Che-Hsin Lee

Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model

Some anaerobic and facultative anaerobic bacteria have been used experimentally as anticancer agents because of their selective growth in the hypoxia regions of solid tumors after systemic administration. We have previously shown the feasibility of using attenuated Salmonella choleraesuis as a gene delivery vector. In this study, we exploited S. choleraesuis carrying thrombospondin-1 (TSP-1) gene for treating primary melanoma and experimental pulmonary metastasis in the syngeneic murine B16F10 melanoma model. Systemic administration of S. choleraesuis allowed targeted gene delivery to tumors. The bacteria accumulated preferentially in tumors over livers and spleens at ratios ranging from 1000:1 to 10,000:1. The level of transgene expression via S. choleraesuis-mediated gene transfer in tumors could reach more than 1800-fold higher than in livers and spleens. Notably, bacterial accumulation was also observed in the lungs with metastatic nodules, but not in healthy lungs. When administered into mice bearing subcutaneous or pulmonary metastatic melanomas, S. choleraesuis carrying TSP-1 gene significantly inhibited tumor growth and enhanced survival of the mice. Immunohistochemical studies in the tumors from these mice displayed decreased intratumoral microvessel density. Taken together, these findings suggest that TSP-1 gene therapy delivered by S. choleraesuis may be effective for the treatment of primary as well as metastatic melanomas.

Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models.

Some anaerobic and facultatively anaerobic bacteria have been used experimentally as anticancer agents because of their selective growth in tumors. In this study, we exploited attenuated Salmonella choleraesuis as a tumoricidal agent and a vector to deliver the endostatin gene for tumor‐targeted gene therapy.

T helper cells promote disease progression of osteoarthritis by inducing macrophage inflammatory protein-1γ

OBJECTIVEnImmune cells are involved in the pathogenesis of osteoarthritis (OA). We examined the effects of T helper (Th) cells, which induce the expression of macrophage inflammatory protein (MIP-1γ), on the progression of OA.nnnDESIGNnUsing anterior cruciate ligament-transection (ACLT), we induced OA in one hind-leg knee joint of B6 mice. The CD4(+) T cells from splenocytes and synovium were flow-cytometrically and immunochemically evaluated, respectively. The knee joints were histologically assessed for manifestations of OA. MIP-1γ levels and nuclear factor-κB (NF-κB) in the knee joints were measured using enzyme-linked immunosorbent and immunoblotting assays, respectively; osteoclastogenesis was detected by tartrate-resistant acid phosphatase (TRAP) staining. The inflammatory responses and MIP-1γ expression were examined using immunohistochemistry.nnnRESULTSnThe number of CD4(+) T cells and the expression of interferon-γ (IFN-γ) increased during OA onset (30 days after ACLT) and then decreased at a later stage of OA (90 days after ACLT). Tissue damage induced by CD4(+) T cells was evident at the later stage. The activation of CD4(+) T cells induced the expression of MIP-1γ and NF-κB. The expression of MIP-1γ can be detected in synovium which CD4(+) T cells were infiltrated. The increased MIP-1γ expression caused an increase in the number of osteoclasts in joints. The regulation of CD4(+) T cells was accompanied by increased macrophage infiltration and matrix metalloproteinase (MMP)-9 expression. Histopathological examinations revealed that CD4(+) T cell knockout (CD4(-/-)) mice had less expression of MIP-1γ and slower cartilage degeneration than control mice had.nnnCONCLUSIONSnCD4(+) T cells were activated during the onset of OA, but cartilage damage was more prominent at a later stage. CD4(+) T cells were involved in the pathogenesis of OA: they induced MIP-1γ expression and subsequent osteoclast formation.

Intraarticular gene transfer of thrombospondin-1 suppresses the disease progression of experimental osteoarthritis

In osteoarthritis, angiogenesis, which occurs in the osteochondral junction and synovium, may accelerate inflammation and contribute to the severity of the disease. We used anterior cruciate ligament‐transection (ACLT) to investigate the therapeutic effect of an angiogenesis inhibitor, thrombospondin‐1 (TSP‐1), in a rat model of osteoarthritis. Osteoarthritis was induced in Wistar rats in the knee of one hind leg. After ACLT, AdTSP‐1 (adenoviral vector encoding mouse TSP‐1) was intraarticularly injected into the knee joints. Transgene expression, angiogenesis, and inflammatory responses in the knee joints were examined. They were also assessed morphologically, radiographically, and histologically for manifestations of disease. The levels of TSP‐1 peaked on day 3 and were substantially maintained for at least 9 days after AdTSP‐1 infection. Adenovirus‐mediated gene expression was detected in the synovial membrane and chondrocytes. TSP‐1 gene transfer induced transforming growth factor‐β (TGF‐β) production, but it reduced microvessel density, macrophage infiltration, and interleukin‐1β (IL‐1β) levels. Gross morphological and histopathological examinations revealed that rats treated with AdTSP‐1 had less severe osteoarthritis than controls. In vivo adenovirus‐mediated TSP‐1 gene transfer significantly reduced microvessel density, inflammation, and suppressed the progression of osteoarthritis. This study provides potential applications of TSP‐1 gene delivery for treating osteoarthritis. Published by Wiley Periodicals, Inc. J Orthop Res 28:1300–1306, 2010

Salmonella choleraesuis as an anticancer agent in a syngeneic model of orthotopic hepatocellular carcinoma.

Some anaerobic and facultative anaerobic bacteria represent novel therapeutic agents that have been recently applied in cancer therapy. Previously, we found that Salmonella choleraesuis in combination with cisplatin could retard tumor growth in the murine subsutaneous hepatocellular carcinoma (HCC) model. In this regard, we investigated the antitumor activity of S. choleraesuis in the ML‐1 orthotopic tumor model. Systemically administered S. choleraesuis accumulated within not only subcutaneous but also orthotopic tumors for at least 30 days, forming tumor‐to‐normal tissue ratios exceeding 1,000–10,000 to 1. The antitumor effects of S. choleraesuis were evaluated in mice bearing subcutaneous and orthotopic ML‐1 tumors. Compared with the control treatment, S. choleraesuis significantly prolonged the animal survival, reduced the tumor size, as well as upregulated interferon (IFN)‐γ and induced IFN‐inducible chemokines CXCL10 (IP‐10) productions. Furthermore, immunohistochemical staining of the tumors revealed decreased intratumoral microvessel density, increased infiltration of neutrophils, CD4+ and CD8+ T cells, and induced cell death in tumor microenvironment. In conclusion, these results suggest that tumor‐targeted therapy using S. choleraesuis, which exerts tumoricidal and antiangiogenic activities, represents a potential strategy for the treatment of HCC.

Adenovirus-Mediated Kallistatin Gene Transfer Ameliorates Disease Progression in a Rat Model of Osteoarthritis Induced by Anterior Cruciate Ligament Transection

In osteoarthritis (OA), inflammation and apoptosis are two important factors contributing to disease progression. As kallistatin can suppress inflammatory responses and reduce cell apoptosis, we investigated the therapeutic effect of kallistatin gene transfer in the rat model of OA by anterior cruciate ligament transection (ACLT). OA was induced in Wistar rats by ACLT in the knee of one hind limb. Adenoviral vector encoding human kallistatin (AdHKBP) was injected intraarticularly into the knee joints after ACLT. The viral effect on tissue was evaluated. The inflammatory responses and transgene expression were determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Apoptosis of chondrocytes was quantified by TUNEL assay. The effects of kallistatin in combination with hyaluronic acid (HA) on the medial femoral condyles and synovia were also assessed histologically. Inflammation trigged by the vectors was limited. Expression of human kallistatin after intraarticular injection was identified. Kallistatin gene transfer reduced the levels of interleukin-1beta and tumor necrosis factor-alpha in joints. Examination of gross morphology revealed that rats treated with AdHKBP had reduced severity of OA compared with control rats treated with adenoviral vector encoding green fluorescent protein (AdGFP). The protective effect of kallistatin on cartilage was accompanied by a decrease in apoptotic cells. Intraarticular administration of AdHKBP, when in conjunction with HA, significantly improved knee joint histologic scores. These results suggest that local administration of adenoviral vectors encoding kallistatin significantly suppressed OA progression, accompanied by reduction of inflammatory response and apoptosis. Thus, kallistatin gene therapy may be a potential treatment for OA.

Intra-articular lentivirus-mediated delivery of galectin-3 shRNA and galectin-1 gene ameliorates collagen-induced arthritis

Different members of the galectin family may have inhibitory or stimulatory roles in controlling immune responses and regulating inflammatory reactions in autoimmune diseases such as rheumatoid arthritis (RA). A hypothetical model of a cross talk between galectin-1 and galectin-3 has been established in the circumstance of rheumatoid joints. As galectin-3 is a positive regulator and galectin-1 is a negative regulator of inflammation and autoimmune responses, in this study we evaluated the effects of local knockdown of galectin-3 or overexpression of galectin-1 on ameliorating collagen-induced arthritis (CIA) in rats. Lentiviral vectors encoding galectin-3 small hairpin RNA (shRNA) and galectin-1, as well as two control vectors expressing luciferase shRNA and green fluorescent protein, were individually injected intra-articularly into the ankle joints of rats with CIA, and their treatment responses were monitored by measuring the clinical, radiological and histological changes. Our results show that both knockdown of galectin-3 and overexpression of galectin-1 induced higher percentages of antigen-induced T-cell death in the lymph node cells from arthritic rats. Furthermore, these treatments significantly reduced articular index scores, radiographic scores and histological scores, accompanied with decreased T-cell infiltrates and reduced microvessel density in the ankle joints. Our findings implicate galectin-3 and galectin-1 as potential therapeutic targets for the treatment of RA.

Toll-like receptor 4 signaling promotes tumor growth.

Chronic inflammation is a potential risk factor for tumor progression. The molecular mechanisms linking chronic inflammation and tumor growth have proven elusive. Herein, we describe a new role for Toll-like receptor 4 (TLR4) in tumor-associated macrophages (TAMs) in promoting tumor growth. TAMs can remodel tumor microenvironment and promote tumor growth. With the use of mice lacking TLR4 signaling, we show that TLR4 signaling influences tumor growth and that TLR4 signaling is a critical upstream activator of nuclear factor-kappa B (NF-κB) in TAMs. TLR4-deficient TAMs produce neither proinflammatory cytokines nor angiogenic factors, and activate no NF-κB activity in tumor cells. Furthermore, using macrophage/tumor cell coculture system and adoptive transfer of macrophages with functional TLR4 macrophages to TLR4-deficient mice bearing tumors, we demonstrate an essential role for TLR4 signaling in inducing NF-κB activity in tumor cells and enhancing tumor growth. Antibody neutralization experiments reveal that TAMs are stimulated by heat shock proteins derived from tumor cells through TLR4, leading to production of growth factors, which may in turn promote tumor growth via NF-κB signal pathway. Therefore, this signaling cascade may represent a therapeutic target in cancer.

Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer.

OBJECTIVEnThe phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is known to be activated in rheumatoid arthritis (RA) synovial tissue, which impacts cell growth, proliferation, survival, and migration. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a negative regulator of PI 3-kinase signaling, thus blocking Akt activation. The aim of this study was to examine the effect of PTEN gene transfer in rats with collagen-induced arthritis (CIA).nnnMETHODSnAdenoviral vectors encoding human PTEN (AdPTEN) or beta-galactosidase (AdLacZ) were injected intraarticularly into rats with CIA, and their treatment responses were monitored by measures of clinical, radiographic, and histologic changes. The expression of phosphorylated Akt, total Akt, vascular endothelial growth factor (VEGF), proinflammatory cytokines, and chemokines, as well as the extent of microvessel density in the ankle joints were determined.nnnRESULTSnAdPTEN treatment reduced Akt phosphorylation and decreased VEGF production in human RA synovial fibroblasts. Compared with AdLacZ treatment of the rats with CIA, AdPTEN treatment significantly reduced ankle circumference, articular index scores, radiography scores, and histology scores, and also decreased microvessel density and levels of VEGF and interleukin-1beta. Furthermore, PTEN gene transfer led to down-regulation of Akt activation and increased apoptosis in the ankle joints.nnnCONCLUSIONnThis study is the first to demonstrate the in vivo effect of intraarticular gene delivery of PTEN on amelioration of arthritis symptoms in rats with CIA, which involved antiangiogenic, antiproliferative, and antiinflammatory effects of PTEN via inhibition of the PI 3-kinase/Akt signaling pathway. Our findings also implicate the PI 3-kinase/Akt pathway as a therapeutic target for the treatment of RA or other inflammatory diseases.

Suppression of collagen-induced arthritis by intra-articular lentiviral vector-mediated delivery of Toll-like receptor 7 short hairpin RNA gene

Knockdown of Toll-like receptors (TLRs) is a novel therapeutic strategy in treating patients with rheumatoid arthritis (RA). We examined the effects of lentiviral vector-mediated delivery of TLR7 short hairpin RNA gene (Lt.shTLR7) on collagen-induced arthritis (CIA). After being immunized on days 0 and 7, Sprague–Dawley rats received intra-articular (i.a.) injection of Lt.shTLR7 or scramble control vector on days 7 and 10. The therapeutic effects were evaluated by measuring ankle circumferences, articular index, and radiographic and histological scores on killing on day 16. Microvessel densities, vascular endothelial growth factor (VEGF) levels, pro-inflammatory cytokine concentrations and T-cell numbers within the synovial tissues were measured. Moreover, VEGF and pro-inflammatory cytokine concentrations in culture supernatants from TLR7-transfected synovial fibroblasts (SFs) stimulated with imiquimod or endogenous ligands were examined. There were significant reduction in ankle circumferences, articular indexes, and radiographic and histological scores. Microvessel densities, VEGF concentrations, interleukin (IL)-1β and IL-6 levels and T-cell densities within synovial tissues were significantly lower. Induction of VEGF, IL-1β and IL-6 production from stimulated SFs was significantly suppressed. Taken together, these data demonstrate the effects of i.a. lentiviral vector-mediated delivery of shTLR7 RNA gene on inhibition of CIA, and implicate the manipulation of TLR7 as a potential therapeutic strategy in RA patients.