Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-? and IFN-?. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor,Toll-like receptor 9,then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.

Extracellular signal-regulated kinase3 (ERK3) is distinguished from other ERK family members especially in its molecular biological characteristics including the big intron between exons in its gene structure, the serine189 mono-phosphorylated site and C-terminal extention of its kinase structure. The specially activating phosphorylation site of serine189 indicates that all MEKs, which phosphorylate serine/threonine double phosphorylation sites of MAPKs, are unable to activate ERK3. The C-terminal extension involves both subcellular localization of ERK3 and binding to intact cyclin D3, which can profoundly affect cell cycle regulation. According to update reports, ERK3 signal pathway in the regulation of cell cycle might be as follows: Ras→B-Raf→ERK3kinase→ERK3→decrease of CDK compounds of G1-phase→increase of the inhibiting factor (retinoblastoma protein) of S-phase→blockage of S-phase of cell cycle→cell differentiation entry while cell proliferation arrest. Moreover, the activation of ERK3 signaling pathway is also associated with cell differentiation, embryonic development, insulin secretion and cancer diseases.

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL. [

NF-?B is thought of as a genetic switch to control expressions of many target genes and directly participates in pathogenesis of infection, inflammation, stress, immunoresponse, cellular apoptosis, toxic shock and tumor as well as cell-cycle regulation and cell differentiation. The overactivation of NF-?B is intimately involved in many human diseases. Various therapeutic strategies against NF-?B, to date, include anti-inflammatory drugs, antioxidants, immunosuppressive agents, inhibitors of protease and proteasome, prostaglandings, nitric oxide, IL-10, microbial products, synthetic inhibitors, antisense oligonucleotides and decoy deoxyoligonucleotides. Studies are underway to develop NF-?B member-specific and cell type-specific drugs that can inhibit the activation of NF-?B only in target cells and that may become a novel way to treat the human diseases.

AIM: To evaluate the effect of staphylococcal enterotoxin B (SEB) or staphylococcal enterotoxin C (SEC) combining with dendritic cells (DC) on T cell functions and in vitro anti-HcaF tumor cytotoxicity of activated T cells. METHODS: S-100 protein expression in DC was detected by immune histochemistry staining. The expressions of I-E~? and CD80 molecules on DC, the expression of CD69 molecule on T cells and the production of IL-2 and TNF-? by T cells were determined with flow cytometry. The proliferation of T cells and its cytotoxicity to HcaF tumor cells were detected by MTT assay. RESULTS: In vitro experiments showed that isolated DC expressed high level of S-100 protein. SEB or SEC-induced DC highly expressed I-E~? and CD80 molecules and that SEB or SEC-induced DC promoted the activation and proliferation of T cells. 100 ?g/L of SEB or SEC was the most effective concentrations to induce T cells to secret IL-2 and TNF-?. The T cells activated by SEB or SEC combined with DC showed significant cytotoxicity to HcaF cells, appearing a stronger role than tumor antigen combined with DC. There was no difference in the role for T lymphocytes between both SEB and SEC. CONCLUSIONS: The results indicate that SEB or SEC combined with DC is an effective way to enhance T cell functions, producing stronger cytotoxicity to HcaF tumor cells than tumor antigen-loaded DC used at present, which offers a forceful evidence for the possibility of superantigen SEB or SEC combining with DC to be applied to clinical tumor immunotherapy.

AIM: To study the relation between prolon ge d survival of skin allograft by chuan-ke-zhi (CKZ, drug of Chinese herbal) and C D4+CD25+ regulatory T cells (CD4+CD25+ Tr) in mice. METHODS: Skin allograft and isograft model in mice were establi shed and CKZ was administered by intraperitoneal injection. To observe its influ ence on survival of the graft, three color fluorescent staining together with fl ow cytometry was used to analyze the change of CD4+CD25+ Tr. RESULTS: The survival of skin allograft in CKZ group was signifi cantly prolonged compared to control group, (19.5?2.3) days and (10.2?2.2) days, respectively, P0.05). CONCLUSION: CKZ has an effect of prolonging the survival of skin allograft. Enhancement of CD4+CD25+ Tr might be one of the mechanisms under lying its immunosuppressive effect.

0.05) compared with control group at 1 h and 3 h; while ~FL 1 in DEX group at 5 h (660.91?72.95) was significant lower (P

AIM: To study the effects of ultraviolet (UV) on mitochondrial functions and apoptosis in HaCaT cells.METHODS: After irradiation by UV at low dose(UVA 2 J/cm~2,UVB 10 mJ/cm~2) and high dose(UVA 6 J/cm~2,UVB(30 mJ/cm~2),) HaCaT cells were cultured for 15 hours.Flow cytometry was used to measure mitochondrial membrane potential,mitochondrial mass and apoptotic rate.Annexin V-FITC/PI staining of apoptotic cells was analyzed by laser confocal microscopy.RESULTS: After UV irradiation,cell proportion with low mitochondrial membrane potential increased with irradiation doses.The proportion of control group,low dose group and high dose group were 7.94%?1.02%,25.87%?4.55% and 39.27%?5.32%,respectively.Cells proportion with low mitochondrial mass increased with irradiation doses.The proportion of control group,low dose group and high dose group were 15.19%?1.58%,40.36%?4.41% and 68.79%?5.46%,respectively.The hypodiploid peaks of DNA content analysis represented the apoptotic rate of HaCaT cells.The apoptotic rate of control group,low dose group and high dose group were 1.82%?0.51%,30.16%?5.47% and 58.49%?5.98%,respectively.To analyze the cells apoptosis by staining with annexin V-FITC and PI,the results were consistent with those of DNA content analysis.Cells in control group showed almost no positive staining cells.Single annexin V-FITC positive cells in low dose group and double positive cells in high dose group were predominant,respectively.CONCLUSION: UV irradiation induces HaCaT cell mitochondrial depolarization,as well as mitochondrial mass loss.These changes are related to cell apoptosis.

AIM: To study the changes of mitochondrial membrane potential(△?m) and mitochondrial mass in apoptosis of Jurkat cells induced by camptothecin(CPT).METHODS: Jurkat cells were treated with CPT.Annexin V-FITC/propidium iodine(PI) double stainig was used to detected early stage of apoptosis and PI staining for analyzing the cell cycle.Jurkat cells were stained by annexin V-PE/DiOC_6(3) to detect changes of △?m.The mitochondrial mass was measured by cytometry with NAO staining.RESULTS: 6 h after treated with 10 ?mol/L CPT,the rate of early apoptotic cells(22.59?1.04)% had significantly difference compared with control group(3.93?0.73)%(P0.05).Apoptotic peak appeared obviously after treated with CPT,the percentage of late apoptotic cells(13.58?0.97)% had distinctly difference compared with control group(3.18?0.51)%(P

AIM: To investigate the feasibility and infection efficiency of MSCs with replication-deficient adenovirus containing delivered gene, and whether enhanced green fluorescence protein (EGFP) gene track the change during rMSCs differentiating neuron-like cells. METHODS: Rat marrow mesenchymal stem cells (rMSCs) were expanded in low density in vitro . Under the control of CMV promoter, pAd-EGFP-Vector was constructed by homologous recombination in E.coil BJ 5183, and the recombinant virus was produced in HEK 293 packaging cell line. rMSCs infected with Ad-EGFP were observed and analyzed with fluorescence microscope. Infection efficiency was assessed by microscopical scoring and flow cytometrics. After withdrawing serum and exposure to ?-mercaptoethanol medium, rMSCs infected with Ad-EGFP was induced to differentiate into neuron-like cells. As a control, the plasmid of pTrack-EGFP also was transfected into rMSCs to evaluate transfection efficiency.RESULTS: The results showed that Adenovirus vector (AdVec) delivered EGFP gene with high efficiency to marrow mesenchymal stem cells. Gene expression analysis showed that 36%?2 % of rMSCs infected with recombinant adenovirus expressed the transgene of EGFP at high levels. However, the transfection of plasmid pTrack-EGFP using routine method of lipofectamin mixed with plasmid DNA (pTrack-EGFP) was not easily successful and the transfection efficiency was much lower. rMSCs infected with Ad-EGFP in different passage could differentiate into typical morphology alike neural cells after withdrawing serum and exposure to ?-mercaptoethanol medium. Immuno-staining with neuron-specific enolase (NSE), a neuronal marker, was strong positive, which suggested that rMSCs infected with Ad-EGFP had the potential to differentiate into neurons or neuron-like cells. CONCLUSION: The AdVec system can deliver target gene into MSCs and EGFP gene carried by AdVec can track the change during rMSCs differentiating into neuron-like cells.