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author:("Xia, deqing")
1.  Managing Local Swelling Following Intratumoral Electro-Chemo-Gene Therapy 
Delivering genes and other materials directly into the tumor tissue causes specifically localized and powerfully enhanced efficacy of treatments; however, these specific effects can cause rapid, drastic changes in the appearance, texture, and consistency of the tumor. These changes complicate clinical response measurements which can confound the results and render recurring treatments difficult to perform and clinical response measurements nearly impossible to accurately obtain. One of these complicating issues is local swelling. Here, we will demonstrate how swelling caused by intratumoral gene treatments can confound the clinical results and impede further treatments, and we will demonstrate an easy technique to help to overcome this potential hurdle.
PMCID: PMC4098855  PMID: 24510827
canine; oral tumor; intratumoral; electroporation; gene therapy; chemotherapy; chemogene therapy; immune therapy; IL12
2.  Safety and Efficacy of Tumor-targeted Interleukin 12 Gene Therapy in Treated and Non-treated, Metastatic Lesions 
Current gene therapy  2014;15(1):44-54.
The ability to control the immune system to actively attack tumors would be a marvelous weapon to combat the incessant attack of cancer. Unfortunately, safe and effective methods are not yet available. To overcome the impediments to this control, tumor-targeted (tt) Interleukin (IL) 12 plasmid DNA can be safely delivered to accessible tumors, and these treatments can induce antitumor immune responses in both the treated and untreated tumors. Here, electroporation-mediated ttIL12 pDNA treatments are shown to be safe and well tolerated in a dose escalation study in canines bearing naturally-occurring neoplasms. The final patient received treatment with up to 3,800 μg pDNA distributed throughout five separate squamous cell carcinoma tumors, doses equivalent to those administered in a Phase I trial with wildtype IL12 pDNA. Not a single severe adverse event occurred in any patient at any of the five dose levels, and only minor, transient changes were noted in any tested parameter. Clinical response analysis and immune marker mRNA detection of treated and non-treated lesions confirmed that the ttIL12 pDNA treatments in only a few tumors could elicit antitumor immune responses in the treated lesions as well as distant metastatic lesions. These observations and results prove that ttIL12 pDNA can be safely administered at clinical levels, and these treatments can effect both treated and non-treated, metastatic lesions.
PMCID: PMC4280356  PMID: 25429465
Cancer; Canine; Electroporation; Gene therapy; Immunotherapy; Interleukin 12; Tumor-targeting
3.  CD8+T cell–specific induction of NKG2D receptor by doxorubicin plus interleukin-12 and its contribution to CD8+T cell accumulation in tumors 
Molecular Cancer  2014;13:34.
Increased infiltration of CD8+T cells into tumors has a positive impact on survival. Our previous study showed that doxorubicin (Dox) plus interleukin-12 (IL-12) boosted the accumulation of CD8+T cells in tumors and had a greater antitumor effect than did either agent alone. The purpose of this study was to determine the impact of NKG2D expression on CD8+T cell infiltration and antitumor efficacy.
Tumor-bearing mice were administered Dox, IL-12 plasmid DNA, or both via intraperitoneal injection or intramuscular electroporation. The induction of NKG2D on CD8+T cells and other lymphocytes was analyzed via flow cytometry, and NKG2D-positive CD8+T cell–specific localization in tumors was determined by using immunofluorescence staining in various types of immune cell–depleted mice.
The combination of Dox plus IL-12 specifically increased expression of NKG2D in CD8+T cells but not in other types of immune cells, including NK cells, which naturally express NKG2D. This induced NKG2D expression in CD8+T cells was associated with increased accumulation of CD8+T cells in murine tumors. Administration of NKG2D-blocking antibody or CD8+T cell–depletion antibody abrogated the NKG2D+CD8+T cell detection in tumors, whereas administration of NK cell–depletion antibody had no effect. Increased NKG2D expression in CD8+T cells was associated with increased antitumor efficacy in vivo.
We conclude that Dox plus IL-12 induces NKG2D in CD8+T cells in vivo and boosts NKG2D+CD8+T-dependent antitumor immune surveillance. This discovery reveals a novel mechanism for how chemoimmunotherapy synergistically promotes T cell–mediated antitumor immune surveillance.
PMCID: PMC3938086  PMID: 24565056
Interleukin-12; Doxorubicin; Tumor-infiltrating lymphocytes; NKG2D+CD8+T cells
4.  Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells 
Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly.
We used Rae-1–overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti–Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining.
Our cell line–based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.
PMCID: PMC3916315  PMID: 24495546
GPI-anchored protein Rae-1; Monoclonal antibody; Hybridomas; Streamlined screening strategy
5.  IL30—A Novel Anti-inflammatory Cytokine Candidate for Prevention and Treatment of Inflammatory Cytokine-Induced Liver Injury 
Hepatology (Baltimore, Md.)  2012;55(4):1204-1214.
The liver is the major metabolic organ and is subjected to constant attacks from chronic viral infection, uptake of therapeutic drugs, life behavior (alcoholic), and environmental contaminants, all of which result in chronic inflammation, fibrosis, and ultimately, cancer. Therefore, there is an urgent need to discover effective therapeutic agents for the prevention and treatment of liver injury; the ideal drug being a naturally occurring biological inhibitor. Here, we establish the role of IL30 as a potent anti-inflammatory cytokine which can inhibit inflammation-induced liver injury. In contrast, IL27, which contains IL30 as a subunit, is not hepatoprotective. Interestingly, IL30 is induced by the pro-inflammatory signal such as IL12 through IFNγ/STAT1 signaling. In animal models, administration of IL30 via a gene therapy approach prevents and treats both IL12-, IFNγ-, and Concanavalin A -induced liver toxicity. Likewise, immunohistochemistry analysis of human tissue samples revealed that IL30 is highly expressed in hepatocytes yet barely expressed in inflammation-induced tissue such as fibrous/connective tissue. These novel observations reveal a novel role of IL30 as a therapeutic cytokine that suppresses pro-inflammatory cytokine-associated liver toxicity.
PMCID: PMC3295919  PMID: 22105582
IL12; IFNγ; IL27p28 (IL30); EBI3; TCCR; ConA
6.  Competitive DNA transfection formulation via electroporation for human adipose stem cells and mesenchymal stem cells 
Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use.
We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells.
Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.
PMCID: PMC3388581  PMID: 22512891
Electroporation; Formulation; Stem cells; Transfection; Cell therapy
7.  WSX1 Expression in Tumors Induces Immune Tolerance via Suppression of Effector Immune Cells 
PLoS ONE  2011;6(4):e19072.
Crosstalk between tumor cells and the cognate microenvironment plays a crucial role in tumor initiation and progression. However, only a few genes are known to affect such a crosstalk. This study reveals that WSX1 plays such a role when highly expressed in tumor cells. The expression of WSX1 in Lewis Lung Carcinoma (LLC) and the melanoma cell line AGS induces the death of T cells and inhibits the production of the effector cytokine IFNγ from NK and T cells, resulting in the promotion of tumor growth. These pro-tumorigenic properties of WSX1 are independent of IL27. This key observation reveals a new pathway of tumor-host interaction, which will ultimately lead to better strategies in immune therapy to reverse tumor tolerance.
PMCID: PMC3084744  PMID: 21559505
8.  Enhancement of Reporter Gene Detection Sensitivity by Insertion of Specific Mini-Peptide-Coding Sequences 
Cancer gene therapy  2009;17(2):131-140.
Two important aspects for gene therapy are to increase the level of gene expression and track the gene delivery site and expression, and a sensitive reporter gene may be one of the options for preclinical studies and possibly for human clinical trials. We report the novel concept of increasing the activity of the gene products. With the insertion of the mini-peptide-coding sequence CWDDWLC into the plasmid DNA of a SEAP reporter gene, we observed vast increases in the enzyme activity in vitro in all murine and human cell lines used. Also, in vivo injection of this CWDDWLC-SEAP encoding gene resulted in the same increases in reporter gene activity, but these increases did not correspond to alterations in the level of the gene products in the serum. Minor sequence changes in this mini-peptide negate the activity increase of the reporter gene. We report the novel concept of increasing the activity of gene products as another method to improve the reporting sensitivity of reporter genes. This improved reporter gene could complement any improved vector for maximizing the reporter sensitivity. Also, this strategy has the potential to be used to discover peptides that improve the activity of therapeutic genes.
PMCID: PMC2808434  PMID: 19713998
Reporter gene; SEAP; peptide; vector; in vivo
9.  Expression of WSX1 in tumors sensitizes IL27-signaling independent NK cell surveillance 
Cancer research  2009;69(13):5505-5513.
It is well known that the interleukin (IL) 27 receptor WSX1 is expressed in immune cells and induces an IL27-dependent immune response. Opposing this conventional dogma, this study reveals a much higher level of WSX1 expression in multiple types of epithelial tumor cells when compared to normal epithelial cells. Expression of exogenous WSX1 in epithelial tumor cells suppresses tumorigenecity in vitro and inhibits tumor growth in vivo. Different from the role of WSX1 in immune cells, the antitumor activity of WSX1 in epithelial tumor cells is independent of IL27 signaling but is mainly dependent on NK cell surveillance. Deficiency of either the IL27 subunit Epstein–Barr virus-induced gene 3 (EBI3) or the IL27 receptor WSX1 in the host animals had no effect on tumor growth inhibition induced by WSX1 expression in tumor cells. Expression of WSX1 in epithelial tumor cells enhances NK cell cytolytic activity against tumor cells, while the absence of functional NK cells impairs the WSX1-mediated inhibition of epithelial tumor growth. The underlying mechanism by which WSX1 expression in tumor cells enhances NK cytolytic activity is dependent on upregulation of NKG2D ligand expression. Our results reveal an IL27-independent function of WSX1—sensitizing NK cell-mediated antitumor surveillance via an NKG2D-dependent mechanism.
PMCID: PMC2706921  PMID: 19549909
Innate Immunity; NK cell; IL27; Interleukin; TCCR; Tumor; WSX1

Results 1-9 (9)