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BMC Biotechnology (1)
Experimental Physiology (1)
Experimental and molecular pathology (1)
The Journal of Clinical Investigation (1)
Li, Yonghai (4)
Rostami, Abdolmohamad (2)
Zhang, Guang-Xian (2)
Chu, Niansheng (1)
Ciric, Bogoljub (1)
Curtis, Mark (1)
Fitzgerald, Denise C. (1)
Fong, Peying (1)
Ganta, Suhasini (1)
Gran, Bruno (1)
Hu, Aihua (1)
Jiang, Zhilong (1)
Kaplan, David E (1)
Li, Hongmei (1)
Ma, Cungen (1)
Scholler, Nathalie (1)
Siegel, Donald L (1)
Yang, Jingxian (1)
Yu, Shuo (1)
Zhao, Zhao (1)
Year of Publication
Endogenous surface expression of ΔF508 CFTR mediates cAMP-stimulated Cl− current in CFTR ΔF508/ΔF508 pig thyroid epithelial cells
The Cystic Fibrosis Transmembrane Conductance Regulator, CFTR, is both an anion channel and a regulator of other transport proteins. CFTR gene mutations underlie the human disease, Cystic Fibrosis (CF). The most common CFTR mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic CFTRΔF508/ΔF508 pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP-elevating agents by increasing anion transport, a process hinging on functional CFTR. To assess whether endogenous levels of ΔF508 CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn CFTR+/+ (WT) and CFTRΔF508/ΔF508 (ΔF) pig thyroids and the stimulated, secretory components of short-circuit current (Isc) compared. Surface biotinylation studies assessed the surface presentation of ΔF508 CFTR. Baseline Isc levels of both wt and ΔF pThECs consisted of an amiloride-sensitive component. In ΔF pThECs, this mirrored previous measurements in CFTR−/− (KO) pThECs. Surprisingly, elevation of cAMP transiently increased Isc to peak levels ~ 65% those achieved by wt. In contrast, KO pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508 CFTR expression was ~ 9% that of WT, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ~ 4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508 CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wt and ΔF508-CFTR regulate additional, thyroid transporters, and together coordinate the overall Isc response.
CFTR; mutation; chloride secretion; membrane surface
Validation of glypican-3-specific scFv isolated from paired display/secretory yeast display library
Siegel, Donald L
Kaplan, David E
Glypican-3 (GPC3) is a heparan-sulfate proteoglycan frequently expressed on the cell membrane of malignant hepatocytes in hepatocellular carcinoma. The capacity for screening potential antibodies in vitro using human hepatocellular lines is critical to ensure binding to this highly post-translationally modified glycophosphatidylinositiol-linked protein. We hypothesized that we could utilize a recently described paired display/secretory yeast library to isolate human-derived scFv against glypican-3 for potential diagnostic and/or therapeutic application.
Using two different biotinylated antigen targets, a synthesized 29mer fragment GPC3550-558 and a truncated GPC3368-548 fused with glutathione S-transferase (GST) we enriched the yeast display library to greater than 30% target-specific yeast with both positive selection and depletion of streptavidin- and GST-specific clones. After cloning of scFv cDNA from the enriched sub-library, scFv specificity was validated by ELISA for binding to recombinant protein from prokaryotic and eukaryotic sources and ultimately naturally presented human protein on the cell membrane of human hepatocellular cell lines. Specificity was confirmed using non-expressing cell lines and shRNA knockdown. Ultimately, five unique scFv with affinity EC50 ranging from 5.0-110.9nM were identified.
Using a paired display/secretory yeast library, five novel and unique scFvs for potential humoral or chimeric therapeutic development in human hepatocellular carcinoma were isolated and characterized.
Adult neural stem cells expressing IL-10 confer potent immunomodulation and remyelination in experimental autoimmune encephalitis
Fitzgerald, Denise C.
The Journal of Clinical Investigation
Adult neural stem cells (aNSCs) derived from the subventricular zone of the brain show therapeutic effects in EAE, an animal model of the chronic inflammatory neurodegenerative disease MS; however, the beneficial effects are modest. One critical weakness of aNSC therapy may be an insufficient antiinflammatory effect. Here, we demonstrate that i.v. or i.c.v. injection of aNSCs engineered to secrete IL-10 (IL-10–aNSCs), a potent immunoregulatory cytokine, induced more profound functional and pathological recovery from ongoing EAE than that with control aNSCs. IL-10–aNSCs exhibited enhanced antiinflammatory effects in the periphery and inflammatory foci in the CNS compared with control aNSCs, more effectively reducing myelin damage, a hallmark of MS. When compared with mice treated with control aNSCs, those treated with IL-10–aNSCs demonstrated differentiation of transplanted cells into greater numbers of oligodendrocytes and neurons but fewer astrocytes, thus enhancing exogenous remyelination and neuron/axonal growth. Finally, IL-10–aNSCs converted a hostile environment to one supportive of neurons/oligodendrocytes, thereby promoting endogenous remyelination. Thus, aNSCs engineered to express IL-10 show enhanced ability to induce immune suppression, remyelination, and neuronal repair and may represent a novel approach that can substantially improve the efficacy of neural stem cell–based therapy in EAE/MS.
Inducible IL-23p19 expression in human microglia via p38 MAPK and NF-kB signal pathways
Experimental and molecular pathology
Activated microglia can release a variety of proinflammatory cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). IL-23, a novel proinflammatory cytokine, is required for the induction of experimental autoimmune encephalomyelitis. Previously we demonstrated that IL-23 is expressed in MS lesions and that microglia are one cellular source of IL-23 in MS patients. In the present study we investigated the inducible expression and regulation of p19, a key subunit of IL-23, in human microglia. We demonstrated the inducible expression of IL-23p19 by lipopolysaccharide-stimulated microglial cells. Using signaling pathway-specific inhibitors, we showed that blocking p38 MAP kinase or NF-κB signaling pathway significantly reduced p19 expression in microglia. The regulatory role of p38 MAP kinase in p19 expression was further confirmed by decreased expression in microglia transduced with dominant negative p38. We concluded that the p38 MAP kinase and NF-κB signaling pathways play an important role in regulation of IL-23p19 expression on human microglia, and are thus potential therapeutic targets in the treatment of MS.
IL-23; microglia; multiple sclerosis; experimental autoimmune encephalomyelitis
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