Preventive effects of Mycobacterium tuberculosis DNA vaccines on the mouse model with latent tuberculosis infection.

Background: About a quarter of the world's population with latent tuberculosis infection (LTBI) are the main source of active tuberculosis. Bacillus Calmette Guerin (BCG) cannot effectively control LTBI individuals from developing diseases. Latency-related antigens can induce T lymphocytes of LTBI individuals to produce higher IFN-γ levels than tuberculosis patients and normal subjects. Herein, we firstly compared the effects of M. tuberculosis (MTB) ag85ab and 7 latent DNA vaccines on clearing latent MTB and preventing its activation in the mouse LTBI model. Methods: A mouse LTBI model was established, and then immunized respectively with PBS, pVAX1 vector, Vaccae vaccine, ag85ab DNA and 7 kinds of latent DNAs (including rv1733c, rv2660c, rv1813c, rv2029c, rv2628, rv2659c and rv3407) for three times. The mice with LTBI were injected with hydroprednisone to activate the latent MTB. Then, the mice were sacrificed for the bacterial count, histopathological examination, and immunological evaluation. Results: Using chemotherapy made the MTB latent in the infected mice, and then using hormone treatment reactivated the latent MTB, indicating that the mouse LTBI model was successfully established. After the mouse LTBI model was immunized with the vaccines, the lung colony-forming units (CFUs) and lesion degree of mice in all vaccines group were significantly decreased than those in the PBS group and vector group (P<0.0001, P<0.05). These vaccines could induce antigen-specific cellular immune responses. The number of IFN-γ effector T cells spots secreted by spleen lymphocytes in the ag85ab DNA group was significantly increased than those in the control groups (P<0.05). In the splenocyte culture supernatant, IFN-γ and IL-2 levels in the ag85ab, rv2029c, and rv2659c DNA groups significantly increased (P<0.05), and IL-17A levels in ag85ab and rv2659c DNA groups also significantly increased (P<0.05). Compared with the PBS and vector groups, the proportion of CD4+CD25+FOXP3+ regulatory T cells in spleen lymphocytes of ag85ab, rv2660c, rv2029c, and rv3407 DNA groups were significantly reduced (P<0.05). Conclusions: MTB ag85ab and 7 kinds of latent DNA vaccines showed immune preventive efficacies on a mouse model of LTBI, especially the rv2659c, and rv1733c DNA. Our findings will provide candidates for the development of new multi-stage vaccines against TB.

Effects of Mycobacterium vaccae vaccine in a mouse model of tuberculosis: protective action and differentially expressed genes.

BACKGROUND: Tuberculosis is a leading cause of death worldwide. BCG is an effective vaccine, but not widely used in many parts of the world due to a variety of issues. Mycobacterium vaccae (M. vaccae) is another vaccine used in human subjects to prevent tuberculosis. In the current study, we investigated the potential mechanisms of M. vaccae vaccination by determining differentially expressed genes in mice infected with M. tuberculosis before and after M. vaccae vaccination. METHODS: Three days after exposure to M. tuberculosis H37Rv strain (5 × 105 CFU), adult BALB/c mice randomly received either M. vaccae vaccine (22.5 µg) or vehicle via intramuscular injection (n = 8). Booster immunization was conducted 14 and 28 days after the primary immunization. Differentially expressed genes were identified by microarray followed by standard bioinformatics analysis. RESULTS: M. vaccae vaccination provided protection against M. tuberculosis infection (most prominent in the lungs). We identified 2326 upregulated and 2221 downregulated genes in vaccinated mice. These changes could be mapped to a total of 123 signaling pathways (68 upregulated and 55 downregulated). Further analysis pinpointed to the MyD88-dependent TLR signaling pathway and PI3K-Akt signaling pathway as most likely to be functional. CONCLUSIONS: M. vaccae vaccine provided good protection in mice against M. tuberculosis infection, via a highly complex set of molecular changes. Our findings may provide clue to guide development of more effective vaccine against tuberculosis.

Immunotherapeutic effects of Mycobacterium tuberculosis rv3407 DNA vaccine in mice.

Tuberculosis (TB) is a major global public health problem. Latent TB infection (LTBI) is a major source of active TB. New vaccines to treat LTBI are urgently demanded. In this study, the gene encoding latency-associated antigen Rv3407 of Mycobacterium tuberculosis (MTB) rv3407 DNA vaccine was used to prepare and the immunogenicity and therapeutic effects were evaluated. Normal mice were immunized intramuscularly three times at two-week intervals with sterile water for injection, plasmid vector pVAX1, M. vaccae vaccine, ag85a DNA or rv3407 DNA. TB-infected mice were immunized intramuscularly three times at two-week intervals with phosphate-buffered saline (PBS) and rv3407 DNA. The normal mice immunized with rv3407 DNA or ag85a DNA showed higher levels of interferon-gamma (IFN-γ) in stimulated spleen lymphocyte culture supernatants, and had more Th1 cells and an elevated ratio of Th1/Th2 immune cells in whole blood, indicating that a Th1-type immune response was predominant. The levels of anti-Ag85A or anti-Rv3407 IgG antibody were significantly increased in the ag85a DNA and rv3407 DNA groups compared to the sterile water for injection, vector, and M. vaccae groups (p < .0001). Compared with the PBS group, the rv3407 DNA group had pulmonary bacterial loads that were lower by 0.56 log10 (p < .01). The mice vaccinated with rv3407 DNA developed antigen-specific cellular and humoral responses. The rv3407 DNA is a potential DNA vaccine candidate against TB.

[Impact of uterine fibroid embolization with danazol alginate microsphere on ovarian function and subsequent pregnancy].

OBJECTIVE: To investigate the impact of danazol alginate microspheres used for uterine arterial embolization (UAE) on ovarian function and subsequent pregnancy using rabbit as a model. METHODS: A total of 32 female rabbits were divided into 3 groups: a control group, danazol alginate microspheres (DKMG) group and alginate microspheres (KMG) group. Basal serum estradiol (E(2)), follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) levels before UAE and 1 - 3 months after UAE were compared for all rabbits. In breeding field all rabbits mated after UAE. Estrus, and pregnancy rate were observed by veterinary. RESULTS: There were no significant changes from baseline FSH, LH, E(2), T levels measured at 1, 2 and 3 months after UAE (P > 0.05). The total pregnancy rate of DKMG or KMG group was 0 within 2 - 4 months after UAE. Compared to the control group (4/8), the difference was statistically significant (P < 0.05); the total pregnancy rate of DKMG, KMG and control groups within 5 - 7 months after UAE, respectively 17% (2/12), 25% (3/12) and 5/8 (P > 0.05); the total pregnancy rate was 42% (5/12), 50% (6/12) and 6/8 respectively within 8 - 10 months after UAE, there were also no significant differences between the three groups (P > 0.05). CONCLUSIONS: There is no obvious effect of danazol alginate microspheres used for uterine arterial embolization on ovarian function in rabbits. After UAE some animals are able to achieve pregnancies, while harmful effects are observed on short term pregnant rate.

Immunogenicity and therapeutic effects of recombinant Ag85AB fusion protein vaccines in mice infected with Mycobacterium tuberculosis.

The immune function of tuberculosis (TB) patients is disordered. By using immune regulators to assist chemotherapy for TB the curative effect might be improved. In this study, a vaccine containing Mycobacterium tuberculosis (M. tuberculosis) recombinant Ag85AB fusion protein (rAg85AB) was constructed and evaluated. The mice were immunized intramuscularly three times at two-week intervals with Ag85AB fusion protein combined with Corynebacterium parvum adjuvant (rAg85AB+CP). In comparison to control mice that received either CP alone or saline, the mice that received rAg85AB+CP had significantly higher number of T cells secreting IFN-γ and higher levels of specific antibodies of IgG, IgG1 and IgG2a isotypes in sera. The specific antibodies also had higher ratios of IgG2a to IgG1, indicating a predominant Th1 immune response. To test for immunotherapy of TB, M. tuberculosis infected mice were given three intramuscular doses of 20µg, 40µg or 60µg of rAg85AB in rAg85AB+CP, or phosphate-buffered saline (PBS), or CP or Mycobacterium phlei (M. Phlei) F.U.36. Compared with the PBS group, 20µg, 40µg and 60µg rAg85AB+CP and M. phlei F.U.36 groups reduced the pulmonary bacterial loads by 0.13, 0.15, 0.42 and 0.40 log10, and the liver bacterial loads by 0.64, 0.64, 0.53 and 0.61 log10, respectively. Pathological changes of lungs were less, and the lesions were limited to a certain extent in 40µg and 60µg rAg85AB+CP and M. phlei F.U.36 groups. These results showed that rAg85AB+CP had immunotherapeutic effect on TB, significantly increasing the cellular immune response, and inhibiting the growth of M. tuberculosis.

Therapeutic effects of traditional Chinese medicine Niubeixiaohe in mouse tuberculosis models.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine Niubeixiaohe (NBXH) is an effective anti-tuberculosis prescription, which is made up of Bulbus Fritillariae Cirrhosae, Rhizoma Bletillae, Radix Platycodonis, Fructus Arctii, Herba Houttuyniae and Glutinous rice. In this study, NBXH powder (I) and three kinds of NBXH extracts (II, III, and IV) were prepared. The water decoction of NBXH had been used to treat TB in clinic sixteen years suggested that it was effective to treat TB. AIM OF THE STUDY: This study evaluated the effects of different processing products of NBXH on mouse TB model in vivo and provide a new Chinese medicine for the clinical treatment of TB. MATERIALS AND METHODS: In this study, 120 female BALB/c mice infected with Mycobacterium tuberculosis H37Rv, were treated with distilled water, M. vaccae vaccine, the low, middle and high doses of NBXH I, the low, middle and high doses of NBXH II, the low, middle and high doses of NBXH III, the low, middle and high doses of NBXH IV for 12 weeks, respectively. RESULTS: The body weights of mice in all NBXH groups were higher than that in the water group. The weight indexes of the spleens in M. vaccae group, the middle dose of NBXH II group, the low dose of NBXH IV group and in the high dose of NBXH IV group were significantly lower than that in the water group(P<0.05). Compared with the water group, the spleen colony counts in the low dose of NBXH I group, the high dose of NBXH II group, the low dose of NBXH III group and the high dose of NBXH IV group reduced by 0.43, 0.46, 0.73, 0.58 logs (P<0.05), respectively. But the lung colony counts had no significant difference between each group. Pulmonary general pathology and histopathology displayed that the lung lesions in treatment groups were improved at certain degree, especially in the low dose of NBXH IIIand IV groups, in which their areas of the lesions were less than 50%, and the half normal lung structure in half of the mice could be observed. CONCLUSION: Powder and three extracts of traditional Chinese medicine NBXH all had anti-tuberculosis therapeutic effects on mouse tuberculosis model, and this study provided a base for the further development of Chinese patent medicine NBXH. Also, this is the first report on comprehensive experimental research of NBXH extracts coming from six kinds of traditional Chinese medicine.

An LOH and mutational investigation of the ST7 gene locus in human esophageal carcinoma.

Frequent loss of heterozygosity (LOH) on human chromosome 7q31 has been reported in numerous malignancies. Suppressor of tumorigenicity 7 (ST7) has been identified as a candidate tumor suppressor gene in this region. To identify whether 7q31 and genetic alterations of ST7 were involved in human esophageal carcinogenesis, we performed LOH mapping of a 5.4 cM region at 7q31-q35 in 43 primary esophageal carcinomas, as well as mutational analyses of the ST7 gene in tumors with LOH in this region. Of 43 tumors, 12 (28%) showed LOH at 7q31-q35. These included four (22%) of 18 squamous cell carcinomas and eight (32%) of 25 adenocarcinomas. The peak LOH locus was D7S480, lying 4.2 Mb telomeric to ST7 and showing LOH in eight of 37 informative tumors, or 22%. No mutations were found in the entire coding or flanking intronic regions of the ST7 gene among 12 tumors with 7q-LOH. In addition, quantitative RT-PCR analyses of ST7 mRNA expression levels in 11/13 normal-tumor pairs failed to show more than a 50% decrease in tumor ST7 mRNA relative to matched normal tissues. These data suggest that LOH at 7q31-q35 is involved in the origin or progression of at least a subset of esophageal carcinomas, but that ST7 is not the target gene of this somatic event.

[Immune regulation effect of rat dendritic cells phagocytosing photochemotherapy-treated allogeneic cells on syngeneic T cells].

The aim of this study was to investigate the immune regulatory effect of dendritic cells phagocytosing photochemotherapy-treated allogeneic spleen lymphocytes on syngeneic T cells. DA rat spleen lymphocytes were treated with 8-methoxypsoralen plus UVA irradiation (PUVA). LEW rat bone marrow-derived DCs were co-cultured with PUVA-treated DA spleen lymphocytes (PUVA-SP), and the surface markers (MHC-II, CD86 and CD40) of treated DC were detected by flow cytometry. CFSE-labeled PUVA SP were incubated with LEW DCs and the phagocytosis of DCs on PUVA-SP was observed by using fluorescent microscope. The ability of DC phagocytosing allogeneic PUVA-SP (PUVA-SP DC) to stimulate the proliferation of LEW T cells was analyzed by mixed leukocyte reactions (MLR). The production of IL-4, IL-10, IL-2, IFN-gamma in MLR culture supernatant was determined by luminex method. The results indicated that the PUVA treatment effectively induced early apoptosis of DA rat spleen lymphocytes. After co-culture, DC efficiently phagocytosed allogeneic PUVA-SP and still maintained an immature phenotype with low levels of MHC II, CD40 and CD86. PUVA-SP DC induced LEW T cell hyporesponsiveness to DA rat antigen, and led to skewing of T cell cytokine expression toward Th2 (IL-10 and IL-4). It is concluded that the PUVA-SP DC effectively down-regulate T cell response to alloantigen and induce Th2 immune deviation in vitro.

The treatment of mice infected with multi-drug-resistant Mycobacterium tuberculosis using DNA vaccines or in combination with rifampin.

The problems of tuberculosis (TB) and its drug resistance are very severe in China. New therapeutic agents or regimens to treat multi-drug-resistant tuberculosis (MDR-TB) are urgently needed. In this study, the effects of Ag85A DNA or ESAT6/Ag85A chimeric DNA vaccines alone or in combination with rifampin (RFP) were studied for the treatment of mice with MDR-TB. Eighty female BALB/c mice infected with Mycobacterium tuberculosis clinical isolate HB361, which was resistant to high level of RFP, and low level of isoniazid (INH), were treated with the saline, plasmid vector pVAX1, RFP, HSP65 DNA, Ag85A DNA, Ag85A DNA combined with RFP, chimeric ESAT6/Ag85A DNA, chimeric ESAT6/Ag85A DNA combined with RFP, respectively. Different effects of DNA vaccines for the treatment of MDR-TB were demonstrated in this study. Compared with saline group, Ag85A DNA vaccine alone or Ag85A DNA in combination with rifampin group reduced the pulmonary and splenic bacterial loads by 0.58, 0.82 and 0.51, 0.69 logs, respectively. The pathological changes of lungs were also slight and the lesions were limited in comparison with that of the control mice in which the lesions were extensive and more necrotic changes were observed. Interestingly, the chimeric Ag85A/ESAT6 DNA vaccine showed the lower effect for the treatment of MDR-TB. Ag85A DNA vaccine played a main role for the treatment of TB and MDR-TB. We believe that this is the first report of the use of DNA vaccine in the treatment of MDR-TB, and that these data suggest that DNA vaccine was effective for the treatment of MDR-TB which might have the potential contribution for resolving this problem in developing countries.