Inhibition of both EGFR and IGF1R sensitized prostate cancer cells to radiation by synergistic suppression of DNA homologous recombination repair.

Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.

Genistein sensitizes bladder cancer cells to HCPT treatment in vitro and in vivo via ATM/NF-κB/IKK pathway-induced apoptosis.

Bladder cancer is the most common malignant urological disease in China. Hydroxycamptothecin (HCPT) is a DNA topoisomerase I inhibitor, which has been utilized in chemotherapy for bladder cancer for nearly 40 years. Previous research has demonstrated that the isoflavone, genistein, can sensitize multiple cancer cell lines to HCPT treatment, such as prostate and cervical cancer. In this study, we investigated whether genistein could sensitize bladder cancer cell lines and bladder epithelial cell BDEC cells to HCPT treatment, and investigated the possible underlying molecular mechanisms. Genistein could significantly and dose-dependently sensitize multiple bladder cancer cell lines and BDEC cells to HCPT-induced apoptosis both in vitro and in vivo. Genistein and HCPT synergistically inhibited bladder cell growth and proliferation, and induced G2/M phase cell cycle arrest and apoptosis in TCCSUP bladder cancer cell and BDEC cell. Pretreatment with genistein sensitized BDEC and bladder cancer cell lines to HCPT-induced DNA damage by the synergistic activation of ataxia telangiectasia mutated (ATM) kinase. Genistein significantly attenuated the ability of HCPT to induce activation of the anti-apoptotic NF-κB pathway both in vitro and in vivo in a bladder cancer xenograft model, and thus counteracted the anti-apoptotic effect of the NF-κB pathway. This study indicates that genistein could act as a promising non-toxic agent to improve efficacy of HCPT bladder cancer chemotherapy.

[Effects of Mycobacterium tuberculosis Hsp16.3 protein on the autophagy function of mice macrophages].

AIM: To investisate the inhibition of Hsp-16.3 on the autophagosomes formation of macrophages. METHODS: Mouse RAW264.7 macrophages were induced by rapamycin (50 ng/µL) following infection with M.tuberculosis H37Rv strains, thereafter, co-incubated with Hsp16.3 protein (25 µg/mL). The effects of Hsp16.3 protein on the autophagosomes formation was observed with transmission electron microscope. The expression of autophagy-related genes (atg8) for macrophages was detected by Western blotting. RESULTS: It was found that rapamycin-induced autophagy of macrophages infected with M.tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes. Hsp16.3 protein inhibits autophagosome formation and affects M.tuberculosis survival inside infected macrophages. Furthermore, Hsp16.3 protein significantly increased M.tuberculosis colony forming units (CFU), and decreased the expression of microtubule-associated protein light chain-3 (LC3) expression level (P<0.05). CONCLUSION: The results showed that Hsp16.3 protein inhibits the formation of autophagosomes by regulating the expression of LC3 protein.

Expression of Mycobacterium tuberculosis ferric uptake regulator A gene in Escherichia coli and generation of monoclonal antibodies to FurA.

Ferric uptake regulator A of Mycobacterium tuberculosis (MTB), which belongs to the Fur superfamily, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the pro-drug isoniazid. The feature and role of FurA in oxidative stress contribute to research on the pathogenesis of mycobacteria. In this study, four novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurA protein as immunogen. The furA gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pRSET-A and effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as soluble form in cell lysates and was purified via Ni-NTA purification system. Using the fusion protein to immunize BALB/c mice, four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. As shown by Western blot analysis and cell fluorescence microscopy assay, the four antibodies could recognize the FurA protein, respectively. Then we assessed the effect of iron on the expression of FurA in MTB H37Rv and we concluded that iron does not affect FurA expression. These results suggest that the antibodies against FurA may provide a powerful tool for elucidating FurA biofunctions and regulation mechanism in the pathogenesis of tuberculosis.

Mechanisms involved in the blood-testis barrier increased permeability induced by EMP.

The blood-testis barrier (BTB) plays an important role in male reproductive system. Lots of environmental stimulations can increase the permeability of BTB and then result in antisperm antibody (AsAb) generation, which is a key step in male immune infertility. Here we reported the results of male mice exposed to electromagnetic pulse (EMP) by measuring the expression of tight-junction-associated proteins (ZO-1 and Occludin), vimentin microfilaments, and transforming growth factor-beta (TGF-beta3) as well as AsAb level in serum. Male BALB/c mice were sham exposed or exposed to EMP at two different intensities (200kV/m and 400kV/m) for 200 pulses. The testes were collected at different time points after EMP exposure. Immunofluorescence histocytochemistry, western blotting, laser confocal microscopy and RT-PCR were used in this study. Compared with sham group, the expression of ZO-1 and TGF-beta3 significantly decreased accompanied with unevenly stained vimentin microfilaments and increased serum AsAb levels in EMP-exposed mice. These results suggest a potential BTB injury and immune infertility in male mice exposed to a certain intensity of EMP.

[The immunological responses induced by Mycobacterium tuberculosis heat shock protein 16.3 and its synthetic peptide in mice].

OBJECTIVE: To evaluate the immune responses and resistance against Mycobacterium tuberculosis (MTB) infection in the mice induced by HSP16.3 of MTB and its synthetic peptide. METHODS: BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16.3 protein and synthetic peptide were both 50 microg each time. A single dose of BCG (5 x 10(6) CFU/mouse) was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay (ELISA). Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16.3 stimulation, and the remaining cells were used for IFN-gamma level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 10(5) colony forming units (CFU) of MTB H(37)Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-t test. RESULTS: The level of specific antibody to HSP16.3 protein and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups (HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA) were higher than the BCG group. The indexes of spleen lymphocyte proliferation (SI) of the 3 experiment groups (3.13 +/- 0.18, 3.21 +/- 0.21 and 2.40 +/- 0.15) were significantly higher than the BCG group (1.67 +/- 0.12) and the saline group (1.04 +/- 0.09) respectively. The SI of HSP16.3 protein + DDA + MPL group (3.13 +/- 0.18) and synthetic peptide + DDA + MPL group (3.21 +/- 0.21) were higher than the synthetic peptide + IFA group (2.40 +/- 0.15). The IFN-gamma levels induced among the 3 experiment groups [(182 +/- 6), (194 +/- 9) and (179 +/- 8) mg/L] were lower than the BCG group [(275 +/- 10) mg/L], but higher than the saline group [(71 +/- 3) mg/L]. The IFN-gamma level induced among the 3 experiment groups did not show any marked difference. Although the protection induced by HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA all showed resistance against MTB H(37)Rv infection in the spleens or lungs (the bacterial logarithmic loads of spleen: 6.74 +/- 0.14, 6.60 +/- 0.13 and 6.81 +/- 0.28; the bacterial logarithmic loads of lung: 5.81 +/- 0.21, 5.74 +/- 0.27 and 6.65 +/- 0.32), none of them was better than the conventional BCG (the bacterial logarithmic loads of spleen and lung: 5.95 +/- 0.17 and 5.62 +/- 0.23). CONCLUSIONS: Both HSP16.3 and its synthetic peptide can be considered as TB vaccine candidates or effective components in TB vaccines.

[Effect of autophagy in MTB's infection and the expression of its related gene].

AIM: To study the effect of autophagy in MTB's infection and the expression of its related gene. METHODS: The formation of autophagy was induced by Rapamycin and observed by the transmission electron microscope. The cleaning role of autophagy to the MTB H37Rv virulent strain after its formation was detected by clone forming unit (CFU). Realtime PCR was used to detect the mRNA of the autophagy related gene was expressed. RESULTS: The RAW264.7 cell could form autophagosome under the induction of the Rapamycin, and it had the determinate cleaning role to the H37Rv strain in the cell after which formed. The mRNA of atg5, atg8 and atg12 which participated the formation of autophagy were expressed more, but the expression of atg7 had no change. CONCLUSION: Autophagy participated the process of immune response of anti-MTB. Atg5, atg8 and atg12 were the important molecule which control the formation of autophagy when MTB infected.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

OBJECTIVE: To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. METHODS: The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. RESULTS: The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. CONCLUSIONS: Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Effect of electromagnetic pulse exposure on permeability of blood-testicle barrier in mice.

OBJECTIVE: To study the effect of electromagnetic pulse (EMP) exposure on the permeability of blood-testicle barrier (BTB) in mice. METHODS: Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval. The mice were injected with 2% Evans Blue solution through caudal vein at different time points after exposure, and the permeability of BTB was monitored using a fluorescence microscope. The testis sample for the transmission electron microscopy was prepared at 2 h after EMP exposure. The permeability of BTB in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer. RESULTS: After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and sertoli cells, but also between sertoli cells and primary spermatocyte or secondary spermatocyte. In contrast, lanthanum nitrate in control group was only found in the testicle sertoli cells between seminiferous tubule and near seminiferous tubule wall. CONCLUSION: EMP exposure could increase the permeability of BTB in the mice.

[Immune response induced by Rpf domain from Micrococcus luteus in mice].

AIM: To investigate the immunobiology of Rpf domain from Micrococcus luteus. METHODS: BALB/c mice were immunized with Rpf domain three times at 2-week interval. ELISA was used to detect the title of the anti-Rpf domain antibody titer in the immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index (SI) was measured by MTT colorimetry. The levels of secreted IFN-gamma, IL-10 and IL-12 upon specific antigen stimulation was detected by ELISA. The Rpf domain immunized BALB/c mice were intravenously infected with 10(5) CFU MTB H37Rv. The number of CFU in the spleens was determined four weeks after final injection. RESULTS: The titer of the specific antibody in sera of the immunized BALB/c mice was 1:128 000. The SI of Rpf domain immunized group (2.10+/-0.12) was significantly higher than that of saline immunized group (0.90+/-0.21). The lever of IFN-gamma, IL-10 and IL-12 levels in culture supernatant of spleen lymphocytes from the fusion protein immunized mice was (1 126+/-36) ng/L, (368+/-13) ng/L and (289+/-14) ng/L, respectively, which was markedly higher than that of saline immunized group (P<0.01). Compared with normal saline immunized mice (6.64+/-0.13) four weeks after final injection, dramatic reduction in MTB replication was observed in the spleen (5.03+/-0.11) from the BALB/c mice immunized with fusion proteins. CONCLUSION: Rpf domain can be used as a candidate for a new TB vaccine.

[Preparation and characterization of monoclonal antibodies against Micrococcus luteus Rpf domain].

AIM: To express Micrococcus luteus Rpf domain in prokaryotic cells and prepare monoclonal antibodies against Rpf domain. METHODS: The gene encoding Micrococcus luteus Rpf domain was amplified from genome of Micrococcus luteus by polymerase chain reaction(PCR), and inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf domain gene was subcloned into the expression vector pPro-EXHT and transfected into E.coli DH5alpha. After induced by IPTG, the bacteria controlled by T7 promoter expressed the fused Micrococcus luteus Rpf domain protein with a hexahistidine tail at its N-terminal and the target protein was purified under denaturing conditions. Using this protein as antigen to immunize the BALB/c mice and prepare monoclonal antibodies against Micrococcus luteus Rpf domain. Then specifities and relative affinities of mAbs were identified by ELISA. RESULTS: The fusion protein was purified by metal chelate affinity chromatography under denaturing condition. Three cloned mAbs were prepared from the mice immunized by Rpf domain. All of them could recognize Rpf domain. specifically. CONCLUSION: The prepared mAbs against Rpf domain have strong specificity with high titers, which provides useful tools for further study of the function of Rpf domain in TB prevention.

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65 (Hsp65) with human interleukin-2 against Mycobacterium tuberculosis in BALB/c mice.

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin-2 (hIL-2) in BALB/c mice. We showed that the DNA vaccine pcDNA-Hsp65-hIL-2 could induce high levels of antigen-specific antibody, IFN-gamma, CD4(+) and CD8(+) T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.

Short hairpin RNA targeting survivin inhibits growth and angiogenesis of glioma U251 cells.

Survivin is a novel tumor-associated gene, its overexpression mostly associates with carcinogenesis and development. Nevertheless, the precise role of survivin in initiation and progression of gliomas is still not completely clear. We constructed here three short hairpin RNA (shRNA) targeting survivin plasmid vectors and introduced them into glioma U251 cells. The three shRNAs were efficiently and specifically able to knockdown the survivin expression in transiently transfected U251 cells. The stable transfectants expressing the shRNA having the strongest inhibitory effect against survivin exhibited decreased cell growth, increased spontaneous apoptosis, mitotic catastrophe and cell cycle arrest. Furthermore, in nude mice xenografts, the stable transfectants presented decreased de novo glioma formation and reduced development of angiogenesis. Results from this study indicate that survivin plays an important role in malignant proliferation, antiapoptosis and angiogenesis of gliomas, which may become an attractive target for gene therapy of gliomas, while RNA interference (RNAi) mediated by shRNA may become a new promising strategy for cancer gene therapy.

Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice.

BACKGROUND: Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis. METHODS: Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 microl normal saline containing 10(6) CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses. RESULTS: There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P < 0.05). The elicited IFN-gamma level of rBCG group was (1993 +/- 106) pg/ml, which was also significantly higher than that in BCG group ((1463 +/- 105) pg/ml, P < 0.05). The splenocyte proliferation index of rBCG group reached 4.34 +/- 0.31, which was higher than that of BCG group (3.79 +/- 0.24, P < 0.05). CONCLUSION: rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.

Biphasic effect of androgens on prostate cancer cells and its correlation with androgen receptor coactivator dopa decarboxylase.

The aim of this study was to explore the mechanism underlying the dual effect of androgen on prostate cancer cells and further explore its correlation with dopa decarboxylase (DDC), an androgen receptor (AR) coactivator and a traditional neuroendocrine differentiation (NED) marker. Cell proliferation and cycling after treatment with synthetic nonmetabolizable androgen R1881 was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method and flow cytometry. Differential gene expression was analyzed by cDNA microarrays. DDC expression during the dual effect of R1881 was further explored with microarray, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and enzyme activity assays. Proliferation of LNCaP cells was inhibited by 1 nM R1881 but stimulated by 0.1 nM R1881. Compared with the untreated cells, 320 (2.26%; 170 up-regulated, 150 down-regulated) and 4608 (32.65%; 2046 up-regulated, 2562 down-regulated) genes were found to be expressed differentially in the 1 nM and 0.1 nM R1881-treated cells, respectively. The results were partially confirmed by RT-PCR and Western blot. The DDC gene was down-regulated in the 1 nM R1881-treated cells and up-regulated in 0.1 nM R1881- and 30 nM hydroxyflutamide-treated cells. The enzymatic activity of DDC in the latter 2 groups was also strengthened. Meanwhile, the NED markers CgA and synaptophysin were not affected by these AR activators. R1881 had a dose-dependent biphasic effect on LNCaP cell proliferation. AR coactivator DDC was respectively down- and up-regulated in high and low concentrations of R1881. DDC up-regulation by exogenous AR activators is not accompanied by up-regulation of definitive NED markers.

[Construction and expression of eukaryotic coexpression plasmid containing human MUC1 gene and GM-CSF gene].

AIM: To construct an eukaryotic coexpression plasmid containing the coding region of human MUC1 tandem repeats gene and GM-CSF gene and to identify its expression in COS-7 cell. METHODS: MUC1 tandem repeats gene was obtained by synthesizing the segments. After identified by restriction endonuclease digestion analysis and DNA sequencing, MUC1 tandem repeats gene and GM-CSF gene were cloned into eukaryotic expression vector pcDNA3.1(+) to construct recombinant plasmid pcDNA3.1(+)-MUC1-GM-CSF. Then the recombinant plasmid was transfected into COS-7 cell by electroporation and the expression of target gene was detected by immunoflourescence and ELISA. RESULTS: Restriction analysis and DNA sequencing showed that the recombinant plasmid contained the coding region of human MUC1 tandem repeats gene and GM-CSF gene. The expression of MUC1 and GM-CSF was detected. CONCLUSION: The suuessful construction and expression of recombinant plasmid pcDNA3.1(+)-MUC1-GM-CSF lay a foundation for further development of DNA vaccine against breast cancer.

[Inhibition of growth and angiogenesis of U251 cell xenograft in vivo by short hairpin RNA targeting survivin gene].

OBJECTIVE: To observe the impact of specific short hairpin RNA (shRNA) targeting survivin gene on tumorigenesis and angiogenesis of human brain glioblastoma U251 cells in vivo of nude mice. METHODS: U251 cells, U251-SR cells transfected stably with shRNA eukaryotic expression vector pWH1-SR targeting survivin gene, and U251-P cells transfected stably with blank pWH1 vector, were inoculated respectively into subcutaneous tissue in flank of 15 nude mice (each group 5 mice), and the tumor growth status was observed and measured. Protein expressions of survivin, proliferating cell nuclear antigen (PCNA) and factor VIII related antigen (F VIII RAg) were investigated by immunohistochemistry SABC method, apoptotic cells were screened by TUNEL method, furthermore proliferative index (PI), apoptotic index (AI) and microvessel density (MVD) were measured respectively in each group of tumor specimens. RESULTS: Comparing with those in U251 and U251-P groups, in U251-SR group, the tumorigenesis time delayed, tumor grew slowly, both tumor volume and tumor weight decreased significantly (P < 0.01 for both); Survivin protein expression was down-regulated markedly; PI and MVD decreased significantly, whereas AI increased remarkably (P < 0.01 for all). CONCLUSIONS: The specific shRNA targeting survivin gene can inhibit significantly tumorigenesis and angiogenesis of U251 cells in vivo.

[Immune response and protective efficacy induced by fusion protein ESAT6-CFP10 of M.tuberculosis in mice].

AIM: To study murine humoral and cellular immune response induced by fusion protein ESAT6-CFP10 and to examine its protective efficacy against M. tuberculosis (MTB) in mice. METHODS: BALB/c mice were immunized subcutaneously on the back with fusion protein ESAT6-CFP10 that was transferred to nitro cellulose (NC) membrane beforehand. Stimulation index (SI) of the spleen lymphocytes of the immunized mice was measured by MTT colorimetry. The level of IFN-gamma and IL-2 and CTL upon antigen-specific stimulation were detected. The vaccinated BALB/c mice were intravenously infected with MTB H37Rv (10(5) CFU/mouse). Four weeks later the number of CFU in spleens was determined. RESULTS: The titer of serum specific antibody in BALB/c mice immunized with fusion protein ESAT6-CFP10 was 1:6,400. The SI of fusion protein immunized group (1.90+/-0.15) was significantly higher than that of saline-immunized group (0.9+/-0.15). The level of IFN-gamma and IL-2 induced by the fusion protein was 1.792+/-19 ng/L and 0.211+/-11 ng/L respectively, which was significantly higher than that of saline-immunized group and lower than that of BCG-immunized group. The specific killing activity of splenocytes was 36%. Compared with the saline-immunized mice (bacterial load was 6.51+/-0.13), MTB number (bacterial load was 5.24+/-0.15) was reduced dramatically in the spleens of BALB/c mice immunized with the fusion protein, but the protective efficacy of the mice immunized with BCG was higher than that of ESAT6-CFP10 vaccinated group. CONCLUSION: Fusion protein ESAT6-CFP10 can be used as a candidate for novel vaccines.

[Immunity characteristics induced by a genetic vaccine expressing Ag85B-ESAT6 fusion protein].

OBJECTIVE: To evaluate the humoral and cell-mediated immune responses induced by a genetic vaccine expressing the Ag85B-ESAT6 fusion protein, and to investigate its protective effect against Mycobacterium tuberculosis (MTB) challenge. METHODS: Fifty BALB/c mice were randomized into 5 groups and subjected to the following treatments respectively: immunization with normal saline, BCG, pcDNA3, A(Z)-pcDNA3-E(F) and E(Z)-pcDNA3-A(F) for 3 times at 2-week intervals. The stimulation index (SI) of the splenic lymphocytes from the immunized mice was measured by the methyl thiazolyl tetrazolium (MTT) method, and the level of secreted IFN-gamma upon antigen-specific stimulation was detected by ELISA. The immunized mice were intravenously infected with 10(5) colony forming unit (CFU) of MTB H(37)Rv. The numbers of MTB CFU in spleens were determined 4 weeks later. RESULTS: The specific antibody titers in the sera of mice immunized with plasmid A(Z)-pcDNA3-E(F) and E(Z)-pcDNA3-A(F) were 1:1,000 and 1:1,500 respectively, and the SI was 2.2 and 2.4 respectively, while the SI of the normal saline group and the plasmid pcDNA3 immunized group was only 0.9 and 1.1 respectively. The IFN-gamma concentrations in cultured supernatant of splenic lymphocytes from mice immunized with plasmid A(Z)-pcDNA3-E(F) [(5.48 +/- 0.38) ng/ml] and E(Z)-pcDNA3-A(F) [(5.76 +/- 0.51) ng/ml] were significantly higher than those of the normal saline group [(0.50 +/- 0.25) ng/ml] and the plasmid pcDNA3 immunized group [(1.20 +/- 0.33) ng/ml, P < 0.05], but were not significantly different with that of the BCG immunized group [(5.55 +/- 0.31) ng/ml]. Compared with plasmid pcDNA3 immunized group, the bacterial load (lg, CFU/g) in spleen was 6.08 +/- 0.25 which dramatically reduced in mice immunized with recombinant plasmids, but the protective efficacy of mice immunized with plasmid A(Z)-pcDNA3-E(F) (4.63 +/- 0.11) or E(Z)-pcDNA3-A(F) (4.50 +/- 0.32) was lower than that of the BCG vaccination group (4.09 +/- 0.27). CONCLUSION: The cell-mediated immune response induced by genetic vaccine expressing the Ag85B-ESAT6 fusion protein was similar to that induced by BCG immunization.