Murine Disease Models Research Articles

Sulfide:quinone oxidoreductase ameliorates neurodegeneration in a murine model of Parkinson's disease

Sulfide:quinone oxidoreductase ameliorates neurodegeneration in a murine model of Parkinson's disease

CXCR4 Blockade Targets Progression of Primary T Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL), a malignant neoplasm of immature T cells, is particularly aggressive leukemia with no targeted therapies. Large-scale sequencing efforts to define the genetic make-up of tumors both at diagnosis and during relapse have provided little actionable information for therapeutic development or predict relapse. T-ALL is reliant on the growth factors and chemokines from the bone marrow (BM) microenvironment for its growth, survival, and BM retention. To identify possible vulnerabilities, we have previously examined the role of CXCL12, a chemokine produced by the BM niche, in maintaining NOTCH1 (Notch1-DE) mutation-driven T-ALL 1. Targeting CXCL12/CXCR4 signaling after leukemic onset reduced the T-ALL burden in murine disease models, suggesting that the CXCL12-CXCR4 could be a viable therapeutic target in human T-ALL.To test the therapeutic role of CXCR4 in human T-ALL, we generated CXCR4 knockout T-ALL cell lines using CRISPR-Cas9 editing and characterized their growth and survival in vitro and in vivo. Although the effect of CXCR4 targeting on leukemic growth and apoptosis in vitro was modest (Figure 1A-B), animals engrafted with CXCR4 knockout T-ALL cell lines survived significantly longer than mice injected with control leukemic cells (Figure 1C). To investigate the clinical efficacy of targeting the CXCR4/CXCL12 axis in primary human T-ALL, we generated T-ALL patient-derived xenografts (PDXs) mouse models from 5 T-ALL and 2 Early T-cell precursor acute ALL (ETP-ALL) primary samples. Remarkably, treating PDX mice with MDX1338, a fully humanized anti-CXCR4 antibody2,3, significantly extended survival from all tested PDX models independently of their mutational landscape (Figure 1D-E). This increased survival was accompanied by mobilization of leukemic cells into peripheral blood, suggesting that CXCR4 targeting leads to leukemic disengagement from the BM niche, affecting their survival. Furthermore, BM imaging revealed disengagement of leukemic cells from the BM niche. Collectively, this study provides a pre-clinical rationale for therapeutic interventions targeting CXCR4 in human T-ALL. Figure 1: CXCR4 antagonism suppresses human T-ALL in vivo. A-B) Growth curve (A) and level of Annexin V (B) of human T-ALL cell lines CRISPR/Cas9-edited with sgCXCR4 or control sgOR2W5. C) Kaplan-Meier survival curves of recipient mice transduced with sgOR2W5 or two independent sgCXCR4 are plotted. D) Schematic of treatment with aCXCR4 Ab (MDX-1338) or isotype control. E-F) Kaplan-Meier survival graph (left) and peripheral blood levels of hCD45+ of NSG mice engrafted with primary adult (E) and pediatric (F) T-ALL samples and treated with aCXCR4 Ab or isotype control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased Hemoglobin Affinity for Oxygen with GBT021601 Reduces Pulmonary Hypertension and Lung Vascular Inflammation in a Murine Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is caused by a single nucleotide change in the β-globin gene coding sequence that results in the production of hemoglobin S (HbS). Under conditions of red blood cell (RBC) deoxygenation, HbS polymerizes and leads to RBC sickling, resulting in hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Despite improvements in treatment options, SCD remains a debilitating condition, and most patients experience progressive end-organ damage with associated complications and repeated hospitalizations. Pulmonary hypertension (PH), which is associated with increased mortality, is a common complication in patients with SCD, with nearly 10% of the SCD population reporting PH. PH in patients with SCD is multifactorial and may be caused by hemolysis, nitric oxide scavenging, and/or tissue hypoxia. Pharmacological increase of the concentration of oxygenated HbS (oxyHbS) in RBCs by voxelotor, a first-generation HbS polymerization inhibitor, reduces RBC sickling and preserves oxygen (O2) delivery by reducing anemia and improving blood rheology. Limited data are currently available on whether inhibiting HbS polymerization by increasing oxyHbS concentration may improve end-organ damage and associated complications, including PH. Objective: To examine the effects of GBT021601, a small-molecule, investigational, second-generation HbS polymerization inhibitor, on hemoglobin (Hb)-O2 affinity, PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Methods: Townes HbSS mice treated with GBT021601 (75 mg/kg orally once daily for 14 days) were exposed to acute hypoxia (8% O2) for 1 hour and reoxygenated for 72 hours. Untreated HbAA Townes mice were used as controls. Lung inflammation was assessed in terms of accumulation of inflammatory cells, activation of nuclear factor kappa B subunit 1, and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. To evaluate the impact of GBT021601 treatment on Hb-O2 affinity and anemia, p50 (partial O2 pressure at which Hb is 50% saturated with O2) and hematocrit levels were measured in SCD mice. In vivo pulmonary hemodynamic measurements included pulmonary artery pressure and right atrial pressure, whereas right ventricular hypertrophy was assessed using the Fulton index, which is the ratio of right ventricular weight to the weight of the left ventricle plus septum. Results: The total number of inflammatory cells and the specific numbers of lymphocytes, macrophages, and neutrophils were lower in untreated HbAA Townes mice and in HbSS Townes mice treated with GBT021601 compared with vehicle-dosed HbSS mice (Figure, panel A). Chronic treatment with GBT021601 increased Hb-O2 affinity, reducing the p50 in SCD mice from 32 mm Hg to 14 mm Hg. The treatment significantly reduced anemia, increasing hematocrit from 27.2% in the vehicle-dosed HbSS mice to 38.5% in the mice treated with GBT021601. Chronic GBT021601 treatment in HbSS mice also reduced pulmonary artery and right atrial pressure compared with those in untreated HbSS mice. Additionally, GBT021601 reduced right ventricular hypertrophy in HbSS mice, as evidenced by a reduction in the Fulton ratio (Figure, panel B). Conclusions: GBT021601 reduced PH, right ventricular dysfunction, and lung inflammation in a murine model of SCD. Our findings suggest that reducing RBC sickling, anemia, and hemolysis by increasing HbS affinity for O2 plays a critical role in modulating the immune system in PH and lung vasculature. These findings highlight the potential therapeutic efficacy of GBT021601 based on its ability to increase HbS affinity for O2 and prevent HbS polymerization and RBC sickling to reduce PH in SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

GBT021601 Prevents Microvascular Dysfunction and Improves Hypoxia-Reoxygenation-Induced Vaso-Occlusion in a Murine Model of Sickle Cell Disease

Background: In sickle cell disease (SCD), a single nucleotide change in the β-globin gene coding sequence results in the production of hemoglobin S (HbS), which polymerizes when deoxygenated and leads to red blood cell (RBC) sickling, hemolysis, anemia, diminished microvascular blood flow, vascular endothelial cell activation, vaso-occlusion, and ischemia. Pharmacological increase of the oxygenated HbS concentration in RBCs has been shown to be a well-tolerated and effective approach to inhibiting HbS polymerization, reducing RBC sickling, and improving oxygen (O2) delivery by reducing anemia and improving blood rheology. GBT021601, a potent second-generation small-molecule HbS polymerization inhibitor, is currently under investigation and has previously been shown to increase hemoglobin (Hb), improve hemolysis, and improve RBC health in a murine model. However, little is known regarding its impact on vaso-occlusion and microvascular dysfunction. Objective: To examine the effects of GBT021601 on SCD-induced microvascular dysfunction, capillary perfusion and density, Hb-O2 oxygen affinity, and Hb and hematocrit levels in a murine model of SCD. Methods: Capillary perfusion was measured by functional capillary density (FCD, a marker of capillary perfusion defined as the number of capillaries with RBCs in transit) in a murine model of SCD with an implanted dorsal window chamber (Figure). HbSS Townes mice were treated with GBT021601 (75 mg/kg) by oral gavage daily for 14 days and were compared with untreated HbSS and HbAA Townes mice. Results: Chronic treatment of HbSS mice with GBT021601 led to increased Hb-O2 affinity, evidenced by a reduction in blood p50 (partial O2 pressure at which Hb is 50% saturated with O2) from 32 mm Hg to 14 mm Hg. GBT021601 significantly reduced anemia by increasing total Hb and hematocrit from 8.3 g/dL and 26% in untreated HbSS mice to 11.2 g/dL and 37% in GBT021601-treated HbSS mice, respectively (P<0.01). Moreover, GBT021601 treatment prevented microvascular dysfunction (reduced RBC velocities and blood flow) in the arterioles and venules of HbSS mice. Increased capillary density is indicative of greater oxygen transport. Challenging HbSS mice with hypoxia (8% O2 for 1 hour) followed by reoxygenation (21% O2) induced vaso-occlusion, causing reduced microvascular blood flow and FCD in untreated mice. However, in GBT021601-treated HbSS mice, microvascular blood flow and FCD were significantly higher than those in untreated HbSS mice. Unlike in untreated HbSS mice, microvascular blood flow and FCD returned to baseline values after 72 hours of reoxygenation in treated HbSS mice. These results suggest that by increasing HbS-O2 affinity and thereby inhibiting HbS polymerization, GBT021601 may attenuate hypoxia-induced vaso-occlusion and impaired capillary perfusion. Furthermore, GBT021601 treatment in HbSS mice reduced both circulating adhesion markers and leukocyte-endothelium interactions in venules after hypoxia and reoxygenation. Conclusions: GBT021601 increased microvascular perfusion, reduced vaso-occlusion, and improved the hypoxia-induced reduction in capillary perfusion in HbSS mice, suggesting that chronic GBT021601 treatment prevents SCD-induced microvascular dysfunction and mitigates the rebound vaso-occlusion induced by hypoxia and reoxygenation in a murine model of SCD. Funding: Global Blood Therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Leukaemia and the CNS Niche: Insights on Bidirectional Communication from a Model of Infant Leukaemia

Infant leukaemia, an acute leukaemia within the first year of life, is a rare, aggressive entity with a poor response to intensive therapy. MLL-AF4+ infant leukaemia, the most common genetic subtype, has been shown to have: an in-utero origin, a lack of co-driver mutations, and lineage plasticity. Central nervous system (CNS) disease, typically a leukaemic infiltrate of the leptomeninges, is particularly common in these patients compared with non-infant paediatric ALL, and presents a serious clinical problem. With increasing understanding of the diverse immune cell landscape in the meninges, we sought to use our recently developed fully murine infant leukaemia model to explore the interactions between leukaemia and this microenvironment. We previously identified two microRNAs (miR128a and miR130b) that are upregulated in MLL-AF4+ infant and paediatric leukaemia patient samples. Overexpression of these microRNAs individually in mouse foetal liver (FL) Lin-Sca1+cKit+ (LSK) cells resulted in microRNA-dependent lineage-specific acute leukaemias in the context of Mll-AF4 with prominent CNS infiltration (Malouf et al. Blood 2021). To identify potential niche-specific leukaemia-propagating cells in our Mll-AF4+ mir128a+ infant proB-ALL model, we performed serial transplants using sorted leukaemia progenitor populations (LSK IL7R+ and Lin-Sca1-cKit+ (LK/CLP)) from mice that had previously succumbed to leukaemia. We observed that these leukaemia progenitors were functionally identical with indistinguishable output. This suggests the leukaemia-initiating cells in this model can emerge from different cell populations, consistent with our current understanding of ALL-initiating cells in human disease. CNS leukaemia developed in these mice with features that also mirror human disease: an immunophenotypically polyclonal infiltrate of the leptomeninges, sparing the brain parenchyma. To complement the functional data, we transcriptionally validated the infant proB-ALL model for two key features: foetal cell-of-origin and infant-specific expression signatures. Published RNA sequencing of murine FL and adult bone marrow (BM) lymphoid-primed multipotent progenitors (LMPP) - a putative cell of origin for infant leukaemia - were reanalysed (Symeonidou et al. Cell Reports 2021 data set). BM-derived leukaemia cells from our model are more similar to FL than adult BM LMPPs. Using a published MLL-AF4+ patient leukaemia data set we showed that cells from our model are also transcriptionally closer to infant than non-infant paediatric MLL-AF4+ ALL patient samples (Figure 1)(Andersson et al. Nature Genetics 2015 data set). Transcriptomic differences were identified by RNA sequencing of leukaemia progenitors from the BM and CNS niche. Statistically significant Gene Ontology terms of particular interest based on these differences are summarised in Figure 2. Genes upregulated in CNS-derived cells included cell adhesion molecules, immune modulators and cell growth factor receptors. These suggest a CNS-specific adaptive response to the microenvironment in a bidirectional manner. These novel observations may represent infant leukaemia-specific adaptations or more generic CNS leukaemia interactions uncovered through the use of an immunocompetent fully murine disease model. In either scenario, the implications of bidirectional communication between leukaemia and the CNS niche are significant for the development of much-needed novel biomarkers and therapies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Developmental disruption and restoration of brain synaptome architecture in the murine Pax6 neurodevelopmental disease model

Neurodevelopmental disorders of genetic origin delay the acquisition of normal abilities and cause disabling phenotypes. Nevertheless, spontaneous attenuation and even complete amelioration of symptoms in early childhood and adolescence can occur in many disorders, suggesting that brain circuits possess an intrinsic capacity to overcome the deficits arising from some germline mutations. We examined the molecular composition of almost a trillion excitatory synapses on a brain-wide scale between birth and adulthood in mice carrying a mutation in the homeobox transcription factor Pax6, a neurodevelopmental disorder model. Pax6 haploinsufficiency had no impact on total synapse number at any age. By contrast, the molecular composition of excitatory synapses, the postnatal expansion of synapse diversity and the acquisition of normal synaptome architecture were delayed in all brain regions, interfering with networks and electrophysiological simulations of cognitive functions. Specific excitatory synapse types and subtypes were affected in two key developmental age-windows. These phenotypes were reversed within 2-3 weeks of onset, restoring synapse diversity and synaptome architecture to the normal developmental trajectory. Synapse subtypes with rapid protein turnover mediated the synaptome remodeling. This brain-wide capacity for remodeling of synapse molecular composition to recover and maintain the developmental trajectory of synaptome architecture may help confer resilience to neurodevelopmental genetic disorders.

Abstract 12174: Inflammation-Dependent Hydroxyeicosatetraenoic Acids Dysregulate Macrophage Resolution Function

Introduction and Hypothesis: Macrophage efferocytosis is a critical step in the resolution of inflammation. Dysregulated macrophage efferocytosis has been implicated in the pathogenesis of chronic inflammatory disorders including atherosclerosis and IBD. Nonetheless, the nature of possible dysregulating mechanisms remains incompletely understood. We reported that non-enzymatic lipid peroxidation and the mucosal levels of monohydroxylated PUFAs including 12- and 15-hydroxyeicosatetraenoic acid (HETE) correlated with disease in murine models of IBD. HETEs can form enzymatically and non-enzymatically and can signal through the leukotriene (LT) receptor BLT2. LTB4-BLT1/2 signaling can suppress macrophage efferocytosis capacity. We hypothesized that non-enzymatically derived mucosal HETEs correlate with disease in a murine Crohn’s disease (CD) model and in IBD patient biopsies; and that HETEs suppress murine and human macrophage efferocytosis capacity. Methods: We exploited the stereospecificity of enzymatic HETE formation to determine by chiral-LC-MS/MS the levels of non-enzymatically derived HETEs in intestine from a murine myeloid Cox2 knock-out model of Crohn’s disease (CD) and in human IBD patient biopsies. We assessed the effect of racemic HETEs on the efferocytosis capacity of murine BMDMs and human THP1 macrophages for apoptotic MPRO neutrophils. We performed proteomics using accurate mass LC-MS/MS followed by GO enrichment analysis. Results: Non-enzymatically derived mucosal 5-, 12-, and 15HETE were elevated with disease: a) by 3-4 fold in the murine CD model (p&lt;0.01 to p&lt;0.0001); and b) by 2-3 fold in ileal and colonic biopsies from areas of active inflammation in CD and ulcerative colitis patients compared to remission controls (p&lt;0.05). Racemic HETEs suppressed efferocytosis of apoptotic neutrophils by murine (12- and 15-HETE: p&lt;0.0001) and human (12-HETE: p&lt;0.0001) macrophages. Racemic 12-HETE altered the proteome of efferocytic BMDM by inducing 200 proteins while silencing 200 more (&lt;1% FDR), and by suppressing pathways associated with efferocytosis. Conclusion: Inflammation-dependent HETEs may suppress macrophage efferocytosis in chronic inflammatory disorders including IBD and atherosclerosis.

Abstract 15266: Effect of Nicotine Exposure to Induced Pluripotent Stem Cell Derived Endothelial Cell Therapy in a Murine Model of Peripheral Artery Disease

Introduction: Clinical solutions for peripheral artery disease (PAD) remain elusive due to the many challenges of surgical intervention. Stem cell therapy offer a promising option, but barriers exist to their clinical translatability. Among these include lifestyle choices like tobacco use that may influence the efficacy of stem cell therapy. The objective of this study aims to examine the potential effects of nicotine on human induced pluripotent stem cell derived endothelial cells (iPSC-ECs) therapy in the context of PAD. Hypothesis: Chronic nicotine exposure negatively affects the therapeutic potential of implanted iPSC-ECs in a murine model of PAD. Methods: Mice were implanted with an osmotic pump for controlled exposed to nicotine or saline (control) for 28 days. Plasma cotinine was measured to confirm nicotine exposure. After 28 days of nicotine or saline exposure, the mice underwent induction of hindlimb ischemia and before receiving an injection of 10 6 iPSC-ECs. A second dose of cells were injected to the ischemic limb on day 7 post-surgery. All mice were monitored for blood perfusion recovery using laser Doppler spectroscopy imaging. Survival of the transplanted cells in vivo were tracked by bioluminescence imaging. The mice receiving iPSC-ECs were also imaged for cell survival using bioluminescence imaging. At day 14 post-surgery, mice were euthanized, and the gastrocnemius muscle and thigh muscle were harvested for histological analysis. Results: The average cotinine concentration in sera was ~200 ng/mL, which is within physiological range for a tobacco user. Blood perfusion recovery at day 14 showed trends of nicotine treated groups have hindered perfusion recovery, compared to those treated with saline. Importantly, nicotine exposure trended with poorer perfusion recovery after cell therapy. Bioluminescent imaging showed iPSC-ECs persisted in vivo for all groups without significant difference. Conclusions: Nicotine exposure was associated with poorer blood perfusion recovery after iPSC-EC therapy, compared to control saline exposure, suggesting that nicotine may have adverse effects on iPSC-EC therapy for treatment of PAD.

Trimethylamine N-Oxide Reduces Neurite Density and Plaque Intensity in a Murine Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common aging-associated neurodegenerative disease; nevertheless, the etiology and progression of the disease is still incompletely understood. We have previously shown that the microbially-derived metabolite trimethylamine N-oxide (TMAO) is elevated in the cerebrospinal fluid (CSF) of individuals with cognitive impairment due to AD and positively correlates with increases in CSF biomarkers for tangle, plaque, and neuronal pathology. We assessed the direct impact of TMAO on AD progression. To do so, transgenic 5XFAD mice were supplemented with TMAO for 12 weeks. Neurite density was assessed through quantitative brain microstructure imaging with neurite orientation dispersion and density imaging magnetic resonance imaging (MRI). Label-free, quantitative proteomics was performed on cortex lysates from TMAO-treated and untreated animals. Amyloid-β plaques, astrocytes, and microglia were assessed by fluorescent immunohistochemistry and synaptic protein expression was quantified via western blot. Oral TMAO administration resulted in significantly reduced neurite density in several regions of the brain. Amyloid-β plaque mean intensity was reduced, while plaque count and size remained unaltered. Proteomics analysis revealed that TMAO treatment impacted the expression of 30 proteins (1.5-fold cut-off) in 5XFAD mice, including proteins known to influence neuronal health and amyloid-β precursor protein processing. TMAO treatment did not alter astrocyte and microglial response nor cortical synaptic protein expression. These data suggest that elevated plasma TMAO impacts AD pathology via reductions in neurite density.

RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4

Chromosomal translocation generates the MLL-AF4 fusion gene, which causes acute leukemia of multiple lineages. MLL-AF4 is a strong oncogenic driver that induces leukemia without additional mutations and is the most common cause of pediatric leukemia. However, establishment of a murine disease model via retroviral transduction has been difficult owning to a lack of understanding of its regulatory mechanisms. Here, we show that MLL-AF4 protein is post-transcriptionally regulated by RNA-binding proteins, including those of KHDRBS and IGF2BP families. MLL-AF4 translation is inhibited by ribosomal stalling, which occurs at regulatory sites containing AU-rich sequences recognized by KHDRBSs. Synonymous mutations disrupting the association of KHDRBSs result in proper translation of MLL-AF4 and leukemic transformation. Consequently, the synonymous MLL-AF4 mutant induces leukemia in vivo. Our results reveal that post-transcriptional regulation critically controls the oncogenic activity of MLL-AF4; these findings might be valuable in developing novel therapies via modulation of the activity of RNA-binding proteins.

Genomic and Phenotypic Trait Variation of the Opportunistic Human Pathogen Aspergillus flavus and Its Close Relatives.

Fungal diseases affect millions of humans annually, yet fungal pathogens remain understudied. The mold Aspergillus flavus can cause both aspergillosis and fungal keratitis infections, but closely related species are not considered clinically relevant. To study the evolution of A. flavus pathogenicity, we examined genomic and phenotypic traits of two strains of A. flavus and three closely related species, Aspergillus arachidicola (two strains), Aspergillus parasiticus (two strains), and Aspergillus nomiae (one strain). We identified >3,000 orthologous proteins unique to A. flavus, including seven biosynthetic gene clusters present in A. flavus strains and absent in the three nonpathogens. We characterized secondary metabolite production for all seven strains under two clinically relevant conditions, temperature and salt concentration. Temperature impacted metabolite production in all species, whereas salinity did not affect production of any species. Strains of the same species produced different metabolites. Growth under stress conditions revealed additional heterogeneity within species. Using the invertebrate fungal disease model Galleria mellonella, we found virulence of strains of the same species varied widely; A. flavus strains were not more virulent than strains of the nonpathogens. In a murine model of fungal keratitis, we observed significantly lower disease severity and corneal thickness for A. arachidicola compared to other species at 48 h postinfection, but not at 72 h. Our work identifies variations in key phenotypic, chemical, and genomic attributes between A. flavus and its nonpathogenic relatives and reveals extensive strain heterogeneity in virulence that does not correspond to the currently established clinical relevance of these species. IMPORTANCE Aspergillus flavus is a filamentous fungus that causes opportunistic human infections, such as aspergillosis and fungal keratitis, but its close relatives are considered nonpathogenic. To begin understanding how this difference in pathogenicity evolved, we characterized variation in infection-relevant genomic, chemical, and phenotypic traits between strains of A. flavus and its relatives. We found extensive variation (or strain heterogeneity) within the pathogenic A. flavus as well as within its close relatives, suggesting that strain-level differences may play a major role in the ability of these fungi to cause disease. Surprisingly, we also found that the virulence of strains from species not considered to be pathogens was similar to that of A. flavus in both invertebrate and murine models of disease. These results contrast with previous studies on Aspergillus fumigatus, another major pathogen in the genus, for which significant differences in infection-relevant chemical and phenotypic traits are observed between closely related pathogenic and nonpathogenic species.

High-Fructose/High-Fat Diet Downregulates the Hepatic Mitochondrial Oxidative Phosphorylation Pathway in Mice Compared with High-Fat Diet Alone.

Both high-fat diet (HFD) alone and high-fructose plus HFD (HFr/HFD) cause diet-induced non-alcoholic fatty liver disease in murine models. However, the mechanisms underlying their impacts on inducing different levels of liver injury are yet to be elucidated. This study employed a proteomic approach to elucidate further on this issue. Adult male C57BL/6J mice were allocated to the HFD or the HFr/HFD group. After feeding for 12 weeks, all mice were euthanized and samples were collected. The proteomic profiles in liver tissues were analyzed using liquid chromatography-tandem mass spectrometry followed by canonical pathway analysis. We demonstrated that the mitochondrial oxidative phosphorylation (OXPHOS) pathway was the most significantly downregulated canonical pathway in the HFr/HFD group when compared with the HFD group. Within the OXPHOS pathway, the HFr/HFD group demonstrated significant downregulation of complexes I and III and significant upregulation of complex IV when compared with the HFD group. Moreover, the HFr/HFD group had lower protein levels of NADH: ubiquinone oxidoreductase subunits S3, S6, A5, and A12 in complex I (p < 0.001, =0.03, <0.001, and <0.001, respectively), lower protein level of cytochrome C in complex III (p < 0.001), and higher protein level of cytochrome C oxidase subunit 2 in complex IV (p = 0.002), when compared with the HFD group. To summarize, we have demonstrated that the hepatic mitochondrial OXPHOS pathway is significantly downregulated in long-term HFr/HFD feeding when compared with long-term HFD feeding. These data support the concept that the hepatic mitochondrial OXPHOS pathway should be involved in mediating the effects of HFr/HFD on inducing more severe liver injury than HFD alone.

Oral administration of human carbonic anhydrase I suppresses colitis in a murine inflammatory bowel disease model

The incidence of inflammatory bowel disease (IBD) is increasing; hence, effective treatments are warranted. The therapeutic effect of human carbonic anhydrase I (hCA I) in IBD remains unknown. Therefore, we investigated whether oral tolerization to hCA I would induce antigen-specific protection from intestinal inflammation in vivo. Severe combined immunodeficient mice received hCA I, keyhole limpet hemocyanin (KLH), or phosphate-buffered saline (PBS) orally for 7 days. Colons and mesenteric lymph nodes (MLNs) were collected 4 weeks after cell transfer. Additionally, the mechanisms underlying the therapeutic effects were investigated. The comparison between the effects of well-established drugs and hCA I oral administration was investigated. Oral administration of hCA I ameliorated colitis remarkably. hCA I reached the cecum and ameliorated colitis more effectively than mesalazine and similarly to prednisolone. Compared with PBS treatment, hCA I treatment reduced interleukin (IL)-17a, IL-6, and retinoic acid-related orphan receptor gamma t (RORγt) expression in the colon or MLNs; moreover, hCA I markedly reduced IL-6, IL-17, and interferon-gamma (IFN-γ) levels in the MLN. Oral administration of hCA I induced immune tolerance and suppressed colitis in vivo. Thus, hCA I administration could be proposed as a new treatment option for IBD.

Assessment of cholesterol homeostasis in the living human brain.

Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, 18F-CHL-2205 (18F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer's disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.

Assessing complex odor discrimination in mice using a novel instrumental patterning task.

Negative patterning tasks are a key tool to unveil the mechanisms by which stimulus representations are acquired-a central concern in Robert Rescorla's research. In these tasks, target stimuli are reinforced when presented individually (A+/B+) but not when presented in compound (AB-). The discrimination of single stimuli from their compound presentation is a challenge for theories of associative learning, because it cannot be explained by the simple accrual of associative strength. The present study examined the conditions under which mice learn this part-whole discrimination in olfactory stimuli using a novel instrumental methodology. In two experiments, reinforcement was contingent on distinct responses depending on whether a set of odor mixtures were presented in isolation or as a compound. Using C57BL/6 mice, Experiment 1 showed a mutual interference between learning a response to individual odors and learning a different response to those odors presented in compound. Using wild-type APP/PS1 mice (a control strain for a murine model of Alzheimer's disease), Experiment 2 replicated this interference and showed that it is stimulus-specific. These experiments show that the instrumental patterning task may not only complement Pavlovian negative patterning tasks but may also motivate its own questions on the representation of complex stimuli. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

A diet-induced murine model for non-alcoholic fatty liver disease with obesity and insulin resistance that rapidly develops steatohepatitis and fibrosis

AbstractNon-alcoholic fatty liver disease (NAFLD) has become the leading cause of chronic liver disease worldwide. Patients with NAFLD often suffer steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The presence of visceral obesity or type 2 diabetes mellitus (T2DM) is a major risk factor and potential therapeutic target for NAFLD. The establishment of animal models with these metabolic comorbidities and with the rapid progression of the disease is needed for developing treatments for NAFLD but remains to be archived. In the present study, KK-Ay mice, widely used as T2DM models, or C57BL6 mice were fed a high-fat, high-fructose, and high-cholesterol diet supplemented with cholic acid (NAFLD diet). The KK-Ay mice fed a NAFLD diet exhibited remarkable obesity and insulin resistance. A prominent accumulation of triglycerides and cholesterol in the liver was observed at 4 weeks. These mice developed steatohepatitis at 4 weeks and fibrosis at 12 weeks. In contrast, C57BL6 mice fed a NAFLD diet remained lean, although they still developed steatohepatitis and fibrosis. In summary, we established a diet-induced murine NAFLD model with the rapid development of steatohepatitis and fibrosis, bearing obesity and insulin resistance. This model could be useful as preclinical models for drug development of NAFLD.

Amiodarone improves anemia in a murine model of sickle cell disease and is associated with increased erythrocyte bis(monoacylglycerol) phosphate

Sickle cell disease (SCD) is associated with altered plasma and erythrocyte lipid profiles. In a previous study, SCD mice with deficiency of proprotein convertase subtilisin/kexin type 9 (PCSK9) were observed to have more severe anemia and increased sickling compared to control SCD mice. Although PCSK9 affects circulating low density lipoprotein (LDL) by regulation of the LDL receptor, the effect of PCSK9 on anemia was independent of LDL receptor expression. In the current study, erythrocyte metabolomics were performed and revealed altered erythrocyte lipid species between SCD mice with and without PCSK9. Of particular interest, the late endosome-specific lipid bis(mono)acylglycerol phosphate (BMP) 44:12 was markedly decreased in erythrocytes from SCD mice deficient in PCSK9 mice relative to control SCD mice. Incubation of sickle erythrocytes with a neutralizing antibody to BMP increased erythrocyte sickling in vitro. In vitro treatment of SCD erythrocytes with amiodarone (1.5 μM) or medroxyprogesterone (6.75 μM), two pharmacologic compounds known to increase BMP, resulted in reduced erythrocyte sickling. Treatment of SCD mice with amiodarone (10 mg/kg) for 2 weeks resulted in increased BMP, improvement in anemia with reduced reticulocytosis, and decreased ex vivo sickling. In conclusion, severity of anemia in SCD is improved with amiodarone treatment, an effect which may be mediated through increased erythrocyte BMP.

Suppressive mechanisms of regulatory B cells in mice and humans.

B cells include immune-suppressive fractions, called regulatory B cells (Bregs), which regulate inflammation primarily through an interleukin 10 (IL-10)-mediated inhibitory mechanism. Several B-cell fractions have been reported as IL-10-producing Bregs in murine disease models and human inflammatory responses including autoimmune diseases, infectious diseases, cancer and organ-transplant rejection. Although the suppressive functions of Bregs have been explored through the hallmark molecule IL-10, inhibitory cytokines and membrane-binding molecules other than IL-10 have also been demonstrated to contribute to Breg activities. Transcription factors and surface antigens that are characteristically expressed in Bregs are also being elucidated. Nevertheless, defining Bregs is still challenging because their active periods and differentiation stages vary among disease models. The identity of the diverse Breg fractions is also under debate. In the first place, since regulatory functions of Bregs are mostly evaluated by ex vivo stimulation, the actual in vivo phenotypes and functions may not be reflected by the ex vivo observations. In this article, we provide a historical overview of studies that established the characteristics of Bregs and review the various suppressive mechanisms that have been reported to be used by Bregs in murine and human disease conditions. We are only part-way through but the common phenotypes and functions of Bregs are still emerging.

Innate immune activation without immune cell infiltration in brains of murine models of Aicardi-Goutières Syndrome.

Chronic inflammation is frequently invoked as a mechanism of neurodegeneration and yet inflammatory cell infiltrates are seldom seen in brains of these disorders. Different disciplines utilize different technologies and methodologies to describe what is immunologically defined as the innate immune response (IIR). We examined murine models of the human neurodegenerative disease Aicardi-Goutières Syndrome, where an IIR is initiated by aberrant RNA metabolism secondary to a mutation in adenosine deaminase acting on RNA gene (ADAR1). We previously showed that these mice demonstrated a deficit in RNA editing that lead to MDA-5 mediated RNA sensing pathway activation of the IIR with massive interferon stimulated gene transcription and translation. As early as 2 weeks of age, in situ hybridization demonstrated that different central nervous system (CNS) cell lineages expressed very high levels of distinct interferon stimulated genes (ISGs) in the absence of interferon and absence of immune cell infiltrates. We have expanded these studies to more completely describe the breadth of ISG expression systemically and in CNS using double label in situ hybridization. Within the CNS aberrant ISG expression was mostly limited to neurons, microglia, ependyma, choroid plexus, and endothelial cells with little expression in oligodendroglia and astrocytes except for STAT1. Wild type controls showed a similar pattern of ISG expression but only in aged mice and at levels minimally detectable by in situ hybridization. Despite months of elevated ISG expression in mutant mice, there was essentially no inflammatory infiltrate, no interferon production and minimal glial reaction. Histomorphological neurodegenerative pathology of ventricular dilatation and deep gray matter mineralization were evident in mutant mice 8-13 months of age but this did not show a spatial relationship to ISG expression. This IIR without immune cell infiltration leads to neurodegeneration through non-canonical pathways that may accentuate normal aging pathways.