Position
Former Research Associate,
Cellular Stress and Inflammation Section
Former Research Associate,
Genetic Engineering and Viral Vector Core
Contact
Biomedical Research Center251 Bayview Boulevard
Suite 200
Baltimore, MD 21224
Research Interests
Core-related Expertise:
- In vitro functional validation and quality assurance (Transfection, Transduction)
- Immunostaining and western blot analysis
- Primary cell isolation and culture (rodent neurons, glia, and hepatocytes)
- Maintainance of cell lines
Publications
Selected Publications
2021
Li, Yazhou; Glotfelty, Elliot J; Karlsson, Tobias; Fortuno, Lowella V; Harvey, Brandon K; Greig, Nigel H
The metabolite GLP-1 (9-36) is neuroprotective and anti-inflammatory in cellular models of neurodegeneration Journal Article
In: J Neurochem, 2021, ISSN: 1471-4159.
@article{pmid34569615,
title = {The metabolite GLP-1 (9-36) is neuroprotective and anti-inflammatory in cellular models of neurodegeneration},
author = {Yazhou Li and Elliot J Glotfelty and Tobias Karlsson and Lowella V Fortuno and Brandon K Harvey and Nigel H Greig},
url = {https://pubmed.ncbi.nlm.nih.gov/34569615/},
doi = {10.1111/jnc.15521},
issn = {1471-4159},
year = {2021},
date = {2021-09-01},
urldate = {2021-09-01},
journal = {J Neurochem},
abstract = {Glucagon-like peptide-1 (GLP-1) is best known for its insulinotropic action following food intake. Its metabolite, GLP-1 (9-36), was assumed biologically inactive because of low GLP-1 receptor (GLP-1R) affinity and non-insulinotropic properties; however, recent studies contradict this assumption. Increased use of FDA approved GLP-1 analogues for treating metabolic disorders and neurodegenerative diseases raises interest in GLP-1 (9-36)'s biological role. We use human SH-SY5Y neuroblastoma cells and a GLP-1R over-expressing variety (#9), in both undifferentiated and differentiated states, to evaluate the neurotrophic/neuroprotective effects of GLP-1 (9-36) against toxic glutamate exposure and other oxidative stress models (via the MTS, LDH or ROS assays). In addition, we examine GLP-1 (9-36)'s signaling pathways, including cyclic-adenosine monophosphate (cAMP), protein kinase-A (PKA), and 5' adenosine monophosphate-activated protein kinase (AMPK) via the use of ELISA, pharmacological inhibitors, or GLP-1R antagonist. Human HMC3 and mouse IMG microglial cell lines were used to study the anti-inflammatory effects of GLP-1 (9-36) against lipopolysaccharide (LPS) (via ELISA). Finally, we applied GLP-1 (9-36) to primary dissociation cultures challenged with α-synuclein or amyloid-β and assessed survival and morphology via immunochemistry. We demonstrate evidence of GLP-1R, cAMP, PKA, and AMPK-mediated neurotrophic and neuroprotective effects of GLP-1 (9-36). The metabolite significantly reduced IL-6 and TNF-α levels in HMC3 and IMG microglial cells, respectively. Lastly, we show mild but significant effects of GLP-1 (9-36) in primary neuron cultures challenged with α-synuclein or amyloid-β. These studies enhance understanding of GLP-1 (9-36)'s effects on the nervous system and its potential as a primary or complementary treatment in pathological contexts.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glucagon-like peptide-1 (GLP-1) is best known for its insulinotropic action following food intake. Its metabolite, GLP-1 (9-36), was assumed biologically inactive because of low GLP-1 receptor (GLP-1R) affinity and non-insulinotropic properties; however, recent studies contradict this assumption. Increased use of FDA approved GLP-1 analogues for treating metabolic disorders and neurodegenerative diseases raises interest in GLP-1 (9-36)'s biological role. We use human SH-SY5Y neuroblastoma cells and a GLP-1R over-expressing variety (#9), in both undifferentiated and differentiated states, to evaluate the neurotrophic/neuroprotective effects of GLP-1 (9-36) against toxic glutamate exposure and other oxidative stress models (via the MTS, LDH or ROS assays). In addition, we examine GLP-1 (9-36)'s signaling pathways, including cyclic-adenosine monophosphate (cAMP), protein kinase-A (PKA), and 5' adenosine monophosphate-activated protein kinase (AMPK) via the use of ELISA, pharmacological inhibitors, or GLP-1R antagonist. Human HMC3 and mouse IMG microglial cell lines were used to study the anti-inflammatory effects of GLP-1 (9-36) against lipopolysaccharide (LPS) (via ELISA). Finally, we applied GLP-1 (9-36) to primary dissociation cultures challenged with α-synuclein or amyloid-β and assessed survival and morphology via immunochemistry. We demonstrate evidence of GLP-1R, cAMP, PKA, and AMPK-mediated neurotrophic and neuroprotective effects of GLP-1 (9-36). The metabolite significantly reduced IL-6 and TNF-α levels in HMC3 and IMG microglial cells, respectively. Lastly, we show mild but significant effects of GLP-1 (9-36) in primary neuron cultures challenged with α-synuclein or amyloid-β. These studies enhance understanding of GLP-1 (9-36)'s effects on the nervous system and its potential as a primary or complementary treatment in pathological contexts.
2020
Grzanna, Mark W; Secor, Erica J; Fortuno, Lowella V; Au, Angela Y; Frondoza, Carmelita G
In: Cartilage, vol. 11, no. 1, pp. 108–116, 2020, ISSN: 1947-6043.
@article{pmid29938530,
title = {Anti-Inflammatory Effect of Carprofen Is Enhanced by Avocado/Soybean Unsaponifiables, Glucosamine and Chondroitin Sulfate Combination in Chondrocyte Microcarrier Spinner Culture},
author = {Mark W Grzanna and Erica J Secor and Lowella V Fortuno and Angela Y Au and Carmelita G Frondoza},
url = {https://pubmed.ncbi.nlm.nih.gov/29938530/},
doi = {10.1177/1947603518783495},
issn = {1947-6043},
year = {2020},
date = {2020-01-01},
urldate = {2020-01-01},
journal = {Cartilage},
volume = {11},
number = {1},
pages = {108--116},
abstract = {OBJECTIVE: Osteoarthritis is a painful, chronic joint disease affecting man and animals with no known curative therapies. Palliative nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used but they cause adverse side effects prompting the search for safer alternatives. To address this need, we evaluated the anti-inflammatory activity of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) with or without the NSAID carprofen.
DESIGN: Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1β (10 ng/mL) for another 24 hours. Production of PGE, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA.
RESULTS: Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1β induced significant increases in PGE, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone.
CONCLUSIONS: The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
OBJECTIVE: Osteoarthritis is a painful, chronic joint disease affecting man and animals with no known curative therapies. Palliative nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used but they cause adverse side effects prompting the search for safer alternatives. To address this need, we evaluated the anti-inflammatory activity of avocado/soybean unsaponifiables (ASU), glucosamine (GLU), and chondroitin sulfate (CS) with or without the NSAID carprofen.
DESIGN: Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1β (10 ng/mL) for another 24 hours. Production of PGE, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA.
RESULTS: Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1β induced significant increases in PGE, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone.
CONCLUSIONS: The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.
DESIGN: Canine chondrocytes were propagated in microcarrier spinner culture and incubated with (1) control medium, (2) ASU (8.3 µg/mL) + GLU (11 µg/mL) + CS (20 µg/mL) combination for 24 hours; and/or carprofen (40 ng/mL). Cultures were next incubated with control medium alone or IL-1β (10 ng/mL) for another 24 hours. Production of PGE, IL-6, IL-8, and MCP-1 (also known as CCL-2) were measured by ELISA.
RESULTS: Chondrocytes proliferated in microcarrier spinner culture and produced type II collagen and aggrecan. Stimulation with IL-1β induced significant increases in PGE, IL-6, IL-8, and MCP-1 production. The increases in production were suppressed by carprofen as well as [ASU+GLU+CS]. The combination of carprofen and [ASU+GLU+CS] reduced PGE production significantly more than either preparation alone. The inhibitory effect of carprofen on IL-6, IL-8, and MCP-1 production was significantly less than that of [ASU+GLU+CS], whereas the combination did not reduce the production of these molecules significantly more than [ASU+GLU+CS] alone.
CONCLUSIONS: The potentiating effect of [ASU+GLU+CS] on low-dose carprofen was identified in chondrocyte microcarrier spinner cultures. Our results suggest that the combination of low-dose NSAIDs like carprofen with [ASU+GLU+CS] could offer a safe, effective management for joint pain.
2019
Back, Susanne; Necarsulmer, Julie; Whitaker, Leslie R; Coke, Lamarque M; Koivula, Pyry; Heathward, Emily J; Fortuno, Lowella V; Zhang, Yajun; Yeh, Grace C; Baldwin, Heather A; Spencer, Morgan D; Mejias-Aponte, Carlos A; Pickel, James; Hoffman, Alexander F; Spivak, Charles E; Lupica, Carl R; Underhill, Suzanne M; Amara, Susan G; Domanskyi, Andrii; Anttila, Jenni E; Airavaara, Mikko; Hope, Bruce T; Hamra, Kent F; Richie, Christopher T; Harvey, Brandon K
Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats. Journal Article
In: Neuron, vol. 102, no. 1, pp. 105–119, 2019, ISSN: 1097-4199 (Electronic); 0896-6273 (Linking).
@article{Back:2019aac,
title = {Neuron-Specific Genome Modification in the Adult Rat Brain Using CRISPR-Cas9 Transgenic Rats.},
author = {Susanne Back and Julie Necarsulmer and Leslie R Whitaker and Lamarque M Coke and Pyry Koivula and Emily J Heathward and Lowella V Fortuno and Yajun Zhang and Grace C Yeh and Heather A Baldwin and Morgan D Spencer and Carlos A Mejias-Aponte and James Pickel and Alexander F Hoffman and Charles E Spivak and Carl R Lupica and Suzanne M Underhill and Susan G Amara and Andrii Domanskyi and Jenni E Anttila and Mikko Airavaara and Bruce T Hope and Kent F Hamra and Christopher T Richie and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30792150},
doi = {10.1016/j.neuron.2019.01.035},
issn = {1097-4199 (Electronic); 0896-6273 (Linking)},
year = {2019},
date = {2019-04-03},
urldate = {2019-04-03},
journal = {Neuron},
volume = {102},
number = {1},
pages = {105--119},
address = {Molecular Mechanisms of Cellular Stress and Inflammation Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.},
abstract = {Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Historically, the rat has been the preferred animal model for behavioral studies. Limitations in genome modification have, however, caused a lag in their use compared to the bevy of available transgenic mice. Here, we have developed several transgenic tools, including viral vectors and transgenic rats, for targeted genome modification in specific adult rat neurons using CRISPR-Cas9 technology. Starting from wild-type rats, knockout of tyrosine hydroxylase was achieved with adeno-associated viral (AAV) vectors expressing Cas9 or guide RNAs (gRNAs). We subsequently created an AAV vector for Cre-dependent gRNA expression as well as three new transgenic rat lines to specifically target CRISPR-Cas9 components to dopaminergic neurons. One rat represents the first knockin rat model made by germline gene targeting in spermatogonial stem cells. The rats described herein serve as a versatile platform for making cell-specific and sequence-specific genome modifications in the adult brain and potentially other Cre-expressing tissues of the rat.
Campbell, Lee A; Coke, Lamarque M; Richie, Christopher T; Fortuno, Lowella V; Park, Aaron Y; Harvey, Brandon K
Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus. Journal Article
In: Mol Ther, vol. 27, no. 1, pp. 151–163, 2019, ISSN: 1525-0024 (Electronic); 1525-0016 (Linking).
@article{Campbell:2019ab,
title = {Gesicle-Mediated Delivery of CRISPR/Cas9 Ribonucleoprotein Complex for Inactivating the HIV Provirus.},
author = {Lee A Campbell and Lamarque M Coke and Christopher T Richie and Lowella V Fortuno and Aaron Y Park and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/30389355},
doi = {10.1016/j.ymthe.2018.10.002},
issn = {1525-0024 (Electronic); 1525-0016 (Linking)},
year = {2019},
date = {2019-01-02},
urldate = {2019-01-02},
journal = {Mol Ther},
volume = {27},
number = {1},
pages = {151--163},
address = {Intramural Research Program, National Institute on Drug Abuse, Biomedical Research Center, Suite 200, 251 Bayview Boulevard, Baltimore, MD 21224, USA. Electronic address: lee.campbell@nih.gov.},
abstract = {Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Investigators have utilized the CRISPR/Cas9 gene-editing system to specifically target well-conserved regions of HIV, leading to decreased infectivity and pathogenesis in vitro and ex vivo. We utilized a specialized extracellular vesicle termed a "gesicle" to efficiently, yet transiently, deliver Cas9 in a ribonucleoprotein form targeting the HIV long terminal repeat (LTR). Gesicles are produced through expression of vesicular stomatitis virus glycoprotein and package protein as their cargo, thus bypassing the need for transgene delivery, and allowing finer control of Cas9 expression. Using both NanoSight particle and western blot analysis, we verified production of Cas9-containing gesicles by HEK293FT cells. Application of gesicles to CHME-5 microglia resulted in rapid but transient transfer of Cas9 by western blot, which is present at 1 hr, but is undetectable by 24 hr post-treatment. Gesicle delivery of Cas9 protein preloaded with guide RNA targeting the HIV LTR to HIV-NanoLuc CHME-5 cells generated mutations within the LTR region and copy number loss. Finally, we demonstrated that this treatment resulted in reduced proviral activity under basal conditions and after stimulation with pro-inflammatory factors lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha). These data suggest that gesicles are a viable alternative approach to deliver CRISPR/Cas9 technology.
2018
Frondoza, Carmelita G; Fortuno, Lowella V; Grzanna, Mark W; Ownby, Stacy L; Au, Angela Y; Rashmir-Raven, Ann M
α-Lipoic Acid Potentiates the Anti-Inflammatory Activity of Avocado/Soybean Unsaponifiables in Chondrocyte Cultures Journal Article
In: Cartilage, vol. 9, no. 3, pp. 304–312, 2018, ISSN: 1947-6043.
@article{pmid29156944,
title = {α-Lipoic Acid Potentiates the Anti-Inflammatory Activity of Avocado/Soybean Unsaponifiables in Chondrocyte Cultures},
author = {Carmelita G Frondoza and Lowella V Fortuno and Mark W Grzanna and Stacy L Ownby and Angela Y Au and Ann M Rashmir-Raven},
url = {https://pubmed.ncbi.nlm.nih.gov/29156944/},
doi = {10.1177/1947603516686146},
issn = {1947-6043},
year = {2018},
date = {2018-01-01},
urldate = {2018-01-01},
journal = {Cartilage},
volume = {9},
number = {3},
pages = {304--312},
abstract = {Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE) play major roles in the pathogenesis of osteoarthritis (OA). Although current pharmacologic treatments reduce inflammation, their prolonged use is associated with deleterious side effects prompting the search for safer and effective alternative strategies. The present study evaluated whether chondrocyte production of PGE can be suppressed by the combination of avocado/soybean unsaponifiables (ASU) and α-lipoic acid (LA). Design Chondrocytes from articular cartilage of equine joints were incubated for 24 hours with: (1) control media, (2) ASU, (3) LA, or (4) ASU + LA combination. Cells were activated with lipopolysaccharide (LPS), interleukin 1β (IL-1β) or hydrogen peroxide (HO) for 24 hours and supernatants were immunoassayed for PGE. Nuclear factor-kappa B (NF-κB) analyses were performed by immunocytochemistry and Western blot following 1 hour of activation with IL-1β. Results LPS, IL-1β, or HO significantly increased PGE production. ASU or LA alone suppressed PGE production in LPS and IL-1β activated cells. Only LA alone at 2.5 µg/mL was inhibitory in HO-activated chondrocytes. ASU + LA inhibited more than either agent alone in all activated cells. ASU + LA also inhibited the IL-1β induced nuclear translocation of NF-κB. Conclusions The present study provides evidence that chondrocyte PGE production can be inhibited by the combination of ASU + LA more effectively than either ASU or LA alone. Inhibition of PGE production is associated with the suppression of NF-κB translocation. The potent inhibitory effect of ASU + LA on PGE production could offer a potential advantage for a combination anti-inflammatory/antioxidant approach in the management of OA.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Objective Pro-inflammatory mediators such as prostaglandin E-2 (PGE) play major roles in the pathogenesis of osteoarthritis (OA). Although current pharmacologic treatments reduce inflammation, their prolonged use is associated with deleterious side effects prompting the search for safer and effective alternative strategies. The present study evaluated whether chondrocyte production of PGE can be suppressed by the combination of avocado/soybean unsaponifiables (ASU) and α-lipoic acid (LA). Design Chondrocytes from articular cartilage of equine joints were incubated for 24 hours with: (1) control media, (2) ASU, (3) LA, or (4) ASU + LA combination. Cells were activated with lipopolysaccharide (LPS), interleukin 1β (IL-1β) or hydrogen peroxide (HO) for 24 hours and supernatants were immunoassayed for PGE. Nuclear factor-kappa B (NF-κB) analyses were performed by immunocytochemistry and Western blot following 1 hour of activation with IL-1β. Results LPS, IL-1β, or HO significantly increased PGE production. ASU or LA alone suppressed PGE production in LPS and IL-1β activated cells. Only LA alone at 2.5 µg/mL was inhibitory in HO-activated chondrocytes. ASU + LA inhibited more than either agent alone in all activated cells. ASU + LA also inhibited the IL-1β induced nuclear translocation of NF-κB. Conclusions The present study provides evidence that chondrocyte PGE production can be inhibited by the combination of ASU + LA more effectively than either ASU or LA alone. Inhibition of PGE production is associated with the suppression of NF-κB translocation. The potent inhibitory effect of ASU + LA on PGE production could offer a potential advantage for a combination anti-inflammatory/antioxidant approach in the management of OA.
2017
Wallace, Veronica J; Cimbro, Raffaello; Rubio, F Javier; Fortuno, Lowella V; Necarsulmer, Julie C; Koivula, Pyry P; Henderson, Mark J; DeBiase, Lindsay M; Warren, Brandon L; Harvey, Brandon K; Hope, Bruce T
Neurons Internalize Functionalized Micron-Sized Silicon Dioxide Microspheres Journal Article
In: Cell Mol Neurobiol, vol. 37, no. 8, pp. 1487–1499, 2017, ISSN: 1573-6830.
@article{pmid28260198,
title = {Neurons Internalize Functionalized Micron-Sized Silicon Dioxide Microspheres},
author = {Veronica J Wallace and Raffaello Cimbro and F Javier Rubio and Lowella V Fortuno and Julie C Necarsulmer and Pyry P Koivula and Mark J Henderson and Lindsay M DeBiase and Brandon L Warren and Brandon K Harvey and Bruce T Hope},
url = {https://pubmed.ncbi.nlm.nih.gov/28260198/},
doi = {10.1007/s10571-017-0479-z},
issn = {1573-6830},
year = {2017},
date = {2017-11-01},
urldate = {2017-11-01},
journal = {Cell Mol Neurobiol},
volume = {37},
number = {8},
pages = {1487--1499},
abstract = {Microparticles have potential as neuron-specific delivery platforms and devices with many applications in neuroscience, pharmacology, and biomedicine. To date, most literature suggests that neurons are not phagocytic cells capable of internalizing microparticles larger than 0.5 μm. We report that neurons transport fluorescently labeled silica microspheres with diameters of 1-2 μm into neurons in vitro and in rat brain without having overt effects on cell viability. Using flow cytometry, fluorescence-activated cell sorting, and confocal and electron microscopy, we first found that SH-SY5Y human neuroblastoma cells internalized 1-μm silicon microspheres with surface charges of -70 mV (hydroxyl and carboxyl), -30 mV (amino), and +40 mV (ammonio). Uptake was rapid, within 2-4 h, and did not affect cell viability 48 h later. Flow cytometry assays indicate that SH-SY5Y cells internalize 1- and 1.5-μm microspheres at the same rate over a 24-h incubation period. Electron microscopy confirms that SH-SY5Y cells internalize 1-, 1.5-, and 2-μm microspheres. Confocal microscopy demonstrated that primary cortical neurons also internalized 1-, 1.5-, and 2-μm amino microspheres within 4 h. Finally, we injected 1-μm amino microspheres into rat striatum and found microspheres inside neurons. Overall, neurons can internalize microspheres up to 2 μm in diameter with a range of surface chemical groups and charges. These findings allow a host of neuroscience and neuroengineering applications including intracellular microdevices within neurons.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microparticles have potential as neuron-specific delivery platforms and devices with many applications in neuroscience, pharmacology, and biomedicine. To date, most literature suggests that neurons are not phagocytic cells capable of internalizing microparticles larger than 0.5 μm. We report that neurons transport fluorescently labeled silica microspheres with diameters of 1-2 μm into neurons in vitro and in rat brain without having overt effects on cell viability. Using flow cytometry, fluorescence-activated cell sorting, and confocal and electron microscopy, we first found that SH-SY5Y human neuroblastoma cells internalized 1-μm silicon microspheres with surface charges of -70 mV (hydroxyl and carboxyl), -30 mV (amino), and +40 mV (ammonio). Uptake was rapid, within 2-4 h, and did not affect cell viability 48 h later. Flow cytometry assays indicate that SH-SY5Y cells internalize 1- and 1.5-μm microspheres at the same rate over a 24-h incubation period. Electron microscopy confirms that SH-SY5Y cells internalize 1-, 1.5-, and 2-μm microspheres. Confocal microscopy demonstrated that primary cortical neurons also internalized 1-, 1.5-, and 2-μm amino microspheres within 4 h. Finally, we injected 1-μm amino microspheres into rat striatum and found microspheres inside neurons. Overall, neurons can internalize microspheres up to 2 μm in diameter with a range of surface chemical groups and charges. These findings allow a host of neuroscience and neuroengineering applications including intracellular microdevices within neurons.
Richie, Christopher T; Whitaker, Leslie R; Whitaker, Keith W; Necarsulmer, Julie; Baldwin, Heather A; Zhang, Yajun; Fortuno, Lowella; Hinkle, Josh J; Koivula, Pyry; Henderson, Mark J; Sun, Wenzhi; Wang, Kai; Smith, Jeffrey C; Pickel, Jim; Ji, Na; Hope, Bruce T; Harvey, Brandon K
In: J Neurosci Methods, vol. 284, pp. 1–14, 2017, ISSN: 1872-678X (Electronic); 0165-0270 (Linking), (*First paper describing LE-Tg (DIO-iRFP)3Ottc rat. *First paper describing LE-Tg (DIO-iRFP)9Ottc rat.).
@article{Richie:2017aa,
title = {Near-infrared fluorescent protein iRFP713 as a reporter protein for optogenetic vectors, a transgenic Cre-reporter rat, and other neuronal studies.},
author = {Christopher T Richie and Leslie R Whitaker and Keith W Whitaker and Julie Necarsulmer and Heather A Baldwin and Yajun Zhang and Lowella Fortuno and Josh J Hinkle and Pyry Koivula and Mark J Henderson and Wenzhi Sun and Kai Wang and Jeffrey C Smith and Jim Pickel and Na Ji and Bruce T Hope and Brandon K Harvey},
url = {https://www.ncbi.nlm.nih.gov/pubmed/28380331},
doi = {10.1016/j.jneumeth.2017.03.020},
issn = {1872-678X (Electronic); 0165-0270 (Linking)},
year = {2017},
date = {2017-04-02},
urldate = {2017-04-02},
journal = {J Neurosci Methods},
volume = {284},
pages = {1--14},
address = {Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD 21224, United States.},
abstract = {BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.},
note = {*First paper describing LE-Tg (DIO-iRFP)3Ottc rat.
*First paper describing LE-Tg (DIO-iRFP)9Ottc rat.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: The use of genetically-encoded fluorescent reporters is essential for the identification and observation of cells that express transgenic modulatory proteins. Near-infrared (NIR) fluorescent proteins have superior light penetration through biological tissue, but are not yet widely adopted. NEW METHOD: Using the near-infrared fluorescent protein, iRFP713, improves the imaging resolution in thick tissue sections or the intact brain due to the reduced light-scattering at the longer, NIR wavelengths used to image the protein. Additionally, iRFP713 can be used to identify transgenic cells without photobleaching other fluorescent reporters or affecting opsin function. We have generated a set of adeno-associated vectors in which iRFP713 has been fused to optogenetic channels, and can be expressed constitutively or Cre-dependently. RESULTS: iRFP713 is detectable when expressed in neurons both in vitro and in vivo without exogenously supplied chromophore biliverdin. Neuronally-expressed iRFP713 has similar properties to GFP-like fluorescent proteins, including the ability to be translationally fused to channelrhodopsin or halorhodopsin, however, it shows superior photostability compared to EYFP. Furthermore, electrophysiological recordings from iRFP713-labeled cells compared to cells labeled with mCherry suggest that iRFP713 cells are healthier and therefore more stable and reliable in an ex vivo preparation. Lastly, we have generated a transgenic rat that expresses iRFP713 in a Cre-dependent manner. CONCLUSIONS: Overall, we have demonstrated that iRFP713 can be used as a reporter in neurons without the use of exogenous biliverdin, with minimal impact on viability and function thereby making it feasible to extend the capabilities for imaging genetically-tagged neurons in slices and in vivo.
2014
Ownby, Stacy L; Fortuno, Lowella V; Au, Angela Y; Grzanna, Mark W; Rashmir-Raven, Ann M; Frondoza, Carmelita G
Expression of pro-inflammatory mediators is inhibited by an avocado/soybean unsaponifiables and epigallocatechin gallate combination Journal Article
In: J Inflamm (Lond), vol. 11, no. 1, pp. 8, 2014, ISSN: 1476-9255.
@article{pmid24678847,
title = {Expression of pro-inflammatory mediators is inhibited by an avocado/soybean unsaponifiables and epigallocatechin gallate combination},
author = {Stacy L Ownby and Lowella V Fortuno and Angela Y Au and Mark W Grzanna and Ann M Rashmir-Raven and Carmelita G Frondoza},
url = {https://pubmed.ncbi.nlm.nih.gov/24678847/},
doi = {10.1186/1476-9255-11-8},
issn = {1476-9255},
year = {2014},
date = {2014-03-01},
urldate = {2014-03-01},
journal = {J Inflamm (Lond)},
volume = {11},
number = {1},
pages = {8},
abstract = {BACKGROUND: Osteoarthritis (OA) is characterized by inflammation, joint immobility, and pain. Non-pharmacologic agents modulating pro-inflammatory mediator expression offer considerable promise as safe and effective treatments for OA. We previously determined the anti-inflammatory effect of an avocado/soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) combination on prostaglandin E2 (PGE2) production and nuclear factor-kappa B (NF-κB) translocation. The aim of this study was to evaluate the effects of ASU + EGCG on pro-inflammatory gene expression.
FINDINGS: Articular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 μg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1β, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1β, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition.
CONCLUSIONS: We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND: Osteoarthritis (OA) is characterized by inflammation, joint immobility, and pain. Non-pharmacologic agents modulating pro-inflammatory mediator expression offer considerable promise as safe and effective treatments for OA. We previously determined the anti-inflammatory effect of an avocado/soybean unsaponifiables (ASU) and epigallocatechin gallate (EGCG) combination on prostaglandin E2 (PGE2) production and nuclear factor-kappa B (NF-κB) translocation. The aim of this study was to evaluate the effects of ASU + EGCG on pro-inflammatory gene expression.
FINDINGS: Articular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 μg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1β, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1β, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition.
CONCLUSIONS: We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.
FINDINGS: Articular chondrocytes from carpal joints of mature horses were pre-incubated for 24 hours with control media alone or ASU (8.3 μg/mL) + EGCG (40 ng/mL), followed by one hour activation with interleukin-1 beta (IL-1β, 10 ng/mL) and tumor necrosis factor-alpha (TNF-α, 1 ng/mL). Total cellular RNA was isolated and real-time PCR performed to measure IL-1β, TNF-α, interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), and interleukin-8 (IL-8) gene expression. Intracellular localization of NF-κB was analyzed by immunohistochemistry and Western blot. Pre-treatment with ASU + EGCG significantly (P < 0.001) decreased gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 in cytokine-activated chondrocytes. Western blot and immunostaining confirmed NF-κB translocation inhibition.
CONCLUSIONS: We demonstrate that ASU + EGCG inhibits cytokine-induced gene expression of IL-1β, TNF-α, IL-6, COX-2, and IL-8 through modulation of NF-κB. Our results indicate that the activity of ASU + EGCG affects a wide array of inflammatory molecules in addition to decreasing PGE2 synthesis in activated chondrocytes. The responsiveness of chondrocytes to this combination supports its potential utility for the inhibition of joint inflammation.
2005
Kim, Jean; Myers, Allen C; Chen, Lieping; Pardoll, Drew M; Truong-Tran, Quynh-Ai; Lane, Andrew P; McDyer, John F; Fortuno, Lowella; Schleimer, Robert P
Constitutive and inducible expression of b7 family of ligands by human airway epithelial cells Journal Article
In: Am J Respir Cell Mol Biol, vol. 33, no. 3, pp. 280–289, 2005, ISSN: 1044-1549.
@article{pmid15961727,
title = {Constitutive and inducible expression of b7 family of ligands by human airway epithelial cells},
author = {Jean Kim and Allen C Myers and Lieping Chen and Drew M Pardoll and Quynh-Ai Truong-Tran and Andrew P Lane and John F McDyer and Lowella Fortuno and Robert P Schleimer},
url = {https://pubmed.ncbi.nlm.nih.gov/15961727/},
doi = {10.1165/rcmb.2004-0129OC},
issn = {1044-1549},
year = {2005},
date = {2005-09-01},
urldate = {2005-09-01},
journal = {Am J Respir Cell Mol Biol},
volume = {33},
number = {3},
pages = {280--289},
abstract = {Activated T cells have been implicated in chronic rhinosinusitis (CRS) and asthma and physically interact with epithelial cells in the airways. We now report that human airway epithelial cells display significant constitutive cell-surface expression of costimulatory ligands, B7-H1, B7-H2, B7-H3, and B7-DC. Expression of B7-H1 and B7-DC was selectively induced by stimulation of either BEAS2B or primary nasal epithelial cells (PNEC) with interferon (IFN)-gamma (100 ng/ml). The combination of IFN-gamma and tumor necrosis factor-alpha (100 ng/ml) selectively induced expression better than IFN-gamma alone. Fluticasone treatment (10(-7) M) reduced the baseline expression and inhibited the induction of B7-H1 and B7-DC in BEAS2B cells. In vitro exposure of PNEC to IFN-gamma also resulted in selective induction of B7-H1 and B7-DC. Monoclonal antibody blockade of B7-H1 or B7-DC enhanced IFN-gamma expression by purified T cells in co-culture experiments, suggesting that these two B7 homologs inhibit T cell responses at the mucosal surface. Immunohistochemical staining of human sinonasal surgical tissue confirmed the presence of B7-H1, B7-H2, and B7-H3 in the epithelial cell layer, especially in samples from patients diagnosed with Samter's Triad, a severe form of CRS. Real-time PCR analysis of sinonasal tissue revealed elevated levels of B7-H1 and B7-DC in CRS compared with controls. These results demonstrate that epithelial cells express functional B7 costimulatory molecules and that expression of selected B7 family members is inducible in vitro and in vivo. Epithelial B7 homologs could play a role in regulation of lymphocytic activity at mucosal surfaces.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Activated T cells have been implicated in chronic rhinosinusitis (CRS) and asthma and physically interact with epithelial cells in the airways. We now report that human airway epithelial cells display significant constitutive cell-surface expression of costimulatory ligands, B7-H1, B7-H2, B7-H3, and B7-DC. Expression of B7-H1 and B7-DC was selectively induced by stimulation of either BEAS2B or primary nasal epithelial cells (PNEC) with interferon (IFN)-gamma (100 ng/ml). The combination of IFN-gamma and tumor necrosis factor-alpha (100 ng/ml) selectively induced expression better than IFN-gamma alone. Fluticasone treatment (10(-7) M) reduced the baseline expression and inhibited the induction of B7-H1 and B7-DC in BEAS2B cells. In vitro exposure of PNEC to IFN-gamma also resulted in selective induction of B7-H1 and B7-DC. Monoclonal antibody blockade of B7-H1 or B7-DC enhanced IFN-gamma expression by purified T cells in co-culture experiments, suggesting that these two B7 homologs inhibit T cell responses at the mucosal surface. Immunohistochemical staining of human sinonasal surgical tissue confirmed the presence of B7-H1, B7-H2, and B7-H3 in the epithelial cell layer, especially in samples from patients diagnosed with Samter's Triad, a severe form of CRS. Real-time PCR analysis of sinonasal tissue revealed elevated levels of B7-H1 and B7-DC in CRS compared with controls. These results demonstrate that epithelial cells express functional B7 costimulatory molecules and that expression of selected B7 family members is inducible in vitro and in vivo. Epithelial B7 homologs could play a role in regulation of lymphocytic activity at mucosal surfaces.
