Autophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis... Show moreAutophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis investigated the function of an regulator of antibacterial autophagy, Damage Regulated Autophagy Modulator 1 (DRAM1) against infection and shows that DRAM1 restricts bacterial growth not only through canonical antibacterial autophagy (xenophagy) but also promotes an autophagy-related pathway, named LC3-associated phagocytosis (LAP). The function of DRAM1 in restricting bacterial proliferation is independent from the recognition of bacteria by xenophagy receptors. Mechanistically, DRAM1 promotes the infection-induced activation of autophagy and LAP as well as the maturation of bacteria-containing vesicles in both pathways. This maturation process, stimulated by DRAM1, involves multiple vesicle fusion steps directing bacteria to lysosomes. Through this maturation process, DRAM1 delivers the cytosolic protein Fau to bacteria-containing vesicles, where it serves as a precursor for antimicrobial peptides. The underlying mechanism may be explained by the discovery of an interaction between DRAM1 and the SNARE protein VTI1B. Overall, the work in this thesis contributes to ongoing research into the potential application of autophagy modulation as a host-directed therapy against infectious diseases. Show less
In this thesis we describe our work regarding the identification of novel biomarkers, host targets and candidate pharmacological compounds for the development of therapies against various... Show moreIn this thesis we describe our work regarding the identification of novel biomarkers, host targets and candidate pharmacological compounds for the development of therapies against various intracellular bacterial infections, focusing primarily on the interplaybetween tuberculosis and diabetes mellitus. We conducted two longitudinal cohort studies in South Africa and Indonesia, and we applied unbiased and selective transcriptomic approaches to identify novel biomarker profiles in tuberculosis patients with concomitant diabetes or hyperglycaemia. Further, we performed experiments with standardized in vitro cellular infection models for drug discovery. We identified potential host targets during Mycobacterium tuberculosis infections and we describe the role of central metabolic pathways bacterial infections in human macrophages. Show less
Every day Tuberculosis (TB) kills approximately three thousand people, a number that is on the rise due to the impact of the current COVID-19 pandemic on essential TB services. The causative agent... Show moreEvery day Tuberculosis (TB) kills approximately three thousand people, a number that is on the rise due to the impact of the current COVID-19 pandemic on essential TB services. The causative agent of TB, Mycobacterium tuberculosis (Mtb), is an ancient pathogen that through its evolution developed complex mechanisms to evade immune surveillance and acquired the ability to establish persistent infection in its hosts. To achieve TB eradication, the discovery of Mtb antigens that effectively correlate with the human response to infection, with the curative host response following TB treatment, and with natural as well as vaccine induced protection is critical. This thesis contributes to this ambitious aim through several findings. First, it uncovers multiple new in vivo expressed Mtb (IVE-TB) antigens by combining Mtb-transcriptomic data with advanced bioinformatics tools and medium throughput cytokine screening. Second, it deepens our understanding of the cellular and humoral immunity to Mtb antigens in latently Mtb infected donors (LTBIs) and TB patients as well as in animal models. Lastly, it demonstrates the feasibility of combining and integrating pre-clinical research of multiple mycobacterial diseases, which are endemic in the same areas and against which vaccines could induce cross-disease protection (i.e., TB and leprosy). Show less
In individuals with tuberculosis-infection – until recently referred to as latent tuberculosis infection – the risk of progression to active tuberculosis (reactivation) varies strongly. Among those... Show moreIn individuals with tuberculosis-infection – until recently referred to as latent tuberculosis infection – the risk of progression to active tuberculosis (reactivation) varies strongly. Among those at increased risk of reactivation are patients with an impaired immune system, e.g. due to immunosuppressive therapy. Therefore, prior to planned immunosuppression, patients are screened for tuberculosis-infection and subsequently treated in case of infection. Current screening methods include the Mantoux test, Interferon-γ release assays (i.e., the QuantiFERON-TB Gold Plus and T-SPOT.TB) and chest X-ray. However, despite screening, cases of reactivation continue to occur – in part due to the lack of a gold standard test for tuberculosis-infection. Therefore, the aims of this thesis were to increase the diagnostic sensitivity for tuberculosis-infection prior to immunosuppression. Using various (novel) methods we showed that approximately two-thirds of all QuantiFERON-TB Gold Plus results just below the manufacturer’s cut-off (in the borderline range) are caused by Mycobacterium tuberculosis-infection, which now warrants preventive treatment in patients with such a result. Furthermore, we quantified the diagnostic accuracy of chest X-ray for tuberculosis-infection and showed that using a novel ultra-low dose CT scanning technique, sensitivity for tuberculosis-infection could be significantly increased by three-fold compared to chest X-ray. Show less
Introduction: The leptin signaling pathway plays an important role as a key regulator of glucose homeostasis, metabolism control and systemic inflammatory responses. However, the metabolic effects... Show moreIntroduction: The leptin signaling pathway plays an important role as a key regulator of glucose homeostasis, metabolism control and systemic inflammatory responses. However, the metabolic effects of leptin on infectious diseases, for example tuberculosis (TB), are still little known. Objectives: In this study, we aim to investigate the role of leptin on metabolism in the absence and presence of mycobacterial infection in zebrafish larvae and mice. Methods: Metabolites in entire zebrafish larvae and the blood of mice were studied using high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and mass spectrometry, respectively. For transcriptome studies of zebrafish larvae, deep RNA sequencing was used. Results: The results show that leptin mutation leads to a similar metabolic syndrome as caused by mycobacterial infection in the two species, characterized by the decrease of 11 amine metabolites. In both species, this metabolic syndrome was not aggravated further when the leptin mutant was infected by mycobacteria. Therefore, we conclude that leptin and mycobacterial infection are both impacting metabolism non-synergistically. In addition, we studied the transcriptomes of lepb(ibl54) mutant zebrafish larvae and wild type (WT) siblings after mycobacterial infection. These studies showed that mycobacteria induced a very distinct transcriptome signature in the lepb(ibl54) mutant zebrafish compared to WT sibling control larvae. Furthermore, lepb(ibl55) Tg (pck1:luc1) zebrafish line was constructed and confirmed this difference in transcriptional responses. Conclusions: Leptin mutation and TB lead non-synergistically to a similar metabolic syndrome. Moreover, different transcriptomic responses in the lepb(ibl54) mutant and TB can lead to the similar metabolic end states. Show less
Doorn, C.L.R. van; Eckold, C.; Ronacher, K.; Ruslami, R.; Veen, S. van; Lee, J.S.; ... ; TANDEM Consortium 2022
Background Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients.... Show moreBackground Globally, the tuberculosis (TB) treatment success rate is approximately 85%, with treatment failure, relapse and death occurring in a significant proportion of pulmonary TB patients. Treatment success is lower among people with diabetes mellitus (DM). Predicting treatment outcome early after diagnosis, especially in TB-DM patients, would allow early treatment adaptation for individuals and may improve global TB control.Methods Samples were collected in a longitudinal cohort study of adult TB patients from South Africa (n = 94) and Indonesia (n = 81), who had concomitant DM (n = 59), intermediate hyperglycaemia (n = 79) or normal glycaemia/no DM (n = 37). Treatment outcome was monitored, and patients were categorized as having a good (cured) or poor (failed, recurrence, died) outcome during treatment and 12 months follow-up. Whole blood transcriptional profiles before, during and at the end of TB treatment were characterized using unbiased RNA-Seq and targeted gene dcRT-MLPA.Findings We report differences in whole blood transcriptome profiles, which were observed before initiation of treatment and throughout treatment, between patients with a good versus poor TB treatment outcome. An eight-gene and a 22-gene blood transcriptional signature distinguished patients with a good TB treatment outcome from patients with a poor TB treatment outcome at diagnosis (AUC = 0.815) or two weeks (AUC = 0.834) after initiation of TB treatment, respectively. High accuracy was obtained by cross-validating this signature in an external cohort (AUC = 0.749).Interpretation These findings suggest that transcriptional profiles can be used as a prognostic biomarker for treatment failure and success, even in patients with concomitant DM. Copyright (C) 2022 The Authors. Published by Elsevier B.V. Show less
New drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder... Show moreNew drugs for use as tuberculosis (TB) treatment are needed due to the constrains of classical antibiotics against TB and the rise of antibiotic-resistant strains, making TB a harder and harder disease to treat. This thesis is focused on using the in vivo whole animalzebrafish embryo model for TB to evaluate potential anti-TB host-directed therapeutics (HDTs) arising from in vitro screens. Although in vitro screens for HDTs using cellular models can be performed at high throughput, a limiting step is the validation in whole animal models and translation of results to clinical applications. Due to the complex infection dynamics of mycobacteria, the use of whole animal models is indispensable in research into TB and the zebrafish model has contributed key findings about host-pathogen dynamics during mycobacterial infection. One of the most promising host targets of HDTs is autophagy, which is recognized as an important host-protective pathway. Boosting autophagy levels using HDTs could be a way to overcome the pathogen’s autophagy evasion strategies and could therefore be a promising therapeutic route. For this thesis we took advantage of the possibilities of the zebrafish embryo model for TB and the zebrafish toolkit to study several autophagy-modulating HDTs as potential anti-TB drugs. Show less
The research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and... Show moreThe research described in this thesis has, using the zebrafish as a model system, shed new light on the intricate relationship between TB and DM2, in particular on the role of leptin, SHP-1 and glucocorticoids.Leptin plays an important role during TB infection and has a huge impact on insulin sensitivity in zebrafish larvae. Similarly to what has been observed in the murine model, leptin deficiency in zebrafish increased the bacterial burden and mortality during the infection, leading to hyperglycemia and the development of insulin resistance. In addition, a novel SHP-1/SHP-2 inhibitor, NSC-87877, was shown to represent a promising anti-diabetic drug that can be used for further DM2 research, as it is able to rescue the phenotype of the leptin-deficient zebrafish and to restore glucose transport to the tissues. In contrast to metformin, NSC-87877 can act at very early developmental stages and inhibits the function of SHP-1 and factors that underlay impaired glucose metabolism, whereas metformin is mostly known to improve insulin sensitivity. Additionally, treatment with the glucocorticoid beclomethasone attenuates the metabolic changes associated with the infection, and transcriptional alterations induced by beclomethasone treatment suggest that genes involved in glucose metabolism, insulin and leptin signaling all play an important role in the modulation of the metabolism.Our data show that zebrafish larvae represent an interesting model system to investigate the complex pathology of TB, and the studies described in this thesis in which this model has been used have provided novel insights into the molecular mechanisms underlying wasting syndrome and the possibilities for adjunctive glucocorticoid therapy to alleviate this metabolic state. Show less
Background: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of... Show moreBackground: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of tuberculosis (TB). We assessed combinations between antibodies and cytokines for diagnosing TB. Methods: Immuoglubulin (Ig) A and IgM antibody titres against selected mycobacterial antigens including Apa, NarL, Rv3019c, PstS1, LAM, "Kit 1" (MTP64 and Tpx)", and "Kit 2" (MPT64, Tpx and 19 kDa) were evaluated by ELISA in plasma samples obtained from individuals under clinical suspicion for TB. Combinations between the antibody titres and previously published cytokine responses in the same participants were assessed for diagnosing active TB. Results: Antibody responses were more promising when used in combination (AUC of 0.80), when all seven antibodies were combined. When anti-"Kit 1"-IgA levels were combined with five host cytokine biomarkers, the AUC increased to 97% (92-100%) with a sensitivity of 95% (95% CI, 73-100%), and specificity of 88.5% (95% CI, 68.7-97%) achieved after leave-one-out cross validation. Conclusion: When used in combination, IgA titres measured with ELISA against multiple Mycobacterium tuberculosis antigens may be useful in the diagnosis of TB. However, diagnostic accuracy may be improved if the antibodies are used in combination with cytokines. Show less
Gela, A.; Murphy, M.; Rodo, M.; Hadley, K.; Hanekom, W.A.; Boom, W.H.; ... ; Delayed BCG Study Team 2022
Background: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CDI, MRI, and butyrophilin 3AI. Such T cells, referred to as donor... Show moreBackground: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CDI, MRI, and butyrophilin 3AI. Such T cells, referred to as donor unrestricted T (DURT) cells, typically express stereotypic T cell receptors. The near-unrestricted nature of DURT cell antigen recognition is of particular interest for vaccine development, and we sought to define the roles of DURT cells, including MRI-restricted MAIT cells, CDth-restricted glucose monomycolate (GMM)-specific T cells, CDth-restricted NKT cells, and gamma delta T cells, in vaccination against Mycobacterium tuberculosis. Methods: We compared and characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in a cohort of vaccinated and unvaccinated infants, as well as before and after BCG-revaccination in adults. Findings: BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CDth-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CDId-restricted NKT cells. By contrast, primary BCG vaccination was associated with increased frequencies of gamma delta T cells as well as a novel subset of CD26(+)CDI6I(+)TRAVI-2(-) IFN-gamma-expressing CD4(+) T cells in infants. Interpretation: Our findings, that most DURT cell populations were not modulated by BCG, do not preclude a role of BCG in modulating other qualitative aspects of DURT cells. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies. Copyright (C) 2022 The Authors. Published by Elsevier B.V. Show less
In this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin... Show moreIn this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin gene on cachexia and diabetes in rodent and zebrafish animal models. 3) how tuberculosis infection and resulting metabolic reprogramming are dependent on leptin signaling in mice and zebrafish larvae. Show less
The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of... Show moreThe zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real-time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models. Show less
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm)... Show moreTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells. Show less
Objectives: IFN gamma-release assays (IGRAs) used for diagnosis of Mycobacterium (M.) tuberculosis infection have limited sensitivity. Alternative cytokines and M. tuberculosis latency-associated... Show moreObjectives: IFN gamma-release assays (IGRAs) used for diagnosis of Mycobacterium (M.) tuberculosis infection have limited sensitivity. Alternative cytokines and M. tuberculosis latency-associated antigens may improve immune-based tests.Methods: Multiplex cytokine analyses was done in culture supernatants after 6-day in vitro restimulation with M. tuberculosis IGRA and latency-associated antigens (i.e. Rv2628, Rv1733) in tuberculosis patients (n = 22) and asymptomatic contacts (AC)s (n = 20) from Ghana.Results: Four cytokines (i.e. IFN gamma, IP-10, IL-22 and IL-6) were significantly increased after IGRA-antigen specific restimulation. IFN gamma, IP-10, and IL-22 correlated positively and showed no differences between the study groups whereas IGRA-antigen induced IL-6 was significantly higher in tuberculosis patients. Using adjusted IGRA criteria, IL-6 showed the highest sensitivity for detection of tuberculosis patients (91%) and ACs (85%) as compared to IFN gamma, IP-10, and IL-22. Rv2628 and Rv1733 restimulation induced significantly higher IFN gamma, IP-10, and IL-22 concentrations in ACs. Combined antigen/cytokine analyses identified study group specific patterns and a combination of Rv2628/Rv1733 induced IFN gamma with IGRA-antigen induced IL-6 was optimal for classification of tuberculosis patients and ACs (AUC: 0.92, p<0.0001).Conclusions: We demonstrate the potency of alternative cytokines, especially IL-6, and latency-associated antigens Rv1733/Rv2628 to improve detection of M. tuberculosis infection and to classify tuberculosis patients and healthy contacts. (C) 2020 The British Infection Association. Published by Elsevier Ltd. All rights reserved. Show less
In this thesis several aspects of complement proteins are described, from circulating levels in blood to their intracellular presence and from autoimmunity to the infectious disease tuberculosis.... Show moreIn this thesis several aspects of complement proteins are described, from circulating levels in blood to their intracellular presence and from autoimmunity to the infectious disease tuberculosis. We explored the local production of complement and we describe in Chapter 2 the production of C1q by chondrocytes. Additionally, studies addressing the potential intracellular C3 role are described in Chapter 3. The potential role of the complement system as biomarker was investigated by addressing the presence and concentrations of C1q in serum of patients with active tuberculosis and controls in Chapter 4. Like C1q, we also investigated the expression and concentration of the natural inhibitor C1-INH in Chapter 5. C1q protein was further analysed as biomarker for tuberculosis in experimental non-human primate models in Chapter 6. In this thesis, a newly identified case of a lupus patient is described with a complex medical history and a compound heterozygous deficiency of C1q in Chapter 7. To better comprehend a possible role of a prominent post-translational modification associated rheumatic disease, carbamylation, the interaction between carbamylated IgG was investigated in relation to the ability to activate the complement system. These studies are described in Chapter 8. Show less
Diabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of... Show moreDiabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of DM is rising, especially in TB endemic countries, it is important to identify the relevant immunological and metabolic processes that underlie TB-DM comorbidity, because such insights will facilitate optimal treatment, diagnosis and prevention. In this thesis, we have started to unravel key factors underlying the association between TBand DM using two approaches. Firstly, we identified and analyzed human macrophage subsets and studied the interactions between these human cells and a major pathogen, Mycobacterium tuberculosis (Mtb), and the specific metabolic changes involved using well-controlled in vitro systems. Next, we employed metabolomics to determine the impact of concurrent TB-DM on circulating metabolites in patient cohorts ex vivo. In this thesis we present evidence derived from in vitro experiments and from ex vivo observational data which collectively suggest a pathogenic role of atherogenic lipid species during TB development. Show less
Background: Medical schools offer students the opportunity to perform international electives. This study aimed to assess health risks among medical students, to tailor institutional guidelines... Show moreBackground: Medical schools offer students the opportunity to perform international electives. This study aimed to assess health risks among medical students, to tailor institutional guidelines.Methods: Multicenter study at Dutch and Belgian universities, among medical students who visited low- or middle-income countries. Students completed four questionnaires: once before the elective and two weeks, three- and six months after return.Results: Data was complete for 479 students (follow-up rate 84%). Most traveled to Surinam (29%) and South-Africa (14%). Half of the students encountered difficulties in adapting to local culture. Almost 40% visited malaria endemic countries. Nearly all (87%) used chemoprophylaxis as prescribed. Definite needle-stick or splash injuries were reported by 7%. All were dealt with adequately in accordance with national guidelines. However, less than half of 24 possible incidents were handled adequately. Two-and-a-half percent had unprotected sex with a new partner. The incidence of travelers' diarrhea (TD) was 46%. In those with TD, the incidence of post-travel new-onset abdominal complaints was 3%. Three percent were involved in a minor traffic accident, 18% were injured during leisure activities, 5% were threatened or experienced physical violence. Only half of the students visiting a highly endemic country were screened for tuberculosis post-travel. For schistosomiasis this was 6%.Conclusions: Students abroad are exposed to medical and non-medical challenges, which should be addressed during pre-travel counseling. Contact details of a professional back home should be provided, so students can confer in case of problems while abroad. Lastly, we recommend a centrally organized post-travel health check. Show less
Background: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify... Show moreBackground: To facilitate better discrimination between patients with active tuberculosis (TB) and latent TB infection (LTBI), whole blood transcriptomic studies have been performed to identify novel candidate host biomarkers. SERPING1, which encodes C1-inhibitor (C1-INH), the natural inhibitor of the C1-complex has emerged as candidate biomarker. Here we collated and analysed SERPING1 expression data and subsequently determined C1-INH protein levels in four cohorts of patients with TB.Methods: SERPING1 expression data were extracted from online deposited datasets. C1-INH protein levels were determined by ELISA in sera from individuals with active TB, LTBI as well as other disease controls in geographically diverse cohorts.Findings: SERPING1 expression was increased in patients with active TB compared to healthy controls (8/11 cohorts), LTBI (13/14 cohorts) and patients with other (non-TB) lung-diseases (7/7 cohorts). Serum levels of C1-INH were significantly increased in The Gambia and Italy in patients with active TB relative to the endemic controls but not in South Africa or Korea. In the largest cohort (n = 50), with samples collected longitudinally, normalization of C1-INH levels following successful TB treatment was observed. This cohort, also showed the most abundant increase in C1-INH, and a positive correlation between C1q and C1-INH levels. Combined presence of increased levels of both C1q and C1-INH had high specificity for active TB (96 %) but only very modest sensitivity 38 % compared to the endemic controls.Interpretation: SERPING1 transcript expression is increased in TB patients, while serum protein levels of C1-INH were increased in half of the cohorts analysed. Show less
The zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the... Show moreThe zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the underlying pharmacological and (patho)physiological processes. In this thesis, we therefore developed and integrated innovative experimental and computational methods for the successful quantification of 1) the internal exposure over time after waterborne drug treatment, 2) disease dynamics and drug-induced changes therein, and 3) between-species differences in disease mechanisms. The state-of-the-art methods that we developed included nanoscale blood sampling, sensitive LC-MS/MS methods for drugs and their isomers and metabolites, and three-dimensional microscopy, integrated with non-linear mixed effects modelling to quantify the pharmacological processes in this small vertebrate. This multidisciplinarity enabled quantification of internal drug exposure-response relationships, contributed to positioning the zebrafish in the preclinical drug development pipeline, and inspired continuous collaborations between experimental and computational scientists. Show less
Tanner, R.; Smith, S.G.; Meijgaarden, K.E. van; Giannoni, F.; Wilkie, M.; Gabriele, L.; ... ; McShane, H. 2019
A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of... Show moreA major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-gamma) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be < 5% coefficient of variation (CV), intermediate precision to be < 20% CV, and inter-site reproducibility to be < 30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development. Show less
