Spatial distribution, source apportionment and potential ecological risk assessment of suspended atmosphere microplastics in different underlying surfaces in Harbin.

Although suspended atmospheric microplastics (SAMPs) have been found to be ubiquitous and have potential impacts on human health, whereas studies related to source apportionment and potential ecological risk assessment in the atmospheric environment are still limited. This study investigated spatial distribution, source apportionment and potential ecological risk of SAMPs in six underlying surfaces of Harbin, China. The results show that all six underlying surfaces existed SAMPs, including polypropylene (PP), polyethylene terephthalate (PET) polyethylene (PE), polystyrene (PS) and polyvinyl chloride (PVC), with approximate 26.13 %, 24.10 %, 23.87 %, 13.51 %, and 12.39 %, respectively. SAMPs abundances from filtered air were relatively high and averaged 1.76 n/m3. The SAMPs mainly contained fibrous (59.01 %), fragmented (30.18 %), and granular (10.81 %) with transparent (62.39 %), black 13.74 %), red (7.43 %), white (6.53 %), blue, and yellow (3.60 %), and particle size ranged from 1.3 to 518 µm. In addition, source apportionment of SAMPs shows that SAMPs were originated from five emission sources including living source (19.53 %), construction source (12.08 %), transportation source (47.25 %), industrial source (5.11 %), and agricultural source (16.13 %) in Harbin. A significant correction was observed between SAMPs abundances and human activity (R = 0.68, P = 0.66), atmospheric humidity (R = -0.40, P = 0.02), and wind direction (R = 0.22, P = 0.04) in different underlying surface. Furthermore, potential ecological hazardous single index (EI) of PVC and PS were higher than PP, PET, and PS in the construction land, cultivated land, forest land, grassland, water area, and unused land. An estimation of the potential ecological risk index (RI) from SAMPs using Positive Matrix Factorization (PMF) model indicated that Harbin presented a minor ecological risk with average 16.59 of RI index of microplastics in environments. In conclusion, data in this study indicate that SAMPs are existed in atmospheric environments, which have possible risks for human health via inhalation.

Treatment of nanofiltration concentrate of landfill leachate using advanced oxidation processes incorporated with bioaugmentation.

Advanced oxidation processes have been broadly applied in wastewater treatment, but few studies have focused on its degradative effect on refractory organic contaminants in membrane concentrates of landfill leachate. In this study, the treatment effects of advanced oxidation processes including electrocoagulation (EC), ozone (OZ), anodic oxidation (AO) and electro-Fenton (EF) incorporated with genetically engineered nitrifying bacteria Rhodococcus erythropolis expressing Nirs and AMO (rRho-NM) on nanofiltration concentrate (NFC) of old landfill leachate were investigated in a lab-scale experiment. The results showed that advanced oxidation processes degraded the refractory organic contaminants including coagulation-resistant substances (CRS), humic acid (HA), fulvic acid (FvA), macro molecular organics (MMOs) and benzene ring compounds (BRCs) and increased the biodegradability in NFC of old landfill leachate. Compared to activated sludge (AS), rRho-NM exhibited an excellent removal performance for total organic carbon (TOC), ammonia nitrogen (NH4-N), total nitrogen (TN), biochemical oxygen demand (BOD) and chemical oxygen demand (COD) for advanced oxidation processes-treated NFC of old landfill leachate. Advanced oxidation processes incorporated with bioaugmentation demonstrated an outstanding degradation performance for removing refractory organic contaminants, TOC, NH4-N, TN, BOD, COD and heavy metal in NFC of old landfill leachate. In addition, OZ incorporated with rRho-NM (OZ-rRho-NM) showed the optimal removal efficacy in reduction of refractory organic contaminants, TOC, NH4-N, TN, BOD and COD, the shortest hydraulic retention time (HRT) and the minimum energy consumption in NFC of landfill leachate. Furthermore, the cheapest treatment cost for NFC could be achieved by EC incorporated with rRho-NM (EC-rRho-NM). More impressively, rRho-NM remained stable in expressing Nirs and AMO genes, increased nitrification and denitrification rate, and improved MBR effluent quality in the treatment of NFC. In conclusion, this work provides new insights into the application of advanced oxidation processes incorporated with bioaugmentation using rRho-NM for the treatment of NFC of old landfill leachate.

Landfill leachate treatment through the combination of genetically engineered bacteria Rhodococcus erythropolis expressing Nirs and AMO and membrane filtration processes.

This study developed a process of genetically engineered bacteria Rhodococcus erythropolis expressing Nirs and AMO combined with membrane bioreactor (MBR), nanofiltration (NF) and reverse osmosis (RO) membrane (pRho-NA-MNR) for advanced treatment of landfill leachate. Results demonstrated that pRho-NA-MNR presented higher removal rate of chemical oxygen demand (COD), biological oxygen demand (BOD), ammonia nitrogen (N-NH4), total nitrogen (TN) and total organic carbon (TOC) than activated sludge (AS-MNR) system. Administration of pRho-NA increased nitrification by converting N-NH4 to nitrite (N-NO2) and Nitrate (N-NO3), and promoting denitrification by converting N-NO2 to nitrogen (N2) in the landfill leachate treatment, promoted the pH control, increased sludge activity and effluent yield, shortened phase length adaptation under alternating aerobic-anoxic conditions. pRho-NA increased the nitration and denitrifying rate in the aerobic and anaerobic stage in the system by increasing Cyt cd1 and Cyt c expression in the activated sludge. Nitrogen removal by nitrification and denitrification was positively correlated to the concentration of Nirs and AMO expression. Treatment with pRho-NA promoted pollutant removal efficiency of membrane bioreactor, nanofiltration and reverse osmosis membrane processes in landfill leachate. In conclusion, data suggest that pRho-NA-MNR facilitates the formation of granular sludge and enhances comparable removal of nitrogen and organic compounds, indicating the practice of this process should be considered in landfill leachate treatment system.

Advanced treatment of sewage by membrane bioreactor associate with genetically engineered autotrophic nitrifying bacteria.

This study introduced a process of MBR combing with genetically engineered bacteria of expressing nirs and ppk genes (GEB-Nirs/PPK) for advanced treatment of sewage in scenic area. An industrial scale application was staged anaerobic digestion, aerobic digestion. Over more than 150 days of continuous operation, TMP in this system was less than 0.18 bar. With a membrane flux of 6.48 m3/h, TMP remained below 0.2 bar and effluent remained above 70 m3 during continuous operation. Average COD and BOD removals averaged 94.2% and 93.6%, and were obtained with average effluent COD and BOD below 10.4 mg/L and 4.2 mg/L, respectively. The TN and TP removals averaged 98.8% and 94.3%, and never higher than 3.2 mg/L and 0.2 mg/L, respectively, in the processing system. In conclusion, these results indicate that the process of MBR associate with genetically engineered autotrophic nitrifying bacteria is of high-efficiency for advanced treatment of sewage.

Simvastatin induces breast cancer cell death through oxidative stress up-regulating miR-140-5p.

Statins, a class of hyperlipidemic drugs, are widely used cholesterol lowering drugs that selectively inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which is the rate-limiting enzyme in cholesterol biosynthesis, leading to decreasing of cholesterol biosynthesis. Statins exert anti-tumoral effects on various cancer, including breast cancer. However, the molecular mechanisms for the actions were not fully elucidated. The purpose of this study was to elucidate the effects of statins on proliferation and apoptosis in the ER-negative breast cancer cell line MDA-MB-231. Our results showed that simvastatin increased the expression of miR-140-5p in a dose dependent manner via activating transcription factor NRF1, reduced cell proliferation and induced apoptosis, and we also found that SLC2A1 was a new target of miR-140-5p. In conclusion, data in this study shed light on the potential anti-tumoral effects of simvastatin in breast cancer and presents a highly promising therapeutic option, using drug and miRNA for combined treating cancers.

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.

Fibroblast growth factor 21 protects mouse brain against D-galactose induced aging via suppression of oxidative stress response and advanced glycation end products formation.

Fibroblast growth factor 21 (FGF21) is a hormone secreted predominantly in the liver, pancreas and adipose tissue. Recently, it has been reported that FGF21-Transgenic mice can extend their lifespan compared with wild type counterparts. Thus, we hypothesize that FGF21 may play some roles in aging of organisms. In this study d-galactose (d-gal)-induced aging mice were used to study the mechanism that FGF21 protects mice from aging. The three-month-old Kunming mice were subcutaneously injected with d-gal (180mg·kg(-1)·d(-1)) for 8weeks and administered simultaneously with FGF21 (1, 2 or 5mg·kg(-1)·d(-1)). Our results showed that administration of FGF21 significantly improved behavioral performance of d-gal-treated mice in water maze task and step-down test, reduced brain cell damage in the hippocampus, and attenuated the d-gal-induced production of MDA, ROS and advanced glycation end products (AGEs). At the same time, FGF21 also markedly renewed the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total anti-oxidation capability (T-AOC), and decreased the enhanced total cholinesterase (TChE) activity in the brain of d-gal-treated mice. The expression of aldose reductase (AR), sorbitol dehydrogenase (SDH) and member-anchored receptor for AGEs (RAGE) declined significantly after FGF21 treatment. Furthermore, FGF21 suppressed inflamm-aging by inhibiting IκBα degradation and NF-κB p65 nuclear translocation. The expression levels of pro-inflammatory cytokines, such as TNF-α and IL-6, decreased significantly. In conclusion, these results suggest that FGF21 protects the aging mice brain from d-gal-induced injury by attenuating oxidative stress damage and decreasing AGE formation.

Fibroblast growth factor (FGF21) protects mouse liver against D-galactose-induced oxidative stress and apoptosis via activating Nrf2 and PI3K/Akt pathways.

FGF21 is recently discovered with pleiotropic effects on glucose and lipid metabolism. However, the potential protective effect of FGF21 against D-gal-induced injury in the liver has not been demonstrated. The aim of this study is to investigate the pathophysiological role of FGF21 on hepatic oxidative injury and apoptosis in mice induced by D-gal. The 3-month-old Kunming mice were subcutaneously injected with D-gal (180 mg kg(-1) d(-1)) for 8 weeks and administered simultaneously with FGF21 (5 or 1 mg kg(-1) d(-1)). Our results showed that the administration of FGF21 significantly alleviated histological lesion including structure damage, degeneration, and necrosis of hepatocytes induced by D-gal, and attenuated the elevation of liver injury markers, serum AST, and ALP in a dose-dependent manner. FGF21 treatment also suppressed D-gal-induced profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level in the liver, and restored the activities of antioxidant enzymes SOD, CAT, GSH-Px, and T-AOC. Moreover, FGF21 treatment increased the nuclear abundance of Nrf2 and subsequent up regulation of several antioxidant genes. Furthermore, a TUNEL assay showed that D-gal-induced apoptosis in the mouse liver was significantly inhibited by FGF21. The expression of caspase-3 was markedly inhibited by the treatment of FGF21 in the liver of D-gal-treated mice. The levels of PI3K and PBK/Akt were also largely enhanced, which in turn inactivated pro-apoptotic signaling events, restoring the balance between pro- and anti-apoptotic Bcl-2 and Bax proteins in the liver of D-gal-treated mice. In conclusion, these results suggest that FGF21 protects the mouse liver against D-gal-induced hepatocyte oxidative stress via enhancing Nrf2-mediated antioxidant capacity and apoptosis via activating PI3K/Akt pathway.

Fibroblast growth factor 21 (FGF21) ameliorates collagen-induced arthritis through modulating oxidative stress and suppressing nuclear factor-kappa B pathway.

It has been demonstrated that circulating FGF21 levels are elevated in the serum and synovial fluid of patients with rheumatoid arthritis (RA). The aim of this study is to investigate efficacy of FGF21 for treatment of RA and the molecular mechanisms of the therapeutic effect on collagen-induced arthritis (CIA). Mice with CIA were subcutaneously administered with FGF21 (5, 2 or 1mg·kg(-1)·d(-1)), IL-1ß antibody (5mg·kg(-1)·d(-1)), IL-17A antibody (5mg·kg(-1)·d(-1)) and dexamethasone (DEX) (1mg·kg(-1)·d(-1)), respectively. The effects of treatment were determined by arthritis severity score, histological damage and cytokine production. The activation of NF-κB was analyzed by Western blotting. We also detected the levels of oxidative stress parameters. Our results showed that FGF21 had beneficial effects on clinical symptom and histological lesion of CIA mice. Similar to antibody and DEX, FGF21 treatment alleviated the severity of arthritis by reducing humoral and cellular immune responses and down-regulating the expression of pro-inflammatory cytokines. FGF21 treatment also reduced the expression of TNF-α, IL-1ß, IL-6, IFN-γ and MMP-3 and increased level of IL-10 in the spleen tissue or the plasma of CIA mice in a dose-dependent manner. Furthermore, FGF21 inhibited IκBα degradation and NF-κB p65 nuclear translocation and induced significant changes of oxidative stress parameters (MDA, SOD, CAT, GSH-PX and GSH) in the plasma. FGF21 exerts therapeutic efficacy for RA through antioxidant reaction and inhibiting NF-κB inflammatory pathway. This study provides evidence that FGF21 may be a promising therapeutic agent for RA patients.

Recombinant Newcastle Disease virus Expressing IL15 Demonstrates Promising Antitumor Efficiency in Melanoma Model.

Recombinant Newcastle Disease Virus (rNDV) has shown oncolytic therapeutic effect in preclinical studies. Previous data indicate that rNDV carrying IL2 has shown promise in cancer therapy. Due to the significant side effects of IL2, IL15 has been introduced into cancer therapy. A number of studies have suggested that IL15 efficiently enhances the activities of CTL and NK cells and inhibits the tumor recurrence and metastasis. Furthermore, IL15 is less toxic than IL2. Therefore, we hypothesize that a recombinant NDV expressing IL15 would be a promising agent for the treatment of malignant tumors. The human IL15 gene or IL2 gene was incorporated into the genome of lentogenic LaSota strain at the position between the HN and L genes (namely rNDV-IL15 or rNDV-IL2). The two viruses efficiently infected tumor cells and expressed IL15 or IL2 protein. Melanoma tumor-bearing mice were treated by intra-tumoral (i.t.) injection of rNDV-IL15 or rNDV-IL2. Both rNDV-IL15 and rNDV-IL2 effectively suppressed tumor growth compared with rNDV. The 120-day survival rate of rNDV-IL15- treated group was 12.5% higher than that of rNDV-IL2 group, although the difference was not statistically significant, both recombinant viruses had strong abilities to induce CD41 T cell and CTL cell responses. However, rNDV-IL15 significantly induced more IFN-γ release and stimulated more CD81 T cells infiltration in the tumor sites compared with rNDV-IL2. In the tumor re-challenged experiment, the survival rates of rNDV-IL15 group and rNDV-IL2 group were statistically higher than that of PBS group. The survival rate of rNDV-IL15 group was 26.67% higher than that of rNDV-IL2 group although the difference was not statistically significant. In conclusion, rNDV-IL15 is a promising antitumor agent against melanoma.

TNF-related Apoptosis-inducing Ligand Delivered by rNDV is a Novel Agent for Cancer Gene Therapy.

Recombinant Newcastle disease virus (rNDV) as antitumor agent has been shown to be effective for cancer therapy. And TNF-related apoptosis-inducing ligand (TRAIL) also has been demonstrated potentially cancer-therapeutic effects. In this study, we constructed TRAIL delivered by rNDV (rNDV-TRAIL) and investigated whether TRAIL would generate the potential synergistic therapeutic effects with rNDV for cancer therapy. In vitro experiments indicated that TRAIL expressed by rNDV demonstrated good biological activity. TRAIL significantly enhanced inducing apoptosis of rNDV in death receptor expression cancer cell lines. Experiments in malignant melanoma-bearing mice demonstrated that expression of TRAIL delivered by rNDV significantly inhibited the tumor growth and prolonged the survival of treated animals compared to control. In conclusion, oncolytic capacity of rNDV was augmented by TRAIL and the inherent anti-neoplastic properties of NDV were enhanced by the introduction of therapeutic TRAIL gene.

[The synergism and mechanism of action of rClone30-hDR5 in combination with TRAIL on HCC].

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.

[Effect of FGF-21 on learning and memory ability and antioxidant capacity in brain tissue of D-galactose-induced aging mice].

This study aims to investigate the effects of fibroblast growth factor 21 (FGF-21) on learning and memory abilities and antioxidant capacity of D-galactose-induced aging mice. Kunming mice (37.1 +/- 0.62) g were randomly divided into normal control group, model group and FGF-21 high, medium and low dose groups (n = 8). Each group was injected in cervical part subcutaneously with D-galactose 180 mg x kg(-1) x d(-1) once a day for 8 weeks. At the same time, FGF-21-treated mice were administered with FGF-21 by giving subcutaneous injection in cervical part at the daily doses of 5, 2 and 1 mg x kg(-1) x d(-1). The normal control group was given with normal saline by subcutaneous injection in cervical part. At seventh week of the experiment, the learning and memory abilities of mice were determined by water maze and jumping stand tests. At the end of the experiment, the mice were sacrificed and the cells damage of hippocampus was observed by HE staining in each group. Reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidant capacity (T-AOC) in the brain of mice were determined. The results showed that different doses of FGF-21 could reduce the time reaching the end (P < 0.01 or P < 0.05) and the number of touching blind side (P < 0.01 or P < 0.05) in the water maze comparing with the model group. It could also prolong the latency time (P < 0.05) and decrease the number of errors (P < 0.01 or P < 0.05) in the step down test. The result of HE staining showed that FGF-21 could significantly reduce brain cell damage in the hippocampus. The ROS and MDA levels of three different doses FGF-21 treatment group reduced significantly than that of the model group [(5.58 +/- 1.07), (7.78 +/- 1.92), (9.03 +/- 1.77) vs (12.75 +/- 2.02) pmol (DCF) x min(-1) x mg(-1), P < 0.01 or P < 0.05], [(2.92 +/- 0.71), (4.21 +/- 0.81), (4.41 +/- 0.97) vs (5.62 +/- 0.63) nmol x mg(-1) (protein), P < 0.01]. Comparing with the model group, the activities of SOD, GPx, CAT and T-AOC of the three different doses FGF-21 treatment groups were also improved in a dose-dependent manner. This study demonstrates that FGF-21 can ameliorate learning and memory abilities of D-galactose induced aging mice, improve the antioxidant abilities in brain tissue and delay brain aging. This finding provides a theoretical support for clinical application of FGF-21 as a novel therapeutics for preventing aging.

Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease.

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.

Genetically engineered Newcastle disease virus expressing interleukin-2 and TNF-related apoptosis-inducing ligand for cancer therapy.

Recombinant Newcastle disease virus (rNDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. In this study, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of inserting both interleukin-2 (IL-2) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) delivered by rNDV. We demonstrated that rNDV expressing TRAIL (rNDV-TRAIL) or both human IL-2 and TRAIL (rNDV-IL-2-TRAIL) significantly enhanced inherent anti-neoplastic of rNDV by inducing apoptosis. And we showed that apoptosis-related genes mRNA expression was increased after treated with rNDV-TRAIL or rNDV-IL-2-TRAIL compared with rNDV and rNDV-IL-2. We also demonstrated that both rNDV-IL-2 and rNDV-IL-2-TRAIL induced proliferation of the CD4(+) and CD8(+) in treated mice and elicited expression of TNF-α and IFN-γ antitumor cytokines. These mice treated with oncolytic agents exhibited significant reduction in tumor development compared with mice treated with the parental virus. In addition, experiments in both hepatocellular carcinoma and melanoma-bearing mice demonstrated that the genetically engineered rNDV-IL-2-TRAIL exhibited prolonged animals' survival compared with rNDV, rNDV-IL-2, and rNDV-TRAIL. In conclusion, the immunotherapy and oncolytic virotherapy properties of NDV can be enhanced by the introduction of IL-2 and TRAIL genes, whose products initiated a broad cascade of immunological affects and induced tumor cells apoptosis in the microenvironment of the immune system.

[Antitumor efficacy of the recombinant Newcastle disease virus rNDV-IL15 on melanoma models].

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.

Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy.

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, several clinical trials have reported that mesogenic NDV is a safe and effective agent for human cancer therapy. Interleukin 2 (IL2) is a cytokine that stimulates T cell propagation to trigger innate and adaptive immunity. IL2 has been used for cancer therapy and has achieved curative effects. In this study, a recombinant NDV LaSota strain expressing human interleukin 2 (rLaSota/IL2) was generated. The ability of rLaSota/IL2 to express human IL2 was detected in the infected tumor cells. In addition, the activity of IL2 was analyzed. The antitumor potential of rLaSota/IL2 was studied by xenograph mice carrying H22 and B16-F10 cells. Tumor-specific CD4(+) and CD8(+) T cells and MHC II were also analyzed in the two tumor-bearing models. Our study showed that rLaSota/IL2 significantly stimulated tumor-specific cytotoxic T-lymphocyte (CTL) responses and increased regulatory CD4(+) and cytotoxic CD8(+) T cells proliferation. The treatment with rLaSota/IL2 led to tumor regression in tumor-bearing mice and prolonged the survival of tumor-bearing mice. Furthermore, tumor challenging experiments demonstrated that rLaSota/IL2 invoked mice a unique capacity to remember a pathogen through the generation of memory T cells, which protect the host in the event of reinfection and form adaptive immune system. The result indicates that tumor-infiltrating CD4(+) T regulatory cells may denote the effective regression of tumors. Taken together, rLaSota/IL2 has potential for immunotherapy and oncolytic therapy of cancers and may be an ideal candidate for clinical application in future cancer therapy.

Chimeric anti-IL-17 full-length monoclonal antibody is a novel potential candidate for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, primarily manifesting as inflammatory arthritis. It is associated with chronic inflammation of the synovial joints, mostly in the hands and feet, as well as with systemic extra-articular inflammation. The chimeric anti-interleukin (IL)-17 full-length monoclonal antibody (CMa17Aab) targets IL-17A, which is an important cytokine in the pathogenesis of RA and other inflammatory disorders. In this study, we investigated whether CMa17Aab exerts therapeutic effects in a mouse model of type II collagen-induced arthritis (CIA). Mice with CIA were subcutaneously injected with the humanized CMa17Aab antibody. The effects of treatment were assessed by estimating the arthritis severity score, the extent of histological damage and bone destruction, the autoreactive humoral and cellular immune responses and the production of cytokines. Treatment with CMa17Aab exerted beneficial effects in the mice with CIA as regards clinical and histological parameters. Compared with the controls, treatment with CMa17Aab resulted in a significant alleviation of the severity of the symptoms of arthritis, by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and downregulating the expression of IL-6, IL-8, matrix metalloproteinase (MMP)-3, IL-17, IL-1ß, tumor necrosis factor (TNF)-α, receptor activator for nuclear factor-κB ligand (RANKL) and interferon (IFN)-γ in inflamed tissues. In conclusion, our study demonstrates that treatment with CMa17Aab exerts beneficial effects in mice with CIA, by preventing joint inflammation, cartilage destruction and bone damage. These preliminary results suggest that CMa17Aab is an important regulator in RA, and that it may represent a novel therapeutic agent that may prove useful in the treatment of this disease.

[Antitumor activity of the recombinant rClone30-CD/5-FC system].

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.

Enhancement of anti-tumor activity of Newcastle disease virus by the synergistic effect of cytosine deaminase.

This study was conducted to investigate enhancement of anti-tumor effects of the lentogenic Newcastle disease virus Clone30 strain (NDV rClone30) expressing cytosine deaminase (CD) gene against tumor cells and in murine groin tumor-bearing models. Cytotoxic effects of the rClone30-CD/5-FC on the HepG2 cell line were examined by an MTT method. Anti-tumor activity of rClone30-CD/5-FC was examined in H22 tumor-bearing mice. Compared to the rClone30-CD virus treatment alone, NDV rClone30-CD/5-FC at 0.1 and 1 MOIs exerted significant cytotoxic effects (P<0.05) on HepG2 cells. For treatment of H22 tumor-bearing mice, recombinant NDV was injected together with 5-FC given by either intra-tumor injection or tail vein injection. When 5-FC was administered by intra-tumor injection, survival for the rClone30-CD/5-FC-treated mice was 4/6 for 80 days period vs 1/6 , 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC and saline alone, respectively. When 5-FC was given by tail vein injection, survival for the rClone30-CD/5-FC-treated mice was 3/6 vs 2/6 , 0/6 and 0/6 for the mice treated with rClone30-CD, 5-FC or saline alone, respectively. In this study, NDV was used for the first time to deliver the suicide gene for cancer therapy. Incorporation of the CD gene in the lentogenic NDV genome together with 5-FC significantly enhances cell death of HepG2 tumor cells in vitro, decreases tumor volume and increases survival of H22 tumor-bearing mice in vivo.