The recombinant Newcastle disease virus Anhinga strain expressing human TRAIL exhibit antitumor effects on a glioma nude mice model.

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. However, the potential oncolytic ability of the recombinant newcastle disease virus (NDV) Anhinga strain carried with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has not been fully explored at present. In the present study, the recombinant NDV/Anh-TRAIL that secretes soluble TRAIL was constructed and the experiment results suggested NDV/Anh-TRAIL as a promising candidate for glioma therapy. Growth kinetic and TRAIL secreted quantity of recombinant NDV/Anh-TRAIL virus were measured. Cytotoxic and cell apoptosis were analyzed for its anti-glioma therapy in vitro. Nude mice were used for the in vivo evaluation. Both tumor volume and mice behavior after injection were observed. The recombinant virus replicated with the same kinetics as the parental virus and the highest expression of TRAIL (77.8 ng/L) was found at 48 hours. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole and flow cytometry data revealed that the recombinant NDV/Anh-TRAIL (56.1 ± 8.2%) virus could induce a more severe apoptosis rate, when compared with the NDV wild type (37.2 ± 7.0%) and mock (7.0 ± 1.8%) groups (P < .01), in U251 cells. Furthermore, in the present animal study, the average tumor volume was smaller in the NDV/Anh-TRAIL group (97.21 mm3 ), when compared with the NDV wild type (205.03 mm3 , P < .05) and PBS (310.30 mm3 , P < .01) groups.

Recombinant Newcastle disease virus expressing P53 demonstrates promising antitumor efficiency in hepatoma model.

BACKGROUND: Numerous studies have demonstrated that the NDV-mediated gene therapy is a promising new approach for treatment of cancers. P53 plays a vital role in tumor suppression and surveillance. Therefore, we hypothesize that a recombinant NDV expressing P53 would be an ideal agent for the hepatoma therapy. RESULTS: In the essay, the human P53 gene was incorporated into the genome of a lentogenic strain (named rNDV-P53), which did not affect viral replication kinetics and magnitude in HepG2 cells. Compared to the vehicle virus, rNDV-P53 increased cell growth suppressor ratio and early apoptosis by 2 folds, and decreased the mitochondrial membrane potential in HepG2 cells. In vivo studies, treatment with rNDV-P53 reduced tumor volume of tumor-bearing mice by more than 4 folds, tumor weight by more than 5 folds comparing with rNDV. The 120-day survival rate of rNDV-P53-treated mice was 75 %, survival rate of rNDV-treated mice was 12.5 %. TUNEL analysis showed a significant increase in the apoptosis rate in the tumor tissues of rNDV-P53-treated mice than that of rNDV-treated mice. Moreover, serum chemistries revealed an insignificant change of blood urea nitrogen (BUN), creatinine levels, alanine aminotransferase (ALT) and aspartate transaminase (AST) in rNDV-P53-treated group compared to normal mice, suggesting treatment with the recombinant virus was not toxic. CONCLUSION: rNDV-P53 is a potent candidate for carcinoma therapy especially for hepatocarcinoma.

Recombinant Newcastle disease virus (NDV/Anh-IL-2) expressing human IL-2 as a potential candidate for suppresses growth of hepatoma therapy.

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical study and are currently approved for clinical trials. NDV Anhinga strain which is a mesogenic strain should be classified as lytic strain and has a therapeutic efficacy in hepatocellular cancer. In this study, we evaluated the capacity of NDV Anhinga strain to elicit immune reaction in vivo and the possibility for using as a vaccine vector for expressing tumor therapeutic factors. Interleukin-2 (IL-2) could boost the immune response against the tumor cells. Therefore, we use NDV Anhinga strain as backbone to construct a recombinant virus (NDV/Anh-IL-2) expressing IL-2. The virus growth curve showed that the production of recombinant NDV/Anh-IL-2 was slightly delayed compared to the wild type. The NDV/Anh-IL-2 strain could express soluble IL-2 and effectively inhibit the growth of hepatocellular carcinoma in vivo. 60 days post-treatment, mice which were completely cured by previous treatment were well protected when rechallenged with the same tumor cell. From the H&E-stained sections, intense infiltration of lymphocyte was observed in the NDV Anhinga strain treated group, especially in NDV/Anh-IL-2 group. The NDV Anhinga strain could not only kill the tumor directly, but could also elicit immune reaction and a potent immunological memory when killing tumor in vivo. In conclusion, the Anhinga strain could be an effective vector for tumor therapy; the recombinant NDV/Anh-IL-2 strain expressing soluble IL-2 is a promising candidate for hepatoma therapy.

The influence of cigR gene on the pathogenicity of Salmonella paratyphi A in vitro and in vivo.

Salmonella Paratyphi A is the causative agent of paratyphoid fever A which is a serious threat to human health in many countries. The cigR gene located in Salmonella pathogenicity island 3 is a type III secretion system 2 effector gene. However, the influence of cigR gene on the pathogenicity of Salmonella Paratyphi A remains unclear. Here, a cigR gene deletion mutant of Salmonella Paratyphi A was constructed and its pathogenic changes were also evaluated. It was found that both the growth and biochemical features have not changed after the loss of cigR, but the absence of cigR significantly enhanced the replication and/or survival ability in phorbol-12-myristate-13-acetate (PMA)-differentiated human macrophage THP-1 cells and in mouse; the proliferative activity and apoptosis of PMA-differentiated THP-1 cell were significantly decreased and increased, respectively, after the lack of cigR gene; and the mutant showed increased virulence to a mouse infection model by decreased half-lethal dose (LD50) value and enhanced the proliferation ratio of bacteria in vivo. These results demonstrated that CigR is an anti-virulence factor and plays an important role in the pathogenicity of Salmonella Paratyphi A.

Correction: Identification of Optimal Insertion Site in Recombinant Newcastle Disease Virus (rNDV) Vector Expressing Foreign Gene to Enhance Its Anti-Tumor Effect.

[This corrects the article DOI: 10.1371/journal.pone.0164723.].

Recombinant Newcastle disease virus expressing human TRAIL as a potential candidate for hepatoma therapy.

Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently proved for clinical trials. We have previously reported, for the first time, NDV Anhinga strain has an efficient cancer therapeutic efficacy in hepatoma. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. In this study, we have showed administration of a recombinant NDV Anhinga strain expressing soluble TRAIL (NDV/Anh-TRAIL) results in an efficient suppression of hepatocellular carcinoma without significant toxicity. The results show that recombinant NDV Anhinga strain expressing soluble TRAIL is a promising candidate for hepatoma therapy.

Efficacy of a combination of high and low dosage of PEGylated FGF-21 in treatment of diabetes in db/db mice.

The aim of this study is to explore a new method-high dose starting and low dose maintaining for PEGylated Fibroblast growth factor 21 (pFGF-21) treatment. Db/db mice were initially treated with pFGF-21 of high dose, then treated with pFGF-21 of low doses. The mice were treated with pFGF-21 at initial dose of 1.0mg/kg for 14days, then treated with pFGF-21 at maintenance doses of 0.125/0.250/0.375/0.500mg/kg for 30days. The hypoglycemic and hypolipidemic effects of pFGF-21 of different maintenance doses were compared. The pharmacological efficacy of the maintenance doses of pFGF-21 was evaluated by blood glucose levels, oral glucose tolerance test, glycosylated hemoglobin levels, insulin levels, body weight, lipid profile parameters, the mRNA expressions of glycolysis-related genes, the mRNA expressions of gluconeogenesis-related genes and the mRNA expressions of lipid metabolism-related genes. Results showed that in comparison to the mice treated only with initial dose, the treatment with pFGF-21 at maintenance doses of 0.125/0.250/0.375/0.500mg/kg exhibited favorable efficacy in lowering blood glucose levels and glycosylated hemoglobin levels, thus alleviating insulin resistance and improving dyslipidemia. However, among all of the maintenance doses, the dose of 0.125mg/kg was less effective than the other maintenance doses. These results suggest that using the treatment method of high dose of PEGylated FGF-21 in the start and low dose maintaining results in favorable control of the glycolipid metabolic balance. This study provides a new method for PEGylated FGF-21 treatment which is beneficial to promote the clinical application of PEGylated FGF-21.

Identification of Optimal Insertion Site in Recombinant Newcastle Disease Virus (rNDV) Vector Expressing Foreign Gene to Enhance Its Anti-Tumor Effect.

Recombinant Newcastle disease virus (rNDV) is tumor selective and intrinsically oncolytic, which has been developed as a vector to express exogenous genes to enhance its oncolytic efficacy. Our previous studies found that insertion sites of foreign gene in rNDV vector affected its expression and anti-tumor activities. However, the optimal insertion site for foreign genes remains unknown. In this study, we inserted the enhanced green fluorescence protein (EGFP) and IL2 genes into four different intergenic regions of the rNDV using reverse genetics technology. Recombinants rNDV-EGFPs and rNDV-IL2s were successfully rescued, which displayed the similar growth kinetics with parental virus. Both EGFP mRNA and protein levels were most abundant in HepG2 cells, when EGFP gene was inserted between the NP/P site of the rNDV. Similarly, the IL-2 expressed by HepG2 cells infected with rNDV-IL2 was highest, when IL2 was inserted into NP/P site. To test whether these rNDVs that express higher foreign genes could induce stronger anti-tumor response, we treated the H22-oxter-tumor-bearing C57BL/6J mice with rNDV-IL2s and then examined the oncolytic efficacy. The results showed that rNDV-IL2-NP/P had the strongest inhibition of murine hepatoma carcinoma tumors. The splenocytes isolated from the mice treated with rNDV-IL2-NP/P reached the highest degrees of CD4+ T and CD8+ T cells. In addition, animals' survival rate in rNDV-IL2-NP/P-treated group was higher than that of other groups. Taken together, these results demonstrate that NP and P gene junction in rNDV is the optimal insertion site for foreign genes expression to enhance rNDV's anti-tumor effects.

Recombinant Newcastle Disease Virus Encoding IL-12 and/or IL-2 as Potential Candidate for Hepatoma Carcinoma Therapy.

Interleukins as immunomodulators are promising therapeutic agents for cancer therapy. Previous studies showed that there was an improved antitumor immunity in tumor-bearing mice using recombinant Newcastle disease virus carrying for interleukin-2. Interleukin-12 is a promising antitumor cytokine too. So we investigated and compared the antitumor effect of genetically engineered Newcastle disease virus strains expressing both interleukin-12 and/or interleukin-2 (rClone30-interleukin-2, rClone30-interleukin-12, and rClone30-interleukin-12-interleukin-2). In vitro studies showed that rClone30s could efficiently infect tumor cells and express interleukin-12 and/or interleukin-2. 3-(4,5-Dimethylthiazol-2-y)-2,5-diphenyl-tetrazolium bromide results showed rClone30s possessed strong cytotoxic activities against multiple tumor cell lines (U251, HepG2, A549, and Hela). Animal studies showed that rClone30-interleukin-12-interleukin-2 was more effective in inhibition of murine hepatoma carcinoma tumors, with the mean tumor volume (day 14) of 141.70 mm(3) comparing 165.67 mm(3) of rClone30-interleukin-12 group, 210.47 mm(3) of rClone30-interleukin-2 group, 574.70 mm(3) of rClone30 group, and 1206.83 mm(3) of phosphate-buffered saline group. Moreover, the rClone30-interleukin-12-interleukin-2 treated mice secreted more interferon γ (333.518 pg/mL) and its downstream cytokine interferon-γ induced protein 10 (16.006 pg/mL) in tumor than the rClone30-interleukin-12 group (interferon γ: 257.548 pg/mL; interferon-γ induced protein 10: 13.601 pg/mL), rClone30-interleukin2 group (interferon γ: 124.601 pg/mL; interferon-γ induced protein 10: 9.779 pg/mL), or rClone30 group (interferon γ: 48.630 pg/mL; interferon-γ induced protein 10:1.650 pg/mL). For the survival study, rClone30-interleukin12-interleukin2 increased the survival rate (12 of 16) of the tumor-bearing mice versus 11 of 16 in rClone30-interleukin-12 group, 10 of 16 in rClone30-interleukin-2 group, 7 of 16 in Clone30 group, and 0/16 in phosphate-buffered saline group, respectively. To determine whether the mice treated with recombinant virus developed protective immune response, the mice were rechallenged with the same tumor cells. The results showed that viral-treated mice were significantly protected from rechallenge. These results suggest that expressing both interleukin-2 and/or interleukin-12 could be ideal approaches to enhance the antitumor ability of Newcastle disease virus, and rClone30-interleukin-12-interleukin-2 is slightly superior over rClone30-interleukin-12 and rClone30-interleukin-2 alone.

Fibroblast growth factor 21 (FGF21) inhibits macrophage-mediated inflammation by activating Nrf2 and suppressing the NF-κB signaling pathway.

Our previous report has shown that FGF21 has anti-inflammatory properties in a collagen-induced arthritis (CIA) model. In this study, the underlying molecular mechanisms of action were also investigated using RAW 264.7 cells, a murine monocyte-macrophage. RAW 264.7 cells were pre-incubated with various concentrations (2000, 500, 100ng/ml) of FGF21 and stimulated with LPS to induce oxidative stress and inflammation. The result of flow cytometry showed that ß-Klotho, FGF21 specific receptor, was expressed in murine splenic macrophages and RAW 264.7. In vitro, FGF21 reduced the expression of TNF-α, IL-1ß, IL-6 and IFN-γ and increased the level of IL-10 in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. FGF21 also suppressed profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level, and restored the activities of antioxidant enzymes SOD and GSH-Px in LPS-stimulated RAW 264.7 macrophages. Moreover, FGF21 inhibited LPS-induced nuclear factor-κB (NF-κB) activation, including degradation of I-κB and nuclear translocation of p65. In addition, the result of Western blot and real-time PCR showed that FGF21 induced heme oxygenase-1 (HO-1) expression and increased the nuclear transcription factor-E2-related factor 2 (Nrf2) levels in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. In conclusion, the results suggest that macrophages are the targets for the anti-inflammatory effects of FGF21, and FGF21 exerted an anti-inflammatory effect mainly via enhancing Nrf2-mediated anti-oxidant capacity and suppressing NF-κB signaling pathway.

Insulin sensitizes FGF21 in glucose and lipid metabolisms via activating common AKT pathway.

Previous studies reveal that fibroblast growth factor 21 (FGF21) sensitizes insulin to achieve a synergy in regulating glucose metabolism. Here, we report that insulin sensitizes FGF21 in regulating both glucose and lipid metabolisms. db/db diabetic mice were subcutaneously administrated once a day for 6 weeks. Effective dose of insulin (1 U) could control blood glucose level of the db/db mice for maximum of 2 h, increased the body weight of the db/db mice and did not improve serum lipid parameters. In contrast, effective dose of FGF21 (0.5 mg/kg) could maintain blood glucose of the db/db mice at normal level for at least 24 h, repressed the weight gain of the mice and significantly improved lipid parameters. Ineffective doses of FGF21 (0.125 mg/kg) and insulin had no effect on blood glucose level of the db/db mice after 24 h administration, body weight or lipid parameters. However, combination of the two ineffective doses could maintain blood glucose level of the db/db mice for at least 24 h, suppressed weight gain and significantly improved lipid parameters. These results suggest that insulin sensitizes FGF21 in regulating both glucose and lipid metabolism. The results aimed to study the molecular basis of FGF21 sensitization indicates that combination of the two ineffective doses increased the mRNA expression of glut1, glut4, ß-Klotho, sirt1, pgc-1α, ucp-1 and AKT phosphorylation, decreased fasn. The results demonstrate that insulin sensitizes FGF21 through elevating the phosphorylation of common gene Akt and amplifying FGF21 downstream signaling, including increasing expression of glut1 sirt1, pgc-1α, ucp-1, and decreasing fasn expression. In summary, we reports herein for the first time that insulin sensitizes FGF21 to achieve a synergy in regulating glucose and lipid metabolism. Along with previous studies, we conclude that the synergistic effect between FGF21 and insulin is realized through mutual sensitization.

Newcastle disease virus chimeras expressing the Hemagglutinin- Neuraminidase protein of mesogenic strain exhibits an enhanced anti-hepatoma efficacy.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, many researchers have reported that lentogenic NDV is a safe and effective agent for human cancer therapy. It had been demonstrated that the amino acid sequence of the fusion protein cleavage site is a major factor in the pathogenicity and anti-tumor efficacy of rNDV. However, the role of Hemagglutinin-Neuraminidase (HN) gene that contributes to virulence and anti-tumor efficacy remains undefined. To assess the role of HN gene in virus pathogenicity and anti-tumor efficacy, a reverse genetic system was developed using the lentogenic NDV Clone30 strain to provide backbone for gene exchange. Chimeric virus (rClone30-Anh(HN)) created by exchange of the HN gene of lentogenic strain Clone30 with HN gene of mesogenic strain produce no significant changes in virus pathogenicity as assessed by conducting the mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays. In vitro, infection with chimeras could induce the formation of syncytium relative significantly in HepG2 cells. Furthermore, chimeras was shown to induce the cell apoptosis via MTT and Annexin V-PI assays, reduce mitochondrial membrane potential and increase the mRNA transcription level of caspase 3. In vivo, ICR mice carrying tumor of hepatoma H22 cells were treated via intratumoral injection of chimeric virus. The treatment of chimera shows an obvious suppression in tumor volume. These results suggest that it could be an ideal approach to enhance the antitumor ability of Newcastle disease virus and highlighted the potential therapeutic application of rClone30-Anh(HN) as a viral vector to deliver foreign genes for treatment of cancers.

Recombinant Newcastle disease virus Anhinga strain (NDV/Anh-EGFP) for hepatoma therapy.

Hepatocellular carcinoma remains one of the most common malignant tumors in the world. Newcastle disease virus (NDV) has been proved to be an efficient oncolytic agent. NDV tumor killing efficacy is not only dependening on the NDV strain but the type of tumor targeted. It is significant to discover more effective and safe oncolytic strains. We investigated the effectiveness of genetically engineered NDV Anhinga strain in hepatoma treatment. The modified virus containing an insertion of enhanced green fluorescent protein (EGFP), named NDV/Anh-EGFP. The antitumor efficacy of the recombinant virus on hepatoma was examined both in vivo and in vitro. NDV Anhinga strain, which could be classified as a lytic strain, is an effective oncolytic agent on hepatoma. There was no significant difference in the TCID50 and growth capability between the recombinant NDV and the parental. NDV/Anh-EGFP can obviously inhibit hepatocarcinoma development in vitro and in vivo. We demonstrate Anhinga strain could become a potent candidate for clinical carcinoma therapy especially for hepatocarcinoma.