High-Risk HPV16 E6 Activates the cGMP/PKG Pathway Through Glycosyltransferase ST6GAL1 in Cervical Cancer Cells.

Alterations in glycosylation regulate fundamental molecular and cellular processes of cancer, serving as important biomarkers and therapeutic targets. However, the potential association and regulatory mechanisms of E6 oncoprotein on glycosylation of cervical cancer cells are still unclear. Here, we evaluated the glycomic changes via using Lectin microarray and determined the corresponding enzymes associated with endogenous high-risk HPV16 E6 expression in cervical cancer cells. α-2,6 sialic acids and the corresponding glycosyltransferase ST6GAL1 were significantly increased in E6 stable-expressing HPV- cervical cancer C33A cells. Clinical validation further showed that the expression of ST6GAL1 was significantly increased in patients infected with high-risk HPV subtypes and showed a positive association with E6 in cervical scraping samples. Interfering ST6GAL1 expression markedly blocked the oncogenic effects of E6 on colony formulation, proliferation, and metastasis. Importantly, ST6GAL1 overexpression enhanced tumorigenic activities of both E6-positive and E6-negative cells. Mechanistical investigations revealed that E6 depended on activating YAP1 to stimulate ST6GAL1 expression, as verteporfin (inhibitor of YAP1) significantly suppressed the E6-induced ST6GAL1 upregulation. E6/ST6GAL1 triggered the activation of downstream cGMP/PKG signaling pathway and ODQ (inhibitor of GMP production) simultaneously suppressed the oncogenic activities of both E6 and ST6GAL1 in cervical cancer cells. Taken together, these findings indicate that ST6GAL1 is an important mediator for oncogenic E6 protein to activate the downstream cGMP/PKG signaling pathway, which represents a novel molecular mechanism and potential therapeutic targets for cervical cancer.

Neutralization of IL-10 produced by B cells promotes protective immunity during persistent HCV infection in humanized mice.

Chronic HCV infection can lead to cirrhosis and is associated with increased mortality. Interleukin (IL)-10-producing B cells (B10 cells) are regulatory cells that suppress cellular immune responses. Here, we aimed to determine whether HCV induces B10 cells and assess the roles of the B10 cells during HCV infection. HCV-induced B10 cells were enriched in CD19hi and CD1dhi CD5+ cell populations. HCV predominantly triggered the TLR2-MyD88-NF-κB and AP-1 signaling pathways to drive IL-10 production by B cells. In a humanized murine model of persistent HCV infection, to neutralize IL-10 produced by B10 cells, mice were treated with pcCD19scFv-IL-10R, which contains the genes coding the anti-CD19 single-chain variable fragment (CD19scFv) and the extracellular domain of IL-10 receptor alpha chain (sIL-10Ra). This treatment resulted in significant reduction of B10 cells in spleen and liver, increase of cytotoxic CD8+ T-cell responses against HCV, and low viral loads in infected humanized mice. Our results indicate that targeting B10 cells via neutralization of IL-10 may offer a novel strategy to enhance anti-HCV immunotherapy.

Detection of circulating Mycobacterium tuberculosis-specific DNA by droplet digital PCR for vaccine evaluation in challenged monkeys and TB diagnosis.

Mycobacterium tuberculosis (M. tb) is emerging as a more serious pathogen due to the increased multidrug-resistant TB and co-infection of human immunodeficiency virus (HIV). The development of an effective and sensitive detection method is urgently needed for bacterial load evaluation in vaccine development, early TB diagnosis, and TB treatment. Droplet digital polymerase chain reaction (ddPCR) is a newly developed sensitive PCR method for the absolute quantification of nucleic acid concentrations. Here, we used ddPCR to quantify the circulating virulent M. tb-specific CFP10 (10-kDa culture filtrate protein, Rv3874) and Rv1768 DNA copy numbers in the blood samples from Bacille Calmette-Guerin (BCG)-vaccinated and/or virulent M. tb H37Rv-challenged rhesus monkeys. We found that ddPCR was more sensitive compared to real-time fluorescence quantitative PCR (qPCR), as the detection limits of CFP10 were 1.2 copies/µl for ddPCR, but 15.8 copies/µl for qPCR. We demonstrated that ddPCR could detect CFP10 and Rv1768 DNA after 3 weeks of infection and at least two weeks earlier than qPCR in M.tb H37Rv-challenged rhesus monkey models. DdPCR could also successfully quantify CFP10 and Rv1768 DNA copy numbers in clinical TB patients' blood samples (active pulmonary TB, extrapulmonary TB (EPTB), and infant TB). To our knowledge, this study is the first to demonstrate that ddPCR is an effective and sensitive method of measuring the circulating CFP10 and Rv1768 DNA for vaccine development, bacterial load evaluation in vivo, and early TB (including EPTB and infant TB) diagnosis as well.

Generation and application of ssDNA aptamers against glycolipid antigen ManLAM of Mycobacterium tuberculosis for TB diagnosis.

The development of effective Mycobacterial antigen diagnostic reagents remains a high priority. Mannose-capped lipoarabinomannan (ManLAM) is a lipoglycan serving as a major cell wall component. ManLAM is also an early released antigen in the blood circulation system during Mycobacteria tuberculosis (M.tb) infection and is a perfect target antigen for TB diagnosis. In this study, ssDNA aptamers "antibodies" against ManLAM of the predominant clinical epidemic M.tb Beijing genotype strains were generated by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. The selected single aptamer T9 demonstrated the highest specificity and binding affinity, with an equilibrium dissociation constant (Kd) of 668 ± 159 nmol/L. We further detected ManLAM antigens in serum and sputum samples from active pulmonary tuberculosis (aPTB) patients, extrapulmonary TB (EPTB) patients and healthy donors by using a T9 based enzyme-linked oligonucleotide assay (ELONA). The results showed that the specificity and sensitivity were 95.31% and 83.00% (for 100 aPTB serum samples), 98.70% and 92.71% (for 96 aPTB sputum samples), and 94.44% and 88.71% (for 62 EPTB serum samples), respectively. A good correlation was observed between the T9 aptamer-based ELONA and the clinical T-SPOT.TB. Thus, T9 based ELONA has potentials for diagnosis of TB, including inactive TB, smear-negative TB, EPTB, and TB with immunodeficiency, and assist the diagnosis of LTBI albeit it could not distinguish LTBI and active TB.