Objective: The interferon-γ (IFN-γ) release assays still fail to meet the diagnostic needs of etiology negative tuberculosis (TB) patients. The purpose of this paper is to explore the value of the IL-2-enzyme-linked immunospot (ELISPOT) assay in the auxiliary diagnosis of TB. Methods: We recruited 127 TB patients, 49 non-TB patients with other diseases (NTB), and 198 healthy people, whose peripheral blood mononuclear cells were isolated and stimulated by Mtb-specific antigen CFP-10/ESAT-6 fusion protein to study the changes of T cells secreting IFN-γ and Interleukin-2 (IL-2) by ELISPOT method. Results: The combination of IFN-γ and IL-2 had an area under the curve (AUC) of 0.867 (IFN-γ: 0.835, IL-2: 0.866) in the diagnosis of TB and could increase the sensitivity of IFN-γ detection alone from 79.5% to 86.6%. The cut-off value for IL-2 and IFN-γ was 16. IL-2 could increase the positive diagnostic rates of IFN-γ-ELISPOT (-) in the etiologically positive and negative ATB patients by 9.4% and 7.1%. In the ATB group, the positive rates and the numbers of spot-forming cells (SFC) of IFN-γ and IL-2 showed significant differences compared to the normal control subjects and NTB group (P<0.05). And SFCs of IFN-γ-ELISPOT were significantly higher than that of IL-2-ELISPOT in ATB patients. Before the treatment, the SFC values of the etiologically positive TB patients were higher than those of the etiologically negative patients. However, this situation was totally reversed after anti-TB treatment. After effective treatment, the positive rates and SFC values of IFN-γ and IL-2 in TB patients decreased and exhibited a decreasing and increasing trend in etiologically positive and negative TB patients, respectively. Conclusions: The combination of IL-2 and IFN-γ could improve the diagnostic sensitivity of single-factor detection in TB patients and these two cytokines could be used as effective biomarkers for the treatment monitoring in TB patients.

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 M. tuberculosis (MTB) H37Rv-infected BALB/c mice were treated with PBS, 10μg, 50μg, 100μg, and 200μg ag85a/b DNA vaccine delivered by IM and EP three times at two-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from 3 mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive PCR (RT-qPCR) and the GEO database. After MTB infection, most of the up-regulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine, for example, Iapp, Scg2, Chga, Amy2a5, etc, and most of the down-regulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of alveolar epithelial cell, for example, Sftpc, Sftpd, Pdpn, etc. Most of the abnormally up-regulated or down-regulated DE genes in the TB model group were recovered in the 100μg and 200μg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway down-regulated and Rap1 signal pathway up-regulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action target and mechanism of IM and EP are highly consistent. Tuberculosis infection caused rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB.