Rag2 Deficiency Enhances Susceptibility to Systemic Mouse Adenovirus Type 1 Infection.

INTRODUCTION: Recombination-activating gene (Rag) 1 and Rag2, which are essential in V(D)J recombination, play a crucial role in B- and T-cell maturation. METHOD: We investigated the effects of Rag2 deficiency in clustered regularly interspaced short palindromic repeats/Cas9-mediated FVB-Rag2 knockout (KO) and wild-type (WT) mice infected with mouse adenovirus type 1 (MAV-1) via the intranasal route. RESULTS: MAV-1 infection caused more severe histopathological changes in FVB-Rag2 KO mice than in WT mice. FVB-Rag2 KO mice exhibited moderate to severe inflammation on day 4 and severe inflammation on day 8 post infection. In contrast, WT mice showed mild inflammation on day 4 and mild to severe inflammation on day 8 post infection, including interstitial pneumonia and inflammatory cell infiltration in the lungs and liver. Viral loads in the spleen and kidneys were significantly higher in FVB-Rag2 KO mice than in WT mice on day 8 post infection. Levels of cytokines and chemokines, including macrophage inflammatory protein-1α, induced protein 10, interferon (IFN)-α, IFN-γ, and tumor necrosis factor alpha, were upregulated in the spleens of FVB-Rag2 KO mice compared with those of WT mice. The upregulation of several cytokines occurred concurrently with the histopathological changes. MAV-1 infection induced more severe systemic infection in FVB-Rag2 KO mice than in WT mice. CONCLUSION: In mice, Rag2 deficiency induces inflammatory cell recruitment via the upregulation of cytokine and chemokine levels. The MAV-1 infection model can be utilized to assess the efficacy and safety of therapeutic agents for human adenoviral diseases.

Lentivirus-Mediated VEGF Knockdown Suppresses Gastric Cancer Cell Proliferation and Tumor Growth in vitro and in vivo.

PURPOSE: Gastric cancer has a high mortality rate worldwide. Although treatments, such as molecular-targeted therapy, have been introduced, the resulting long-term survival and prognosis remain unsatisfactory. Downregulation of the target genes using lentivirus-mediated short hairpin RNA (shRNA) can be an effective therapeutic strategy for patients with gastric cancer. Overexpressed vascular endothelial growth factor A (VEGF) in human gastric cancer cells can be an effective novel therapeutic target for human gastric cancer. Thus, this study aimed to evaluate the therapeutic effects of lentivirus-mediated knockdown of VEGF gene expression in human gastric cancer growth. MATERIALS AND METHODS: Specific shRNA sequences targeting VEGF were designed to construct a lentiviral expression vector. After human gastric carcinoma cells (cell line NCI-N87) were infected with the lentiviral vector, the therapeutic effects of the lentivirus-mediated shRNA targeting VEGF were analyzed both in vitro and in vivo. RESULTS: Stable suppression of VEGF gene expression in NCI-N87 cells using shRNA (ShVEGF) showed significant inhibition of cell proliferation, clonogenicity, and cell motility. ShVEGF also showed increased G0/G1 cell cycle arrest and apoptosis. In addition, in vivo results from nude mice xenografted ShVEGF showed significant inhibition of tumor growth. Assessing the therapeutic effects of intratumoral injection of lentivirus-targeting VEGF (Virus_VEGF) revealed that it significantly inhibited tumor growth compared to that in the Virus_Scramble or saline injection control groups. CONCLUSION: The constructed ShVEGF showed significant inhibition of NCI-N87 gastric cancer cell growth both in vitro and in vivo. These experimental results suggest a novel therapeutic strategy for patients with gastric cancer using lentivirus-mediated shRNA targeting VEGF.

Comparison of the virulence of Streptococcus pneumoniae in ICR mouse stocks of three different origins.

Streptococcus pneumoniae causes many people to suffer from pneumonia, septicemia, and other diseases worldwide. To identify the difference in susceptibility of and treatment efficacy against S. pneumoniae in three ICR mouse stocks (Korl:ICR, A:ICR, and B:ICR) with different origins, mice were infected with 2 × 106, 2 × 107, and 2 × 108 CFU of S. pneumoniae D39 intratracheally. The survival of mice was observed until three weeks after the infection. The three stocks of mice showed no significant survival rate difference at 2 × 106 and 2 × 107 CFU. However, the lung and spleen weight in the A:ICR stock was significantly different from that in the other two stocks, whereas the liver weight in B:ICR stock was significantly lower than that in the other two stocks. Interestingly, no significant CFU difference in the organs was observed between the ICR stocks. The level of interferon gamma inducible protein 10 in Korl:ICR was significantly lower than that in the other two stocks. The level of granulocyte colony stimulating factor in B:ICR was significantly lower than in the other two stocks. However, tumor-necrosis factor-alpha and interleukin-6 levels showed no significant difference between the ICR stocks. In the vancomycin efficacy test after the S. pneumoniae infection, both the single-dose and double-dose vancomycin-treated groups showed a significantly better survival rate than the control group. There was no significant survival difference between the three stocks. These data showed that Korl:ICR, A:ICR, and B:ICR have no susceptibility difference to the S. pneumoniae D39 serotype 2.

Radioprotective effect of newly synthesized toll-like receptor 5 agonist, KMRC011, in mice exposed to total-body irradiation.

Exposure to ionizing radiation leads to severe damages in radiosensitive organs and induces acute radiation syndrome, including effects on the hematopoietic system and gastrointestinal system. In this study, the radioprotective ability of KMRC011, a novel toll-like receptor 5 (TLR5) agonist, was investigated in C57BL6/N mice exposed to lethal total-body gamma-irradiation. In a 30-day survival study, KMRC011-treated mice had a significantly improved survival rate compared with control after 11 Gy total-body irradiation (TBI), and it was found that the radioprotective activity of KMRC011 depended on its dosage and repeated treatment. In a 5-day short-term study, we demonstrated that KMRC011 treatment stimulated cell proliferation and had an anti-apoptotic effect. Furthermore, KMRC011 increased the expressions of genes related to DNA repair, such as Rad21, Gadd45b, Sod2 and Irg1, in the small intestine of lethally irradiated mice. Interestingly, downregulation of NF-κB p65 in the mouse intestine by KMRC011 treatment was observed. This data indicated that KMRC011 exerted a radioprotective activity partially by regulating NF-κB signaling. Finally, peak expression levels of G-CSF, IL-6, IFN-γ, TNF-α and IP-10 induced by KMRC011 treatment were different depending on the route of administration and type of cytokine. These cytokines could be used as candidate biomarkers for the evaluation of KMRC011 clinical efficacy. Our data indicated that KMRC011 has radioprotective activity in lethally irradiated mice and may be developed as a therapeutic agent for radioprotection.

Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout.

Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as NeoR cassettes and their characterization. By eliminating any possible effects of adding a NeoR cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2-/-, B6-Rag2-/-, and BALB/c-Prkdc-/- mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45+DX-5+ natural killer cells. However, B6-Il2rg-/- mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg-/- mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.