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Long-term cardiovascular assessment in women with preeclampsia
No full textBackground: Preeclampsia (PE) is one of the most common medical multi-organ, pregnancy-specific disorder. PE is associated with endothelial dysfunction, elevated blood pressure, and inflammation. In addition to vascular dysfunction, preeclampsia is associated with cardiac remodeling and left ventricular (LV) dysfunction. Whether these cardiovascular changes eventually resolve remains unclear.Aims: This thesis is aimed to study the long-term effects of PE on the cardiovascular system in women with a history of PE-complicated pregnancy.Material and Methods: Studies I and II examined 15 women with a history of PE (mean age 39±4 years) and 16 matched healthy women with an uncomplicated pregnancy (41±3 years) 10-12 years following the index pregnancy. Assessment of medical and family history, physical examination, anthropometric measurements, and biochemical markers were evaluated. In Study I forearm flow-mediated vasodilatation (FMD), pulse wave analyses (PWA), 24-h ambulatory blood pressure measurement (ABPM), plasma concentrations of glucose metabolism, lipid metabolism, inflammatory markers, and vascular function were assessed. In Study II echocardiography including Tissue Doppler Imaging and two-dimensional speckle-tracking echocardiography for myocardial strain imaging was used for evaluation of systolic and diastolic, left ventricular (LV), and right ventricular function (RV). LV global strain, atrial size, indices of ventricular-arterial coupling (VAC), and concentrations of N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) were analyzed.Results: In Study, I endothelial function assessed as hyperemia, hyperemia-induced FMD, and as responses to glyceryl trinitrate were similar in both groups (+10.7±5.4 % vs +9.0±4.7 %, and +29.1±9.7 % vs +25.1±8.8 %). ABPM showed slightly higher blood pressure (24-h 117±11/75±8 mm Hg vs 112±11/71±9 mm Hg) and reduced systolic and diastolic night/day ratios (0.81±0.06 vs 0.76±0.05, and 0.88±0.04 vs 0.84±0.04; both p=Conclusion: A long-term follow-up (10-12 years after index pregnancy) of women with a history of PE-complicated pregnancy showed normalized endothelial function despite higher blood pressure and impaired glucose tolerance in comparison to women without a previous PE. Extensive echocardiographic examinations could not demonstrate significant alterations in systolic or diastolic LV function or VAC. We suggest that pre-existing risk factors may be more important for future cardiovascular complications than myocardial and vascular damage occurring during pregnancy in women with PE. This highlights the importance of early treatment of risk factors, integrating adequate preventive strategies, and long- term surveillance with close monitoring.List of scientific papersI. Östlund E, Al-Nashi M, Hamad RR, Larsson A, Eriksson M, Bremme K, Kahan T. Normalized endothelial function but sustained cardiovascular risk profile eleven years following a pregnancy complicated by preeclampsia. Hypertens Res. 2013;36(12):1081-7. https://doi.org/10.1038/hr.2013.81 II. Al-Nashi M, Eriksson MJ, Östlund E,Bremme K, Kahan T. Cardiac structure and function and ventricular-arterial interaction 11 years following a pregnancy complicated with preeclampsia. J Am Soc Hypertens. 2016;10(4):297-306. https://doi.org/10.1016/j.jash.2016.01.012 </p
Antibiotics in pregnancy : utilization, determinants and safety
No full textAntibiotics are indispensable in maternal health. Crucial for treating and preventing bacterial infections ranging from common ailments to life-threatening conditions, antibiotics are inevitably prescribed to pregnant women frequently. This thesis aimed to advance knowledge on antibiotic use during pregnancy, by examining utilization and its underlying factors; and by addressing safety concerns regarding prenatal exposures on the foetus.In Study I, we comprehensively described trends and patterns in antibiotic use among women who gave birth in Sweden through a population-based drug utilization study. Prescriptions for systemic antibiotics were filled in 20.7% of 1434431 pregnancies overall, decreasing from 24.7% in 2007 to 18.0% in 2019. Phenoxymethylpenicillin, nitrofurantoin, amoxicillin and cefadroxil use were most prevalent. Intravaginal and intestinal antibiotic preparations were used to a substantially lower extent than systemic antibiotics.In Study II, we conducted a population-based cohort study to evaluate how sociodemographic, medical, obstetric and healthcare utilization characteristics predicted antibiotic use during pregnancy. While the magnitude of associations was found to be modest for the majority of these characteristics, pre-existing morbidities, preconception polypharmacy and maternal age below 20 years most strongly predicted the use of systemic antibiotics, broad-spectrum antibiotics and multiple antibiotic courses.In Study III, we investigated the association between first trimester tetracycline exposure and risks of major congenital malformations. In this population-based cohort study including 6340 exposed infants, we found that the risks of major congenital malformations (MCMs) overall, for all 12 organ system subgroups, and the 16 individual malformations selected based on prior safety signals and/or fulfilment of prespecified statistical power criteria, were not significantly higher than among propensity score-matched unexposed controls.In Study IV, using a population-based cohort design, we investigated the associations between prenatal antibiotic exposure and infections during infancy, and whether such associations differed by antibiotic class. Compared to 1052361 unexposed infants, 294654 infants prenatally exposed to antibiotics had slightly higher rates of antimicrobial prescription fills, incident infections in specialist care and infection-related deaths, but these associations were attenuated in the sibling analyses. No differential associations with infections were found for antibiotics commonly used during pregnancy.In conclusion, this thesis found no concerning patterns in antibiotic utilization among pregnant women in Sweden, nor in their safety. Tetracyclines and any antibiotic use were associated with no increased risks of MCMs and with minimal infection risks in infancy, respectively. Antibiotic prescribing for pregnant women should, nonetheless, be guided by risk-benefit assessments and the latest evidence to ensure optimal perinatal outcomes.List of scientific papersI. Nakitanda AO, Odsbu I, Pasternak B, Karlsson P, Pazzagli L. Antibiotic use during pregnancy in Sweden: a nationwide utilization study covering 2007-2019. Acta Obstetricia et Gynecologica Scandinavica. 2024;103(3):531-539. https://doi.org/10.1111/aogs.14741 II. Nakitanda AO, Pazzagli L, Pasternak B, Odsbu I. Predictors of antibiotic use during pregnancy in Sweden: a population-based cohort study. [Submitted]III. Nakitanda AO, Odsbu I, Cesta CE, Pazzagli L, Pasternak B. First trimester tetracycline exposure and risk of major congenital malformations: a population-based cohort study. [Submitted]IV. Nakitanda AO, Kieler H, Odsbu I, Rhedin S, Almqvist C, Pasternak B, Pazzagli L. In-utero antibiotic exposure and subsequent infections in infancy: a registerbased cohort study with sibling analysis. American Journal of Obstetrics & Gynecology MFM. 2023;5(4):100860. https://doi.org/10.1016/j.ajogmf.2023.100860 </p
Iron deficiency in heart failure
No full textBackground: Iron plays a central role in several vital processes in the human body and is fundamental for organs with high energy demands, like the heart. Iron deficiency (ID) is common in patients with heart failure (HF) and independently associated with a poor prognosis. The overall aim of this doctoral project was to examine potential causes of ID in patients with HF, assess pathological changes in the myocardial iron metabolism, investigate time-dependent changes of ID in HF, and study the role of ID in new-onset HF.Methods and results: Study I. An interventional, non-randomised, open label study where oral iron absorption was assessed in 30 patients with chronic HF and ID, and 12 age- and sex-matched controls without HF and ID at Södersjukhuset, Sweden. Two hours after intake of an oral dose of iron, patients with HF had higher absolute increase of S-iron than controls (median increase 83.8 [interquartile range 61.5;128.5] µg/dL vs 47.5 [30.7;61.5] µg/dL, p = 0.001).Study II. An observational cross-sectional study including 69 patients undergoing an elective coronary bypass graft operation (CABG) at the Karolinska University Hospital in Sweden. Tissue samples from myocardial and skeletal muscle were analysed regarding expression of genes involved in the regulation of the intracellular iron metabolism. Expression of the proteins transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) was increased in both myocardial and skeletal muscle biopsies from patients with absolute ID (ferritin Study III. An observational prospective cohort of patients with new-onset HF. Among 482 patients for whom there were iron data at baseline, 34% had ID. The prevalence of ID after 12 months was 32% among those who did not receive i.v. iron and had complete iron data at both baseline and at 12 months (n=368). After 12 months, 19% had persistent ID, 13% had developed ID, 11% had resolved ID, and 57% never had ID. Anaemia at baseline was the strongest independent predictor of ID one year after diagnosis (odds ratio (OR) 3.91, 95% confidence interval (CI) 1.88-8.13, p Study IV. A population-based cohort study of individuals who underwent blood sampling through occupational health checkups or out-patient care in the county of Stockholm during 1985–1996. Individuals who had at least three values of S-iron or transferrin saturation (TSAT) and no previous HF diagnosis (n = 127037 and 125482, respectively) were analysed. During a median follow-up of 24.9 years (interquartile range 19.4-27.2), 9324 (7.3%) individuals in the S-iron cohort and 9209 (7.3%) in the TSAT cohort were diagnosed with new-onset HF. The individuals within the lowest quartile of S-iron (Conclusions: Iron absorption was increased in patients with chronic HF and ID compared with in controls without HF and ID. Expression of the iron importers TfR1 and DMT1 were increased in biopsies from myocardial and skeletal muscle in patients with absolute ID undergoing elective CABG, irrespective of left ventricular function. About one third of the patients with new-onset HF had ID both at the time of HF diagnosis and after one year, though a quarter of the patients changed their ID status. Patients with anaemia, HF hospitalisation, female sex, HF with preserved ejection fraction or diabetes mellitus at baseline were more likely to have ID after one year. Low S-iron and TSAT were independently associated with incident HF.List of scientific papersI. Cabrera CC, Ekström M, Linde C. Persson H, Hage C, Eriksson MJ, Wallén H, Persson B, Tornvall P, Lyngå P. Increased iron absorption in patients with chronic heart failure and iron deficiency. Journal of Cardiac Failure. 2020;26(5);440-443. https://doi.org/10.1016/j.cardfail.2020.03.004 II. Cabrera CC, Frisk C, Löfström U, Lyngå P, Linde C, Hage C, Persson H, Eriksson M.J, Wallén H, Persson B, Ekström M. Relationship between iron deficiency and expression of genes involved in iron metabolism in human myocardium and skeletal muscle. International Journal of Cardiology. 2023, May 15;379:82-88. https://doi.org/10.1016/j.ijcard.2023.03.032 III. Cabrera CC, Ekström M, Tornvall P, Löfström U, Frisk C, Linde C, Hage C, Persson H, Eriksson MJ, Wallén H, Persson B, Lyngå P. Iron deficiency in new onset heart failure – association with clinical factors and quality of life. [Accepted]IV. Cabrera CC, Leander K, Vikström M, Jonsson M, Hammar N, Ekström M, Lyngå P, Tornvall P. Association between low serum iron and transferrin saturation and incident heart failure. [Submitted]</p
Pharmacovigilance of mass drug administration as preventive chemotherapy to control and eliminate lymphatic filariasis in Tanzania
No full textMass drug administration (MDA) campaigns are usually conducted on an annual basis by many countries globally where neglected tropical diseases (NTDs), including lymphatic filariasis (LF), are endemic. Such campaigns are organized and executed by the Neglected Tropical Diseases Control Programmes (NTDCP) or the National Programmes for Elimination of LF (NPELF) whichever is applicable. During such campaigns, drugs are normally distributed randomly to at-risk populations to halt the transmission of diseases. It has been proven through research and evidence gathered through the World Health Organization (WHO) and NTDCP that such a move helps to stop the spreading of LF in affected communities. Nevertheless, in these campaigns, individuals subjected to such preventive chemotherapy are in all cases not tested for LF diagnosis before drug administration. Depending on disease prevalence, elimination status, distribution coverage, and availability of resources, such campaigns may be conducted once or twice a year.During or after these campaigns evidence shows that, the concept of pharmacovigilance is not being considered in most of the cases and that drugs are distributed randomly without collecting safety information to be certain that the same are not causing any harm to exposed individuals. Pharmacovigilance (i.e., the science and activities relating to the detection, assessment, understanding, and prevention of adverse effects or any other medicine or vaccine-related problem) is still weak in many resource-constrained countries. Such countries are to-date grappling to set up systems that would allow for the effective collection of data on drug-related adverse events (AEs) and reactions (ADRs). Underreporting is one of the mounting challenges and National Medicines Regulatory Authorities (NMRAs) do not have adequate resources or machinery to counter the status quo. Long-term and chronic undesirable events due to prolonged use of MDA drugs including those affecting the liver and kidneys are even of more concern.In this PhD programme, we began by conducting a study to determine the prevalence and correlates of LF infection in the Mkinga district which is located in the Tanga region on the offshores of the Indian Ocean - the northeastern part of Tanzania (Paper I). The rationale of conducting such a study was to find out the level of antigenemia and microfilaremia as the same have an impact on the safety profile of drugs used for NTDs including LF (i.e., ivermectin and albendazole – IA). In this cross-sectional community-based survey, a total of 4115 individuals (49.7% males, 35.2% children) were screened for circulating filarial antigens (CFA), microfilaremia (mf), and disease manifestations in 15 villages between November 2018 and January 2019. MDA uptake in the previous year (2017) was also assessed. The overall prevalence of CFA-positivity was 5.8% (239/4115; 95% CI: 5.1–6.6), with significant heterogeneity seen between villages (range 1.2% to 13.5%). CFApositivity was higher in males (8.8%) than females (3.3%) and correlated with increasing age (p Following the prevalence study, we went on to conduct the safety surveillance study (Paper II) to document AEs that occurred following IA MDA. In this study, we identified the type, incidence, and associated risk factors. Around 9,640 eligible individuals received single-dose IA combination preventive chemotherapy and treatment-associated AEs were actively monitored through house-to-house visits on day 1, day 2, and day 7 of MDA. After MDA, 9288 participants (96.3%) were followed up of whom 442 reported 719 MDA-associated AEs. The incidence of experiencing one or more types of MDA-associated AE was 4.8% (95% CI = 4.3– 5.2%); this was significantly higher among those with pre-MDA clinical events than those without (8.5% versus 4.1%, p To further ascertain the safety of MDA drugs in blood, kidneys, and liver, we extracted data to measure the haematological and biochemical parameters to determine their changing patterns (Paper III). In so doing we also examined their relationship with those who had experienced AEs in the previous safety study. In this nested analytical prospective study, we assessed data amongst 499 eligible individuals whose blood samples were collected before and after MDA. We measured Complete Blood Count (CBC) and renal and liver function tests at days 0 and 7 following MDA. After a comprehensive assessment of all blood indices as well as kidney and liver surrogate markers, we demonstrated that haematological and biochemical changes may occur following IA MDA. The median values of haematological parameters, including RBC, Hb, and HCT decreased while MCH, MCHC, and monocytes increased significantly in both CFA-positive and negative individuals (pIn Paper IV, a pharmacokinetic (PK) study was conducted to determine the PK properties of IVM in humans. The study was done to characterize the disposition of IVM and determine predictors of its PK for dose optimization during MDA. This was also a nested study in a bigger safety study in which data was evaluated amongst 468 individuals. PK samples were collected at 0, 2, 4, and 6 hours from individuals weighing > 15 Kg receiving IVM (3-, 6-, 9-, or 12 mg) and ALB (400 mg) during an MDA campaign. Individual characteristics were assessed including demographics, laboratory/clinical parameters, and genetic variations of selected drug-metabolizing enzymes and transporters. IVM plasma concentrations were quantified by LC-MS/MS and analysed using population-pharmacokinetic (POPPK) modelling. A two-compartment model with transit absorption kinetics, and allometrically scaled oral clearance (CL/F) and central volume (Vc/F) were adopted. Fitting of the model to the current data identified a 48% higher bioavailability for the 3 mg dose compared to other doses and further identified a subpopulation with 97% higher mean transit time (MTT). The final estimates for CL/F, Vc/F, inter-compartment clearance (Q), peripheral volume (Vp), MTT, and absorption rate constant (Ka) for a 70 Kg person (on a dose other than 3 mg) were 7.7 L/h, 147 L, 20.4 L/h, 207 L, 1.5 h, and 0.71/h, respectively. Simulations indicated that weight-based dosing provides comparable exposure across weight bands, but height-based dosing with a capping IVM dose at 12 mg for individuals with height > 160cm under-doses those weighing > 70 Kg. The variability in IVM PK is partly explained by body weight and dose. The established POPPK model can be used for IVM dose optimization. Height-based pole dosing may result in varying IVM exposure of individuals in different weight bands; hence use of weighing scales for IVM dosing during MDA is recommended.In Paper V, we investigated the efficacy of IA in clearing mf and reducing CFA levels in individuals. This community-based prospective study assessed the efficacy of MDA drugs in mf clearance and CFA reduction on days 7- and 6 months following MDA. The study was done in the same Mkinga district, Tanga region between November 2018 and June 2019. The status of mf and CFA on day 7 and six-month post-MDA was monitored. The primary efficacy outcomes were the clearance rates of mf on day 7 and six months, and CFA at 6 months of post-MDA. Out of 4,115 individuals screened, 239 (5.8%) tested positive for CFA, with 11 (4.6%) also positive for mf. The McNemar test revealed a significant improvement in mf clearance on day 7 following MDA (p=0.02). Out of 183 CFA-positive individuals who were available at 6-month follow-up, 160 (87.4%) remained CFA positive, while 23 became CFA negative. The CFA clearance rate at 6 months post-MDA was 12.6% (95% CI = 8.52 – 18.5%). There was no significant association of variability in IVM plasma exposure (Cmax and AUC) with post-MDA mf or CFA clearance status. We concluded that preventive chemotherapy with IA effectively clears mf within a week, but the same drugs are ineffective in clearing CFA at six months post-MDA. We recommended alternative drug combinations targeting adult worms as IA is not macrofilaricidal.In conclusion, LF has not been fully eliminated from the study area and the prevalence of the disease is still above the threshold recommended by WHO. MDA drugs are relatively safe for use during MDA. However, chronic illnesses, previous clinical manifestations of LF, being female, or having pre-existing clinical symptoms are significant predictors of AEs. Individuals exposed to MDA might experience changes in haematological and biochemical parameters after drug intake. The NTD programme should consider these findings when monitoring individuals taking part in future MDA campaigns. POPPK modelling can be used for IVM dose optimization and whenever possible, the programme should consider using weighing balances rather than height-poles for dose estimation when administering MDA drugs to individuals in endemic communities. Despite that the MDA drugs are still efficacious, alternative combinations targeting adult worms should be sought by the NTD programme.List of scientific papersI. Fimbo AM, Minzi OMS, Mmbando BP, Barry A, Nkayamba AF, Mwamwitwa KW, Malishee A, Seth MD, Makunde WH, Gurumurthy P, Lusingu JPA, Kamuhabwa AAR, Aklillu E. Prevalence and Correlates of Lymphatic Filariasis Infection and Its Morbidity Following Mass Ivermectin and Albendazole Administration in Mkinga District, North-Eastern Tanzania. J Clin Med. 2020 May 21;9(5):1550. https://doi.org/10.3390/jcm9051550 II. Fimbo AM, Minzi OM, Mmbando BP, Gurumurthy P, Kamuhabwa AAR, Aklillu E. Safety and Tolerability of Ivermectin and Albendazole Mass Drug Administration in Lymphatic Filariasis Endemic Communities of Tanzania: A Cohort Event Monitoring Study. Pharmaceuticals. 2022 May 12;15(5):594. https://doi.org/10.3390/ph15050594 III. Fimbo AM, Rajabu Hussein Mnkugwe, Eulambius Mathias Mlugu, Peter Kunambi, Bruno P. Mmbando, Omary MS Minzi, Appolinary A.R. Kamuhabwa and Eleni Aklillu. Surveillance of haematological and biochemical changes following mass Ivermectin and Albendazole administration for the control of lymphatic filariasis in endemic communities of Tanzania. [Submitted]IV. Fimbo AM, Mlugu EM, Kitabi EN, Kulwa GS, Iwodyah MA, Mnkugwe RH, Kunambi PP, Malishee A, Kamuhabwa AAR, Minzi OM, Aklillu E. Population pharmacokinetics of ivermectin after mass drug administration in lymphatic filariasis endemic communities of Tanzania. CPT Pharmacometrics Syst Pharmacol. 2023 Dec;12(12):1884-1896. https://doi.org/10.1002/psp4.13038 V. Fimbo AM, Rajabu Hussein Mnkugwe, Eulambius Mathias Mlugu, Peter P. Kunambi, Bruno P. Mmbando, Omary MS Minzi, Appolinary A. R. Kamuhabwa and Eleni Aklillu. Efficacy of ivermectin and albendazole combination in suppressing transmission of lymphatic filariasis following mass administration in Tanzania: A Prospective Cohort Study. [Manuscript]</p
Airway hyperresponsiveness and viral recognizing toll-like receptors
No full textCommon colds caused by viruses are, in healthy individuals, usually self-limited and with relatively mild symptoms. In asthmatic individuals, respiratory viruses can burst the defense immune system and trigger asthma exacerbations, including causing airway hyperresponsiveness (AHR). AHR is defined as the predisposition of the airways to contract excessively in response to stimuli that would produce little or no effect in healthy persons. The mechanism by which infections alter AHR is unclear. Toll-like receptors (TLRs) are at the forefront of our microbial defense and studies suggest that TLRs might have a role in the development of AHR. The present thesis investigates the impact of TLR ligands (TLR 3, 4, 7, and 9).The presented data are derived from experiments using TLR ligands in mice and guinea pig models of AHR with and without concomitant local inflammation. The latter was induced by ovalbumin (OVA) exposure. The result is presented in five papers. The first paper explored the relationship between microbial stimulation and the development of AHR by applying either poly(I:C) activating TLR3 or LPS triggering TLR4, representing viral and bacterial-induced interactions, respectively, intranasally in vivo. The second paper repeated these experiments in mice with pre-established, OVA-induced allergic lung inflammation. The activation of TLR3 or TLR4, caused AHR, with local inflammatory profiles characterized by inflammatory cell recruitment and cytokine release that appeared to be receptor specific. Paper III explored the effects of concomitant TLR3 and TLR4 stimulation. Four days of consecutive treatment in naïve mice triggered an AHR along with an increased influx of inflammatory cells and enhanced release of proinflammatory cytokines including TNFα in bronchioalveolar lavage fluid (BALF). In mice, with OVA-induced allergic airway inflammation, the combined TLR3 and TLR4 stimulation caused a further increase of AHR in the peripheral lung. Treatment with TNFα-blocking antibody infliximab given to allergic mice blunted tissue damping (G) and tissue elastance (H) without affecting the influx of the cells in BALF. This inhibitory effect in the peripheral airways was not found in naïve mice. Paper IV evaluated how in vivo administration of the viral TLR agonists TLR7 or TLR9, given to mice with allergic lung inflammation, affects AHR and airway inflammation. The challenge with the TLR7 agonist reduced AHR and airway inflammation in asthmatic mice, whereas TLR9 activation showed the opposite effect with a more profound inflammation. Paper V used an in vivo model to investigate whether the TLR7 agonist Imiquimod could induce bronchorelaxation by acting directly on airway smooth muscle. Imiquimod was shown to relax guinea pig airways pre-contracted with histamine within seconds. Unexpectedly, the bronchodilatory effect was found to be independent of TLR7. A quinolone moiety in imiquimod also found in other bronchodilatory compounds, like quinine and chloroquine, may contribute to the bronchodilatory property.To conclude; Challenge with TLR agonists can increase AHR in allergic airways which is confirming that TLRs can play a pivotal role in the responsiveness following microbial infection in asthmatics. An exacerbated AHR does not need to be associated with increased inflammation and the results suggest that at least part of the effect can be a direct-acting effect on the ASM. Blocking of TNFα showed a therapeutic effect on peripheral airways in allergic animals during exposure to viral and bacterial TLR agonists. This indicates TNFα blockade as a treatment option. Challenge with a viral TLR7 agonist reduced AHR and airway inflammation in asthmatic mice. Opposite effects of viral TLR9 agonist call attention to the complexity of TLR interaction. TLR7 agonists with quinoline moieties can be of interest in the development of drugs to treat asthma as they can reduce AHR and induce bronchodilatation.List of scientific papersI. Magnus Starkhammar, Susanna Kumlien Georén, Linda Swedin, Sven-Erik Dahlén, Mikael Adner, Lars-Olaf Cardell. Intranasal Administration of poly (I:C) and LPS in BALB/c Mice Induces Airway Hyperresponsiveness and Inflammation via Different Pathways. PLoS ONE. Vol. 7 Issue 2, p. e32110, 2012. https://doi.org/10.1371/journal.pone.0032110 II. Magnus Starkhammar, Olivia Larsson, Susanna Kumlien Georén, Marina Leino, Sven-Erik Dahlén, Lars-Olaf Cardell, Mikael Adner. Toll-like Receptor Ligands LPS and Poly (I:C) Exacerbates Airway Hyperresponsiveness in a Model of Airway Allergy in Mice, Independently of Inflammation. PLoS ONE. Vol. 16 Issue 8, p. e104114, 2014. https://doi.org/10.1371/journal.pone.0104114 III. Magnus Starkhammar, Susanna Kumlien Georén, Sven-Erik Dahlén, Lars-Olaf Cardell, Mikael Adner. TNFα-blockade stabilizes local airway hyperresponsiveness during TLR-induced exacerbations in murine model of asthma. Respiratory Research. Vol. 16 Issue 1, 2015. https://doi.org/10.1186/s12931-015-0292-5 IV. Mikael Adner, Magnus Starkhammar, Susanna Kumlien Georén, Sven-Erik Dahlén, Lars-Olaf Cardell. Toll-like receptor (TLR) 7 decreases and TLR9 increases the airway responses in mice with established allergic inflammation. European Journal of Pharmacology. Vol. 718 Issue 1-3, pp. 544-551, 2013. https://doi.org/10.1016/j.ejphar.2013.09.004 V. Olivia Larsson. Martijn Manson, Magnus Starkhammar, Barbara Fuchs, Mikael Adner, Susanna Kumlien Georén, Lars-Olaf Cardell. The TLR7 agonist imiquimoid induces bronchodilation via a nonneuronal TLR7-independent mechanism: a possible role for quinoline in airway dilation. American Journal of Physiology: Lung Cellular and Molecular Physiology. Vol. 310 Issue 11, pp. L1121-L1129, 2016. https://doi.org/10.1152/ajplung.00288.2015 </p
Multimodal and multiscale brain networks : understanding aging, Alzheimer’s disease, and other neurodegenerative disorders
No full textThe human brain can be modeled as a complex network, often referred to as the connectome, where structural and functional connections govern its organization. Several neuroimaging studies have focused on understanding the architecture of healthy brain networks and have shed light on how these networks evolve with age and in the presence of neurodegenerative disorders. Many studies have explored the brain networks in Alzheimer’s disease (AD), the most common type of dementia, using various neuroimaging modalities independently. However, most of these studies ignored the complex and multifactorial nature of AD.The aim of this thesis was to investigate and analyze the brain’s multimodal and multiscale network organization in aging and in AD by using different multilayer brain network analyses and different types of data. Additionally, this research extended its scope to incorporate other dementias, such as Lewy body dementias, allowing for a comparison of these disorders with AD and normal aging. These comparisons were made possible through the application of protein co-expression networks.In Study I, we investigated sex differences in healthy individuals using multimodal brain networks. To do this we used resting-state functional magnetic resonance imaging (rs-fMRI) and diffusion-weighted imaging (DWI) data from the Human Connectome Project (HCP) to perform multilayer and deep learning analyses. These analyses identified differences between men's and women's underlying brain network organization, showing that the deep-learning analysis with multilayer network metrics (area under the curve, AUC, of 0.81) outperforms the classification using single-layer network measures (AUC of 0.72 for functional networks and 0.70 for anatomical networks). Furthermore, we integrated the multilayer brain networks methodology and neural network models into a software package that is easy to use by researchers with different backgrounds and is also easily expandable for researchers with different levels of programming experience.Then, we used the multilayer brain networks methodology to study the interaction between sex and age on the functional network topology using a large group of people from the UK Biobank (Study II). By incorporating multilayer brain network analyses, we analyzed both positive and negative connections derived from functional correlations, and we obtained important insights into how cognitive abilities, physical health, and even genetic factors differ between men and women as they age. Age and sex were strongly associated with multiplex and multilayer measures such as the multiplex participation coefficient, multilayer clustering, and multilayer global efficiency, accounting for up to 89.1%, 79.9%, and 79.5% of the variance related to age, respectively. These results indicate that incorporating separate layers for positive and negative connections within a complex network framework reveals sensitive insights into age- and sex-related variations that are not detected by traditional metrics. Furthermore, our functional metrics exhibited associations with genes that have previously been linked to processes related to aging.In Study III, we assessed whether multilayer connectome analyses could offer new perspectives on the relationship between amyloid pathology and gray matter atrophy across the AD continuum. Subjects from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) were divided into four groups based on cerebrospinal fluid (CSF) amyloid-β (Aβ) biomarker levels and clinical diagnosis. We compared the different groups using weighted and binary multilayer measures that assess the strength of the connections, the modularity, as well as the multiplex segregation and integration of the brain connectomes. Across Aβ-positive (Aβ+) groups, we found widespread increases in the overlapping connectivity strength and decreases in the number of identical connections in both layers. Moreover, the brain modules were reorganized in the mild cognitive impairment (MCI) Aβ+ group and an imbalance in the quantity of couplings between the two layers was found in patients with MCI Aβ+ and AD Aβ+.Using a subsample from the same database, ADNI, we analyzed rs-fMRI data from individuals at preclinical and clinical stages of AD (Study IV). By dividing the time series into different time windows, we built temporal multilayer networks and studied the modular organization across time. We were able to capture the dynamic changes across different AD stages using this temporal multilayer network approach, obtaining outstanding areas under the curve of 0.90, 0.92 and 0.99 in the distinction of controls from preclinical, prodromal, and clinical AD stages, respectively, on top and beyond common risk factors. Our results not only improved the discrimination between various disease stages but, importantly, they also showed that dynamic multilayer functional measures are associated with memory and global cognition in addition to amyloid and tau load derived from positron emission tomography. These results highlight the potential of dynamic multilayer functional connectivity measures as functional biomarkers of AD progression.In Study V, we used in-depth quantitative proteomics to compare post-mortem brains from three key brain regions (prefrontal cortex, cingulate cortex, and the parietal cortex) directly related to the disease mechanisms of AD, Parkinson’s disease with dementia (PDD), dementia with Lewy bodies (DLB) in prospectively followed patients and older adults without dementia. We used covariance weighted networks to find modules of protein sets to further understand altered pathways in these dementias and their implications for prognostic and diagnostic purposes.In conclusion, this thesis explored the complex world of brain networks and offered insightful information about how age, sex, and AD influence these networks. We have improved our understanding of how the brain is organized in different imaging modalities and different time scales, as well as developing software tools to make this methodology available to more researchers. Additionally, we assessed the connections among various proteins in different areas of the brain in relation to health, Alzheimer's disease, and Lewy body dementias. This work contributes to the collective effort of unraveling the mysteries of the human brain organization and offers a foundation for future research to understand brain networks in health and disease.List of scientific papersI. Gómez-Ruiz E*, Canal-Garcia A*, Chang YW*, Zhao H, Zufiria-Gerbolés B, Sun J, Veréb D, Westman E, Mijalkov M, Pereira JB, Volpe G. Brain Connectivity Analysis with Multilayer Graphs and Deep Learning. * Authors contributed equally to this work as first authors. [Manuscript]II. Mijalkov M, Veréb D, Jamialahmadi O, Canal-Garcia A, Gómez-Ruiz E, VidalPiñeiro M, Romeo S, Volpe G, Pereira JB. 2023. Sex differences in multilayer functional network topology over the course of aging in 37543 UK Biobank participants. Network Neuroscience. 7(1):351-76. https://doi.org/10.1162/netn_a_00286 III. Canal-Garcia A, Gómez-Ruiz E, Mijalkov M, Chang YW, Volpe G, Pereira JB, Alzheimer’s Disease Neuroimaging Initiative. 2022. Multiplex Connectome Changes across the Alzheimer’s Disease Spectrum Using Gray Matter and Amyloid Data. Cerebral Cortex. 32(16):3501-3515. https://doi.org/10.1093/cercor/bhab429 IV. Canal-Garcia A, Veréb D, Mijalkov M, Westman E, Volpe G, Pereira JB, Alzheimer’s Disease Neuroimaging Initiative. 2024. Dynamic multilayer functional connectivity detects preclinical and clinical Alzheimer’s disease. Cerebral Cortex. bhad542. https://doi.org/10.1093/cercor/bhad542 V. Canal-Garcia A, Branca RM, Francis PT, Winblad B, Lehtiö J, Nilsson P, Aarsland D, Pereira JB, Bereczki E. Comparative brain regional specific proteomic signatures of Alzheimer’s disease and Lew body dementias. [Manuscript]</p
Fibrotic lung disease : early signs, treatment and outcomes
No full textIdiopathic pulmonary fibrosis (IPF) has gained a lot of attention by the research community in recent decades. Thanks to enhanced understanding on pathophysiological mechanisms behind the fibrosing process together with two approved treatments that decelerate the progress, the interest within the field has increased.However, despite the success, IPF is still recognized as a devastating disease with inevitably progressive loss of lung function, disabling symptoms, impaired health-related quality of life (HRQoL) and a poor, unpredictable prognosis. While large controlled clinical trials have increased knowledge and enabled new treatments for IPF, they have failed to show a beneficial effect on HRQoL.Many needs of the individual IPF patients are unmet. By gathering patient data in a registry, we can track patient characteristics, disease behavior and HRQoL longitudinally in a real-world healthcare setting with its strengths and limitations. By doing so, we can truly see how IPF is managed.The aim of this thesis is to explore the disease pathway and management of IPF. The thesis is built around four studies. As an important quality index of IPF management, early diagnosis was highlighted and discussed by estimating a high prevalence of interstitial lung abnormalities, potential pre-stage for IPF, in a large population-based sample (study I). Study II and III gave an overview on current important gaps in IPF care. IPF was diagnosed at a moderate stage when its symptoms already impair HRQoL. Patients received antifibrotic treatment with a delay, and a significant proportion of patients were not treated at all. Most importantly, national differences were observed: only one third of Finnish IPF patients received treatment between 2014 and 2017, while two-thirds of Swedish IPF patients were treated during the same period. Additionally, study III demonstrated that antifibrotic treatment may stabilize HRQoL in IPF, especially symptoms related to progressive loss of lung function. In study IV, the prognostic value of six-minute walking test was confirmed.In summary, many gaps in IPF care are identified and discussed, raising further questions for future studies.List of scientific papersI. Pesonen I, Johansson F, Johnsson Å, Blomberg A, Boijsen M, Brandberg J, Cederlund K, Egesten A, Emilsson ÖI, Engvall JE, Frølich A, Hagström E, Lindberg E, Malinovschi A, Stenfors N, Swahn E, Tanash H, Themundo R, Torén K, Vanfleteren LEGW, Wollmer P, Zaigham S, Östgren CJ, Sköld M. High prevalence of interstitial lung abnormalities in middle-aged never smokers. ERJ Open Research. 2023 9: 00035-2023. https://doi.org/10.1183/23120541.00035-2023 II. Pesonen I, Carlson L, Murgia N, Kaarteenaho R, Skold CM, Myllarniemi M, Ferrara G. Delay and inequalities in the treatment of idiopathic pulmonary fibrosis: the case of two Nordic countries. Multidiscip Respir Med. 2018, 13: 14. https://doi.org/10.1186/s40248-018-0126-7 III. Pesonen I, Carlson L, Kalafatis D, Ferrara G, Sköld M. Health-related quality of life in idiopathic pulmonary fibrosis – a study from a Swedish registry. [Manuscript]IV. Pesonen I, Gao J, Kalafatis D, Carlson L, Sköld M and Ferrara G. Six minute walking test outweighs other predictors of mortality in idiopathic pulmonary fibrosis. A real-life study from the Swedish IPF registry. Respiratory Medicine: X 2020, 2. https://doi.org/10.1016/j.yrmex.2020.100017</p
New beginnings, new challenges : health & housing of asylum seekers and refugees in their early post-migration period in Sweden
No full textAsylum seekers and recently resettled refugees are at an increased risk of poor mental health. Besides pre-migration experiences, the early post-migration period in host countries presents numerous challenges that can negatively impact their mental health and well-being, including challenges such as poor housing, and socioeconomic difficulties. Additionally, Sweden has adopted more restrictive migration policies, which risk leading to a growing tension between mental health needs and the policy objectives of reducing migration. Gaining a deeper understanding of day-to-day challenges faced during the early post- migration period is crucial to effectively address and mitigate their potential adverse impact on the mental health of asylum seekers and refugees.Study I, a cross-sectional population-based survey, utilized the generic EQ-5D- 5L scale to assess the health-related quality of life index value in a study population of 1,215 individuals from Syria who recently resettled in Sweden. The results showed that the most frequently reported problem on the EQ-5D-5L scale was depression/anxiety, and a low index value was associated with being a woman, older age, and low social support. Study II is a qualitative study based on semi-structured interviews with fourteen asylum seekers at two accommodation centers in Sweden, exploring their experiences of living in these centers. The results indicated that their experiences were heavily influenced by the uncertainty of the asylum process and the constraints imposed by limited resources and housing conditions, often described as living a frozen life. This frozen life was a source of constant worry, leading to concerns about potential long-term effects on their health. Despite these challenges, the asylum seekers highlighted care practices that arose spontaneously among the residents, reflecting a shared concern for each other's well-being. Study III, a qualitative study utilizing the same data collection process used in Study II, explored the experiences of asylum seekers during the COVID-19 pandemic. The findings revealed that the living conditions at the centers shaped how the pandemic was experienced. The asylum seekers reported feeling increasingly excluded from society, a sentiment reinforced by a pandemic response from authorities that was perceived as lacking understanding or care for their unique situation. Study IV is a register-based prospective longitudinal cohort study that includes all adult asylum seekers who received residence permits between 2010 and 2012. The study investigated the association between housing type during the asylum process (institutional or self-organized) and the prescriptions of antidepressants or anxiolytic medication, as well as specialized in- and outpatient visits with diagnoses of CMDs, over a five-year follow-up period after being granted refugee status. The results indicated that individuals who had lived in institutional housing were at greater risk of having more prescriptions for antidepressants or anxiolytic medication, as well as a higher likelihood of specialized in- and outpatient visits with diagnoses of CMDs, compared to those who had lived in self-organized housing.The thesis emphasizes the importance of post-migration living conditions in shaping the mental health of asylum seekers and refugees in Sweden, with a particular focus on the asylum process and housing as key factors associated with distress. It also suggests that collective institutional accommodation tends to be more harmful to mental health than self-organized housing. Overall, the findings advocate for context-sensitive interventions addressing individual, community, and structural factors, with a focus on improving housing conditions, alleviating day-to-day challenges, and strengthening social support networks to prevent long-term mental health issues. Additionally, the thesis also calls for a transparent and fast-tracked asylum process.List of scientific papersI. Gottvall, M., Sjölund, S., Arwidson, C., & Saboonchi, F. (2020). Health-related quality of life among Syrian refugees resettled in Sweden. Quality of Life Research. https://doi.org/10.1007/s11136-019-02323-5II. van Eggermont Arwidson, C., Holmgren, J., Gottberg, K., Tinghög, P., & Eriksson, H. (2022). Living a frozen life: a qualitative study on asylum seekers' experiences and care practices at accommodation centers in Sweden. Conflict and Health 2022, 16(1):1-47. https://doi.org/10.1186/s13031-022-00480-yIII. van Eggermont Arwidson, C., Holmgren, J., Tinghög, P., Eriksson, H., & Gottberg, K. (2024). (Over)crowded house: exploring asylum seekers' experiences of the COVID-19 pandemic while living at accommodation centers in Sweden. BMC Public Health, 24(1): 622. https://doi.org/10.1186/s12889-024-18089-6IV. van Eggermont Arwidson, C., Holmgren, J., Gottberg, K., & Tinghög, P. Housing during the asylum process and its association with healthcare utilization for common mental disorders among refugees in Sweden: A nationwide cohort study. [Submitted]</p
Immune mechanisms controlling tuberculosis-diabetes co-morbidity
No full textTuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (M. tuberculosis), remains a leading global health concern, responsible for millions of infections annually. Despite extensive scientific efforts, the biological and immunological mechanisms that predispose certain individuals to develop active TB, rather than containing the infection in a latent state, are not fully understood. Notably, the incidence of active TB is significantly higher among individuals with diabetes mellitus (DM), which suggests a profound interaction between metabolic disorders and immune responses in the progression of M. tuberculosis infection.The immune defense against M. tuberculosis is mainly mediated by macrophages, a major target of infection. Macrophages are activated by CD4+ Th1 cells, which respond to mycobacterial antigens by secreting cytokines such as interferon- gamma (IFN-y), thereby enhancing the macrophage ability to contain or kill the intracellular bacteria.This thesis presents an in-depth study of the molecular factors that modulate the immune responses of macrophages and T cells to M. tuberculosis infection. Through a series of controlled experiments using M. tuberculosis-infected in vitro and in vivo models, we investigate how these cells orchestrate a defense against the bacterium and how their efficacy is compromised under diabetic conditions.In DM, the high glucose levels cause alterations in the carbohydrate metabolism. These result in elevated levels of methylglyoxal (MGO), a reactive compound that is a byproduct of glycolysis. MGO glycates various cellular macromolecules altering their function, in turn accounting for DM pathogenesis. In paper I, we found that MGO irreversibly binds to thioredoxin reductase 1 (TXNRD1), a mammalian selenoprotein, transforming it into a NADPH oxidase. This modification is crucial for the activation of NRF2, a typically cytoprotective transcription factor. We showed that NRF2 activation suppressed the secretion of IL-1B and nitric oxide (NO). Deficiency of the secretion of these molecules accounts for the enhanced intracellular proliferation of M. tuberculosis. The overexpression of NRF2 by genetic deficiency of its inhibitor KEAP1, was also sufficient to reduce the inflammatory responses in bone marrow-derived macrophages (BMMs) and promoted the intracellular growth of M. tuberculosis. Using various specific inhibitors of TXNRD1, including the FDA-approved auranofin, we replicated the effects of MGO on M. tuberculosis-infected BMMs. Treatment of M. tuberculosis- infected mice with MGO and auranofin increased the expression of antioxidant genes, hampered the production of protective molecules by macrophages and increased the bacterial levels in lungs. Altogether we propose that alterations of carbohydrate metabolism in DM account for the increased risk for TB in DM patients by targeting a TXNRD1-NRF2 redox pathway.In paper II, we extend the exploration of host defense against M. tuberculosis under hyperglycemic conditions and attempt to define a role for hypoxia- inducible factor-1 (HIF-1), a key regulator of responses to hypoxia. We found that M. tuberculosis infection significantly stabilized the expression of HIF-la protein and the downstream responses of HIF-1 in BMMs and mice, enhancing immune and metabolic functions. Treatment with deferoxamine (DFO), a hypoxia mimetic, further amplified these HIF-1-dependent responses, reducing bacterial loads both in vitro and in vivo. Conversely, exposure to high glucose or MGO attenuated these responses and increased M. tuberculosis burden in BMMs. Similar trends were observed in hyperglycemic Leprdb/db mice, which exhibited higher bacterial loads and reduced expression of HIF-1-targeted genes in the lungs compared to controls. Importantly, the negative effects of high glucose and MGO were reversible with DFO treatment. Additionally, BMMs lacking Hif1a showed diminished protective immune and glycolytic responses after mycobacterial infection under both high glucose treatment and normal conditions. Our findings suggest targeting HIF-1 as a novel strategy to mitigate TB progression in diabetic patients.In paper III, we investigated the role of HIF-1 in T cells during M. tuberculosis infection. We found that mice with HIF-1 stabilization in T cells, due to genetic deficiency of its inhibitor VHL (Vhl cKO), showed a higher susceptibility to M. tuberculosis, with increased bacterial loads and exacerbated lung pathology. The heightened vulnerability was associated with fewer M. tuberculosis-specific T cells in the lungs, which exhibited diminished proliferation, altered transcriptional profiles, and increased expression of inhibitory receptors. By contrast, mice with HIF-1 deficiency in T cells exhibited similar responses to those of wild-type controls during M. tuberculosis infection. In addition to these findings, the study revealed that Vhl cKO mice presented blunted T-cell responses following immunization with the BCG, the only available vaccine against TB, suggesting a broader defect in T-cell functionality beyond natural infection scenarios. Moreover, our results underscore the critical role of VHL in regulating MYC activity, which is pivotal for T-cell activation, growth, expansion, and survival upon TCR signaling. The absence of VHL led to significant alterations in these processes, highlighting the intricate link between cellular oxygen-sensing mechanisms and T- cell-mediated immunity.In conclusion, we found that:1. MGO binds and inhibits TXNRD1 reductase and converts it to a NADPH- oxidase that mediates NRF2 activation, impairing the macrophage control of M. tuberculosis.2. While HIF-1 is protective in macrophages against M. tuberculosis, its function and expression are diminished under conditions of hyperglycemia and carbonyl stress.3. Stabilization of HIF-1 in T cells disrupts the control of M. tuberculosis infection in mice by impairing T cell activation.List of scientific papersI. Hanxiong Li, Ruining Liu*, Gokul Raj Kathamuthu*, Radosveta Gencheva, Axel Tobias Scholz, Mohammad Alzrigat, Lucia Coppo, Elias S.J. Arnér, Martin E. Rottenberg. The inhibition of TXNRD1 by methylglyoxal impairs the intracellular control of Mycobacterium tuberculosis. [Manuscript]II. Terán G*, Li H*, Catrina SB, Liu R, Brighenti S, Zheng X, Grünler J, Nylén S, Carow B, Rottenberg ME. High Glucose and Carbonyl Stress Impair HIF-1-Regulated Responses and the Control of Mycobacterium tuberculosis in Macrophages. Mbio. 2022 Sep 19:e01086-22. https://doi.org/10.1128/mbio.01086-22III. Liu R, Muliadi V, Mou W, Li H, Yuan J, Holmberg J, Chambers BJ, Ullah N, Wurth J, Alzrigat M, Schlisio S, Carow B, Larsson LG, Rottenberg ME. HIF-1 stabilization in T cells hampers the control of Mycobacterium tuberculosis infection. Nature communications. 2022 Sep 5;13(1):1-20. https://doi.org/10.1038/s41467-022-32639-9* Equal contribution</p
Exploration of novel therapeutic targets in neuroblastoma
No full textNeuroblastoma is a malignancy of the sympathetic nervous system occurring in early childhood. It accounts for approximately 6% of all childhood cancers and is a highly heterogeneous disease. While patients with low- and intermediate-risk disease have a good prognosis, high-risk disease remains a therapeutic challenge. High-risk neuroblastoma often presents with metastatic spread and therapy resistance already at diagnosis. Consequently, there is a great need for improved treatment options to improve patient survival but also to reduce side effects and chronic health conditions. Human neuroblastomas bear recurrent genetic alternations in neuritogenesis-associated genes such as teneurins (TENM1- TENM4) and components of Rho/Rho-associated Coiled-Coil Kinase (ROCK1 and ROCK2) signaling, pathways associated with differentiation and proliferation. This thesis aims to identify and characterize novel targets and therapeutic approaches for neuroblastoma and to investigate the microenvironmental factors.In Paper I, the efficacy of the ROCK1/2 inhibitor RKI-1447 was assessed in vitro and in vivo using preclinical neuroblastoma models. Treatment with RKI-1447 repressed tumor growth by inhibiting N-MYC in vitro and in the transgenic TH- MYCN mouse model. A combinational drug screen revealed RKI-1447 to be a beneficial combinational partner for bromodomain and extra terminal domain (BET) inhibitors. Synergistic effects between RKI-1447 and the BET inhibitor ABBV- 075 (Mivebresib) were confirmed in different neuroblastoma models, including a zebrafish-embryo xenograft model. Furthermore, our studies demonstrated that BET inhibitor treatment increased the phosphorylation of ROCK targets, which could be prevented by combinational treatment, proposing a possible mechanism of action.With the aim of gaining insight into neuroblastoma bone marrow metastases, the bone marrow microenvironment of neuroblastoma patients with and without metastases was compared, using single-cell RNA-sequencing, in Paper II. The results indicated a niche-dependent remodeling of immune cell populations and shifted transcriptomic profiles. Several immune cell populations were enriched in the metastatic bone marrow, including tumor-associated neutrophils, macrophages, and exhausted T cells. Additionally, an increased number of regulatory T cells as well as a decreased number of B cells was observed. Furthermore, this work identified potential therapeutic interventions, including markers of poor clinical prognosis occurring in malignant cells, and communications pathways between tumor and immune cells.Paper III explores the function of teneurins (TENMs) in neuroblastoma tumorigenesis. Transient knockdown of TENM4 significantly decreased proliferation in all investigated neuroblastoma cell lines, in contrast to knockdown of TENM1, TENM2 and TENM3. To verify these results, we generated TENM4-/- clones from the neuroblastoma cell line SK-N-BE(2) using CRISPR-Cas9; the clones demonstrated a neuronal differentiation-like morphology with reduced clonogenic capacity and proliferation compared to wild-type cells. Gene set enrichment analysis of RNA-sequencing data from neuroblastoma cells with TENM4-knockdown identified key components including induced differentiation, inhibited mTOR signaling, and epithelial to mesenchymal transition, as TENM4 targets. TENM4-/- cells did not lead to tumor formation when grafted into nude mice as opposed to wild-type cells. Furthermore, using a CRISPR-Cas13d-model with a TENM4 knockdown under a doxycycline-inducible promoter, we observed a delay until tumors were established in mice receiving doxycycline, compared to the control counterparts. Finally, we detected a significantly higher number of TENM4-positive tumor cells in high-risk vs. non-high-risk and MYCN-amplified vs. non-MYCN-amplified human neuroblastomas.This thesis covers various topics, from an explorative study searching for novel therapeutic targets in a common site of neuroblastoma metastasis, the bone marrow microenvironment, to interventional studies on novel molecular targets, using genetic inhibition or pharmacological drugs. I hope this research has increased our understanding of neuroblastoma and can support further studies on novel therapies in neuroblastoma.List of scientific papersI. Pepich A, Tümmler C, Ajamieh SA, Treis D, Boje AS, Vellema Q, Tsea I, Åkerlund E, Seashore-Ludlow B, Fard SS, Kogner P, Johnsen JI, Wickström M. The ROCK-1/2 inhibitor RKI-1447 blocks N-MYC, promotes cell death, and emerges as a synergistic partner for BET inhibitors in neuroblastoma. Cancer Letters. 2024 Nov 28;605: 217261. https://doi.org/10.1016/j.canlet.2024.217261II. Mei S, Alchahin AM, Embaie BT, Gavriliuc IM, Verhoeven BM, Zhao T, Li X, Jeffries NE, Pepich A, Sarkar H, Olsen TK, Wickström M, Stenman J, Reina-Bedoya O, Kharchenko PV, Saylor PJ, Johnsen JI, Sykes DB, Kogner P, and Baryawno N. Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature. JCI Insight. 2024 Feb 15;9(6): e173337. https://doi.org/10.1172/jci.insight.173337III. Andonova* T, Abu Ajamieh* S, Pepich A, Olsen TK, Tümmler C, Liu M, Thankaswamy-Kosalai S, Kanduri C, Djos A, Fransson S, Martinsson T, Baryawno N, Wilhem M, Näsman A, Kogner P, Johnsen JI, Wickström M. Inhibition of Teneurin 4 expression suppresses growth and induces differentiation in neuroblastoma. [Manuscript] *These authors contributed equally to the manuscript and share primary authorship.</p