192 results on '"Zheng, Changyu"'
Search Results
152. Adenovirus-mediated p16INK4 gene transfer significantly suppresses human breast cancer growth.
- Author
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Campbell, Islay, Magliocco, Anthony, Moyana, Terence, Zheng, Changyu, and Xiang, Jim
- Subjects
TUMOR suppressor genes ,BREAST cancer ,GENE therapy ,ADENOVIRUS diseases ,CANCER cells - Abstract
The p16
INK4 tumor suppressor gene encodes a protein that inhibits cyclin-dependent kinase 4, and its homologous deletion is common in human breast cancer. p16INK4 gene transfer has been reported to be efficacious in inducing growth inhibition of various human tumors such as brain, lung, prostate, and esophageal cancers. However, the efficiency of the p16INK4 gene with regard to growth inhibition of human breast cancer has not been studied extensively. To examine its tumor-suppressive function and its potential in breast cancer gene therapy, the wild-type p16INK4 gene was expressed in an adenovirus-mediated gene delivery system and introduced into breast cancer cell lines that do not express p16INK4 protein. Expression of the introduced p16INK4 blocked tumor cell entry into the S phase of the cell cycle, induced tumor cell apoptosis, and inhibited tumor cell proliferation both in vitro and in vivo. These results strongly suggest that p16INK4 is a tumor suppressor gene and suggest that it has potential utility in breast cancer gene therapy. [ABSTRACT FROM AUTHOR] more...- Published
- 2000
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153. Effect of clodronate on macrophage depletion and adenoviral-mediated transgene expression in salivary glands.
- Author
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Songlin Wang, Baum, Bruce J., Kagami, Hideaki, Zheng, Changyu, O'Connell, Brian C., and Atkinson, Jane C.
- Subjects
TRANSGENES ,SALIVARY glands ,IMMUNE response ,VIRUSES ,GENE expression ,LIVER ,MACROPHAGES - Abstract
Expression of transgenes from adenoviral vectors is short-lived in salivary glands, in part because of immune responses to the virus and/or transgene product. Previous studies demonstrated that depletion of macrophages with multilamellar liposomes containing clodronate (Cl
2 MBP) increases adenoviral-mediated trans- gene expression in the liver. This technique was tested in salivary glands. Rats were treated with Cl2 MBP-liposomes or control liposomes by femoral vein, intra- peritoneal, or carotid artery injections. Thereafter, a recombinant adenovirus, AdCMVluciferase, was instilled intraductally in submandibular glands (SMGs), or delivered to the liver via femoral vein injection. Marked depletion (>94%) of liver maorophages and increased levels of luciferase activity in the Liver (45-fold higher than controls) were present in animals receiving Cl2 MBP-liposomes. In contrast, the same treatment never depleted more than 41% of SMG macrophages nor increased luciferase activity in SMGs, regardless of the route of administration. In conclusion, while macrophage depletion with Cl2 MBP-liposomes is associated with markedly increased adanoviral-mediated transgene expression, this strategy was ineffective for salivary glands. [ABSTRACT FROM AUTHOR] more...- Published
- 1999
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154. Evaluation of Salivary Gland Acinar and Ductal Cell-Specific Promoters In Vivo with Recombinant Adenoviral Vectors
- Author
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Zheng, Changyu, Hoque, A.T.M. Shamsul, Braddon, Virginia R., Baum, Bruce J., and O'Connell, Brian C.
- Abstract
Adenoviral vectors efficiently deliver exogenous genes to salivary glands. There are two general epithelial cell types, with very different functions, in salivary glands - acinar and ductal. To determine if gene expression can be restricted in vivo to either general cell type using a relatively cell/tissue-specific promoter in conjunction with adenovirus-mediated gene transfer, we tested the human amylase and kallikrein promoters. For initial studies the sensitive reporter gene luciferase was used in two adenoviral constructs. The adenovirus AdAMY-luc contains the human salivary gland amylase promoter (-1003 to +2)(AMY1C) and AdKALL-luc contains the human tissue kallikein promoter (-315 to -1)(KLK1). The adenovirus AdKALL-hAQP1 was also used to test a therapeutic gene, human aquaporin-1 (hAQP1), potentially of importance in treating surviving ductal cells in irradiation-damaged glands. Luciferase expression after AdAMY-luc delivery in vivo directly to the parotid, submandibular, and sublingual glands, as well as to the lungs, and intravenously via the femoral vein, was restricted to the three salivary glands and the pancreas. AdKALL-luc delivery via the same routes resulted in a more general distribution of luciferase expression, although greatest luciferase activity was seen in salivary glands and lung. Luciferase activity after AdAMY-luc delivery was proportionally greater (~14-fold) in acinar cells, whereas luciferase activity after AdKALL-luc delivery was proportionally greater (~9-fold) in ductal cells. The expression of hAQP1 after AdKALL-hAQP1 gene transfer was mainly observed in ductal cells in vivo. AdKALL-hAQP1 was as useful as AdCMV-hAQP1 in increasing salivary flow rates of irradiated rats. This study demonstrates that adenoviral vectors containing the relatively cell/tissue-specific AMY1C or KLK1 promoters may be useful for targeting therapeutic gene expression in salivary glands. more...
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- 2001
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155. 877. Long-Term Transgene Expression with a Second Generation Hybrid AdenoRetroviral Vector
- Author
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Zheng, Changyu, Zhang, Weitan, Vitolo, Joseph M., and Baum, Bruce J.
- Subjects
- *
ADENOVIRUSES - Abstract
An abstract of the article "Long-Term Transgene Expression with a Second Generation Hybrid AdenoRetroviral Vector," by Changyu Zheng, Weitan Zhang, Joseph M. Vitolo, Bruce J. Baum and colleagues is presented. more...
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- 2005
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156. STIM2 enhances receptor-stimulated Ca2+signaling by promoting recruitment of STIM1 to the endoplasmic reticulum–plasma membrane junctions
- Author
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Ong, Hwei Ling, de Souza, Lorena Brito, Zheng, Changyu, Cheng, Kwong Tai, Liu, Xibao, Goldsmith, Corinne M., Feske, Stefan, and Ambudkar, Indu S.
- Abstract
STIM2 increases physiological responses to agonist stimulation by promoting clustering of STIM1 and activation of Orai1.
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- 2015
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157. Gastric Cancer Actively Remodels Mechanical Microenvironment to Promote Chemotherapy Resistance via MSCs-Mediated Mitochondrial Transfer.
- Author
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He X, Zhong L, Wang N, Zhao B, Wang Y, Wu X, Zheng C, Ruan Y, Hou J, Luo Y, Yin Y, He Y, Xiang AP, and Wang J
- Abstract
Chemotherapy resistance is the main reason of treatment failure in gastric cancer (GC). However, the mechanism of oxaliplatin (OXA) resistance remains unclear. Here, we demonstrate that extracellular mechanical signaling plays crucial roles in OXA resistance within GC. We selected OXA-resistant GC patients and analyzed tumor tissues by single-cell sequencing, and found that the mitochondrial content of GC cells increased in a biosynthesis-independent manner. Moreover, we found that the increased mitochondria of GC cells were mainly derived from mesenchymal stromal cells (MSCs), which could repair the mitochondrial function and reduce the levels of mitophagy in GC cells, thus leading to OXA resistance. Furthermore, we investigated the underlying mechanism and found that mitochondrial transfer was mediated by mechanical signals of the extracellular matrix (ECM). After OXA administration, GC cells actively secreted ECM in the tumor microenvironment (TEM), increasing matrix stiffness of the tumor tissues, which promoted mitochondria to transfer from MSCs to GC cells via microvesicles (MVs). Meanwhile, inhibiting the mechanical-related RhoA/ROCK1 pathway could alleviate OXA resistance in GC cells. In summary, these results indicate that matrix stiffness could be used as an indicator to identify chemotherapy resistance, and targeting mechanical-related pathway could effectively alleviate OXA resistance and improve therapeutic efficacy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.) more...
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- 2024
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158. Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's disease.
- Author
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Gupta S, Yamada E, Nakamura H, Perez P, Pranzatelli TJ, Dominick K, Jang SI, Abed M, Martin D, Burbelo P, Zheng C, French B, Alevizos I, Khavandgar Z, Beach M, Pelayo E, Walitt B, Hasni S, Kaplan MJ, Tandon M, Magone MT, Kleiner DE, Chiorini JA, Baer A, and Warner BM more...
- Subjects
- Humans, Female, Interferons, Piperidines pharmacology, Piperidines therapeutic use, Middle Aged, Male, Pyrimidines pharmacology, Pyrimidines therapeutic use, Adult, Inflammation, Pyrroles pharmacology, Pyrroles therapeutic use, Epithelial Cells drug effects, Sjogren's Syndrome drug therapy, Sjogren's Syndrome immunology, Janus Kinase Inhibitors pharmacology, Janus Kinase Inhibitors therapeutic use, Signal Transduction drug effects, Janus Kinases metabolism, STAT Transcription Factors metabolism, Leukocytes, Mononuclear drug effects, Leukocytes, Mononuclear immunology, Leukocytes, Mononuclear metabolism, Salivary Glands, Minor immunology
- Abstract
Objectives: Inflammatory cytokines that signal through the Janus kinases-signal transducer and activator of transcription (JAK-STAT) pathway, especially interferons (IFNs), are implicated in Sjögren's disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signalling and the effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been fully investigated., Methods: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single-cell (sc) RNA sequencing (RNAseq), immunofluorescence (IF) microscopy and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi., Results: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (eg, focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell type-specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFN-β, which were normalised by JAKi without cytotoxicity., Conclusions: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalises this aberrant signalling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a phase Ib/IIa randomised controlled trial to treat SjD with tofacitinib was initiated., Competing Interests: Competing interests: BMW has Cooperative Research Award and Development Agreements (CRADA) from Pfizer and Mitobridge (a subsidiary of Astellas Pharma). NIAMS has CRADAs with AstraZeneca and Bristol Myers Squibb. These CRADA did not financially support the experimental results presented herein., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.) more...
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- 2024
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159. GZMK+CD8+ T cells Target A Specific Acinar Cell Type in Sjögren's Disease.
- Author
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Pranzatelli TJF, Perez P, Ku A, Matuck B, Huynh K, Sakai S, Abed M, Jang SI, Yamada E, Dominick K, Ahmed Z, Oliver A, Wasikowski R, Easter QT, Baer AN, Pelayo E, Khavandgar Z, Kleiner DE, Magone MT, Gupta S, Lessard C, Farris AD, Burbelo PD, Martin D, Morell RJ, Zheng C, Rachmaninoff N, Maldonado-Ortiz J, Qu X, Aure M, Dezfulian MH, Lake R, Teichmann S, Barber DL, Tsoi LC, Sowalsky AG, Tyc KM, Liu J, Gudjonsson J, Byrd KM, Johnson PLF, Chiorini JA, and Warner BM more...
- Abstract
Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2 - seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK +CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease. more...
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- 2024
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160. Association of G protein-coupled receptor 78 with salivary dysfunction in male Sjögren's patients.
- Author
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Tanaka T, Guimaro MC, Nakamura H, Perez P, Ji Y, Michael DG, Afione SA, Zheng C, Goldsmith C, Swaim WD, Pedersen AML, and Chiorini JA
- Subjects
- Animals, Female, Humans, Male, Mice, Case-Control Studies, Lysosomes metabolism, Mice, Transgenic, Salivary Glands, Minor metabolism, Apoptosis, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Sjogren's Syndrome metabolism, Sjogren's Syndrome genetics
- Abstract
Objective: Sjögren's disease (SjD) has a strong sex bias, suggesting an association with sex hormones. Male SjD represents a distinct subset of the disease, but the pathogenic mechanisms of male SjD is poorly characterized. The aim of this study is to identify initiating events related to the development of gland hypofunction and autoimmunity in male SjD patients., Materials and Methods: Human minor salivary glands were transcriptomically analyzed with microarrays to detect differentially expressed genes in male SjD patients. Identified genes were tested on their involvement in the disease using conditional transgenic mice and gene-overexpressing cells., Results: GPR78, an orphan G protein-coupled receptor, was overexpressed in the salivary glands of male SjD patients compared with male healthy controls and female SjD patients. Male GPR78 transgenic mice developed salivary gland hypofunction with increased epithelial apoptosis, which was not seen in control or female transgenic mice. In cell culture, GPR78 overexpression decreased lysosomal integrity, leading to caspase-dependent apoptotic cell death. GPR78-induced cell death in vitro was inhibited by treatment with estradiol., Conclusion: GPR78 overexpression can induce apoptosis and salivary gland hypofunction in male mice through lysosomal dysfunction and increased caspase-dependent apoptosis in salivary gland epithelium, which may drive disease in humans., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Oral Diseases published by Wiley Periodicals LLC.) more...
- Published
- 2024
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161. Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's Disease.
- Author
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Gupta S, Yamada E, Nakamura H, Perez P, Pranzatelli TJF, Dominick K, Jang SI, Abed M, Martin D, Burbelo P, Zheng C, French B, Alevizos I, Khavandgar Z, Beach M, Pelayo E, Walitt B, Hasni S, Kaplan MJ, Tandon M, Teresa Magone M, Kleiner DE, Chiorini JA, Baer AN, and Warner BM more...
- Abstract
Objectives: Inflammatory cytokines that signal through the JAK- STAT pathway, especially interferons (IFNs), are implicated in Sjögren's Disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signaling and effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been reported., Methods: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single cell (sc) RNA sequencing (RNAseq), immunofluorescence microscopy (IF), and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi., Results: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (e.g., focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell-type specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFNβ, which were normalized by JAKi without cytotoxicity., Conclusions: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalizes this aberrant signaling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a Phase Ib/IIa randomized controlled trial to treat SjD with tofacitinib was initiated., Competing Interests: Potential Conflicts of Interest: BMW has Cooperative Research Award and Development Agreements [CRADA] from Pfizer, Inc., and Mitobridge, Inc. (A subsidiary of Astellas Pharma, Inc.). NIAMS has CRADAs with Astra Zeneca and Bristol Myers Squibb. These CRADA did not financially support the experimental results presented herein. more...
- Published
- 2023
- Full Text
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162. Gingipain of Porphyromonas gingivalis manipulates M1 macrophage polarization through C5a pathway.
- Author
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Hou Y, Yu H, Liu X, Li G, Pan J, Zheng C, and Yu W
- Subjects
- Adhesins, Bacterial isolation & purification, Animals, Antigens, CD metabolism, Cysteine Endopeptidases isolation & purification, Cytokines metabolism, Down-Regulation drug effects, Escherichia coli metabolism, Gingipain Cysteine Endopeptidases, Inflammation pathology, Inflammation Mediators metabolism, Lipopolysaccharides pharmacology, Macrophages drug effects, Mice, RAW 264.7 Cells, Up-Regulation drug effects, Adhesins, Bacterial pharmacology, Cell Polarity drug effects, Complement C5a metabolism, Cysteine Endopeptidases pharmacology, Macrophages cytology, Macrophages metabolism
- Abstract
Gingipains secreted by Porphyromonas gingivalis (P. gingivalis, Pg) play an important role in maintaining macrophage infiltrating. And, this study is to evaluate effects of gingipain on M1 macrophage polarization after exposure to Porphyromonas gingivalis (P. gingivalis, Pg) and if these effects are through complement component 5a (C5a) pathway. Mouse RAW264.7 macrophages were exposed to gingipain extracts, Escherichia coli lipopolysaccharides (Ec-LPS), Pg-LPS with or without the C5aR antagonist: PMX-53 for 24 h. Then, gene expressions and protein of IL-12, IL-23, iNOS, IL-10, TNF-α, IL-1β, and IL-6 were determined by qRT-PCR and ELISA assays. Surface markers CD86 for M1 and CD206 for M2 were also evaluated by flow cytometry. The results show that gingipain extracts alone increased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1β, and IL-6, but not IL-10. Gingipain extracts plus Ec-LPS decreased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1β, and IL-6 in which Ec-LPS induced increase. For gingipain extracts plus Pg-LPS-treated RAW264.7, macrophages, gingipain extracts enhanced expressions of IL-12 and IL-23 in which Pg-LPS induced increase, but not iNOS and IL-10 while gingipain extracts decreased expressions of TNF-α, IL-1β, and IL-6 in which Pg-LPS induced increase. Interestingly, PMX-53 increased expressions of IL-12, IL-23, and iNOS when RAW264.7 macrophages were treated with gingipain extracts plus Ec-LPS or Pg-LPS and PMX-53, while PMX-53 decreased expressions of TNF-α, IL-1β, and IL-6. Changes of CD86-positive macrophages were consistent with cytokine changes. Our data indicate that gingipain is a critical regulator, more like a promoter to manipulate M1 macrophage polarization in order to benefit P. gingivalis infection through the C5a pathway. more...
- Published
- 2017
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163. Gene Therapy of Salivary Diseases.
- Author
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Baum BJ, Afione S, Chiorini JA, Cotrim AP, Goldsmith CM, and Zheng C
- Subjects
- Adenoviruses, Human genetics, Animals, Cell Line, Dependovirus, Disease Models, Animal, Gene Order, Gene Transfer Techniques, Genetic Vectors administration & dosage, Genetic Vectors genetics, Genetic Vectors isolation & purification, Humans, Parvovirinae genetics, Salivary Glands metabolism, Transduction, Genetic, Transgenes, Genetic Therapy methods, Salivary Gland Diseases genetics, Salivary Gland Diseases therapy
- Abstract
For many years, our research group worked to develop gene transfer approaches for salivary gland disorders that lacked effective conventional therapy. The purpose of this chapter is to describe and update key methods used in this process. As described in our original chapter from the 2010 volume, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model. more...
- Published
- 2017
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164. Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice.
- Author
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Lai Z, Yin H, Cabrera-Pérez J, Guimaro MC, Afione S, Michael DG, Glenton P, Patel A, Swaim WD, Zheng C, Nguyen CQ, Nyberg F, and Chiorini JA
- Subjects
- Adult, Aged, Animals, Aquaporin 5 metabolism, Bone Morphogenetic Protein 6 genetics, Bone Morphogenetic Protein 6 metabolism, Cell Line, Cell Membrane Permeability, Down-Regulation, Female, Gene Silencing, Humans, Lacrimal Apparatus metabolism, Male, Mice, Inbred C57BL, Mice, Inbred NOD, Middle Aged, Salivary Glands metabolism, Sjogren's Syndrome genetics, Water metabolism, Young Adult, Aquaporin 1 genetics, Aquaporin 5 genetics, Genetic Therapy, Sjogren's Syndrome therapy
- Abstract
Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Currently, no effective treatments exist for Sjögren's syndrome, and there is a limited understanding of the physiological mechanisms associated with xerostomia and hyposalivation. The present work revealed that aquaporin 5 expression, a water channel critical for salivary gland fluid secretion, is regulated by bone morphogenetic protein 6. Increased expression of this cytokine is strongly associated with the most common symptom of primary Sjögren's syndrome, the loss of salivary gland function. This finding led us to develop a therapy in the treatment of Sjögren's syndrome by increasing the water permeability of the gland to restore saliva flow. Our study demonstrates that the targeted increase of gland permeability not only resulted in the restoration of secretory gland function but also resolved the hallmark salivary gland inflammation and systemic inflammation associated with disease. Secretory function also increased in the lacrimal gland, suggesting this local therapy could treat the systemic symptoms associated with primary Sjögren's syndrome. more...
- Published
- 2016
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165. Effects of human vascular endothelial growth factor on reparative dentin formation.
- Author
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Zhang J, Liu X, Yu W, Zhang Y, Shi C, Ni S, Liu Q, Li X, Sun Y, Zheng C, and Sun H
- Subjects
- Animals, Calcification, Physiologic drug effects, Cell Differentiation drug effects, Child, Dental Pulp cytology, Gene Expression Profiling, Gene Expression Regulation drug effects, Humans, Male, Rats, Wistar, Tooth, Deciduous cytology, Dentin metabolism, Vascular Endothelial Growth Factor A pharmacology, Wound Healing drug effects
- Abstract
It is a challenge for dentists to save dental pulp in patients with pulp disease without resorting to root canal therapy. Formation of tertiary dentin to maintain pulp vitality is a key odontoblast response to dental pulp injury. Vascular endothelial growth factor (VEGF) is the most potent angiogenic and vasculogenic factor involved in tertiary dentin formation. It was hypothesized that VEGF may be used to treat pulp diseases such as pulpitis. To explore this hypothesis, the first step was to assess whether VEGF affects dental pulp cells to promote reparative dentin formation. In the current study, an AdCMV‑hVEGF vector was constructed to deliver hVEGF into dental pulp cells of exfoliated deciduous teeth (hDPCs) in vitro and dental pulp cells in a rat model in vivo. The collected data clearly demonstrated that hVEGF increased alkaline phosphatase and mineralization by enzymatic activity. RT‑qPCR data demonstrated that hVEGF significantly increased the expression levels of genes commonly involved in osteogenesis/odontogenesis. Data from the in vivo assays indicated that hVEGF enhanced pulp cell proliferation and neovascularization, and markedly increased formation of reparative dentin in dental pulp. The in vitro and in vivo data suggest that hVEGF may have potential clinical applications, thus may aid in the development of novel treatment strategies for dental pulpitis. more...
- Published
- 2016
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166. Essential role of carbonic anhydrase XII in secretory gland fluid and HCO3 (-) secretion revealed by disease causing human mutation.
- Author
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Hong JH, Muhammad E, Zheng C, Hershkovitz E, Alkrinawi S, Loewenthal N, Parvari R, and Muallem S
- Subjects
- Adolescent, Animals, Carbonic Anhydrases genetics, Cells, Cultured, Child, Chloride-Bicarbonate Antiporters metabolism, Glycosylation, HEK293 Cells, HeLa Cells, Homozygote, Humans, Mice, Pancreatic Ducts cytology, Pancreatic Juice metabolism, Phenotype, Protein Processing, Post-Translational, Salivary Glands cytology, Xerostomia metabolism, Xerostomia pathology, Young Adult, Bicarbonates metabolism, Carbonic Anhydrases metabolism, Mutation, Missense, Pancreatic Ducts metabolism, Saliva metabolism, Salivary Glands metabolism, Xerostomia genetics
- Abstract
Key Points: Fluid and HCO3 (-) secretion is essential for all epithelia; aberrant secretion is associated with several diseases. Carbonic anhydrase XII (CA12) is the key carbonic anhydrase in epithelial fluid and HCO3 (-) secretion and works by activating the ductal Cl(-) -HCO3 (-) exchanger AE2. Delivery of CA12 to salivary glands increases salivation in mice and of the human mutation CA12(E143K) markedly inhibits it. The human mutation CA12(E143K) causes disease due to aberrant CA12 glycosylation, and misfolding resulting in loss of AE2 activity., Abstract: Aberrant epithelial fluid and HCO3 (-) secretion is associated with many diseases. The activity of HCO3 (-) transporters depends of HCO3 (-) availability that is determined by carbonic anhydrases (CAs). Which CAs are essential for epithelial function is unknown. CA12 stands out since the CA12(E143K) mutation causes salt wasting in sweat and dehydration in humans. Here, we report that expression of CA12 and of CA12(E143K) in mice salivary glands respectively increased and prominently inhibited ductal fluid secretion and salivation in vivo. CA12 markedly increases the activity and is the major HCO3 (-) supplier of ductal Cl(-) -HCO3 (-) exchanger AE2, but not of NBCe1-B. The E143K mutation alters CA12 glycosylation at N28 and N80, resulting in retention of the basolateral CA12 in the ER. Knockdown of AE2 and of CA12 inhibited pancreatic and salivary gland ductal AE2 activity and fluid secretion. Accordingly, patients homozygous for the CA12(E143K) mutation have a dry mouth, dry tongue phenotype. These findings reveal an unsuspected prominent role of CA12 in epithelial function, explain the disease and call for caution in the use of CA12 inhibitors in cancer treatment., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.) more...
- Published
- 2015
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167. STIM2 enhances receptor-stimulated Ca²⁺ signaling by promoting recruitment of STIM1 to the endoplasmic reticulum-plasma membrane junctions.
- Author
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Ong HL, de Souza LB, Zheng C, Cheng KT, Liu X, Goldsmith CM, Feske S, and Ambudkar IS
- Subjects
- Acinar Cells metabolism, Analysis of Variance, Animals, Bacterial Proteins, Blotting, Western, Gene Knockdown Techniques, Genetic Vectors genetics, HEK293 Cells, Humans, Luminescent Proteins, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Mutagenesis, Site-Directed, RNA, Small Interfering genetics, Saliva cytology, Sequence Analysis, DNA, Stromal Interaction Molecule 1, Stromal Interaction Molecule 2, Calcium Signaling physiology, Cell Adhesion Molecules metabolism, Cell Membrane metabolism, Endoplasmic Reticulum metabolism, Membrane Proteins metabolism, Neoplasm Proteins metabolism
- Abstract
A central component of receptor-evoked Ca(2+) signaling is store-operated Ca(2+) entry (SOCE), which is activated by the assembly of STIM1-Orai1 channels in endoplasmic reticulum (ER) and plasma membrane (PM) (ER-PM) junctions in response to depletion of ER Ca(2+). We report that STIM2 enhances agonist-mediated activation of SOCE by promoting STIM1 clustering in ER-PM junctions at low stimulus intensities. Targeted deletion of STIM2 in mouse salivary glands diminished fluid secretion in vivo and SOCE activation in dispersed salivary acinar cells stimulated with low concentrations of muscarinic receptor agonists. STIM2 knockdown in human embryonic kidney (HEK) 293 cells diminished agonist-induced Ca(2+) signaling and nuclear translocation of NFAT (nuclear factor of activated T cells). STIM2 lacking five carboxyl-terminal amino acid residues did not promote formation of STIM1 puncta at low concentrations of agonist, whereas coexpression of STIM2 with STIM1 mutant lacking the polybasic region STIM1ΔK resulted in co-clustering of both proteins. Together, our findings suggest that STIM2 recruits STIM1 to ER-PM junctions at low stimulus intensities when ER Ca(2+) stores are mildly depleted, thus increasing the sensitivity of Ca(2+) signaling to agonists., (Copyright © 2015, American Association for the Advancement of Science.) more...
- Published
- 2015
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168. Assessment of transfection of AdCMV-EGFP to rat submandibular gland cells.
- Author
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Liu C, Miao L, Sun W, Wu X, Yan F, Sun H, and Zheng C
- Subjects
- Animals, Cell Proliferation, Genetic Therapy, Rats, Rats, Wistar, Submandibular Gland cytology, Submandibular Gland enzymology, alpha-Amylases metabolism, Adenoviridae genetics, Genetic Vectors genetics, Green Fluorescent Proteins genetics, Promoter Regions, Genetic genetics, Submandibular Gland metabolism, Transfection methods
- Abstract
We evaluated the efficiency of transfecting adenoviral vectors encoding enhanced green fluorescent protein (AdCMV-EGFP) into rat submandibular gland cells and the effects of gene transfer on cell proliferation and secretory function. Isolated submandibular gland cells were transfected with different titers (or multiplicity of infection, MOI) of AdCMV-EGFP. The transfection efficiency was evaluated by quantifying EGFP-positive cells by inverted fluorescence microscopy, cell proliferation by MTT assay, and cell secretory activity by measuring α-amylase in culture medium. A transfection efficiency of up to 70.8% was achieved in submandibular gland cells. MTT assay showed that increased viral titers resulted in significant inhibition of cell proliferation, which occurs on day 5 post-transfection. Simultaneously, the amylase levels started to reduce with a significant decrease on day 7 after transfection. The results show that AdCMV-EGFP transfection of submandibular gland cells at higher MOI results in cytotoxicity, decreased cell proliferation, and secretory function. However, the lower adenoviral titers (e.g., 200 particles/cell) could be an efficient and safe labeling tool for gene transfer to submandibular gland cells. more...
- Published
- 2015
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169. Antitumor effect of TRAIL on oral squamous cell carcinoma using magnetic nanoparticle-mediated gene expression.
- Author
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Miao L, Liu C, Ge J, Yang W, Liu J, Sun W, Yang B, Zheng C, Sun H, and Hu Q
- Subjects
- Animals, Apoptosis genetics, Carcinoma, Squamous Cell pathology, Carcinoma, Squamous Cell therapy, Cell Line, Tumor, Gene Expression, Humans, Male, Mice, Mice, Inbred BALB C, Mouth Neoplasms pathology, Mouth Neoplasms therapy, Polyethyleneimine chemistry, Promoter Regions, Genetic genetics, Telomerase genetics, Transfection, Carcinoma, Squamous Cell genetics, Drug Carriers chemistry, Genetic Therapy methods, Magnetite Nanoparticles chemistry, Mouth Neoplasms genetics, TNF-Related Apoptosis-Inducing Ligand genetics
- Abstract
We developed a new magnetic nanovector to improve the efficiency and targeting of transgene therapy for oral squamous cell carcinoma (OSCC). Positively charged polymer PEI-modified Fe(3)O(4) magnetic nanoparticles were tested as gene transfer vectors in the presence of a magnetic field. The Fe(3)O(4) nanoparticles were prepared by a co-precipitation method and had good dispersibility in water. These nanoparticles modified by PEI were combined with negatively charged pACTERT-EGFP via electrostatic interaction. The transfection efficiency of the magnetic nano-gene vector with the magnetic field was determined by a fluorescence-inverted microscope and flow cytometry. The results showed significant improvement compared with the control group (p < 0.05). The magnetic complexes also exhibited up to 6-times higher transfection efficiency compared with commonly used PEI or lipofectin. On the basis of these results, the antitumor effect with suicide gene therapy using pACTERT-TRAIL in vitro and vivo was evaluated. In vitro apoptosis was determined with the Annexin V-FITC Apoptosis Detection Kit. The results suggested that PEI-modified Fe(3)O(4) nanoparticles could mediate the killing of Tca83 cells. Furthermore, treatment with pACTERT-TRAIL delivered by magnetic nanoparticles showed a significant cytostatic effect through the induction of apoptosis in a xenograft model. This indicates that magnetic nano-gene vectors could improve the transgene efficiency for Tca83 cells and could exhibit antitumor functions with the plasmid pACTERT-TRAIL. This may be a new way to treat OSCC. more...
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- 2014
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170. Efficiently engineered cell sheet using a complex of polyethylenimine-alginate nanocomposites plus bone morphogenetic protein 2 gene to promote new bone formation.
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Jin H, Zhang K, Qiao C, Yuan A, Li D, Zhao L, Shi C, Xu X, Ni S, Zheng C, Liu X, Yang B, and Sun H
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- Animals, Bone Morphogenetic Protein 2 metabolism, Bone Substitutes chemical synthesis, Equipment Design, Equipment Failure Analysis, Glucuronic Acid chemistry, Hexuronic Acids chemistry, Male, Nanocomposites chemistry, Nanocomposites ultrastructure, Osteogenesis drug effects, Osteogenesis physiology, Rats, Rats, Wistar, Skull Fractures diagnosis, Tissue Engineering instrumentation, Treatment Outcome, Alginates chemistry, Bone Morphogenetic Protein 2 genetics, Mesenchymal Stem Cell Transplantation instrumentation, Mesenchymal Stem Cells physiology, Polyethyleneimine chemistry, Skull Fractures therapy, Tissue Scaffolds
- Abstract
Regeneration of large bone defects is a common clinical problem. Recently, stem cell sheet has been an emerging strategy in bone tissue engineering. To enhance the osteogenic potential of stem cell sheet, we fabricated bone morphogenetic protein 2 (BMP-2) gene-engineered cell sheet using a complex of polyethylenimine-alginate (PEI-al) nanocomposites plus human BMP-2 complementary(c)DNA plasmid, and studied its osteogenesis in vitro and in vivo. PEI-al nanocomposites carrying BMP-2 gene could efficiently transfect bone marrow mesenchymal stem cells. The cell sheet was made by culturing the cells in medium containing vitamin C for 10 days. Assays on the cell culture showed that the genetically engineered cells released the BMP-2 for at least 14 days. The expression of osteogenesis-related gene was increased, which demonstrated that released BMP-2 could effectively induce the cell sheet osteogenic differentiation in vitro. To further test the osteogenic potential of the cell sheet in vivo, enhanced green fluorescent protein or BMP-2-producing cell sheets were treated on the cranial bone defects. The results indicated that the BMP-2-producing cell sheet group was more efficient than other groups in promoting bone formation in the defect area. Our results suggested that PEI-al nanocomposites efficiently deliver the BMP-2 gene to bone marrow mesenchymal stem cells and that BMP-2 gene-engineered cell sheet is an effective way for promoting bone regeneration. more...
- Published
- 2014
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171. Irbit mediates synergy between ca(2+) and cAMP signaling pathways during epithelial transport in mice.
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Park S, Shcheynikov N, Hong JH, Zheng C, Suh SH, Kawaai K, Ando H, Mizutani A, Abe T, Kiyonari H, Seki G, Yule D, Mikoshiba K, and Muallem S
- Subjects
- Animals, Antiporters metabolism, Biological Transport, Cyclic AMP-Dependent Protein Kinases physiology, Cystic Fibrosis Transmembrane Conductance Regulator physiology, Epithelium metabolism, Inositol 1,4,5-Trisphosphate biosynthesis, Inositol 1,4,5-Trisphosphate Receptors physiology, Mice, Pancreatic Ducts metabolism, Phosphorylation, Salivary Ducts metabolism, Sulfate Transporters, Adenosylhomocysteinase physiology, Calcium metabolism, Cyclic AMP physiology, Signal Transduction physiology
- Abstract
Background & Aims: The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts., Methods: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses., Results: Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation., Conclusions: Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.) more...
- Published
- 2013
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172. Antitumor effect of human TRAIL on adenoid cystic carcinoma using magnetic nanoparticle-mediated gene expression.
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Miao L, Zhang K, Qiao C, Jin X, Zheng C, Yang B, and Sun H
- Subjects
- Apoptosis, Humans, Microscopy, Electron, Transmission, Spectroscopy, Fourier Transform Infrared, Carcinoma, Adenoid Cystic physiopathology, Gene Expression, Magnetics, Nanoparticles, TNF-Related Apoptosis-Inducing Ligand physiology
- Abstract
To overcome treatment limitations of adenoid cystic carcinoma, we developed a novel treatment combining gene therapy and nanotechnology. In this study, we created a plasmid, pACTERT-TRAIL, which used the human telomerase reverse transcriptase promoter, a tumor-specific promoter, to drive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). A Fe(3)O(4)-PEI-plasmid complex (FPP) was generated, in which the iron oxide nanoparticles were modified by positively charged polyethylenimine (PEI) to enable them to carry the negatively charged plasmid. In vitro transfection assays showed that efficiency of magnetofection (i.e., FPP transfection) was sixfold higher compared to PEI alone or Lipofectamine 2000 (hereafter referred to as lipofectin) (P < 0.05). Importantly, apoptotic assays demonstrated that FPP-mediated TRAIL gene transfer could efficiently induce apoptosis of SACC-83 cells in vitro and in vivo. These results demonstrate that magnetofection of the plasmids driven by the tumor-specific promoter hTERT provides an effective way to deliver therapeutic genes for the treatment of adenoid cystic carcinoma in the future., From the Clinical Editor: In this novel study addressing adenoid cystic carcinoma, the authors created a plasmid to drive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Following that, a Fe(3)O(4)-PEI-plasmid complex (FPP) was generated, in which the iron oxide nanoparticles were modified by positively charged polyethylenimine (PEI) enabling them to carry the negatively charged plasmid, giving rise to sixfold higher transfection rates compared to standard technology., (Copyright © 2013 Elsevier Inc. All rights reserved.) more...
- Published
- 2013
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173. Using poly(lactic-co-glycolic acid) microspheres to encapsulate plasmid of bone morphogenetic protein 2/polyethylenimine nanoparticles to promote bone formation in vitro and in vivo.
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Qiao C, Zhang K, Jin H, Miao L, Shi C, Liu X, Yuan A, Liu J, Li D, Zheng C, Zhang G, Li X, Yang B, and Sun H
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- Animals, Bone Morphogenetic Protein 2 metabolism, Bone Morphogenetic Protein 2 pharmacology, Cell Line, Drug Delivery Systems, Gene Expression drug effects, Genetic Therapy, Immunohistochemistry, Lactic Acid pharmacology, Male, Mice, Microspheres, Particle Size, Plasmids genetics, Polyethyleneimine pharmacology, Polyglycolic Acid pharmacology, Polylactic Acid-Polyglycolic Acid Copolymer, Rats, Rats, Wistar, Skull drug effects, Skull injuries, Skull pathology, Transfection, Bone Morphogenetic Protein 2 genetics, Lactic Acid chemistry, Nanoparticles chemistry, Osteogenesis drug effects, Polyethyleneimine chemistry, Polyglycolic Acid chemistry
- Abstract
Repair of large bone defects is a major challenge, requiring sustained stimulation to continually promote bone formation locally. Bone morphogenetic protein 2 (BMP-2) plays an important role in bone development. In an attempt to overcome this difficulty of bone repair, we created a delivery system to slowly release human BMP-2 cDNA plasmid locally, efficiently transfecting local target cells and secreting functional human BMP-2 protein. For transfection, we used polyethylenimine (PEI) to create pBMP-2/PEI nanoparticles, and to ensure slow release we used poly(lactic-co-glycolic acid) (PLGA) to create microsphere encapsulated pBMP-2/PEI nanoparticles, PLGA@pBMP-2/PEI. We demonstrated that pBMP-2/PEI nanoparticles could slowly release from the PLGA@pBMP-2/PEI microspheres for a long period of time. The 3-15 μm diameter of the PLGA@pBMP-2/PEI further supported this slow release ability of the PLGA@pBMP-2/PEI. In vitro transfection assays demonstrated that pBMP-2/PEI released from PLGA@pBMP-2/PEI could efficiently transfect MC3T3-E1 cells, causing MC3T3-E1 cells to secrete human BMP-2 protein, increase calcium deposition and gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), SP7 and I type collagen (COLL I), and finally induce MC3T3-E1 cell differentiation. Importantly, in vivo data from micro-computed tomography (micro-CT) and histological staining demonstrated that the human BMP-2 released from PLGA@pBMP-2/PEI had a long-term effect locally and efficiently promoted bone formation in the bone defect area compared to control animals. All our data suggest that our PLGA-nanoparticle delivery system efficiently and functionally delivers the human BMP-2 cDNA and has potential clinical application in the future after further modification. more...
- Published
- 2013
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174. Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.
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Baum BJ, Alevizos I, Zheng C, Cotrim AP, Liu S, McCullagh L, Goldsmith CM, Burbelo PD, Citrin DE, Mitchell JB, Nottingham LK, Rudy SF, Van Waes C, Whatley MA, Brahim JS, Chiorini JA, Danielides S, Turner RJ, Patronas NJ, Chen CC, Nikolov NP, and Illei GG more...
- Subjects
- Aged, Citrates, Gallium, Humans, Male, Middle Aged, Radiation Injuries diagnostic imaging, Radiation Injuries genetics, Radionuclide Imaging, Salivary Gland Diseases diagnostic imaging, Salivary Gland Diseases etiology, Salivary Gland Diseases physiopathology, Adenoviridae genetics, Aquaporin 1 genetics, Aquaporin 1 therapeutic use, DNA, Complementary genetics, Genetic Therapy adverse effects, Radiation Injuries therapy, Salivary Gland Diseases therapy
- Abstract
No conventional therapy exists for salivary hypofunction in surviving head and neck cancer patients with Radiation Therapy Oncology Group late grade 2-3 toxicity. We conducted a phase I clinical trial to test the safety and biologic efficacy of serotype 5, adenoviral-mediated aquaporin-1 cDNA transfer to a single previously irradiated parotid gland in 11 subjects using an open label, single-dose, dose-escalation design (AdhAQP1 vector; four dose tiers from 4.8 × 10(7) to 5.8 × 10(9) vector particles per gland). Treated subjects were followed at scheduled intervals. Multiple safety parameters were measured and biologic efficacy was evaluated with measurements of parotid salivary flow rate. Symptoms were assessed with a visual analog scale. All subjects tolerated vector delivery and study procedures well over the 42-d study period reported. No deaths, serious adverse events, or dose-limiting toxicities occurred. Generally, few adverse events occurred, and all were considered mild or moderate. No consistent changes were found in any clinical chemistry and hematology parameters measured. Objective responses were seen in six subjects, all at doses <5.8 × 10(9) vector particles per gland. Five of these six subjects also experienced subjective improvement in xerostomia. AdhAQP1 vector delivery to a single parotid gland was safe and transfer of the hAQP1 cDNA increased parotid flow and relieved symptoms in a subset of subjects. more...
- Published
- 2012
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175. Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane.
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Qin L, Liu X, Sun Q, Fan Z, Xia D, Ding G, Ong HL, Adams D, Gahl WA, Zheng C, Qi S, Jin L, Zhang C, Gu L, He J, Deng D, Ambudkar IS, and Wang S
- Subjects
- Acids metabolism, Adenoviridae metabolism, Animals, Anions, Biological Transport, Fibroblasts metabolism, Fibroblasts pathology, Intracellular Space metabolism, Mutation genetics, N-Acetylneuraminic Acid metabolism, Nitrate Transporters, Nitrates metabolism, Organic Anion Transporters genetics, Protons, Sialic Acid Storage Disease metabolism, Submandibular Gland cytology, Submandibular Gland metabolism, Sus scrofa, Symporters genetics, Anion Transport Proteins metabolism, Cell Membrane metabolism, Organic Anion Transporters metabolism, Symporters metabolism
- Abstract
In vivo recycling of nitrate (NO(3)(-)) and nitrite (NO(2)(-)) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate-nitrite-NO balance. More than 25% of the circulating NO(3)(-) is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO(3)(-) to NO(2)(-), which enters circulation and leads to NO generation. The transporters for NO(3)(-) in salivary glands have not yet been identified. Here we report that sialin (SLC17A5), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO(3)(-)/H(+) cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO(3)(-) or sialic acid (SA), but not by Br(-), and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO(3)(-)-induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H(+)-dependent NO(3)(-) conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO(3)(-) secretion in saliva after intake of a NO(3)(-)-rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO(3)(-)/H(+) transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis. more...
- Published
- 2012
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176. Assessment of the safety and biodistribution of a regulated AAV2 gene transfer vector after delivery to murine submandibular glands.
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Zheng C, Voutetakis A, Goldstein B, Afione S, Rivera VM, Clackson T, Wenk ML, Boyle M, Nyska A, Chiorini JA, Vallant M, Irwin RD, and Baum BJ
- Subjects
- Animals, Body Weight, Erythropoietin blood, Erythropoietin genetics, Female, Male, Mice, Mice, Inbred BALB C, Risk Assessment, Sex Factors, Sirolimus pharmacology, Submandibular Gland metabolism, Toxicity Tests, Dependovirus genetics, Gene Transfer Techniques adverse effects, Genetic Vectors administration & dosage, Submandibular Gland virology
- Abstract
Clinical gene transfer holds promise for the treatment of many inherited and acquired disorders. A key consideration for all clinical gene transfer applications is the tight control of transgene expression. We have examined the safety and biodistribution of a serotype 2, recombinant adeno-associated viral (AAV2) vector that encodes a rapamycin-responsive chimeric transcription factor, which regulates the expression of a therapeutic transgene (human erythropoietin [hEpo]). The vector, AAV2-TF2.3w-hEpo (2.5 × 10(7)-2.5 × 10(10) particles), was administered once to a single submandibular gland of male and female mice and mediated hEpo expression in vivo following a rapamycin injection but not in its absence. Control (saline treated) and vector-treated animals maintained their weight, and consumed food and water, similarly. Vector delivery led to no significant toxicological effects as judged by hematology, clinical chemistry, and gross and microscopic pathology evaluations. On day 3 after vector delivery, vector copies were not only abundant in the targeted right submandibular gland but also detected in multiple other tissues. Vector was cleared from the targeted gland much more rapidly in female mice than in male mice. Overall, our results are consistent with the notion that administration of the AAV2-TF2.3w-hEpo vector to salivary glands posed no significant risk in mice. more...
- Published
- 2011
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177. Systemic delivery of bioactive glucagon-like peptide 1 after adenoviral-mediated gene transfer in the murine salivary gland.
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Voutetakis A, Cotrim AP, Rowzee A, Zheng C, Rathod T, Yanik T, Loh YP, Baum BJ, and Cawley NX
- Subjects
- Alloxan, Animals, COS Cells, Cell Line, Cell Line, Tumor, Chlorocebus aethiops, Diabetes Mellitus, Experimental chemically induced, Diabetes Mellitus, Experimental genetics, Diabetes Mellitus, Experimental therapy, Dipeptidyl Peptidase 4 metabolism, Genetic Vectors genetics, Glucagon-Like Peptide 1 blood, Glucagon-Like Peptide 1 genetics, Glucose metabolism, Insulin metabolism, Insulin Secretion, Male, Mice, Mice, Inbred BALB C, Adenoviridae genetics, Gene Transfer Techniques, Glucagon-Like Peptide 1 metabolism, Salivary Glands metabolism
- Abstract
An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7-37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic beta-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1's glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis. more...
- Published
- 2010
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178. Salivary epithelial cells: an unassuming target site for gene therapeutics.
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Perez P, Rowzee AM, Zheng C, Adriaansen J, and Baum BJ
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- Animals, Endocrine System Diseases genetics, Epithelial Cells pathology, Erythropoietin genetics, Growth Hormone genetics, Humans, Parathyroid Hormone genetics, Salivary Glands pathology, Secretory Pathway, Upper Gastrointestinal Tract metabolism, Upper Gastrointestinal Tract pathology, Endocrine System Diseases therapy, Epithelial Cells metabolism, Gene Transfer Techniques, Genetic Therapy, Genetic Vectors, Viruses
- Abstract
Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer., (Published by Elsevier Ltd.) more...
- Published
- 2010
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179. Conditional overexpression of TGF-beta1 disrupts mouse salivary gland development and function.
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Hall BE, Zheng C, Swaim WD, Cho A, Nagineni CN, Eckhaus MA, Flanders KC, Ambudkar IS, Baum BJ, and Kulkarni AB
- Subjects
- Animals, Cells, Cultured, Disease Models, Animal, Fibrosis physiopathology, Inflammation physiopathology, Mice, Mice, Transgenic, Salivary Glands pathology, Salivary Gland Diseases physiopathology, Salivary Glands growth & development, Transforming Growth Factor beta1 physiology, Xerostomia physiopathology
- Abstract
Transforming growth factor-beta (TGF-beta) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-beta1 in the salivary gland, we created a transgenic mouse (beta1(glo)) that conditionally overexpresses active TGF-beta1 upon genomic recombination by Cre recombinase. beta1(glo) mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (beta1(glo)/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those beta1(glo)/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-beta signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-beta1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-beta in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome, or with radiation therapy given to head-and-neck cancer patients. more...
- Published
- 2010
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180. Development of a gene transfer-based treatment for radiation-induced salivary hypofunction.
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Baum BJ, Zheng C, Alevizos I, Cotrim AP, Liu S, McCullagh L, Goldsmith CM, McDermott N, Chiorini JA, Nikolov NP, and Illei GG
- Subjects
- Aquaporin 1 genetics, Female, Humans, Male, Radiation Injuries genetics, Xerostomia etiology, Xerostomia genetics, Aquaporin 1 therapeutic use, Gene Transfer Techniques, Radiation Injuries therapy, Xerostomia therapy
- Abstract
A significant long-term side effect of radiation therapy for head and neck cancers is xerostomia, a dry mouth, due to salivary gland damage. Despite continuing efforts to eliminate this problem, many patients continue to suffer. This brief review describes our efforts to develop a gene transfer approach, employing the aquaporin-1 cDNA, to treat patients with existing radiation-induced salivary hypofunction. A Phase I/II clinical trial, using a recombinant adenoviral vector to mediate gene transfer, is currently underway. more...
- Published
- 2010
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181. Gene therapy of salivary diseases.
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Baum BJ, Adriaansen J, Cotrim AP, Goldsmith CM, Perez P, Qi S, Rowzee AM, and Zheng C
- Subjects
- Adenoviridae genetics, Animals, Disease Models, Animal, Genetic Vectors genetics, Humans, Mice, Salivary Gland Diseases genetics, Genetic Therapy methods, Salivary Gland Diseases therapy
- Abstract
For many years, our laboratory has been developing gene transfer approaches for salivary gland disorders that currently lack effective therapy. The purpose of this chapter is to describe key methods used in this developmental process. Specifically, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model. more...
- Published
- 2010
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182. In vivo secretion of the mouse immunoglobulin G Fc fragment from rat submandibular glands.
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Racz GZ, Perez-Riveros P, Adriaansen J, Zheng C, and Baum BJ
- Subjects
- Adenoviridae genetics, Adenoviridae metabolism, Animals, Genetic Vectors genetics, Genetic Vectors metabolism, Human Growth Hormone genetics, Humans, Male, Mice, Mice, Inbred BALB C, Rats, Rats, Wistar, Transgenes, Immunoglobulin Fc Fragments metabolism, Immunoglobulin G metabolism, Submandibular Gland immunology, Submandibular Gland metabolism
- Abstract
Background: Salivary glands have been proposed as target organs for gene therapy. They secrete endogenous, as well as transgenic proteins, in a polarized manner. Transgene-encoded regulated pathway proteins primarily follow the regulated pathway in rat salivary glands and are secreted into saliva in an exocrine manner. Conversely, constitutive pathway proteins generally are secreted more basolaterally and thus follow the endocrine route. In the present study, we studied in vivo the sorting of the mouse immunoglobulin G2b Fc fragment, which is physiologically secreted via the constitutive pathway., Methods: Adenoviral vectors encoding the Fc fragment and human growth hormone were delivered into rat and mouse submandibular glands in vivo to compare their serum-to-saliva distribution. We also compared the intracellular localization of the Fc fragment and growth hormone by confocal microscopy., Results: We found that the Fc fragment was secreted almost entirely into the bloodstream from rat and mouse submandibular glands via a constitutive or constitutive-like pathway. This sorting behaviour is clearly different from that of transgenic human growth hormone, which is secreted in a regulated pathway, both in neuroendocrine cells and as a transgenic protein from salivary gland cells. We also found that simultaneously expressed human growth hormone and the mouse Fc fragment do not appear to influence each other's sorting behaviour. The Fc fragment showed a primarily basal localization, whereas growth hormone showed an apical localization, in rat submandibular gland acinar cells., Conclusions: The results obtained in the present study indicate that the mouse Fc fragment is a useful model protein for examining the basolateral versus apical secretory pathways employed by transgenic secretory proteins in salivary glands., (2009 John Wiley & Sons, Ltd.) more...
- Published
- 2009
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183. Aquaporin-1 gene transfer to correct radiation-induced salivary hypofunction.
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Baum BJ, Zheng C, Cotrim AP, McCullagh L, Goldsmith CM, Brahim JS, Atkinson JC, Turner RJ, Liu S, Nikolov N, and Illei GG
- Subjects
- Adenoviridae genetics, Animals, Aquaporin 1 genetics, Cell Line, Clinical Trials as Topic, Disease Models, Animal, Genetic Therapy adverse effects, Genetic Vectors, Humans, Radiation Injuries etiology, Radiation Injuries genetics, Radiation Injuries metabolism, Radiotherapy adverse effects, Research Design, Salivary Glands radiation effects, Xerostomia etiology, Xerostomia genetics, Xerostomia metabolism, Aquaporin 1 biosynthesis, Gene Transfer Techniques adverse effects, Genetic Therapy methods, Radiation Injuries therapy, Salivary Glands metabolism, Xerostomia therapy
- Abstract
Irradiation damage to salivary glands is a common iatrogenic consequence of treatment for head and neck cancers. The subsequent lack of saliva production leads to many functional and quality-of-life problems for affected patients and there is no effective conventional therapy. To address this problem, we developed an in vivo gene therapy strategy involving viral vector-mediated transfer of the aquaporin-1 cDNA to irradiation-damaged glands and successfully tested it in two pre-clinical models (irradiated rats and miniature pigs), as well as demonstrated its safety in a large toxicology and biodistribution study. Thereafter, a clinical research protocol was developed that has received approval from all required authorities in the United States. Patients are currently being enrolled in this study. more...
- Published
- 2009
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184. Extended transgene expression from a nonintegrating adenoviral vector containing retroviral elements.
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Zheng C, Vitolo JM, Zhang W, Mineshiba F, Chiorini JA, and Baum BJ
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- Animals, Erythropoietin genetics, Female, Genetic Vectors, Humans, Moloney murine leukemia virus genetics, Rats, Rats, Wistar, Retroviridae genetics, Submandibular Gland metabolism, Terminal Repeat Sequences, Adenoviridae genetics, Gene Expression Regulation, Promoter Regions, Genetic, Transgenes
- Abstract
We studied the effects of specific retroviral elements in a first-generation serotype 5 adenoviral (Ad5) vector, AdLTR(2)EF1alpha-hEPO. This vector contains 858 base pair (bp) [251-bp envelope sequence plus 607-bp long-terminal repeat (LTR)] from Moloney murine leukemia virus (MoMLV), upstream of the human elongation factor-1alpha (EF1alpha) promoter and human erythropoietin (hEPO) cDNA, with the LTR sequence downstream of the polyadenylation signal. We compared expression of AdLTR(2)EF1alpha-hEPO with corresponding expressions of two conventional Ad5 vectors, AdEF1alpha-hEPO and AdCMV-hEPO, in vivo in submandibular glands in rats. Both the conventional vectors yielded low serum hEPO levels by day 7, and little change in hematocrits. In contrast, after receiving AdLTR(2)EF1alpha-hEPO, the rats showed elevated hEPO levels and hematocrits for 1-3 months. In vitro studies showed that the integration efficiencies of all the vectors were similar (approximately 10(-3)). Approximately 0.1% of the vector genomes were present 1 year after delivery in the case of each of the three vectors, primarily as intact linear double-strand DNA. The unique results seen with AdLTR(2)EF1alpha-hEPO are partly because of LTR enhancer activity. However, other cis-acting activity, which is not immunomodulatory but nevertheless influences promoter methylation, appears to be involved. A vector such as AdLTR(2)EF1alpha-hEPO may prove useful in clinical applications in which extended, but not "permanent," transgene expression is desirable. more...
- Published
- 2008
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185. Evaluation of promoters for use in tissue-specific gene delivery.
- Author
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Zheng C and Baum BJ
- Subjects
- Adenoviridae genetics, Animals, Cells, Cultured, Gene Expression Regulation, Immunoenzyme Techniques, Luciferases metabolism, Rats, Sequence Deletion, Aquaporin 5 genetics, Gene Transfer Techniques, Genetic Therapy, Genetic Vectors, Promoter Regions, Genetic genetics, Submandibular Gland physiology
- Abstract
Vectors used in gene therapy require an expression cassette. The expression cassette consists of three important components: promoter, therapeutic gene and polyadenylation signal. The promoter is essential to control expression of the therapeutic gene. A tissue-specific promoter is a promoter that has activity in only certain cell types. Use of a tissue-specific promoter in the expression cassette can restrict unwanted transgene expression as well as facilitate persistent transgene expression. Therefore, choosing the correct promoter, especially a tissue-specific promoter, is a major step toward achieving successful therapeutic transgene expression. Ideally, the elements of the natural promoter region, necessary for obtaining the required level of the gene expression while retaining tissue-specificity, should be known. Also, it is important to understand whether interactions occur between the promoter region and the rest of the vector genome that could affect promoter activity and specificity. To assess this, it is helpful to select a suitable vector system that will be used in further gene therapy studies. Second, have one or several candidate tissue-specific promoters available for use. Third, ideally have an in vitro cell model suitable to evaluate tissue-specificity. Fourth, have a convenient in vivo animal model to use. Fifth, select a good reporter gene system. Next, using conventional recombinant DNA techniques create different promoter constructs with the selected vector system. Lastly, have a suitable transfection method to test the plasmid constructs in both the in vitro and the in vivo models. more...
- Published
- 2008
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186. Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral-mediated gene transfer.
- Author
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Yan X, Voutetakis A, Zheng C, Hai B, Zhang C, Baum BJ, and Wang S
- Subjects
- Animals, Antibodies blood, Antibody Formation, Erythropoietin administration & dosage, Erythropoietin blood, Human Growth Hormone administration & dosage, Human Growth Hormone blood, Humans, Male, Organ Specificity, Parotid Gland pathology, Protein Transport, Swine, Tissue Distribution, Adenoviridae genetics, Erythropoietin metabolism, Gene Transfer Techniques, Human Growth Hormone metabolism, Parotid Gland metabolism, Swine, Miniature metabolism, Transgenes
- Abstract
Background: Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig., Methods: Recombinant serotype 5 adenoviral (Ad5; 10(11) particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR)., Results: The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by approximately 10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100 x more than in other tissues., Conclusions: Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs. more...
- Published
- 2007
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187. Female mice are more susceptible to developing inflammatory disorders due to impaired transforming growth factor beta signaling in salivary glands.
- Author
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Nandula SR, Amarnath S, Molinolo A, Bandyopadhyay BC, Hall B, Goldsmith CM, Zheng C, Larsson J, Sreenath T, Chen W, Ambudkar IS, Karlsson S, Baum BJ, and Kulkarni AB
- Subjects
- Activin Receptors, Type I genetics, Activin Receptors, Type I physiology, Animals, Disease Models, Animal, Female, Gene Deletion, Gene Expression Regulation, Genetic Predisposition to Disease, Inflammation genetics, Male, Mice, Mice, Transgenic, Protein Serine-Threonine Kinases, Receptor, Transforming Growth Factor-beta Type I, Receptors, Transforming Growth Factor beta genetics, Receptors, Transforming Growth Factor beta physiology, Salivary Glands pathology, Sjogren's Syndrome genetics, Sjogren's Syndrome pathology, Sjogren's Syndrome physiopathology, Transforming Growth Factor beta genetics, Disease Susceptibility physiopathology, Inflammation physiopathology, Salivary Glands physiopathology, Sex Characteristics, Signal Transduction physiology, Transforming Growth Factor beta physiology
- Abstract
Objective: Transforming growth factor beta (TGFbeta) plays a key role in the onset and resolution of autoimmune diseases and chronic inflammation. The aim of this study was to delineate the precise function of TGFbeta signaling in salivary gland inflammation., Methods: We impaired TGFbeta signaling in mouse salivary glands by conditionally inactivating expression of TGFbeta receptor type I (TGFbetaRI), either by using mouse mammary tumor virus-Cre mice or by delivering adenoviral vector containing Cre to mouse salivary glands via retrograde infusion of the cannulated main excretory ducts of submandibular glands., Results: TGFbetaRI-conditional knockout (TGFbetaRI-coko) mice were born normal; however, female TGFbetaRI-coko mice developed severe multifocal inflammation in salivary and mammary glands and in the heart. The inflammatory disorder affected normal growth and resulted in the death of the mice at ages 4-5 weeks. Interestingly, male TGFbetaRI-coko mice did not exhibit any signs of inflammation. The female TGFbetaRI-coko mice also showed an increase in Th1 proinflammatory cytokines in salivary glands and exhibited an up-regulation of peripheral T cells. In addition, these mice showed an atypical distribution of aquaporin 5 in their salivary glands, suggesting likely secretory impairment. Administration of an adenoviral vector encoding Cre recombinase into the salivary glands resulted in inflammatory foci only in the glands of female TGFbetaRI-loxP-flanked (floxed) mice (TGFbetaRI-f/f mice), but not in those of male and female wild-type mice or male TGFbetaRI-f/f mice., Conclusion: These results suggest that female mice are uniquely more susceptible to developing inflammatory disorders due to impaired TGFbeta signaling in their salivary glands. more...
- Published
- 2007
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188. Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction.
- Author
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Baum BJ, Zheng C, Cotrim AP, Goldsmith CM, Atkinson JC, Brahim JS, Chiorini JA, Voutetakis A, Leakan RA, Van Waes C, Mitchell JB, Delporte C, Wang S, Kaminsky SM, and Illei GG
- Subjects
- Animals, Aquaporin 1 metabolism, Gene Transfer Techniques, Genetic Vectors, Humans, Parotid Gland physiopathology, Parotid Gland radiation effects, Rats, Salivary Glands physiopathology, Swine, Swine, Miniature, Aquaporin 1 genetics, DNA, Complementary genetics, Genetic Therapy, Head and Neck Neoplasms radiotherapy, Radiation Injuries, Experimental therapy, Salivary Gland Diseases therapy, Salivary Glands radiation effects
- Abstract
The treatment of most patients with head and neck cancer includes ionizing radiation (IR). Salivary glands in the IR field suffer significant and irreversible damage, leading to considerable morbidity. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat [C. Delporte, B.C. O'Connell, X. He, H.E. Lancaster, A.C. O'Connell, P. Agre, B.J. Baum, Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci. U S A. 94 (1997) 3268-3273] and miniature pig [Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J Baum, S. Wang. Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands. Mol. Ther. 11 (2005) 444-451] salivary glands approximately 16 weeks following IR resulted in a dose-dependent increase in salivary flow to > or =80% control levels on day 3. A control Ad vector was without any significant effect on salivary flow. Additionally, after administration of Ad vectors to salivary glands, no significant lasting effects were observed in multiple measured clinical chemistry and hematology values. Taken together, the findings show that localized delivery of AdhAQP1 to IR-damaged salivary glands is useful in transiently increasing salivary secretion in both small and large animal models, without significant general adverse events. Based on these results, we are developing a clinical trial to test if the hAQP1 cDNA transfer strategy will be clinically effective in restoring salivary flow in patients with IR-induced parotid hypofunction. more...
- Published
- 2006
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189. Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands.
- Author
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Zheng C and Baum BJ
- Subjects
- Adenoviridae genetics, Animals, DNA, Complementary metabolism, Genes, Reporter, Genetic Therapy instrumentation, Green Fluorescent Proteins metabolism, Immunohistochemistry, In Vitro Techniques, Luciferases metabolism, Male, Mice, Microscopy, Fluorescence, Plasmids metabolism, Rats, Rats, Wistar, Transfection, Transgenes, Viruses genetics, Gene Transfer Techniques, Genetic Vectors, Promoter Regions, Genetic, Salivary Glands metabolism
- Abstract
To optimize vectors for salivary gland gene transfer, we screened viral [cytomegalovirus (CMV; human immediate early), Rous sarcoma virus (RSV), simian virus 40, and Moloney murine leukemia virus long terminal repeat] and mammalian [elongation factor 1alpha (EF1alpha), cytokeratin 18 (K18), cytokeratin 19 (K19), kallikrein (Kall), and amylase (AMY), all human, and rat aquaporin-5 (rAQP5), and derivative elements] promoters driving luciferase activity in vitro and in vivo. In adenoviral vectors, the CMV promoter showed highest activity, with the EF1alpha and RSV promoters slightly less powerful, in rat submandibular glands (SMGs). The K18 2.5-kb, K19 3.0-kb, and rAQP5 0.4-kb and Kall promoters had intermediate activity, while the AMY promoter exhibited lowest activity. To localize transgene expression, enhanced green fluorescence protein was used. The CMV, RSV, EF1alpha, K18 2.5-kb, K19 3.0-kb, rAQP5 0.4-kb, and AMY promoters were not cell-type specific in SMGs; however, the Kall promoter was primarily active in ductal cells. These data will facilitate optimal expression cassette design for salivary gland gene transfer. more...
- Published
- 2005
- Full Text
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190. Intratumoral administration of immature dendritic cells following the adenovirus vector encoding CD40 ligand elicits significant regression of established myeloma.
- Author
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Liu Y, Xia D, Li F, Zheng C, and Xiang J
- Subjects
- Animals, Antigens, CD metabolism, Apoptosis, Cancer Vaccines therapeutic use, Coculture Techniques, Cytotoxicity Tests, Immunologic, Female, Genetic Vectors, Mice, Mice, Inbred BALB C, Multiple Myeloma immunology, Multiple Myeloma pathology, T-Lymphocytes, Cytotoxic, Transgenes physiology, Tumor Cells, Cultured, Adenoviridae genetics, CD40 Ligand therapeutic use, Dendritic Cells immunology, Genetic Therapy, Immunotherapy, Multiple Myeloma therapy
- Abstract
Our previous study showed that J558 myeloma cells engineered CD40L lost their tumorigenicity in syngeneic mice, and the inoculation of J558/CD40L tumor cells further led to the protective immunity against wild tumors. In the present study, we investigated whether the vaccine can exert more efficient antitumor immunity by combination with adenovirus mediated CD40L gene therapy and immature dendritic cells (iDCs). The results demonstrated that intratumoral administration of iDCs 2 days after AdVCD40L injection, not only significantly suppressed the tumor growth, but also eradiated the established tumors in 40% of the mice. The potent antitumor effect produced by the combination therapy correlated with high expression of MHC, costimulatory and Fas molecules on J558 cells, which was derived from CD40L transgene expression. In addition, transgene CD40L expression could dramatically induce J558 cell apoptosis. Effectively capturing apoptotic bodies by iDCs in vivo could induce DC maturation, prime tumor-specific CTLs and tend to Th1-type immune response. Finally, in vivo depletion experimentation suggested both CD4+ and CD8+ T cells were involved in mediating the antitumor immune responses of combined treatment of AdVCD40L and iDCs, with CD8+ T cells being the major effector. These findings could be beneficial for designing strategies of DCs vaccine and CD40L for anticancer immunotherapy. more...
- Published
- 2005
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191. Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics.
- Author
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Voutetakis A, Kok MR, Zheng C, Bossis I, Wang J, Cotrim AP, Marracino N, Goldsmith CM, Chiorini JA, Loh YP, Nieman LK, and Baum BJ
- Subjects
- Animals, Blotting, Southern, DNA Primers, Dependovirus genetics, Enzyme-Linked Immunosorbent Assay, Erythropoietin analysis, Erythropoietin blood, Erythropoietin genetics, Erythropoietin pharmacokinetics, Gene Transfer Techniques, Hematocrit, Humans, Male, Mice, Mice, Inbred BALB C, Polymerase Chain Reaction, Recombinant Proteins analysis, Recombinant Proteins blood, Tissue Distribution, beta-Galactosidase genetics, Bioreactors, Genetic Therapy methods, Salivary Glands physiology
- Abstract
The use of critical-for-life organs (e.g., liver or lung) for systemic gene therapeutics can lead to serious safety concerns. To circumvent such issues, we have considered salivary glands (SGs) as an alternative gene therapeutics target tissue. Given the high secretory abilities of SGs, we hypothesized that administration of low doses of recombinant adeno-associated virus (AAV) vectors would allow for therapeutic levels of transgene-encoded secretory proteins in the bloodstream. We administered 10(9) particles of an AAV vector encoding human erythropoietin (hEPO) directly to individual mouse submandibular SGs. Serum hEPO reached maximum levels 8-12 weeks after gene delivery and remained relatively stable for 54 weeks (longest time studied). Hematocrit levels were similarly increased. Moreover, these effects proved to be vector dose-dependent, and even a dosage as low as 10(8) particles per animal led to significant increases in hEPO and hematocrit levels. Vector DNA was detected only within the targeted SGs, and levels of AAV copies within SGs were highly correlated with serum hEPO levels (r = 0.98). These results show that SGs appear to be promising targets with potential clinical applicability for systemic gene therapeutics. more...
- Published
- 2004
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192. Gene transfer to salivary glands.
- Author
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Baum BJ, Wellner RB, and Zheng C
- Subjects
- Animals, Aquaporins genetics, Aquaporins metabolism, Genetic Therapy trends, Genetic Vectors genetics, Humans, Salivary Gland Diseases metabolism, Salivary Gland Diseases physiopathology, Salivary Glands innervation, Viruses genetics, Gene Transfer Techniques trends, Genetic Therapy methods, Genetic Vectors therapeutic use, Saliva metabolism, Salivary Gland Diseases therapy, Salivary Glands metabolism
- Abstract
This article provides a review of the application of gene transfer technology to studies of salivary glands. Salivary glands provide an uncommon target site for gene transfer but offer many experimental situations likely of interest to the cell biologist. The reader is provided with a concise overview of salivary biology, along with a general discussion of the strategies available for gene transfer to any tissue. In particular, adenoviral vectors have been useful for proof of concept studies with salivary glands. Several examples are given, using adenoviral-mediated gene transfer, for addressing both biological and clinical questions. Additionally, benefits and shortcomings affecting the utility of this technology are discussed. more...
- Published
- 2002
- Full Text
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