Quantitative analysis of serum-based IgG agalactosylation for tuberculosis auxiliary diagnosis.

Tuberculosis (TB) is the leading infectious cause of mortality worldwide, especially in developing countries. However, effective means for TB diagnosis, especially for bacillus-negative (Bn) TB laboratory diagnosis, are urgently needed. In the present study, serum IgG from each tuberculosis patients and healthy controls was purified using affinity chromatography. The samples were then analyzed using mass spectrometry (MS) and ultraperformance liquid chromatography (UPLC) methods. We quantitatively assessed the changes of serum IgG galactosylation in 567 human serum samples including 377 pulmonary TB patients and 190 healthy donors (HDs). We found significantly more agalactosylated (G0) vs monogalactosylated (G1) and digalactosylated (G2) N-glycans of IgG in TB patients, including smear-negative TB patients, than in HDs. The detection rate of TB diagnostic performance by MS for IgG-Gal ratio G0/(G1 + G2 × 2) is 90.48% for bacillus-positive (Bp) and 73.16% for Bn TB patients. Further, combination of MS method with other routine laboratory TB diagnostic methods significantly increased the detection rate to 91.01%-98.39%. Similar results were observed in Mycobacterium tuberculosis (M. tb) infection mouse models. The decrease in galactosylation of IgG in TB patients was also qualitatively confirmed using specific lectin blot assay. Using the above techniques, we can discriminate the content of IgG G0 with terminal N-acetylglucosamine and IgG-Gal ratio G0/(G1 + G2 × 2) between TB patients and HDs. Our data suggest that quantitative analysis of serum-based IgG-Gal ratio G0/(G1 + G2 × 2) could be used for TB auxiliary diagnosis with high effectiveness and feasibility and its combination with other routine laboratory TB diagnostic methods could remarkably improve the detection rate.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Lnc-EST12, which is negatively regulated by mycobacterial EST12, suppresses antimycobacterial innate immunity through its interaction with FUBP3.

Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of intracellular pathogens. However, the role and mechanism of the important lncRNAs in Mycobacterium tuberculosis (M.tb) infection remain largely unexplored. Recently, we found that a secreted M.tb Rv1579c (an early secreted target with a molecular weight of 12 kDa, named EST12) protein activates NLRP3-gasdermin D (GSDMD)-mediated pyroptosis and plays a pivotal role in M.tb-induced immunity. In the present study, M.tb and the EST12 protein negatively regulated the expression of a key lncRNA (named lnc-EST12) in mouse macrophages by activating the JAK2-STAT5a signaling pathway. Lnc-EST12, with a size of 1583 bp, is mainly expressed in immune-related organs (liver, lung and spleen). Lnc-EST12 not only reduces the expression of the proinflammatory cytokines IL-1ß, IL-6, and CCL5/8 but also suppresses the NLRP3 inflammasome and GSDMD pyroptosis-IL-1ß immune pathway through its interaction with the transcription factor far upstream element-binding protein 3 (FUBP3). The KH3 and KH4 domains of FUBP3 are the critical sites for binding to lnc-EST12. Deficiency of mouse lnc-EST12 or FUBP3 in macrophages increased M.tb clearance and inflammation in mouse macrophages or mice. In conclusion, we report a new immunoregulatory mechanism in which mouse lnc-EST12 negatively regulates anti-M.tb innate immunity through FUBP3.

Aptamer-T Cell Targeted Therapy for Tumor Treatment Using Sugar Metabolism and Click Chemistry.

The development of a tumor-targeted immunotherapy is highly required. The most advanced application is the use of CD19 chimeric antigen receptor (CAR)T (CAR-T) cells to B cell malignancies, but there are still side effects including potential carcinogenicity of lentiviral or retroviral insertion into the host cell genome. Here, we developed a nonviral aptamer-T cell targeted strategy for tumor therapy. Tumor cells surface-specific ssDNA aptamers were conjugated to CD3+T cells (aptamer-T cells) using N-azidomannosamine (ManNAz) sugar metabolic cell labeling and click chemistry. We found that the aptamer-T cells could specifically target and bind to tumor cells (such as SGC-7901 gastric cancer cell and CT26 colon carcinoma cell) in vitro and in mice after adoptively transfer in. Aptamer-T cells led to significant regression in tumor volume due to being enriched at tumor microenvironment and producing strong cytotoxicity activities of CD3+T cells with enhanced perforin, granzyme B, CD107a, CD69, and FasL expression. Moreover, aptamer-T displayed even stronger antitumor effects than an anti-PD1 immune-checkpoint monoclonal antibody (mAb) treatment in mice and combination with anti-PD1 yielded synergic antitumor effects. This study uncovers the strong potential of the adoptive nonviral aptamer-T cell strategy as a feasible and efficacious approach for tumor-targeted immunotherapy application.

AAV-expressed G protein induces robust humoral and cellular immune response and provides durable protection from rabies virus challenges in mice.

Rabies is a highly lethal infectious zoonosis caused by rabies virus (RABV), and the mortality rate is almost 100 % once clinical symptoms appear, which poses a huge threat to public health security across the many parts of the word. Vaccination is reported to be the most effective approach to prevent the disease. G protein is the only protein present on the surface of RABV, it also could induce humoral immunity to produce virus neutralizing antibodies (VNA) and stimulate T cells to produce cellular immunity. Adeno-associated viruses (AAVs) have been used as vectors for gene therapy of different human diseases for its low immunogenicity, high safety and long-term stable expression. To develop a safe and effective vaccine, recombinant AAVs containing different kind of G gene were constructed. After intramuscular (i.m.) immunization in mice, all of these rAAV-G vaccines could induce the production of high levels of VNA and effective cellular immune response. Consistently, all of the rAAV-G vaccines could provide protection against lethal RABV challenge. Our results shown that the rAAV-G vaccines could be potential candidates used in the control of RABV infection. In addition, rAAV-G as a vaccine has many advantages of low preparation cost, simple storage and transportation conditions (4 °C storage and transportation), simple immunization program (only one immunization) and so on. Thence, the rAAV-G vaccines could be potential candidates used in the control of RABV infection.