Combined Chibby and ß-Catenin Predicts Clinical Outcomes in Patients with Hepatocellular Carcinoma.

Chibby is an antagonist of ß-catenin and is considered a potential tumor suppressor protein, but the role of Chibby in hepatocellular carcinoma (HCC) has not been characterized. The expression patterns of Chibby and ß-catenin in HCC specimens and paired adjacent noncancerous tissues were measured by Western blotting and immunohistochemistry. The correlations between Chibby expression and clinicopathological parameters were analyzed. Then the biological functions of Chibby were analyzed in vitro. The Chibby protein was significantly downexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and is a risk factor for HCC recurrence and shorter survival. Furthermore, we found that in HCC tissues the high expression of ß-catenin with low expression of Chibby in the nuclei was an independent predictor for disease-free survival (DFS) (p = 0.012) and overall survival (OS) (p = 0.005). Subsequent genetic manipulation in vitro studies revealed that Chibby knockdown induced the expression of ß-catenin and C-myc, cyclin D1 protein, which promoted cell proliferation and invasiveness. In contrast, overexpression of Chibby decreased ß-catenin expression and inhibited the cell proliferation and invasiveness. Our results suggest that low expression of Chibby was associated with advanced tumor-node-metastasis (TNM) stage and poor differentiation. Furthermore, the combination of Chibby and ß-catenin can predict poor prognosis in patients with HCC. Chibby inhibited HCC progression by blocking ß-catenin signaling in vitro. Chibby is a biomarker and may be a potential therapeutic target for HCC.

Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer.

BACKGROUND: Roles of cancer stem cells and early growth response gene 1 (Egr1) in carcinogenesis have been extensively studied in lung cancer. However, the role of Egr1 in the metastasis of lung cancer remains undetermined, especially in regard to stem cell-related pathways. METHODS: Egr1, osteopontin (OPN) and Oct4 expression in human lung cancer was determined by performing immunohistochemistry. Immunoblotting, ELISA, luciferase reporter assay, chromatin immunoprecipitation assay and RT-PCR were performed to validate the regulation of Oct4-Egr1-OPN axis. Moreover, the effect of Oct4-Egr1-OPN axis on lung cancer progression was evaluated by cell migration assay and mice study. RESULTS: We detected Oct4, Egr1, and OPN expression in clinical specimens from 79 lung cancer patients, including 72 adenocarcinomas and 7 squamous cell carcinomas. High expression of Oct4, Egr1, and OPN accounted for 53, 51, and 57% of the patients, respectively. All of the three biomarkers were positively correlated in clinical human lung cancer. Patients with high expression of OPN were significantly associated with shorter disease-free survivals than those with low expression of OPN (p < 0.05). In lung cancer cells, Oct4 transactivated the Egr1 promoter and upregulated Egr1 expression. In a human lung cancer xenograft model, Oct4-overexpressing tumors expressed elevated levels of Egr1. Furthermore, overexpression of Oct4 in lung cancer cells increased the metastatic potential. CONCLUSIONS: Egr1 exerts a promoting effect on cancer metastasis in Oct4-overexpressing lung cancer. Thus, therapeutic strategies targeting the Oct4/Egr1/OPN axis may be further explored for the treatment of lung cancer, especially when lung cancer is refractory to conventional treatment due to cancer stem cells.

BMP-2 restoration aids in recovery from liver fibrosis by attenuating TGF-ß1 signaling.

Transforming growth factor-ß (TGF-ß) plays a central role in hepatic fibrogenesis. This study investigated the function and mechanism of bone morphogenetic protein-2 (BMP-2) in regulation of hepatic fibrogenesis. BMP-2 expression in fibrotic liver was measured in human tissue microarray and mouse models of liver fibrosis induced by bile duct ligation surgery or carbon tetrachloride administration. Adenovirus-mediated BMP-2 gene delivery was used to test the prophylactic effect on liver fibrosis. Primary hepatic stellate cells (HSC), HSC-T6 and clone-9 cell lines were used to study the interplay between BMP-2 and TGF-ß1. Hepatic BMP-2 was localized in parenchymal hepatocytes and activated HSCs and significantly decreased in human and mouse fibrotic livers, showing an opposite pattern of hepatic TGF-ß1 contents. BMP-2 gene delivery alleviated the elevations of serum hepatic enzymes, cholangiocyte marker CK19, HSC activation markers, and liver fibrosis in both models. Mechanistically, exogenous TGF-ß1 dose dependently reduced BMP-2 expression, whereas BMP-2 significantly suppressed expression of TGF-ß and its cognate type I and II receptor peptides, as well as the induced Smad3 phosphorylation levels in primary mouse HSCs. Aside from its suppressive effects on cell proliferation and migration, BMP-2 treatment prominently attenuated the TGF-ß1-stimulated α-SMA and fibronectin expression, and reversed the TGF-ß1-modulated epithelial-to-mesenchymal transition marker expression in mouse HSCs. The mutual regulation between BMP-2 and TGF-ß1 signaling axes may constitute the anti-fibrogenic mechanism of BMP-2 in the pathogenesis of liver fibrosis. BMP-2 may potentially serve as a novel therapeutic target for treatment of liver fibrosis.

MiR-30a-5p Inhibits Epithelial-to-Mesenchymal Transition and Upregulates Expression of Tight Junction Protein Claudin-5 in Human Upper Tract Urothelial Carcinoma Cells.

The involvement of microRNAs (miRNAs) in cancer development and their potential as prognostic biomarkers are becoming increasingly known. However, the signature of miRNAs and their regulatory roles in tumorigenesis of upper tract urothelial carcinoma (UTUC) remain to be elucidated. This study aimed to profile the miRNA expression pattern in UTUC tumor tissues and identify candidate miRNAs with prognostic and/or therapeutic functions. METHODS AND RESULTS: We collected 22 UTUC tissue and adjacent normal tissues samples from patients who underwent nephroureterectomy. The miRNAs signatures of three selected UTUC samples using next-generation sequencing showed that miR-30a-5p was significantly downregulated in UTUC tumors compared to adjacent normal tissues. The differentially-expressed miRNAs were specifically validated by quantitative real-time polymerase chain reaction. In addition, the miRNA expression signatures were analyzed with the transcriptome profile characterized by microarray. Further in vitro studies indicated that overexpression of miR-30a-5p significantly suppressed proliferation, migration, and epithelial-to-mesenchymal transition (EMT) in cultured BFTC-909 UTUC cells. As a potential target gene of miR-30a-5p in the tight junction pathway suggested by the pathway enrichment analysis, the reduced expression of tight junction protein claudin-5 in UTUC cells was demonstrated to be upregulated by miR-30a-5p genetic delivery. CONCLUSIONS: Taken together, our findings demonstrated that miR-30a-5p inhibits proliferation, metastasis, and EMT, and upregulates the expression of tight junction claudin-5 in UTUC cells. Thus, miR-30a-5p may provide a promising therapeutic strategy for UTUC treatment.

Involvement of phosphatase and tensin homolog deleted from chromosome 10 in rodent model of neuropathic pain.

BACKGROUND: Many cancer research studies have extensively examined the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) pathway. There are only few reports that suggest that PTEN might affect pain; however, there is still a lack of evidence to show the role of PTEN for modulating pain. Here, we report a role for PTEN in a rodent model of neuropathic pain. RESULTS: We found that chronic constriction injury (CCI) surgery in rats could elicit downregulation of spinal PTEN as well as upregulation of phosphorylated PTEN (phospho-PTEN) and phosphorylated mammalian target of rapamycin (phospho-mTOR). After examining such changes in endogenous PTEN in neuropathic rats, we explored the effects of modulating the spinal PTEN pathway on nociceptive behaviors. The normal rats exhibited mechanical allodynia after intrathecal (i.t.) injection of adenovirus-mediated PTEN antisense oligonucleotide (Ad-antisense PTEN). These data indicate the importance of downregulation of spinal PTEN for nociception. Moreover, upregulation of spinal PTEN by i.t. adenovirus-mediated PTEN (Ad-PTEN) significantly prevented CCI-induced development of nociceptive sensitization, thermal hyperalgesia, mechanical allodynia, cold allodynia, and weight-bearing deficits in neuropathic rats. Furthermore, upregulation of spinal PTEN by i.t. Ad-PTEN significantly attenuated CCI-induced microglia and astrocyte activation, upregulation of tumor necrosis factor-α (TNF-α) and phospho-mTOR, and downregulation of PTEN in neuropathic rats 14 days post injury. CONCLUSIONS: These findings demonstrate that PTEN plays a key, beneficial role in a rodent model of neuropathic pain.

Hepatoma-derived growth factor stimulates podosome rosettes formation in NIH/3T3 cells through the activation of phosphatidylinositol 3-kinase/Akt pathway.

Hepatoma-derived growth factor (HDGF) stimulates the migration, invasion and metastasis in several types of cancer cells. However, the mechanism underlying HDGF-stimulated migration remains unclear. In this study, we investigated the influence of HDGF on cytoskeleton remodeling and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in non-transformed NIH/3T3 cells. Exogenous HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression and Akt phosphorylation in dose- and time-dependent manners. Application of LY294002, a PI3K inhibitor, attenuated the HDGF-induced migration, dorsal ruffles and podosome rosettes formation. Consistently, the HDGF-overexpressing NIH/3T3 transfectants exhibited significantly increased motility and elevated PI3K/Akt activities, which were repressed by LY294002 or adenovirus-mediated overexpression of endogenous PI3K antagonist, PTEN. In summary, HDGF elicits the activation of PI3K/Akt signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration.

Genetic typing, based on the 56-kilodalton type-specific antigen gene, of Orientia tsutsugamushi strains isolated from chiggers collected from wild-caught rodents in Taiwan.

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.

Development of a Scrub Typhus Diagnostic Platform Incorporating Cell-Surface Display Technology.

Scrub typhus (ST), also known as tsutsugamushi disease and caused by rickettsia Orientia tsutsugamushi, is an underestimated fatal epidemic in the Asia-Pacific region, resulting in a million human infections each year. ST is easily misdiagnosed as clinical diagnosis is based on non-specific skin eschar and flu-like symptoms. Thus, the lack of accurate, convenient, and low-cost detection methods for ST poses a global health threat. To address this problem, we adopted baculovirus surface-display technology to express three variants of TSA56, the major membrane antigen of O. tsutsugamushi, as well as the passenger domain of ScaC (ScaC-PD), on insect Sf21 cell surfaces rather than biosafety level 3 bacteria in an enzyme-linked immunosorbent assay (ELISA). Recombinant TSA56 and ScaC-PD were all properly expressed and displayed on Sf21 cells. Our cell-based ELISA comprising the four antigen-displaying cell types interacted with monoclonal antibodies as well as serum samples from ST-positive field-caught rats. This cell-based ELISA presented high accuracy (96.3%), sensitivity (98.6%), and specificity (84.6%) when tested against the ST-positive rat sera. Results of a pilot study using human sera were also highly consistent with the results of immunofluorescence analyses. By adopting this approach, we circumvented complex purification and refolding processes required to generate recombinant O. tsutsugamushi antigens and reduced the need for expensive equipment and extensively trained operators. Thus, our system has the potential to become a widely used serological platform for diagnosing ST.

Corrigendum: Development of a Scrub Typhus Diagnostic Platform Incorporating Cell-Surface Display Technology.

[This corrects the article DOI: 10.3389/fimmu.2021.761136.].

Low-dose etoposide enhances telomerase-dependent adenovirus-mediated cytosine deaminase gene therapy through augmentation of adenoviral infection and transgene expression in a syngeneic bladder tumor model.

The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 microg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1alpha expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (10(9) plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4(+) and CD8(+) T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers.

Elevated serum anti-endothelial cell autoantibodies titer is associated with lupus nephritis in patients with systemic lupus erythematosus.

BACKGROUND AND PURPOSE: Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disease associated with endothelial dysfunction and the existence of multiple species of autoantibodies. However, the association between endothelial dysfunction and renal manifestations remains unclear in Taiwanese SLE patients. METHODS: Serum samples were collected from SLE patients with biopsy-proven lupus nephritis (n = 32), stable SLE patients (n = 32) and healthy controls (n = 32). The SLE Disease Activity Index (SLEDAI) of SLE patients was scored, and levels of anti-endothelial cell antibodies (AECA) and anti-endothelial activities in serum samples were measured by cell-enzyme-linked immunosorbent assay and crystal violet assay, respectively, using cultured human endothelial EA.hy926 cells. RESULTS: Significantly higher AECA (p<0.001) and anti-endothelial activities (p<0.001) were found in sera from patients with lupus nephritis compared with that from stable SLE patients or controls. Moreover, AECA titers (p<0.001) and anti-endothelial activities (p<0.001) were strongly correlated with SLEDAI scores in these patients. CONCLUSION: The strong correlations of AECA and anti-endothelial activity with lupus nephritis activity support an endothelial origin for renal complications in Taiwanese SLE patients.

Blockade of endothelin-1 release contributes to the anti-angiogenic effect by pro-opiomelanocortin overexpression in endothelial cells.

Pro-opiomelanocortin (POMC) is the precursor of several neuropeptides, such as corticotropin (ACTH), alpha-melanocyte-stimulating hormone (MSH), and the endogenous opioid, beta-endorphin (EP). ACTH-dependent Cushing's syndrome is characterized by ACTH overproduction and is associated with an increased risk of cardiovascular disease. Endothelial dysfunction has been recognized as an early marker of cardiovascular disease. However, the mechanism underlying endothelial dysfunction by ACTH overexpression in Cushing's patients remains elusive. Endothelial cells, the primary cells producing endothelin (ET)-1, are both the source and target of POMC-derived peptides. In the present study, we generated adenovirus vectors (Ad) encoding POMC (Ad-POMC) and green fluorescent protein (GFP; Ad-GFP) to investigate whether POMC gene transfer altered the ET-1 homeostasis and angiogenic functions in human EA.hy926 endothelial cells. Via adenovirus gene delivery, the POMC-transduced EA.hy926 cells released significantly elevated ACTH and beta-EP levels (P < 0.001). In addition, POMC gene delivery significantly decreased the ET-1 release (P < 0.001) without affecting the ET-1 messenger RNA (mRNA) level. Despite no effect on the secretion of matrix metalloproteinases (MMPs) and cell proliferation, POMC gene delivery significantly inhibited the migration (P < 0.01) and tube-forming capability (P < 0.01) of endothelial cells. Moreover, the POMC-induced inhibition of tube formation could be partially reversed by adding exogenous ET-1 (P < 0.05). In summary, the attenuated ET-1 release and angiogenic processes by POMC overexpression may contribute to endothelial dysfunction, thereby providing a link between Cushing's syndrome and cardiovascular diseases.

Inhibition of corneal angiogenesis by local application of vasostatin.

PURPOSE: This study was designed to investigate the effects of the locally supplied endogenous angiogenesis inhibitor vasostatin (VS) on corneal angiogenesis. METHODS: Recombinant VS was expressed and purified. The effects of VS on the proliferation of endothelial cells were investigated using the methyl thiazolyl tetrazolium (MTT) assay in the absence or presence of angiogenic factors such as basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). Corneal neovascularization was induced by implantation of hydron pellets containing bFGF in rat corneal micropockets. The potency of VS to inhibit corneal angiogenesis was investigated by incorporation of VS with bFGF in hydron pellets or topical application of VS containing eye drops to rat eyes implanted with bFGF pellets. The extent of corneal neovascularization was evaluated by microscopic and histological analyses. RESULTS: VS potently inhibited the growth of endothelial cells in the absence or presence of angiogenic factors such as bFGF or VEGF. In the rat corneal micropocket assay, concurrent incorporation of VS abolished the bFGF induced neovascularization. When formulated in a methylcellulose eye drop, VS remained intact and functional in a 4 degrees C solution for more than 7 days. Topical application of VS eye drops potently inhibited bFGF induced neovascularization in rat corneas. CONCLUSIONS: The present study effectively demonstrated the potential feasibility of local application of VS for treatment of corneal angiogenesis.

Field assessment of Orientia tsutsugamushi infection in small mammals and its association with the occurrence of human scrub typhus in Taiwan.

We conducted an extensive study in Taiwan of Orientia tsutsugamushi (OT) infection in small wild mammals. Field trapping was carried out at six districts in eastern and western Taiwan as well as various offshore islands during the period 2006-2010. A total of 1061 specimens representing 11 rodent species were captured. The presence of OT infection was assessed by indirect immunofluorescence assay and polymerase chain reaction assays of 56-kDa type-specific antigen gene. The chigger infestation rate among the animals was 35% (371/1061). Among these, OT was detected in 64% (238/371) of the chiggers from the infested animals and in the spleens from 273 (34.3%) of 797 animals. Excluding animals in the Suncus murinus group, the antibody positive rate of scrub typhus was 69.1% (477 of 690 of serum samples). The prevalence of OT infection in animals from areas with a low incidence of human cases of scrub typhus was significantly lower than that in rodents obtained from regions with a high incidence of human cases of the disease (44.4%±4.0% vs. 71.2%±9.7%, p<0.001). In Taiwan, the prevalence of OT infection in wild rodents is considerably high and appears to correlate positively with the occurrence of scrub typhus in humans.

PTEN overexpression attenuates angiogenic processes of endothelial cells by blockade of endothelin-1/endothelin B receptor signaling.

Arteriovenous (AV) graft is frequently used as vascular access in hemodialysis patients. However, clotting or thrombosis of AV grafts often occurs and requires surgical removal. At present, the molecular pathogenesis underlying thrombosis of AV graft is not clear. The PTEN/Akt signaling has been implicated in the pathogenesis of vascular diseases. In this study, elevated PTEN expression and concomitant Akt inactivation was observed in endothelium of atherosclerotic brachial arteries from hemodialysis patients. To investigate whether PTEN upregulation affects endothelial function, adenovirus-mediated PTEN (Ad-PTEN) overexpression was performed in aorta rings and cultured endothelial cells. It was found that PTEN overexpression potently inhibited the microvessel sprouting in aorta rings and the angiogenic activities of endothelial cells including migration and tube formation. On the contrary, PTEN knockdown by RNA interference promoted the endothelial migration and reversed the Ad-PTEN-induced inhibition of endothelial migration. Expression analysis showed that PTEN overexpression attenuated the expression of endothelin-1 (ET-1) and endothelin B receptor (ETBR) in endothelial cells at transcriptional levels. However, exogenous ET-1 supply only partially reversed the PTEN-induced inhibition of migration and tube formation. This was delineated due to that PTEN overexpression also perturbed endothelial nitric oxide synthase (eNOS) activation and vascular endothelial growth factor (VEGF) release. In summary, PTEN upregulation induces endothelial dysfunction by attenuating the availability and signaling of multiple angiogenic pathways in endothelial cells, thereby may contribute to thrombosis of AV graft.

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-1ß 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.

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

OBJECTIVE: The 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). METHODS: Adenoviral 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. RESULTS: AdPTEN 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. CONCLUSION: This 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.

Molecular detection and characterization of spotted fever group rickettsiae in Taiwan.

Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal(2), respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.

Suppression of choroidal neovascularization by intramuscular polymer-based gene delivery of vasostatin.

The purpose of this study was to evaluate the efficacy of gene delivery of angiogenesis inhibitor, vasostatin (VS), in suppressing experimental model of choroidal neovascularization (CNV). A mammalian expression vector carrying VS, pCMV3-VS, was constructed and evaluated for its ability to produce VS in transfected cells using western blot analysis and a cell viability assay. CNV was induced in Brown Norway rats by fundus argon laser photocoagulation and evaluated by fundus fluorescein angiography (FAG). Ten days post-laser treatment, gene delivery was achieved by intramuscular (IM) injection of poly-(N-vinyl pyrrolidone) (PVP) polymer conjugated with pCMV3-VS (PVP-VS) or a control vector (PVP-vector). Systemic VS expression was analysed by western blot analysis and enzyme linked immunosorbent assay (ELISA), and the extent of CNV was monitored by FAG analysis at different time intervals post-PVP treatment. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Besides, IM injection of PVP-VS, but not PVP-vector, led to elevated VS level in plasma for 30 days. After laser photocoagulation, rats injected with PVP-VS exhibited significantly lower incidence of CNV comparing with animals of control groups (P < 0.01) for at least 42 days. Moreover, rats treated with PVP-VS also showed a significant reduction in the CNV lesions compared with control groups (P < 0.001) for at least 42 days. Above all, no overt adverse effects were observed in rats received PVP-VS. These results demonstrate the potential of IM VS gene delivery for CNV treatment.

Increased endostatin/collagen XVIII expression correlates with elevated VEGF level and poor prognosis in hepatocellular carcinoma.

Liver is the primary source for collagen XVIII, the precursor of angiogenesis inhibitor, endostatin. However, the role of endostatin/collagen XVIII expression during liver carcinogenesis remains elusive. Therefore, we studied its expression in five hepatoma cell lines and 105 hepatocellular carcinoma specimens. The poorly differentiated hepatoma cell lines exhibited increased endostatin/collagen XVIII levels compared with the well-differentiated ones. In hepatoma tissues, endostatin/collagen XVIII expression was detected in various types of liver cells and was significantly stronger in adjacent nontumor tissues than that in tumors (P<0.001). Endostatin/collagen XVIII expression in nontumor tissues correlated with tumor stages (P=0.014) and expression of vascular endothelial growth factor (P=0.007), but not the stages of hepatic fibrosis (P>0.05). Kaplan-Meier analysis showed that patients with higher endostatin/collagen XVIII expression had significantly shorter overall survival (P=0.011) and disease-free survival (P=0.0034). Moreover, endostatin/collagen XVIII level was an independent prognostic factor for tumor recurrence (P=0.034) by multivariate analysis. In conclusion, increased endostatin/collagen XVIII expression correlated with hepatoma progression and predicted poor prognosis for patients with hepatocellular carcinoma.