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PURPOSE: It remains unknown whether local inhibition of Nuclear factor-kappa B (NF-κB) could have therapeutic value in the treatment of allergic rhinitis (AR). This study aimed to evaluate the effect of selective NF-κB inhibition using NF-κB decoy oligodeoxynucleotides (ODNs) for the local treatment of AR in ovalbumin (OVA)-sensitized wild-type mice. METHODS: BALB/c mice were sensitized with OVA and alum, and then challenged intranasally with OVA. NF-κB decoy ODNs were given intranasally to the treatment group, and NF-κB scrambled ODNs were given to the sham treatment group. Allergic symptom scores, eosinophil infiltration, cytokine levels in the nasal mucosa, nasal lavage fluid, and spleen cell culture, serum total and OVA-specific immunoglobulins, as well as intercellular adhesion molecure-1 (ICAM-1) in the nasal mucosa, were analyzed. RESULTS: NF-κB decoy ODNs significantly reduced allergic symptoms and eosinophil infiltration in the nasal mucosa. They also suppressed serum levels of total IgE, OVA-specific IgE, and IgG1. IL-5 and TNF-α levels and the expression of ICAM-1 were decreased in the nasal mucosa of the treatment group compared to the positive control and sham treatment groups. In addition, IL-6 levels were significantly decreased in the nasal lavage fluid of the treatment group. Furthermore, NF-κB decoy ODNs significantly reduced expression of the systemic Th2 cytokines, IL-4 and IL-5 in spleen cell culture. CONCLUSIONS: This study demonstrates for the first time that local NF-κB inhibition using NF-κB decoy ODNs suppressed the allergic response in a murine AR model. This shows the therapeutic potential of local NF-κB inhibition in the control of AR.
Subject(s)
Animals , Mice , Anti-Allergic Agents , Cell Culture Techniques , Cytokines , Eosinophils , Immunoglobulin E , Immunoglobulin G , Immunoglobulins , Intercellular Adhesion Molecule-1 , Interleukin-4 , Interleukin-5 , Interleukin-6 , Nasal Lavage Fluid , Nasal Mucosa , NF-kappa B , Oligodeoxyribonucleotides , Ovalbumin , Ovum , Placebos , Rhinitis, Allergic , SpleenABSTRACT
Objective To investigate whether specific cellular uptake of 99Tcm-survivin-ASODN in nude mice bearing human HCC is influenced by its cytosine content.Methods Three kinds(A1,A2,A3) of synthesized survivin ASODN with three cytosine contents(10％,20％,30％),20 bases per single-strand were prepared.They were labeled with 99Tcm by conjugating with a bifunctional chelator HYNIC,purified through Cellufine GH-25 and then encapsulated with liposome.Antisense gene imaging and the biodistribution of 99Tcm-HYNIC-survivin ASODN in nude mice bearing HCC were performed.The data were analysed by Kruskal-Wallis H test.Results At 4 h post injection,all the 3 labelled compounds showed increased uptake by tumor,liver and kidney.With increase in cytosine content,the uptake increased in kidney (％ID/g:1.50±0.06,2.80±0.09 and 3.96±0.03),and decreased in tumor (％ID/g:2.08±0.08,1.69±0.01 and 1.20±0.09).T/NT in imaging (4.49-4.93,4.12-4.21,3.35-3.85;H=12.50,P＜0.05) and in biodistribution (4.08-4.94,4.02-4.18,3.66-3.85;H=10.82,P＜0.05) were all significantly different.Conclusion ASODN with lower cytosine content shows higher uptake by HCC tumor cells and less stasis in kidneys,thus providing better quality in antisense gene imaging.
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Objective To explore the influence on the expression of elF4E and heparanase by antisense oligodeoxyribonucleotides (ASODN) transfection in human bladder carcinoma BIU-87 cells.Methods After transfecting the 2.5,5.O,7.5 μg/ml eIF4E ASODN into BIU-87 cells,at 24 h,48 h and 72 h,a cell control group (no transfection),a blank control group (empty liposomes) and a non-sense control group (transfected with non-sense ASODN) were established.The expression of eIF4E,HPA and mRNA were detected by in situ hybridration.The expression of eIF4 and HPA protein were detected by immunocytochemistry.SPSS 13.0 statistical software was used for statistical analysis.Results The expression quantity of eIF4E protein and mRNA were lower in the experimental groups ( protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:3.55 ±0.52,3.52 ±0.51,3.22.±0.44 respectively; 5.0 μg/ml group:3.43 ±0.47,3.41 ± 0.46,3.19 ± 0.41 respectively ; 7.5 μg/ml group:2.36 ± 039,2.33 ± 0.37,2.05 ± 0.30 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:3.19 ±0.41,3.13 ±0.39,2.90 ±0.38 respectively ; 5.0 μg/ml group:3.07 ± 0.39,2.94 ± 038,2.27 ± 0.37 respectively ; 7.5 μg/ml group:2.22 ± 037,2.06 ± 0.30,1.95 ± 0.29 respectively.All data were less than those in the control groups (P ＜0.05).The expression quantity of HPA protein and mRNA were lower in experimental groups (protein:2.5 μg/ml ASODN treated 24 h,48 h,72 h were:4.44 ±0.55,4.40 ±0.54,3.99 ±0.52 respectively; 5.0 μg/ml group:4.41 ±0.55,4.21 ±0.53,3.77 ±0.50 respectively; 7.5 μg/ml group:4.02 ±0.52,3.98 ±0.52,2.32 ±0.37 respectively.mRNA:2.5 μg/ml treated 24 h,48 h,72 h were:4.12 ±0.51,3.75 ± 0.50,3.63 ± 0.45 respectively ; 5.0 μg/ml group:4.00 ± 0.51,3.71 ± 0.50,3.54 ± 0.44respectively ; 7.5 μg/ml group:3.87 ± 0.52,3.61 ± 0.49,2.08 ± 0.30 respectively).All data were less than in control groups ( P ＜ 0.05 ).There was a positive correlation on expression of HPA protein and eIF4E protein by inhibitory effect of eIF-4E ASODN (protein r=9.23,mRNA r=9.59,P ＜0.01).Conclusion eIF-4E ASODN might be used to inhibit the expression of eIF-4E gene and HPA gene in bladder cancer BIU-87 cells.
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Objective To investigate the effects of CpG oligodeoxynucleotide (ODN) on cellular immune responses in suckling mice infected with human rotavirus (HRV). Methods Forty ICR suckling mice were randomly divided into four groups: control group. HRV infection group, CpG ODN pretreatment group and CpG ODN treatment group. Suckling mice were sacrificed four days after rotavirus challenge. Small intestine and spleen samples were collected under sterile condition. Thedegree of small intestinal mucosal injures was evaluated with standard scoring criteria. The spleen index was calculated and spleen lymphocyte stimulation index was detected by methyl thiazolyl tetrazolium ( MTT) assays. Levels of gamma interferon (IFN-γ), interleukin-4 ( IL-4) in the supernatant of spleen lymphocyte culture were detected by enzyme linked immunosorbent assay (ELISA). T lymphocyte subsets were analyzed by flow cytometry. The data was compared by one way ANOVA. Results The scores of mucosal injures of mice in HRV infection group, CpG ODN pretreatment group and CpG ODN treatment group were 4. 00 ±1. 31, 2. 75 ±1. 28 and 2. 87 ±0. 99, respectively, and the differences among groups were statistically significant (F=ll. 32,P＜0. 01). The scores of mucosal injures in CpG ODN pretreatment group and CpG ODN treatment group were both lower than that in HRV infection group (P＜0. 05). Compared with control group, spleen lymphocyte stimulation index and the percentage of CD8+ T lymphocytes were higher, the percentage of CD4+ T lymphocyte and ratio of CD4+ /CD8+ T cells were lower, levels of T helper (Th)1 type cytokine IFN-y increased significantly in HRV infection group; while the spleen index, spleen lymphocyte stimulation index, percentages of CD4+ and CD8+ T lymphocytes, IFN-y levels were all increased in CpG ODN pretreatment group and CpG ODN treatment group (P＜0. 05). The spleen index, spleen lymphocyte stimulation index, the percentage of CD4+ T lymphocytes, CD4+/CD8+ ratios and IFN-y levels in CpG ODN pretreatment group and CpG ODN treatment group were all significantly higher than HRV infection group (P＜0. 05). Conclusion CpG ODN potently enhances cellular immune responses in ICR suckling mice infected with HRV and CpG ODN could induce dominant Th1 response.
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Objective: To investigate the effect of antisense oligodeoxynucleotide (ASODN) of Livin mRNA on the proliferation and apoptosis of human lung adenocarcinorna A549 cells. Methods: Livin ASODN was transfected into A549 cells mediated by cationic liposome. The proliferation rates of A549 cells were assessed by MTT method. The transcription of Livin mRNA was detected by RT-PCR. The Livin protein expression was determined by immunohistochemistry and confocal laser scanning microscopy before and after transfection. The apoptotic ratios of A549 cells were examined by acridine orange/ethidium bromide(AO/EB) fluorescent staining. Results: It was observed that A549 cells expressed both Livin α and Livin β simultaneously, and the distribution of Livin protein could be observed in both cytoplasm and nuclei. The proliferation of A549 cells was inhibited significantly by Livin ASODN in a dose-dependent manner (P<0.01). After transfection of Lip-ASODN at a final concentration of 400 nmol/L, the expressions of Uvin were inhibited at mRNA and protein levels and the apoptotic ratio reached (31.25 ± 5.75)%. The difference was significant compared with control group [ (3.23% ± 1.98)%, P<0.01]. Conclusion: Livin ASODN significantly down-regulates the expression of Livin mRNA and effectively inhibites the proliferation and induces the apoptosis of A549 cells. Therefore Livin gene may become a new target for gene therapy for lung cancer.
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Objective To investigate the effect of specialized human tel omerase antisense oligodeoxyribonucleotides (AS-ODN) on the growth inhibition o f well, moderate and poor differentiated gastric cancer cell lines, and to explore its inhibitory mechanism and the correlation between the inhibition ratio of gastric cancer cells and differentiation of the tumor cells. Methods Under the given circumstances, three distinct differentiated gastric cancer cell lines were treated with AS-ODN. The telomerase a ctivities were measured by modified telomeric repeat amplification protocal assa y. The cell viability was detected by Trypan blue test, and the cell apoptosis was determined by cell morphological observation under light and electromicroscope, flow cytometry an d TUNEL assay. Results The telomerase activity and cell growth were apparently suppressed in MK N45 and SGC7901 cells, under defined concentrations of AS-ODN. Whilst, in MKN28 cells, only telomerase activity was suppressed at same concentration . There were no obvious changes in non-antisense oligomers treated group. The apoptotic features of MKN45 and SGC7901 were noticed by microscopic observa tion, TUNEL assay, after three distinct gastric cancer cell lines being continuo usly exposed to 10 ?mol/L AS-ODN for 96 h. Furthermore, the flow cytometric analy sis verified that the average apoptotic rate of MKN45 and SGC7901 was 44.75% and 33.56% respectively, but there were no obvious changes in non-antisense oli gomers treated group (P
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Objective To prepare melanoma antigen n(MAGEn)protein vaccine and to investigate the immune responses and anti-tumor effects of MAGE-n protein vaccine accompanied by CpG-containing oligodeoxynucleotide(CpG-ODN)adjuvant.Methods The DH5? containing the MAGE-n prokaryotic expression plasmid pGEX-MAGE-n was induced and the protein was purified as protein vaccine.The CpG-ODN was synthesized as adjuvant and the C57BL/6 mice were inoculated.The cellular and humoral immune responses were detected by ELISPOT,cytotoxicity assay and enzyme linked immunosorbent assay(ELISA).The antitumor effects were detected through tumor volume and life span.Results The MAGE-n protein accompanied by CpG-ODN could induce strong MAGE-n-specific cellular and humoral immune responses.In the MAGE-n positive B16 tumor model of C57BL/6,the growth velocity of tumor was decreased and the life span was prolonged with the treatment of vaccine.Conclusion MAGE-n protein vaccine accompanied by CpG-ODN adjuvant can induce strong immune responses and anti-tumor effects against MAGE-n positive B16 tumor,which provides a new way for tumor therapy.
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Objective To investigate the effect of suppression of ischemia induced retinal neovascularization by VEGF antisense oligodeoxyribonucleotides. [WT5”HZ]Methods [WT5”BZ]Mouse models of hyperoxia induced ischemic retinopathy were established. Retrobulbar injections were performed with VEGF antisense oligodeoxyribonucleotides or NS in 4 groups:normal control and various doses respectively. The nuclei of new vessel buds extending from the retina into the vitreous in different groups were counted and compared under the light microscope. Results There were plenty of new vessel buds in the eyes of mice in hyperoxic condition., while the number of the nuclei of new vessel buds is less in the murine eyes with retrobulbar injection of VEGF antisense oligodeoxyribonucleotides,especially the nuclei were redused with 59.3% in eyes with large dose. Conclusion The proliferation of retinal new vessel may be suppressed by using the retrobulbar injection of VEGF antisense oligodeoxyribonucleotides.
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The effect of antisense oligodeoxyribonucleotides(oligo[dN]s) on hepatitis B virus(HBV) replication in HepG2 cells harboring a cloned HBV genome was examined. Antisense oligo(dN)s directed at translational initiation sites of S, pre C and P genes of HBV were treated to the cells and the amount of HBsAg and HBV DNA content were measured 72 hours after the treatment. HBsAg expressions in HepG2 cells harboring the HBV genome were inhibited 68%, 53%, and 46% by the treatment with antisense oligo[dN] directed at S, pre C, and P gene loci, respectively, and HBV DNA content in the cells was also reduced by the treatment of each antisense oligo[dN]. The doubling times of the cultured cells treated with 25 micrograms, 50 micrograms, and 100 micrograms of antisense oligo[dN]/ml medium were 43.3, 62.1, and 93.0 hours, respectively, compared with 37.5 hours of the untreated control cells. Cellular DNA synthesis was inhibited by the treatment with 100 micrograms/ml of antisense oligo [dN], however, no significant effect was observed by the treatment with 50 micrograms or less of antisense oligo[dN]/ml. These results suggested that antisense oligo[dN]s specific to the translational initiation sites of S, pre C, and P genes of HBV may have therapeutic potential for the suppression of HBV propagation in chronic HBV infected patients.
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Humans , Cloning, Molecular , DNA, Viral/genetics , Hepatitis B virus/genetics , Hepatoblastoma/pathology , Liver Neoplasms/pathology , Oligonucleotides, Antisense/pharmacology , Thionucleotides/pharmacology , Transfection , Tumor Cells, Cultured/virology , Virus ReplicationABSTRACT
Objective:To study the role and mechanism of c myc ASODNS on cell expression of surface antigen of the tumor cells and susceptibility of target cells to immune cells attack.Methods:c myc mRNA was examined by RT PCR.MTT assay was usd to explore the effects of c myc ASODNS on PG cell sentisentity to lysis by CD 3AK effector cells.The expression of HLA ABC,ICAM 1,c myc protein was examined by flow cytometry.Results:When PG cells were treated with ASODNS(1 ?mol/L) there was a markably reduction of expression of c myc protein.Expression ratio of HLA ABC and ICAM 1 surface antigen expression ratio of PG cells were enhanced from 68.44%,38.44% to 83.16% and 42.09% respectively( P