Jeng-Long Hsieh

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

OBJECTIVE Immune 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. DESIGN Using 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. RESULTS The 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. CONCLUSIONS CD4(+) 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

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.

CD8+ T cell-induced expression of tissue inhibitor of metalloproteinses-1 exacerbated osteoarthritis.

Despites the fact that T cells are involved in the pathogenesis of osteoarthritis (OA) little is known about the roles of CD8+ T cells in this disease. We investigated the effects of CD8+ T cells and the expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) on joint pathology. Using anterior cruciate ligament-transection (ACLT), OA was induced in mice. The knee joints were histologically assessed for manifestations of OA. The CD8+ T cells from splenocytes and synovium were flow-cytometrically and immunochemically evaluated, respectively. Local expression of TIMP-1, matrix metalloproteinase (MMP)-13, and VEGF were examined. Cartilage degeneration was slower in CD8+ T cell knockout mice than in control mice. CD8+ T cells were activated once OA was initiated and expanded during OA progression. More CD8+ T cells from splenocytes expressed TIMP-1 in ACLT-group mice than in Sham-group mice. The number of TIMP-1-expressing CD8+ T cells in OA mice correlated with the disease severity. TIMP-1 expression in cartilage was co-localized with that of MMP-13 and VEGF. TIMP-1 protein was detected in synovium in which angiogenesis occurred. During the pathogenesis of OA, the expression of TIMP-1, VEGF and MMP-13 accompanying with CD8+ T cells activation were increased. Furthermore, inhibiting the expression of TIMP-1 in joints could retard the progression of OA.

Inhibition of cartilage damage by pro-opiomelanocortin prohormone overexpression in a rat model of osteoarthritis

Pro-opiomelanocortin (POMC) is a precursor of various neuropeptides. POMC-derived neuropeptides are potent inflammation inhibitors and immunosuppressants. Evidence that osteoarthritis (OA) is an inflammatory disease is accumulating. We assessed whether intra-articular gene delivery of POMC ameliorates experimentally induced OA in a rat model. OA was induced in Wistar rats by anterior cruciate ligament-transection (ACLT) in the knee of one hind limb. Adenoviral vector encoding human POMC (AdPOMC) was injected intra-articularly into the knee joints after ACLT. The transgene expression and the inflammatory responses were evaluated using immunoblotting, immunohistochemistry and enzyme-linked immunosorbent assay. The treated joints were assessed histologically for manifestations of the disease. Human POMC was expressed in the chondrocytes and synovial membrane after the intra-articular injection. POMC gene transfer reduced nuclear factor-κB activity and the levels of interleukin-1β in HTB-94 chondrosarcoma cells and Raw 264.7 macrophages; it also reduced microvessel density in the synovium. Histological examination showed that symptoms of OA in AdPOMC-treated rats were less severe than in rats treated with either empty adenoviral vector (AdNull) or normal saline. Intra-articular injection of adenoviral vectors expressing POMC significantly suppressed the progression and severity of OA, and reduced inflammatory responses and angiogenesis. POMC gene delivery may offer novel therapeutic approach for treating OA.

Lentiviral small hairpin RNA knockdown of macrophage inflammatory protein-1γ ameliorates experimentally induced osteoarthritis in mice.

Immune cells are involved in the pathogenesis of osteoarthritis (OA). CD4(+) T cells were activated during the onset of OA and induced macrophage inflammatory protein (MIP)-1γ expression and subsequent osteoclast formation. We evaluated the effects of local knockdown of MIP-1γ in a mouse OA model induced by anterior cruciate ligament transection. The mouse macrophage cell lines and osteoclast-like cells generated from immature hematopoietic monocyte/macrophage progenitors of murine bone marrow were cocultured with either receptor activator of NFκB ligand (RANKL) or CD4(+) T cells. The levels of MIP-1γ and RANKL in cells and mice were examined by enzyme-linked immunosorbent assay (ELISA). The osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase and cathepsin K staining. OA was induced in one hind-leg knee joint of B6 mice. Lentiviral vector encoding MIP-1γ small hairpin RNA (shRNA) and control vector were individually injected intra-articularly into the knee joints, which were histologically assessed for manifestations of OA. The expression of MIP-1γ and matrix metalloproteinase (MMP)-13 and the infiltration of CD4(+) T cells, macrophages, and osteoclastogenesis in tissues were examined using immunohistochemistry. CD4(+) T cells were involved in OA by inducing MIP-1γ expression in osteoclast progenitors and the subsequent osteoclast formation. Neutralizing MIP-1γ with a specific antibody abolishes RANKL-stimulated and CD4(+) T-cell-stimulated osteoclast formation. MIP-1γ levels were significantly higher in synovium and the chondro-osseous junction of joints 90 days postsurgery. The number of infiltrated CD4(+) T cells and macrophages and IL-1β expression were reduced in the synovial tissues of mice treated with MIP-1γ shRNA. Histopathological examinations revealed that mice treated with MIP-1γ shRNA had less severe OA than control mice had, as well as decreased osteoclast formation and MMP-13 expression. Locally inhibiting MIP-1γ expression may ameliorate disease progression and provide a new OA therapy.

Gene therapy for bladder cancer using E1B-55 kD-deleted adenovirus in combination with adenoviral vector encoding plasminogen kringles 1–5

Mutations or loss of heterozygosity of p53 are detected in approximately 50% of bladder cancers. E1B-55 kD-deleted adenovirus has been shown to kill tumour cells with defective p53 function while sparing normal cells. Here, we examined the cytolytic effect and replication of E1B-55 kD-deleted adenovirus, designated Ad5WS1, on human bladder cancer cell lines with various p53status. Ad5WS1 caused more severe cytolytic effect and replicated more efficiently in J82 and TCC-SUP bladder cancer cells carrying mutant p53compared with TSGH-8301 and BFTC-905 bladder cancer cells retaining wild-type p53. Introduction of dominant negative p53into BFTC-905 cells rendered them more susceptible to Ad5WS1-induced cytolysis. Furthermore, cells susceptible to lysis caused by Ad5WS1 were not attributable to their greater infectability by adenovirus. Finally, Ad5WS1 suppressed the growth of TCC-SUP bladder tumour xenografts, which could be augmented when combined with replication-defective adenoviral vector encoding kringles 1–5 of plasminogen (K1–5), an angiogenic inhibitor. Taken together, our results show that E1B-55 kD-deleted adenovirus replicates and hence lyses bladder cancer cells with mutant p53much more efficient than those with wild-type p53. Thus, E1B-deleted adenovirus may have therapeutic potential, especially in combination with adenoviral vector expressing K1–5, for the treatment of bladder cancer.

Potent antitumor activity of Oct4 and hypoxia dual-regulated oncolytic adenovirus against bladder cancer

Most solid tumors undergo hypoxia, leading to rapid cell division, metastasis and expansion of a cell population with hallmarks of cancer stem cells (CSCs). Tumor-selective replication of oncolytic adenoviruses may be hindered by oxygen deprivation in tumors. It is desirable to develop a potent oncolytic adenovirus, retaining its antitumor activity even in a hypoxic environment. We have previously generated an Oct4-dependent oncolytic adenovirus, namely Ad9OC, driven by nine copies of the Oct4 response element (ORE) for specifically killing Oct4-overexpressing bladder tumors. Here, we developed a novel Oct4 and hypoxia dual-regulated oncolytic adenovirus, designated AdLCY, driven by both hypoxia response element (HRE) and ORE. We showed that hypoxia-inducible factor (HIF)-2α and Oct4 were frequently overexpressed in hypoxic bladder cancer cells, and HIF-2α was involved in HRE-dependent and Oct4 transactivation. AdLCY exhibited higher cytolytic activities than Ad9OC against hypoxic bladder cancer cells, while sparing normal cells. AdLCY exerted potent antitumor effects in mice bearing human bladder tumor xenografts and syngeneic bladder tumors. It could target hypoxic CD44- and CD133-positive bladder tumor cells. Therefore, AdLCY may have therapeutic potential for targeting hypoxic bladder tumors and CSCs. As Oct4 is expressed in various cancers, AdLCY may be further explored as a broad-spectrum anticancer agent.

A Population-Based 16-Year Study on the Risk Factors of Surgical Site Infection in Patients after Bone Grafting: A Cross-Sectional Study in Taiwan.

AbstractBone grafting is a commonly used orthopedic surgical procedure that will provide bone formation in bone defects or regions of defective bone healing. A major complication following bone grafting is a postoperative recipient graft site infection that is associated with substantial mortality and increased use of medical resources. The purpose of the study was to identify the risk factors associated with infection after bone-grafting surgery.Data from 1,303,347 patients listed in the Taiwan National Health Insurance Research Database (NHIRD) and admitted to hospitals from 1997 through 2012 who underwent primary bone grafting (mean age: 46.57 years old; mean length of hospital stay: 8.04 days) were analyzed. The incidence of infection by age, hospital stay, gender, income, chronic disease (tuberculosis [TB]; diabetes mellitus [DM]; acquired immunodeficiency syndrome [AIDS]), fracture complications (nonunion; delayed union fracture), types of graft and hospital was evaluated.Three percent of the patients developed a postoperative recipient graft site infection. Multivariable analysis revealed that patients were more likely to develop a post bone-grafting surgery infection if they were older, had a longer hospital stay, were male, had a lower income, or had comorbid TB, DM, or AIDS. Patients were more likely to develop an infection if they had a nonunion, an alloplast graft, or treated in a local clinic.Our findings should provide a clinically relevant reference for surgeons who perform bone grafting. Patients should be informed of the potential risks.

The Estrogen Receptor-β Expression in De Quervain's Disease.

Stenosing tenosynovitis of the first dorsal compartment of the wrist (a.k.a. de Quervain’s disease) is common but how estrogen is involved is still unknown. We previously reported that inflammation was involved in the pathogenesis of this ailment. In the present study, we extended our investigation of estrogen receptor (ER)-β expression to determine whether estrogen is involved in the pathogenesis of de Quervain’s. Intraoperative retinaculum samples were collected from 16 patients with the ailment. Specimens were histologically graded by collagen structure and immunohistochemically evaluated by quantifying the expression of ER-β, interleukin (IL)-1β and IL-6 (inflammatory cytokines), cyclooxygenase (COX)-2 (an inflammatory enzyme), and vascular endothelial growth factor (VEGF), and Von Willebrand’s factor (vWF). De Quervain’s occurs primarily in women. The female:male ratio in our study was 7:1. We found that ER-β expression in the retinaculum was positively correlated with disease grade and patient age. Additionally, disease severity was associated with inflammatory factors—IL-1β and IL-6, COX-2, and VEGF and vWF in tenosynovial tissue. The greater the levels of ER-β expression, tissue inflammation, and angiogenesis are, the more severe de Quervain’s disease is. ER-β might be a useful target for novel de Quervain’s disease therapy.