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Characterizing the roles of distinct ventral midbrain progenitor cell types in dopaminergic neuron development
No full textParkinson's disease is a neurodegenerative disorder characterized by the progressive loss of midbrain dopaminergic (mesDA) neurons. With the underlying cause of the disease still unknown, current treatments can only address the symptoms of the disease but not the continual loss of mesDA neurons. Cell replacement therapy aims to replace these lost mesDA neurons and is considered a promising treatment option. However, the widespread clinical application of cell replacement therapy depends on a reliable and standardized supply of transplantable neurons. Advances in stem cell technology offer a renewable source of human mesDA neurons for transplantation but generating high-quality mesDA neurons in vitro requires a detailed understanding of their normal development in the human brain. This thesis consists of three studies that all aim to improve our understanding of mesDA neuron development and apply this knowledge to enhance the in vitro differentiation of mesDA neurons.In paper I, we develop a new protocol for deriving human mesDA neurons from stem cells and introduce a framework for using single-cell RNA sequencing to evaluate the quality and composition of these cell preparations.In paper II, we generate and characterize a fluorescent reporter human stem cell line designed to isolate two ventral midbrain (VM) progenitor cell types from our in vitro cultures differentiated toward a mesDA fate.In paper III, we identify key cell types involved in both human and mouse VM development and focus on two types of floor plate radial glia that contribute to mesDA neuron development in distinct ways. One radial glia was identified as neurogenic and likely giving rise to mesDA neurons, while the other radial glia resembled traditional floor plate radial glia and expressed factors known to influence mesDA neuron development. Finally, we identify a novel transcription factor expressed by the neurogenic radial glia that regulates the timing of mesDA neurogenesis, and extracellular matrix proteins expressed by the other radial glia that improve the yield of mesDA neurons differentiated from stem cells in vitro.In sum, the work presented in this thesis characterizes the individual roles of VM progenitor cell types and their contributions to the mesDA lineage. Furthermore, we demonstrate how knowledge gained from studying VM development can improve the generation of mesDA neurons from human stem cells for potential use in cell replacement therapy for Parkinson's disease.List of scientific papersI. Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation. Kaneyasu Nishimura, Shanzheng Yang, Ka Wai Lee, Emilia Sif Ásgrímsdóttir, Kasra Nikouei, Wojciech Paslawski, Sabine Gnodde, Guochang Lyu, Lijuan Hu, Carmen Saltó, Per Svenningsson, Jens Hjerling-Leffler, Sten Linnarsson, Ernest Arenas. Stem Cell Reports (2023) https://doi.org/10.1016/j.stemcr.2022.10.016II. Generation of a fluorescent hESC reporter line (Kle-033-A-1) for the isolation of distinct midbrain progenitor cell types. Emilia Sif Ásgrímsdóttir, Ernest Arenas. Stem Cell Research (2024) https://doi.org/10.1016/j.scr.2024.103523III. Midbrain dopaminergic neuron development is regulated by two molecularly distinct subtypes of radial glia cells. Emilía Sif Ásgrímsdóttir, Luca Fusar Bassini, Pia Rivetti di Val Cervo, Daniel Gyllborg, Claudia Puigsasllosas Pastor, Kawai Lee, Christopher Grigsby, Saiful Islam, Peter Lönnerberg, Carlos Villaescusa, Carmen Saltó, Sten Linnarsson, Gioele La Manno, Enrique M. Toledo, Ernest Arenas. BioRxiv (2024) https://doi.org/10.1101/2024.03.08.584031 [Manuscript]</p
Uveal melanoma, from primary tumor to macrometastasis
No full textUveal melanoma (UM) is a rare but deadly intraocular malignancy. As the most commonly occurring primary intraocular cancer in adults, it poses significant challenges in prognostication and treatment due to its high propensity for developing late-stage metastasis. Moreover, since a 100% accuracy rate is virtually impossible, improvements can still be made to current clinical tools used for prognostication, cellular composition assessment, detection of dormant micrometastatic tumor cells, and evaluation of early treatment impact. Consequently, the focus of this thesis is to improve the prognostication, highlight important cellular features of both primary tumors and micrometastases, and shine a light on the importance of early treatment of the primary tumor.In paper I, we elaborated on the diagnostic importance of uveal melanoma involvement of the ciliary body, monosomy 3, as well as the size, clinical and histological factors of the tumor. Furthermore, these factors were combined with the American Joint Committee on Cancer and the Cancer Genome Atlas models to develop a new prognostic classification system. Clinicopathological factors of two patient groups, consisting of 1796 patients, were compared between those with, and without, involvement of the ciliary body and monosomy 3. We found that involvement of the ciliary body, tumor diameter, and patient age at diagnosis to be independent predictors of monosomy 3. Moreover, tumor diameter, age at diagnosis, male sex, involvement of the ciliary body, and monosomy 3 independently predicted metastasis. Our proposed prognostic classification system, combining sex, patient age, extraocular extension, involvement of the ciliary body, monosomy 3, tumor size, and 8q (optional), outperformed both the American Joint Committee on Cancer 4 T-categories and the Cancer Genome Atlas groups A-D in validation cohorts.In paper II, we digitally measured and examined the cellular composition of different melanocytes in tumors, nevi, and metastases from enucleated eyes and resected liver tissue retrieved from the archives of Ophthalmic Pathology section at St. Erik Eye Hospital. We identified 4 different variations of melanocytes in normal choroid, primary UMs and liver metastases, which would correspond with previously described normal choroidal melanocytes, and malignant melanocytes of the spindle A, spindle B, and epithelioid type. Contrasting most previous studies performed on tumor cell morphology and staining patterns, where manual assessments were performed, we used the more objective approach of computerized analysis. Previously shown superior to manual assessment regarding reproducibility and prognostic utility. We also analyzed the expression of BRCA1 associated protein-1 (BAP-1), Indoleamine 2,3- dioxygenase (IDO), Insulin-like growth factor-1receptor (IGF-1R), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) as well as examined the area of the nucleus, the nucleus perimeter, nucleus maximum and minimum caliper, nucleus eccentricity, and nucleus to cell area ratio. Consequently, we demonstrated that spindle A cells are more commonly located in the center of primary UM, whilst spindle B cells, together with epithelioid cells, are more common in the base of UM - an area recognized as the initial path for hematogenous spread of tumor cells. No normal melanocytes or spindle A cells were identified in liver metastases, where only spindle B or epithelioid cells were seen. In addition, we demonstrated diverse expressions of BAP-1, IDO, IGF-1R, and TIGIT between the different subsets of melanocytes, highlighting the fact that 4 different cell types, with distinct levels of protein expressions, can be identified during the progression of uveal melanoma.In paper III, we examined the presence of dormant micrometastatic tumor cells in autopsy tissue from patients diagnosed with primary UM, without any clinical or radiological signs of macrometastatic disease. Data from over 4000 patients diagnosed at St. Erik Eye Hospital over a period of 60 years was examined together with data from autopsy registers. In the end, 11 subjects were identified as suitable candidates. Afterwards, tissue blocks obtained at autopsy were collected and examined with hematoxylin and eosin staining (HE) and immunohistochemistry (IHC). Thereafter, we analyzed of a subset of patients with immunomagnetic separation (IMS). Conclusively, we found micrometastases in several distant organs in 5 of 5 patients with coexisting macrometastases, as well as in 5 of 6 patients without. All micrometastases had very low, or no, proliferative activity when examined with the immunohistochemical marker M MIB1-IgG1 antibody against Ki67 (MIB1). With this finding, we suggest that micrometastases are present in practically every patient diagnosed with primary UM, and that macrometastases indeed do develop from micrometastases.In paper IV, we wanted to determine the impacts of delayed treatment on overall survival. As uveal melanoma shows a high metastatic rate, potential effects of delayed treatment regarding prognosis has been under debate. Thus, we performed a retrospective cohort study on 1256 patients diagnosed with UM and collected data on their diagnosis and treatment dates. The patients were split into two different groups based on the interval between diagnosis and treatment, namely a delay in treatment of over, and under, one month. Prognostic effects of the interval between the two patient groups were evaluated. Consequently, we found that patients with a treatment delay of at least one month has both shorter overall survival and disease-specific survival in stages II and III. In addition, there was also an increased hazard ratio for metastatic death in both univariate and competing risk regression models. When examined with a Markov multi-state model, those with a treatment delay of over one month showed a hazard ratio of 1.45 (95% Confidence Interval (CI) 1.12-1.89) for evolution to metastatic death. Interestingly, when performing competing risk incidence analyses based on treatment timing, we were not able to prove any statistically significant disparities in the incidence of any cause of death other than metastases. We conclude that treatment delays of uveal melanoma are linked with reduced overall and disease-specific survival rates, leading to the suggestion that primary treatment should be initiated with the shortest possible delay after diagnosing UM.List of scientific papersI. VT Gill, S Sabazade, C Herrspiegel, et al. A prognostic classification system for uveal melanoma based on a combination of patient age and sex, the American Joint Committee on Cancer and the Cancer Genome Atlas models. Acta Ophthalmologica. 2023;101:34-48. https://doi.org/10.1111/aos.15210II. Stålhammar G, VT Gill. Digital morphometry and cluster analysis identifies four types of melanocyte during uveal melanoma progression. Communications medicine. 2023;3(1):60. https://doi.org/10.1038/s43856-023-00291-z III. VT Gill, E Norrman, S Sabazade, A Karim, E Lardner, G Stålhammar. Multiorgan Involvement of Dormant Uveal Melanoma Micrometastases in Postmortem Tissue From Patients Without Coexisting Macrometastases. American Journal of Clinical Pathology. 2023;160:164-174. https://doi.org/10.1093/ajcp/aqad029IV. A Moghadam, VT Gill, S Sabazade, A Hagström, G Stålhammar. The Prognostic Significance of Treatment Delays on Uveal Melanoma Survival. [Manuscript]All previously published papers were reproduced with permission from the publisher under a creative commons license.</p
Spatial control of protein binding with DNA nanostructures
No full textThe physical and chemical properties of DNA, including its structure predictability thanks to Watson-Crick base pairing, make it into an obvious polymer of choice to use as a biomaterial for the fabrication of complex three dimensional nanostructures. These nanostructures are produced by the technique of DNA origami, where a long single stranded circular DNA, called scaffold, is folded into a pre-designed shape by the hybridization of partially complementary oligonucleotides, the staples. The programmability of DNA origami, i.e. the specific control, by design, of the location of every DNA sequence within the structure, can be harnessed for the positioning of molecules with nanoscale precision. In the present thesis, we have explored different branches of the DNA origami technology, from its functionalization with proteins, its structural characterization and its application for method development, targeted treatment and study of molecular processes taking place at the nanoscale.In Paper I, we develop an approach to quantify the incorporation of proteins in DNA origami using DNA-PAINT, a multiplexed super-resolution imaging method that allows the characterization of structures with single-molecule resolution and high reliability. Even as protein-decorated DNA nanostructures become increasingly important tools for biomedical sciences, the existing strategies to study and maximize protein incorporation provide limited insight into functionalization. With DNA-PAINT, we were able to explore factors influencing incorporation rate such as oligonucleotide quality, protein size, or purification method, rank their impact, and model their combined effects. Thus, we were able to offer a comprehensive view of functionalization efficiency and precise parameters for yield optimization, which can have significant implications for the application of DNA origami in fields beyond academia.In Paper II, we introduce the new method PLASTIQ for assessing DNA origami structural integrity in vivo with a detection sensitivity of 0.01 femtomolar. Despite the potential of DNA origami in therapeutics, the lack of structural assessment methods for in vivo use hampers its clinical initiation. Sampling only 1 ul of blood, PLASTIQ allowed us to monitor the degradation patterns of nanostructures over time. We examined the protective effects of PEGylation and obtained the pharmacokinetic profiles of different origamis. Additionally, we were able to observe differential degradation of structural regions depending on how exposed they were to the environment. Altogether, PLASTIQ is an accurate tool for assessing structural stability, offering valuable insights for advancing DNA origami-based drug development.In Paper III, we present PANMAP, a method based on antigen patterning on DNA nanostructures to measure antibody affinity while taking multivalency into account. Multivalency is fundamental in many biological systems, especially in antibody-antigen interactions where multiple binding sites improve affinity and specificity. However, conventional affinity assays such as ELISA provide only a limited perspective on binding characterization and do not address multivalency. With PANMAP overcoming these limitations, we found that antibody binding equilibrium is influenced by antigen spacing, leading to competitive exclusion at close distances, optimal bivalent binding at intermediate distances, and a monovalent regime at longer distances. Thus, PANMAP enabled a complete profile of multivalency and the binding states that constitute it, potentially providing useful insights into biological processes and engineering applications.In Paper IV, we apply DNA origami displaying Jag1 ligands to stimulate neuroepithelial stem-like cells to study the molecular mechanism leading to Notch receptor activation. The Notch pathway is a highly evolutionarily conserved signaling system that plays a key role in embryonic and nervous system development. However, it is unclear how the activation unravels, with the leading hypothesis being force-driven conformational changes. Here, we demonstrate that Notch triggering can occur without pulling forces and that it instead proceeds upon extended binding. These findings suggest an alternative molecular mechanism for receptor activation, suggesting potential for the design of soluble agonists.In Paper V, we engineer a pH-responsive DNA robotic switch that selectively displays death receptor ligands only in acidic tumor microenvironments to induce apoptosis of cancer cells. As these receptors are responsible for initiating cell death but are ubiquitously expressed on the membranes of most cells, a targeted approach for their use in tumor therapies is desired. The DNA robotic switch hides the ligands, arranged in a hexagonal pattern inside a cavity while at neutral pH, until encountering pH 6.5 where the ligands are revealed, leading to clustering of DR and triggering apoptosis of breast cancer cells. Our results probe the functionality of the nanodevice in vitro and shows the significant tumor volume reduction in mice with human breast cancer xenografts. Overall, this work highlights the potential for targeted cancer treatment using DNA origami.List of scientific papers1. Iris Rocamonde-Lago, Ferenc Fördös, Cagla Sahin, Ian T. Hoffecker & Björn Högberg. Exploring DNA origami protein functionalization using super resolution imaging. [Manuscript]II. Yang Wang*, Iris Rocamonde-Lago*, Janine Waldvogel, Shuya Zang, Igor Baars, Alexander Kloosterman, Boxuan Shen, Ian T. Hoffecker, Qin He & Björn Högberg. DNA origami structural integrity tracked in vivo using proximity ligation. [Manuscript]III. Iris Rocamonde-Lago, Ieva Berzina, Ian T. Hoffecker & Björn Högberg. Profiling of multivalent binding with DNA origami reveals spatial determinants of antigen-antibody interactions. [Manuscript]IV. Ioanna Smyrlaki, Ferenc Fördös, Iris Rocamonde-Lago, Yang Wang, Boxuan Shen, Antonio Lentini, Vincent C. Luca, Björn Reinius, Ana I. Teixeira & Björn Högberg. Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force. Nature Communications, 15(1), 465. https://doi.org/10.1038/s41467-023-44059-4V. Yang Wang, Igor Baars, Ieva Berzina, Iris Rocamonde-Lago, Boxuan Shen, Yunshi Yang, Marco Lolaico, Janine Waldvogel, Ioanna Smyrlaki, Keying Zhu, Robert A. Harris & Björn Högberg. A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns. Nature Nanotechnology, 19(9), 1366-1374. https://doi.org/10.1038/s41565-024-01676-4*Shared first authorship</p
Sex differences in the impact of lower respiratory tract infections on older adults’ health trajectories: a population-based cohort study
No full textAbstract
Background
Lower respiratory tract infections (LRTIs) are a major global health concern, particularly among older adults, who have an increased risk of poorer health outcomes that persist beyond the acute infectious episode. We aimed to investigate the mid-term (up to 7 years) and long-term (up to 12 years) effects of LRTIs on the objective health status trajectories of older adults, while also considering potential sex differences.
Methods
Cohort data of adults aged ≥ 60 years from the Swedish National study of Aging and Care in Kungsholmen (SNAC-K) collected between 2001 and 2016 was analyzed. Information on LRTIs was obtained from the Swedish National Patient Register, and objective health status was assessed using the Health Assessment Tool (HAT) which incorporates indicators of mild and severe disability, cognitive and physical functioning, and multimorbidity. The LRTI-exposed and -unexposed participants were matched using propensity score matching based on an expansive list of potential confounders. Mixed linear models were used to analyze the association between LRTIs and changes in HAT scores.
Results
The study included 2796 participants, 567 of whom were diagnosed with a LRTI. LRTIs were independently associated with an excess annual decline of 0.060 (95% CI: -0.107, -0.013) in the HAT score over a 7-year period. The associations were stronger among males, who experienced an excess annual decline of 0.108 (95% CI: -0.177, -0.039) in up to 7-years follow-up, and 0.097 (95% CI: -0.173, -0.021) in up to 12-years follow-up. The associations were not statistically significant among females in either follow-up period.
Conclusion
LRTIs, even years after the acute infectious period, seem to have a prolonged negative effect on the health of older adults, particularly among males. Preventative public health measures aimed at decreasing LRTI cases among older adults could help in preserving good health and functioning in old age.</p
New developments in computed tomography : low dose techniques, image reconstruction, and spectral imaging
No full textCT is an indispensable modality in any modern radiology department. It has revolutionized medical imaging, and the global frequency of CT scans is steadily increasing. As novel developments in CT hardware and software emerge, it is important to evaluate the clinical implications of these advancements. CT protocols should be optimized to maximize image quality for specific diagnostic tasks while reducing radiation exposure for both patients and radiology personnel in accordance with the ALARA principle. This dissertation has assessed how novel CT technologies related to radiation dose reduction, image reconstruction, and spectral imaging can be implemented in four specific diagnostic tasks: 1) CT-guided musculoskeletal (MSK) biopsies, 2) evaluation of distal extremities in an emergency setting, 3) CT head examinations of trauma patients, and 4) CT abdomen examinations of patients with PDAC.The overarching aim of this thesis was to assess how novel CT technology can be implemented in various CT protocols to reduce radiation dose and to enhance image quality.In Study I, we assessed a low-dose CT protocol for MSK biopsies that utilized several radiation dose reduction methods, including single-shot axial wide scanning, tube current modulation, and an IR algorithm. 57 patients underwent biopsies with the low-dose CT protocol (Group A), and 58 patients underwent biopsies with a standard CT protocol (Group B). A spreadsheet was created for Protocol A to calculate the start and end positions of the control scan, replicating the functionality built into the software of Protocol B. The mean total dose length product (DLP) was significantly lower in Group A compared to Group B (41.5+2.9 vs. 257.4+22.0 mGy*cm; p In Study II, an ultra-low-dose CT (ULD-CT) protocol for distal extremities was evaluated in comparison to digital radiography (DR) in terms of diagnostic accuracy, radiation dose, and workflow efficiency. 207 extremities in 203 consecutive patients from the emergency department were included. Patients underwent DR followed by an ULD-CT protocol that incorporated several radiation dose reduction techniques. DR detected fractures in 73/207 (35.3%) extremities, while ULD-CT detected fractures in 109/207 (52.7%) extremities, resulting in an odds ratio for fracture detection between ULD-CT and DR of 2.0 (95% CI, 1.4-3.0). ULD-CT also yielded additional relevant diagnostic information, such as articular involvement and loose intraarticular bodies. The mean total time, including both the examination time and the time to produce a preliminary report, was shorter for ULD-CT than DR (7.6+2.5 vs. 9.8+4.7 min, p = 0.002). The diagnostic information from ULD-CT led to changes in treatment recommendation for 34/207 (16.4%) extremities, with treatment upgrades in 27/207 (13.0%) and downgrades in 7/207 (3.4%) extremities.In Study III, the image quality between an IR algorithm (ASiR-V) and a DLR algorithm for head CT scans of 94 consecutive trauma patients was compared. Quantitative image quality parameters included image noise, signal-to-noise ratio (SNR) in gray and in white brain matter, and contrast-to-noise ratio (CNR) between gray and white brain matter. Qualitative image quality parameters comprised: image noise, artifacts, brain structures, and the conspicuity of intracranial hemorrhage (ICH). All image quality parameters were compared across four image reconstructions: ASIR-V and three strength levels of DLR, high (DLIR-H), medium (DLIR-M), and low (DLIR-L), for the same CT scan. DLIR-H and DLIR-M demonstrated significantly higher SNR and CNR, and lower image noise than ASiR-V and DLIR-L (p In Study IV, the image quality of MD-iodine images was compared to the image quality of virtual monoenergetic images (VMIs) at different keV levels, for assessing PDAC in the portal venous phase (PVP). From spectral data of 50 PCD-CT examinations (46 patients) with PDAC, MD-iodine images and VMIs at 55 keV and 70 keV were generated. Quantitative and qualitative image quality was compared across these three image reconstructions. Quantitative image quality was evaluated by calculating the CNR between PDAC and normal pancreatic parenchyma. Qualitative image quality parameters included PDAC conspicuity, image noise, sharpness of pancreatic and surrounding structures ("structures"), and overall image quality. MD-iodine had significantly higher CNR compared to 70 keV (p = 0.003), but lower compared to 55 keV (p = 0.049). MD-iodine received significantly lower reader scores for all parameters compared to both VMI reconstructions (p List of scientific papersI. First experiences of a low-dose protocol for CT-guided musculoskeletal biopsies combining different radiation dose reduction techniques.Alagic Z, Alagic H, Bujila R, Srivastava S, Jasim S, Lindqvist M, Wick MC. Acta Radiol. 2020 Jan;61(1):28-36. https://doi.org/10.1177/0284185119847676II. Ultra-low-dose CT for extremities in an acute setting: initial experience with 203 subjects.Alagic Z, Bujila R, Enocson A, Srivastava S, Koskinen SK. Skeletal Radiol. 2020 Apr;49(4):531-539. https://doi.org/10.1007/s00256-019-03309-7III. Deep learning versus iterative image reconstruction algorithm for head CT in trauma.Alagic Z, Diaz Cardenas J, Halldorsson K, Grozman V, Wallgren S, Suzuki C, Helmenkamp J, Koskinen SK. Emerg Radiol. 2022 Apr;29(2):339-352. https://doi.org/10.1007/s10140-021-02012-2IV. Photon-counting detector computed tomography: iodine density versus virtual monoenergetic imaging of pancreatic ductal adenocarcinoma.Alagic Z, Valls Duran C, Suzuki C, Halldorsson K, Svensson-Marcial A, Saeter R, Koskinen SK. Abdom Radiol. 2024 Sep; Online ahead of print. https://doi.org/10.1007/s00261-024-04605-0</p
Biological mechanisms behind health effects of air pollution exposure from childhood to adulthood
No full textBACKGROUND: Early life air pollution exposure impairs respiratory health during childhood and up to young adulthood. The proposed mechanism for air pollution adverse health effects includes systemic inflammation and metabolic dysregulation, which could be explored by the tools of proteomics and metabolomics. However, epidemiological evidence of the role of proteomics and metabolomics in the relationship between air pollution and respiratory health from childhood to young adulthood is mostly unknown.AIM: The aim of the thesis is to elucidate biological mechanisms behind the negative health effects of air pollution exposure in children and young adults.METHODS: All four included studies were based on Swedish birth cohorts. Time- weighted average exposure to air pollutants (i.e., particulate matter with diameter ≤10 um (PM10), ≤2.5 um (PM2.5), and nitrogen dioxide (NO2) or nitrogen oxides (NOx)) was estimated using validated dispersion models.In study I and II, inflammation-related proteins were repeatedly measured in children of the EMIL and Born-into-Life cohorts at 6 months, 1 year and 2 years of age with Olink inflammation panel (n=158). We examined sex-specific longitudinal associations between preceding air pollution exposure and inflammation-related proteins.Building upon Study I, Study II focused on 6-month-old infants with data on inflammation-related proteins, as well as dynamic spirometry (n=82). We defined individual inflammation-related protein profiles and analyzed the interaction between air pollution and the abnormal inflammation-related protein profile on lung function on the additive scale.In Study III, urine metabolomics analyses were carried out at 4 years (n=612) and 24 years of age (N=836) in the BAMSE cohort, using non-targeted screening followed by in-house library annotation. We examined age-specific associations between air pollution and urinary metabolites. Pathway enrichment and interaction with genetic variants were also explored.Based on the results from Study III, Study IV focused on child participants 4 years of age, for whom spirometry was repeatedly measured at 8-, 16- and 24- year-follow-ups (n=584). We grouped metabolites into 11 groups based on the stability, sources and biological types according to Pubchem, and focused on the carnitine profiles related to fatty acids oxidation. We characterized the metabolite profile in each group by counting the number of abnormally low metabolites and the number of abnormally high metabolites. We assessed the interaction between air pollution and metabolic profile in early childhood on lung function in later life.RESULTS: Our results showed that preceding air pollution exposure was longitudinally associated with inflammation-related proteins during early childhood, in age and sex-specific manner. Further, early life air pollution exposure and abnormal inflammation-related protein profiles interact synergistically towards lower lung function in infants. Next, we observed that both early life and recent air pollution exposures were associated with urinary metabolic profiles in children and young adults. In particular, air pollution was associated with upregulated caffeine metabolism in children, likely due to increased enzyme activity. In addition, our data suggest that carnitine profile in the pathway of oxidation of fatty acids may modify the air pollution exposure - lung function association.CONCLUSIONS: Our findings indicate that molecular changes related to air pollution exposure can be detected even in environments with relatively low exposure levels as in Stockholm County. This highlights the public health importance of further reducing urban air pollution levels. The identified air pollution-related inflammatory profiles, and metabolic pathways during childhood provide valuable insights for early identification of susceptible subpopulations and prediction of long-term health consequences.List of scientific papersI. He S, Klevebro S, Baldanzi G, Pershagen G, Lundberg B, Eneroth K, Hedman AM, Andolf E, Almqvist C, Bottai M, Melén E, Gruzieva O. Ambient air pollution and inflammation-related proteins during early childhood. Environ Res. 2022 Dec;215(Pt 2):114364. Epub 2022 Sep 17. https://doi.org/10.1016/j.envres.2022.114364II. He S, Lundberg B, Hallberg J, Klevebro S, Pershagen G, Eneroth K, Melen E, Bottai M,* Gruzieva O.* Joint association of air pollution exposure and inflammation-related proteins in relation to infant lung function. Int J Hyg Environ Health. 2024 Jan;255:114294. Epub 2023 Nov 10. https://doi.org/10.1016/j.ijheh.2023.114294III. He S,* Habchi B,* Chaleckis R, Hernandez-Pacheco N, Bergström A, Merritt AS, Kull I, Eneroth K, Bottai M, Pershagen G, Merid SK, Björkander S, Yu Z, Melén E, Gruzieva O, Wheelock CE,* Klevebro S .* Long-term exposure to air pollution is associated with urinary metabolite shifts in children and young adults in a Swedish birth cohort. [Submitted]IV. He S, Habchi B, Chaleckis R, Hernandez-Pacheco N, Bergström A, Merritt AS, Kull I, Eneroth K, Pershagen G, Björkander S, Yu Z, Wheelock CE, Klevebro S, Bottai M, Melén E,* Gruzieva O.* Air pollution exposure, metabolomic markers, and lung function in children and young adults: findings from a Swedish birth cohort. [Manuscript]* These authors contributed equally</p
Design of miR-34a mimics shifting the targetome towards HNF4
No full textmicroRNAs (miRNAs) regulate gene expression by inhibiting translation or inducing the degradation of messenger RNAs (mRNAs). miR-34a acts as a tumor suppressor, is predicted to have thousands of targets with more than hundreds of them are validated. Dysregulation of miR-34a has been implicated in various diseases.In this thesis, we investigated the structural and functional aspects of miRNA mediated repression, exploring how the structure governs the function of miRNA. We developed a structure-based design approach to increase specificity of miR- 34a. We than used this to design a series of miR-34a mimics aiming at specifically trapping a subset of the targets, centered around hepatocyte nuclear factor 4 alpha (HNF4a), which has been reported to be dysregulated in several cancer types. We used dual-luciferase reporter assay to measure the downregulation efficiency and RNA:RNA binding by SHAPE (RABS) to characterize the structural features. We found that the mimic with an altered tail region exhibited increased down-regulation activity for HNF4a specifically, while having little or no effect on the other targets in the luciferase reporter system. We highlight the critical importance of tail region of miR-34a, where three cytosines are located, with two of them being adjacent. We evaluated the overall effects of the designed miRNA mimics on the transcriptome via RNA sequencing and demonstrated that the mimics were able to capture mRNA targets sharing similar structural features with HNF4a. By designing a mimic to stabilize tail binding of miR-34a:target interaction, we successfully shifted miR-34a targetome, thus creating a specific yet broad interaction, which could provide insights for the design of miRNA-based drugs to treat multigenetic diseases.This thesis includes three projects. In Project I, we established a structural probing methodology RABS (RNA:RNA binding by SHAPE) to characterize the structural features of miRNA binding to its mRNA target. In project II, we investigated a specific miR-34a:HNF4a interaction and designed several miR-34a mimics to increase the specificity to HNF4a. In project III, we designed miRNA based small RNAs, which could shift miRNA targetome to a subset of target mRNAs. We showed the proof of concept for structure-activity relationship design of miRNA- based mimics to modulate target specificity, which may contribute to the concept of miRNA-based drug development.List of scientific papersI. Elnaz Banijamali, Lorenzo Baronti, Walter Becker, Joanna J Sajkowska-Kozielewicz, Ting Huang, Christina Palka, David Kosek, Lara Sweetapple, Juliane Müller, Michael D Stone, Emma R Andersson, Katja Petzold. RNA:RNA interaction in ternary complexes resolved by chemical probing. RNA. 2023 Mar;29(3):317-329 https://doi.org/10.1261/rna.079190.122II. Ting Huang, David Kosek, Rubin Dasgupta, Joanna J Sajkowska- Kozielewicz, Emma R Andersson, Katja Petzold. Design of miR-34a mimics shifting the targetome towards HNF4x for treatment of cancer [Manuscript]III. Ting Huang, Emma R Andersson, Katja Petzold. Design of small RNAs shifting the targetome of microRNA [Manuscript]</p
Unravelling treatment efficacy and failure in systemic lupus erythematosus
No full textSystemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple organ systems with varying severity and presents with unpredictable patterns over time. In the past fifteen years, the management of SLE has advanced notably through the introduction of three new licensed medications, the adoption of a target-driven treatment paradigm, and an increased focus on minimising treatment toxicities and managing comorbidities. Nevertheless, there remains a need to optimise the implementation of systematic monitoring strategies and holistic management plans that address diverse aspects of SLE through tailored interventions.Using multiple data sources and study designs, this thesis investigates the effects of therapeutic interventions, with a focus on B-cell therapy, and lifestyle factors on health-related quality of life (HRQoL), disease activity, and organ damage in patients with SLE.Study I examined the prevalence of poor HRQoL outcomes and severe fatigue amongst individuals classified as treatment responders in two randomised controlled trials of belimumab. Despite significant improvements from baseline, a substantial proportion of these patients continued to experience poor HRQoL, particularly in physical aspects (8-29%), and fatigue (26%). Organ damage contributed to poor physical HRQoL, while belimumab was protective against severe fatigue.Studies II and III are cross-sectional studies evaluating associations between lifestyle factors and various patient-reported outcomes. In study II we explored associations between BMI and HRQoL and fatigue in all patients enrolled in the same two clinical trials of belimumab, and observed that BMI significantly impacted physical aspects of HRQoL, fatigue and social functioning, independently of disease activity, treatment, and organ damage. There was a gradual decline in HRQoL with increasing BMI. In Study III we expanded to associations of obesity and tobacco smoking with a broader array of patient- reported outcomes in patients from the Östergötland county in Sweden. We confirmed similar patterns of diminished HRQoL and fatigue amongst overweight and obese patients. Compared with non-smokers, current smokers reported reduced HRQoL and higher levels of fatigue and pain, with estimates that were consistently larger than those derived from comparisons with former smokers.Study IV is a post-hoc analysis of four randomised controlled trials of belimumab where we estimated the effect of belimumab and antimalarial agents on preventing renal flares. Intravenous belimumab, at both 1 mg/kg or 10 mg/kg doses every fourth week, was associated with a lower risk of renal flares. Similarly, the use of antimalarial agents conferred a protective effect. Importantly, the protective effect of belimumab against renal flares was enhanced when it was co- administered with antimalarial agents.In Study V, we investigated whether belimumab levels in serum were associated with clinical response and safety, as well as the occurrence of anti-drug antibodies against belimumab. In a cohort of 100 belimumab-treated SLE patients followed for up to two years at three Swedish University Hospitals, we found modest associations between belimumab levels and various measures of disease activity, but no clear link with serological activity or safety outcomes. Notably, using a drug-tolerant acid-dissociation radioimmunoassay, we did not detect anti-drug antibody formation following belimumab exposure.Lastly, in Study VI, we developed a novel tool to measure organ damage using healthcare administrative codes and evaluated its accuracy to estimate SDI scores collected from a well-characterised cohort from Sweden. This new index displayed very high sensitivity, specificity and positive predictive value to detect organ damage. When applied to a nationwide cohort of newly diagnosed SLE patients from Sweden, 40% of them developed organ damage within the first five years of diagnosis. Men and older individuals had a higher risk to develop organ damage compared with their counterparts, while no changes in the risk of organ damage were observed across calendar periods over the last decades. Additionally, there was a strong association between early development of RBODI- based organ damage and long-term mortality, underscoring the need for effective strategies to prevent organ damage.List of scientific papersI. Adverse health-related quality of life outcome despite adequate clinical response to treatment in systemic lupus erythematosus. Gomez A, Qiu V, Cederlund A, Borg A, Lindblom J, Emamikia S, Enman Y, Lampa J, Parodis I. Front Med (Lausanne). 2021;8:651249 https://doi.org/10.3389/fmed.2021.651249II. Impact of overweight and obesity on patient-reported health- related quality of life in systemic lupus erythematosus. Gomez A, Butrus F, Johansson P, Åkerström E, Soukka S, Emamikia S, Enman Y, Pettersson S, Parodis I. Rheumatology (Oxford). 2021;60(3):1260-72. https://doi.org/10.1093/rheumatology/keaa453III. Obesity and tobacco smoking are independently associated with poor patient-reported outcomes in SLE: a cross-sectional study. Gomez A, Parodis I, Sjöwall C. Rheumatology International. 2024;44(5):851-61. https://doi.org/10.1007/s00296-024-05546-zIV. Belimumab and antimalarials combined against renal flares in patients treated for extra-renal systemic lupus erythematosus: results from 4 phase III clinical trials. Gomez A, Jägerback S, Sjöwall C, Parodis I. Rheumatology (Oxford). 2024;63(2):338-48. https://doi.org/10.1093/rheumatology/kead253V. Belimumab concentrations and immunogenicity in relation to drug efficacy and safety in systemic lupus erythematosus. Gomez A, Walhelm T, Loeff F, Jonsen A, Nikolopoulos D, van den Broek B, Bengtsson A, de Vries A, Rispens T, Sjöwall C, Parodis I. [Manuscript]VI. Development and evaluation of a register-based organ damage index in systemic lupus erythematosus: a nationwide, population-based study from Sweden. Gomez A, Parodis I, Saleh M, Simard JF, Sjöwall C, Arkema EV. [Manuscript]</p
HLA-DR immunopeptidomics and quantitative lipid mediator profiling for studying lung involvement in immune-mediated diseases
No full textIn this thesis, we developed mass spectrometry-based methodologies for HLA- DR immunopeptidomics and lipid mediator profiling for the study of rheumatoid arthritis (RA) and long-term outcomes of preterm birth respectively.RA is an autoimmune disease affecting the joints. Smoking and a group of alleles of the HLA-DRB1 gene, termed shared epitope (SE) are major risk factors for developing RA. HLA-DR is a protein involved in antigen presentation, and its polymorphism affects peptide binding preference. Evidence suggests that the autoimmunity of RA might be triggered in the lung. To our knowledge, there have been no previous non-targeted studies on the HLA-DR antigen landscape in the RA lung to date.In Paper I, we developed a method for HLA-DR immunopeptidomics analysis of lung immune cells sampled by bronchoalveolar lavage (BAL). We applied this method on treatment-naïve RA patients (n=9) and healthy controls (n=10), as well as RA patients following 6 months of treatment (n=5). As the cohort represented a high genetic variance of HLA-DR types, we deconvoluted peptides to predicted binding HLA-DRB1 alleles. Interestingly, some alleles were predicted to bind more peptides. These included all present SE alleles (*01:01, *04:01, *04:04, *04:05, *10:01) and only one non-SE allele (*04:07). Together with published transcriptomics data, this suggests allele-specific expression profiles. Furthermore, the peptides predicted to bind to SE alleles contained native versions of known citrullinated RA autoantigens, including a-enolase and fibrinogen B. This suggests that future immunogenicity studies on the identified SE-binding peptides might lead to the discovery of novel autoantigens in RA.In the following studies we investigated lipid mediators, many of which have known biological activities in the context of lung diseases. In Paper II, we developed a method to profile octadecanoids in a stereospecific manner, which allows for inference of their biosynthetic route and study of potential stereospecific biological activities. Using chiral super-critical fluid chromatography coupled to tandem mass spectrometry (SFC-MS/MS), we enabled stereo-specific profiling of 103 octadecanoids in less than 13 min. Additionally, we developed a more sensitive achiral method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which includes octadecanoids produced exclusively by autooxidation. Both methods were validated according to EMA guidelines in solvent and surrogate matrix for linearity, lower limit of quantification, accuracy, precision, recovery, and matrix effects.Next, we addressed the limitations of established validation protocols for LC- MS/MS of endogenous compounds, as they often rely on replicate analysis from one pooled sample material. Thereby, they do not account for the inter-individual matrix diversity encountered in cohort studies. In Paper III, we developed a workflow which enables the validation of phenotyping robustness using cohort measurements. The statistical approaches are reflecting those typically applied in downstream cohort studies, such as association and quantile analysis. Therefore, this workflow allows for praxis-oriented assessment of method performance. Applications are shown on a subset of sphingolipids, such as identification of coelutions, evaluation of sample preparation, and selection of the most suitable internal standard per analyte.Lastly, we applied the developed methods to investigate long-term consequences of sustained oxygen support after preterm birth, termed bronchopulmonary dysplasia (BPD). We hypothesized that young adults with a history of BPD might have persisting alterations in their lipid mediator profile of the lung, which might help understand why impaired lung function is frequently observed in these individuals throughout life. In Paper IV, we quantified oxylipins and sphingolipid levels in bronchoalveolar lavage fluid (BALF) of young adults with a history of BPD, as well as three age- and sex-matched control groups. These comprised preterm born individuals without a history of BPD, healthy at-term born, and individuals with mild asthma. Following analysis in three LC-MS/MS methods, a total of 22 oxylipins and 44 sphingolipids were quantified passing quality control criteria. Following noise removal using lasso and ridge regression, groups were compared using multivariate orthogonal projection to latent structures discriminate analysis (OPLS-DA) modeling. These analyses suggest that female groups were primarily differing in their oxylipin profile, while male groups in their sphingolipid profile. The BALF of the female BPD group contained higher levels of 12-HHTrE compared to the other groups, and 9,10-EpOME was increased in both female BPD and asthma group compared to the healthy group. Our previous studies showed higher levels of these analytes in BALF of females with smoking-associated chronic obstructive pulmonary disease (COPD). The male BPD and asthma group showed increased levels of Cer(d18:/16:0), Cer(d18:1/18:0), and DhCer(18:0/16:0). Increased levels of the former two are known predictors of adverse cardiovascular outcomes.Taken together, this PhD thesis provides methodologies to investigate the HLA- DR immunopeptidome and lipid mediator profiles. By applying these methodologies in RA and BPD respectively, we could report novel findings which might aid the understanding the immunological mechanisms driving these diseases, and associated molecular phenotypes in the lung.List of scientific papersI. Benedikt Zöhrer, Ákos Végvári, Martina Bonatti, Iryna Kolosenko, Nicole Wagner, Antonio Checa, Vijay Joshua, C. Magnus Sköld, Karin Lundberg, Lars Klareskog, Vivianne Malmström, Åsa M. Wheelock. Characterization of the HLA-DR immunopeptidome of bronchoalveolar lavage cells in patients with newly diagnosed rheumatoid arthritis and healthy current-smoker controls. [Manuscript]II. Alessandro Quaranta, Benedikt Zöhrer, Johanna Revol-Cavalier, Kurt Benkestock, Laurence Balas, Camille Oger, Gregory S. Keyes, Åsa M. Wheelock, Thierry Durand, Jean-Marie Galano, Christopher E. Ramsden, Mats Hamberg, Craig E. Wheelock. Development of a Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry Platform for the Quantitative Metabolic Profiling of Octadecanoid Oxylipins. Analytical Chemistry. 2022, 94 (42): 14618-14626. https://doi.org/10.1021/acs.analchem.2c02601III. Benedikt Zöhrer, Cristina Gómez, Joaquim Jaumot, Helena Idborg, Signe S. Torekov, Asa M. Wheelock, Craig E. Wheelock, Antonio Checa. Cohort-based strategies as an in-house tool to evaluate and improve phenotyping robustness of LC-MS/MS lipidomics platforms. Analytical and Bioanalytical Chemistry. 2024, 416 (25): 5485-5496. https://doi.org/10.1007/s00216-024-05404-8IV. Benedikt Zöhrer*, Nicole Wagner*, Antonio Checa, Alessandro Quaranta, Martina Bonatti, Iryna Kolosenko, Petra Um-Bergström, Anders Lindén, Eva Berggren-Broström, C. Magnus Sköld, Craig E. Wheelock, Åsa M. Wheelock. *Shared first authorship. Lipid mediator profiles in bronchoalveolar lavage fluid of young adults with a history of bronchopulmonary dysplasia are distinct from those of healthy controls and subjects with mild asthma. [Manuscript]</p
