion Differently.

Circ Res 2021 Feb 23;128(4):513-529. Epub 2020 Dec 23.

Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital, Leipzig, Germany (I.G., S.F., A.E., S.N., M.M.A.-D., R.R., J.M., D.G., R.B., S.K., B.I., K.S.).

Rationale: While thrombin is the key protease in thrombus formation, other coagulation proteases, such as fXa (factor Xa) or aPC (activated protein C), independently modulate intracellular signaling via partially distinct receptors.

Objectives: To study the differential effects of fXa or fIIa (factor IIa) inhibition on gene expression and inflammation in myocardial ischemia-reperfusion injury.

Methods And Results: Mice were treated with a direct fIIa inhibitor (fIIai) or direct fXa inhibitor (fXai) at doses that induced comparable anticoagulant effects ex vivo and in vivo (tail-bleeding assay and FeCl-induced thrombosis). Myocardial ischemia-reperfusion injury was induced via left anterior descending ligation. We determined infarct size and in vivo aPC generation, analyzed gene expression by RNA sequencing, and performed immunoblotting and ELISA. The signaling-only 3K3A-aPC variant and inhibitory antibodies that blocked all or only the anticoagulant function of aPC were used to determine the role of aPC. Doses of fIIai and fXai that induced comparable anticoagulant effects resulted in a comparable reduction in infarct size. However, unbiased gene expression analyses revealed marked differences, including pathways related to sterile inflammation and inflammasome regulation. fXai but not fIIai inhibited sterile inflammation by reducing the expression of proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNFα [tumor necrosis factor alpha]), as well as NF-κB (nuclear factor kappa B) and inflammasome activation. This anti-inflammatory effect was associated with reduced myocardial fibrosis 28 days post-myocardial ischemia-reperfusion injury. Mechanistically, in vivo aPC generation was higher with fXai than with fIIai. Inhibition of the anticoagulant and signaling properties of aPC abolished the anti-inflammatory effect associated with fXai, while inhibiting only the anticoagulant function of aPC had no effect. Combining 3K3A-aPC with fIIai reduced the inflammatory response, mimicking the fXai-associated effect.

Conclusions: We showed that specific inhibition of coagulation via direct oral anticoagulants had differential effects on gene expression and inflammation, despite comparable anticoagulant effects and infarct sizes. Targeting individual coagulation proteases induces specific cellular responses unrelated to their anticoagulant effect.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317219DOI Listing
February 2021

T cell receptor repertoires within liver allografts are different to those in the peripheral blood.

J Hepatol 2021 May 18;74(5):1167-1175. Epub 2020 Dec 18.

Department of Gastroenterology, Hepatology & Endocrinology, Hannover Medical School, Hannover, Germany. Electronic address:

Background & Aims: T cells are the main mediators of allogeneic immune responses. Specific T cell clones can be tracked by their unique T cell receptor (TCR), but specificity and function remain elusive and have not been investigated in human liver biopsies thus far.

Methods: TCR repertoire analysis of CD4, CD8, and regulatory T cells of the peripheral blood and liver graft was performed in 7 liver transplant recipients with either stable course (non-rejector, NR), subclinical cellular rejection (SCR), or acute cellular rejection (ACR) during an observation period from pre-transplant to 6 years post-transplant. Furthermore, donor-reactive T cells, identified by their expression of CD154 and glycoprotein A repetitions predominant (GARP) after allogeneic activation, were tracked longitudinally in peripheral blood and within the liver allograft.

Results: Although overall clonality of the TCR repertoire did not increase in peripheral blood after liver transplantation, clonality of donor-reactive CD4 and regulatory T cells increased and these clones accumulated within the liver graft. Surprisingly, the TCR repertoires between the liver graft and the periphery were distinct and showed only limited overlap. Notably, during ACR, TCR repertoires aligned suggesting either graft-specific homing or release of activated T cells from the graft.

Conclusions: This is the first study comparing TCR repertoires between liver grafts and blood in patients with NR, SCR, and ACR. Moreover, we attribute specificity and function to a subgroup of intragraft T cell populations. Given the limited overlap between peripheral blood and intragraft repertoires, future studies investigating function and specificities of T cells after liver transplantation should focus on the intragraft immune response.

Lay Summary: In solid organ transplantation, T cells are key mediators of the recipient's immune response directed at the transplanted organ. In our study, we characterised the T cell repertoire in a cohort of 7 liver transplant recipients. We demonstrate that donor-specific T cells expand clonally and accumulate in the transplanted liver. Moreover, we show that the composition of T cells in peripheral blood differs from the T cells in the liver allograft, only aligning in the context of acute cellular rejection but not in normal graft or subclinical cellular rejection. This indicates that the intragraft immune response is not mirrored in the peripheral blood. Our findings clarify the importance of protocol liver biopsies in identifying intragraft immune responses for future investigations of allo-directed immune responses.
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http://dx.doi.org/10.1016/j.jhep.2020.12.014DOI Listing
May 2021

Correction to: Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines.

BMC Cancer 2020 Nov 23;20(1):1127. Epub 2020 Nov 23.

Fakultät für Medizin, Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, 81675, München, Germany.

An amendment to this paper has been published and can be accessed via the original article.
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http://dx.doi.org/10.1186/s12885-020-07642-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682055PMC
November 2020

Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology.

J Mol Cell Cardiol 2021 01 28;150:91-100. Epub 2020 Oct 28.

Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany; REBIRTH Excellence Cluster, Hannover Medical School, Hannover, Germany. Electronic address:

Background: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing.

Methods And Results: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level.

Conclusion: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.
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http://dx.doi.org/10.1016/j.yjmcc.2020.10.008DOI Listing
January 2021

Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines.

BMC Cancer 2020 Oct 28;20(1):1039. Epub 2020 Oct 28.

Fakultät für Medizin, Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, 81675, München, Germany.

Background: Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies.

Methods: A time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3.

Results: The Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments.

Conclusions: Taken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.
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http://dx.doi.org/10.1186/s12885-020-07540-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594334PMC
October 2020

Identification of miRNAs associated with dendritic cell dysfunction during acute and chronic hepatitis B virus infection.

J Med Virol 2021 Jun 10;93(6):3697-3706. Epub 2020 Nov 10.

Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India.

The uptake or expression of hepatitis B virus (HBV) proteins by dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA (miRNA) may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease namely, immune active (IA; n = 20); low replicative (LR; n = 20); HBeAg negative (n = 20); acute viral hepatitis (AVH, n = 20) and healthy controls (n = 20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by quantitative polymerase Chain Reaction (qPCR). The miRNA-messenger RNA (mRNA)regulatory networks identified 19 miRNAs and 12 target gene interactions in major histocompatibility complex and other immune pathways. miR-2278, miR-615-3p, and miR-3681-3p were downregulated in the IA group compared to healthy control, miR-152-3p and miR-3613-3p in the LR group compared to IA group and miR-152-3p and miR-503-3p in HBe negative compared to LR group. However, miR-7-1-1-3p, miR-192-5p, miR-195-5p, and miR-32-5p in LR, miR-342-3p, and miR-940 in HBe negative, and miR-34a-5p, miR-130b-3p, miR-221-3p, miR-320a, miR-324-5p, and miR-484 in AVH were upregulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs-mRNAs in DCs seems responsible for an impaired immune response during HBV pathogenesis.
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http://dx.doi.org/10.1002/jmv.26629DOI Listing
June 2021

YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.

Int J Mol Sci 2020 Sep 25;21(19). Epub 2020 Sep 25.

Clinic of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke University, 39120 Magdeburg, Germany.

Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.
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http://dx.doi.org/10.3390/ijms21197076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583764PMC
September 2020

IDH1/2 mutations in acute myeloid leukemia patients and risk of coronary artery disease and cardiac dysfunction-a retrospective propensity score analysis.

Leukemia 2020 Sep 18. Epub 2020 Sep 18.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Carl-Neuberg Strasse 1, 30625, Hannover, Germany.

Clonal hematopoiesis of indeterminate potential (CHIP) is linked to leukemia gene mutations and associates with an increased risk for coronary artery disease and poor prognosis in ischemic cardiomyopathy. Two recurrently mutated genes in CHIP and adult acute myeloid leukemia (AML) encode for isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). Global expression of mutant IDH2 in transgenic mice-induced dilated cardiomyopathy and muscular dystrophy. In this retrospective observational study, we investigated whether mutant IDH1/2 predisposes to cardiovascular disease in AML patients. Among 363 AML patients, IDH1 and IDH2 mutations were detected in 26 (7.2%) and 39 patients (10.7%), respectively. Mutant IDH1 patients exhibited a significantly higher prevalence of coronary artery disease (26.1% vs. 6.4%, p = 0.002). Applying inverse probability-weighting analysis, patients with IDH1/2 mutations had a higher risk for a declining cardiac function during AML treatment compared to IDH1/2 wild type patients [left ventricular ejection fraction pretreatment compared to 10 months after diagnosis: 59.2% to 41.9% (p < 0.001) vs 58.5% to 55.4% (p = 0.27), respectively]. Mechanistically, RNA sequencing and immunostaining in hiPS-derived cardiomyocytes indicated that the oncometabolite R-2HG exacerbated doxorubicin mediated cardiotoxicity. Evaluation of IDH1/2 mutation status may therefore help identifying AML patients at risk for cardiovascular complications during cytotoxic treatment.
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http://dx.doi.org/10.1038/s41375-020-01043-xDOI Listing
September 2020

Fibrosis and Immune Cell Infiltration Are Separate Events Regulated by Cell-Specific Receptor Notch3 Expression.

J Am Soc Nephrol 2020 11 28;31(11):2589-2608. Epub 2020 Aug 28.

Clinic of Nephrology and Hypertension, Diabetes and Endocrinology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany

Background: Kidney injuries that result in chronic inflammation initiate crosstalk between stressed resident cells and infiltrating immune cells. In animal models, whole-body receptor deficiency protects from leukocyte infiltration and organ fibrosis. However, the relative contribution of expression in tissue versus infiltrating immune cells is unknown.

Methods: Chimeric mice deficient for in hematopoietic cells and/or resident tissue cells were generated, and kidney fibrosis and inflammation after unilateral ureteral obstruction (UUO) were analyzed. Adoptive transfer of labeled bone marrow-derived cells validated the results in a murine ear infection model. adhesion assays, integrin activation, and extracellular matrix production were analyzed.

Results: Fibrosis follows UUO, but inflammatory cell infiltration mostly depends upon Notch3 expression in hematopoietic cells, which coincides with an enhanced proinflammatory milieu (., CCL2 and CCL5 upregulation). Notch3 expression on CD45 leukocytes plays a prominent role in efficient cell transmigration. Functionally, leukocyte adhesion and integrin activation are abrogated in the absence of receptor Notch3. Chimeric animal models also reveal that tubulointerstitial fibrosis develops, even in the absence of prominent leukocyte infiltrates after ureteral obstruction. Deleting Notch3 receptors on resident cells blunts kidney fibrosis, ablates NF-B signaling, and lessens matrix deposition.

Conclusions: Cell-specific receptor Notch3 signaling independently orchestrates leukocyte infiltration and organ fibrosis. Interference with Notch3 signaling may present a novel therapeutic approach in inflammatory as well as fibrotic diseases.
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http://dx.doi.org/10.1681/ASN.2019121289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608975PMC
November 2020

Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

Cell 2020 09 5;182(6):1419-1440.e23. Epub 2020 Aug 5.

Department of Infectious Diseases and Respiratory Medicine, Charité, Universitätsmedizin Berlin, Berlin, Germany; German Center for Lung Research (DZL).

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRCD11c inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DR monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
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http://dx.doi.org/10.1016/j.cell.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405822PMC
September 2020

Rare heterozygous GDF6 variants in patients with renal anomalies.

Eur J Hum Genet 2020 12 31;28(12):1681-1693. Epub 2020 Jul 31.

Department of Human Genetics, Hannover Medical School, 30625, Hannover, Germany.

Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis.
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http://dx.doi.org/10.1038/s41431-020-0678-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784874PMC
December 2020

Integrative Bioinformatic Analyses of Global Transcriptome Data Decipher Novel Molecular Insights into Cardiac Anti-Fibrotic Therapies.

Int J Mol Sci 2020 Jul 2;21(13). Epub 2020 Jul 2.

Chair of Medical Informatics, Friedrich-Alexander University (FAU) of Erlangen-Nürnberg, 91058 Erlangen, Germany.

Integrative bioinformatics is an emerging field in the big data era, offering a steadily increasing number of algorithms and analysis tools. However, for researchers in experimental life sciences it is often difficult to follow and properly apply the bioinformatical methods in order to unravel the complexity and systemic effects of omics data. Here, we present an integrative bioinformatics pipeline to decipher crucial biological insights from global transcriptome profiling data to validate innovative therapeutics. It is available as a web application for an interactive and simplified analysis without the need for programming skills or deep bioinformatics background. The approach was applied to an ex vivo cardiac model treated with natural anti-fibrotic compounds and we obtained new mechanistic insights into their anti-fibrotic action and molecular interplay with miRNAs in cardiac fibrosis. Several gene pathways associated with proliferation, extracellular matrix processes and wound healing were altered, and we could identify micro (mi) RNA-21-5p and miRNA-223-3p as key molecular components related to the anti-fibrotic treatment. Importantly, our pipeline is not restricted to a specific cell type or disease and can be broadly applied to better understand the unprecedented level of complexity in big data research.
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http://dx.doi.org/10.3390/ijms21134727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370212PMC
July 2020

Library Preparation for Small RNA Transcriptome Sequencing in Patients Affected by Cerebral Cavernous Malformations.

Methods Mol Biol 2020 ;2152:467-478

International Neuroscience Institute, Hannover, Germany.

Small RNA sequencing by Illumina's Next Generation technology has revolutionized the transcriptome analysis by facilitating massive parallel sequencing of RNA molecules at low cost. Illumina's Next Generation RNA sequencing is ideal for profiling small RNA (microRNAs, snoRNAs, and piRNAs) libraries in the identification of novel biomarkers for better clinical diagnosis. This method offers significant advantages when compared to microarray analysis with the ability to identify novel transcripts, higher sensitivity, specificity, and detection of rare and low-abundance transcripts. Small RNAs, including microRNAs and snoRNAs, belong to the class of small non-coding RNAs with 50-200 nucleotides in length and are involved in post-transcriptional regulation of gene expression. Executing Illumina's Next Generation Sequencing technology, we have recently deciphered microRNAs and snoRNAs expressed in cerebral cavernous malformations (CCMs). Small RNA library preparation is a prerequisite step prior to RNA sequencing for the identification of microRNAs and snoRNAs. Here, we describe stepwise small RNA library preparation starting from total RNA isolated from CCMs patient until library validation using the Illumina TruSeq Small RNA Sample preparation kit. We believe this method will shed light into the functional identification of other novel small non-coding RNAs in CCMs that awaits discovery.
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http://dx.doi.org/10.1007/978-1-0716-0640-7_35DOI Listing
March 2021

Synergistic activity of IDH1 inhibitor BAY1436032 with azacitidine in IDH1 mutant acute myeloid leukemia.

Haematologica 2021 Feb 1;106(2):565-573. Epub 2021 Feb 1.

Hannover Medical School, Hannover, Germany.

Mutant IDH1 (mIDH1) inhibitors have shown single-agent activity in relapsed/refractory AML, though most patients eventually relapse. We evaluated the efficacy and molecular mechanism of the combination treatment with azacitidine, which is currently the standard of care in older AML patients, and mIDH1 inhibitor BAY1436032. Both compounds were evaluated in vivo as single agents and in combination with sequential (azacitidine, followed by BAY1436032) or simultaneous application in two human IDH1 mutated AML xenograft models. Combination treatment significantly prolonged survival compared to single agent or control treatment (P<.005). The sequential combination treatment depleted leukemia stem cells (LSC) by 470-fold. Interestingly, the simultaneous combination treatment depleted LSCs by 33,150-fold compared to control mice. This strong synergy is mediated through inhibition of MAPK/ERK and RB/E2F signaling. Our data strongly argues for the concurrent application of mIDH1 inhibitors and azacitidine and predicts improved outcome of this regimen in IDH1 mutated AML patients.
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http://dx.doi.org/10.3324/haematol.2019.236992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849562PMC
February 2021

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

Circulation 2020 03 17;141(9):751-767. Epub 2020 Jan 17.

Institute of Molecular and Translational Therapeutic Strategies (K.S., M.J., A.F., K.B., L.G.-L., K.X., C. Bär, A.P., A.J., K.Z., S.S., H.J.-P., J.R., K.S., S.D., M.-T.P., F.K., F.P.K., F.K., J.L., L.H., L.S., S.B., J.F., T.T.), Hannover Medical School, Germany.

Background: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis.

Methods: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing.

Results: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds.

Conclusions: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.042559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050799PMC
March 2020

Identification of Ppar-modulated miRNA hubs that target the fibrotic tumor microenvironment.

Proc Natl Acad Sci U S A 2020 01 23;117(1):454-463. Epub 2019 Dec 23.

Department for Molecular Biology, Interfaculty Institute of Cell Biology, Eberhard Karls University Tübingen, 72076 Tübingen, Germany;

Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.
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http://dx.doi.org/10.1073/pnas.1909145117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955372PMC
January 2020

Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

Emerg Microbes Infect 2019 ;8(1):1763-1776

Institute of Virology Muenster, Westfaelische Wilhelms-University, Muenster, Germany.

Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV and However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.
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http://dx.doi.org/10.1080/22221751.2019.1698271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913622PMC
April 2020

Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection.

mBio 2019 12 3;10(6). Epub 2019 Dec 3.

Research Group Biomarkers for Infectious Diseases, TWINCORE Centre for Experimental and Clinical Infection Research, Hannover, Germany

In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB ( = 8), latent TB infection (LTBI;  = 21), and treated LTBI (LTBItt;  = 6) and in uninfected exposed controls (ExC;  = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of infection on chest radiographs. SNORD104 expression decreased during infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like ( = 0.03) but not M1-like ( = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB. Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.
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http://dx.doi.org/10.1128/mBio.01037-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890987PMC
December 2019

Multiplex profiling of inflammation-related bioactive lipid mediators in Toxocara canis- and Toxocara cati-induced neurotoxocarosis.

PLoS Negl Trop Dis 2019 09 26;13(9):e0007706. Epub 2019 Sep 26.

Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Hanover, Germany.

Background: Somatic migration of Toxocara canis- and T. cati-larvae in humans may cause neurotoxocarosis (NT) when larvae accumulate and persist in the central nervous system (CNS). Host- or parasite-induced immunoregulatory processes contribute to the pathogenesis; however, detailed data on involvement of bioactive lipid mediators, e.g. oxylipins or eico-/docosanoids, which are involved in the complex molecular signalling network during infection and inflammation, are lacking.

Methodology/principal Findings: To elucidate if T. canis- and T. cati-induced NT affects the homeostasis of oxylipins during the course of infection, a comprehensive lipidomic profiling in brains (cerebra and cerebella) of experimentally infected C57BL/6J mice was conducted at six different time points post infection (pi) by liquid-chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS). Only minor changes were detected regarding pro-inflammatory prostaglandins (cyclooxygenase pathway). In contrast, a significant increase of metabolites resulting from lipoxygenase pathways was observed for both infection groups and brain regions, implicating a predominantly anti-inflammatory driven immune response. This observation was supported by a significantly increased 13-hydroxyoctadecadienoic acid (HODE)/9-HODE ratio during the subacute phase of infection, indicating an anti-inflammatory response to neuroinfection. Except for the specialised pro-resolving mediator (SPM) neuroprotectin D1 (NPD1), which was detected in mice infected with both pathogens during the subacute phase of infection, no other SPMs were detected.

Conclusions/significance: The obtained results demonstrate the influence of Toxocara spp. on oxylipins as part of the immune response of the paratenic hosts. Furthermore, this study shows differences in the alteration of the oxylipin composition between T. canis- and T. cati-brain infection. Results contribute to a further understanding of the largely unknown pathogenesis and mechanisms of host-parasite interactions during NT.
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http://dx.doi.org/10.1371/journal.pntd.0007706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762062PMC
September 2019