Tuberculosis (TB) has been the leading lethal infectious disease worldwide since 2014, and about one third of the world’s population has a latent TB infection (LTBI). This is largely attributed to the difficulties in diagnosis and treatment of TB and LTBI patients. Exosomes offer a new perspective on investigation of the process of TB infection. In this study, we performed small RNA sequencing to explore small RNA profiles of serum exosomes derived from LTBI and TB patients and healthy controls (HC). Our results revealed distinct miRNA profile of the exosomes in the three groups. We screened 250 differentially expressed miRNAs including 130 specifically expressed miRNAs. Some miRNAs were further validated to be specifically expressed in LTBI (hsa-let-7e-5p, hsa-let-7d-5p, hsa-miR-450a-5p, and hsa-miR-140-5p) and TB samples (hsa-miR-1246, hsa-miR-2110, hsa-miR-370-3P, hsa-miR-28-3p, and hsa-miR-193b-5p). Additionally, we demonstrated four expression panels in LTBI and TB groups, and six expression patterns among the three groups. These specifically expressed miRNAs and differentially expressed miRNAs in different panels and patterns provide potential biomarkers for detection/diagnosis of latent and active TB using exosomal miRNAs. Additionally, we also discovered plenty of small RNAs derived from genomic repetitive sequences, which might play roles in host immune responses along with Mtb infection progresses. Overall, our findings provide important reference and an improved understanding about miRNAs and repetitive region-derived small RNAs in exosomes during the Mtb infectious process, and facilitate the development of potential molecular targets for detection/diagnosis of latent and active tuberculosis.

Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI. Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells. The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our understanding of the molecular basis of Hsp16.3-facilitated Mtb survival in macrophages.

Tuberculosis (TB) has exceeded HIV as the most lethal infectious disease globally for two consecutive years. Moreover, one third of the world’s population is estimated to have latent tuberculosis infection (LTBI). This is mainly because of difficulties associated with diagnosis and treatment for both TB and LTBI patients. Exosomes provide a promising research tool for TB diagnosis and treatment because they are released from various cells containing valuable biochemical information related to disease. In this study, we performed RNA-sequencing analysis on exosomes derived from clinical specimens of healthy controls (HC), active tuberculosis (ATB), and LTBI patients. Our results revealed the distinct gene expression profiles of the exosomes from LTBI and ATB patients. (1) We identified many distinct up-regulated and down-regulated differentially expressed genes (DEGs) in LTBI and ATB samples, and further screened the top-20 DEGs which might provide a potential panel for differentiation of HC, LTBI, and ATB. (2) We classified all the DEGs into six expression patterns, screened the top-20 genes in each pattern, and mainly focused on those highly expressed in LTBI and ATB. (3) Some Mycobacterium tuberculosis (Mtb) RNAs were only enriched in the exosomes of LTBI samples. (4) Pathway and function analysis further indicated down-regulated signaling pathways/immune response and up-regulated apoptosis/necrosis. Our findings indicate the selective packaging of RNA cargoes into exosomes under different stages of Mtb infection, while facilitating the development of potential targets for the diagnosis, prevention and treatment of tuberculosis.

EML4-ALK is a new driver gene of non-small cell lung cancer and a target of crizotinib. The objectives of this study were to determine the frequency of ALK rearrangements in a large cohort of patients with primary lung adenocarcinoma and to analyze the association of ALK rearrangements with clinicopathological characteristics and clinical outcomes. The roles of fluorescence in situ hybridization (FISH), Ventana immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR) in the detection of ALK rearrangements were evaluated. The ALK rearrangement was detected in 430 specimens from individual patients with primary lung adenocarcinoma using FISH and Ventana IHC based on tissue microarrays. The EGFR status was detected in all of the specimens through DNA sequencing. An RT-PCR was performed on 200 of the specimens and confirmed by sequencing. Of the 430 patients, 46 (10.7%) harbored ALK rearrangements. The ALK rearrangements were associated with a younger age and the EGFR wild type in comparison with ALK-negative patients. The sensitivity and specificity of the Ventana IHC were 100% and 98.2%, respectively, and the concordance rate between the FISH and the Ventana IHC was 98.4%. The sensitivity and specificity of RT-PCR were 95.5% and 87.0%, respectively, and the concordance rate between the FISH and the RT-PCR was 89.0%. The Cox analysis indicated that an early stage and EGFR-activating mutations were independently associated with a longer OS. This study demonstrated that ALK rearrangements are associated with a younger age and the EGFR wild type rather than with other clinicopathological factors. Although the FISH and Ventana IHC have better concordance, and RT-PCR is a more sensitive method and can identify different variants or partners, the IHC and RT-PCR need to be further evaluated in clinical trials to identify their roles in guiding patients’ targeted therapy using crizotinib.

Health care workers (HCWs) are at risk of latent tuberculosis infection (LTBI). In China, tuberculosis (TB) is a major public health problem, but the prevalence of LTBI in HCWs especially in the hospital for pulmonary diseases has not been assessed enough. The aim of this study was to determine the prevalence and putative risk factors of LTBI among HCWs in a chest hospital and a TB research institute in China. A cross-sectional study was conducted among HCWs in China in 2012. LTBI was assessed by T-SPOT.TB, and information on HCWs was collected using a standardised questionnaire. Risk factors for LTBI were analyzed by univariate and multivariate regression. The overall prevalence of LTBI among HCWs was 33.6%. Analyzed by job category, the highest prevalence was found among laboratory staff (43.4%). In the different workplaces, the proportion of LTBI was significantly higher among the high risk workplaces (37.4%) compared to the low risk workplaces. The duration of employment had a significant impact on the prevalence of LTBI. Positive T-SPOT.TB test results accounted for 17.6%, 16.8%, 23.5%, 41.8% and 41.6% in groups of ≤2, 3–5, 6–10, 11–20, and >20 working years respectively. In multivariate analysis, job categories (Laboratory staff [2.76 (95% CI: 1.36; 5.60)], technician staff [2.02 (95% CI: 1.12; 3.64)]); working duration as a HCW for 11 to 20 years [3.57 (95% CI: 1.46; 8.71)], and 20 years above [3.41 (95% CI: 1.28; 9.11)]; and the history of household TB contact [2.47 (95% CI: 1.15; 5.33)] were associated with increased risk of LTBI. Prevalence of LTBI estimated by T-SPOT.TB is high among Chinese HCWs and working duration, job category and the history of household TB contact were associated with increased risk. These data highlight adequate infection control measures should be undertaken.