Browsing by Subject "Targeting"

Now showing 1 - 4 of 4
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    Characterization of a unique basolateral targeting domain in the Drosophila TGF-B Type II receptor punt
    (2013-10) Mundt, Melinda G.
    In polarized epithelial cells, differential localization of receptors and ligands can control signaling. Drosophila wing imaginal discs are a polarized epithelial layer in which the TGF-B superfamily ligand Dpp is expressed both apically and basolaterally, yet requires a graded distribution throughout the disc to signal properly. We found that Punt, the type II TGF-B receptor that Dpp signals through, is localized specifically at the basolateral membrane, which limits Dpp signaling to the basolateral surface. In characterizing the sequence of Punt, our lab found a unique basolateral targeting domain, the Punt targeting domain (PTD). The PTD is both necessary and sufficient for basolateral localization. Mutation of the insect-conserved portion and whole PTD results in apical mislocalization but characterization of the PTD shows that there is no minimal sequence within the PTD responsible for function. Furthermore, changes in localization of Punt and the other type II receptor Wit affect fly viability.
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    Development of Antibody Conjugated Nanoparticles for Targeting Cerebrovascular Amyloid Deposits
    (2017-08) Talele, Surabhi
    Cerebral Amyloid Angiopathy (CAA) is characterized by the deposition of amyloid beta (Aβ) proteins in the cerebral vasculature. CAA could independently cause massive lobar hemorrhages and stroke. Moreover, CAA affects majority of Alzheimer’s Disease (AD) patients and is associated with a rapid decline in memory and cognition in AD1. Currently, there is no pre-mortem diagnosis available for CAA, the treatment options are ineffective, and just provide symptomatic relief. Also, the current diagnostic agents do not provide the required spatial resolution and contrast enhancement for the early detection of cerebrovascular amyloid deposits. To overcome these issues, our lab aims to design novel theranostic nanovehicles capable of targeting the cerebrovascular amyloid deposits. These nanovehicles are expected to facilitate the early diagnosis of CAA by enabling the detection of cerebrovascular amyloid with high specificity and sensitivity. The goal of the current study was to optimize the amyloid targeting and contrast enhancement for cerebrovascular amyloid detection by positron emission tomography (PET) and magnetic resonance imaging (MRI). Chitosan-polycarbophil nanoparticles (NPs) were functionalized with the anti-amyloid antibody IgG4.1 using carbodiimide chemistry. We characterized the particles for various physicochemical properties. The NPs were fluorescently labeled with Alexa Fluor 647 to evaluate amyloid protein targeting in vitro using human cerebral microvascular endothelial cell (hCMEC/D3) monolayers and in mouse models in vivo. We optimized the labeling of the NPs with PET isotope 89Zr followed by their serum stability studies prior to in vivo injections. The nanoprobe could also be employed for the early detection of other cerebrovascular diseases, by incorporating appropriate targeting moieties.
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    Intranasal targeting of neuropeptides to the central nervous system: evaluation of pharmacokinetics, pharmacodynamics, and a novel vasoconstrictor formulation.
    (2009-02) Dhuria, Shyeilla V
    Delivery of therapeutics to the central nervous system (CNS) for the treatment of neurological and psychiatric diseases and disorders is a formidable challenge due to the presence of the blood-brain barrier (BBB). The intranasal route of administration is a noninvasive method to bypass the BBB and rapidly target therapeutics to the CNS by utilizing the olfactory and trigeminal neural pathways connecting the nasal mucosa to the brain and spinal cord. Despite the enormous potential of the intranasal method, it is limited by the low efficiency of delivery to the CNS. The overall objective of this research was to assess targeting of neuropeptides to the CNS following intranasal administration by evaluating pharmacokinetics, pharmacodynamics, and a novel vasoconstrictor formulation. To assess if intranasal administration targeted the neuropeptide, hypocretin-1 (HC), to the CNS, pharmacokinetics and targeting were compared over a two hour period following intranasal and intravenous administration of 10 nmol of 125I-HC to anesthetized rats. Results indicated that intranasal administration resulted in less exposure to the blood and peripheral tissues (~10-fold), similar brain concentrations, increased tissue-to-blood concentration ratios (trigeminal nerve, 14-fold; olfactory bulbs, 9-fold), and greater drug targeting efficiency to the brain (5- to 8-fold) compared to an equivalent intravenous dose. Approximately 80% of the brain exposure after intranasal administration was due to direct transport pathways from the nasal passages. Results from these studies indicated that intranasal administration targets HC to the CNS within 30 minutes of dosing, along direct pathways involving the trigeminal and olfactory nerves. To determine if intranasal administration resulted in pharmacodynamic effects in the CNS, food consumption, water intake, and wheel running activity were monitored following intranasal administration of 100 nmol of HC. Further, HC signaling pathways were investigated to understand the molecular mechanisms underlying the behavioral effects of intranasal HC. Intranasal administration of HC increased food consumption and wheel running activity over the first four hours following dosing, but had no effect on water intake. Intranasal HC activated HC signaling pathways in the diencephalon and in the brainstem, which are brain areas involved in the regulation of appetite and locomotor activity. These findings indicate that intranasal HC reaches the CNS in its biologically active form and at concentrations sufficient to affect HC-mediated behaviors and to activate signaling pathways. To determine if a vasoconstrictor nasal formulation could enhance targeting to the CNS, drug targeting was compared 30 minutes following intranasal administration of 125I-labeled neuropeptides (HC; L-Tyr-D-Arg, D-KTP) in the presence and absence of a vasoconstrictor (phenylephrine, PHE). Results showed that intranasal administration of HC or D-KTP with 1% PHE reduced absorption into the blood, increased deposition in the olfactory epithelium, and increased delivery to the olfactory bulbs. Concentrations in the remaining brain regions and in the trigeminal nerve were reduced in the presence of the vasoconstrictor. The dramatic reduction in the blood concentrations contributed to tissue-to-blood ratios that were increased for HC throughout the brain. For D-KTP, ratios were increased in the olfactory bulbs with 1% PHE, and throughout the brain using a higher concentration of 5% PHE. Use of vasoconstrictor formulations could be used with CNS therapeutics having adverse side effects, where systemic exposure would be limited. The key findings of this research are that intranasal administration targets neuropeptides to the CNS compared to intravenous administration, with brain concentrations that are sufficient to affect CNS-mediated behaviors and signaling pathways. Intranasal administration of HC and other neuropeptides has potential for treating CNS diseases involving the hypocretinergic system, including narcolepsy, Alzheimer's disease, and appetite disorders. In addition, use of a vasoconstrictor nasal formulation could improve intranasal treatments by reducing systemic exposure and enhancing delivery to rostral brain areas, which could be important for CNS therapeutics having adverse systemic effects.
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    Salmonella enterica Typhimurium as a tumor-targeting immunotherapy vector
    (2015-08) Drees, Jeremy
    Interest in cancer immunotherapy has grown in recent years due to its potential for significant and durable therapeutic responses. Immune checkpoint blockade has emerged as an immunotherapy as a single agent but has even greater appeal when it is used in combination with other immunostimulatory approaches. However, the dosing of checkpoint blockade and its combinatorial use with other immunotherapies has been limited by systemic immune-related adverse side effects. One way to overcome these adverse effects is to deliver the therapeutic agents specifically to the tumor microenvironment. Salmonella enterica Typhimurium (S. Typhimurium) has been studied for cancer therapy due to its genetic manipulability and tumor-targeting propensity, and in this thesis, the potential of S. Typhimurium as a tumor-targeting immunotherapy vector was investigated. Functional antagonistic single chain antibodies (scFvs) against the immune checkpoints CTLA-4 and PD-L1 were isolated from an immunized chicken library and engineered for secretion from S. Typhimurium. The inherent anti-tumor properties and tumor-targeting capability of S. Typhimurium were then tested in transplanted primary and metastatic tumor models as well as a genetically engineered autochthonous BALB-neuT breast cancer model. In each of these models, S. Typhimurium demonstrated native anti-tumor efficacy; however the bacteria did not adequately colonize the autochthonous tumors of the BALB-neuT model. Disruption of tumor vasculature by treating BALB-neuT mice with a vasculature disrupting agent (VDA) improved the colonization of autochthonous tumors over 1000-fold to levels similar to those observed for transplanted tumors. Subsequent comparison of the tumor targeting capability and efficacy of S. Typhimurium engineered to secrete the antagonistic ?PD-L1 (scFv) versus a control strain showed that secretion of the scFv may further improve the colonization of autochthonous tumors, leading to a greater reduction in tumor burden of treated mice. These findings provide a proof of principle for the expression and delivery of functional immunotherapeutic single chain antibodies using S. Typhimurium, demonstrate S. Typhimurium's native tumoricidal activity independent of tumor-targeting, illustrate the importance of clinically representative tumor models when studying bacterial cancer therapy, and demonstrate the potential of VDA treatment to improve bacterial tumor-targeting. Collectively, this work illustrates S. Typhimurium's promise as a tumor-targeting immunotherapy vector.