Single-cell profiling reveals distinct immune response landscapes in tuberculous pleural effusion and non-TPE.

Background: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and remains a major health threat worldwide. However, a detailed understanding of the immune cells and inflammatory mediators in Mtb-infected tissues is still lacking. Tuberculous pleural effusion (TPE), which is characterized by an influx of immune cells to the pleural space, is thus a suitable platform for dissecting complex tissue responses to Mtb infection. Methods: We employed singe-cell RNA sequencing to 10 pleural fluid (PF) samples from 6 patients with TPE and 4 non-TPEs including 2 samples from patients with TSPE (transudative pleural effusion) and 2 samples with MPE (malignant pleural effusion). Result: Compared to TSPE and MPE, TPE displayed obvious difference in the abundance of major cell types (e.g., NK, CD4+T, Macrophages), which showed notable associations with disease type. Further analyses revealed that the CD4 lymphocyte population in TPE favored a Th1 and Th17 response. Tumor necrosis factors (TNF)-, and XIAP related factor 1 (XAF1)-pathways induced T cell apoptosis in patients with TPE. Immune exhaustion in NK cells was an important feature in TPE. Myeloid cells in TPE displayed stronger functional capacity for phagocytosis, antigen presentation and IFN-γ response, than TSPE and MPE. Systemic elevation of inflammatory response genes and pro-inflammatory cytokines were mainly driven by macrophages in patients with TPE. Conclusion: We provide a tissue immune landscape of PF immune cells, and revealed a distinct local immune response in TPE and non-TPE (TSPE and MPE). These findings will improve our understanding of local TB immunopathogenesis and provide potential targets for TB therapy.

Rapid Surface Shaving for Proteomic Identification of Novel Surface Antigens for Vaccine Development.

The bacterial cell surface (surfaceome) is the first site encountered by immune cells and is thus an important site for immune recognition. As such, the characterization of bacterial surface proteins can lead to the discovery of novel antigens for potential vaccine development. In this chapter, we describe a rapid 5-min surface shaving proteomics protocol where live bacterial cells are incubated with trypsin and surface peptides are "shaved" off. The shaved peptides are subsequently identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several checkpoints, including colony forming unit (CFU) counts, flow cytometry, and a false positive unshaved control, are introduced to ensure cell viability/membrane integrity are maintained and that proteins identified are true surface proteins. The protein topology of shaved peptides can be bioinformatically confirmed for surface location. Surface shaving facilitates identification of surface proteins expressed under different conditions, by different strains as well as highly abundant essential and immunogenic bacterial surface antigens for potential vaccine development.

Development and comparison of novel multiple cross displacement amplification (MCDA) assays with other nucleic acid amplification methods for SARS-CoV-2 detection.

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.

Behavioural and pharmaceutical interventions for the prevention of skin cancers in solid organ transplant recipients: a systematic review of randomised controlled trials.

OBJECTIVES: Solid organ transplant recipients are at increased risk of skin cancer, affecting more than 50% of recipients. We aimed to determine the effectiveness of interventions for behavioural change for sun protection or skin cancer prevention in solid organ transplant recipients. DESIGN: Systematic review. DATA SOURCES: We searched MEDLINE, Embase, the Cochrane Central Register of Controlled Trials (CENTRAL) and CINAHL from inception to November 2019. ELIGIBILITY CRITERIA: We included randomised controlled trials that evaluated the effect of behavioural or pharmaceutical interventions on behavioural change or skin cancer prevention in solid organ transplant recipients. DATA EXTRACTION AND SYNTHESIS: Risks of bias and evidence certainty were assessed using Cochrane and the Grading of Recommendations Assessment Development and Evaluation framework. RESULTS: Twenty trials (n=2295 participants) were included. It is uncertain whether behavioural interventions improve sun protection behaviour (n=3, n=414, standardised mean difference (SMD) 0.89, 95% CI -0.84 to 2.62, I2=98%) and knowledge (n=4, n=489, SMD 0.50, 95% CI 0.12 to 0.87, I2= 76%) as the quality of evidence is very low. We are uncertain of the effects of mammalian target of rapamaycin inhibitors on the incidence of non-melanocytic skin cancer (n=5, n=1080, relative risk 0.46, 95% CI 0.28 to 0.75, I2 =72%) as the quality of evidence is very low. CONCLUSIONS: Behavioural and pharmaceutical preventive interventions may improve sun protective behaviour and knowledge, and reduce the incidence of non-melanocytic skin cancer, but the overall quality of the evidence is very low and insufficient to guide decision-making and clinical practice. PROSPERO REGISTRATION NUMBER: CRD42017063962.