Mucosal gene therapy using a pseudotyped lentivirus vector encoding murine interleukin-10 (mIL-10) suppresses the development and relapse of experimental murine colitis

BACKGROUND: Therapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis. METHODS: Lentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-α and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR. RESULTS: Mucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-α and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected. CONCLUSIONS: Topically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis

The activation mechanism of the aryl hydrocarbon receptor (AhR) by molecular chaperone HSP90

The aryl hydrocarbon receptor is a member of the nuclear receptor superfamily that associates with the molecular chaperone HSP90 in the cytoplasm. The activation mechanism of the AhR is not yet fully understood. It has been proposed that after binding of ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3methylcholanthrene (3-MC), or β-naphthoflavone (β-NF), the AhR dissociates from HSP90 and translocates to the nucleus. It has also been hypothesized that the AhR translocates to the nucleus and forms a complex with HSP90 and other co-chaperones. There are a few reports about the direct association or dissociation of AhR and HSP90 due to difficulties in purifying AhR. We constructed and purified the PAS domain from AhR. Binding of the AhR-PAS domain to β-NF affinity resin suggested that it possesses ligand-binding affinity. We demonstrated that the AhR-PAS domain binds to HSP90 and the association is not affected by ligand binding. The ligand 17-DMAG inhibited binding of HSP90 to GST-PAS. In an immunoprecipitation assay, HSP90 was co-immunoprecipitated with AhR both in the presence or absence of ligand. Endogenous AhR decreased in the cytoplasm and increased in the nucleus of HeLa cells 15. min after treatment with ligand. These results suggested that the ligand-bound AhR is translocated to nucleus while in complex with HSP90.We used an in situ proximity ligation assay to confirm whether AhR was translocated to the nucleus alone or together with HSP90. HSP90 was co-localized with AhR after the nuclear translocation. It has been suggested that the ligand-bound AhR was translocated to the nucleus with HSP90. Activated AhR acts as a transcription factor, as shown by the transcription induction of the gene CYP1A1 8. h after treatment with β-NF

Polychlorinated biphenyl (118) activates osteoclasts and induces bone resorption in goldfish

To analyze the effect of polychlorinated biphenyl (PCB) 118 on fish bone metabolism, we examined osteoclastic and osteoblastic activities, as well as plasma calcium levels, in the scales of PCB (118)-injected goldfish. In addition, effect of PCB (118) on osteoclasts and osteoblasts was investigated in vitro. Immature goldfish, in which the endogenous effects of sex steroids are negligible, were used. PCB (118) was solubilized in dimethyl sulfoxide at a concentration of 10 ppm. At 1 and 2 days after PCB (118) injection (100 ng/g body weight), both osteoclastic and osteoblastic activities, and plasma calcium levels were measured. In an in vitro study, then, both osteoclastic and osteoblastic activities as well as each marker mRNA expression were examined. At 2 days, scale osteoclastic activity in PCB (118)-injected goldfish increased significantly, while osteoblastic activity did not change significantly. Corresponding to osteoclastic activity, plasma calcium levels increased significantly at 2 days after PCB (118) administration. Osteoclastic activation also occurred in the marker enzyme activities and mRNA expressions in vitro. Thus, we conclude that PCB (118) disrupts bone metabolism in goldfish both in vivo and in vitro experiments. © 2012 The Author(s)

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner

Correlation between anti-S IgG and neutralizing antibody titers against three live SARS-CoV-2 variants in BNT162b2 vaccine recipients

We analyzed serially collected serum samples from healthy adults who underwent BNT162b2 vaccination to elucidate the association between spike (S)-IgG antibody titers determined by ELISA using the WHO international standard (NIBSC code 20/136) and neutralizing antibody titers against three live SARS-CoV-2 variants. This study included 53 health care workers who received two doses of the BNT162b2 vaccine. S-IgG and nucleocapsid (N)-IgG antibody titers were measured by ELISA. Neutralizing (NT) antibody responses against three variants (Wuhan D614 G: KUH003, Alpha, and Delta) were evaluated before and after the first and second vaccination. N-IgG were not detected in any serum samples. S-IgG antibody titers remarkably increased after two BNT162b2 vaccine doses in all participants. S-IgG antibody titers were strongly correlated with NT titers against three variants of live viruses: KUH003 (r = 0.86), Alpha (r = 0.72), and Delta (r = 0.84). Serum samples from participants after one dose of BNT162b2 neutralized Alpha efficiently (median titer, 113.0), but median NT titers against KUH003 and Delta variants were lower, 57.0 and 28.0, respectively (p < .01). Two doses of the BNT162b2 vaccine elicited a strong immune response in this study. The second dose was required for induction of a strong booster effect. Serum collected from BNT162b2 vaccine recipients contained significantly lower neutralizing activity against Delta than that of against KUH003 (p < .0001) and Alpha (p < .0001). If a new variant emerges, live virus-based NT titers should be examined in serum obtained from vaccine recipients to evaluate vaccine efficacy for protection against infection

Therapeutic Efficacy of Replication-Competent Retrovirus Vector–Mediated Suicide Gene Therapy in a Multifocal Colorectal Cancer Metastasis Model

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro , multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26.FLuc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition