Changhong Shi

A review of murine models of latent tuberculosis infection

Abstract The mechanisms of latency and the causes of reactivation of Mycobacterium tuberculosis remain poorly understood; an important reason for this gap in knowledge is the absence of a standardized animal model of latent tuberculosis infection (LTBI). A complete LTBI model should incorporate 2 aspects of LTBI: a persistent infection model with a low bacterial load and a latent infection model that is modified from the Cornell model. Many parameters must be carefully considered to establish an LTBI model, including the inoculating dose, the route of infection, the time interval between infection and the initiation of antibiotic therapy, and the genetic background of the host animal. The responsiveness of this mouse model of LTBI can be assessed through the integrated use of indices, including Karnofsky performance status, bacterial load in spleen and lungs, induced levels of interferon-gamma and tumour necrosis factor-alpha, expression of interleukin (IL)-10 and IL-4 in tissues, specific antigen load in organs, time required for hormone-induced TB relapse, expression level of dormancy genes, and CD4 T-cell count.

GLP-1 C-terminal structures affect its blood glucose lowering-function

Glucagon‐like peptide‐1 (GLP‐1), which is an endogenous insulinotropic peptide that can stimulate islet cells to secret insulin, is a promising new drug candidate for the treatment of type 2 diabetes. However, due to the very short half‐life of this peptide, the clinical value of GLP‐1 is restricted. A GLP‐1 peptide analog that had been altered by deletion of five amino acids from the C‐terminus (sGLP‐1) was selected and investigated in vivo for the therapeutic effect on GK rats with type II DM (T2DM). The results revealed that sGLP‐1 exhibited decreased blood glucose‐lowering ability compared to GLP‐1 in the first week, as measured after once‐daily administration. However, after drug administration for 2 weeks, the blood glucose‐lowering effect of sGLP‐1 became superior to that of GLP‐1. sGLP‐1 reduced apoptosis of the old islets, enhanced insulin production, and promoted new islets replication. sGLP‐1 is a shorter but more efficient GLP‐1 analog for type 2 diabetes management. Because sGLP‐1 prolonged the proliferation and recovery of islet cells, the ability to maintain blood glucose (BG) within a normal range was still present 2 weeks after drug withdrawal. These results confirmed the importance of the C‐terminus of GLP‐1 molecule, and further demonstrated that GLP‐1 (7–37) can be truncated till the 32nd amino acid to have a better long‐term BG lowing function. This result may imply for the presence of glucagon family clearance receptors in vivo and demonstrates that the C‐terminus participates in GLP‐1 clearance. Copyright

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‐γ, 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.

Immunotherapeutic efficacy of recombinant Mycobacterium smegmatis expressing Ag85B–ESAT6 fusion protein against persistent tuberculosis infection in mice

The application of immunotherapy in combination with chemotherapy is considered an effective treatment strategy against persistent Mycobacterium tuberculosis (Mtb) infection. In this study, we constructed a novel recombinant Mycobacterium smegmatis (rMS) strain that expresses Ag85B and ESAT6 fusion protein (AE–rMS). Immunization of C57BL/6 mice with AE–rMS generated mainly Th1-type immune responses by strongly stimulating IFN-γ- and IL-2-producing splenocytes and increasing antigen-specific cytotoxic T lymphocyte (CTL) activity. To test the immunotherapeutic efficacy of AE–rMS, a persistent tuberculosis infection (PTBI) model was established via tail-vein injection of C57BL/6 mice with 1 × 104 colony forming units (CFU) of Mtb strain H37Rv in combination with concurrent chemotherapy drugs isoniazid (INH) and pyrazinamide (PZA). PTBI mice immunized with AE–rMS showed high levels of IFN-γ secreted by splenocytes and decreased bacteria loads in lung. Treatment with only the anti-tuberculosis (anti-TB) drugs RFP and INH (RI), decreased bacteria loads to low levels, with the Th1-type immune response further attenuated. Moreover, AE–rMS, when combined with RI treatment, further reduced the bacteria load as well as the pathological tissue damage in lung. Together, these results demonstrated the essential roles of AE–rMS-induced Th1-type responses, providing an effective treatment strategy by combining AE–rMS and RI for persistent TB.

Monoclonal Antibodies Against a Mycobacterium tuberculosis Ag85B-Hsp16.3 Fusion Protein

The secreted Mycobacterium tuberculosis (MTB) proteins, Ag85B and Hsp16.3, have been the focus of intensive research in recent years. These proteins have high sensitivity in bacterium-negative tuberculosis (TB) patients, and are valuable for the rapid diagnosis of bacterium-negative TB. Fusion proteins including multiple antigens such as Ag85B and Hsp16.3 provide improved sensitivity and specificity for serological diagnosis of active TB compared with a single antigen. Many studies have shown that the production of MAbs recognizing a specific repertoire of M. tuberculosis antigens and the tests based on monoclonal antibodies have been found to be valuable in positive detection of TB, particularly for smear-positive pulmonary TB. A number of MAbs are currently used for serodiagnosis of TB. Therefore, an Ag85B-Hsp16.3 fusion protein was expressed and purified using an E. coli system in this study. Three Ag85B-Hsp16.3 fusion protein-specific MAbs were generated by routine murine hybridoma techniques. The titer, specificity, and relative affinity of all three MAbs were determined by ELISA and the serological responses were analyzed. The levels of antigens in a proportion of TB patients were shown to be significantly higher than those in healthy controls. The sensitivity and specificity of the currently available detection systems is likely to be improved by the employment of a combination of these MAbs with others that are already in use.

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.