Lineage fate of ductular reactions in liver injury and carcinogenesis.

Ductular reactions (DRs) are observed in virtually all forms of human liver disease; however, the histogenesis and function of DRs in liver injury are not entirely understood. It is widely believed that DRs contain bipotential liver progenitor cells (LPCs) that serve as an emergency cell pool to regenerate both cholangiocytes and hepatocytes and may eventually give rise to hepatocellular carcinoma (HCC). Here, we used a murine model that allows highly efficient and specific lineage labeling of the biliary compartment to analyze the histogenesis of DRs and their potential contribution to liver regeneration and carcinogenesis. In multiple experimental and genetic liver injury models, biliary cells were the predominant precursors of DRs but lacked substantial capacity to produce new hepatocytes, even when liver injuries were prolonged up to 12 months. Genetic modulation of NOTCH and/or WNT/β-catenin signaling within lineage-tagged DRs impaired DR expansion but failed to redirect DRs from biliary differentiation toward the hepatocyte lineage. Further, lineage-labeled DRs did not produce tumors in genetic and chemical HCC mouse models. In summary, we found no evidence in our system to support mouse biliary-derived DRs as an LPC pool to replenish hepatocytes in a quantitatively relevant way in injury or evidence that DRs give rise to HCCs

Immune system and peripheral nerves in propagation of prions to CNS.

Prions are not only unique in the way they replicate. Also the sequence of events triggered by peripheral prion infection, generically termed 'peripheral pathogenesis', sets prions aside from all other known pathogens. Whereas most bacteria, parasites, and viruses trigger innate and adaptive immune responses, the mammalian immune system appears to be remarkably oblivious to prions. Transmissible spongiform encephalopathies (TSEs) do not go along with inflammatory infiltrates, and antibodies to the prion protein are not typically raised during the course of the disease. On the other hand, there is conspicuous involvement of lymphoid organs, which accumulate sizeable concentrations of the infectious agent early during disease. Moreover, various states of immune deficiency can abolish peripheral pathogenesis and prevent 'take' of infection when prions are administered to peripheral sites. Here, we critically re-visit the current evidence for an involvement of the immune system in prion diseases, and will attempt to trace the elaborate mechanisms by which prions, upon entry into the body from peripheral sites, reach the brain

Early and rapid engraftment of bone marrow-derived microglia in scrapie

Prion neuroinvasion is accompanied by maximal activation of microglia, the significance of which for pathogenesis is unknown. Here, we used bone marrow (BM) cells expressing GFP (green fluorescent protein) to study the turnover of microglia in mouse scrapie. We found that >or=50% of all brain microglia were replaced by BM-derived cells before clinical disease onset. In terminally sick mice, microglia density increased threefold to fourfold. Hence BM-derived microglia rapidly and efficaciously colonize the brain in scrapie. Whereas reconstitution of wild-type mice with prion protein-deficient (Prnp(o/o)) BM did not alter scrapie pathogenesis, Prnp(o/o) mice transplanted with wild-type BM cells were resistant to peripherally administered prions despite high levels of infectivity in the spleen. Cerebellar homogenates from prion-inoculated Prnp(o/o) mice reconstituted with >10% of wild-type microglia failed to infect transgenic mice overexpressing the cellular prion protein. Hence, in contrast to previous reports, microglia are not competent for efficient prion transport and replication in vivo

Repetitive Immunization Enhances the Susceptibility of Mice to Peripherally Administered Prions

The susceptibility of humans and animals to prion infections is determined by the virulence of the infectious agent, by genetic modifiers, and by hitherto unknown host and environmental risk factors. While little is known about the latter two, the activation state of the immune system was surmised to influence prion susceptibility. Here we administered prions to mice that were repeatedly immunized by two initial injections of CpG oligodeoxynucleotides followed by repeated injections of bovine serum albumin/alum. Immunization greatly reduced the required dosage of peripherally administered prion inoculum necessary to induce scrapie in 50% of mice. No difference in susceptibility was observed following intracerebral prion challenge. Due to its profound impact onto scrapie susceptibility, the host immune status may determine disease penetrance after low-dose prion exposure, including those that may give rise to iatrogenic and variant Creutzfeldt-Jakob disease

Aerosols Transmit Prions to Immunocompetent and Immunodeficient Mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrPC, efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrPC selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrPSc and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories

Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body’s vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor–β (TGFβ) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)–dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)+ perivascular cells. In turn, genetic deletion of Lrg1 hampered growth of lung metastasis. Postsurgical adjuvant administration of an LRG1-neutralizing antibody delayed metastatic growth and increased overall survival. This study has established a systems map of early primary tumor-induced vascular changes and identified LRG1 as a therapeutic target for metastasis

Intraepithelial and Interstitial Deposition of Pathological Prion Protein in Kidneys of Scrapie-Affected Sheep

Prions have been documented in extra-neuronal and extra-lymphatic tissues of humans and various ruminants affected by Transmissible Spongiform Encephalopathy (TSE). The presence of prion infectivity detected in cervid and ovine blood tempted us to reason that kidney, the organ filtrating blood derived proteins, may accumulate disease associated PrPSc. We collected and screened kidneys of experimentally, naturally scrapie-affected and control sheep for renal deposition of PrPSc from distinct, geographically separated flocks. By performing Western blot, PET blot analysis and immunohistochemistry we found intraepithelial (cortex, medulla and papilla) and occasional interstitial (papilla) deposition of PrPSc in kidneys of scrapie-affected sheep. Interestingly, glomerula lacked detectable signals indicative of PrPSc. PrPSc was also detected in kidneys of subclinical sheep, but to significantly lower degree. Depending on the stage of the disease the incidence of PrPSc in kidney varied from approximately 27% (subclinical) to 73.6% (clinical) in naturally scrapie-affected sheep. Kidneys from flocks without scrapie outbreak were devoid of PrPSc. Here we demonstrate unexpectedly frequent deposition of high levels of PrPSc in ovine kidneys of various flocks. Renal deposition of PrPSc is likely to be a pre-requisite enabling prionuria, a possible co-factor of horizontal prion-transmission in sheep

mTOR regulates MAPKAPK2 translation to control the senescence-associated secretory phenotype

Senescent cells secrete a combination of factors collectively known as the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence and activates an immune surveillance response, but it can also show pro-tumorigenic properties and contribute to age-related pathologies. In a drug screen to find new SASP regulators, we uncovered the mTOR inhibitor rapamycin as a potent SASP suppressor. Here we report a mechanism by which mTOR controls the SASP by differentially regulating the translation of the MK2 (also known as MAPKAPK2) kinase through 4EBP1. In turn, MAPKAPK2 phosphorylates the RNA-binding protein ZFP36L1 during senescence, inhibiting its ability to degrade the transcripts of numerous SASP components. Consequently, mTOR inhibition or constitutive activation of ZFP36L1 impairs the non-cell-autonomous effects of senescent cells in both tumour-suppressive and tumour-promoting contexts. Altogether, our results place regulation of the SASP as a key mechanism by which mTOR could influence cancer, age-related diseases and immune responses

ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Detection and Localisation of PrPSc in the Liver of Sheep Infected with Scrapie and Bovine Spongiform Encephalopathy

Prions are largely contained within the nervous and lymphoid tissue of transmissible spongiform encephalopathy (TSE) infected animals. However, following advances in diagnostic sensitivity, PrPSc, a marker for prion disease, can now be located in a wide range of viscera and body fluids including muscle, saliva, blood, urine and milk, raising concerns that exposure to these materials could contribute to the spread of disease in humans and animals. Previously we demonstrated low levels of infectivity in the liver of sheep experimentally challenged with bovine spongiform encephalopathy. In this study we show that PrPSc accumulated in the liver of 89% of sheep naturally infected with scrapie and 100% of sheep challenged with BSE, at both clinical and preclinical stages of the disease. PrPSc was demonstrated in the absence of obvious inflammatory foci and was restricted to isolated resident cells, most likely Kupffer cells