Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.Peer Reviewe

Primary glia cells from bank vole propagate multiple rodent-adapted scrapie prions

Since the beginning prion research has been largely dependent on animal models for deciphering the disease, drug development or prion detection and quantification. Thereby, ethical as well as cost and labour-saving aspects call for alternatives in vitro. Cell models can replace or at least complement animal studies, but their number is still limited and the application usually restricted to certain strains and host species due to often strong transmission barriers. Bank voles promise to be an exception as they or materials prepared from them are uniquely susceptible to prions from various species in vivo, in vitro and in cell-free applications. Here we present a mainly astrocyte-based primary glia cell assay from bank vole, which is infectible with scrapie strains from bank vole, mouse and hamster. Stable propagation of bank vole-adapted RML, murine 22L and RML, and hamster 263K scrapie is detectable from 20 or 30 days post exposure onwards. Thereby, the infected bank vole glia cells show similar or even faster prion propagation than likewise infected glia cells of the corresponding murine or hamster hosts. We propose that our bank vole glia cell assay could be a versatile tool for studying and comparing multiple prion strains with different species backgrounds combined in one cell assayPeer Reviewe

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.Peer Reviewe

Bartonella Adhesin A Mediates a Proangiogenic Host Cell Response

Bartonella henselae causes vasculoproliferative disorders in humans. We identified a nonfimbrial adhesin of B. henselae designated as Bartonella adhesin A (BadA). BadA is a 340-kD outer membrane protein encoded by the 9.3-kb badA gene. It has a modular structure and contains domains homologous to the Yersinia enterocolitica nonfimbrial adhesin (Yersinia adhesin A). Expression of BadA was restored in a BadA-deficient transposon mutant by complementation in trans. BadA mediates the binding of B. henselae to extracellular matrix proteins and to endothelial cells, possibly via β1 integrins, but prevents phagocytosis. Expression of BadA is crucial for activation of hypoxia-inducible factor 1 in host cells by B. henselae and secretion of proangiogenic cytokines (e.g., vascular endothelial growth factor). BadA is immunodominant in B. henselae–infected patients and rodents, indicating that it is expressed during Bartonella infections. Our results suggest that BadA, the largest characterized bacterial protein thus far, is a major pathogenicity factor of B. henselae with a potential role in the induction of vasculoproliferative disorders

Propagation of CJD Prions in Primary Murine Glia Cells Expressing Human PrPc

There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments

Endogene neuronale Vorläuferzellen unterdrücken das Wachstum von Glioblastomen

GesamtdissertationNeural precursor cells (NPCs) migrate towards glioblastoma in vivo and in vitro. They are attracted by glioma cells independent of other cell types or surrounding tissue. Young animals display prolonged survival after glioma inoculation compared to older animals due to the larger amount of precursors, which accumulate around the tumour. Young animals are naturally equipped with a greater number of NPCs. Additionally subventricular proliferation is even more restrained by the presence of a tumour only in adult animals. This further reduction of dividing precursors in adult mice solely refers to diminished proliferation rates since whole cell number and cell death rate was not affected by glioblastoma. Survival times of old animals can be aligned to the one of young mice by applying exogenous precursor cells. Proliferative capacity of NPCs is determined by the age of the subventricular zone and is an intrinsic and stable attribute. Neural precursors as well as NPC-conditioned medium directly induce glioma cell death. The transcription factor activating transcription factor-3 (ATF-3) is necessary and sufficient for the induction of cell death. Differential gene expression and morphological changes after administration of NPC-conditioned medium point to glioma cell death induced by endoplasmic reticulum stress.Neuronale Vorläuferzellen migrieren spezifisch zu Glioblastomen in vivo und in vitro. Dieser Prozess findet unabhängig von anderen Zelltypen oder umgebendem Gewebe statt. Junge Mäuse verfügen nach Inokulierung von Gliomzellen über eine längere Überlebenszeit im Vergleich zu adulten Tieren. Dies lässt sich auf die erhöhte Anzahl von Vorläuferzellen, die um den Tumor herum akkumulieren, zurückführen. Junge Tiere besitzen von Natur aus eine höhere Anzahl von neuronalen Vorläuferzellen. Dazu kommt, dass die subventrikuläre Proliferation nur in adulten Tieren zusätzlich durch den Tumor negativ beeinflusst wird. Diese Reduktion von sich teilenden subventrikulären Vorläuferzellen in adulten Mäusen beruht ausschließlich auf einer verminderten Proliferationsrate da sowohl Gesamtzellzahl und Zelltodrate vom Tumor unbeeinflußt blieben. Es zeigte sich, dass adulte Tiere nach Gabe von exogenen Vorläuferzellen die Überlebenszeit junger Tiere erreichen. Das proliferative Potential von neuronalen Vorläuferzellen wird durch das Alter der subventrikulären Zone determiniert und stellt eine intrinsische und stabile Eigenschaft dar. Neuronale Vorläuferzellen sowie Vorläuferzell-konditioniertes Medium induzieren Gliomzelltod unabhängig von äußeren Faktoren in vitro. Der Transkriptionsfaktor activating transcription factor-3 (ATF-3) ist für die Induktion von Gliomzelltod notwendig und hinreichend. Differentielle Genexpression und Morphologieänderung in Gliomzellen, induziert durch Vorläuferzell-konditioniertes Medium, lassen Zelltod durch endoplasmatischen Stress vermuten

Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics