Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor ...activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor β-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.
Non-catechol-based high-affinity selective dopamine D
receptor (D1R) agonists were recently described, and candidate PET ligands were selected on the basis of favorable properties. The objective of ...this study was to characterize in vivo in nonhuman primates 2 novel D1R agonist PET radiotracers, racemic
F-MNI-800 and its more active atropisomeric (-)-enantiomer,
F-MNI-968.
Ten brain PET experiments were conducted with
F-MNI-800 on 2 adult rhesus macaques and 2 adult cynomolgus macaques, and 8 brain PET experiments were conducted with
F-MNI-968 on 2 adult rhesus macaques and 2 adult cynomolgus macaques. PET data were analyzed with both plasma-input-based methods and reference-region-based methods. Whole-body PET images were acquired with
F-MNI-800 from 2 adult rhesus macaques for radiation dosimetry estimates.
F-MNI-800 and
F-MNI-968 exhibited regional uptake consistent with D1R distribution. Specificity and selectivity were demonstrated by dose-dependent blocking with the D
antagonist SCH-23390.
F-MNI-968 showed a 30% higher specific signal than
F-MNI-800, with a nondisplaceable binding potential of approximately 0.3 in the cortex and approximately 1.1 in the striatum. Dosimetry radiation exposure was favorable, with an effective dose of about 0.023 mSv/MBq.
F-MNI-968 has significant potential as a D1R agonist PET radiotracer, and further characterization in human subjects is warranted.
To interrogate whether altered function of the hippocampal-mPFC circuit underlies the deficit in fear extinction recall in rats subjected to single-prolonged stress (SPS), changes in brain ...region-specific metabolic rate were measured in male rats (control and SPS treated). Brain region metabolic rates were quantified using uptake of 14C-2-deoxyglucose (14C-2DG) during fear memory formation, fear memory extinction and extinction recall. Control and SPS rats had similar regional brain activities at baseline. During extinction recall, 14C-2DG uptake decreased in hippocampal regions in control rats, but not in SPS rats. SPS rats also exhibited a significant deficiency in fear extinction recall, replicating a previously reported finding. Reduced hippocampal activity during fear extinction recall in control animals may reflect reduction in fear overgeneralization, thereby enabling discrimination between distinct contexts. In contrast, persistent levels of hippocampal activity in SPS-exposed male animals during fear extinction recall may reflect the dysfunctional persistence of fear overgeneralization. Future studies in females can test gender-specificity of these effects, with appropriate attention to luteal dependent effects on extinction of fear learning. Detailed knowledge of regional brain activities underlying stress-induced deficits in extinction recall may help identify therapeutic targets in PTSD.
Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis. In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive ...disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most multiple sclerosis patients. We have generated a TCR transgenic mouse that expresses the α- and β-chains of a myelin oligodendrocyte glycoprotein (MOG) 35-55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4(+) and CD8(+) T cells that recognize MOG and produce proinflammatory cytokines, allowing for the first time to our knowledge the simultaneous examination of myelin-reactive CD4(+) and CD8(+) T cells in the same host. 1C6 CD8(+) T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4(+) T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an IgH specific for MOG, the mice develop spontaneous EAE with high incidence, but surprisingly the disease pattern does not resemble the neuromyelitis optica-like disease observed in mice bearing CD4(+) T cells and B cells reactive to MOG on the C57BL/6 background. Collectively, our data show that although myelin-reactive CD8(+) T cells contribute to disease, disease is primarily driven by myelin-reactive CD4(+) T cells and that the coexistence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.
Purpose
Tc-99m methylene diphosphonate (
99m
TcMDP) is an
in vivo
bone imaging agent that also accumulates in injured skeletal muscle cells. The objective of this study was to investigate if
99m
...TcMDP could be used to detect muscle injury in the
mdx
mouse model of Duchenne muscular dystrophy (DMD).
Procedures
Static whole-body single-photon emission computed tomography/computed tomography (CT) scans were acquired at 2 h post-injection of
99m
TcMDP in two cohorts of animals at different sites: one cohort of mice at 6, 15, and 19 weeks of age, and a separate cohort at 16 weeks. The second cohort was also imaged with high-resolution CT at 8 weeks.
Results
mdx
mice had higher
99m
TcMDP uptake and significantly higher
99m
TcMDP concentrations in muscle than controls.
Conclusions
Higher uptake of
99m
TcMDP in muscle of
mdx
mice agrees with histological reports of muscle calcification in
mdx
mice, and suggests the potential translational use of
99m
TcMDP imaging for tracking DMD progression and therapeutic response.
Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and ...pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-
GluR1
and anti-
Gabra1
ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-
Gabra1
ASOs also reduced binding of the GABA
A
receptor PET ligand
18
F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.
Multimodal in vivo and ex vivo imaging demonstrates the CNS pharmacology of antisense oligonucleotides administered to the intrathecal space.
IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity ...of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment. See related Spotlight by Silva, p. 544.
18FFPEB is a positron emission tomography tracer which, in preclinical studies, has shown high specificity and selectivity toward the metabotropic glutamate receptor 5 (mGluR5). It possesses the ...potential to be used in human studies to evaluate mGluR5 function in a range of neuropsychiatric disorders, such as anxiety and Fragile X syndrome. To define optimal scan methodology, healthy human subjects were scanned for 6 hours following either a bolus injection (n = 5) or bolus-plus-constant-infusion (n = 5) of 18FFPEB. Arterial blood samples were collected and parent fraction measured by high-performance liquid chromatography (HPLC) to determine the metabolite-corrected plasma input function. Time activity curves were extracted from 13 regions and fitted by various models to estimate VT and BPND. 18FFPEB was well fitted by the two-tissue compartment model, MA1 (t∗ = 30), and MRTM (using cerebellum white matter as a reference). Highest VT values were observed in the anterior cingulate and caudate, and lowest VT values were observed in the cerebellum and pallidum. For kinetic modeling studies, VT and BPND were estimated from bolus or bolus-plus-constant-infusion scans as short as 90 minutes. Bolus-plus-constant-infusion of 18FFPEB reduced intersubject variability in VT and allowed equilibrium analysis to be completed with a 30-minute scan, acquired 90–120 minutes after the start of injection.
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