Molecular targeting of hepatocyte growth factor by an antagonist, NK4, in the treatment of rheumatoid arthritis

INTRODUCTION: Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice. METHODS: Arthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4(+ )T cells stimulated with allogeneic spleen cells. RESULTS: The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4(+ )T cells stimulated with allogeneic spleen cells. CONCLUSIONS: These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4(+ )T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA

Efficient pH and dissolved CO2 conditions for indoor and outdoor cultures of green alga Parachlorella

Efficient pH and dissolved CO2 conditions for indoor (50–450 mL scale) and outdoor (100–500 L scale) culture of a green alga BX1.5 strain that can produce useful intracellular lipids and extracellular polysaccharides were investigated for the first time in Parachlorella sp. The cultures harvested under 26 different conditions were analysed for pH, dissolved CO2 concentration, and the biomass of extracellular polysaccharides. The BX1.5 strain could thrive in a wide range of initial medium pH ranging from 3 to 11 and produced valuable lipids such as C16:0, C18:2, and C18:3 under indoor and outdoor culture conditions when supplied with 2.0% dissolved CO2. Particularly, the acidic BG11 medium effectively increased the biomass of extracellular polysaccharides during short-term outdoor cultivation. The BG11 liquid medium also led to extracellular polysaccharide production, independent of acidity and alkalinity, proportional to the increase in total sugars derived from cells supplied with high CO2 concentrations. These results suggest Parachlorella as a promising strain for indoor and outdoor cultivation to produce valuable materials

Combinatorial anti-angiogenic gene therapy in a human malignant mesothelioma model

Anti-angiogenic gene therapy represents a promising strategy for cancer; however, it has rarely been tested in malignant mesothelioma, a highly aggressive tumor associated with asbestos with poor prognosis. In the present study, we investigated whether anti-angiogenic factors such as angiostatin, endostatin and the soluble form of vascular endothelial growth factor receptor 2 (sFlk1) were able to inhibit endothelial cell proliferation via lentivirus-mediated gene transfer into malignant mesothelioma cells in culture. We also assessed whether a dual-agent strategy had greater therapeutic benefit. Human malignant pleural mesothelioma MSTO-211H cells were transduced using lentiviral vectors that individually expressed angiostatin, endostatin and sFlk1 and linked to enhanced green fluorescent protein (EGFP) marker gene expression via an internal ribosome entry site. The lentivirus expressing EGFP alone was used as a control. The resultant cells designated as MSTO-A, MSTO-E, MSTO-F and MSTO-C were confirmed by western blot analysis and fluorescence microscopy to stably express the corresponding proteins. No differences were observed in the in vitro growth rates between any of these cells. However, co-culture of MSTO-A, MSTO-E and MSTO-F showed significant suppression of human umbilical endothelial cell growth in vitro compared with that of MSTO-C. Furthermore, a combination of any two among MSTO-A, MSTO-E and MSTO-F significantly enhanced efficacy. These results suggest that combinatorial anti-angiogenic gene therapy targeting different pathways of endothelial growth factor signaling has the potential for greater therapeutic efficacy than that of a single-agent regimen

Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors.

Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression

Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors.

Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression

A Case of X-Linked Hypophosphatemic Rickets with Dentin Dysplasia in Mandibular Third Molars

X-linked hypophosphatemic rickets (XLH) is a disease characterized by impaired bone mineralization, and its dental features include gingival abscesses and large pulp spaces due to dentin dysplasia. A 20-year-old woman with XLH was referred to oral surgery for extraction of mandibular third molars. She was diagnosed with XLH at approximately 1 year of age and was treated thereafter. There was no history of gingival abscesses, and panoramic radiographic and computed tomographic examinations revealed no evidence of dentin dysplasia. However, histopathological examination of the extracted teeth showed dentin dysplasia, including interglobular dentin. In this XLH patient, dentin dysplasia was revealed histologically even though no obvious abnormality was found on visual and radiographic examinations. These findings suggest that in patients with XLH, oral management must take dentin dysplasia of the permanent teeth into consideration even if the patient’s general condition is well controlled with conventional therapy

Dual-vector prodrug activator gene therapy using retroviral replicating vectors.

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes