Therapeutic potential of xtr-miR-22-3p from Plastrum testudinis in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is marked by a block at the promyelocyte stage. Treatments like ATRA and ATO face resistance and relapse issues. Plastrum testudinis, a traditional Chinese medicine, may offer therapeutic potential. This study investigated xtr-miR-22-3p from P. testudinis for treating APL. High expression of xtr-miR-22-3p was confirmed, with target prediction indicating interactions with key genes, including PML. xtr-miR-22-3p reduced HL-60 leukemia cell growth, altered the cell cycle, and selectively inhibited HL-60 proliferation while promoting BMSC growth, suggesting its potential as a targeted APL therapy.

The effect of miR-381 on proliferation and prognosis of breast cancer by altering CCNA2 expression.

BACKGROUND: MiR-381 can regulate the expression of cyclin A2 (CCNA2) to inhibit the proliferation and migration of bladder cancer cells, but whether miR-381 has the same function in breast cancer is not well know. METHODS: The over express or silence miR-381 expressing cell lines were constructed by lentivirus infection to reveal the biological functions of miR-381 in vitro. The expression of miR-381 and CCNA2 in 162 breast cancer patients were detected to further reveal their impact and predictive value on progression-free survival (PFS) and overall survival (OS). RESULTS: After transfection of MDA-MB-231 and MCF-7 cells with miR-381 mimics, the expression of miR-381 was effectively up-regulated and CCNA2 was effectively down-regulated, while the opposite results were observed in tumour cell which transfected with miR-381 inhibitors. After transfection of cell lines with miR-381 mimics, tumour cell activity was significantly reduced, while the opposite results were observed in tumour cell which transfected with miR-381 inhibitors. The area under curves (AUCs) of miRNA-381 and CCNA2 for predicting PFS and OS were 0.711, 0.695, 0.694 and 0.675 respectively. Cox regression analysis showed that miRNA-381 ≥ 1.65 2-ΔΔCt and CCNA ≥ 2.95 2-ΔΔCt were the influence factors of PFS and OS, the hazard ratio (HR) values were 0.553, 2.075, 0.462 and 2.089, respectively. CONCLUSION: miR-381 inhibitors breast cancer cells proliferation and migration by down-regulating the expression of CCNA2, both of them can predict the prognosis of breast cancer.

miR-381 can regulate the expression of cyclin A2 and inhibit the proliferation and migration of bladder cancer cells, but whether miR-381 has the same function in breast cancer is not well know. We analysed the levels of miR-381 and cyclin A2 in breast cancer patients and breast cancer cells to reveal the mechanism of miR-381 affecting the expression of cyclin A2. We found miRNA-381 affects the proliferation and migration of breast cancer cells by down-regulating the expression of cyclin A2. The expression of serum miR-381 and cyclin A2 have important values in predicting the prognosis of breast cancer. Our findings provide mechanistic insights into how miR-381 regulates the proliferation and migration of breast cancer, as well as a new target for clinical treatment. Future research may focus on how to improve patient prognosis by up-regulating expression of miR-381 and down-regulating the expression of cyclin A2.

Protein Engineering of PhUGT, a Donor Promiscuous Glycosyltransferase, for the Improved Enzymatic Synthesis of Antioxidant Quercetin 3-O-N-Acetylgalactosamine.

Quercetin 3-O-N-acetylgalactosamine (Q3GalNAc), a derivative of dietary hyperoside, had never been enzymatically synthesized due to the lack of well-identified N-acetylgalactosamine-transferase (GalNAc-T). Herein, PhUGT, an identified flavonoid 3-O-galactosyltransferase from Petunia hybrida, was demonstrated to display quercetin GalNAc-T activity, transferring a N-acetylgalactosamine (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) to the 3-OH of quercetin to form Q3GalNAc with a low conversion of 11.7% at 40 °C for 2 h. Protein engineering was thus performed, and the resultant PhUGT variant F368T got an enhanced conversion of 75.5% toward UDP-GalNAc. The enzymatically synthesized Q3GalNAc exhibited a comparable antioxidant activity with other quercetin 3-O-glycosides. Further studies revealed that PhUGT was a donor promiscuous glycosyltransferase (GT), recognizing seven sugar donors. This finding overturned a previous notion that PhUGT exclusively recognized UDP-galactose (UDP-Gal). The reason why PhUGT was mistaken for a UDP-Gal-specific GT was demonstrated to be a shorter reaction time, in which many quercetin 3-O-glycosides, except hyperoside, could not be effectively synthesized. The fact that the microbial cell factory expressing PhUGT could yield an array of Q3Gs further confirmed the donor promiscuity of PhUGT. This study laid a foundation for the scale production of Q3GalNAc and provided a potent biocatalyst capable of glycodiversifying quercetin as well.

CCT4 suppression inhibits tumor growth in hepatocellular carcinoma by interacting with Cdc20.

BACKGROUND: The chaperonin containing t-complex (CCT) proteins play an important role in cell cycle-related protein degradation in yeast and mammals. The role of the chaperonin containing t-complex 4 (CCT4), one subtype of CCT proteins, in the progress of hepatocellular carcinoma (HCC) was not fully elucidated. Here, we aimed to explore the mechanisms of CCT4 in HCC. METHODS: In this study, we used the UALCAN platform to analyze the relationship between CCT4 and HCC, and the association of CCT4 with the overall survival (OS) of HCC patients was also analyzed. CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot (WB) assay. Lentivirus vector was used to knock down the CCT4 expression, and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines. Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect the cell proliferation, and flow cytometry (FCM) was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells. Co-immunoprecipitation (co-IP) assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC. Data were calculated from at least three replicate experiments and expressed as mean ± standard deviation. Student's t test, paired t test, and Kaplan-Meier analysis were used to compare across different groups. RESULTS: We found CCT4 was upregulated in HCC tissues compared with normal tissues, and its high expression was associated with poor prognosis (P < 0.001). CCT4 was significantly increased in HCC tumor tissues compared with normal tissues (0.98 ±â€Š0.12 vs. 0.23 ±â€Š0.05, t = 7.73, P < 0.001). After being transfected with CCT4 short-hairpin RNA (shRNA), CCT4 was decreased in mRNA level and protein level in both Huh7 (mRNA level: 0.41 ±â€Š0.07 vs. 1.01 ±â€Š0.11, t = 8.09, P = 0.001; protein level: 0.61 ±â€Š0.03 vs. 0.93 ±â€Š0.07, t = 7.19, P = 0.002) and Hep3b cells (mRNA level: 0.55 ±â€Š0.11 vs. 1.04 ±â€Š0.15, t = 4.51, P = 0.011; protein level: 0.64 ±â€Š0.10 vs. 0.95 ±â€Š0.08, t = 4.32, P = 0.012). CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7 (OD value of 3 days: 0.60 ±â€Š0.14 vs. 0.97 ±â€Š0.16, t = 3.13, P = 0.036; OD value of 4 days: 1.03 ±â€Š0.07 vs. 1.50 ±â€Š0.12, t = 5.97, P = 0.004) and Hep3b (OD value of 3 days: 0.69 ±â€Š0.14 vs. 1.10 ±â€Š0.11, t = 3.91, P = 0.017; OD value of 4 days: 1.12 ±â€Š0.12 vs. 1.48 ±â€Š0.13, t = 3.55, P = 0.024) cells. EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7 (EdU positive rate: [31.25 ±â€Š3.41]% vs. [58.72 ±â€Š3.78]%, t = 9.34, P = 0.001) and Hep3b cells (EdU positive rate: [44.13 ±â€Š7.02]% vs. [61.79 ±â€Š3.96]%, t = 3.79, P = 0.019). FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells (apoptosis rate of Huh7: [9.10 ±â€Š0.80]% vs. [3.66 ±â€Š0.64]%, t = -9.18, P = 0.001; apoptosis rate of Hep3b: [6.69 ±â€Š0.72]% vs. [4.20 ±â€Š0.86]%, t = -3.84, P = 0.018). We also found that CCT4 could regulate anaphase-promoting complex (APC)Cdc20 activity via interacting with Cdc20. Furthermore, CCT4 knockdown induced securin (0.65 ±â€Š0.06 vs. 0.44 ±â€Š0.05, t = -4.69, P = 0.009) and B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (Bim; 0.96 ±â€Š0.06 vs. 0.61 ±â€Š0.09, t = -5.65, P = 0.005) accumulation. The upregulation of securin inhibited cell growth by downregulating cyclin D1 (0.65 ±â€Š0.05 vs. 1.04 ±â€Š0.07, t = 8.12, P = 0.001), and the accumulation of Bim inhibited Bcl-2 (0.77 ±â€Š0.04 vs. 0.87 ±â€Š0.04, t = 3.00, P = 0.040) and activated caspase 9 (caspase 9: 0.77 ±â€Š0.04 vs. 0.84 ±â€Š0.05, t = 1.81, P = 0.145; cleaved caspase 9: 0.64 ±â€Š0.06 vs. 0.16 ±â€Š0.07, t = 1.81, P = 0.001), which led to elevated apoptosis. CONCLUSIONS: Overall, these results showed that CCT4 played an important role in HCC pathogenesis through, at least partly, interacting with Cdc20.

Polyphenols from Securidaca inappendiculata alleviated acute lung injury in rats by inhibiting oxidative stress sensitive pathways.

Objective: Securidaca inappendiculata is a medicinal plant frequently used in the treatment of inflammatory diseases in south China. In this study, we aimed to explore its bioactive constituent which contributes to the anti-inflammatory activity. Methods: Polyphenol-enriched and polyphenol-deprived fractions (PRF and PDF, respectively) were separated from the ethanolic extract by HPD300 macroporous resin-based method, and their anti-inflammatory activities were investigated on a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model in rats. The possible mechanism of action in alleviating acute inflammation was studied using RAW264.7 cells. Results: Both Folin-Ciocalteu and 1H nuclear magnetic resonance (NMR) analyses showed that polyphenolic content in PRF was approximately 10 times higher than that of PDF, and this observation reflected in their antioxidative capacities. PRF but not PDF significantly decreased the level of malondialdehyde, suppressed the expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, and improved the severity of ALI in rats. PRF at 10 µg/mL effectively downregulated the expression of proteins NAMPT, HMGB1, TLR4, and p-p65, and scavenged the intracellular reactive oxygen species (ROS) in LPS-primed RAW264.7 cells. N-acetyl-L-cysteine exhibited similar inhibitory effects on ROS production and NAMPT-mediated TLR4/NF-κB activation in vitro, whereas nicotinamide mononucleotide antagonized all the changes induced by PRF during cotreatments. Conclusion: As an antioxidant, PRF exhibited potent anti-inflammatory activity under both in vivo and in vitro conditions by downregulating NAMPT and TLR4/NF-κB. Accordingly, polyphenols were identified as important bioactive constituents in S. inappendiculata targeting oxidative stress-sensitive pro-inflammatory pathways.

Effect of exogenous phytohormones treatment on glycyrrhizic acid accumulation and preliminary exploration of the chemical control network based on glycyrrhizic acid in root of Glycyrrhiza uralensis

ABSTRACT One-year-old Glycyrrhiza uralensis Fisch. ex DC, Fabaceae, was treated with three exogenous phytohormones in June and July, namely gibberellin, auxin (indole-3-acetic acid), methyl jasmonate at different concentrations. Control plants were treated with water. Roots of controls and hormones-treated G. uralensis plants were harvested at different times, and the contents of seven main chemical components were determined. Root glycyrrhizic acid content of plants treated in June increased significantly compared with controls, and the difference was significant. As for plants treated in July, root glycyrrhizic acid content increased in which sprayed with appropriate concentrations of hormones, but the effects of hormones were more evident in plants treated in June coincided with the vigorous growth period than those treated in July. Gibberellin at 40 mg/l and auxin at 40 mg/l applied in the two treatment periods significantly promoted the accumulation of glycyrrhizic acid in G. uralensis root. Treatment with methyl jasmonate at 100 and 25 mg/l in June and July, respectively, also increased glycyrrhizic acid content significantly. The determination of major active compositions indicated that liquiritin, isoliquiritin, isoliquiritin apioside and liquiritin apioside contents were positively related to glycyrrhizic acid content. The study preliminarily found phytohormones and the main chemical components associated with glycyrrhizic acid content, and these discoveries could provide a basis for establishing a chemical control network with glycyrrhizic acid as the core, confirming the secondary product metabolic pathways in the network and completely uncovering synthesis mechanism underlying glycyrrhizic acid-combined functional gene polymorphism.

[Effects of superphosphate addition on NH3 and greenhouse gas emissions during vegetable waste composting].

To study the effects of superphosphate (SP) on the NH, and greenhouse gas emissions, vegetable waste composting was performed for 27 days using 6 different treatments. In addition to the controls, five vegetable waste mixtures (0.77 m3 each) were treated with different amounts of the SP additive, namely, 5%, 10%, 15%, 20% and 25%. The ammonia volatilization loss and greenhouse gas emissions were measured during composting. Results indicated that the SP additive significantly decreased the ammonia volatilization and greenhouse gas emissions during vegetable waste composting. The additive reduced the total NH3 emission by 4.0% to 16.7%. The total greenhouse gas emissions (CO2-eq) of all treatments with SP additives were decreased by 10.2% to 20.8%, as compared with the controls. The NH3 emission during vegetable waste composting had the highest contribution to the greenhouse effect caused by the four different gases. The amount of NH3 (CO2-eq) from each treatment ranged from 59.90 kg . t-1 to 81.58 kg . t-1; NH3(CO2-eq) accounted for 69% to 77% of the total emissions from the four gases. Therefore, SP is a cost-effective phosphorus-based fertilizer that can be used as an additive during vegetable waste composting to reduce the NH3 and greenhouse gas emissions as well as to improve the value of compost as a fertilizer.

[Sequence analysis and identification of a chloroplast matK gene in Rhei Rhizoma from different botanical origins].

Rhei Rhizoma is a Chinese medicine with multiple botanical origins. There is a problem to identify it with conventional methods. To compare the characteristics of chloroplast matK gene sequences of different Rheum species and authenticate inspected species, the matK gene sequences of different species from different origins were amplified, cloned, and sequenced. Genomic DNA of Rheum plants was extracted using modified DNA extracted Kit and matK gene sequences were analyzed by ContingExpress, DNAman and MEGA5.0. The length of matK gene sequences of Rheum palmatum, R. tanguticum and R. officinale were 1 518 bp containing 57 variable loci. According to the mutation sites, R. palmatum, R. tanguticum and R. officinale were divided into different genotypes separately. Based on the established method according to the loci 587, 707, 838, we successfully identified the genuine Rheum species from its adulterants.

Comparative study of the immune effect of an Edwardsiella tarda antigen in two forms: subunit vaccine vs DNA vaccine.

Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent of a systematic fish disease called edwardsiellosis, which affects a wide range of marine and freshwater fish. E. tarda vaccines in various forms have been reported by a number of research groups; however, comparative studies on the immune mechanisms of these vaccines are lacking. In this report, we identified a new E. tarda vaccine candidate, Eta2, and analyzed in a comparative manner the immune response induced by Eta2 in two different forms: purified recombinant subunit vaccine and DNA vaccine. Eta2 is a protein of 178 residues and shares high levels of sequence identities with the OmpH family of outer membrane protein chaperones of several bacterial species. Recombinant Eta2 (rEta2) purified from Escherichia coli was highly protective against E. tarda challenge in a Japanese flounder (Paralichthys olivaceus) model and produced relative percent of survival rates of 83% and 78%, respectively, at 4- and 8-week post-vaccination (p.v.). Eta2 as a DNA vaccine in the form of plasmid pCEta2 also induced strong protective immunity at 4- and 8-week p.v. Immunological analysis indicated that (i) rEta2 and pCEta2 enhanced head kidney macrophage activation at 1- and, for pCEta2, 7-day p.v.; (ii) rEta2 and pCEta2 induced similar patterns of serum antibody production, however, the antibodies induced by rEta2 were of much higher levels and afforded stronger passive immunoprotection upon naïve flounder than those induced by pCEta2; (iii) both rEta2 and pCEta2 upregulated the expression of specific and nonspecific immune factors which include, in the case of pCEta2 but not rEta2, interferon, interferon-induced Mx protein, and CD8α; however, the induction patterns effected by rEta2 and pCEta2 were different. While high levels of interleukin 1ß (IL-1ß), natural killer cell enhancing factor, Mx, MHC Iα, and IgM inductions were observed in pCEta2-vaccinated fish, only IL-1ß, complement C3, and IgM inductions were highly induced in rEta2-vaccinated fish. Taken together, these results indicate that both rEta2 and pCEta2 induce specific and nonspecific immunities, however, pCEta2 induces both B cell and T cell responses, whereas rEta2 induces mainly humoral response.

Construction and analysis of an experimental Streptococcus iniae DNA vaccine.

Streptococcus iniae is a severe aquaculture pathogen that can also infect humans and animal. A putative secretory antigen, Sia10, was identified from a pathogenic S. iniae strain by in vivo-induced antigen technology. Using turbot as an animal model, the immunoprotective effect of Sia10 was examined as a DNA vaccine in the form of plasmid pSia10, which expresses sia10 under the cytomegalovirus immediate-early promoter. In fish vaccinated with pSia10, transcription of sia10 was detected in muscle, liver, spleen, and kidney at 7, 14, 21, 28, 35, 42, and 49 days post-vaccination. In addition, production of Sia10 protein was also detected in the muscle tissues of pSia10-vaccinated fish. Fish vaccinated with pSia10 exhibited a relative percent survival (RPS) of 73.9% and 92.3%, respectively, when challenged with high and low doses (producing a cumulative mortality of 92% and 52%, respectively, in the control groups) of S. iniae. Immunological and transcriptional analyses showed that vaccination with pSia10 (i) induced much stronger chemiluminescence response and significantly higher levels of nitric oxide production and acid phosphatase activity in head kidney macrophages; (ii) caused the production of specific serum antibodies, which afforded apparent immunoprotection when transferred passively into naïve fish; and (iii) upregulated the expression of the genes encoding proteins that are possibly involved in both innate and adaptive immune responses. Taken together, these results indicated that pSia10 is an effective vaccine candidate and may be used in the control of S. iniae infection in aquaculture.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class IIalpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection.

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC IIalpha, IL-1beta, Mx, and MHC Ialpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections.

[Killing effect of double suicide genes mediated by retroviral vector on k562 cells].

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.

[Experimental study on immunotherapy with dendritic cell in leukemic mice model].

OBJECTIVE: To explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT). METHODS: Mature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia. RESULTS: Mature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05). CONCLUSION: Immunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.

[Application of pseudotyped lentivirus-mediated double suicide genes to allogeneic bone marrow transplantation in mice].

OBJECTIVE: To explore the feasibility and efficiency of cytosine deaminase (CD)/thymidine kinase (TK) gene-modified donor T cells used in allogeneic bone marrow transplantation (allo-BMT) as an approach to mitigate GVHD without compromising engraftment. METHODS: The pseudotyped lentivirus vectors containing CD and TK double suicide genes were transfected with lipofectine to donor T cells. Lethally irradiated 615 leukemia mice were transplanted with BALB/c bone marrow plus CD(+)TK(+)T cells. GVHD prophylaxis was by administration of ganciclovir (GCV) and 5-Fluoride cytosine (5-FC). RESULTS: The pseudotyped lentivirus-mediated gene transfer system could efficiently transfer CD and TK double suicide genes into donor T cells. Administration of GCV and 5-FC to the mice could markedly potentiate the CFU-S and CFU-GM yields and raise the number of peripheral white blood cells. 1 x 10(7) CD(+)TK(+) allogeneic T cells caused GVHD of a similar magnitude and time course to that of fresh, naive T cells after allo-BMT. Administration of GCV and 5-FC in mice received CD(+)TK(+)T cells reduced the severity of GVHD and resulted in significantly longer survival as compared with non-administration mice, and the effect was stronger than that of administration of GCV or 5-FC alone. CONCLUSION: Administration of CD + TK gene-modified donor T cells to recipient in allo-BMT might be an approach to mitigate GVHD without compromising alloengraftment.

[Killing effect of double suicide genes mediated by lentivirus on lymphoma cells].

BACKGROUND & OBJECTIVE: The lentiviral vectors can integrate interest genes into genome of the target cells that allow for stable transgenic expression even in non-dividing cells without evoking an immune response of the host. All the features have promised them to be used in vivo gene therapy. This study was designed to explore the killing effect of double suicide genes mediated by lentivirus on lymphoma cells (Raji). METHODS: The three plasmids expressed lentivirus, packaging plasmid pCMV 8.2, envelope plasmid pCMV.VSVG and target plasmid (pHR'CS. GFP as control group, pHR'CS.CDglytk as experiment group) were packaged into 293T cells using lipofectine method. Supernatant was harvested and concentrated. The Raji cells were infected with the concentrated virus. The gene integration and expression were confirmed by fluorescence microscopy and RT-PCR. After prodrug GCV or/and 5-FC administration, MTT method was used to detect the growth inhibition rate (GIR) of Raji cells for evaluating the killing effect of CD and HSV-tk double suicide genes on Raji cells. RESULTS: The three plasmids were effectively transferred into 293T cells. Green fluorescence on the cell was observed through fluorescence microscopy and a lot of virus particles were observed through transmission electronic microscopy. Double suicide genes mediated by lentivirus were effectively and stably expressed in Raji cells. The GIR of Raji cells using GCV or 5-FC was 51% or 50%, respectively, and it was apparently higher than that of untransfected cells(P< 0.01). When using GCV and 5-FC together, the GIR was 73%, which was apparently higher than that of group using GCV or 5-FC alone (P< 0.01). CONCLUSION: Double suicide genes mediated by lentiviral vector could transfect lymphoma cells effectively and stably. The double suicide gene system enhanced killing effect remarkably on lymphoma cells than CD/5FC or HSV-tk/GCV system alone.

[Toxic effects of HSV-TK and CD-TK suicidal gene systems on prostate carcinoma cells].

BACKGROUND & OBJECTIVE: Suicidal gene therapy is one of promising gene therapies. In order to assess the value of suicidal gene therapy on human prostate carcinoma, the authors studied the toxic effects of HSV-TK gene and CD-TK fusion gene systems on prostate carcinoma cell line PC-3m. METHODS: HSV-TK gene and CD-TK fusion gene were separately transfected into PC-3m cells through retrovirus vectors. Reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate successful transfection and transcription of suicidal genes. The toxic effects of GCV, 5-FC, and both of them on transfected PC-3m cells were explored by MTT assay; non-transfected PC-3m cells were used as control. RESULTS: Significantly cytotoxic activity of GCV was observed and 50% inhibitory concentration(IC(50)) was 8.34 microg/ml,the bystander effect of GCV was modest,while the bystander effect of 5-Fc was significant,it began to show when the percent of tansfected PC-3m cells in mixed cells was 5%. Simultaneous treatment with two prodrugs on CD-TK expressing cells resulted in additive or synergistic toxicity,coefficient of drug interaction(CDI) was under 1. CONCLUSION: CD-TK fusion suicidal gene system has significant toxic effect on PC-3m cells in vitro, which was superior to HSV-TK alone gene system.

[Tegument viral protein 22 enhanced cell-killing effect of the herpes simplex virus thymidine kinase/ganciclovir system on ovarian cancer in vivo].

OBJECTIVE: To explore the enhanced cell-killing effect in vivo of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapeutic system using tegument viral protein (VP22) intercellular trafficking. METHODS: Human ovarian epithelial cancer cell line 3AO was infected by lentivirus containing HSV-TK and HSV-VP22-TK respectively. Tumors were induced in nude mice by subcutaneous injection of the mixture of 90% 3AO cells and 10% 3AO cells carried with HSV-TK (3AO/TK) or HSV-VP22-TK (3AO/VP22-TK). Nude mice injected with 3AO cells were used as blank control. When the volume of tumor was 150 mm(3), GCV was administered at 10 mg/kg or 50 mg/kg intraperitoneally. RESULTS: There were significant differences, in the tumor volume and weight between 3AO/TK group and 3AO/VP22-TK group after administration of 10 mg/kg GCV (P < 0.01), and the later was more efficient than the former. But there was no significant difference after administration of 50 mg/kg GCV (P > 0.05). The tumor inhibition rates in 3AO/VP22 group and 3AO/VP22-TK group were 37.7% and 91.5% respectively after administration of 10 mg/kg GCV (P < 0.01), and were 81.8% and 96.7% respectively after administration of 50 mg/kg GCV (P > 0.05). CONCLUSION: These results clearly indicate that VP22 enhances the efficiency of the suicide gene transfer, thereby increases the cell-killing effect on tumor in vivo.