Murine Strains Research Articles

Identification of polo-like kinase 1 as a therapeutic target in murine lupus.

IntroductionThe signalling cascades that contribute to lupus pathogenesis are incompletely understood. We address this by using an unbiased activity‐based kinome screen of murine lupus.MethodsAn unbiased activity‐based kinome screen (ABKS) of 196 kinases was applied to two genetically different murine lupus strains. Systemic and renal lupus were evaluated following in vivo PLK1blockade. The upstream regulators and downstream targets of PLK1 were also interrogated.ResultsMultiple signalling cascades were noted to be more active in murine lupus spleens, including PLK1. In vivo administration of a PLK1‐specific inhibitor ameliorated splenomegaly, anti‐dsDNA antibody production, proteinuria, BUN and renal pathology in MRL.lpr mice (P < 0.05). Serum IL‐6, IL‐17 and kidney injury molecule 1 (KIM‐1) were significantly decreased after PLK1 inhibition. PLK1 inhibition reduced germinal centre and marginal zone B cells in the spleen, but changes in T cells were not significant. In vitro, splenocytes were treated with anti‐mouse CD40 Ab or F(ab’)2 fragment anti‐mouse IgM. After 24‐h stimulation, IL‐6 secretion was significantly reduced upon PLK1 blockade, whereas IL‐10 production was significantly increased. The phosphorylation of mTOR was assessed in splenocyte subsets, which revealed a significant change in myeloid cells. PLK1 blockade reduced phosphorylation associated with mTOR signalling, while Aurora‐A emerged as a potential upstream regulator of PLK1.ConclusionThe Aurora‐A → PLK1 → mTOR signalling axis may be central in lupus pathogenesis, and emerges as a potential therapeutic target.

3097 – RNA EDITOR-EXONUCLEASE AXIS SCULPTS THE TRANSCRIPTOME DURING TERMINAL ERYTHROID DIFFERENTIATION

Erythropoiesis is a committed process by which erythroid progenitors become mature red blood cells (RBCs). Reticulocytes are terminal-staged, immature RBCs which contain selected residual RNA after enucleation. Normally, reticulocytes are efficiently processed and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated from iPS or CD34+ hematopoietic stem/progenitor cells in vitro, erythroid cells tend to arrest at the reticulocyte stage. This is the major bottleneck for the complete recapitulation of erythropoiesis in vitro, and a hindrance to modeling hemoglobinopathies in cell cultures. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases 7/4, official names are ZCCHC6/11) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is then recognized by exoribonucleases and targeted for decay. We posited that this is the machinery behind RNA degradation that accompanies terminal erythropoiesis. Utilizing both constitutional and erythroid-specific conditional murine knockout models, we found that blood from TUTase Zcchc6 RNA editor knockout embryos indeed exhibit reticulocytosis with a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease, Dis3l2, phenocopied the RNA decay defect of the Zcchc6 KO. Furthermore, knockout models did not show signs of hemolysis suggesting a cell intrinsic and niche-independent role for the TUTase7-Dis3l2 axis in promoting red blood cell maturation. Modulating the expression of this axis holds promise for enhancing in vitro erythropoiesis and modeling of hemoglobinopathies. Future mechanism studies will also reveal a post-transcriptional regulatory machinery governing maturation from reticulocytes to erythrocytes.

Combining mass spectrometry and genetic labeling in mice to report TRP channel expression

Transient receptor potential (TRP) ion channels play important roles in fundamental biological processes throughout the body of humans and mice. TRP channel dysfunction manifests in different disease states, therefore, these channels may represent promising therapeutic targets in treating these conditions. Many TRP channels are expressed in several organs suggesting multiple functions and making it challenging to untangle the systemic pathophysiology of TRP dysfunction. Detailed characterization of the expression pattern of the individual TRP channels throughout the organism is thus essential to interpret data such as those derived from systemic phenotyping of global TRP knockout mice. Murine TRP channel reporter strains enable reliable labeling of TRP expression with a fluorescent marker. Here we present an optimized method to visualize primary TRP-expressing cells with single cell resolution throughout the entire organism. In parallel, we methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest. The TRP protein expression data are then correlated with the GFP reporter expression data. The combined methodological approach presented here can be adopted to generate expression data for other genes of interest and reporter mice.•We present an optimized method to systemically characterize gene expression in fluorescent reporter mouse strains with a single cell resolution.•We methodically combine systemic gene expression profiling with an adjusted mass spectrometry protocol to document acute protein levels in selected organs of interest in mice.

The Unique Genetic and Histological Characteristics of DMBA-Induced Mammary Tumors in an Organoid-Based Carcinogenesis Model.

Here, we report a model system using in vitro 7,12-dimethylbenz[a]anthracene (DMBA; 0.6 μM)-treated mammary tissue-derived organoids generated from heterozygous BALB/c-Trp53 knockout mice to induce tumors after injection into the nude mouse subcutis. In parallel, a single oral dose of DMBA (50 mg/kg bodyweight) to the same murine strain induced mammary adenocarcinomas, characterized by biphasic structures differentiated into luminal and myoepithelial lineages and frequent Hras mutations at codon 61. In the present study, the genetic and histological characteristics of DMBA-induced tumors in the organoid-based model were evaluated to validate its similarities to the in vivo study. The organoid-derived tumors were low-grade adenocarcinomas composed of luminal and basal/myoepithelial cells. When the organoid-derived carcinomas were passaged to other nude mice, they partly progressed to squamous cell carcinomas (SCCs). Whole exome sequencing revealed no mutations at Hras codon 61 in the organoid-derived tumors. However, various mutations were detected in other genes such as Tusc3 and Tgfbr2, which have been reported as cancer-associated or homeostatic squamous cell genes. The most common mutational pattern observed in these genes were the G:C to T:A transversions and G:C to A:T transitions, which are not typical of the mutations caused by DMBA treatment. In conclusion, DMBA exhibited carcinogenicity in the both the ex vivo and in vivo mammary carcinogenesis models, albeit with distinct histological and genetical alterations. Further studies are needed to clarify whether organoid-based carcinogenesis models generated following chemical treatment in vitro could be applied to the clarification of the novel mode of action of chemical carcinogenesis.

Inhibiting ACSL1-Related Ferroptosis Restrains Murine Coronavirus Infection.

Murine hepatitis virus strain A59 (MHV-A59) was shown to induce pyroptosis, apoptosis, and necroptosis of infected cells, especially in the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59 is unknown. We utilized murine macrophages and a C57BL/6 mice intranasal infection model to address this. In primary macrophages, the ferroptosis inhibitor inhibited viral propagation, inflammatory cytokines released, and cell syncytia formed after MHV-A59 infection. In the mouse model, we found that in vivo administration of liproxstatin-1 ameliorated lung inflammation and tissue injuries caused by MHV-A59 infection. To find how MHV-A59 infection influenced the expression of ferroptosis-related genes, we performed RNA-seq in primary macrophages and found that MHV-A59 infection upregulates the expression of the acyl-CoA synthetase long-chain family member 1 (ACSL1), a novel ferroptosis inducer. Using ferroptosis inhibitors and a TLR4 inhibitor, we showed that MHV-A59 resulted in the NF-kB-dependent, TLR4-independent ACSL1 upregulation. Accordingly, ACSL1 inhibitor Triacsin C suppressed MHV-A59-infection-induced syncytia formation and viral propagation in primary macrophages. Collectively, our study indicates that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serve as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.

Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

Akt Plays Differential Roles during the Life Cycles of Acute and Persistent Murine Norovirus Strains in Macrophages.

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

IMMU-47. THE ANTIGENIC PROFILE OF MURINE AND HUMAN MEDULLOBLASTOMA

Abstract BACKGROUND Cancer immunogenomics represents a complementary approach to the application of genomics in developing novel immunotherapies. We performed a multi-faceted computer algorithm, the Open Reading Frame Antigen Network (O.R.A.N.), on medulloblastoma transcription profiles and predicted antigens across a broad array of antigen classes. METHODS Patient-specific HLA haplotypes were called via customized Optitype and Phlat algorithms. Preclinical models- sonic hedgehog driven (Ptch1) and Group 3 MYC-driven (NSC) medulloblastoma were derived from C57BL6 murine strain with known MHC haplotypes. Only expressed mutations such as single nucleotide variations, small indels, gene fusions, and personalized TAAs were used for antigenic epitope predictions. Patient-specific or murine tumor associated antigens (TAA) were selected only if expressed &amp;gt;1 transcript per million (TPM) in tumor and the standardized expression across a human tissue database (29 organs or sub-regions, n=9,141) or a mouse normal tissue database (ENCODE, n=99) was below 1 TPM, respectively. TAA sequences were passed through eight MHC class I and four MHC class II affinity algorithms. All epitopes were screened against a customized human or murine proteomic library to guarantee that epitopes were not shared by other expressed isoforms or genes. Immune deconvolution with single cell RNASeq integration was leveraged for teasing out medulloblastoma immunologic landscape. RESULTS MB patients harbor MHC-I restricted 1.9 SNV, 0.1 Indel, 0.5 gene fusions and MHC-II restricted 2.5 SNV, 0.1 Indel and 0.5 gene fusion. 79.4% patients have at least 1 neoantigen. 88.2% patients have at least one immunogenic TAA. Importantly, cancer testis antigens and previously unappreciated neurodevelopmental antigens were found expressed across all medulloblastoma subgroups. We predicted 6 neoantigens and 14 TAAs for murine NSC tumor and 19 neoantigens and 13 TAAs for Ptch1 tumor. CONCULSION: Using a custom antigen prediction pipeline, we identified potential human and murine tumor rejection antigens with important implications for development of medulloblastoma cellular therapies.

Dynamin-2 Deficiency Causes Neutropenia and Dysplastic Bone Marrow Changes in an Age and Sex Dependent Manner in Mice

▪The dynamins are a family of ubiquitously expressed proteins with GTPase activity and are known for their role in membrane remodeling and intracellular trafficking. However, their exact role within various hematopoietic lineages is incompletely understood. In humans, most of the clinical cases with cytopenia in Charcot-Marie-Tooth (CMT) disease due to dynamin-2 mutations are associated with neutropenia, and CMT patients may suffer from impaired wound healing. Of interest, pregnancy notably worsens CMT disease, possibly due to hormonal changes. Antiprogesterone treatment was successfully given in a CMT rat model, and similar treatment is being considered for human CMT. We previously reported that inhibiting dynamin (DNM) activity impairs migration capability in mature megakaryocytes.We obtained the conditional deletion of Dnm2 and targeted its deletion in hematopoietic tissues with the vav-cre murine strain. Homozygous deletion of Dnm2 in blood tissues appears embryonic lethal. None of the pups born showed a Vav-cre/Dnm-2 fl/fl genotype, whereas a third of the pups born had a Vav-cre/Dnm-2 fl/wt (Dnm2 het) genotype. Bone marrow cells from the heterozygous female mice (Dnm2 het) had 35% to 50% decreased Dnm2 expression in comparison with age-matched controls (CTRL). Evaluation of the complete blood counts demonstrated that Dnm2 het female mice developed leukopenia which was detected from 40 weeks of age (average granulocyte-monocyte counts: CTRL 532/mm3 vs. Dnm2 het 300/mm3; p=0.0164). Neutropenia was unequivocal at 65 weeks of age (average neutrophil counts, CTRL 700/mm3 vs. Dnm2 het 343/mm3, p=0.016). Dnm2 het showed a trend for higher platelet counts than controls, but this was non-statistically significant. Further analysis of hematopoietic lineage maturation by flow cytometry indicated that lineage-negative cells and granulocyte-monocyte progenitors were decreased in Dnm2 het mice (average bone marrow lineage-negative cells: CTRL 2.8x10E6 vs. Dnm2 het 1.97x10E6, p =0.0056; average granulocyte-monocyte progenitors: CTRL 1.35x10E6 vs. Dnm2 het 0.85x10e6, p=0.01), along with a relative increase of common lymphoid progenitors and of megakaryocyte/erythrocyte progenitors in the bone marrow. Immunohistochemical staining for mature neutrophils with Ly6G showed an overall decreased number of mature granulocytes in the bone marrow of Dnm2 het mice (average Ly6G-positive cells: CTRL 20% vs. Dnm2 het 29%, p=0.0026). A linear pattern of distribution of Ly6G positive bone marrow cells along blood vessels was observed in fewer mice in the Dnm2 het group than in the CTRL group (25% vs. 59%, p=0.02), indicating that the migration pattern within the bone marrow is altered in the Dnm2 het group (see Figure). In addition, Dnm2 het mice developed splenomegaly (average spleen weight: Dnm2 het 146 mg vs. CTRL 99 mg, p=0.006), which was secondary to a marked florid reactive germinal center hyperplasia. Some of the Dnm2 het mice, including 5 mice whose pregnancy occurred in middle-age (p=0.005 when comparing with CTRL or young Dnm2 het mice) and 2 non-pregnant older mice, showed physical signs of distress with markedly reduced activity, poor grooming, ruffled furs, and hunched posture. Both non-pregnant sick mice showed a marked decrease in Ly6G positive mature neutrophils at 0.3% of total marrow cells (Figure), and the bone marrow from one mouse was completely effaced by immature myeloid precursors, consistent with the development of acute myeloid leukemia. In addition, a third of Dnm2 hetmice showed no distress but displayed morphological bone marrow abnormalities including megakaryocytic dysmorphology.In summary, female mice with loss of Dnm2 in the hematopoietic compartment develop persistent neutropenia as they age, with decreased granulocyte progenitor production and with migration defects. These abnormalities are associated with a risk for developing megakaryocytic dysplasia, and acute myeloid leukemia. These findings might also suggest a mechanism for chronic idiopathic neutropenia, which has a predominance in middle-aged women. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A TRPV6 expression atlas for the mouse

The transient receptor potential vanilloid 6 (TRPV6) channel is highly Ca2+-selective and has been implicated in mediating transcellular Ca2+ transport and thus maintaining the Ca2+ balance in the body. To characterize its physiological function(s), a detailed expression profile of the TRPV6 channel throughout the body is essential. Capitalizing on a recently established murine Trpv6-reporter strain, we identified primary TRPV6 channel-expressing cells in an organism-wide manner. In a complementary experimental approach, we characterized TRPV6 expression in different tissues of wild-type mice by TRPV6 immunoprecipitation (IP) followed by mass spectrometry analysis and correlated these data with the reporter gene expression. Taken together, we present a TRPV6 expression atlas throughout the entire body of juvenile and adult mice, providing a novel resource to investigate the role of TRPV6 channels in vivo.

In Vitro Evaluation of the Virucidal Activity of Different Povidone-Iodine Formulations Against Murine and Human Coronaviruses.

IntroductionPolyvinylpyrrolidone–iodine (PVP-I) demonstrates broad-spectrum anti-infective activity and is available in different formulations for oral rinse and topical use in medical and personal care settings. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need to supplement available preventive strategies.MethodsWe assessed virucidal activity of PVP-I formulations, including 0.5% (w/v) solution, 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution, versus placebos when challenged with coronaviruses in two in vitro studies. Murine coronavirus strain A59 (American Type Culture Collection [ATCC]® VR-764™), human coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), human coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF), and human coronavirus strain 229E (ATCC® VR-740™) were used as surrogates for SARS-CoV-2. Both studies used the American Society for Testing and Materials in vitro time-kill method.ResultsAll active PVP-I formulations in study 1 demonstrated virucidal activity at 15 s, with mean log10 reduction of greater than 4.56 or greater than 99.99% inactivation; a cytotoxic effect against the National Collection of Type Cultures clone 1469 host cells was observed with 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution. Active PVP-I formulations in study 2 demonstrated effective virucidal activity against coronaviruses in less than 15 s; log10 reduction in viral titer for each coronavirus strain was consistently higher for 10.0% (w/v) solution and 0.5% (w/v) solution versus 7.5% (w/v) scrub.ConclusionBoth studies demonstrated in vitro virucidal activity of PVP-I formulations when challenged with SARS-CoV-2 surrogate coronaviruses. Although promising, further investigations are needed to evaluate SARS-CoV-2 inactivation.

Strain Estimation of the Murine Right Ventricle Using High-Frequency Speckle-Tracking Ultrasound

Right ventricular (RV) strain measurements from ultrasound via speckle-tracking techniques are being used more frequently as a non-invasive diagnostic tool for a variety of cardiopulmonary pathologies. However, despite the clinical utility of ultrasound RV strain measurements, quantification of RV strain in rodents remains difficult owing to unique image artifacts and non-standardized methodologies. We demonstrate here a simple approach for measuring RV strain in both mice and rats using high-frequency ultrasound and automated speckle tracking. Our results show estimated peak RV free-wall longitudinal strain values (mean ± standard error of the mean) in mice (n = 15) and rats (n = 5) of, respectively, −10.38% ± 0.4% and −4.85% ± 0.42%. We further estimated the 2-D Green–Lagrange strain within the RV free wall, with longitudinal components estimated at −5.7% ± 0.48% in mice and −2.1% ± 0.28% in rats. These methods and data may provide a foundation for future work aimed at evaluating murine RV strain levels in different disease models.

Neurochemical abnormalities in the hippocampus of male rats displaying audiogenic seizures, a genetic model of epilepsy

BackgroundEpilepsy is a disorder characterized by recurrent seizures that affects 1% of the population. However, the neurochemical alterations observed in epilepsy are not fully understood. There are different animal models of epilepsy, such as genetic or drug induced. In the present study, we utilize Wistar Audiogenic Rats (WAR), a murine strain that develops seizures in response to high intensity audio stimulation, in order to investigate abnormalities in glutamatergic and GABAergic systems. MethodsSynaptosomes and glial plasmalemmal vesicles were prepared from hippocampus and cortex, respectively. Glutamate and GABA release and uptake were assayed by monitoring the fluorescence and using L-[3H]-radiolabeled compounds. Glutamate and calcium concentration in the synaptosomes were also measured. The expression of neuronal calcium sensor 1 (NCS-1) was determined by western blot. ResultsGlutamate and GABA release evoked by KCl was decreased in WAR compared to control Wistar rats. Calcium independent release was not considerably different in both groups. The total amount of glutamate of synaptosomes, as well as glutamate uptake by synaptosomes and GPV were also decreased in WAR in comparison with the controls. In addition, [Ca2+]i of hippocampal synaptosomes, as well as NCS-1 expression in the hippocampus, were increased in WAR in comparison with controls. ConclusionIn conclusion, our results suggest that WAR have important alterations in the glutamatergic and GABAergic pathways, as well as an increased expression of NCS-1 in the hippocampus and inferior colliculus. These alterations may be linked to the spreading of hyperexcitability and recruitment of various brain regions.

Caerin 1.1 and 1.9 Peptides from Australian Tree Frog Inhibit Antibiotic-Resistant Bacteria Growth in a Murine Skin Infection Model.

ABSTRACTThe host defense peptide caerin 1.9 was originally isolated from skin secretions of an Australian tree frog and inhibits the growth of a wide range of bacteria in vitro. In this study, we demonstrated that caerin 1.9 shows high bioactivity against several bacteria strains, such as Staphylococcus aureus, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and Streptococcus haemolyticus in vitro. Importantly, unlike the antibiotic Tazocin, caerin 1.9 does not induce bacterial resistance after 30 rounds of in vitro culture. Moreover, caerin 1.1, another peptide of the caerin family, has an additive antibacterial effect when used together with caerin 1.9. Furthermore, caerin 1.1 and 1.9 prepared in the form of a temperature-sensitive gel inhibit MRSA growth in a skin bacterial infection model of two murine strains. These results indicate that caerin 1.1 and 1.9 peptides could be considered an alternative for conventional antibiotics.IMPORTANCE Antibiotic-resistant bacteria cause severe problems in the clinic. We show in our paper that two short peptides isolated from an Australian frog and prepared in the form of a gel are able to inhibit the growth of antibiotic-resistant bacteria in mice, and, unlike antibiotics, these peptides do not lead to the development of peptide-resistant bacteria strains.

Efficient interspecies transmission of synthetic prions.

Prions are comprised solely of PrPSc, the misfolded self-propagating conformation of the cellular protein, PrPC. Synthetic prions are generated in vitro from minimal components and cause bona fide prion disease in animals. It is unknown, however, if synthetic prions can cross the species barrier following interspecies transmission. To investigate this, we inoculated Syrian hamsters with murine synthetic prions. We found that all the animals inoculated with murine synthetic prions developed prion disease characterized by a striking uniformity of clinical onset and signs of disease. Serial intraspecies transmission resulted in a rapid adaptation to hamsters. During the adaptation process, PrPSc electrophoretic migration, glycoform ratios, conformational stability and biological activity as measured by protein misfolding cyclic amplification remained constant. Interestingly, the strain that emerged shares a strikingly similar transmission history, incubation period, clinical course of disease, pathology and biochemical and biological features of PrPSc with 139H, a hamster adapted form of the murine strain 139A. Combined, these data suggest that murine synthetic prions are comprised of bona fide PrPSc with 139A-like strain properties that efficiently crosses the species barrier and rapidly adapts to hamsters resulting in the emergence of a single strain. The efficiency and specificity of interspecies transmission of murine synthetic prions to hamsters, with relevance to brain derived prions, could be a useful model for identification of structure function relationships between PrPSc and PrPC from different species.

Immune control of Mycobacterium tuberculosis is dependent on both soluble TNFRp55 and soluble TNFRp75.

Tuberculosis presents a global health challenge, and tumour necrosis factor (TNF) signalling is required for host immunity against Mycobacterium tuberculosis (Mtb). TNF receptor shedding, however, compromises effective immunity by reducing bioactive TNF through the formation of inactive complexes. In this study, we first compared the effect of total soluble TNF receptors using a transgenic p55ΔNS /p75-/- murine strain on host protection during a low-dose aerosol Mtb H37Rv challenge. We report that the presence of membrane-bound TNFRp55 alone in the absence of TNFRp75 results in superior control of a primary Mtb infection where p55ΔNS /p75-/- hyperactive dendritic cells displayed an increased capacity to induce a hyperactive Mtb-specific CD4+ T-cell response. p55ΔNS /p75-/- dendritic cells expressed a higher frequency of MHCII and increased MFIs for both CD86 and MHCII, while CD4+ T cells had higher expression of CD44 and IFN-γ. Next, the relative contributions of soluble TNFRp55 and soluble TNFRp75 to host protection against either primary Mtb infection or during reactivation of latent tuberculosis were delineated by comparing the experimental outcomes of control C57BL/6 mice to transgenic p55ΔNS /p75-/- , p55ΔNS and p75-/- mouse strains. We found that soluble TNFRp55 is redundant for immune regulation during the chronic stages of a primary Mtb infection. However, TNFRp55 together with soluble TNFRp75 has a crucial role in immune regulation of reactivation of latent tuberculosis.

Recovery of Nucleic Acids of Enteric Viruses and Host-Specific Bacteroidales from Groundwater by Using an Adsorption-Direct Extraction Method.

In this study, the adsorption-elution method was modified to concentrate viral particles in water samples and investigate the contamination of groundwater with norovirus genogroup II (NoV GII), rotavirus A (RVA), and Pepper mild mottle virus (PMMoV). The mean recovery rate of a murine norovirus strain, which was inoculated into groundwater samples collected from a deep well, was the highest (39%) when the viral RNA was directly extracted from the membrane instead of eluting the adsorbed viral particles. This adsorption-direct extraction method was applied to groundwater samples (20 liters) collected from deep wells used for the public drinking water supply (n = 22) and private wells (n = 9). RVA (85 copies/liter) and NoV GII (35 copies/liter) were detected in water samples from a deep well and a private well, respectively. PMMoV was detected in 95% and 89% of water samples from deep wells and private wells, respectively, at concentrations of up to 990 copies/liter. The modified method was also used to extract bacterial DNA from the membrane (recovery rate of inoculated Escherichia coli K-12 was 22%). The Bacteroidales genetic markers specific to ruminants (BacR) and pigs (Pig2Bac) were detected in samples from a deep well and a private well, respectively. The modified virus concentration method has important implications for the management of microbiological safety in the groundwater supply. IMPORTANCE We investigated the presence of enteric viruses and bacterial genetic markers to determine fecal contamination in groundwater samples from deep wells used for the public drinking water supply and private wells in Japan. Groundwater is often subjected to chlorination; malfunctions in chlorine treatment result in waterborne disease outbreaks. The modified method successfully concentrated both viruses and bacteria in 20-liter groundwater samples. Norovirus genogroup II (GII), rotavirus A, Pepper mild mottle virus, and Bacteroidales genetic markers specific to ruminants and pigs were detected. Frequent flooding caused by increased incidences of extreme rainfall events promotes the infiltration of surface runoff containing livestock wastes and untreated wastewater into wells, possibly increasing groundwater contamination risk. The practical and efficient method developed in this study will enable waterworks and the environmental health departments of municipal/prefectural governments to monitor water quality. Additionally, the modified method will contribute to improving the microbiological safety of groundwater.

Abstract 128: Mapping spatial cellular dynamics of bone marrow niches in metastatic breast cancer

Abstract The study of dormancy and reactivation of disseminated tumor cells (DTCs) in the distant organs are critical in in metastatic cancer research. During early stages of the disease, breast cancer cells have high chance to metastasize and settle in other organs such as bone. It is important to understand the spatial distribution of tumor cells under distinct microenvironments and how these niches determine the fates of tumor cells in homeostatic and pathological settings. In this work, we proposed a deep imaging systems biology (DISB) framework by integrating multiple components, including in vivo spontaneous tissue metastases models, tissue clearing, deep imaging, digital reconstruction, image quantitation, and graphic-theoretic analysis, to study the spatial distribution and pathophysiology of DTCs and related stroma cells in organs over time. Within the DISB framework, genetically engineered fluorescent reporter murine strains, which highlight specific bone marrow lineages, were used as the dimensional maps of diversified perivascular and endosteal niches. Metastases asynchronously occur in immune competent mice after resection of syngeneic primary tumors with or without adjuvant therapies, mimicking the natural course of metastatic spreading in clinics. Long bones carrying metastatic tumor cells were collected and subjected to the BABB (Benzoic Acid Benzyl Benzoate) clearing methodology. The spatial distribution of cancer and bone cells in the tumor tissue was captured by whole slide confocal imaging and digitally reconstructed. We developed deep learning image computing model to segment, quantitate, and obtain the spatial distribution and correlations among different components in the niche, such as cancer cells, vessels, and normal tissue cells over different bone specimens at various time points. We applied the DISB framework to image and trace specific bone marrow lineages (Olympus FluoView FV1000), and the results indicated that the spatial distribution of tumor cells in such spontaneous metastases models is significantly different from those in experimental metastases models. DTCs are mainly in close proximity to the endothelium, but such distribution is likely a random process. Of interest, the proliferating micrometastases were observed in close association with the endosteal vessels, suggesting the potential correlation of osteogenesis and metastatic growth in bone. In addition, adjuvant chemotherapies likely did not shift tumor cells across different bone marrow niches. Combined image-omics and graph-theoretic analysis on various cell types and vessels provide a new powerful new approach to understand the mechanism leading to diverse behaviors of tumor and various types of stroma cells in homeostatic and pathological settings in various bone marrow microenvironments under different perturbations or developmental stages. Citation Format: Tiancheng He, Weijie Zhang, Jiasong Li, Jianting Sheng, Xiang Zhang, Stephen T. Wong. Mapping spatial cellular dynamics of bone marrow niches in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 128.

EPCT-22 SAFETY AND EFFICACY OF INTRAVENTRICULAR IMMUNOVIROTHERAPY WITH ONCOLYTIC HSV-1 G207 FOR TREATMENT OF LEPTOMENINGEAL DISEASE

Leptomeningeal metastatic disease (LMD) occurs in 30–50% of newly diagnosed and recurrent pediatric malignant cerebellar tumors and 20–45% of malignant supratentorial tumors. Radiation and chemotherapy often cause substantial long-term neurotoxicity and outcomes remain poor for patients with LMD. At recurrence, LMD is generally minimally responsive to conventional therapies. Immunovirotherapy with engineered oncolytic HSV-1 G207 has emerged as a promising treatment for children with high-grade brain tumors. G207 infects and kills tumor cells while sparing normal cells and stimulates a robust anti-tumor immune response. Intratumoral G207 inoculation demonstrated safety and preliminary efficacy in a pediatric Phase 1 trial in recurrent/progressive high-grade glioma (NCT02457845), and a Phase 2 trial (NCT04482933) is forthcoming. Additionally, a Phase 1 trial of intratumoral G207 in recurrent/progressive malignant pediatric cerebellar tumors is ongoing (NCT03911388). While intratumoral inoculation delivers G207 directly to a primary tumor, it requires neurosurgical procedures thereby limiting repeat doses. Thus, we sought to establish the safety and efficacy of intraventricular G207. Utilizing an immunocompetent, HSV-sensitive murine strain, we determined that a standard 1x107 plaque-forming units (PFU) dose of G207 resulted in damage to the ependymal lining. However, interferon induction with an intraventricular low-dose (1x104 PFU) of G207 or polyinosinic-polycytidylic acid (poly I:C), a toll-like receptor 3 agonist, three days prior to standard treatment dose protected the ependymal lining. This approach enabled safe delivery of multiple subsequent doses. Importantly, with these protective measures, G207 significantly prolonged survival in pediatric patient-derived xenograft models and an immunocompetent murine LMD model of group 3 medulloblastoma, the most aggressive and fatal subtype. Collectively, these data indicate that toxicity from intraventricular G207 can be safely mitigated prior to a therapeutic dose, and that intraventricular G207 effectively targets group 3 medulloblastoma including LMD. These findings provide support for clinical translation of intraventricular G207.

Potential genetic basis of B cell hyperactivation in murine lupus models.

Lupus B cells not only produce autoantibodies against nuclear antigens but also provide co-stimulation to T cells. However, there is still a lack of comprehensive understanding of the mechanism underlying lupus B cell hyperactivation. This study focuses on the detection of B cell activation status, analysis of early BCR signaling response, DNA sequencing, and quantity determination of BCR signaling regulators in murine lupus models. Our result showed that there is a B cell hyperactivation with a significant elevation of B cell activation markers, and a BCR signaling hyperactivity with an abnormal increase of phosphorylated BCR signaling molecules and cytoplasmic calcium in the early response to BCR crosslinking in B6.Sle1/2/3 lupus mouse. Whole exome sequencing identified a multiple point mutation in the exon of many BCR signaling regulators in common murine lupus models, MRL/lpr, NZM2410, BXSB, NZB, and NZW strains. cNDA sequencing confirmed FcγR2b, Ly9, Pirb, Siglecg, and CD22 BCR signaling regulator variants in B6.Sle1/2/3 lupus mouse, but surface protein expression of these regulators on B cells showed an abnormal increase. Our findings support that these BCR signaling regulator variants are potential causative genes of B cell hyperactivation in murine lupus models through their possible functional reduction.