Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model.

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.

Change-Plane Analysis for Subgroup Detection and Sample Size Calculation.

We propose a systematic method for testing and identifying a subgroup with an enhanced treatment effect. We adopts a change-plane technique to first test the existence of a subgroup, and then identify the subgroup if the null hypothesis on non-existence of such a subgroup is rejected. A semiparametric model is considered for the response with an unspecified baseline function and an interaction between a subgroup indicator and treatment. A doubly-robust test statistic is constructed based on this model, and asymptotic distributions of the test statistic under both null and local alternative hypotheses are derived. Moreover, a sample size calculation method for subgroup detection is developed based on the proposed statistic. The finite sample performance of the proposed test is evaluated via simulations. Finally, the proposed methods for subgroup identification and sample size calculation are applied to a data from an AIDS study.

[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.

Sequential advantage selection for optimal treatment regime.

Variable selection for optimal treatment regime in a clinical trial or an observational study is getting more attention. Most existing variable selection techniques focused on selecting variables that are important for prediction, therefore some variables that are poor in prediction but are critical for decision-making may be ignored. A qualitative interaction of a variable with treatment arises when treatment effect changes direction as the value of this variable varies. The qualitative interaction indicates the importance of this variable for decision-making. Gunter, Zhu and Murphy (2011) proposed S-score which characterizes the magnitude of qualitative interaction of each variable with treatment individually. In this article, we developed a sequential advantage selection method based on the modified S-score. Our method selects qualitatively interacted variables sequentially, and hence excludes marginally important but jointly unimportant variables or vice versa. The optimal treatment regime based on variables selected via joint model is more comprehensive and reliable. With the proposed stopping criteria, our method can handle a large amount of covariates even if sample size is small. Simulation results show our method performs well in practical settings. We further applied our method to data from a clinical trial for depression.

[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.

Production and characterization of monoclonal antibody against Mycobacterium tuberculosis RpfB domain.

RpfB, one of the five resuscitation-promoting factors (Rpfs) produced by Mycobacterium tuberculosis (MTB), plays an important role in the resuscitation and growth of the dormant MTB. RpfB is likely the target antigen recognized by the host immune system. Studies have shown that Rpf genes exist in many bacteria and their encoded proteins all contain Rpf-like domain. It is likely that this domain has biological characteristics and immunogenicity similar to that of the complete Rpf protein. Therefore, RpfB domain protein from M. tuberculosis was selected for this study. Mice were subcutaneously immunized three times over 2-week intervals. Mice splenocytes were then isolated and fused with SP20 cells. Hybridoma colonies were screened for monoclonal antibody (MAb) against RpfB domain. ELISA was used to examine the titer, specificity, and relative affinity of the antibody. The ability of produced anti-RpfB monoclonal antibody to recognize other proteins in the Rpf family and to inhibit the growth of Mycobacterium tuberculosis and Micrococcus luteus was examined. Our results showed that three anti-RpfB MAbs were successfully generated. All three MAbs can recognize RpfB domain specifically and can effectively inhibit the promoting effect of RpfB domain on the growth of MTB H37Ra strain and M. luteus at 1:1000 dilution, indicating that anti-RpfB domain MAbs may inhibit the reactivation of dormant or latent MTB in vivo. Therefore, they may be able to prevent the recurrence of the occult infection. The production of anti-RpfB domain MAbs provides a powerful experimental tool to further study the biological and immunological characteristics of the RpfB domain and to evaluate the possibility of using RpfB domain as a candidate component for tuberculosis subunit vaccine.

[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.

[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.