[Immunoprophylaxis of recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis].

AIM: To evaluate immunoprophylaxis of recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis. METHODS: BALB/c mice were immunized with recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis. ELISA was used to detect the anti-MPT64 antibody titers and subtype in immunized mice sera. Splenocytes of immunized mice were separated and used to detect IFN-gamma and IL-12 levels, splenocytic proliferation, counts of CD4(+) and CD8(+) T cell percentage, cytolytic T lymphocyte specific lysis. The bacteria numbers in vaccination animal's lung and spleen were counted by colony forming units (CFUs) on plate. RESULTS: Recombinant Mycobacterium vaccae secreted MPT64 of Mycobacterium tuberculosis could induce high level of specific IgG antibodies, stronger T cell proliferation response, production of IFN-gamma and IL-12, CD4(+) and CD8(+) cell percentages and CTL effect in mice. And an efficacy protection against Mycobacterium tuberculosis was also observed in mice model. CONCLUSION: Recombinant Mycobacterium vaccae secreted MPT64 could induce high level humoral and cell mediated immune responses in mice and could be used as a candidate of new vaccine against TB.

[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.

[Sexual hormone levels in semen and germ cell apoptosis].

OBJECTIVE: To explore the relationship between sexual hormones in semen and germ cell apoptosis in male population. METHODS: Sixty-six infertile patients and thirty fertile males were selected randomly. The levels of folicle stimulating hormone ( FSH), prolactin (PRL), luteinizing hormone (LH), and testosterone (T) in semen were measured by ELISA. Terminal deoxynucleotidyl transferase mediated UTP nick end labeling (TUNEL) was used for the detection of germ cell apoptosis. RESULTS: The levels of FSH, LH, PRL, T in thirty fertile men were (1.63 +/- 0.15) U/L, (2.18 +/- 0.21) U/L, (6.34 +/- 0.30) nmol/L, (1.85 +/- 0.11) nmol/L, respectively, and germ cell apoptosis rate was (4.61 +/- 1.23)%. FSH, LH, PRL, T levels in infertile group were (1.25 +/- 0.18) U/L, (1.76 +/- 0.32) U/L, (5.86 +/- 0.13) nmol/l, (1.45 +/- 0.13) nmol/, respectively, and germ cell apoptosis rate was (18.36 +/- 2.04)%. There were significant differences in all parameters between infertile group and fertile group. The levels of FSH, LH, PRL, T were negatively correlated with germ cell apoptosis rates( r = -0.88, -0.93, -0.90, -0.98). The volume of apoptotic germ cell decreased, and chromatin was compacted to form cell-membrane blebs and apoptotic bodies. CONCLUSION: Low concentration of sexual hormones may increase the apoptosis of germ cells, which can induce male infertility.

[Sodium nitroprusside facilitates human sperm capacitation and acrosome reaction].

OBJECTIVE: To explore the effect of nitric oxide (NO) on human sperm capacitation and acrosome reaction (AR). METHODS: Different concentrations of sodium nitroprusside (SNP) were added to the sperm suspension from 48 healthy fertile men, and the suspension was incubated in 1 x Earle at 37 degrees C for 1 hour. Progesterone was used to induce AR for 15, 30, 45 and 60 min, and then acid phosphatase (ACP) activity in the suspension before and after capacitation and at different time of AR was measured by p-nitrophenyl sodium phosphate assay. In the meantime, sperm motile parameters were assayed by CASA to observe sperm capacitation and AR. RESULTS: ACP activity and sperm motile parameters increased in the 50 approximately 100 nmol/L NO concentration group, showed no significant variation in the 150 approximately 200 nmol/L group, and decreased in the 250 approximately 300 nmol/L group. CONCLUSION: NO can facilitate sperm capacitation, AR and sperm motile parameters in low concentration and suppress them in high concentration. ACP activity assay of sperm is an objective and reliable method to evaluate sperm capacitation and AR in whole sperm population.

[Adriamycin thermotherapy through hepatic artery for model of VX2 carcinoma in rabbit liver].

BACKGROUND & OBJECTIVE: It was reported that heating can enhance sensitivity of rabbit VX2 cell to adriamycin and increase intracellular concentration of adriamycin. This study was designed to evaluate the anti-tumor effects of interventional hyperthermia and interventional chemotheramotherapy on VX2 carcinoma in rabbit liver. METHODS: VX2 carcinoma cells were surgically implanted into the right liver lobe of 60 male New Zealand white rabbits, which were randomly divided into 4 groups(15 rabbits per group). To inject physiological saline(37 degrees C), adriamycin (37 degrees C), physiological saline(60 degrees C), and adriamycin (60 degrees C) in different groups via hepatic artery of the rabbits with liver cancer. One week later, to observe the volume of tumor, the serum level of aspartate transaminase(AST), and observe the survival period of VX2 rabbits. RESULTS: In group of ADM(60 degrees C), the tumor growth rate (0.53 +/- 0.21)% was significantly lower than group 2(1.09 +/- 0.26)%, group 3(3.32 +/- 1.28)%, and group 4(3.48 +/- 1.17)% (P < 0.05, P < 0.05, P < 0.01, respectively). The survival period of adriamycin (60 degrees C) group (50.0 +/- 2.0)d was significantly higher than the untreated control group (40.5 +/- 3.0)d, (P < 0.05). The serum level of AST of TNP-470 with lipiodol group was not higher than the other treated groups(P > 0.05), but being significantly higher than the untreated control group after treated(P < 0.05). CONCLUSION: Adriamycin (60 degrees C) greatly decreases the tumour growth rate, and prolongs the survival period.