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Lung-Ji Chang

Publications and source records attributed to Lung-Ji Chang.

At least 19 recordsLinked to original sources
PubMed2006

Induction of primary anti-HIV CD4 and CD8 T cell responses by dendritic cells transduced with self-inactivating lentiviral vectors.

In this study, we demonstrate that a minimal self-inactivating (SIN) lentiviral vector (LV) that does not encode any human immunodeficiency virus (HIV) genes is able to induce HIV-specific CD4 and CD8 T cell responses after transduction of dendritic cells (DCs). The LV-DC-primed T cells displayed HIV-specific lytic degranulation, as illustrated by acquisition of CD107a/b expression on the cell surface and up-regulation of active caspase 3. HIV-specific cytotoxic T lymphocyte (CTL) response was consistently detected using different assays, and T cell receptors specific to three prominent HIV epitopes, SL9 (Gag peptide: SLYNTVATL), IV9 (Pol peptide: ILKEPVHGV), and MA10 (In peptide: MASDFNLPPV) were detected using HLA-A0201 peptide-tetramers. These results demonstrate that DCs transduced with the minimal SIN-LV can efficiently induce HIV-specific CD4 and CD8 T cell responses. Since LVs are popular gene transfer tools, our results have fundamental implications for future LV applications and DC vaccine development.

CD4-Positive T-Lymphocytes↗
PubMed2006

Efficient generation of transgenic rats through the male germline using lentiviral transduction and transplantation of spermatogonial stem cells.

Spermatozoa produced from spermatogonial stem cells (SSCs) are the vehicle by which genes of a male are passed to the next generation. A single SSC has the ability to self-renew and produce thousands of spermatozoa; therefore, it is an ideal target for genetic modification to efficiently generate transgenic animals in mammalian species. Rats are an important model organism for biological research; however, gene function studies have been difficult because of a limited ability to generate transgenic animals. Transgenic rat production through SSCs offers a means to overcome this obstacle. Because SSCs divide slowly both in vivo and in vitro, lentiviral vectors may be an ideal method for introducing stable genetic modification. Using a lentiviral vector, an enhanced green fluorescent protein (eGFP) transgene was introduced into the genome of cultured rat SSCs, which were microinjected into testes of immunodeficient mice to assess transduction efficiency. Approximately 40% of rat SSCs exposed to the lentiviral vector overnight carried the eGFP transgene and generated colonies of spermatogenesis. When transduced SSCs were transplanted into recipient rat testes, in which endogenous germ cells had been decreased but not eliminated by busulfan treatment, approximately 6% of offspring were transgenic. The transgene was stably integrated into the donor SSC genome and transmitted to and expressed by progeny in subsequent generations. Thus, lentiviral transduction of SSCs followed by transplantation is an effective means for generating transgenic rats through the male germline, and this approach may be applicable to other species in which existing methods are inadequate or not applicable.

Animals↗
PubMed2006

Derivation and large-scale expansion of multipotent astroglial neural progenitors from adult human brain.

The isolation and expansion of human neural cell types has become increasingly relevant in restorative neurobiology. Although embryonic and fetal tissue are frequently envisaged as providing sufficiently primordial cells for such applications, the developmental plasticity of endogenous adult neural cells remains largely unclear. To examine the developmental potential of adult human brain cells, we applied conditions favoring the growth of neural stem cells to multiple cortical regions, resulting in the identification and selection of a population of adult human neural progenitors (AHNPs). These nestin(+) progenitors may be derived from multiple forebrain regions, are maintainable in adherent conditions, co-express multiple glial and immature markers, and are highly expandable, allowing a single progenitor to theoretically form sufficient cells for approximately 4x10(7) adult brains. AHNPs longitudinally maintain the ability to generate both glial and neuronal cell types in vivo and in vitro, and are amenable to genetic modification and transplantation. These findings suggest an unprecedented degree of inducible plasticity is retained by cells of the adult central nervous system.

Adolescent↗
PubMed2006

Role of Pax4 in Pdx1-VP16-mediated liver-to-endocrine pancreas transdifferentiation.

Although Pdx1-VP16 expression induces hepatic cell transdifferentiation into pancreatic precursor cells (WB-1), these incompletely reprogrammed cells fail to become glucose-sensitive insulin-producing cells in the absence of the activation of late-stage pancreatic transcription factors. As Pax4 promotes late-stage beta-cell differentiation and maturation, we generated lentiviral vector (LV) containing mouse Pax4 gene and developed two hepatic cell lines expressing Pax4 in the absence (WB-2 cells) or presence (WB-1A cells) of Pdx1-VP16, via LV-mediated gene transfer. Functional Pax4 protein expression in WB-2 and WB-1A cells was confirmed by electrophoretic mobility shift assay and Pdx1-VP16 protein expression in WB-1 and WB-1A cells was confirmed by Western blotting. Activation of Pax4 resulted in the expression of the late-stage transcription factors, including Pax6, Isl-1, and MafA, and generated a gene expression profile for WB-1A cells similar to that of functional rat insulinoma INS-1 cells. Insulin abundance in WB-1A cells was demonstrated by immunostaining. WB-1A cells exhibited glucose-responsive insulin release in vitro, and caused a rapid reversal of hyperglycemia following cell transplantation into streptozotocin-induced diabetic mice. Intraperitoneal glucose tolerance test showed a normal glucose response in WB-1, and WB-1A transplanted mice similar to that of normal mice. Removal of transplanted WB-1A cells resulted in a return of hyperglycemia, confirming that they were responsible for the observed normoglycemia. The explanted WB-1A cells exhibited strong insulin staining comparable to native islet beta-cells. These studies indicate that activation of Pax4 in Pdx1-VP16-expressing cells reprograms pancreatic precursor-like WB-1 cells into glucose-responsive, more mature insulin-producing cells.

Animals↗
PubMed2006

Technical hurdles in a pilot clinical trial of combined B7-2 and GM-CSF immunogene therapy for glioblastomas and melanomas.

OBJECTIVE: Malignant glioblastomas and melanomas continue to have a dismal prognosis despite advances in conventional therapy. This has led to investigations of novel treatment strategies including immunogene therapy. We report a pilot clinical trial of combined B7-2 and GM-CSF immunogene therapy for gliomas and melanomas and discuss technical hurdles encountered. METHODS: Patients with recurrent malignant gliomas or medically refractory melanomas were vaccinated with irradiated autologous tumor cells transduced with B7-2 and GM-CSF genes using a retroviral vector. Patients were monitored for toxicity, inflammatory/immune reactions, and clinical status. RESULTS: Vaccine preparation was attempted from 116 malignant glioma and 32 melanoma specimens. Adequate vaccines could only be prepared for five glioblastoma and three melanoma patients. Six patients (three recurrent glioblastomas and three melanomas) were actually vaccinated. Minor toxicities included flu-like symptoms (3/6), injection site erythema (4/6), and asymptomatic elevations in liver enzymes (3/6). Most patients showed evidence of an inflammatory response but specific anti-tumor immunity was not demonstrated. All six patients have died, although three patients with minimal residual disease at treatment had prolonged recurrence-free intervals after vaccination. CONCLUSIONS: Combined B7-2 and GM-CSF immunogene therapy for glioblastomas and melanomas using autologous tumor cells has many technical pitfalls hindering large scale application and evaluation. As a result, this pilot study was too limited to draw meaningful conclusions regarding safety or anti-tumor immunity. While immunotherapy has been promising in pre-clinical studies, alternate strategies will be required to bring these benefits to patients.

Adult↗
PubMed2006

An effective cancer vaccine modality: lentiviral modification of dendritic cells expressing multiple cancer-specific antigens.

Viral modification of dendritic cells (DCs) may deliver a "danger signal" critical to the hypo-reactive DCs in cancer patients. Using three highly differentially expressed hepatoma tumor-associated antigens (TAAs): stem cell antigen-2 (Sca-2), glycoprotein 38 (GP38) and cellular retinoic acid binding protein 1 (RABP1), we explored the therapeutic potential of the DCs modified with lentiviral vectors (LVs). Preventive and therapeutic injection of the LV-TAA-DC vaccine into tumor-bearing mice elicited a strong anti-tumor response and extended survival, which was associated with tumor-specific interferon-gamma and cytotoxic T cell responses. In vivo elimination of the LV-TAA-DCs by a co-expressed thymidine kinase suicide gene abrogated the therapeutic effect. The modification of DCs with LVs encoding multiple TAAs offers a great opportunity in cancer immunotherapy.

Animals↗
PubMed2006

CpG sites preferentially methylated by Dnmt3a in vivo.

Dnmt3a and Dnmt3b are two major de novo DNA methyltransferases essential for embryonic development in mammals. It has been shown that Dnmt3a and Dnmt3b have distinct substrate preferences for certain genomic loci, including major and minor satellite repeats. However, the exact target CpG sites where Dnmt3a and Dnmt3b catalyze DNA methylation remains largely unknown. To identify a CpG site that is specifically methylated by Dnmt3a or Dnmt3b, we screened methylated genomic loci by methylation sensitive restriction fingerprinting using genomic DNA from wild-type, Dnmt3a null, Dnmt3b null, and Dnmt3a-Dnmt3b double null ES cells. Interestingly, one of the CpG sites was preferentially methylated in wild-type and Dnmt3b null ES cells but not in Dnmt3a null or Dnmt3a-Dnmt3b double null ES cells, suggesting that the site-specific methylation was Dnmt3a-dependent. Sequencing results revealed that the isolated CpG site is located within the 1st exon of the G isoform of fibroblast growth factor (Fgf-1.G) on mouse chromosome 18. Exogenous expression of Dnmt3a but not Dnmt3b in the double null ES cells restored DNA methylation of this CpG site. When we examined alternative transcription initiation sites, we determined that another CpG site in the 5'-flanking region of the Fgf-1.A isoform was also methylated specifically by Dnmt3a. Using chimeric constructs between Dnmt3a and Dnmt3b, we further determined that the NH(2)-terminal regulatory domain of Dnmt3a was responsible for establishing its substrate specificity. These results indicate that certain CpG sites within the Fgf-1 gene locus are preferentially methylated by Dnmt3a but not by Dnmt3b. Selective methylation by a specific member of Dnmt3 may therefore play a role in the orchestration of gene expression during embryonic development.

Animals↗
PubMed2006

Reprogramming liver-stem WB cells into functional insulin-producing cells by persistent expression of Pdx1- and Pdx1-VP16 mediated by lentiviral vectors.

Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone single-cell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1- or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia.

Animals↗
PubMed2006

Lentiviral vector-mediated gene transfer in embryonic stem cells.

The major limitations in gene transduction to embryonic stem (ES) cells are (1) low efficiency of gene delivery and (2) suppression of gene expression after integration into the host genome. A human immunodeficiency virus type I (HIV-1)-based lentiviral vector has been demonstrated to be an excellent tool for stable and efficient gene expression in ES cells. Here, we introduce a protocol for lentiviral vector-mediated transgene expression in murine ES cells. Using lentiviral vectors expressing LacZ, green fluorescent protein, and Cre recombinase, we demonstrate the efficiency and utility of the vectors in ES cell study.

Animals↗
PubMed2005

De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'-deoxycytidine.

The deoxycytidine analog 5-aza-2'-deoxycitidine (5-aza-dC) is a potent chemotherapeutic agent effective against selective types of cancer. The molecular mechanism by which 5-aza-dC induces cancer cell death, however, is not fully understood. It has been accepted that the mechanism of toxicity is due to the covalent binding between the DNA methyltransferase (Dnmt) and 5-aza-dC-substituted DNA. In order to define which member of the Dnmt family plays a dominant role in the cytotoxicity, we examined the effect of 5-aza-dC on cell growth and apoptosis in various Dnmt null mutant embryonic stem (ES) cells. Of interest, Dnmt3a-Dnmt3b double null ES cells were highly resistant to 5-aza-dC when compared to wild type, Dnmt3a null, Dnmt3b null, or Dnmt1 null ES cells. The cellular sensitivity to 5-aza-dC correlated well with the expression status of Dnmt3 in both undifferentiated and differentiated ES cells. When exogenous Dnmt3a or Dnmt3b was expressed in double null ES cells, the sensitivity to 5-aza-dC was partially restored. These results suggest that the cytotoxic effect of 5-aza-dC may be mediated primarily through Dnmt3a and Dnmt3b de novo DNA methyltransferases. Further, the ability to form Dnmt-DNA adducts was similar in Dnmt1 and Dnmt3, and the expression level of Dnmt3 was not higher than that of Dnmt1 in ES cells. Therefore, Dnmt3-DNA adducts may be more effective for inducing apoptosis than Dnmt1-DNA adducts. These results imply a therapeutic potential of 5-aza-dC to cancers expressing Dnmt3.

Animals↗
PubMed2005

Development of immunoglobulin G enzyme-linked immunosorbent assay for the serodiagnosis of severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel SARS-associated coronavirus (SARS-CoV). The clinical characteristics are high fever, rapidly progressive diffuse pneumonitis and respiratory distress. It is highly infectious through intimate contact or direct contact with infectious body fluids. Outbreaks within communities and hospitals have been reported. Development of rapid and reliable diagnostic tools is urgently needed. We developed an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), using whole virus antigen of SARS-CoV. Eighty-six serum samples collected from patients who were hospitalized for other causes were examined to determine the cut-off O.D. value. The cut-off O.D. value was defined as 0.175 by calculating the mean O.D. value of the 86 sera plus 3 standard deviations. To determine the sensitivity and specificity of the ELISA, 56 positive sera and 204 negative sera were tested. The sensitivity was 96.4% and the specificity was 100%. The results suggest that the IgG ELISA using whole virus antigen of SARS-CoV has a high sensitivity and specificity in detecting SARS IgG antibodies. This IgG ELISA is a powerful tool for serodiagnosis of SARS.

Antigens, Viral↗
PubMed2005

Mycoplasma-mediated alterations of in vitro generation and functions of human dendritic cells.

While tumor cell-derived factors have been demonstrated to hamper the in vitro differentiation and maturation of dendritic cells (DCs) from hematopoietic stem cells, their effects on DC differentiation from CD14+ plastic-adherent monocytic precursors have been controversial. To address this issue, we examined the effects of the culture supernatants from six tumor cell lines on in vitro DC differentiation and maturation from monocytes. Two tumor cell supernatants, MDA468 and 293T, were found to be able to affect the in vitro differentiation of DCs from monocytic precursors, leading to the generation of a distinct type of DC with markedly reduced expression of DC-SIGN, downregulation of CD11c, HLA-DR and CD1a, and upregulation of CD123, HLA-ABC, CD80, CD40, CD86, CD54, CD83, CD25 and CCR7. Functionally, these DCs exhibited reduced phagocytosis and enhanced allostimulatory capacity. Further investigation demonstrated that the changes in DC phenotype and functions were due to the presence of mycoplasmas in these two cell lines; eradication of mycoplasmas completely abolished the observed effects, and importantly, pure mycoplasmas in the absence of tumor cell supernatants were able to produce the same effects. Since mycoplasmas are common contamination agents in routine tissue culture, our results caution that many reported effects of DCs in culture warrant re-evaluation. The distinct effects of mycoplasmas on DC differentiation described in this report could potentially benefit future development of DC-based vaccination and therapeutic applications.

Biomarkers↗
PubMed2005

Genetic engineering of human embryonic stem cells with lentiviral vectors.

Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

Cell Differentiation↗
PubMed2005

Improving erectile function by silencing phosphodiesterase-5.

PURPOSE: We investigated whether phosphodiesterase-5 (PDE5) can be down-regulated by a specific small interfering RNA (siRNA) and whether this would improve erectile function. MATERIALS AND METHODS: A PDE5 siRNA encoding oligonucleotide was inserted into pSUPER-retro vector, resulting in the siRNA expressing construct, pPDE5-silencer. The construct was packaged into oncoretroviral particles and then transduced into rat cavernous smooth muscle cells (CSMCs). Cells were examined for PDE5 expression by reverse transcriptase-polymerase chain reaction, Western blotting and immunofluorescence microscopy. Cells were then treated with 10 microM sodium nitroprusside (SNP) and examined for cyclic guanosine monophosphate (cGMP) at 0, 10, 30, 60 and 240 minutes. The siRNA expressing cassette was transferred from the oncoretrovirus to lentivirus, which was then injected into rat penises. Three months later erectile function was examined by electrostimulation and PDE5 expression in cavernous smooth muscle was determined by immunohistochemistry. RESULTS: CSMCs transfected with pPDE5-silencer (CSMC plus siRNA) showed an 88.2% decrease in PDE5 compared with CSMCs transfected with control vector (CSMCs plus vector). Within 10 minutes of SNP treatment cells (CSMCs, CSMCs plus vector and CSMCs plus siRNA) showed similar sharp increases in cGMP. However, while cGMP levels in CSMCs and CSMCs plus vector returned to almost baseline in 1 hour, the cGMP level in CSMCs plus siRNA remained high even 4 hours after SNP treatment. Rats injected with the siRNA expressing lentivirus showed increased electrostimulated erectile function, as measured by peak intracorporeal pressure and the intracorporeal pressure increase, compared with rats injected with control lentivirus. PDE5 expression was decreased in the siRNA treated cavernous smooth muscle. CONCLUSIONS: PDE5 expression could be decreased by siRNA, resulting in prolonged cGMP accumulation and improved erection.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2005

Dynamic DNA methylation and histone modifications contribute to lentiviral transgene silencing in murine embryonic carcinoma cells.

Embryonic stem cells are subjected to a dynamic genome regulation during development. Here we report that the ectopic lentiviral transgenes are quickly silenced in murine embryonic carcinoma P19 cells. The silencing was correlated with CpG hypermethylation in the transgene promoter. Using high-resolution sodium bisulfite genome sequencing, we detected distinct DNA methylation kinetics in different proviral regions. DNase I sensitivity and chromatin immunoprecipitation assays revealed condensed chromatin structure and histone code switch during silencing. Longitudinal analysis of nonsilenced and silenced identical single-cell clones revealed that the silencing was coupled with CpG methylation in the promoter, as well as a global histone H3 deacetylation. Interestingly, the primer binding site and the packaging signal region appeared to serve as a DNA methylation initiation center which was rapidly hypermethylated regardless of transgene silencing and chromatin modifications. Analysis of cellular genes 45 to 50 kbp upstream and downstream of the integration site indicated that transcriptional activities of the flanking host genes were not affected. Genetic modifications of stem cells have great therapeutic potentials and our results picture a dynamic embryonic genome response to ectopic transgene integration that may have important implications in the future safety and efficacy modifications of stem cells.

Animals↗
PubMed2004

Alteration of T cell immunity by lentiviral transduction of human monocyte-derived dendritic cells.

BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells that play important roles during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 derived lentiviral vectors (LVs) transduce DCs at high efficiency but their effects on DC functions have not been carefully studied. Modification of DCs using LVs may lead to important applications in transplantation, treatment of cancer, autoimmune and infectious diseases. RESULTS: Using DCs prepared from multiple blood donors, we report that LV transduction of DCs resulted in altered DC phenotypes and functions. Lentiviral transduction of DCs resulted in down-regulation of cell surface molecules including CD1a, co-stimulatory molecules CD80, CD86, ICAM-1, and DC-SIGN. DCs transduced with LVs displayed a diminished capacity to polarize naive T cells to differentiate into Th1 effectors. This impaired Th1 response could be fully corrected by co-transduction of DCs with LVs encoding interleukin-12 (IL-12), interferon-gamma (IFN-gamma), or small interfering RNA (siRNA) targeting IL-10. CONCLUSIONS: DCs transduced with LVs in vitro displayed diminished Th1 functions due to altered DC phenotypes. Our study addresses an important issue concerning lentiviral infection and modification of DC functions, and provides a rational approach using LVs for immunotherapy.

Base Sequence↗
PubMed2004

Functional characterization of hepatoma-specific stem cell antigen-2.

Identification of tumor-specific antigens and genetic pathways may lead to potential diagnostic and therapeutic applications in cancer treatment. cDNA microarray has been used in cancer gene profiling, but the broad spectrum of data accruing and narrow signal-to-noise range of this technology have limited its use in rapid identification of highly differentially expressed tumor genes. Here, we used a modified suppression subtractive hybridization (SSH) method to isolate a small number of highly differentially expressed genes from murine hepatoma cells. For functional analysis of these hepatoma-specific genes, we employed the small interference RNA (siRNA)-mediated gene silencing method with lentiviral vectors, which have the advantages of high delivery efficiency and long lasting effect. Stem cell antigen-2 (Sca-2) was identified as one of the highest differentially expressed tumor antigens. Lentiviral siRNA successfully suppressed >90% of Sca-2 expression and the suppression lasted longer than 3 mo. Interestingly, inhibition of Sca-2 induced rapid hepatoma cell apoptosis, and the survival Sca-2-negative hepatoma cells exhibited high sensitivity to extrinsic tumor necrosis factor alpha (TNF-alpha) apoptosis signal but not intrinsic apoptosis signal. Analysis of TNF receptor 1 (TNFR1) by flow cytometry and Western blotting indicated that Sca-2 expression downregulated cell surface but not de novo synthesis of TNFR1 in the hepatoma cells. Together, our results suggested that Sca-2 was a signal transducer situated at the nexus of surface molecules regulating death receptor-mediated apoptosis. The technology illustrated that this method can deduce a small number of highly differentially expressed tumor genes that may have diagnostic and therapeutic potential.

Animals↗