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Exploring T cell mediated immunotherapy against therapy resistant leukemic stem cells
No full textThe cancer stem cell (CSC) model suggests that cancers depend on continuous replenishment from rare and distinct CSCs, but their existence has been challenging to prove for many malignancies. The CSCs in myeloid leukemias, known as leukemic stem cells (LSCs) have been well characterised and shown to selectively escape treatment, thus representing the cellular source of relapse. Relapse following initial periods of clinical remission represents the most significant threat to leukemic patients as the available treatment options at this stage are sparse due to the development of resistance. There is therefore a need for strategies facilitating earlier detection of an impending relapse to initiate preemptive treatments early, as well as for new targeted therapies to eliminate LSCs.The cellular identity of LSCs was recently assigned to the hematopoietic stem cell (HSC) compartment in patients with low to intermediate risk myelodysplastic syndromes (MDS), for which the only curative treatment option is allogeneic stem cell transplantation (allo-HSCT). Detailed characterisation of LSCs in terms of phenotype, function, and treatment resistance is thus now possible, with the aim to ultimately identify novel targets allowing for targeted elimination of LSCs. In study I, we performed LSC-directed screening for measurable residual disease (MRD) in patients with MDS after allo-HSCT. By flow cytometric purification of the hematopoietic stem and progenitor cells, the MRD-sensitivity was enhanced 97- fold compared to conventional screening methods on unfractionated bone marrow cells. Consequently, in our patient cohort, targeted screening of the leukemia initiating cells led to the detection of impending relapses on average ten months before clinical diagnosis. In the clinic, this would allow for prescribing preventative treatments earlier while disease burden remains low which could improve outcome.A potentially effective cancer treatment option is adoptive T cell therapy, using T cells that carry cancer-specific T cell receptors (TCR). TCR T cells can, unlike chimeric antigen receptor (CAR) T cells, recognise intracellular antigens but despite the theoretical large range of targetable antigens, very few are immunogenic. Consequently, no TCR T cell therapy is yet clinically approved for treatment against hematological malignancies, highlighting the need for new potential targets. In study II we showed that terminal deoxynucleotidyl transferase (TdT), which is normally transiently expressed during early B and T cell development and overexpressed in >80% of patients with B and T acute lymphoblastic leukemia (ALL), is a promising target for TCR based immunotherapy in context of HLA-A*02:01 (HLA-A2). T cells targeting TdT, identified through the blood of healthy donors, showed very efficient elimination of TdT+ leukemic cells, while sparing healthy hematopoietic stem and progenitor cells and mature lymphocytes in clinically relevant mouse models. Thus, TdT TCR T cells are a novel and promising immunotherapy option for patients with B- and T-ALL.Myeloid leukemias are, to a greater extent than the lymphoid leukemias, characterised by somatic recurrent mutations which generate cancer-specific neoantigens representing a group of unique attractive therapeutic targets. In study III, an HLA-A2 restricted TCR with high specificity against a recurrent neoantigen generated from the FLT3-D835Y mutation in acute myeloid leukemia (AML) was identified and shown to exhibit great anti-leukemic effect restricted to the FLT3-D835Y mutated cells while non-mutated cells were spared. Interestingly, the FLT3-D835Y TCR T cells also had the potential to eliminate the LSCs in vitro as shown by loss of leukemia initiating formation in mice following co-cultures.The results from study II and III revealed the therapeutic potential of TCR T cells, therefore, in study IV we proposed TCR T cells as a targeted therapy against LSCs. Identification of antigens that mediate specific elimination of all LSCs across many patients has been challenging due to large inter- and intra-patient heterogeneity. Myeloproliferative leukemia protein (MPL) has an important role in lifelong maintenance of HSCs and is therefore expressed on all HSCs and consequently also all LSCs in patients with low to intermediate risk MDS. We suggest TCR T cells targeting MPL presented on HLA-A2 as an immunotherapeutic approach with clinical relevancy to treat an impending relapse following an haploidentical allo- HSCT. MPL TCR T cells would potentially also represent a novel research tool to study the dependency of HSCs and LSCs in normal and malignant hematopoiesis.List of scientific papersI. Identification and surveillance of rare relapse-initiating stem cells during complete remission post-transplantation. M. Dimitriou, T. Mortera-Blanco, M. Tobiasson#, S. Mazzi#, M. Lehander#, K. Högstrand, M. Karimi, G. Walldin, M. Jansson, S. Vonlanthen, P. Ljungman, S. Langermeijer, T. Yoshizato, E. Hellström-Lindberg, P.S. Woll and SE.W. Jacobsen*. Blood, (2024) 143 (11): 953– 966. *Corresponding authors. #Equal contribution. https://doi.org/10.1182/blood.2023022851 II. T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes. M. Ali#, E. Giannakopoulou#, Y. Li, M. Lehander, S. Virding Culleton, W. Yang, C. Knetter, M. Odabasi, R. Chand Bollineni, X. Yang, Zs. Foldvari, M-L. Böschen, E. Taraldsrud, E. Strønen, M. Toebes, A. Hillen, S. Mazzi, A. de Ru, G. Janssen, A. Kolstad, G. Tjønnfjord, B. Lie, M. Griffioen, S. Lehmann, L. Osnes, J. Buechner, KG. Garcia, T. Schumacher, P. van Veelen, M. Leisegang, SE. W. Jacobsen#, P. S. Woll# and J. Olweus*. Nature Biotechnology. 2022. Apr;40(4):488-498. Epub 2021 Dec 6. *Corresponding authors. #Equal contribution. https://doi.org/10.1038/s41587-021-01089-x III. A T-cell receptor targeting a recurrent driver mutation in FLT3 mediates elimination of primary human acute myeloid leukemia in vivo. E. Giannakopoulou, M. Lehander, S. Virding Culleton, W. Yang, Y. Li, T. Karpanen, T. Yoshizato, E. Rustad, M. Milek Nielsen, R. Chand Bollineni, T. Tran, M. Delic-Sarac, TJ. Gjerdingen, K. Douvlataniotis, M. Laos, M. Ali, A. Hillen, S. Mazzi, D. Chin, A. Mehta, J. Sejerø Holm, A. Kai Bentzen, M. Bill, M. Griffioen, T. Gedde-Dahl, S. Lehmann, SE. W. Jacobsen*,#, P. S. Woll*,# and J. Olweus*. Nature Cancer. 2023 Oct;4(10):1474-1490. Epub 2023 Oct 2. *Corresponding authors. #Equal contribution. https://doi.org/10.1038/s43018-023-00642-8 IV. MPL as a target antigen for T cell receptor mediated elimination of leukemic stem cells. M. Lehander*, S. Virding Culleton#, Z. Földvári#, A. Titov# C. Knetter, O. Chowdhury, A. Hillen, E. Chari, F. Grasso, W. Yang, M. Brennan, J. Zeun, J. Olweus, and SE.W. Jacobsen and P.S. Woll*. *Corresponding authors. #Equal contribution. [Manuscript]</p
Health consequences of gender-based harassment at work
No full textBackground: Experiences at work with gender-based violence and harassment (GBVH) from superiors, co-workers or third parties are common. This includes a range of verbal or physical behaviors that can be of a sexual nature and can be one incidence or a pervasive part of the job. Decades of research show associations of sexual harassment with reduced mental and physical health, but many suffer severe methodological limitations, so that the magnitude of the consequences for workers’ health still is unclear. Also, non-sexual sexist experiences, here called gender harassment, have gained less attention despite being more common.Aims: The studies aimed to further our understanding of different kinds of GBVH as occupational health hazards. Studies I-III investigated the health outcomes long-term sickness absence (study I), psychotropic medication use (study II) and alcohol-related morbidity and mortality (study III). Study IV, a systematic review, assessed the evidence from published results for the prospective association of GBVH with different health and occupational outcomes.Methods: Studies I-III are prospective cohort studies. Information about sexual harassment from a superior or colleague, sexual harassment from a third party (not used in study I) and gender harassment from a superior or colleague was obtained from three survey items from the Swedish Work Environment Survey (SWES). SWES is a cross-sectional survey and conducted biannually on a representative sample of the Swedish working population. Data from several cohorts was pooled (Study I: cohorts 1999-2013, N: N=64 297, Study II: cohorts 2007-2013, N=23 449, study III: cohorts 1995-2013, N: 86 033). Information about the respective outcomes, and demographic and occupational information were linked from multiple registers. The continuous follow-up ranged from 1 year to 20 years in the respective studies.In study I, relative rates (RR) of long-term sickness absence in the year after baseline were determined using modified Poisson regression. In study II, hazard ratios (HR) of incident use of psychotropic medication and in study III, the HR of alcohol-related morbidity or mortality were estimated with Cox proportional hazard models.In study IV, bibliographic databases were systematically searched for prospective studies concerning exposure to GBVH in the work context and a health or manifest occupational outcome. After quality assessments, the results from the eligible studies with medium or high quality were grouped in clusters with similar exposures and outcomes and summarized in a narrative synthesis.Findings: Study I showed weak statistically significant associations of reoccurring sexual and gender harassment with long-term sickness absence (RRs ranged from 1.04 to 1.06). Study II showed statistically significant associations of sexual and gender harassment with incident psychotropics use (HRs ranged from 1.20 to 1.31) and study III with alcohol-related morbidity and mortality (HRs ranged from 1.5 to 2.88).In the systematic review, results from 29 eligible studies were summarized. They investigated mostly sexual violence and harassment (SVH). There were too few studies about physical health or occupational outcomes to synthesize the results and no consistent evidence for a prospective association of SVH with sickness absence. There was consistent evidence of an association of SVH with subsequent poor mental health and indications of an association with hazardous substance use.There was no consistent evidence of gender differences in the association of the different kinds of GBVH with the investigated health outcomes in any of the four studies of the thesis. Furthermore, there was no consistent evidence of a difference in the association of sexual harassment from a member of the work organization compared to harassment from a third party with the investigated health outcomes.Conclusions: Experiences with GBVH, both of a sexual and non-sexual nature are a risk factor for mental health and harm from alcohol use. If GBVH contributes to long-term sickness absence is unclear. Women and men appear to be similar in their susceptibility to the health effects of GBVH. Sexual harassment appears to be harmful regardless if it stems from a member of the organization or a third party.More prospective and longitudinal studies with precise concepts and reliable assessments of different kinds of GBVH and objective health outcomes are needed to fully comprehend the health impact of work-related GBVH.List of scientific papersI. Blindow K, Bondestam F, Johansson G, Bodin T, Westerlund H, Nyberg A. Sexual and gender harassment in Swedish workplaces: A prospective cohort study on implications for long-term sickness absence. Scandinavian Journal of Work, Environment & Health. 2021. https://doi.org/10.5271/sjweh.3971 II. Blindow KJ, Paulin J, Hanson LM, Johnell K, Nyberg A. Sexual and gender harassment and use of psychotropic medication among Swedish workers: a prospective cohort study. Occupational and Environmental Medicine. 2022;79(8):507-13. https://doi.org/10.1136/oemed-2021-108087 III. Blindow KJ, Thern E, Hernando-Rodriguez JC, Nyberg A, Magnusson Hanson LL. Gender-based harassment in Swedish workplaces and alcohol-related morbidity and mortality: A prospective cohort study. Scandinavian Journal of Work, Environment & Health. 2023;49(6):395-404. https://doi.org/10.5271/sjweh.4101 IV. Blindow KJ, Cedstrand E, Elling DL, Hagland M, Bodin, T. Genderbased violence and harassment at work and health and occupational outcomes. A systematic review of prospective studies. [Manuscript]</p
“Should i stay, or should i go” : teachers’ motivation to stay at their workplace
No full textThe overarching aim of this thesis was to increase the knowledge on the relationship between teachers’ psychosocial work environment and teachers’ retention intention. This was done by exploring the psychosocial work factors and mechanisms that facilitate teacher retention in four studies.The first study in this thesis was a cross-sectional study that explored the individual and contextual factors associated with Swedish teachers' intention to remain in the profession (n= 5903). The main finding in study 1 was the importance of teachers’ work-related health for their retention intention and that transactional work stress models such as the JD-R model are needed to understand how psychosocial work factors influence teachers’ retention intention.The second study was a qualitative interview study teachers (n= 8) that explored the specific aspects of teachers’ psychosocial work environment that facilitated teacher retention in a positive deviant case: a school that has the characteristics of a hard-to-staff school but that has had a low turnover rate over time. The analysis of the teachers’ narratives pointed to the importance of the teachers’ surrounding social context and suggested that their decision to remain at their school was due to being embedded in a protective professional community. The teachers’ decision to remain at the school was attributed to the teacher social capital found within this protective community which provided them with a sense of security and belonging, recognition and appreciation.The third study was a prospective longitudinal study that examined the longitudinal influence of three job demands (quantitative demands, emotional demands, work pace) and four job resources (possibilities for development, social support from supervisor, social support from colleagues, recognition) measured at baseline with exhaustion, work engagement, and retention intention measured at two timepoints (12-months and 24-months; n= 308). Moreover, the third study also explored the buffering effect of each job resource on the relationship between each job demand and exhaustion at 12-months and 24-months. The main findings in study 3 were that the influence of the health-impairing process on teachers’ retention intention is stronger than that of the motivational process and that the buffering hypothesis proposed by the JD-R model needs further development.Lastly, the fourth study examined the relationship between psychosocial safety climate, job demands, and job resources measured at the school-level with teacher turnover at 12-months and 24-months using objective turnover data from 14 schools in two Swedish municipalities. The main findings in study 4 were that the examination of school-level teacher turnover requires the consideration of labour market dynamics, and that psychosocial safety climate is a viable predictor of teacher turnover.Therefore, the combined findings in this thesis point to the importance of teachers’ work-related health; the management of psychosocial risk factors; and protective job resources such as collegial relationships for teachers’ retention intention.List of scientific papersI. Casely-Hayford, J., Björklund, C., Bergström, G., Lindqvist, P., & Kwak, L. (2022). What makes teachers stay? A cross-sectional exploration of the individual and contextual factors associated with teacher retention in Sweden. Teaching and Teacher Education. 113. https://doi.org/10.1016/j.tate.2022.103664 II. Casely-Hayford, J., Lindqvist, P., Björklund, C., Bergström, G., Kwak, L. Enculturating a Protective Professional Community—Processes of Teacher Retention in a Swedish Hard-to-Staff School. (2024). Education Sciences. 14(1), 114. https://doi.org/10.3390/educsci14010114 III. Casely-Hayford, J., Björklund, C., Bergström, G., Lindqvist, P., & Kwak, L. A longitudinal study of health-impairing and motivational processes in the psychosocial work environment and the subsequent influence on teachers’ retention intentions. [Submitted]IV. Casely-Hayford, J., Toropova, A., Björklund, C., Bergström, G., Lindqvist, P., & Kwak, L. Are schools with a better work environment better at retaining teachers? A repeated cross-sectional study examining schools’ organizational and social risk factors and teacher retention. [Manuscript]</p
Translational studies of glucocerebrosidase in Parkinson´s disease
No full textA number of genetic variants have been linked to Parkinson’s disease (PD). Among these, mutations in the GBA1 gene are identified as one of the most common risk factors for developing PD. The connection between increased PD risk and individuals with GBA1 mutations was first recognized in the 1990s, but even today, the mechanism remains unclear. GBA1 gene encodes glucocerebrosidase (GCase), a lysosomal hydrolase degrading glucosylceramide (GlcCer). The enzyme activity of GCase is reduced in PD patients, particularly among GBA1 mutation carriers. However, compromised GCase activity alone does not lead to disease development, adding complexity to its contribution to the greater risk of PD. Therefore, this thesis aims to untangle the intricate connection between GBA1 variants and PD pathogenesis.In this thesis, we take advantage of patient-derived induced pluripotent stem cells (iPSCs) as a tool for exploring the role of the GBA1 mutations in PD pathogenesis. Firstly, we develop an optimized protocol for efficiently generating midbrain dopaminergic (mDA) neurons from iPSCs. The established protocol is validated to produce mDA neurons with high reproducibility across several iPS cell lines.Using GBA1-PD patient-derived iPSCs as a reference for human samples, we investigate GBA1-specific secretome alterations in the cerebrospinal fluid (CSF) of GBA1-PD patients. The comparison of CSF and iPSC-derived mDA neurons allows us to excerpt the CSF proteins attributed to mDA neuronal populations. Notably, the most significantly altered protein in the CSF of GBA1-PD patients, FKBP4, is upregulated in the GBA1-PD mDA neurons. Our identification of FKBP4, a member of the immunophilin protein family, suggests an involvement of immune systems in GBA1-associated PD.Glycosphingolipids (GSLs) are brain-enriched lipids metabolized by lysosomal glycosidases, including GCase. Herein, we report that GBA1-PD mDA neurons exhibit decreased levels of α-2,3SpG, a neolacto-series GSL, compared with its isogenic control neurons. Also, we show a 1.39-fold elevation of α-synuclein release in GBA1-PD mDA neurons. These results highlight the alterations in GSL and α-synuclein secretion specific in GBA1-PD mDA neurons.Prosaposin (PSAP) is a precursor protein of saposin C, an essential activator of GCase. To investigate the potential of PSAP/saposin C as a therapeutic target for PD, we employ gene overexpression systems and evaluate the role of PSAP in α-synuclein pathology. Human neuroblastoma SH-SY5Y cells stably overexpressing PSAP display enhanced GCase activities with a concomitant decrease in intracellular/extracellular α-synuclein levels. small interfering RNA-mediated knockdown of endogenous PSAP shows the opposite effect on α-synuclein levels, indicating the involvement of PSAP in α-synuclein regulation. Furthermore, we demonstrate that saposin C detaches α-synuclein from an artificial lipid bilayer membrane containing GlcCer, leading us to postulate that observed α-synuclein regulation of PSAP might be attributed to saposin C’s ability to interfere with α-synuclein-to-lipid membrane interaction.To conclude, this thesis contributes to elucidating the missing link in our understanding of the molecular mechanisms underlying the elevated risk of PD among GBA1 mutation carriers, providing valuable insights into the pathogenesis of GBA1-associated PD.List of scientific papersI. Kojima R, Paslawski W, Lyu G, Arenas E, Zhang X, Svenningsson P. Secretome Analyses Identify FKBP4 as a GBA1-Associated Protein in CSF and iPS Cells from Parkinson’s Disease Patients with GBA1 Mutations. Int J Mol Sci. 2024 Jan 4;25(1):683. https://doi.org/10.3390/ijms25010683 II. Kojima R, Wallom KL, Lyu G, Paslawski W, Zhang X, Arenas E, Platt F, Svenningsson P. Altered sialyl(α2-3)paragloboside levels in GBA1 N409S (N370S) Parkinson’s disease iPSC-derived midbrain dopaminergic neurons. [Manuscript]III. Kojima R, Zurbruegg M, Li T, Paslawski W, Zhang X, Svenningsson P. Prosaposin Reduces a-Synuclein in Cells and Saposin C Dislodges it from Glucosylceramide-enriched Lipid Membranes. J Mol Neurosci. 2022 Nov;72(11):2313-2325. https://doi.org/10.1007/s12031-022-02066-y </p
Fast multi-contrast magnetic resonance imaging of the brain : a diagnostic performance study
No full textBackground: Fast MRI is beneficial in time-critical situations like ischemic stroke and motion-prone patients and to meet the increasing clinical demand. Two fast and motion-robust, single-scan multi-contrast MRI techniques, EPIMix and NeuroMix, have been developed by the MR-physics group at the Department of Neuroradiology, Karolinska University Hospital. EPIMix visualizes the whole brain with echo-planar-based two-dimensional axial images in just above one minute. The improved method NeuroMix renders, in addition to echo-planar images, even fast spin echo and three-dimensional images in 1.5-2.5 minutes, providing higher image resolution and fewer artifacts.Purpose: Evaluation of the diagnostic performance of EPIMix and NeuroMix against a reference standard.Population: Patients with suspected brain pathology (Paper I) or suspected acute ischemic stroke (Paper II-IV).Methods: In all studies, a consecutive paired cross-over multi-reader multicase study design was used to measure diagnostic accuracy. MRI of the brain with both cMRI (5-20 min) and EPIMix (1.5 min) or NeuroMix (2.5 min) were acquired and analyzed by blinded readers. The primary outcome, including sensitivity and specificity against the reference standard, was evaluated by the area under the receiver operating characteristic curve (AUC) and compared by DeLong’s test. Interrater and intrarater agreements were evaluated using kappa statistics. EPIMix sensitivity was further tested for non-inferiority compared to the reference standard in Paper III.Main results:Paper I: In a prospective cohort of 82 patients, the diagnostic performance to categorize brain MRI as abnormal or normal was high for EPIMix with 93-95% sensitivity, 100% specificity and an AUC of .99. Disease categorization was congruent between EPIMix and clinical routine MRI in 90% (Reader 2) and 93% (Reader 1). Image quality was rated lower for EPIMix (P .02 compared to cMRI). The interrater agreement was almost perfect; κ =.90 for EPIMix. Paper III: In a prospective cohort of 118 included patients, 30 (25%) had acute infarctions. EPIMix was non-inferior to cMRI with 90–100% sensitivity. Specificity was 98–100% for EPIMix. AUC was .94-1.00 (DeLong P > .15 compared to cMRI). Paper IV: In a retrospective cohort of 44 patients, including patients in the reperfusion treatment window of stroke, 34 (77%) had acute infarction. The sensitivity and specificity of infarct detection were 94–100% and 90–100%, and AUC > .95 for Neuromix (DeLong P > .15 compared to cMRI). Relative signal intensity in the infarcted area for NeuroMix strongly correlated with cMRI (R = .73 for rDWI, R =.83 for rT2-FLAIR). Inter- and intrarater agreement were almost perfect, κ > .88 for NeuroMix.Conclusions: The fast MRI techniques EPIMix and NeuroMix have high diagnostic performance for the detection of brain pathology and acute ischemic stroke.List of scientific papersI. Diagnostic performance of a new multicontrast one minute full brain exam (EPIMix) in neuroradiology: A prospective study. Falk Delgado A, Kits A, Bystam J, Kaijser M, Skorpil M, Sprenger T, Skare S. Journal of Magnetic Resonance Imaging. 2019; 50(6):1824-1833. https://doi.org/10.1002/jmri.26742 II. One-minute multi-contrast echo planar brain MRI in ischemic stroke - a retrospective observational study of diagnostic performance. Kits A, De Luca F, Kolloch J, Müller S, Mazya MV, Skare S, Falk Delgado A. Journal of Magnetic Resonance Imaging. 2021 Oct;54(4):1088-1095. https://doi.org/10.1002/jmri.27641 III. A 78 Seconds Complete Brain MRI Examination in Ischemic Stroke: A Prospective Cohort Study. Af Burén S, Kits A, Lönn L, De Luca F, Sprenger T, Skare S, Falk Delgado A. Journal of Magnetic Resonance Imaging. 2022 Sep;56(3):884-892. https://doi.org/10.1002/jmri.28107 IV. 2.5-Minute Fast Brain MRI with Multiple Contrasts in Acute Ischemic Stroke. Kits A, Al-Saadi J, De Luca F, Janzon F, Mazya V M, Lundberg J, Sprenger T, Skare S, Falk Delgado A. Neuroradiology. 2024 May; 66(5):737-747. https://doi.org/10.1007/s00234-024-03331-0 </p
Automated external defibrillator-equipped drones for facilitating early defibrillation in out-of-hospital cardiac arrest
No full textBackground: Each year, approximately 3.8 million out-of-hospital cardiac arrests (OHCAs) occur globally. Survival rates are low (8–12%) and have not increased during the past decade. It is known that early cardiopulmonary resuscitation (CPR) and defibrillation increase the chance of survival in this patient group, and if given within the first three to five minutes, the chance of survival can be as high as 50– 70%. Unfortunately, the time taken for emergency medical services (EMS) to arrive is increasing in many countries. The number of publicly available automated external defibrillators (AEDs) has increased in recent years, but they are very seldom used. A reason for this could be that most public AEDs are placed in public locations, whereas approximately 70% of all OHCAs occur in residential locations. A promising novel method for delivering AEDs to the location of an OHCA is the use of AED-equipped drones, and previous studies have shown that AED-equipped drones have the theoretical potential to decrease the time to AED arrival and their use compared with standard procedures. Studies showing real-life feasibility, however, are missing. The overall aim of the current work and the included studies was to investigate, describe and evaluate how AED-equipped drones may facilitate early defibrillation in cases of OHCA.Specific aims and methods: Study I was a registry-based retrospective study. Data were obtained from the Swedish registry for cardiopulmonary resuscitation (2010–2018) and analysed using a geographical information system (GIS). The aim was to identify areas in Sweden with a high incidence of OHCA and prolonged ambulance response times (> eight minutes), to identify optimal locations for placing AED-equipped drones. Study II was a feasibility study aiming to investigate if it is feasible to deliver AEDs using drones to patients with suspected OHCA. This was a prospective interventional study where three AED-equipped drones were dispatched and deployed in cases of suspected OHCA in western Sweden during a 4-month study period in 2020. Study III was a prospective interventional study with the aim of evaluating if AED-drones could deliver AEDs before ambulance arrival in cases of suspected OHCA. AED-drones were dispatched to cases of suspected OHCA in western Sweden over a study period of 11 months (2021–2022), and up to five AED-drones were active over the study period. Times for AED-delivery by drones were compared with ambulance response times. Study IV was an observational study including data from drone flights with AED-deliveries before EMS arrival in cases of suspected OHCA in Sweden between 2020 and 2023. The aim was to evaluate the characteristics and changes over time of dispatcher referral rate of bystanders to retrieve drone-delivered AEDs in cases of suspected OHCA. Based on experiences from Studies II and III, a bundle of interventional directives aimed to support dispatcher referral to AEDs was implemented at the dispatch centre in 2022. Emergency calls were then audited and evaluated using a case report form (CRF) based on a modified version of the Cardiac Arrest Registry to Enhance Survival (CARES).Results: In Study I it was found that by using only relatively few AED-equipped drones, the proportion of historical OHCA cases that could have received an AED on scene within eight minutes increased substantially. By using 61 AED-equipped drones, all high-incidence areas (>100 OHCAs in 2010–2018) would have been covered (all patients would have received an AED within eight minutes, either by ambulance or drone), resulting in a coverage of 58.2%. Moreover, the study revealed that to increase the proportion of coverage, the number of AED-drones needed to be increased rapidly; for example, if going from 80% to 100% coverage, the number of drones had to increase from 366 to 2408. In Study II it was found that it is possible to implement AED-drones in the dispatch and EMS system and that it is feasible to deliver AEDs by using drones (11/12 cases; 92%). There was also a trend indicating that drones arrived prior to ambulance arrival in the majority of cases (7/11; 64%) with a median time benefit of 01:52 (min:sec, interquartile range (IQR) 01:35–04:54) compared with ambulance response times. Results from Study III showed that dispatch of AED-drones was possible in both daylight and darkness, during summer and winter. The drones delivered AEDs before ambulance arrival in 37 out of 55 cases (67%). In cases where the drone arrived first, a median time benefit of 03:14 (IQR 01:42–05:42) was seen. Moreover, drone-delivered AEDs were attached in 6/18 (33%) true OHCA cases and used to defibrillate two patients. One of these patients survived to 30 days. In Study IV, a total of 99 cases of AED-delivery using drones to suspected OHCAs before EMS arrival were included. Of these, dispatcher-referral of callers occurred in 25 cases (25%). Referral in confirmed EMS-treated OHCAs occurred in 21/37 cases (57%). The AED-referral rate in cases of confirmed EMS-treated OHCAs rose from 7/18 (39%) before bundle implementation (June 2020–May 2022) to 14/19 (74%) after bundle implementation (June 2022–May 2023). Of the EMS-treated OHCAs, 16/37 (43%) AEDs were attached, and 4/37 (11%) of the cases were defibrillated. The most common reasons for non-referral in EMS-treated OHCA were single bystander, distraught/unwilling caller and call ended early. The median time benefit for drones compared with the EMS was greater in the group of referred cases (03:00 vs. 01:54).Conclusion: Altogether, the studies in this thesis show that it is feasible to implement AED-equipped drones in the chain of survival as part of the emergency medical response in cases of suspected OHCA. Drones can be deployed and deliver AEDs in the proximity of real-life cases of suspected OHCA in an automated fashion in various conditions (summer and winter, in daylight and in darkness). By using this novel method, an important time benefit compared with the standard EMS response can be introduced, and this significant time benefit creates a time window that makes the use of AEDs possible in cases of OHCA. Nevertheless, the clinical effect of the early AED-delivery is dependent on knowledge of CPR in the community, and the performance of dispatch centres during dispatcher-assisted cardiopulmonary resuscitation (DA-CPR). The AED-drone systems can be further optimized regarding performance, such as by introducing drones that can fly faster, have a longer range, and can fly in rougher conditions. Additionally, human factors, such as communication between dispatchers and laypersons/callers, could be improved so that more drone-delivered AEDs are used by laypersons during the first minutes of an OHCA.List of scientific papersI. Schierbeck S, Nord A, Svensson L, Rawshani A, Hollenberg J, Ringh M, Forsberg S, Nordberg P, Hilding F, Claesson A. National coverage of Out-of-Hospital Cardiac Arrests using Automated External Defibrillator-equipped drones - A geographical information system analysis. Resuscitation. 163, June 2021, 136-145. https://doi.org/10.1016/j.resuscitation.2021.02.040 II. Schierbeck S, Hollenberg J, Nord A, Svensson L, Nordberg P, Ringh M, Forsberg S, Lundgren P, Axelsson C, Claesson A. Automated External Defibrillators delivered by drones to patients with suspected Out-of-Hospital Cardiac Arrest. European Heart Journal. Volume 43, Issue 15, 14 April 2022, Pages 1478–1487. https://doi.org/10.1093/eurheartj/ehab498 III. Schierbeck S, Nord A, Svensson L, Ringh M, Nordberg P, Hollenberg J, Lundgren P, Folke F, Jonsson M, Forsberg S, Claesson A. Drones can deliver Automated External Defibrillators before ambulance arrival in suspected Out-of-Hospital Cardiac Arrests - A real-life prospective observational study. The Lancet Digital Health. Volume 5, Issue 12, December 2023, E862-E871. https://doi.org/10.1016/S2589-7500(23)00161-9 IV. Schierbeck S, Nord A, Svensson L, Ringh M, Nordberg P, Forsberg S, Riva G, Jonsson M, Claesson A. Dispatcher referral of bystanders to retrieve drone-delivered Automated External Defibrillators in suspected Out-of-Hospital Cardiac Arrest. [Manuscript]Appendix: Schierbeck S, Svensson L, Claesson A. Use of a drone-delivered Automated External Defibrillator in an Out-of-Hospital Cardiac Arrest. New England Journal of Medicine. May 2022; 386:1953-1954. https://doi.org/10.1056/NEJMc2200833 </p
From cytologic features to serum markers: advancing prognostication of uveal melanoma
No full textUveal melanoma (UM) is the most common primary intraocular malignancy in adults. As surveys show, almost all patients diagnosed with UM want prognostic information. However, the tools available today for early prognostication in UM are limited; beyond what information can be obtained by tumor size and location, current methods typically rely on tumor tissue obtained from enucleated eyes or invasive biopsies, or on repeated sampling of peripheral plasma or serum. Therefore, improving the toolbox for prognostication of aggressive vs. less aggressive disease is the focus of this dissertation.In paper I, we explored the scope of digital image analysis of cytologic features in uveal melanoma. We know that cytologic features such as shape and size of tumor cells can predict metastatic death in uveal melanoma. Currently, however, cytologic analysis is conducted manually by visual inspection of pathologists, which is time-consuming and impacts its reproducibility. In this study, we analyzed twelve morphometric variables in a large number of uveal melanoma cells. We then correlated the results with BRCA associated protein-1 (BAP-1) expression and BAP-1 gene mutation status, monosomy 3, gene expression classification as well as patient survival. We demonstrated that digital image analysis of variables describing the shape and size of tumor nuclei correlated to BAP-1 status, to monosomy 3, and to gene expression class. Mean time consumption per tumor was less than 2.5 min. Thus, digital image analysis is a fast and highly reproducible technique, that for the first time allows to objectively quantify cytologic tumor features in large tumors and gives prognostic information on survival in UM.In paper II, we explored if biopsies from posterior UM hold the same prognostic information as tissue from enucleated specimens. We classified BAP-1 expression in transvitreal biopsies of posterior UM and correlated our results with BAP-1 expression in subsequent biopsies. We found that BAP-1 expression in transvitreal biopsies was concordant with BAP-1 in enucleated specimens. Moreover, BAP-1 expression in transvitreal biopsies identified patients with poor metastasis-free survival. Thus, transvitreal biopsies can be a good prognostic tool for patients that do not undergo enucleation. The results also indicate the need to further study BAP-1 expression in even smaller UMs, to infer the tumor size at which loss of BAP-1 starts to occur, and to correlate it with the early seeding of micrometastases.In paper III, we explored prognostic testing on serum samples obtained from patients at diagnosis of uveal melanoma. We first screened for cancer-related proteins by protein profiling in order to find potential biomarkers. Second, we performed ELISA to evaluate the serum levels of the best candidates. Third, receiver operating characteristics were used to define thresholds for metastatic risk. This led to a prognostic test (serUM-Px) that stratifies patients into low, intermediate, and high-risk categories which we tested in a training cohort and validation cohort. We found that serUM-Px is a prognostic test, based on a single peripheral venous blood sample at the time of UM diagnosis, which can be used to stratify patients into low, intermediate and high metastatic risk categories. Thus, this test predicts metastases many years in advance without the need for biopsy or repeated sampling.In paper IV, we wanted to assess the prognostic utility of Tenascin C (TNC) in uveal melanoma. Therefore, we collected peripheral blood samples from 82 patients with small posterior uveal melanomas between 1996 and 1999 as described above for paper III. TNC concentrations were examined 2021. RNA sequencing data of TNC from an additional 80 larger tumors were collected. These levels were subsequently linked with the cumulative incidence of metastasis-related death through competing risk data analysis. In our analysis we observed no significant disparities in tumor size, age at diagnosis, visual acuity, serum protein levels, or treatment modality between patients with above or below median serum or tumor TNC levels. However, above median TNC RNA was associated with BAP1 mutation, monosomy 3, and epitheloid cytomorphology. The competing risk analysis indicated increased metastatic mortality in patients with above median TNC and primary tumor TNC RNA compared to their below median counterparts. We conclude that TNC is a prognostic biomarker in uveal melanoma that exhibits elevated levels in peripheral blood and tumors at diagnosis in patients destined for metastatic death.List of scientific papersI. Herrspiegel C, See TRO, Mendoza PR, Grossniklaus HE, Stålhammar G. Digital morphometry of tumor nuclei correlates to BAP-1 status, monosomy 3, gene expression class and survival in uveal melanoma. Experimental Eye Research. 2020;193:107987. https://doi.org/10.1016/j.exer.2020.107987 II. Herrspiegel C, Kvanta A, Lardner E, et al. Nuclear expression of BAP-1 in transvitreal incisional biopsies and subsequent enucleation of eyes with posterior choroidal melanoma. British Journal of Ophthalmology. 2021;105(4):582-586. https://doi.org/10.1136/bjophthalmol-2020-316498 III. Herrspiegel C, Plastino F, Lardner E, Seregard S, Williams PA, André H, Stålhammar G. A serum protein signature at the time of Uveal Melanoma diagnosis predicts long-term patient survival. BMC Cancer. 2023 Mar 27;23(1):277. The two first authors contributed equally to the work in Paper III. https://doi.org/10.1186/s12885-023-10757-x IV. Herrspiegel C, Plastino F, André H, Stålhammar G. Levels of Tenascin C in Peripheral Blood and Primary Tumors at the time of Uveal Melanoma Diagnosis Correlate with Long-Term Patient Survival. The two first authors contributed equally to the work in Paper IV. [Accepted] https://doi.org/10.1016/j.jcjo.2023.12.002 </p
Engineering cell communication from nanoscale to tissue levels towards therapeutic applications
No full textCell communication which is vital for the proper function, development, and survival of multicellular organisms is often disrupted in diseases. This thesis aims to enhance our understanding of cell communication by employing advanced tools that facilitate the manipulation and analysis of biological systems, spanning from the nanoscale to tissue levels, towards therapeutic applications.In Paper I, we utilized a microfluidic device to artificially create neuromuscular junctions and gain insights into nerve-muscle communication. We simulated endocrine signaling and the formation of neuromuscular junctions by using myotubes derived from primary mouse myoblasts and motor neurons derived from embryonic stem cells. Transducing skeletal muscle with PGC-1α1, increased the neuromuscular junction formation and size. Neurturin emerged as a mediator in the PGC-1α1-depentent retrograde signaling from muscle to motor neurons. This discovery may pave the way for potential therapies in diseases where neuromuscular junctions are affected early on.In Paper II, we employed DNA origami nanotechnology to harness the spatial organization of insulin receptors and control multivalent receptor activation. This innovative approach involved assembling insulin into nanoclusters on the surface of DNA origami nanostructures. Beyond in vitro assessments, we extended our investigation to in vivo studies, utilizing a zebrafish model of diabetes. Our findings not only demonstrate the effectiveness of insulin nanoclusters but also highlight the applicability of DNA origami nanostructures in the field of nanomedicine.In Paper III, we monitored the biodistribution and clearance dynamics of fluorescently labelled DNA origami nanostructures in live zebrafish embryos. We coupled advanced imaging techniques with single-cell RNA sequencing to gain insight into the interactions of DNA nanostructures with biological systems in vivo. This work serves as a guide for evaluating DNA-origami based nanomedicines in animal models.In Paper IV, we introduce a new method for monitoring the stability of DNA origami nanostructures by using the proximity ligation assay. We were able to detect the preservation of proximity between selected regions when nanostructures were bound to cultured cells as a validation of nanostructure integrity. This approach holds great potential for applications both in vitro and in vivo.List of scientific papersI. Richard Mills*, Hermes Taylor-Weiner*, Jorge C. Correia, Leandro Z. Agudelo, Ilary Allodi, Christina Kolonelou, Vicente Martinez-Redondo, Duarte M. S Ferreira, Susanne Nichterwitz, Laura H. Comley, Vanessa Lundin, Eva Hedlund, Jorge L. Ruas, Ana I. Teixeira. Neurturin is a PGC-11-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation. Mol Metab. 7, 12–22 (2018). *These authors contributed equally to this work. https://doi.org/10.1016/j.molmet.2017.11.001 II. Joel Spratt*, José M. Dias*, Christina Kolonelou, Georges Kiriako, Enya Engström, Ekaterina Petrova, Christos Karampelias, Igor Cervenka, Natali Papanicolaou, Antonio Lentini, Björn Reinius, Olov Andersson, Elena Ambrosetti, Jorge L. Ruas, Ana I. Teixeira. Multivalent insulin receptor activation using insulin–DNA origami nanostructures. Nat Nanotechnol. (2023). *These authors contributed equally to this work. https://doi.org/10.1038/s41565-023-01507-y III. Christina Kolonelou, Lars Bräutigam, Steven Edwards, Enya Engström, José M. Dias, Joel Spratt, Christos Karampelias, Stefan Wennmalm, Hjalmar Brismar, Olov Andersson, Ana I. Teixeira. Biodistribution of DNA-origami nanostructures in live zebrafish embryos with single-cell resolution. [Manuscript]IV. Christina Kolonelou, Ana I. Teixeira. A proximity ligation assay-based method to analyse the integrity of DNA origami nanostructures. [Manuscript]</p
Polycystic ovary syndrome and maternal obesity : does it programme transgenerational dysfunction?
No full textAs one of the most prevalent syndromes among reproductive-aged women, polycystic ovary syndrome (PCOS) manifests with endocrine, reproductive and metabolic disturbances. However, the etiology of PCOS is yet to be fully understood. Here in this thesis, we set out to understand whether and how PCOS can be transmitted to the future generations. More importantly, we wanted to address if the transmission of PCOS-related reproductive and metabolic phenotypes achieves a transgenerational pattern. With the help of Swedish national register-based studies and Chilean case-control studies, we obtained unique databases to investigate the disease inheritance in daughters and sons of women with and without PCOS. The use of two different PCOS mouse models further allowed us to study the transgenerational inheritance of PCOS.Study I of this thesis focused on female offspring of women with PCOS or PCOS- like mouse models. We demonstrated a five-fold increased risk of daughters of women with PCOS to be diagnosed with the syndrome. In mice exposed to androgens during late pregnancy with or without diet-induced obesity, we showed a transgenerational inheritance of irregular estrous cycles, pregnancy complications and metabolic disturbances, including increased adiposity and liver steatosis, in female offspring. We also identified preserved common gene signatures between mouse oocytes derived from all generations and serum and adipose tissue of women and daughters of women with PCOS, indicating a role of oocyte epigenetics in mediating this transgenerational transmission. It is not only the female offspring that are affected by the hyperandrogenic in utero environment. Study II revealed an increased likelihood of obesity diagnosis in sons of women with PCOS. This was further supported by mouse transgenerational studies, where great grandsons also suffer from reproductive and metabolic disturbances attributed to maternal androgen exposure, although maternal obesity elicited a wider range of alterations in phenotypes and sperm small non- coding RNAs. Study III set out to understand the molecular changes upon gestational androgen exposure. Impaired placenta development with decreased trophoblast lineage formation capacity was confirmed both in pregnant mice and human trophoblast organoids, thus identifying a potential underlying cause for PCOS-associated pregnancy complications. The findings of Study I and II led to the aim of Study IV, to identify the role of germ cells in mediating PCOS inheritance without the impact of the maternal intrauterine environment. Here a transgenerational inheritance of metabolic alterations, including increased adiposity and liver steatosis in both female and male lineages were observed. Importantly, we showed that donor exercise effectively rescued the abnormal metabolic phenotypes across the generations. Although the identification of the epigenetic mechanism underlying this inheritance is ongoing, our phenotypic data provided evidence supporting the role of germ cells in mediating PCOS transmission.List of scientific papersI. Risal, S., Pei, Y., Lu, H., Manti, M., Fornes, R., Pui, H.P., Zhao, Z., Massart, J., Ohlsson, C., Lindgren, E., Crisosto, N., Maliqueo, M., Echiburú, B., Ladrón de Guevara, A., Sir-Petermann, T., Larsson, H., Rosenqvist, M.A., Cesta, C.E., Benrick, A., Deng, Q. and Stener-Victorin, E. 2019. Prenatal androgen exposure and transgenerational susceptibility to polycystic ovary syndrome. Nature Medicine. 25(12), pp.1894-1904. https://doi.org/10.1038/s41591-019-0666-1 II. Risal, S., Li, C., Luo, Q., Fornes, R., Lu, H., Eriksson, G., Manti, M., Ohlsson, C., Lindgren, E., Crisosto, N., Maliqueo, M., Echiburú, B., Recabarren, S., Sir-Petermann, T., Benrick, A., Brusselaers, N., Qiao, J., Deng, Q. and Stener-Victorin, E. 2023. Transgenerational transmission of reproductive and metabolic dysfunction in the male progeny of polycystic ovary syndrome. Cell Reports Medicine. 4(5). https://doi.org/10.1016/j.xcrm.2023.101035 III. Lu, H., Jiang, H., Li, C., Derisoud, E., Zhao, A., Eriksson, G., Lindgren, E., Pui, H.P., Risal, S., Pei, Y., Maxian, T., Ohlsson, C., Benrick, A., Haider, S., Stener-Victorin, E. and Deng, Q. Dissecting the impact of maternal androgen exposure on offspring health through targeting the androgen receptor in developmental programming. [Submitted]IV. Lu, H., Li, C., Pei, Y., Dekanski, A., Manti, M., Risal, S., Ohlsson, C., Lindgren, E., Benrick, A., Deng, Q. and Stener-Victorin, E. Epigenetic transgenerational germline inheritance of polycystic ovary syndrome. [Manuscript]</p
Pyrocarbon in shoulder hemi arthroplasty
No full textPyrolytic carbon, or pyrocarbon (PyC), has biocompatibility, a low friction coefficient and good wear resistance, making it a material of great potential for use in orthopaedics. PyC shoulder implants have shown promising mid-term results, but there is a shortage of research that support expectations of reduced glenoid wear in shoulder hemi arthroplasty (HA), and a lack of randomised trials comparing PyC to other options of shoulder arthroplasty.This thesis synthesizes findings from four studies focusing on shoulder arthroplasty outcomes and PyC, using data from the Swedish Shoulder Arthroplasty Registry (SSAR), and clinical trials. The studies investigate various implants and describe patient-reported outcome measures (PROMs), implant stability, and revision rates across different arthroplasty types.For study 1 we utilized data on 1140 shoulders from the SSAR to compare the results after resurfacing hemi arthroplasty (HA) and stemmed HA. Younger patients were shown to have higher revision rate irrespective of implant type. Patient-reported outcome was better for patients with primary osteoarthritis (OA) when compared to patients with secondary OA.In study 2 we evaluated the reliability, validity, and responsiveness of the Swedish translation of the Western Ontario Osteoarthritis of the Shoulder index (WOOS), affirming its suitability for assessing clinical outcomes in shoulder arthroplasty patients. We could also show that WOOS is a stable and consistent tool for longitudinal outcome measurement.Study 3 compares the performance of pyrocarbon (PyC) versus CobaltChromium (CoCr) resurfacing implants in a randomised controlled trial (RCT). Findings suggest that PyC implants may offer advantages in terms of reduced glenoid erosion and lower risk of revision compared to CoCr implants, although larger studies are needed to confirm these results.In study 4, extracting data from SSAR, we analysed results after stemmed PyC HA (n=101) and compared them to results after total shoulder arthroplasty (TSA) (n=142). We noted comparable hazard ratios (HR) for revision when adjusted for confounding factors. The results from PROMs for TSA were superior to PyC HA.In conclusion, PyC HA appears to be a safe alternative in shoulder arthroplasty surgery. Our results show comparable or better outcomes than other options for HA. In comparison to TSA, PyC HA have lower outcome in PROMs but similar risk of revision. The patient demographics differ between the groups, clouding interpretation of outcomes.List of scientific papersI. Ödquist M, Hallberg K, Rahme H, Salomonsson B, Rosso A. Lower age increases the risk of revision for stemmed and resurfacing shoulder hemi arthroplasty. Acta Orthop. 2018 Feb;89(1):3-9. https://doi.org/10.1080/17453674.2017.1411081 II. Hallberg K, Salomonsson B. Validity, reliability, and responsiveness of the Swedish version of Western Ontario Osteoarthritis of the Shoulder index. BMC Musculoskelet Disord. 2022 Apr 11;23(1):351. https://doi.org/10.1186/s12891-022-05300-1 III. Hallberg K, Stark A, Ross M, Salomonsson B, Sköldenberg O. A comparison of migration patterns, glenoid erosion and clinical outcomes between a metal and a pyrocarbon resurfacing shoulder arthroplasty. A prospective single-blind randomized trial using radiostereometric analysis. [Manuscript]IV. Hallberg K, Stark A, Salomonsson B, Sköldenberg O. Pyrocarbon Stemmed Hemi Arthroplasty compared to Anatomical Total Shoulder Arthroplasty. Short-to-Mid-term Outcomes from the Swedish Shoulder Arthroplasty Registry. [Manuscript]</p