Electroencefalografische karakterisatie van comorbiditeiten in neurodegeneratieve ziekten: van epilepsie tot insomnia - Jan Van Erum (18/12/2019)
Jan Van Erum
- 18 december 2019
- Promotoren: Peter Paul De Deyn en Debby Van Dam
Neurodegenerative diseases are often associated with comorbidities that worsen the disease phenotype. As our society might benefit from more effective therapeutic strategies for these comorbidities, we sought to provide a better characterization of the underlying mechanisms that play a role in their development. In this doctoral thesis, we focused on two comorbidities in particular, namely epilepsy and sleep disturbances.
Epilepsy is more prevalent in certain neurodegenerative diseases. Patients are more prone to the development of seizures and epilepsy than aged control individuals. As many of these neurodegenerative disorders are characterized by the presence of pathologic forms of tau and neurofibrillary tangles, studying the effects of tau pathology on the development of epilepsy might prove rewarding. To gain more insight into the relation between tau pathology and epileptogenesis, the Tau58/4 tauopathy mouse model was subjected to a seizure susceptibility analysis. We reported increased seizure susceptibility in young Tau58/4 mice, which are still in an early pathologic stage, but not in old Tau58/4 mice with full-blown tau pathology. Therefore, we hypothesized a crucial role for pre-fibrillar hyperphosphorylated tau in increasing the sensitivity towards PTZ-induced seizures.
Aside from epilepsy, many patients also suffer from other comorbidities, including sleep and circadian rhythm disturbances. In contrast to epilepsy, sleep abnormalities are among the first noticeable symptoms of the disease. To gain a better understanding of how certain dementia-specific pathologic proteins interact with sleep-regulating brain centers and influence cognition, two different transgenic mouse models were studied. We found that, at advanced stages of pathology, both models display severe sleep deficits. In APP23 mice, the sleep and activity profile hints towards a hyperactive condition during the dark phase. In Tau58/4 mice sleep time was reduced during both photoperiods. Most remarkably, disturbances in rapid eye movement sleep were already present from a young age. Based on the pathologic spread in the APP23 model and the spectral data of the EEG, we concluded that Aβ probably has toxic effects at the level of the cortex, resulting in disrupted patterns of cortical activation and sleep loss. By contrast, tau pathology might be more detrimental for the central regulation of sleep, regulated by sleep centers in the brainstem, because sleep disturbances are photoperiod-independent and arise around the time pathologic tau starts to accumulate in pontine regions. To conclude, Aβ and tau obviously have differential effects on sleep in these two distinct models.
De regeling en ontregeling van kleine heat shock eiwitten en een geassocieerd co-chaperone in gezondheid en ziekte - Elias Adriaenssens (13/12/2019)
- 13 december 2019
- Promotoren: Vincent Timmerman en Stuart Maudsley
Small heat shock proteins are molecular chaperones which prevent misfolded proteins from aggregating. Many aspects regarding the molecular mechanisms of small heat shock proteins, both in health and disease, remain to be unraveled.
In this thesis we found that mammalian small heat shock proteins are targeted to the mitochondrial intermembrane space. While small heat shock proteins of plants are targeted to virtually every membrane-enclosed compartment, this is the first example of mammalian small heat shock proteins to have an organelle-localization. This previously unknown mitochondrial function of HSPB1 is disturbed by CMT disease-causing mutations. One of the most severe HSPB1 mutations is located in a conserved IxI/V motif in the C-terminal domain. This mutation is known to cause the formation of extremely large oligomers, which we found to prevent HSPB1 from being imported into mitochondria.
The underlying structural changes, associated to the formation of these large mutant oligomers, were so far poorly characterized. In this thesis we identified an unexpected mechanism underlying the structural changes. The substitution of Pro182 with Leu allows the IxI/V peptide to sample a larger conformational space and the peptide therefore finds itself less frequently in the required conformation to bind the hydrophobic cleft. As a consequence, other IxI/V containing proteins can bind more to HSPB1, explaining the altered protein-protein interactions.
Finally, the co-chaperone BAG3 forms a protein scaffold which binds both small heat proteins and Hsp70. As such, it brings both arms of protein surveillance together. In this thesis we studied three different mutations in the IPV-motif of BAG3, which is where small heat shock proteins bind BAG3, and found that they induce aggresome formation. As a result of that, mutant BAG3 traps the small heat shock proteins and Hsp70 at the aggresome which leads to a general collapse in the chaperone-network.
Evaluatie van intermitterende preventieve behandeling tijdens de zwangerschap (IPTp) in het district Chókwè, Zuid-Mozambique: dekking en effect op de zwangerschap en parasitologische uitkomsten - Paulo Arnaldo (10/12/2019)
- 10 december 2019
- Promotoren: Luc Kestens en Anna Rosanas-Urgell
Malaria in pregnancy (MiP) is an important public health problem in Sub-Saharan Africa. It is known to be the most common and preventable cause of harmful outcomes to both mothers and developing foetuses in malaria-endemic areas. In stable transmission areas, MiP typically does not cause clinical symptoms and is usually not detected. This thesis aimed to evaluate the community coverage of IPTp-SP and factors potentially related to low IPTp-SP among women with non-institutional and institutional deliveries; investigate the factors associated with malaria infection and adverse pregnancy outcomes among pregnant women at delivery; explore the perceptions, views, experiences and behaviors of pregnant women and health workers on accessing IPTp-SP for malaria prevention in pregnancy and assess the frequency of dhfr/dhps mutations in P. falciparum isolates collected from pregnant women, analyze the association between mutant haplotypes with parasitological and pregnancy outcomes and, investigate the effect of IPTp-SP on the carriage of asexual and sexual stages.
We found that less than a half of the women reported taking the recommended ≥ 3 doses of SP during their last pregnancy. This coverage remained well below the national’s target of 80% of pregnant women receiving ≥ 3 doses of IPTp-SP. This low IPTp-SP coverage was associated with non-institutional births, late first ANC visit, lower awareness about IPTp and the level of education attained by pregnant women.
Pregnant women were not aware of the risks of MiP or the benefits of its prevention. Delays in accessing antenatal care, irregular attendance of visits, and insufficient time for proper antenatal care counselling by health workers may explain inadequate IPTp delivery.
We detected a prevalence of 16.8% of maternal malaria infections at delivery, 7.7%, 20.8% and 1.1% of low birth weight, preterm delivery and stillbirths, respectively. Maternal malaria infections were mainly asymptomatic and more likely to be acquired by young mothers living in rural areas. The prevalence of malaria infections did not differ significantly between women receiving < 3 and ≥ 3 SP doses. Low birth weight prevalence did not significantly differ between women receiving < 3 and those receiving ≥ 3 SP doses. However, babies born to women in their first pregnancy were at higher risk of being underweight at birth than those born from mothers with multiple pregnancies.
We report the persistence of quintuple mutated parasites in the study area, while we provided the first evidence of the occurrence of super-resistant P. falciparum parasites (carrying the sextuple mutant parasites), although not associated with adverse pregnancy outcomes. The study further shows an important burden of submicroscopic gametocyte carriers infected with mutant parasites, indicating that pregnant women could constitute a non-negligible human reservoir of malaria transmission. Overall, these findings indicate that, in addition of a suboptimal IPTp-SP coverage in the study area, the emergence of highly-resistant parasites is a threat to IPTp-SP effectiveness for malaria prevention during pregnancy. Therefore, although efforts to improve the coverage of IPTp are important, alternatives to SP for IPTp, such as dihydroartemisinin-piperaquine should continue to be tested (specifically in the study area) and new interventions against MiP (e.g. community delivery of IPTp or screening and treatment of pregnant women) should be explored in Mozambique.
Zoektocht naar nieuwe genen betrokken bij frontotemporale dementie: uitdagingen bij gen identificatie in het tijdperk van nieuwe-generatie sequentiëring - Yalda Baradaran-Heravi (22/11/2019)
- 22 november 2019
- Promotoren: Julie van der Zee en Christine Van Broeckhoven
Frontotemporal dementia (FTD) is a leading cause of early-onset dementia (EOD). Like for other dementias, no effective treatments exist. The clinical and biological complexity of dementia has long been underestimated. To improve diagnosis and therapy development, we need a better understanding of the underlying disease causes, to which we aimed to contribute by studying the genetic etiology of FTD. Major insights into the pathological mechanisms underlying FTD have come from genetic studies, and have allowed identification of key genes and proteins in FTD. Nevertheless, half of the patients with a positive family history remain unexplained, suggesting underlying causes in other not yet identified genes. Despite availability of massive sequencing technologies, gene hunting remains challenging, particularly because of the difficulties in rare-variant interpretation (the so-called variants of uncertain significance, VUS).
It is in this context that our research is situated, where we aimed to contribute to the further unravelling of the genetic etiology and molecular signatures of FTD, by family-based gene-hunting, validation of candidate genes in extended patient cohorts, and elucidation of the driving molecular mechanisms. One of these molecular mechanisms is the role of aberrant RNA-binding proteins (RBPs) in perturbing stress granule (SG) dynamics and seeding of toxic protein aggregation. Mutations in a growing number of RBP coding genes have been shown to interfere with SG formation, and cause or influence disease. We propose that aggregation-prone RBPs should be considered candidate genes, and offer opportunities for gene prioritization when mining sequencing data of unresolved FTD and related amyotrophic lateral sclerosis (ALS) patients. In line with this, we provide first genetic evidence for a causal implication of a novel RBP gene, RBM45, in the FTD-ALS spectrum. In a second family-based exome gene-hunting effort, we present two possible candidate genes that require further follow-up to demonstrate causality. Sharing these genetic findings, even though preliminary or affecting just a handful of patients, is of importance to move the field forward, as they may trigger replication studies and present converging evidence with other related research.
Epigenetische profilering van ongunstige levensstijlfactoren en nutraceutische interventies bij gezondheid en ziekte - Ken Declerck (13/11/2019)
- 13 november 2019
- Promotor: Wim Vanden Berghe
We characterized DNA methylation profiles in blood and saliva samples associated with early adverse conditions, exemplified by children prenatally exposed to pesticides or orphan children grown up in institutions, suffering from chronic stress. In addition, methylation changes were localized in functionally relevant genes which may underly the different health outcomes.
In the second part of the thesis, an atherosclerosis-associated methylation profile in blood samples was determined. Differentially methylated regions were identified in BRCA1 and CRISP2 genes in both, blood samples as well as aortic atherosclerotic plaque tissues. These DMRs were further associated with subclinical atherosclerosis measures, supporting their usefulness as early diagnostic markers. In addition, a similar DNA methylation profile could be observed in blood and brain tissues of Alzheimer’s disease patients. Interestingly, the latter signature is driven by changes in blood immune cell type composition and immune cell infiltration.
In the final part of the thesis, we demonstrated widespread epigenetic effects of cardio-protective flavanols in vitro. Interestingly, despite substantial inter-individual epigenetic variation in response to a flavanol diet intervention, individual changes may affect common cell adhesion pathways. Furthermore, we identified reciprocal atherosclerosis and flavanol diet specific epigenetic changes, which involve systemic immunological changes in blood cell types.
In the final chapter of the thesis, an immunomodulatory echinacea extract was found to activate the interferon kinase signaling pathway involving changes in endogenous retroviral gene expression and DNA methylation profiles.
In conclusion, this work demonstrates the applicability of DNA methylation signatures of blood and saliva as health or disease biomarker, but also points to limitations and challenges which must be overcome in future studies to fully exploit the potential of epigenetics in personalized health applications.
Tuberculose in Mali: uitdagingen voor de diagnose, fylogenetica en behandeling - Bassirou Diarra (08/10/2019)
- 8 oktober 2019
- Promotoren: Leen Rigouts en Bouke de Jong
Drug-resistance is threatening the global control of tuberculosis (TB), the leading infectious cause of death worldwide, with incomplete understanding of predictors of poor treatment outcome. First-line TB drugs are more powerful and less toxic compared to most second-line drugs, further stressing the importance of early identification of rifampicin-resistant (RR) TB and preventing the transmission of de novo resistance. If feasible, WHO recommends “universal drug-susceptibility testing”, i.e. the search and identification of RR among new TB patients.
As universal drug-susceptibility testing is not yet implemented in Mali, we conducted a cohort study in Bamako to detect RR-TB early on during first-line treatment, and monitor RR-TB treatment. As conventional microscopy by either Ziehl-Neelsen or auramine is less sensitive, and does not distinguish between live and dead bacilli, mycobacterial culture is required for treatment monitoring. As an alternative to culture, which is not widely available, we applied fluorescein di-acetate (FDA) vital stain for detection of live mycobacteria in monitoring treatment success. Moreover, we estimated the population structure of mycobacterial strains circulating in Bamako, and determined clinical characteristics and outcome by lineage.
Our data shows that TB patients in Bamako present with a high bacillary burden, thus with advanced disease. Increased sensitization of the population about TB symptoms and training of clinicians may contribute to earlier diagnosis and treatment initiation. Among new TB patients, the level of primary RR was moderately high at 2.6% (95% CI: 1.7-3.7). All RR were caused by mutations within the RR determining region of the rpoB gene, suggesting that the rapid Xpert MTB/RIF assay serves the diagnostic algorithm well.
A high proportion (64%) of auramine-based treatment failures were FDA negative, reflecting continued expectoration of death bacilli. However, FDA was insufficiently able to identify early RR-TB or predict culture-confirmed treatment failure.
Regarding drug-resistant TB, we identified multiple challenges, including evidence of nosocomial transmission, low coverage of monthly monitoring and attrition. Neither HIV nor diabetes mellitus were identified as risk factors for acquiring drug-resistance.
As for circulating mycobacterial strains, we found that all first-six lineages of human importance were represented in Bamako, in addition to 0.8% M. bovis. The most widely represented lineages were Lineage 4 (Euro-American) (57%), and Lineage 6 (M. africanum) (22.9%). Lineage 6-diseased patients - compared to Lineage 4 - showed slower disease progression, higher smear-microscopy grades, and slower smear conversion by auramine and FDA. Hence, more efficacious treatment regimens are needed for Lineage 6 patients.
Hoe verscheidenheid in Mycobacterium tuberculosis complex stammen de diagnose, het ziektebeeld en uitkomst van behandeling kan beïnvloeden - Chakirath N'Dira Sanoussi (07/10/2019)
Chakirath N'Dira Sanoussi
- 7 oktober 2019
- Promotoren: Leen Rigouts en Bouke de Jong
Tuberculosis (TB) is caused by bacteria of the Mycobacterium tuberculosis complex (MTBC) which comprises 7 human-adapted phylogenetic lineages, including the two M. africanum lineages (West-African 1 (L5) and 2 (L6)) that are geographically restricted to West and Central-Africa, and cause up to 40% of TB in West-Africa. This thesis aimed to increase the understanding of L5 epidemiology, diagnosis and genomic characteristics, and included a nationwide prospective cohort study in Benin, the country with the highest prevalence of L5 worldwide.
We identified numerous novel epidemiological associations that shed new light on this distinct MTBC member and suggested technical advances for improved unbiased TB diagnosis and molecular epidemiological studies. We found that L5 is under-represented in positive cultures and identified a decreased performance of the rapid MPT64-antigen-based assay for the identification of L5 as a MTBC member among positive cultures, likely due to an L5-wide non-synonymous SNP (I43N) in the mpt64 gene. This risks misclassifying of positive L5 cultures as non-tuberculous mycobacteria. To overcome the culture bias observed, direct spoligotyping was used for lineage determination. The countrywide distribution of lineages differed significantly by patient’s treatment history, with M. africanum present in 39.2% of new and 26.3% of previously-treated patients (31.1% and 21% respectively for L5 alone). In Cotonou, over 10 years, the L5 prevalence among new patients significantly declined by 9.4 % (1.2-17.6) as did L1, while the L4 prevalence increased by 16% (7.4-24.6), and the L6 prevalence remained similar.
To facilitate such multicentric studies/surveys, cetylpyridinium chloride, OMNIgene.SPUTUM and ethanol were compared for up to 28-day storage of sputum in ambient temperature, with an algorithm proposed to help the user in the choice.
Using comparative genomics, we identified differences in gene content not only between L5 and the currently used M. tuberculosis reference genome H37Rv (L4), but also among L5 strains (complete genomes from Benin, Gambia, Nigeria). Some genes were absent in L5, and based on their function, can partly explain the reduced growth in culture and other suggested characteristics of M. africanum.
In conclusion, direct genotyping should be used for MTBC population structure studies, and the algorithm for TB diagnostics testing in M. africanum- endemic countries could be improved, favoring direct diagnostics for unbiased results. The high within-L5 gene content variability suggests the pangenome of MTBC may be larger than previously thought, implying a reference-free de novo genome assembly approach may be preferable over the currently used H37Rv-based genome analyses.
De bijdrage van zeldzame genetische varianten tot de complexe etiologie van neurodegeneratieve hersenziekten - Federica Perrone (27/09/2019)
- 27 september 2019
- Promotoren: Christine Van Broeckhoven en Julie van der Zee
One of the major attempts of medical research is to entirely comprehend the neurodegenerative brain diseases (NBDs) etiology. These widespread disorders have an enormous impact on patients and families. Available therapeutic strategies are only able to attenuate symptoms. Neurodegeneration in the brain therefore progresses towards an irremediable loss of neurons. The patients’ condition severely worsening to a state of incapability to perform everyday life activities. The most common disease NBDs subtypes are Alzheimer’s disease (AD) and Parkinson’s disease, followed by less frequent known as frontotemporal dementia and Amyotrophic Lateral Sclerosis.
The past three decades have witnessed major advances in understanding NBDs molecular etiology. Initial genetic and association studies on affected individuals have discovered causal and risk genes underlying the biological mechanisms of neurodegeneration. Unfortunately, these mutated genes could only explain a small proportion of the affected patients. After whole exome and genome sequencing tools became available, an unprecedented rate of novel NBDs genes has been discovered. A large number of rare variants has therefore been identified of which contribution to disease remains still unclear. To date, functional research aiming to demonstrate the effect of genetic mutations on the protein function is scarce and these variants of uncertain significance (VUS) remain uncharacterized. This leaves a large fraction of the genetic NBDs etiology unexplained.
The work performed during this PhD aimed to clarify whether rare variants in NBD genes, identified through next generation sequencing, contribute to the disease etiology. To achieve this, the main NBD disease genes were screened in Belgian early-onset dementia patients with unclear clinical symptoms. A more accurate diagnosis was given to patients with a known pathogenic mutation identified. VUS were also observed, two of which were further characterized using patient biomaterials. Results from this study highlighted the NBDs genetic heterogeneity and corroborated the hypothesis of an NBDs disease continuum, further supported by the screening of two recently identified ALS genes (TUBA4A, CHCHD10) in Belgian FTD-ALS patients. Finally, the established AD disease genes (APP, PSEN1, PSEN2) were screened in Belgian AD patients. Both known pathogenic mutations and VUS were discovered. Cerebral spinal fluid was available for a subset of mutation carriers to further investigate a possible pathogenic role for amyloid-β 43 peptide in AD pathogenesis, which has recently raised attention in this field.
The work performed during this PhD has provided evidence for the importance of deciphering the possible pathogenic effect of the VUS in the NBDs.
Karakterisatie van klinische respiratoir syncytieel virus isolaten en het effect van protease inhibitoren op vroege virale infectie - Winke Van der Gucht (27/09/2019)
Winke Van der Gucht
- 27 september 2019
- Promotoren: Peter Delputte en Paul Cos
Respiratory syncytial virus (RSV) is the principal viral cause of serious lower respiratory tract infections in infants and is the primary cause of hospitalization during the first year of life. RSV disease severity in young children displays great variety, ranging from mild cases with common cold-like symptoms to severe cases in need of hospitalization, respiratory support, additional hydration and tube-feeding. The factors attributing to the observed differences in disease severity can be divided into host factors, such as genetic and environmental background, and virus-intrinsic factors.
Multiple studies have indicated that genetic variations in the host inflammatory immune response to RSV are responsible for the wide range of disease severity. In contrast, the role of factors intrinsic to the virus itself in contributing to disease has only partially been researched. No vaccine or therapeutic has been market approved to prevent or treat RSV infections and associated disease, therefore, treatment is currently limited to supportive care. Research on vaccines has been severely hampered by a legacy of vaccine enhanced respiratory disease. Blocking RSV entry by small molecules is an attractive therapeutic pathway, but fully understanding the entry mechanisms of RSV into the host cell is important for their development. Two theories currently describe the RSV fusion event, fusion at the cell surface and fusion within endosomes. Questions remain about the event that triggers the fusion of the viral envelope with the cell membrane and two main propositions have been made: spontaneous conversion of the metastable prefusion F protein conformation to the stable postfusion F protein conformation, or post-translational modifications by furin-like proteases.
Firstly, this thesis focuses on the isolation and phenotypical and genetic characterization of recent clinical isolates. RSV was isolated from nasal samples derived from sick children and characterized in vitro. These experiments showed that the clinical isolates differ from laboratory strains isolated in 1961, and from each other on several characteristics. Additionally, these clinical isolates were then continuously cultured to assess their adaptation, which was found limited after 30 passages in most cases.
Secondly, this thesis aimed to gain more insight in the role of host proteases during the RSV entry process. RSV infected cells were first treated with several protease inhibitors to examine their effects on viral entry and the replication cycle of RSV. Serine protease AEBSF was found to inhibit the RSV infection during early entry, and the process was further elucidated using electron microscopy showing that fusion was inhibited.
Predictieve biomerkers voor de functionele genezing van hiv - Pieter Pannus (19/09/2019)
- 19 september 2019
- Promotor: Guido Vanham
Although combination antiretroviral therapy (cART) is very effective at suppressing viral replication and preventing disease progression, it is not curative due to the existence of a latent viral reservoir in resting memory CD4+ T cells. As a result, HIV infected patients need to take life-long antiretroviral therapy. Besides the high economic cost, it is proving to be very challenging to get all HIV positive people on treatment, especially in low -and middle income countries. In addition, although the pill burden has been reduced to one pill per day, daily treatment continues to affect quality of life and may still have a long-term impact on the patients’ health. Therefore, a cure for HIV is needed.
It is becoming increasingly clear that achieving a ‘sterilizing cure’, where a patient becomes completely virus-free, will be very difficult. In contrast, achieving a ‘functional cure’, where a patient is still infected but suppresses the virus without the need for drugs after a period of therapy, might be more realistic. This state of “post-treatment control” has been observed in a small proportion of patients and has become an important focus of HIV cure research. The main goal of this doctoral thesis was to find immunologic and/or viral biomarkers which are predictive of functional cure with the help of three clinical trials.
In the first trial we hypothesized that virally suppressed patients on cART who have a very small viral reservoir (measured as the amount of proviruses) and very little proviral transcriptional activity (measured as the amount of cell-associated viral RNA), would be more likely to have delayed viral rebound after treatment interruption as compared to the general HIV positive population on cART. Sixteen such patients interrupted therapy but all experienced rapid viral rebound within two to eight weeks. This observation is consistent with the findings from other recent trials and affirms that it is unlikely that post-treatment control can be achieved by merely reducing the size of the reservoir, and that an immune intervention is required.
In the second trial we set up a sensitive and specific assay to measure the in vitro viral inhibitory activity (VIA) of CD8+ T cells, which was to be used in the third (therapeutic vaccination) trial. In addition, we explored associations between VIA and clinical, viral and immune parameters. Interestingly, we found positive associations of VIA with expression of immune exhaustion markers PD1 and CD160, as well as with CD57, a marker of replicative senescence, and HLA-DR, a marker of immune activation, on terminally differentiated memory CD8+ T cells. While we did not show that these cells are responsible for the VIA we measured, it indicates that a distinct activation state of fully differentiated memory cells might play an important role in suppressing viral replication.
In the third and last trial, we assessed the immunogenicity of an innovative therapeutic vaccination strategy and its capacity to induce delayed viral rebound after treatment interruption in virally suppressed patients on cART. Study participants received three intra-nodal injections of a naked mRNA vaccine. The mRNA construct coded for a set of rationally selected, conserved, immune subdominant HIV peptides (vaccine) and three dendritic cell activation stimuli (adjuvant). Unfortunately, the construct was found to contain an error which likely compromised the translation of the mRNA, thereby invalidating the findings of the trial.
In conclusion, the findings from this doctoral work contribute to the growing consensus that the induction of a functional cure will require an immune intervention (e.g. therapeutic vaccination) on top of a sufficiently reduced viral reservoir. Unfortunately, we were not able to verify the potential of our own therapeutic vaccine due to a serious error in the mRNA construct which was produced by one of our private partners.
Strategieën voor het verbeteren van differentiële diagnose en het voorzien van biomerkers voor viscerale leishmaniasis - Bruno Cesar Bremer Hinckel (28/06/2019)
Bruno Cesar Bremer Hinckel
- 28 juni 2019
- Promotoren: Jean-Claude Dujardin en Xaveer Van Ostade
Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the Leishmania donovani complex. Without treatment, VL is fatal. Although diagnostic techniques, mainly based on the detection of anti-Leishmania antibodies are available, invasive procedures such as microscopy from spleen or bone marrow aspirates are still required for the diagnosis of seronegative VL suspects, for the detection of recurrent cases and to confirm cure after successful treatment. The rK39, the most widely used antigen for VL diagnosis has been an essential point-of-care tool for the detection of active VL cases, although it cannot be used as a test of cure due to antibody persistance (IgG) even after succeessful chemotherapy. Furthermore, this test has a decreased sensitivtiy in Eastern Africa, where the direct agglutination test (DAT) is still an intergral part of the algorithm to diagnose active VL cases. Previous investigations showed the potential of IgG1 as a biomarker of post-chemotherapeutic relapse for VL in rapid diagnostic tests (RDTs) sensitised with crude lysate antigen (CLA).
We employed in silico tools to search for desired protein features in a large number of L. donovani antigens detected by human IgG1 in western blots. We then employed prediction algorithms to profile epitopes from the shortlisted proteins. We screened a panel of high-scoring peptide epitopes in a high-throughput manner using arrays, with low reagent consumption. The most reactive peptide was adapted to RDTs, showing promising results of both sensitivity and specificity. This peptide has the potential of replacing the CLAs in IgG1 RDTs.
Using cure paired serum samples from India we evaluated the potential of IgG1 against the rK39 to monitor treatment outcome. ELISAs with rK39 and IgG1-specific conjugate gave a far more discriminative decrease in post-treatment antibody response when compared to IgG. Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 response in patients who had successful treatment. Furthermore, both IgG1 rK39 RDTs and ELISAs showed a highly significant difference in test outcome between cured patients and those who relapsed. RDTs were more sensitive than corresponding ELISAs.
Considering VL diagnosis in eastern Africa, we showed that the pool of Leishmania antigens located at the surface of the parasite (membrane proteins - MPs) represent an interesting target for additional investigations. This protein extract might contain the DAT antigen while the identification of specific antigens would enable the development of an RDT with improved sensitivity for active cases detection in eastern Africa.
Nieuwe mutationele mechanismen in ABCA7 beïnvloeden de ziekte van Alzheimer: een derde generatie sequencing project - Arne De Roeck (20/06/2019)
Arne De Roeck
- 20 juni 2019
- Promotor: Kristel Sleegers
Dementia is the fifth leading cause of death in the world. To find a therapy, we need a better understanding of the underlying disease causes, to which we aimed to contribute by studying the genetics of Alzheimer’s disease (AD), the predominant form of dementia. In particular, we focused our work on the identification and characterization of complex genetic variants in the ATP Binding Cassette Subfamily A Member 7 (ABCA7) gene.
Our research group was one of the first to demonstrate a strong AD risk increasing effect of rare ABCA7 premature termination codon (PTC) variants. We first expanded on this striking finding by next-generation sequencing (NGS) analysis of coding ABCA7 variants in early-onset AD patients. We identified novel PTC variants and observed an overall fivefold PTC enrichment in patients. Because of incomplete disease penetrance and a wide onset age range, we characterized these PTC mutations on the transcript level by long-read sequencing on a MinION sequencer. We detected frequent transcript alterations which can rescue a deleterious PTC effect and are a possible target for therapeutic interventions.
NGS, however, has limitations. By analysis of DNA sequences that are disregarded by conventional NGS processing, we were able to identify a highly polymorphic intronic ABCA7 variable number tandem repeat (VNTR). Subsequent Southern blotting revealed that VNTR expansions had a striking fourfold AD risk increasing effect. In downstream analyses we demonstrated that increasing VNTR lengths correlated with reduced ABCA7 expression and increased alternative splicing of a defunct ABCA7 isoform. Furthermore, we observed reduced amyloid β levels in cerebrospinal fluid of AD patients carrying long VNTR lengths.
Subsequently we aimed to develop a novel genotyping method based on long-read sequencing to enable improved characterization of this ABCA7 VNTR and other tandem repeats. We made use of the recently released high-throughput PromethION sequencing platform, and were the first to establish human whole genome long-read sequencing using only a single flow cell per individual. Finally, we developed “NanoSatellite”, a dynamic time warping-based tandem repeat calling algorithm, which demonstrated accurate length estimation for all ABCA7 VNTR lengths, including clinically relevant expansions, and detection of tandem repeat motif interruptions.
In conclusion, we contributed to the establishment of ABCA7 as a strong risk factor of AD, which opens opportunities for novel drug targets and smarter AD clinical trials. In the process, we established novel methods, which will hopefully enable other researchers to study complex alterations in our genome and transcriptome.
Klaarheid in het benevelde brein: tau pathologie belicht door microscopische beeldvorming van het intact brein - Jan Detrez (06/06/2019)
- 6 juni 2019
- Promotoren: Winnok De Vos en Jean-Pierre Timmermans
De neuropathologie van de ziekte van Alzheimer wordt onder meer getypeerd door een stereotypisch, progressief spreidingspatroon van tau pathologie doorheen het brein. Het is echter nog niet geweten hoe deze spreiding tot stand komt en in welke mate ze samenhangt met cellulaire en functionele defecten. De klassieke manier om pathologie op celniveau te bestuderen in het brein is gebaseerd op het gebruik van dunne coupes, wat gepaard gaat met substantieel verlies van materiaal en wat analytische procedures bemoeilijkt. Met de opkomst van weefselklaring en light sheet microscopie is het mogelijk geworden integrale organen met cellulaire resolutie te visualiseren. We hebben deze aanpak getest met verschillende transgene muismodellen en hebben aangetoond dat een gerichte injectie van tau fibrillen in een Tau.P301L muismodel een connectoom-afhankelijk spreidingspatroon van tau pathologie veroorzaakt dat lijkt op het patroon dat men ziet in Alzheimerpatiënten. We hebben verder gezien dat dit geassocieerd is met een specifiek microgliaal fenotype, gekenmerkt door een uitgestrekt cellichaam en opgezwollen soma. Verder hebben we aangetoond dat een co-injectie met een antilichaam dat het aggregatiedomein van tau bindt, de tau pathologie vertraagt.
Een nadeel van whole-brain microscopie is dat het enkel op gefixeerd weefsel kan gebeuren, waardoor het geen functionele informatie kan geven. Om dit op te lossen, hebben we resting state functionele MRI uitgevoerd, wat toelaat om een functionele dimensie toe te voegen aan de volumetrische microscopische data. Verrassend genoeg, zagen we ondanks een duidelijke tau pathologie, geen significante effecten op de resting state netwerken, wat suggereert dat de accumulatie van tau pathologie an sich niet noodzakelijk een invloed heeft op functionele connectiviteit.
Samengevat hebben we een whole-brain microscopische aanpak op punt gezet en inzicht verworven in de progressieve ophoping van tau pathologie. De combinatie met rsfMRI heeft het bovendien mogelijk gemaakt om morfologische met functionele data te koppelen, wat een innovatief platform biedt voor preklinische studies, ook buiten het AD veld.
Ontrafelen van de nucleaire functie van mutant tyrosyl-tRNA synthetase geassocieerd met de ziekte van Charcot-Marie-Tooth - Sven Bervoets (03/06/2019)
- 3 juni 2019
- Promotoren: Albena Jordanova en Marie-Luise Petrovic-Erfurth
Aminoacyl-tRNA synthetasen (aaRS) katalyseren de ligatie van tRNAs met hun respectievelijke aminozuur, de eerste stap in de translatie van proteïnen, wat hen onmisbaar maakt in elk organisme. Ondanks hun expressie in elke cel, veroorzaken dominante mutaties in deze enzymen een perifere neuropathie. Deze ongeneesbare ziekte tast de langste motorische en sensorische neuronen in het menselijke lichaam aan, wat leidt tot progressief verlies van motorische capaciteiten, gevoel, en uiteindelijk beweging. Patiënten met dominante aaRS mutaties vertonen een gelijkaardig klinisch ziektebeeld, wat suggereert dat er een gedeeld ziektemechanisme is. Echter, dit pathomechanisme dient nog steeds opgehelderd te worden.
Eerst dacht men dat het verlies van de aaRS enzymatische activiteit aan de basis lag van de pathologie. Recent werk toont echter aan dat een toxische, gain-of-function bijdraagt aan de neuropathie. Buiten hun centrale rol in de translatie van proteïnen hebben aaRSs een aantal niet-canonieke functies verkregen gerelateerd met cellulaire compartimenten waar eiwittranslatie niet plaatsvindt. Alle aaRSs met een rol in een perifere neuropathie zijn gelokaliseerd in de nucleus, maar het verband tussen hun nucleaire aanwezigheid en de perifere neuropathie is nog niet onderzocht.
Deze studie focust op de nucleaire rol van tyrosyl-tRNA synthetase (TyrRS). Dit aaRS leidt tot een subtype van een perifere neuropathie, genaamd Dominante Intermediaire Charcot-Marie-Tooth type C (DI-CMTC). Conformatieveranderingen in het mutante TyrRS resulteren in een ongewone interactie met de transcriptiemachinerie, wat een toxische, gain-of-function ondersteunt. Als gevolg komt een set van specifieke genen verkeerd tot expressie. De transcriptionele veranderingen betreffen voornamelijk genen gerelateerd aan mitochondriën. Daarenboven vertonen cellulaire en Drosophila modellen van DI-CMTC fragmentatie van mitochondriën. Exclusie van het mutante proteïne uit de nucleus, zowel genetisch als farmacologisch, voorkomt de ziektefenotypes in de modellen voor TyrRS-geïnduceerde perifere neuropathie. In conclusie, onze data identificeren de nucleus als de basis van TyrRS-geïnduceerde perifere neuropathie, wat mogelijk een gedeeld pathomechanisme is voor aaRS-geïnduceerde neurodegeneratie. Ons werk effent de weg naar de ontwikkeling van nieuwe therapeutische strategieën om perifere neuropathieën te behandelen.
Regulatoire T-cellen bij HIV-infectie en bij gevaccineerden - Raquel José Matavele Chissumba (08/05/2019)
Raquel José Matavele Chissumba
- 8 mei 2019
- Promotoren: Luc Kestens en Jani Ilesh
The human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Globally, more than 36 million individuals are infected with HIV-1. During HIV-1 infection, high levels of systemic immune activation occurs which stimulates massive viral replication. Unfortunately, there is not an effective vaccine yet against HIV-1.
Evidence from previous efficacy trials of HIV-1 vaccine candidates, shows that a good HIV-1 should induce pronounced HIV-specific memory T and B cell responses, but at the same time, needs to limit responses that favor infection. A higher risk of HIV-1 acquisition was observed in participants of a previous HIV-1 efficacy trial which was associated to higher levels of immune activation and augmented expression of HIV binding molecules in vector specific CD4 T cells. Regulatory T cells (Tregs) are a heterogeneous population of CD4 T cells with the potential to suppress exacerbated immune activation. Furthermore, Tregs also participate in mechanisms of immune memory development. However, Tregs can also suppress the development of protective antigen-specific immune responses. Little is known regarding Tregs during early infection by HIV and particularly during vaccination against HIV-1. Tregs share some differentiation pathways with Th17 cells and reciprocal development between these two CD4 T cells populations has been demonstrated.
We aimed to assess the frequencies and phenotypic alterations of Tregs, in terms of expression of activation markers, suppression markers and HIV-1 binding molecules, during HIV-infection and HIV vaccination. We also assessed how these alterations correlated with indicators of HIV disease progression and vaccine-induced immune responses.
Our findings suggest that during HIV-1 infection, an increase of Tregs in relation to total CD4 T cells correlates with systemic immune activation. Certain subsets of Tregs like those expressing the transcription factor Helios may have a beneficial role in mechanisms controlling the levels of viral replication. However higher levels of Helios expressing Tregs can be associated with deficiency in production of antibodies against HIV-1. During DNA-HIVIS/MVA-CMDR-HIV/±CN54rgp140 vaccination, pre- and post-vaccination Tregs proportions, their activation status, the Th17/Tregs ratio and other host factors affecting Tregs relative abundance, may have an impact on the magnitude of HIV vaccine-induced immune responses. Furthermore, the DNA-HIVIS/MVA-CMDR-HIV/±CN54rgp140 does not induce increased susceptibility to HIV-1 infection of Tregs and total CD4 T cells.
Therapeutic and prophylactic HIV-1 vaccine trials should consider the evaluation of Tregs for a better understanding of the mechanistic impact of these experimental interventions.
Biologische markers voor de differentiële diagnose van de ziekte van Alzheimer en Lewy body dementie - Sylvie Slaets (08/05/2019)
- 8 mei 2019
- Promotoren: Sebastiaan Engelborghs en Peter Paul De Deyn
De incorporatie van geneesmiddelenresistentiestudies in de zoektocht naar nieuwe anti-leishmaniale geneesmiddelen - Aya Hefnawy (11/04/2019)
- 11 april 2019
- Promotoren: Jean Claude Dujardin en Dirk Valkenborg
Visceral leishmaniasis (VL) is a neglected tropical disease that is lethal if left untreated. Drug resistance (DR) is a major problem for VL in the Indian sub continent (ISC). New chemotherapeutic agents are needed and there is no implementation of DR studies into the industrial R&D pipeline. In this study we reviewed the utility of DR studies for two compounds, antimonials (SSG) and miltefosine (MIL), both of which are no longer used as first line treatment options for VL in the ISC. We investigated how implementation of DR studies could have extended their use. Based on that we recommended the implementation of DR studies in an early stage of the R&D process through two approaches.
(A) Testing the efficacy of the newly discovered compounds on clinically isolated resistant strains.
(B) Utilizing DR studies to understand mechanisms of resistance to new compounds and possibly identify their mode of action. These two recommendations were tested in this PhD thesis. In collaboration with GSK, newly identified compounds from the Leishbox were tested on recently isolated ISC strains including an SSG-Resistant one. Only 45 percent of the compounds showed panactivity. This finding shows the importance of validation of the results of High-Throughput Screens (HTS) with a panel of recent clinical isolates of different geographical origin and isolates with demonstrated resistance to currently used compounds. For approach B one of the panactive compounds of the Leishbox with a promising profile was chosen (further-called compound X).
Resistance to X was selected in a stepwise manner in promastigotes. The time to develop resistance to compound X was found to be longer than developing resistance to MIL or SSG. X-Resistance was stable in the absence of drug pressure and amastigotes were also found to be resistant to X. Genomic and metabolomic characterization was performed to understand molecular adaptations of X-resistant parasites. In contrast to vast metabolic variations between X-resistant and X-sensitive parasites, only few genomic changes have been identified including a SNP in the gene encoding dynamin-1 like protein. Both these genomic and metabolic adaptations could give cues to the mechanism of resistance to X.
This work highlights the importance of academic and industrial collaboration. DR in Leishmania is an inevitable threat to any new drug candidate so implementing DR studies is the next logical step in the industrial drug discovery pipeline.
Het bestuderen van de functies van Leishmania in vitro gedurende de biologische cyclus: de zoektocht naar een slaaptoestand van amastigoten - Marlene Jara Portocarrero (27/03/2019)
Marlene Jara Portocarrero
- 27 maart 2019
- Promotoren: Jean Claude Dujardin en Paul Cos
Leishmaniasis encompasses mild to fatal diseases caused by the infection with protists of the genera Leishmania. Parasitological evidence suggests that Leishmania amastigotes can remain in tissues of infected individuals as subclinical infections. These latent infections represent a threat as the onset or recrudescence of the disease is unpredictable. Latent infection in other microorganisms are associated with a downregulated physiological stage called quiescence. Although quiescence is a known process for bacteria it has been scarcely studied among protists.
The goal of this thesis was to unravel if Leishmania amastigotes can be quiescent. Our first hypothesis was that Leishmania infection can produce long term persistent but subclinical infections after the chemotherapy and clinical cure of mucosal leishmaniasis. Accordingly, we found that 41.2 % of the patients had subclinical persistence of a low number of parasites on their healed mucosa 6 to 18 months after treatment. The results highlight that Leishmania overcome the host immune system and chemotherapy to remain in their host. These subclinical infections resemble the well-known latent infections of Mycobacterium tuberculosis characterized by quiescent stages.
Secondly, we hypothesized that Leishmania amastigotes should share common parasitological and molecular features of others quiescent microorganism. We found that the steady infection index of amastigotes in vitro was associated with downregulation in the content of proteins, kDNA minicircle and with a dramatic drop in rDNA expression. Metabolomics showed amastigotes are characterized by a downregulation in the levels of biosynthetic molecules as amino acids and polyamines. Altogether, these results showed that amastigotes are endowed with molecular features of quiescence.
Thirdly, we hypothesized that the expression of GFP within the rDNA locus (rGFP expression) could be a biosensor to monitor the potential diversity of quiescent stages among amastigotes at single cell level. We found a positive and stage specific relation between rGFP expression and the proliferative condition of Leishmania. Moreover, rGFP expression showed that among amastigotes different quiescent stages may coexist; the population could include shallow and deep quiescent stages that may be sorted by their levels rGFP expression.
In summary this work supports the occurrence of quiescence among Leishmania amastigotes and offers the use of rGFP expression as a tool for further molecular and metabolic characterization of quiescent subpopulations. This may contribute to the understanding of the mechanisms that drive the maintenance of this clinically and epidemiologically relevant stage.