| 1. |
- Andersson, Blanka, et al.
(författare)
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A novel mycobacterial growth inhibition assay employing live-cell imaging of virulent M. tuberculosis and monitoring of host cell viability
- 2020
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Ingår i: Tuberculosis. - : CHURCHILL LIVINGSTONE. - 1472-9792 .- 1873-281X. ; 124
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Tidskriftsartikel (refereegranskat)abstract
- Our aim was to develop a Mycobacterium tuberculosis (Mtb) growth inhibition assay (MGIA) as a summary estimate of host immune control of virulent Mtb. Mycobacterial growth inhibition (MGI) using previously frozen human PBMCs infected with H37Rv was assessed by live-cell imaging (Incucyte (c)) complemented by imaging flow cytometry analysis of phagocytosis. MGI measured as relative fluorescence units (RFU) was calibrated to time to positive culture (TTP) in BACTEC 960 MGIT. At a MOI (multiplicity of infection) of 5, there was a wide range of MGI of blood donors (1.1*10(6)-2.7*10(6) RFU, n = 14). Intra- and inter-assay variability were at most 17.5 and 20.7 CV%. Cell viability at day 5 was 57 and 62% monitored by the LDH and Draq7 assays respectively. There was a strong correlation between a readout for Mtb growth using CFU counts or TTP compared to RFU (r2 >= 0.96). Our MGIA enabling live-cell imaging and monitoring of cell viability was able to detect a wide range of Mtb growth inhibition by PBMCs and was calibrated to several readout options for bacterial growth. This MGIA may be valuable as a surrogate marker of host immunity in a personalized medicine approach.
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| 2. |
- Borgstrom, E. W., et al.
(författare)
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CD4(+) T cell proliferative responses to PPD and CFP-10 associate with recent M. tuberculosis infection
- 2020
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Ingår i: Tuberculosis. - : Elsevier BV. - 1472-9792 .- 1873-281X. ; 123
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Tidskriftsartikel (refereegranskat)abstract
- Interferon-gamma release assays cannot differentiate latent from active tuberculosis (TB), nor identify the recently infected with increased risk of active disease. The objective of this study was to identify biomarkers of recent infection following exposure to tuberculosis, to increase the positive predictive value for incipient TB. Contacts to patients with pulmonary TB were tested repeatedly with interferon-gamma release assays and flow-cytometry. Proliferative CD4(+) T cell responses to purified protein derivative (PPD) and 11 M. tuberculosis antigens were analysed. The individual probability of recent and remote infection was estimated using clinical data in a novel mathematical model and compared with CD4(+) responses in a prediction model. The most specific prediction of recent infection was high CD4(+) proliferative responses to CFP-10 and PPD and a low CD4(+) response to ESAT-6. CD4(+) proliferative responses to Rec85a, Rec85b and Rv1284 were also observed in recent infection, but did not reach significance in the prediction model. Conclusions: High CD4(+) proliferative responses to CFP-10 and PPD and a low response to ESAT-6 may be used as biomarkers to improve positive predictive values for recent LTBI and thus, increased risk of incipient TB. Rec85a, Rec85b and Rv1284 are also of interest to study further in this context.
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| 3. |
- de Sousa, Nuno Rufino, et al.
(författare)
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A fieldable electrostatic air sampler enabling tuberculosis detection in bioaerosols
- 2020
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Ingår i: Tuberculosis. - : Elsevier BV. - 1472-9792 .- 1873-281X. ; 120
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Tidskriftsartikel (refereegranskat)abstract
- Tuberculosis (TB) infects about 25% of the world population and claims more human lives than any other infectious disease. TB is spread by inhalation of aerosols containing viable Mycobacterium tuberculosis expectorated or exhaled by patients with active pulmonary disease. Air-sampling technology could play an important role in TB control by enabling the detection of airborne M. tuberculosis, but tools that are easy to use and scalable in TB hotspots are lacking. We developed an electrostatic air sampler termed the TB Hotspot DetectOR (THOR) and investigated its performance in laboratory aerosol experiments and in a prison hotspot of TB transmission. We show that THOR collects aerosols carrying microspheres, Bacillus globigii spores and M. bovis BCG, concentrating these microparticles onto a collector piece designed for subsequent detection analysis. The unit was also successfully operated in the complex setting of a prison hotspot, enabling detection of a molecular signature for M. tuberculosis in the cough of inmates. Future deployment of this device may lead to a measurable impact on TB case-finding by screening individuals through the aerosols they generate.
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| 4. |
- Kalsum, Sadaf, et al.
(författare)
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A high content screening assay for discovery of antimycobacterial compounds based on primary human macrophages infected with virulent Mycobacterium tuberculosis
- 2022
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Ingår i: Tuberculosis. - : CHURCHILL LIVINGSTONE. - 1472-9792 .- 1873-281X. ; 135
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Tidskriftsartikel (refereegranskat)abstract
- Drug resistance in Mycobacterium tuberculosis is an emerging threat that makes the discovery of new candidate drugs a priority. In particular, drugs with high sterilizing activity within host cells are needed to improve efficacy and reduce treatment duration. We aimed to develope and validate a High Content Screening assay based on Mycobacterium tuberculosis-infected primary human monocyte-derived macrophages as its natural reservoir. Infected primary human monocyte-derived macrophages were exposed to control antibiotics or tested compounds on 384 well plates. Intracellular bacterial growth and macrophage numbers were evaluated using an ImageXpress High Content Screening system and Z-factor was calculated to assess the reproducibility. The combination of isoniazid and rifampicin as a positive control rendered a Z-factor above 0.4, demonstrating suitability of the assay for screening and compound profiling purposes. In a validation experiment, isoniazid, rifampicin, moxifloxacin and levofloxacin all effectively inhibited intracellular growth as expected. Finally, a pilot screening campaign including 5700 compounds from diverse libraries resulted in the identification of three compounds with confirmed antimycobacterial activity in the low micromolar range and low host cell toxicity. The assay represents an attractive screening platform for both academic research on host-pathogen mechanisms in tuberculosis and for the identification and characterization of novel antimycobacterial compounds.
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| 5. |
- Kiflie, Amare, et al.
(författare)
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Differential effects of asymptomatic Ascaris lumbricoides, Schistosoma mansoni or hook worm infection on the frequency and TGF-beta-producing capacity of regulatory T cells during active tuberculosis
- 2021
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Ingår i: Tuberculosis. - : CHURCHILL LIVINGSTONE. - 1472-9792 .- 1873-281X. ; 131
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Tidskriftsartikel (refereegranskat)abstract
- Helminth induced expansion of regulatory T cells (Tregs) may take part in suppressing protective host responses during tuberculosis (TB), although Tregs functionality and link to TB disease severity remains unexplored. We investigated the species-specific effect of helminths on frequency and TGF-beta producing capacity of Tregs, and possible connection to TB disease severity. 89 pulmonary TB patients (PTB) and 69 community controls (CCs) from Gondar, Ethiopia, were included. Clinical disease severity was graded by TB score, and flow cytometry used to characterize Treg frequency and functionality measured as their TGF-beta-producing capacity. In helminth positive PTB patients (Helminth+PTB+) compared to helminth negative PTB or CCs, TGF-beta(+) Tregs were significantly increased mainly in hookworm coinfection whereas S. mansoni increased TGF-beta(+) Tregs in CCs. Treatment of TB and helminths decreased TGF-beta(+) Tregs in Helminth+PTB+ at 2 months follow-up. There were no overall differences in the frequency of Tregs in CCs or PTB unless stratification on TB disease severity was performed. At inclusion Helminth+PTB+ had increased frequency of Tregs already at low disease severity, and TGF-beta(+) Tregs correlated to intermediate-to-high disease severity. In conclusion, helminth specific increase of TGF-beta(+) Tregs in PTB patients was correlated to TB disease severity and was restored following anti-helminth treatment.
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| 6. |
- Kurtz, Sherry L., et al.
(författare)
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Whole genome profiling refines a panel of correlates to predict vaccine efficacy against Mycobacterium tuberculosis
- 2020
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Ingår i: Tuberculosis. - : Elsevier. - 1472-9792 .- 1873-281X. ; 120
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Tidskriftsartikel (refereegranskat)abstract
- New vaccines are needed to combat the public health threat posed by M. tuberculosis (M. tb), but no correlates have been defined to aid vaccine development. Using mouse models, we previously developed an in vitro system that measures the ability of M. tb-immune lymphocytes to control bacterial replication during co-culture with M. tb-infected macrophages. We demonstrated that the degree of in vitro growth control by lymphocytes from mice given vaccines of varying efficacy reflected the relative degree of in vivo protection against lethal challenge. Further, using targeted analyses of gene expression in lymphocytes recovered from co-cultures, we found mediators whose relative expression also correlated with in vitro and in vivo outcomes. Here we advanced those findings by employing genome-wide expression analyses. We first screened splenocytes recovered from co-cultures by microarray, revealing additional genes whose expression correlated with protection. After applying pathway analyses to down-select gene candidates, we used both splenocytes and peripheral blood lymphocytes to validate microarray findings by qRT-PCR. We then subjected data from top candidates to rigorous statistical analyses. Resulting correlate candidates, including CXCL9, IFN-γ, and CCL5, significantly predicted protection with high specificity. These findings therefore refine and extend a panel of relevant immune correlates to advance vaccine development.
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| 7. |
- Rabe, Hardis, et al.
(författare)
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In vitro stimulation with nontuberculous mycobacteria induced a stronger cytokine response in leukocytes isolated from individuals with latent tuberculosis compared to those isolated from active tuberculosis or cystic fibrosis patients
- 2024
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Ingår i: Tuberculosis. - : Churchill Livingstone. - 1472-9792 .- 1873-281X. ; 147
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium tuberculosis and opportunistic environmental non-tuberculous mycobacteria (NTM) can cause severe infection. Why latent tuberculosis infection advances to active disease, and why some individuals with cystic fibrosis (CF) develop pulmonary infections with NTM is still poorly understood. The aim of this study was to investigate the effector function of peripheral blood mononuclear cells (PBMC) from individuals with active or latent tuberculosis, individuals with CF with or without pulmonary NTM-infection and healthy controls, by measuring cytokine response to in vitro stimulation with different species of NTMs. The cytokine concentrations of IL-17A, IL-22, IL-23, IL-10, IL12p70 and IFN-γ were measured in PBMC-culture supernatants after stimulation with NTMs. PBMCs from individuals with latent tuberculosis infection showed strong IL-17A, IL-22, and IFN-γ responses compared to individuals with active tuberculosis or CF. IL-10 production was low in both tuberculosis groups compared to the CF groups and controls. This study suggests that IL-17A and IL-22 might be important to keep tuberculosis in a latent phase and that individuals with CF with an ongoing NTM infection seem to have a low cytokine response. © 2024 The Authors
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| 8. |
- Tesfaye, Fregenet, et al.
(författare)
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Alternative biomarkers for classification of latent tuberculosis infection status in pregnant women with borderline Quantiferon plus results
- 2020
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Ingår i: Tuberculosis. - : Elsevier BV. - 1472-9792. ; 124
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Tidskriftsartikel (refereegranskat)abstract
- Borderline interferon-gamma (IFN-γ) results (near the cut-off level 0.35 IU/ml) occur in QuantiFERON (QFT) assays. We investigated the performance of alternative biomarkers for classification of latent tuberculosis infection (LTBI) status in pregnant women with borderline QFT IFN-γ responses. Pregnant women (n = 96) were identified from a cohort study in Ethiopia, based on QFT-Plus IFN-γ results (QFT-low: <0.20 IU/ml, n = 33; QFT-borderline: 0.20–0.70 IU/ml, n = 31; QFT-high: >0.70 IU/ml, n = 32), including 12 HIV-positive individuals in each group and with 20 HIV-negative non-pregnant women from the same cohort with QFT IFN-γ <0.20 IU/ml as controls. Concentrations of 8 markers (IL-1ra, IL-6, IL-8, IP-10, MCP-1, MCP-2, osteopontin and resistin) were measured in whole blood QFT supernatants, stimulated separately with TB1 and TB2 antigens. K-nearest neighbor analysis (KNN) was used to classify participants with regard to likelihood of LTBI. Concentrations of MCP-2, IP-10 and IL-1ra were higher in QFT-borderline compared to QFT-low participants in both antigen stimulations (p < 0.001). KNN classification indicated high likelihood of LTBI in 13/31 (42%) women with QFT-borderline IFN-γ results. MCP-2, IP-10 and IL-1ra expressed in whole blood after TB antigen stimulation may be considered as alternative biomarkers for classification of LTBI status in pregnant women with borderline QFT IFN-γ results.
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