AIM: To study the effect and mechanism of BCG on human nature killer cells. METHODS: PBMC or purified NK cells were isolated from normal human peripheral blood with negative anti-Mycobacterium tuberculosis antibody and cultured with BCG, IL-12, BCG plus IL-12 and BCG plus anti-IL-12Rβ1 mAb (2B10), respectively. The levels of IFN-γ and IL-12p40 in the culture supernatants were measured by ELISA. The frequency of IFN-γ and granzyme B producing cells were analyzed by ELISpot. The cytolytic activity was detected by MTT reduction assay. The surface expression of IL-12Rβ1 on NK cells was detected by flow cytometry. RESULTS: BCG significantly induced IFN-γ production by PBMC in a dose-dependent manner. When PBMC was stimulated with BCG, the frequency of granzyme B producing cells was higher than that in unstimulated PBMC (P<0.05). BCG enhanced the cytotoxic activity of PBMC. BCG alone didn' t induce IFN-γ production by purified NK cells, but it can augment IL-12-induced IFN-γ production by purified NK cells. The cytotoxic activities of BCG-stimulated and unstimulated purified NK cells were not significantly different (P>0.05). BCG induced IL-12 production by PBMC in a dose-dependent manner and enhanced IL-12Rβ1 expression on different subsets of NK cells. Blocking the effect of IL-12 by anti-IL-12Rβ1 mAb (2B10) inhibited BCG-induced IFN-γ production and granzyme B releasing by PBMC. CONCLUSION: BCG can indirectly promote biologic activity of NK cells and the production of endogenous IL-12 combined with up-regulation IL-12Rβ1 expression on the surface of NK cells is a part of the mechanisms of IL-12 on human NK cells.

AIM: To analyze the capability of cytomegaIovirus (CMV)-infected human embryonic lung fibroblasts (HELFs) to induce immune response. METHODS: HELFs were infected with cytomegalovirus and stained with antibody against HLA-A2 molecular, the expression of HLA-A2 was detected by FCM. The infected HELFs were incubated with individual pp65 peptide NLVPMVATV. While the uninfected and unloaded infected HELFs served as control respectively. After PBMC was added to the differently treated HELFs and incubated, the immune response was measured with IFN-γ release as readout. RESULTS: The expression of HLA-A molecular on infected fibroblasts diminished markedly compared with that on the uninfected. The peptides expressed on the infected HELFs together with those pulsed externally induced a stronger response than the infected HELFs alone. CONCLUSION: Although CMV can downregulate the expression of MHC Ⅰ on the infected cells, it can not decrease the capacity of cells to present peptides loaded externally, and therefore still induce immune response to some extent.

AIM: To explore the different effect and mechanism of arsenic sulfide on telemorase activity and hTERT-mRNA expression in CML cell lines-KS62 and APL cell lines-NB4. METHODS: Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR. Flow cytometry was used to analyze the cell cycle and apoptosis. RESULTS: 0.15-0.6 mg/L arsenic sulfide (72 h)can induce apoptosis and inhibit telomerase activity and hTERT-mRNA expression in NB4 cell. The concentration of arsenic sulfide with the same effect on K562 cell was 0.3-3 mg/U 0.3 mg/L arsenic sulfide (72 h) can cause the proportion of the NB4 cell in G2/M phase increased, but for K562 cell, The concentration of arsenic sulfide was 1.5 mg/L. CONCLUSION: Telomerase system may be one.of the pathway for arsenic sulfide inducing apoptosis of NB4 and K562 cell; G2/M phrase arrest may have correlation with decrease of telomerase activity; The sensitivity of NB4 and K562 call for arsenic sulfide is different, the mechanism of it need to study more.

im To study the structure-function relationship of hTNFα . Methods We compared the cytotoxicity, receptor binding ability and toxicity in animal body of wild type(wt)hTNFα with its mutants including R2K-, N30S-, R32W-, L157F-hTNFα , and two multi-site mutants(R32W-L157F-hTNFα and R2K-N30S-R32W-L157F-hTNFα ). Results We found that the two multi-site mutants remained similar cytotoxicity to several human tumor cell lines as wild type hTNFα . However, their cytotoxicity to L929 cells were decreased sharply as compared with those of wt hTNFα . The two multi-site hTNFα mutants had lower binding activity with hTR75 than hTR55. We also found that compared with the wild type, the LD50 of the mutant R32W-L157F-hTNFα was decreased about 300 fold and the dose of mutant R2K-N30S-R32W-L157F-hTNFα resulted in 30％ death was 700 folds lower than LD50 of wt hTNFα . To certify the systematic toxicity of the mutant R2K-N30S-R32W-L157F-hTNFα , we assayed its toxicity to monkeys and found that its systematic toxicity was lower than that of wt hTNFα. Conclusion A 4-site mutants(R2k-N30S-R32W-L157F-hTNFα )of hTNFα is obtained, which the mutant may possess potential application value in clinical therapy.

AIM: DNA vaccines expressing protective domain of surface protective antigen A（spaA）of Erysipelothrix rhusiopathiae have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in murine models. METHODS: This paper report using a DNA vaccine expressing a fusion of the spaA protein and various elements, such as a secretion leader sequence from the highly expressed human gene encoding α1-antitrypsin (AAT), a highly soluble and stably folded domain from the rat cartilage oligomerization matrix protein (COMP), and three copies of the complement component, C3d3, to enhance the titers of neutralizing spaＡ-specific antibody. RESULTS: Analysis of titers of the antibody raised in vaccinated mice at different time points indicated that immunizations with the DNA expressing pcDNA3-AAT-COMP-spaAN-3C3d((pcD-ACSC)) had higher titers than pcDNA3-spaA_N(pcD-S) at weeks 4. Furthermore, the immune protective efficacy of the spaA-chimeras was demonstrated by lethal challenge with a virulent homologous strain 1249 against immunized mice. CONCLUSION: These results suggest that using a plasmid vector containing a strong heterologous signal sequence that mediate efficient antigen secretion in vivo and a fused piece of sequence improving antigens solubility, as well as C3d3, genetic adjuvant, could enhance the antibody responses level. This approach might be an efficient way to improve the antibody level of spaA_N DNA vaccination.

AIM: We investigated the numbers and proportions of lymphocyte subsets in advanced ovarian cancer patients undergoing chemotherapy, so as to identify whether there is immune reconstitution after chemotherapy, and seek rational chemo-immunotherapy strategies in ovarian cancer treatment. METHODS: Blood samples from each ovarian cancer patient were obtained before (S_0) and at day 5-7 (S_1), day 12-14 (S_2) and day 25-28 (S_3) after chemotherapy in 13 patients. Flow cytometry technique was employed to analyse the numbers, proportions of CD3~+, CD4~+, CD8~+, CD4~+CD25~+ Treg and memory-like phenotype lymphocyte subsets. RESULTS: Lymphopenia was observed at S1 after chemotherapy, but lymphocytes were found gradually recovered after S1. The numbers of CD3~+, CD4~+, CD8~+T cells and CD4~+CD25~+ Treg cells reduced to the lowest on S_1. The proportions of CD8~+ T cells and CD4~+CD25~+ Treg cells reduced to the lowest on S_1 and S_2 respectively. The proportions of CD45RO~+ memory T cells increased significantly on S2 while the proportion of CD8~+CD45RA~+ T cells decreased remarkably on S_2. CONCLUSION: Paclitaxel and carboplatin induce lymphopenia at day 5-7 after chemotherapy, then comes the temporary immune reconstitution. It probably turns out that the window period during immune reconstitution offers a best opportunity for cancer immunotherapy.

Aim To obtain the HCC cell lines which could coexpressed the B7.1 and SEA. Methods The positive clones expressing the B7.1 and SEA were screened by immunohistochemical staining. The amount of SEA in culture supernatant was detected by ELISA. Results HCC cell clones coexpressing B7.1 and SEA were obtained, and expression amount of SEA in culture supernatant reached 10～ 14× 10-8g/L. Conclusion The co-rec-ogenition immune effective system of SEA and B7.1 on HCC cells is established.

AIM: To investigate the effects of intermittent hypoxic exposure and normoxic convalescence on the parameter of erythrocyte and serum hypoxia inducible factor1 alpha (HIF-1α) and erythropoietin (EPO)levels. METHODS: Rat models of intermittent hypoxic exposure were established, combined with the clinical research on volunteers experiencing the intermittent plateau work. Blood samples for red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (HCT) were collected, serum HIF-1α and EPO levels were measured using enzymelinked immunosorbentassy. RESULTS: RBC counts, Hb concentration and HCT were significantly higher than the normoxic group (P < 0.05), after exposure of rats to hypoxia from 7 to 28 days. Compared with the normoxic group, serum HIF-1α levels were higher in the group of IH3, 7, 14 days, and EPO had a corresponding increase in the group of IH3, 7 days. Then, a decrease was observed in parameter of erythrocyte and serum HIF-1α and EPO levels after 14 days normoxic convalescence treat. In volunteers studies, RBC counts in 8 monthes group and Hb concentration in 2 years group were significantly higher than the plain group (P < 0.05). The change of HCT was nearly the same as RBC, and HCT in 2 years group was higher than the plain group (P < 0.05). Compared with the plain group, EPO had no significant differences in any of plateau group. CONCLUSION: Intermittent hypoxic exposure can enhance serum hypoxia inducible factor-1 alpha and erythropoietin levels and the generation of red blood cells, which leads to an increase in hemoglobincon concentration and hematocrit. The results have changed with the hypoxic exposure period prolonged. Normoxic convalescence after intermittent hypoxic exposure can make the related indexes reduced, and contribute to the organism recovery.

AIM: To investigate the immune mechanisms for Periplocin from Cortex Periplocae (CPP) in tumor-bearing mice. METHODS: H_(22) tumor-bearing model BALB/c mice were applied to evaluated in vivo immunoregulatory effect of CPP. The influence of different dose CPP (0.25, 0.50 and 1.00 mg/kg) on immune organs in tumorbearing mice were observed. T cell subsets of mice spleen were detected by flow cytometry. MTT assay was used to determine the influence of CPP on lymphocyte proliferation of mice spleen stimulated by ConA. The levels of TNF-α, IL-2 and IL-12 in serum from mice were detected by means of ELISA. RESULTS: Thymus index and spleen index of H_(22) tumor-bearing model control mice became less than that of normal mice (P < 0.05). Compared to both model and normal control groups, thymus index and spleen index of H_(22) tumor-bearing mice treated with CPP increased obviously (P < 0.05). CPP had no influence on the number of CD8~+ T cells, but up-regulated markedly the number of CD3~+, CD4~+ T cells and the ratio of CD4~+/CD8~+ in tumorbearing mice. In CPP-treated mice, the percentage of CD3~+, CD4~+ T cells were not different from normal mice (P<0.05), the ratio of CD4~+/CD8~+ was higher than that of normal mice (P < 0.05). CPP enhanced obviously lymphocyte proliferation of mice spleen induced by ConA, the SI scores were even higher than that of nornal mice. The levels of TNF-α, IL-2 and IL-12 in serum from CPP-treated mice, increased significantly compared to model control group (P<0.05) in a dose-dependent manner, were similar to or higher than that of normal mice. CONCLUSION: CPP protected immune organs of tumor-bearing mice, increased obviously the percentage of CD4~+ and CD4~+/ CD8~+ among the T cell line, and enhanced lymphocyte proliferation of mice spleen significantly, stimulated the production of TNF-α, IL-2 and IL-12. The results suggested that CPP possessed potent immunoregulatory effect.

AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by i-onizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferasy reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation, mediated gene therapy.