Mouse Model In Order Research Articles

Nicorandil treatment improves survival and spatial learning in aged granulin knockout mice.

Mutations in the human granulin (GRN) gene are associated with multiple diseases, including dementia disorders such as frontotemporal dementia (FTD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). We studied a Grn knockout (Grn-KO) mouse model in order to evaluate a potential therapeutic strategy for these diseases using nicorandil, a commercially available agonist for the ABCC9/Abcc9-encoded regulatory subunit of the "K+ATP" channel that is well-tolerated in humans. Aged (13 months) Grn-KO and wild-type (WT) mice were treated as controls or with nicorandil (15 mg/kg/day) in drinking water for 7 months, then tested for neurobehavioral performance, neuropathology, and gene expression. Mortality was significantly higher for aged Grn-KO mice (particularly females), but there was a conspicuous improvement in survival for both sexes treated with nicorandil. Grn-KO mice performed worse on some cognitive tests than WT mice, but Morris Water Maze performance was improved with nicorandil treatment. Neuropathologically, Grn-KO mice had significantly increased levels of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytosis but not ionized calcium binding adaptor molecule 1 (IBA-1)-immunoreactive microgliosis, indicating cell-specific inflammation in the brain. Expression of several astrocyte-enriched genes, including Gfap, were also elevated in the Grn-KO brain. Nicorandil treatment was associated with a subtle shift in a subset of detected brain transcript levels, mostly related to attenuated inflammatory markers. Nicorandil treatment improved survival outcomes, cognition, and inflammation in aged Grn-KO mice.

Syngeneic Mouse Models for Pre-Clinical Evaluation of CAR T Cells.

Chimeric antigen receptor (CAR) T cells have revolutionized the treatment of hematological malignancies. Unfortunately, this improvement has yet to be translated into the solid tumor field. Current immunodeficient models used in pre-clinical testing often overestimate the efficacy of CAR T cell therapy as they fail to recapitulate the immunosuppressive tumor microenvironment characteristic of solid tumors. As CAR T cell monotherapy is unlikely to be curative for many solid tumors, combination therapies must be investigated, for example, stromal remodeling agents and immunomodulators. The evaluation of these combination therapies requires a fully immunocompetent mouse model in order to recapitulate the interaction between the host's immune system and the CAR T cells. This review will discuss the need for improved immunocompetent murine models for the pre-clinical evaluation of CAR T cells, the current use of such models and future directions.

Modified Impedance Sensing System Determination of Virulence Characteristics of Pathogenic Bacteria Klebsiella Species.

An impedance sensing system is a family of biosensors that measure changes in electrical impedance to perform their functions. Physical and chemical changes in the impedance of the sensing element, such as changes in the concentration of a target analyte or changes in the physical properties of the sensing element, can result in changes in the impedance of the sensing element. Many impedance biosensors have been developed for the detection of pathogens in the past few decades. Several types of biosensors have been developed for the detection of infections, including transduction elements, biorecognition components, and electrochemical approaches. In this review, we discuss the characteristics and pathogenic factors associated with 2,3-butanediol-producing Klebsiella pneumoniae collected using impedance sensors. An impedance sensing system was introduced as a great method for monitoring the virulence factors of Klebsiella spp. in situ. Klebsiella pneumoniae produces virulence factors, including capsules, lipopolysaccharides, fimbriae, and siderophores, as part of its pathogenesis. It is possible to examine virulence factors' pathogenic characteristics in vitro and in vivo using real tissues or mouse models in order to conduct experiments. For the monitoring of virulence factors in situ, a novel alternative method has been developed to mimic the environment of real tissues. For the purpose of developing tissue-mimicking models, mucin and mannose were used to modify the surface of gold electrodes. These components are known to contribute to the adhesion of pathogens to epithelial cells in mammals.

Exploration of the skeletal phenotype of the Col1a1 +/Mov13 mouse model for haploinsufficient osteogenesis imperfecta type 1.

Osteogenesis Imperfecta is a rare genetic connective tissue disorder, characterized by skeletal dysplasia and fragile bones. Currently only two mouse models have been reported for haploinsufficient (HI) mild Osteogenesis Imperfecta (OI); the Col1a1 +/Mov13 (Mov13) and the Col1a1 +/-365 mouse model. The Mov13 mice were created by random insertion of the Mouse Moloney leukemia virus in the first intron of the Col1a1 gene, preventing the initiation of transcription. Since the development of the Mov13 mice almost four decades ago and its basic phenotypic characterization in the 90s, there have not been many further studies. We aimed to extensively characterize the Mov13 mouse model in order to critically evaluate its possible use for preclinical studies of HI OI. Bone tissue from ten heterozygous Mov13 and ten wild-type littermates (WT) C57BL/6J mice (50% males per group) was analyzed at eight weeks of age with bone histomorphometry, micro computed tomography (microCT), 3-point bending, gene expression of different collagens, as well as serum markers of bone turnover. The Mov13 mouse presented a lower bone strength and impaired material properties based on our results of 3-point bending and microCT analysis respectively. In contrast, no significant differences were found for all histomorphometric parameters. In addition, no significant differences in Col1a1 bone expression were present, but there was a significant lower P1NP concentration, a bone formation marker, measured in serum. Furthermore, bone tissue of Mov13 mice presented significantly higher expression of collagens (Col1a2, Col5a1 and Col5a2), and bone metabolism markers (Bglap, Fgf23, Smad7, Edn1 and Eln) compared to WT. Finally, we measured a significantly lower Col1a1 expression in heart and skin tissue and also determined a higher expression of other collagens in the heart tissue. Although we did not detect a significant reduction in Col1a1 expression in the bone tissue, a change in bone structure and reduction in bone strength was noted. Regrettably, the variability of the bone phenotype and the appearance of severe lymphoma in adult Mov13 mice, does not favor their use for the testing of new long-term drug studies. As such, a new HI OI type 1 mouse model is urgently needed.

Assessing kidney development and disease using kidney organoids and CRISPR engineering.

The differentiation of human pluripotent stem cells (hPSCs) towards organoids is one of the biggest scientific advances in regenerative medicine. Kidney organoids have not only laid the groundwork for various organ-like tissue systems but also provided insights into kidney embryonic development. Thus, several protocols for the differentiation of renal progenitors or mature cell types have been established. Insights into the interplay of developmental pathways in nephrogenesis and determination of different cell fates have enabled the in vitro recapitulation of nephrogenesis. Here we first provide an overview of kidney morphogenesis and patterning in the mouse model in order to dissect signalling pathways that are key to define culture conditions sustaining renal differentiation from hPSCs. Secondly, we also highlight how genome editing approaches have provided insights on the specific role of different genes and molecular pathways during renal differentiation from hPSCs. Based on this knowledge we further review how CRISPR/Cas9 technology has enabled the recapitulation and correction of cellular phenotypes associated with human renal disease. Last, we also revise how the field has positively benefited from emerging technologies as single cell RNA sequencing and discuss current limitations on kidney organoid technology that will take advantage from bioengineering solutions to help standardizing the use of this model systems to study kidney development and disease.

Effects of cerebral amyloid angiopathy on the brain vasculome.

β‐amyloid (Aβ) deposits in brain blood vessel walls underlie the vascular pathology of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Growing evidence has suggested the involvement of cerebrovascular dysfunction in the initiation and progression of cognitive impairment in AD and CAA patients. Therefore, in this study, we assessed the brain vasculome in a mouse model in order to identify cerebrovascular pathways that may be involved in AD and CAA vascular pathogenesis in the context of aging. Brain endothelial cells were isolated from young and old wild‐type mice, and young and old transgenic mice expressing Swedish mutation in amyloid precursor protein and exon 9 deletion in presenilin 1 (APPswe/PSEN1dE9). Microarray profiling of these endothelial transcriptomes demonstrated that accumulation of vascular Aβ in the aging APPswe/PSEN1dE9 mouse is associated with impaired endothelial expression of neurotransmitter receptors and calcium signaling transductors, while the genes involved in cell cycle and inflammation were upregulated. These results suggest that the vascular pathology of AD and CAA may involve the disruption of neurovascular coupling, reactivation of cell cycle in quiescent endothelial cells, and enhanced inflammation. Further dissection of these endothelial mechanisms may offer opportunities to pursue therapies to ameliorate vascular dysfunction in the aging brain of AD and CAA patients.

P328: DYNAMIC EVOLUTION OF TCF3-PBX1 LEUKEMIAS AT THE SINGLE-CELL LEVEL UNDER CHEMOTHERAPY PRESSURE

Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While research has been focused on high-risk patients, the biology of low-to-intermediate-risk patients has so far been inadequately investigated and can lead to overtreatment with severe side effects and under treatment with risk of relapse. The translocation t(1;19) codes for chimeric fusion protein TCF3-PBX1, which is associated with intermediate risk ALL. Using our previous generated TCF3-PBX1 conditional knock-in mice, we established a model to study in vivo chemotherapy resistance. Aims: We hypothesize that chemotherapy and microenvironment play a crucial role in development of resistance in TCF3-PBX1 leukemias by influencing transcriptional regulation, activation of signaling pathways and the hierarchical structure of leukemic cells. In this project we aim to characterize dynamic changes of TCF3-PBX1 leukemia cells in different tissues under chemotherapy pressure. Methods: Recipient C57/BL6 healthy mice were sub-lethally irradiated, transplanted with mouse TCF3-PBX1 leukemia cells and treated with vehicle (n=15), prednisolone (n=13) and daunorubicin (n=15) for 20 days. Mice were monitored for signs of disease regularly and circulating GFP+ TCF3-PBX1 leukemia cells were assessed by flow cytometry. Sick mice were sacrificed, leukemia cells were isolated from five different organs (bone marrow, spleen, lymph nodes, spinal cord and brain) and characterized by immunophenotyping, sanger sequencing, bulk RNA sequencing (bulk RNAseq) of GFP+ sorted cells, single cell RNA sequencing and mass cytometry (CYTOF). Results: All mice transplanted with TCF3-PBX1 leukemia cells and treated with vehicle succumbed to disease with a median survival of about 70 days. We optimized chemotherapy drug concentration and transplanted cell dose, so 60% of mice treated with prednisolone or daunorubicin survived at least 150 days. Leukemic infiltration was showed by histological stainings in analyzed tissues including central nervous system (CNS) (spinal cord, brain) and GFP+ leukemia cells were quantified by flow cytometry. No major differences were observed in immunophenotype of TCF3-PBX1 leukemia and variant allele frequency (VAF) of the known PTPN11 mutation in analyzed tissues or depending on in vivo treatments. Bulk RNAseq of FACS-sorted GFP+ TCF3-PBX1 leukemia cells were clustered and CNS cells separated from other tissues. scRNAseq revealed additional heterogeneity within each tissue based on signaling pathway and cell cycle activity. Interleukin signaling, regulation of apoptosis pathways, and signaling by the B cell receptor (BCR) were regulated based on pathway analysis in CNS compared to other tissues. Hence, we elucidated the hierarchical structure of hematopoietic cells, interaction of leukemic cells with the microenvironment, and changes in signaling response to chemotherapy in vivo treatment by mass spectrometry (CyTOF) to validate the identified altered signaling pathway activities at protein level. Summary/Conclusion: We have developed a mouse model in order to characterize in vivo chemotherapy resistance depending on niche. Global transcriptomics and phospho-proteomics at the single-cell level might elucidate novel mechanisms of chemotherapy resistance suitable for pharmacological therapies.

Abstract 240: Role Of Smooth Muscle-derived Vascular Progenitor Cells In Atherosclerosis

Purpose: Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Emerging evidence implicates the outer layer of the blood vessel, the adventitia, in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis progression by facilitating inflammatory cell infiltration. Our group previously identified a unique population of multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and reside in the vessel adventitia, where they are poised to respond to vascular injury. We hypothesized that in the setting of atherosclerosis, AdvSca1-SM cells contribute to VV expansion to drive disease progression. Methods: We generated a highly specific lineage tracing mouse model in order to track AdvSca1-SM cells in vivo even if they differentiate into other cell types. Lineage tracing mice were placed on either normal chow or Western diet for 8 or 16 weeks, then vascular tissue was analyzed using IF microscopy, scRNA-Seq, and flow cytometry. Results: scRNA-Seq and flow cytometry revealed that AdvSca1-SM cells in atherosclerosis primarily differentiate into mature SMCs, modulated SMCs, and myofibroblasts. Contrary to our preliminary data, AdvSca1-SM cells very rarely differentiate into endothelial cells. Additionally, despite previous evidence of SMCs gaining a macrophage-like phenotype in atherosclerosis, we identified only rare instances of AdvSca1-SM cells contributing to macrophage populations. Conclusions: As in our findings in acute vascular injury, AdvSca1-SM cells in atherosclerosis predominantly differentiate into SMCs and myofibroblasts. Future studies on advanced lesions will define the functional role of AdvSca1-SM cells in atherosclerotic plaque progression.

CC16 Deficiency in the Context of Early-Life Mycoplasma pneumoniae Infection Results in Augmented Airway Responses in Adult Mice.

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16-/-) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults (early life infection model; ELIM) and compared to adult mice infected for only 3 days (adult infection model; AIM). CC16-/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16-/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16-/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16-/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16-/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Prec-P-01 - Preclinical investigation of new targeted combination therapies in cutaneous T-cell lymphoma

Prec-P-01 - Preclinical investigation of new targeted combination therapies in cutaneous T-cell lymphoma

6.28 Identifying Networks Underlying Sleep Disruption in Autism Spectrum Disorder Mouse Models

Sleep alterations are very common in autism spectrum disorder (ASD) and impact the individual with ASD as well as their families. Little is known about the etiology and neural circuit underpinnings of disrupted sleep in ASD. This study examines sleep architecture across 2 ASD mouse models in order to understand how brain network dynamics give rise to perturbed sleep-wake states to better understand the etiology.

Intravenous Administration of Human Amniotic Mesenchymal Stem Cells in the Subacute Phase of Cerebral Infarction in a Mouse Model Ameliorates Neurological Disturbance by Suppressing Blood Brain Barrier Disruption and Apoptosis via Immunomodulation

Neuro-inflammation plays a key role in the pathophysiology of brain infarction. Cell therapy offers a novel therapeutic option due to its effect on immunomodulatory effects. Amniotic stem cells, in particular, show promise owing to their low immunogenicity, tumorigenicity, and easy availability from amniotic membranes discarded following birth. We have successfully isolated and expanded human amniotic mesenchymal stem cells (hAMSCs). Herein, we evaluated the therapeutic effect of hAMSCs on neurological deficits after brain infarction as well as their immunomodulatory effects in a mouse model in order to understand their mechanisms of action. One day after permanent occlusion of the middle cerebral artery (MCAO), hAMSCs were intravenously administered. RT-qPCR for TNFα, iNOS, MMP2, and MMP9, immunofluorescence staining for iNOS and CD11b/c, and a TUNEL assay were performed 8 days following MCAO. An Evans Blue assay and behavioral tests were performed 2 days and several months following MCAO, respectively. The results suggest that the neurological deficits caused by cerebral infarction are improved in dose-dependent manner by the administration of hAMSCs. The mechanism appears to be through a reduction in disruption of the blood brain barrier and apoptosis in the peri-infarct region through the suppression of pro-inflammatory cytokines and the M2-to-M1 phenotype shift.

Autophagy-related gene 7 deficiency caused by miR-154-5p overexpression suppresses the cell viability and tumorigenesis of retinoblastoma by increasing cell apoptosis.

BackgroundRetinoblastoma is a rare cancer of the retina that accounts for 3% of all childhood cancers. The aim of this study was to illuminate the oncogenic role and potential molecular mechanisms of the microRNA miR-154-5p and autophagy-related gene 7 (ATG7) in retinoblastoma, and to establish a nude mouse model in order to explore new therapeutic horizons for the disease.MethodsQuantitative reverse transcription-polymerase chain reaction and western blot were performed to detect the expression levels of miR-154-5p and ATG7. The targeting relationship between miR-154-5p and ATG7 was analyzed by employing the luciferase reporter assay. MiR-154-5p mimic and pcDNA-ATG7 were transfected, either alone or in combination, into Y79 cells. The subsequent in vitro experiments involved four groups: the control group, miR-154-5p group, ATG7 group, and miR-154-5p + ATG7 group. Orthotopic xenograft models were established by injecting BALB/c athymic nude mice with treated and untreated Y79 cells.ResultsY79 cells were transfected with miR-NC or miR-154-5p. Compared to those in the control group, the mRNA expression levels of miR-154-5p were increased in the miR-154-5p mimic group; in contrast, decreases were observed in the mRNA and protein expression levels of ATG7. Y79 cells were transfected with PcDNA or pcDNA-ATG7. The mRNA expression level of ATG7 was increased in pcDNA-ATG7 group. MiR-154-5p was found to have an element complementary to the three prime untranslated region of ATG7. Overexpression of miR-154-5p inhibited Y79 cells proliferation and migration, and promoted Y79 cells apoptosis via targeting of ATG7. In the in vivo experiment, the tumors of the miR-154-5p group of mice were significantly reduced in weight. Tumor growth and the protein levels of Survivin were both suppressed when miR-154-5p was overexpressed in vivo; however, cell apoptosis and the protein levels of p21 were promoted. In the miR-154-5p group, the expression levels of miR-154-5p were upregulated compared to those in the control group, but the ATG7 expression level was downregulated.ConclusionsMiR-154-5p overexpression downregulated ATG7, which inhibited cell proliferation and apoptosis in vitro, as well as tumor formation in vivo.

Meniscal and ligament modifications in spontaneous and post-traumatic mouse models of osteoarthritis

BackgroundOsteoarthritis (OA) is a whole joint disease that affects all joint tissues, with changes in the articular cartilage (AC), subchondral bone and synovium. Pathologies in menisci and ligaments, however, are rarely analysed, although both are known to play vital roles in the mechanical stability of the joint. The aim of our study was to describe the pathological changes in menisci and ligament during disease development in murine spontaneous and post-traumatic surgically induced OA and to quantify tissue mineralisation in the joint space using micro-computed tomography (μCT) imaging during OA progression.MethodsKnees of Str/ort mice (spontaneous OA model; 26–40 weeks) and C57CBA F1 mice following destabilisation of medial meniscus (DMM) surgery (post-traumatic OA model; 8 weeks after DMM), were used to assess histological meniscal and ligament pathologies. Joint space mineralised tissue volume was quantified by μCT.ResultsMeniscal pathological changes in Str/ort mouse knees were associated with articular cartilage lesion severity. These meniscal changes included ossification, hyperplasia, cell hypertrophy, collagen type II deposition and Sox9 expression in the fibrous region near the attachment to the knee joint capsule. Anterior cruciate ligaments exhibited extracellular matrix changes and chondrogenesis particularly at the tibial attachment site, and ossification was seen in collateral ligaments. Similar changes were confirmed in the post-traumatic DMM model. μCT analysis showed increased joint space mineralised tissue volume with OA progression in both the post-traumatic and spontaneous OA models.ConclusionsModifications in meniscal and ligament mineralisation and chondrogenesis are seen with overt AC degeneration in murine OA. Although the aetiology and the consequences of such changes remain unknown, they will influence stability and load transmission of the joint and may therefore contribute to OA progression. In addition, these changes may have important roles in movement restriction and pain, which represent major human clinical symptoms of OA. Description of such soft tissue changes, in addition to AC degradation, should be an important aspect of future studies in mouse models in order to furnish a more complete understanding of OA pathogenesis.

The Ldb1 Transcriptional Co‐Regulator is Required for Establishment, Development, and Survival of the Pancreatic Endocrine Lineage

Pancreatic islets are comprised of hormone secreting cell types that are vital regulators of glucose metabolism. Specifically, the pancreatic β cell is indispensable for glucose control and its dysfunction is central to diabetes mellitus. β cell development is regulated by transcription factor (TF) cascades, mediating differentiation of progenitors into mature insulin producing β cells. Our prior studies show that TF, Islet1 (Isl1), interacts with a transcriptional co‐regulator, Ldb1, to regulate β cell maturation from embryonic day (E) 18.5 onward. However, Ldb1 is also expressed in early stages of pancreas development, before Isl1 is present (as early as E10.5). The earlier Ldb1+ cell types include Pdx1+ multipotent progenitor cells (MPCs) and endocrine progenitors expressing the TF Neurogenin3 (Ngn3). MPC progeny will populate the entire pancreas (endocrine and exocrine), while Ngn3+ endocrine progenitors (Isl1−) are fated to become islet cells. Our hypothesis is that Ldb1 has Isl1 independent roles in maintaining progenitor identity in these requisite populations during early pancreatic development. To test this we generated a whole pancreas knockout of Ldb1 (Ldb1Δpanc) and observed severe developmental and postnatal phenotypes. At E13.5, Ldb1Δpanc mice exhibit disorganized progenitor pools, suggesting early defects in endocrine identity. At E15.5, Ldb1Δpanc mice had a significant reduction of Ngn3+ progenitors and Pdx1HI immunoreactivity, a mark of presumptive β cells. Ldb1Δpanc mice die by postnatal day 7 (P7) with severe hyperglycemia and hypoinsulinemia due to drastic islet hormone cell reduction. Interestingly, total pancreatic mass remained unchanged in Ldb1Δpanc neonates, suggesting that Ldb1 impacts are islet specific in the pancreas. Considering these observations, we generated a new model of Ldb1 loss specifically in Ngn3+ islet progenitors, termed Ldb1Δendo. We confirmed loss of Ldb1 in endocrine clusters and observed postnatal hyperglycemia, with a reduction of islet cells in neonates, similar to that seen in Ldb1Δpanc mice. Chromatin immunoprecipitation in vivo and in vitro highlights that Ldb1 imparts control on Pdx1 through occupation of the Pdx1 Area I–II regulatory domains, and that Ngn3 control also occurs via direct occupation. We are now further assessing the developmental phenotype, examining markers of proliferation, apoptosis, and altered cell identity through lineage tracing in the Ldb1Δendo model. Concomitantly, our published work revealed that Ldb1 and Isl1 interact in complex with the single stranded DNA binding protein co‐regulator, SSBP3, which helps stabilize this transcriptional complex. To examine the functional relationship between SSBP3, Isl1, and Ldb1 in vivo, we developed a new SSBP3 floxed mouse model in order to generate pancreas wide loss of SSBP3 (SSBP3Δpanc). We observed preliminary reductions in key islet mRNAs along with hyperglycemia in neonates at P1. Our work provides insight into the transcriptional complexes dictating how islet progenitors adopt their cell fate. Enhancing our understanding of this differentiation process can contribute to therapeutic β and islet cell generation strategies to help the growing diabetic population.Support or Funding InformationNIH‐NIDDK R01DK111483‐01NIH‐F31DK120217NIH‐NIGMS T32GM008111

CMT2Q-causing mutation in the Dhtkd1 gene lead to sensory defects, mitochondrial accumulation and altered metabolism in a knock-in mouse model

Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the ‘dehydrogenase E1 and transketolase domain containing 1’ (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1Y486* point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1Y486* knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.

Integrated lipidomic and transcriptomic analyses identify altered nerve triglycerides in mouse models of prediabetes and type 2 diabetes.

ABSTRACTPeripheral neuropathy (PN) is a complication of prediabetes and type 2 diabetes (T2D). Increasing evidence suggests that factors besides hyperglycaemia contribute to PN development, including dyslipidaemia. The objective of this study was to determine differential lipid classes and altered gene expression profiles in prediabetes and T2D mouse models in order to identify the dysregulated pathways in PN. Here, we used high-fat diet (HFD)-induced prediabetes and HFD/streptozotocin (STZ)-induced T2D mouse models that develop PN. These models were compared to HFD and HFD-STZ mice that were subjected to dietary reversal. Both untargeted and targeted lipidomic profiling, and gene expression profiling were performed on sciatic nerves. Lipidomic and transcriptomic profiles were then integrated using complex correlation analyses, and biological meaning was inferred from known lipid-gene interactions in the literature. We found an increase in triglycerides (TGs) containing saturated fatty acids. In parallel, transcriptomic analysis confirmed the dysregulation of lipid pathways. Integration of lipidomic and transcriptomic analyses identified an increase in diacylglycerol acyltransferase 2 (DGAT2), the enzyme required for the last and committed step in TG synthesis. Increased DGAT2 expression was present not only in the murine models but also in sural nerve biopsies from hyperlipidaemic diabetic patients with PN. Collectively, these findings support the hypothesis that abnormal nerve-lipid signalling is an important factor in peripheral nerve dysfunction in both prediabetes and T2D.This article has an associated First Person interview with the joint first authors of the paper.

Abstract PR04: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract High-grade serous ovarian cancer (HGSOC) is the most frequent and most aggressive histologic subtype of ovarian cancer. The cornerstone of the existing treatment of HGSOC is DNA-damaging chemotherapy; however, practically all patients eventually develop a progressive disease and the 5-year survival is only 40%. Immunotherapy would seem to be an attractive alternative treatment to chemotherapy, yet existing immunotherapies perform poorly in ovarian cancer, with only ~10% of patients responding to checkpoint blockade. Why this is the case remains poorly understood and there is a pressing need to understand the underlying biology of immune evasion in ovarian cancer. One critical area of interest is the role of homology-dependent DNA repair (HR) in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. Hence, we have modeled the biology of ovarian cancer using patient-relevant mutational landscapes in an immune-proficient, syngeneic mouse model in order to help us identify the contribution of common driver mutations to the immune repertoire in the tumor microenvironment, and thus to responses of HGSOC tumors to immunotherapy. We hypothesize that the immune composition and gene expression signatures of the resulting tumors will vary based on the combination of genetic alterations and the DNA repair proficiency of the transformed cells. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in HR-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune /stromal analysis using flow cytometry, scRNAseq transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean Smith, David Pepin, Raghav Mohan, Ferenc Reinhardt, Tian Xia, Tony E. Chavarria, Esmee Hoefsmit, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR04. doi:10.1158/1535-7163.TARG-19-PR04

Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model

ObjectivesKlebsiella pneumoniae is an important emerging pathogen of humans and animals leading to serious clinical consequences. Increased antibiotic use has promoted the emergence of carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains. Recently, phage therapy has gained momentum as a possible alternative against emerging antimicrobial resistance. This study was performed to assess the therapeutic effects of a novel lytic phage (VTCCBPA43) in a pneumonic mouse model in order to explore the efficacy of phage therapy against virulent K. pneumoniae infection. MethodsThe tailed phage VTCCBPA43 was assessed for its growth kinetics, in vitro host range, and temperature and pH sensitivity. Protein constituents were analysed by SDS-PAGE and nLC-MS/MS. Therapeutic efficacy was observed 2 h post-challenge with virulent K. pneumoniae in a BALB/c mouse model. ResultsPhage VTCCBPA43 was found to be highly temperature-tolerant (up to 80 °C). It was most active at pH 5, had a burst size of 172 PFU/mL and exhibited a narrow host range. It was identified as a KP36-like phage by shotgun proteomics. Following intranasal application of a single dose (2 × 109 PFU/mouse) post-challenge with virulent K. pneumoniae, the presence of biologically active phage in vivo and a significant reduction in the lung bacterial load at all time points was observed. A reduction in lesion severity suggested overall beneficial effects of VTCCBPA43 phage therapy in the pneumonic mouse model. ConclusionThis research represents the first in vivo evidence of effective phage therapy against K. pneumoniae infection by the intranasal route.

Periodontal treatment prevents arthritis in mice and methotrexate ameliorates periodontal bone loss

Recent studies indicate a causal relationship between the periodontal pathogen P. gingivalis and rheumatoid arthritis involving the production of autoantibodies against citrullinated peptides. We therefore postulated that therapeutic eradication P. gingivalis may ameliorate rheumatoid arthritis development and here turned to a mouse model in order to challenge our hypothesis. F1 (DBA/1 x B10.Q) mice were orally inoculated with P. gingivalis before collagen-induced arthritis was provoked. Chlorhexidine or metronidazole were orally administered either before or during the induction phase of arthritis and their effects on arthritis progression and alveolar bone loss were compared to intraperitoneally injected methotrexate. Arthritis incidence and severity were macroscopically scored and alveolar bone loss was evaluated via microcomputed tomography. Serum antibody titres against P. gingivalis were quantified by ELISA and microbial dysbiosis following oral inoculation was monitored in stool samples via microbiome analyses. Both, oral chlorhexidine and metronidazole reduced the incidence and ameliorated the severity of collagen-induced arthritis comparable to methotrexate. Likewise, all three therapies attenuated alveolar bone loss. Relative abundance of Porphyromonadaceae was increased after oral inoculation with P. gingivalis and decreased after treatment. This is the first study to describe beneficial effects of non-surgical periodontal treatment on collagen-induced arthritis in mice and suggests that mouthwash with chlorhexidine or metronidazole may also be beneficial for patients with rheumatoid arthritis and a coexisting periodontitis. Methotrexate ameliorated periodontitis in mice, further raising the possibility that methotrexate may also positively impact on the tooth supporting tissues of patients with rheumatoid arthritis.