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Development of novel and accessible treatments for trichotillomania and skin-picking disorder
No full textBackground: Trichotillomania (TTM) and Skin-picking disorder (SPD) are psychiatric conditions characterized by recurrent and excessive hair-pulling and skin-picking, respectively. These behaviors result in significant hair loss or skin lesions and often consume substantial amounts of time, leading to significant distress and functional impairment. Prevalence rates vary across studies but have been demonstrated to be as high as 3.5% for TTM and 5% for SPD.The recommended first-line treatment for TTM and SPD is behavior therapy (BT). Unfortunately, healthcare practitioners often lack sufficient knowledge about these disorders, limiting the availability of treatments, particularly for individuals in geographically distant areas. Group therapy offers a potential solution, allowing caregivers to treat more people in the same timeframe. Further enhancement of availability can be achieved through online delivery. While therapist-supported internet-delivered behavior therapy (iBT) has been extensively studied for related disorders, treatments specifically for TTM and SPD have only been explored through unguided or minimally supported online interventions, yielding modest effect-sizes.Objective: The thesis aimed to develop and evaluate formats for delivering BT to increase availability and accessibility for these disorders. Specifically, we aimed to: 1) test the feasibility and preliminary efficacy of ACT-enhanced group behavior therapy (AEGBT) for TTM and SPD in an open pilot study, 2) test the feasibility and preliminary efficacy iBT for TTM and SPD in an open pilot study, 3) explore the participants experiences of iBT for TTM and SPD in a qualitative study, and 4) test the feasibility and efficacy of iBT for adult patients with SPD compared to a wait-list condition in a randomized trial.Methods: In Study I, based on the original protocol for individual face-to-face therapy we developed AEGBT and conducted an open pilot study with 40 adult participants with TTM and/or SPD in mixed diagnosis groups to test the feasibility and preliminary efficacy of the treatment. In Study II, we developed iBT and conducted an open pilot study with 25 adult participants with TTM and/or SPD to test the feasibility and preliminary efficacy of the treatment. Both Study I and II were conducted in routine psychiatric care. In Study III, we explored the participants’ experiences of undergoing iBT in a qualitative study. In Study IV, conducted in an academic setting, we randomized 70 participants with SPD to either iBT or a waitlist of equal duration.Results: Study I demonstrated high group attendance and minimal treatment drop-out. AEGBT produced significant decreases in hair-pulling and skin-picking severity from pre- to post-treatment, with moderate to large within-group effects from d = 0.77 to 1.24. Symptom reduction was sustained for SPD participants, but not for those with TTM at the 12-month follow-up. The utilization of a group format allowed therapists to efficiently manage 25% more patients in comparison to an individual treatment format. Study II reported high levels of participant satisfaction and iBT credibility, reflected in high average module completion and few participants ending treatment prematurely. iBT resulted in significant decreases in hair-pulling and skin-picking severity with effect sizes ranging from d = 0.89 to 1.75. Similar to Study I, long-term efficacy favored SPD participants. Study III, identified five over-arching themes, unveiling that participants perceived iBT as beneficial and efficacious, albeit time-consuming, leading to stress in some. The treatment's flexibility was appreciated by some participants, while others expressed a need for increased support. Study IV demonstrated a significantly greater improvement in SPD symptoms in the iBT group compared to the control group at post-treatment, with a between-group effect-size in the large range (bootstrapped d = 1.3). The improvement compared to pre-treatment remained significant at the 6-month follow-up.Conclusions: In conclusion, both AEGBT and iBT proved to be effective, feasible, and safe treatment approaches for TTM and SPD in routine psychiatric care. iBT, while advantageous, presented challenges for participants. Tailoring the treatment to individual needs or blending iBT with face-to-face treatment could enhance efficacy and applicability. Additionally, iBT demonstrated preliminary efficacy and sustained long-term benefits for SPD compared to a passive control condition. Directions for future research includes evaluating AEGBT and iBT in studies with larger sample sizes and active control conditions alongside cost-effectiveness analyses comparing face-to-face behavior therapy versus these novel treatment approaches. Furthermore, in order to enable nationwide access to BT for individuals with TTM and SPD, evaluations of the implementation of these approaches in regular healthcare is needed.List of scientific papersI. Asplund, M., Rück, C., Lenhard, F., Gunnarsson, T., Bellander, M., Delby, H., & Ivanov, V. Z. (2021). ACT-enhanced group behavior therapy for trichotillomania and skin-picking disorder: A feasibility study. J Clin Psychol. https://doi.org/10.1002/jclp.23147 II. Asplund, M., Lenhard, F., Andersson, E., & Ivanov, V. Z. (2022). Internet-delivered acceptance-based behavior therapy for trichotillomania and skin-picking disorder in a psychiatric setting: A feasibility trial. Internet interventions: the application of information technology in mental and behavioural health. 30, 100573-100573. https://doi.org/10.1016/j.invent.2022.100573 III. Asplund, M., Lenhard, F., Rück, C., Forsberg, L., & Ivanov, V. Z. (2024). Experiences of internet-delivered acceptance-enhanced behavior therapy for trichotillomania and skin-picking disorder in a psychiatric setting: a qualitative study. [Manuscript]IV. Asplund, M., Lenhard, F., Rück, C., Andersson, Erik., Grimlund, T., Nilsson, M., Sarachu Nilsson, M., Sundh, L., & Ivanov, V. Z. (2024). Internet-delivered acceptance-enhanced behavior therapy for skin-picking disorder: A randomized controlled trial. [Manuscript]</p
DNA tools for imaging and manipulation of biological samples
No full textSpatial organization within biological systems is important for understanding mechanisms underlying health and disease. This thesis investigates the role of spatial distribution at cellular and molecular levels, utilizing DNA-based tools to elucidate its impact on receptor activation and creating methods for studying spatial organization of cells. The research of this thesis is structured into three studies, two focusing on clustering receptors with the aim of inducing apoptosis, one focusing on imaging and mapping cells with their transcriptomic contents.In Paper I, we investigate the spatial clustering of Death Receptor 5 (DR5) on cellular surfaces, examining its significance in the induction of apoptosis. The study demonstrates that a hexagonal arrangement of DR5-binding peptides on DNA origami structures can induce apoptosis. We identify that a 6 nm spacing in this hexagonal shape is the optimal peptide spacing for apoptosis induction. This finding highlights the significance of spatial organization in receptor signaling and introduces DNA origami as a potential platform for cancer therapy.Paper II expands on the foundation laid in Paper I with the development of a pH- sensitive DNA origami structure capable of selective DR5 activation in acidic tumor environments by using Triplex Forming Oligos (TFOs). This structure dynamically presents DR5-binding peptides in response to pH variations, thereby concentrating apoptotic activity within tumor sites where pH is lower than that of the normal physiological value. We demonstrate that the TFO forms a triplex at pH 6.5, but not pH 7.4, and can do so repeatedly. Furthermore, we demonstrate that this structure can induce apoptosis in cancer cells at pH 6.5, but not at pH 7.4, and does not induce apoptosis in other cell types at pH 7.4 either. Finally, we also show that injection of this structure into mice with growing tumors creates a reduction in tumor growth. The efficacy of this approach in inducing targeted apoptosis underscores the therapeutic potential of responsive DNA origami drug delivery systems.In paper III, we propose a novel method for spatial transcriptomics that enables the mapping of DNA spatial organization without the use of optics. Through PCR and DNA sequencing, this technique allows us to generate a spatial map of biological samples, overlayed with transcriptomic data to illustrate the spatial distribution of gene expression. We demonstrate the capacity of this method to create pure sequencing libraries. Furthermore, we show that this technique can reconstruct spatial location of artificially placed targets, preserving their relative spatial relations to each other. This method offers a potential new way for examining spatial dimensions of cellular function and gene expression.This thesis presents DNA-based methods in which the spatial organization of biological samples can be investigated or manipulated. The studies contribute to the advancement of targeted therapeutic interventions and spatial transcriptomics, offering insights that could inform future research and applications in biotechnology and medicine.List of scientific papersI. Yang Wang, Igor Baars, Ferenc Fördös, Björn Högberg. Clustering of Death Receptor for Apoptosis Using Nanoscale Patterns of Peptides. ACS Nano. 2021, 15(6), 9614−9626. https://doi.org/10.1021/acsnano.0c10104 II. Yang Wang, Igor Baars, Iris Rocamonde Lago, Yunshi Yang, Ieva Berzina, Keying Zhu, Marco Lolaico, Boxuan Shen, Robert A Harris, Björn Högberg. Peptide-pattern display from DNA origami driven by cancer acidity for selective apoptosis. Nature Nanotechnology. [Accepted]III. Igor Baars, Alexander Kloosterman, Marco Lolaico, Jakub Horváth, Anna Andersson, and Björn Högberg. Spatial reconstruction of a patterned sample using in situ PCR and DNA sequencing. [Manuscript]</p
In search of therapeutic candidates for tandem repeat disorders
No full textTriplet-repeat disorders, including Friedreich's Ataxia (FRDA) and Huntington's disease (HD), are genetic conditions characterized by the expansion of specific trinucleotide sequences within affected genes. In FRDA, the expansion of GAA•TTC repeats within the FXN gene leads to a reduction in Frataxin expression, causing debilitating symptoms. Conversely, HD is caused by an expansion of CAG•CTG repeats within the HTT gene, resulting in the production of toxic mRNA and protein. This Ph.D. thesis investigated the molecular mechanisms underlying repeat expansions in FRDA and HD, presenting innovative therapeutic strategies.In paper I, an innovative experimental system was established to analyze largescale repeat expansions in mammalian cells. Using a shuttle plasmid, the study demonstrated that LNA-DNA mixmers and PNA oligomers effectively reduce GAA•TTC repeat expansions. Additionally, it reveals that these repeats block replication fork progression, providing insight into potential therapeutic targets.Paper II focused on increasing Frataxin expression in patient-derived cells using single-strand Locked Nucleic Acid (LNA)-DNA mixmers. By targeting repeatexpanded chromosomal DNA, we achieved significant increase of FXN mRNA and protein expression. The study emphasizes the impact of oligonucleotide design on treatment effectiveness, implying the potential of chemically modified oligonucleotides (ONs) in upregulating FXN expression.In paper III, Minicircles (MCs) are introduced as non-viral DNA vectors for Frataxin gene expression. By using oligonucleotides MC production is optimized, demonstrating the potential for Frataxin expression in patient-derived fibroblasts. This research offers insights into novel gene expression methods, highlighting the efficiency and safety of MCs.In paper IV, the efficacy of Anti-gene Oligonucleotides (AGOs) in Huntington's disease (HD) was investigated. HD is another trinucleotide repeat disorder characterized by CAG•CTG expansions within the HTT gene. Screening different AGO sequences and designs revealed parameters influencing HTT downregulation. High LNA content and palmitoylated lipophilic moieties enhance AGOs' potency. RNA sequencing confirms significant downregulation of HTT and reveals regulatory effects on genes involved in immune response, mRNA processing, and neurogenesis.Together, these studies advance the understanding of triplet-repeat disorders like FRDA and HD, offering innovative therapeutic strategies and hope for affected individuals.List of scientific papersI. Anastasia Rastokina*, Jorge Cebrián*, Negin Mozafari*, Nicholas H Mandel, C I Edvard Smith, Massimo Lopes, Rula Zain#, Sergei M Mirkin#. Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers. Nucleic Acid Research. 2023, 51, 16. *Joint first authors. #Co-senior authors. https://doi.org/10.1093/nar/gkad441 II. Negin Mozafari, Salomé Milagres, Tea Umek, Cristina S. J. Rocha, Claudia Marina Vargiu, Fiona Freyberger, Osama Saher, Pontus Blomberg, Per T. Jørgensen, C. I. Edvard Smith, Jesper Wengel, Rula Zain. Anti-gene oligonucleotides targeting Friedreich’s ataxia expanded GAA•TTC repeats increase Frataxin expression. [Submitted]III. Negin Mozafari, Maureen Fredriks, Karin E. Lundin, Jesper Wengel, C. I. Edvard Smith, Pontus Blomberg. and Rula Zain. Triplexmediated purification of Frataxin expressing Minicircle DNA. [Manuscript]IV. Osama Saher, Eman M. Zaghloul, Tea Umek, Daniel W. Hagey, Negin Mozafari, Mathias B. Danielsen, Alaa S. Gouda, Karin E. Lundin, Per T. Jørgensen, Jesper Wengel, C.I. Edvard Smith and Rula Zain. Chemical modifications and design influence the potency of Huntingtin Anti-Gene oligonucleotides. NUCLEIC ACID THERAPEUTICS. 2023, 33,2. https://doi.org/10.1089/nat.2022.0046 </p
Race against time : performance of emergency medical dispatch centres in out-of-hospital cardiac arrest
No full textBackground: Out-of-hospital cardiac arrest (OHCA) is an acute medical condition where the heart suddenly stops beating, thus causing blood circulation to cease in the body with irreversible brain damage and death if not treated. Every year, about 6000 people in Sweden experience OHCA and the survival rate in 2022 was 12%. OHCA is the most urgent medical situation handled by emergency medical dispatch centres (EMDCs). However, EMDCs can greatly affect the person’s chance of survival through early recognition, rapid dispatch of Emergency Medical Services (EMS) and other resources, initiation of dispatcher-assisted cardiopulmonary resuscitation (DA-CPR) and referral to the nearest available automated external defibrillator (AED). The chances of survival decrease by approximately 10% per minute that passes without cardiopulmonary resuscitation (CPR) and defibrillation, therefore continuous optimization of time delays at the EMDC may be crucial for survival.Aim, methods and results: The overall aim of this thesis was to evaluate the handling and performance of EMDCs and dispatchers during emergency 112 calls concerning OHCA in accordance with the American Heart Association (AHA) performance goals for DA-CPR and to investigate whether a machine learning (ML) model could be used to recognize OHCA in emergency calls.The aim of study I was to investigate whether an ML model can recognize OHCA to a greater extent and at an earlier stage compared with dispatchers. Before the study, the ML model was trained to understand Swedish and to recognize OHCA in emergency calls. In this study, the dispatcher's performance was compared with the ML model for 851 OHCA calls. Of all cases of OHCA recognized, ML recognized 36% (n = 305) within 1 min compared with 25% (n = 213) by dispatchers. The proportion of recognized OHCA was 86% for ML and 84% for dispatchers. The median time to recognition was 72 s for ML and 94 s for dispatchers. Comparing the time to OHCA recognition identified by both ML and dispatchers, ML was on average 28 s (p In study II, the aim was to describe the performance of EMDCs in OHCA calls in accordance with the AHA performance goals for EMDCs regarding OHCA call handling and estimate the probability of 30-day survival related to time to recognition, time to dispatch, and time to first DA-CPR directed chest compression. The study included 936 OHCA calls, of which 79% (AHA goal, 75%) were recognized by the dispatchers. Of all cases of recognizable OHCA, 85% were recognized by dispatchers (AHA goal, 95%). DACPR chest compressions were provided in 61% (AHA goal, 75%) of OHCA calls. The median time to recognition was 113 s (AHA goal, The aim of study III was to investigate the ability of Swedish EMDCs to answer medical emergency calls and dispatch an ambulance in the event of an OHCA in accordance with the AHA performance goals in a 1-step (immediate triage at the EMDC) and a 2- step (call transferred from initial EMDC to a regional EMDC for triage) procedure over 10 years and to assess whether delays are associated with 30-day survival. A total of 9,174,940 medical emergency calls were answered with a median answer delay of 7.3 s. A total of 45,367 OHCA calls were answered with a median answer delay of 7.2 s (AHA goal, 100 s (AHA goal, minimal acceptable) (p = 0.0013). Outcome data in the 2-step procedure were not available for analysis.In study IV, the aim was to compare Swedish dispatchers’ performance during OHCA calls after dispatchers were trained and supported by a new decision support system STEP (safety, security, efficiency, precision) versus a previous criteria-based dispatch (CBD) system. The study included 958 OHCA calls of which 82% were recognized in STEP compared with 79% in CBD (p = 0.550, AHA goal, 75%). Of the cases of recognizable OHCA, 92% were recognized in STEP compared with 85% in CBD (p Conclusions: The results from study I showed that an ML model was able to recognize a larger proportion of OHCAs within the first minute compared with dispatchers and thus has the potential to be a decision support tool during live emergency 112 calls regarding OHCA. Study II showed that improvement measures are needed at Swedish EMDCs to achieve the AHA goals for OHCA call handling. More lives can potentially be saved if EMDCs optimize handling of OHCA calls in accordance with the AHA goals. Study III showed that most emergency calls were answered within the AHA's performance goals. When an ambulance was dispatched within the AHA's high-performance goal in response to OHCA calls, survival rates were higher compared with calls when dispatch was delayed. The results from study IV showed that when supported by STEP, the new decision support system, dispatchers performed better in five of six performance measures compared with when dispatchers were supported by a CBD system. Highperformance OHCA handling is a continuous process with room for improvement over time.List of scientific papersI. Byrsell F, Claesson A, Ringh M, Svensson L, Jonsson M, Nordberg P, Forsberg S, Hollenberg J, Nord A. Machine learning can support dispatchers to better and faster recognize out-of-hospital cardiac arrest during emergency calls: A retrospective study. Resuscitation. 2021;162:218–26. https://doi.org/10.1016/j.resuscitation.2021.02.041 II. Byrsell F, Claesson A, Jonsson M, Ringh M, Svensson L, Nordberg P, Forsberg S, Hollenberg J, Nord A. Swedish dispatchers’ compliance with the American Heart Association performance goals for dispatch-assisted cardiopulmonary resuscitation and its association with survival in out-of-hospital cardiac arrest: A retrospective study. Resuscitation Plus. 2022;9:100190. https://doi.org/10.1016/j.resplu.2021.100190 III. Byrsell F, Jonsson M, Claesson A, Ringh M, Svensson L, Riva G, Nordberg P, Forsberg S, Hollenberg J, Nord A. Swedish emergency medical dispatch centres’ ability to answer emergency medical calls and dispatch an ambulance in response to out-of-hospital cardiac arrest calls in accordance with the American Heart Association performance goals: An observational study. Resuscitation. 2023;189:109896. https://doi.org/10.1016/j.resuscitation.2023.109896 IV. Byrsell F, Claesson A, Ringh M, Svensson L, Jonsson M, Riva G, Nordberg P, Forsberg S, Berglund E, Hollenberg J, Nord A. A new Swedish decision support system increase dispatchers’ performance in out-of-hospital cardiac arrest calls compared with a previous criteria-based dispatch system. [Manuscript]</p
The motives and ethical justifiability of treating patients with borderline personality disorder under compulsory care
No full textThis thesis aims to investigate what motives psychiatrists have for detaining patients with BPD under compulsory care. Also, to examine hospital staff’s experiences of treating patients with BPD under involuntary care and improve the inpatient mental health care for these patients. Finally, to ethically deliberate on if or when compulsory care is justified for patients with BPD, and thereby provide ethical guidance to psychiatrists when deciding on such care. The thesis consists of five studies. Here are the abstracts:Study I: A qualitative study based on semi-structured interviews with twelve Swedish psychiatrists to investigate psychiatrists' motives for practising compulsory care of patients with BPD. Results: The qualitative data resulted in three themes: (1) patients with BPD are perceived as difficult, (2) there are medical and non-medical motives for compulsory care of patients with BPD, and (3) patients with BPD have decision competence and sometimes demand to be taken into compulsory care. Conclusion: The interviewed psychiatrists' judgements and values, rather than clinical and legal directives, were decisive in their practice of compulsory care.Study II: A normative study on the ethical justifiability of treating patients with BPD compulsorily on their demand, as Ulysses contracts. We scrutinize the arguments commonly used in favour of such Ulysses contracts: (1) the patient lacks free will, (2) Ulysses contracts as self-paternalism, (3) the patient lacks decision competence, (4) Ulysses contracts as a defence of the authentic self, and (5) Ulysses contracts as a practical solution in emergencies. Conclusion: Ulysses contracts including compulsory care should not be used for this group of patients.Study III: A questionnaire study distributed to all psychiatrists and psychiatric residents working in psychiatric emergency units or inpatient care in Sweden. The aim was to investigate their motives for treating patients with BPD under compulsory care. Results: The psychiatrists’ views were divided on when it was justified to treat patients with BPD under compulsory care. Conclusion: The large variations in doctors’ opinions indicate that the care of borderline personality disorder patients is arbitrary. Further, the assessed risk of harm seems to increase the use of compulsory care.Study IV: A questionnaire study, the respondents being nurses and psychiatric aides employed at psychiatric hospital wards in Sweden. The study aimed to investigate the healthcare staff’s experiences of treating patients with BPD under compulsory care. Results: Most respondents experienced that more than a week’s compulsory admission either increased (68%) or had no effect (26%) on self-harm behaviour. A majority (69%) considered the compulsory admissions to be too long at their wards. Respondents recommended care plans with discharge dates and around three-day-long voluntary admissions to reduce compulsory hospital admissions. Discussion: These findings imply that many patients with BPD are regularly forced to receive psychiatric care that inadvertently can make them self-harm more.Study V: A normative study on if and when compulsory care is ethically justified for patients with BPD. Analysis of commonly used arguments, which defend the use of compulsory care for patients with BPD: (1) the patients lack decision competence, (2) the patients lack authenticity, (3) compulsory care is suicide-preventive, (4) compulsory care safeguards the doctor against litigation, (5) compulsory care is a practical solution in emergencies, and (6) it is better for the caregiver to ‘err on the safe side’. Conclusion: Compulsory care is rarely ethically justified, save for exceptional situations when the clinician has probable reason to believe that the patient lacks decision capacity, e.g., by suffering from a severe mental co-morbidity, and stands to benefit from such care.List of scientific papersI. Lundahl A, Helgesson G, Juth N. Psychiatrists’ motives for practising in-patient compulsory care of patients with borderline personality disorder (BPD). International Journal of Law and Psychiatry. 2018, May-Jun; 58: 63-71. https://doi.org/10.1016/j.ijlp.2018.03.005 II. Lundahl, A., Helgesson, G. & Juth, N. Against Ulysses contracts for patients with borderline personality disorder. Medicine, Health Care and Philosophy. 2020; 23: 695–703. https://doi.org/10.1007/s11019-020-09967-y III. Lundahl A, Hellqvist J, Helgesson G, Juth N. Psychiatrists’ motives for compulsory care of patients with borderline personality disorder – a questionnaire study. Clinical Ethics. 2022, 17(4): 377390. https://doi.org/10.1177/14777509211040190IV. Lundahl A, Torenfält M, Helgesson G, Juth N. Patients with borderline personality disorder and the effects of compulsory admissions on self-harm behaviour: a questionnaire study. Nordic Journal of Psychiatry. 2023, Jul; 77(5): 498-505. https://doi.org/10.1080/08039488.2023.2166106 V. Lundahl A, Helgesson G, Juth N. Is compulsory care ethically justified for patients with borderline personality disorder? Clinical Ethics. 2024; 19(1): 35-46. https://doi.org/10.1177/14777509231216036</p
Lineage, cell state and cell identity in the developing human brain
No full textOrganisms are complex systems consisting of thousands of distinct types of cells that perform a wide array of functions, yet for the most part share the same DNA. To regulate the expression of genes, the genome contains a vast amount of complex regulatory sequences that bind transcription factors and control the activation of the transcriptional machinery. This gene regulatory process is key in the diversification of cell types and development in general. The brain is a particularly complex tissue consisting of thousands of distinct neurons. In this thesis I present two papers and a preliminary data section that are all aimed at developing and applying high throughput single-cell methods for the description of cellular heterogeneity in the brain.Paper I describes the development of a novel probe-based spatial transcriptomics method that increases throughput and extends application of smFISH to high autofluorescence tissues. This is achieved through capture of RNAs on a slide through electrophoretic transfer, followed by removal of tissue.In Paper II we used scATAC-seq and single-cell multiomics to generate an atlas of chromatin accessibility and gene expression in the first trimester developing human brain. Using this atlas, we identified key transcription factors in the development of the nervous system, for instance members of the NFI family that play a role in maturation in separate branches of the tree of neural lineages. We also trained a machine learning model to predict enhancer specificity between different immature neuron types and used this model to explain temporal dynamics in the regulation of Purkinje neuron genes. Finally, we used our dataset to predict cell types that might be involved in psychiatric disorders, finding that midbrain-derived GABAergic neurons might be disproportionately at risk in major depressive disorder.Finally, in the preliminary data section we introduce a new protocol for combined long term live imaging and in situ sequencing of human tissue samples. We applied this method to the first trimester cortex and to patient-derived glioblastoma samples. In the glioblastoma samples we were able to track nearly 1,000 cells that we could then also annotate based on gene expression. Interestingly we found that many cells were migrating along a blood vessel and that tumor cells that are similar to neural progenitor cells tended to move greater total distances than other tumor cells.List of scientific papersI. Scalable in situ single-cell profiling by electrophoretic capture of mRNA using EEL FISH. Lars E. Borm, Alejandro Mossi Albiach, Camiel C. A. Mannens, Jokubas Janusauskas, Ceren Özgün, David Fernández-García, Rebecca Hodge, Francisca Castillo, Charlotte R. H. Hedin, Eduardo J. Villablanca, Per Uhlén, Ed S. Lein, Simone Codeluppi and Sten Linnarsson. Nature Biotechnology. 41, 222–231 (2023).https://doi.org/10.1038/s41587-022-01455-3II. Chromatin accessibility during human first trimester neurodevelopment. Camiel C.A. Mannens, Lijuan Hu, Peter Lönnerberg, Marijn Schipper, Caleb Reagor, Xiaofei Li, Xiaoling He, Roger A. Barker, Erik Sundström, Danielle Posthuma, Sten Linnarsson. Nature.https://doi.org/10.1038/s41586-024-07234-1</p
From vaccination to infection : mechanistic studies of immune responses to SARS-CoV-2
No full textThe emergence of a novel coronavirus, SARS-CoV-2, sparked a global health crisis, leading to the COVID-19 pandemic. Development and deployment of vaccines against COVID-19 elevated population-level immunity and reduced COVID-19-related morbidity and mortality worldwide. However, recurrent infections are frequent and the immune mechanisms conferring lasting protection remain elusive. In this thesis, I focused on characterizing how adaptive immune responses to COVID-19 vaccines evolve over time in terms of magnitude, quality and durability, and what immune mechanisms contribute to lasting protection from SARS-CoV-2 infection. The work was performed in a non-human primate (NHP) model, whose physiological and immunological similarity to humans provides essential translational insights about SARS-CoV-2 infection and prophylaxis.In Paper I, I characterized the innate and adaptive immunity after unmodified mRNA vaccination. A low dose of COVID-19 mRNA vaccine elicited a type I interferon-related innate immune activation but did not elicit detectable virus-specific adaptive responses with a single dose. Two boost immunizations, one and six months after prime, were required for induction of robust B and T cell immunity. Increased avidity of serum antibodies after each dose indicated significant affinity maturation of the B cell response. Mechanistically, the low dose of mRNA vaccine disseminated to fewer draining lymph node clusters compared to a higher dose, suggesting that poor immunogenicity after the first vaccination is a result of restricted priming.In Paper II, I evaluated the evolution of immune responses over three immunizations with a protein subunit COVID-19 vaccine, and their capacity to provide durable protection from SARS-CoV-2 infection. Each immunization expanded the neutralizing breadth towards different SARS-CoV-2 variants, while simultaneously promoting affinity maturation of virusspecific B cells. Vaccine-elicited immunity, particularly after three immunizations, conferred significant protection from SARS-CoV-2 infection. Both virus-specific neutralizing antibodies in the blood before infection as well as anamnestic immune responses in the lungs early after infection were associated with reduced viral replication, underscoring the importance of mucosal immunity for effective defense against respiratory viruses.In Paper III, I continued to follow the NHPs from the previous study to investigate the protective effects of combined infection- and vaccine-derived immunity, also known as hybrid immunity. Pre-existing immunity, whether hybrid or a result of a past infection, reduced viral loads in the respiratory tract after a reinfection with a highly divergent forward drift SARS-CoV-2 variant XBB.1.5. Reinfection, but not intramuscular boost vaccination, significantly expanded lung tissueresident memory T cells and mucosal antibodies, which were associated with protection from infection in the lower respiratory tract. This positions infection-elicited tissue-resident memory T cells as one of the key players able to reduce recurrent infection rates at the mucosal sites.Collectively, these studies provide a deeper understanding of immunity in response to COVID-19 vaccines and immunological mechanisms critical for control of SARS-CoV-2 infection, with possible implications for development of vaccines against respiratory viruses.List of scientific papersI. Lenart K, Hellgren F, Ols S, Yan X, Cagigi A, Cerveira RA, Winge I, Hanczak J, Mueller SO, Jasny E, Schwendt K, Rauch S, Petsch B, Loré K. A third dose of the unmodified COVID-19 mRNA vaccine CVnCoV enhances quality and quantity of immune responses. Molecular Therapy Methods & Clinical Development. 2022 Dec 8; 27:309-323. https://doi.org/10.1016/j.omtm.2022.10.001 II. Lenart K, Arcoverde Cerveira R*, Hellgren F*, Ols S, Sheward DJ, Kim C, Cagigi A, Gagne M, Davis B, Germosen D, Roy V, Alter G, Letscher H, Van Wassenhove J, Gros W, Gallouët AS, Le Grand R, Kleanthous H, Guebre-Xabier M, Murrell B, Patel N, Glenn G, Smith G, Loré K. Three immunizations with Novavax's protein vaccines increase antibody breadth and provide durable protection from SARS-CoV-2. NPJ Vaccines. 2024 Jan 20; 9(1):17. *Equal contribution. https://doi.org/10.1038/s41541-024-00806-2 III. Lenart K, Feuerstein H, Perez Vidakovics L, McInerney G, Guebre-Xabier M, Trost JF, Eriksson I, Smith G, Patel N, Loré K. SARS-CoV-2 reinfection but not booster vaccination expands lung tissue-resident memory CD8 T cells. [Manuscript]</p
Unraveling predictive indicators for therapeutic response in HER2-positive breast cancer
No full textBreast cancer is a highly heterogeneous disease, exhibiting significant diversity both in its biological subtypes and clinical manifestations. Biologically directed therapies have revolutionized the treatment landscape for breast cancer in the past three decades, offering more targeted and personalized approaches and have significantly changed the treatment paradigm. This shift in treatment conception has indeed resulted in improved outcomes for patients, driven by extensive research efforts aimed at personalized biomarker exploration.The aim of my thesis was to identify biomarkers for optimizing therapy selection in HER2- positive breast cancer.In paper I, we concentrated on current evidence regarding the dynamics of tumor-infiltrating lymphocytes (TILs) during neoadjuvant treatment and examined the fluctuating patterns of TILs, correlating them with treatment prediction and survival outcomes. We observed a consistent decrease in TILs levels after neoadjuvant therapy (NAT) across all breast cancer subtypes, with a numerically larger decrease noted in HER2-positive breast cancer. In TNBC patients, increased TILs during treatment were associated with better disease-free survival (DFS) or recurrence-free survival (RFS), as indicated by pooled hazard ratios from univariate analyses of four eligible studies. However, due to insufficient studies, this analysis was limited to TNBC. Additionally, we identified eight studies reporting on-treatment TILs counts, which was uniformly increased compared to baseline levels. Increased on-treatment TILs compared to bassline were positively associated with pathological complete response (pCR) status in seven out of total eight studies, but no pooled analysis was done due to data heterogeneity. These findings suggest that dynamic monitoring of TILs may serve as a flexible and economical biomarker for treatment de-escalation and future trial design, particularly in HER2-positive and TNBC patients.In paper II, a comprehensive analysis was conducted to assess the predictive and prognostic significance of baseline and serial levels of serum thymidine kinase (sTK1) in patients with HER2-positive early breast cancer enrolled in the PREDIX HER2 trial. At baseline, no association was found between serum thymidine kinase 1 (sTK1) levels and clinicopathological characteristics such as age, tumor grade, and Ki-67 status. We observed a dramatic increase in TK1 activity in all patients after two cycles of treatment, although neither baseline sTK1 levels nor sTK1 levels at subsequent on-treatment time points were associated with pathological complete response (pCR) status. Furthermore, there was no significant effect of baseline or cycle 2 sTK1 activity on event-free survival (EFS). For patients with residual disease (non-pCR), a higher sTK1 activity at the end of treatment visit appeared to be linked with longer survival time, though the association did not reach statistical significance. Our study provides evidence of sTK1 activity dynamics in a prospective phase II trial, although no prognostic association was identified.In paper III, we profiled intrinsic molecular subtypes in longitudinally collected tissue material obtained from patients enrolled in the PREDIX HER2 trial and explored their association with treatment response and long-term outcomes. The PAM50 intrinsic subtypes were determined using an SSP-based method. The results revealed that the majority of patients at baseline were classified as HER2-enriched (HER2-E) subtype (55%), as expected. Approximately 40% of patients were categorized as Luminal A or Luminal B types, while the remaining were classified as basal-like (BL) subtype. The baseline HER2-E subtype showed a significant association with better pCR and EFS. Under treatment, intrinsic subtypes exhibited temporal plasticity, with the majority of patients experiencing a subtype switch. Specifically, 71 out of 93 HER2-E patients transitioned to a non-HER2-E subtype from baseline to on-treatment. The present study highlights the potential utility of PAM50 intrinsic molecular subtypes for prognostication in HER2-positive breast cancer, and a prospective validation clinical trial is ongoing. Further exploration of the clinical implications associated with subtype switching during treatment is needed.In summary, our research provides a comprehensive biomarker exploration aimed at predicting treatment response and adding prognostic value in patients with early HER2- positive breast cancer. Through these investigations, we aim to enhance our understanding of breast cancer heterogeneity and improve treatment escalation and de-escalation strategies for achieving better patient outcomes.List of scientific papersI. Yajing Zhu, Evangelos Tzoras, Alexios Matikas, Jonas Bergh, Antonios Valachis, Ioannis Zerdes, Theodoros Foukakis. Expression patterns and prognostic implications of tumor-infiltrating lymphocytes dynamics in early breast cancer patients receiving neoadjuvant therapy: A systematic review and meta-analysis. Frontiers in Oncology. 12 (2022): 999843. https://doi.org/10.3389/fonc.2022.999843 II. Yajing Zhu, Ioannis Zerdes, Alexios Matikas, Ivette Raices Cruz, Mattias Bergqvist, Ellinor Elinder, Ana Bosch, Henrik Lindman, Zakaria Einbeigi, Anne Andersson, Lena Carlsson, Ann Charlotte Dreifaldt, Erika Isaksson Friman, Mats Hellstrom, Hemming Johansson, Kang Wang, Jonas C. S. Bergh, Thomas Hatschek, Theodoros Foukakis. The role of serum thymidine kinase 1 activity in neoadjuvant-treated HER2-positive breast cancer: biomarker analysis from the Swedish phase II randomized PREDIX HER2 trial. Breast Cancer Research and Treatment. (2024): 1-10. https://doi.org/10.1007/s10549-023-07200-x III. Yajing Zhu, Emmanouil Sifakis, Kang Wang, Ioannis Zerdes, Jonas Bergh, Thomas Hatschek, Alexios Matikas, Theodoros Foukakis. Intrinsic molecular subtype changes during and after neoadjuvant HER2-targeted therapy: an exploratory analysis of the Swedish PREDIX HER2 trial. [Manuscript]</p
High-capacity in vitro models for the assessment of pain-related mechanisms
No full textChronic pain affects one in five people. Yet, new efficient drugs with fewer adverse effects remain an unmet need for chronic pain. In vitro models that closer recapitulate relevant disease mechanisms are suggested as a way forward to support drug development and to decrease the failure rate of drug candidates during the transition from preclinical to clinical studies. While current more physiologically relevant in vitro models have shown the possibility to replicate certain aspects of chronic pain diseases, these models are often complex and do not allow the automated experiments at scale which are required in a drug screening context. Therefore, in this thesis methods to create scalable in vitro models that closer resemble in vivo pain transmission were developed and evaluated. Moreover, high-capacity in vitro models were applied for mechanism of action studies.In study I, a high-capacity microchannel plate (MC-plate) was characterized for the application in neuroscience research. The MC-plate parallelizes 96 compartmentalized cell cultures in a conventional 384-well format and was demonstrated to be compatible with primary rodent dissociated neuronal cultures of the peripheral and central nervous system, as well as human induced pluripotent stem cell (iPSC)-derived sensory neurons. In addition, the plate design allowed selective axonal growth to adjacent compartments and the fluidic integrity supported localized treatment.The MC-plate was further used to develop an in vitro co-culture model of dissociated dorsal root ganglia (DRG) neurons and spinal cord (SC) neurons (Study II). The cell populations were cultured in spatially separated compartments, to attempt to replicate the pain pathway. In this way, the axons of DRG neurons were growing through the microchannels, thus providing the possibility to model synaptic connections of the first pain transmission point in the nervous system. The functional synaptic connections between the two cell types were assessed with electric field stimulation (EFS). EFS was applied to DRG neurons and induced a transient calcium influx in SC neurons in a timed manner. These results were validated using antagonists targeting receptors of excitatory synaptic transmission. The timed signals in the SC neuronal compartments were fully or partially inhibited by the AMPA receptor antagonist NBQX and NMDA receptor antagonist MK801, respectively, confirming that synaptic mechanisms are involved in the observed signal.In study III, a novel technique of cell adhesion-based bioprinting was evaluated and used as an alternative method to create spatially defined DRG cultures. Several cell lines were successfully patterned in 2D and 3D using the bioprinting method based on cell-surface interactions. Moreover, dissociated DRG neurons were printed and cultured for up to 9 days in 2D.In study IV, the mechanisms contributing to pain relief, observed in patients with rheumatoid arthritis (RA) when treated with the JAK/STAT antagonist baricitinib, were studied using both in vitro and in vivo techniques. A previously established high-capacity in vitro DRG model, in a conventional 384-well format, was used to investigate the direct effect of baricitinib on neuronal excitability and morphology. In the in vitro experiments, baricitinib decreased excitability of DRG neurons. Baricitinib also altered the morphology of satellite glial cells (SGCs) which was quantified as a decrease in length of glial fibrillary acidic protein (GFAP) positive extensions of SGCs. Moreover, combining the in vitro results with in vivo experiments of a collagen antibody-induced arthritis model confirmed baricitinib targeting JAK/STAT and SGCs but it also indicated adaptor-associated protein kinase 1 (AAK1) as an additional target in DRGs that contributes to the observed analgesic effect.In summary, this thesis evaluated methods to create and apply scalable in vitro methods to contribute to mechanistic studies and drug screening in chronic pain in a drug discovery context.List of scientific papersI. A Microfluidic High-Capacity Screening Platform for Neurological Disorders. Lydia Moll, Johan Pihl, Mattias Karlsson, Paul Karila, and Camilla I. Svensson. ACS Chemical Neuroscience. 2024,15 (2), 236-244. https://doi.org/10.1021/acschemneuro.3c00409 II. A high-capacity in vitro model of synaptically connected peripheral and central neurons for pain research. Lydia Moll, Christina Nodin, Johan Pihl, Mattias Karlsson, Paul Karila, and Camilla I. Svensson. [Manuscript]III. 3D micro‑organisation printing of mammalian cells to generate biological tissues. Gavin D. M. Jeffries, Shijun Xu, Tatsiana Lobovkina, Vladimir Kirejev, Florian Tusseau, Christoffer Gyllensten, Avadhesh Kumar Singh, Paul Karila, Lydia Moll, Owe Orwar. Scientific Reports. 2020, 10, 19529. https://doi.org/10.1038/s41598-020-74191-w IV. Characterization of the antinociceptive effect of baricitinib in the collagen antibody-induced arthritis mouse model. Nils Simon*, Resti Rudjito*, Lydia Moll, Katalin Sandor, Juan Antonio Vazquez Mora, Carlos Morado Urbina, Sven David Arvidsson, Qing Luo, Qiaolin Deng, Arisai Martínez Martínez, Jens Gammeltoft Gerwien, Paul Karila, Venkatesh Krishnan, Juan Miguel Jiménez-Andrade, Camilla I Svensson. * Contributed equally [Manuscript]</p
Targeting the ovary : mapping mechanisms to link endocrine-disrupting chemicals to female fertility
No full textIn recent decades, growing attention has been paid to endocrine-disrupting chemicals (EDCs) as mounting evidence suggests that they can cause detrimental reproductive outcomes in animals and humans. Even though epidemiological studies show the link between EDC exposure and adverse reproductive outcomes such as sub-fertility, longer time-to-pregnancy, and decreased ovarian reserve in women, the underlying mechanisms are not fully elucidated. This is challenging for chemical regulation as we need mechanistic information to properly identify EDCs that may cause detrimental effects. In this thesis, we aim to map the mechanisms linking EDCs to female infertility, identify potential biomarkers of exposure using in vitro culture systems, and carry out cross-species comparisons using rat models.Paper I characterized the effects of in vitro culture alone on ovarian cortical tissue using transcriptomic profiling and investigated the feasibility of using this model to study the effects of exposure to EDCs on the human ovary. RNA sequencing (RNA-seq) revealed a marked change induced by tissue fragmentation and culture itself, including changes in energy metabolism (i.e., glycolysis). Follicles were activated to grow during the culture, which could be explained by the disruption of the Hippo signaling, and partially, by upregulation of the glycolysis pathway. Papers II-III investigated the effects of selected chemicals (pharmaceuticals: DES, KTZ; persistent organic pollutants (POPs): HCB, DDE, PCB156, PCB180, PFOS, and their mixture) on ovarian cells and tissue in culture. All exposures affected follicle growth in culture. Additionally, exposure to POPs resulted in increased follicle atresia in in vitro culture. Using transcriptomic profiling, we found disruption of lipid biology and energy homeostasis as well as increased oxidative stress as potential novel mechanisms connecting chemical exposures to disrupted folliculogenesis. Furthermore, we identified stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) as potential biomarkers of chemical exposure.Papers IV-V investigated the reproductive outcomes induced by DES and KTZ exposure in rats and explored the changes in endpoint sensitivity during pubertal and adult exposure. Moreover, we also developed and assessed the feasibility of using surface photo counting (SPC) as a fast tool to prioritize chemical exposure groups for further histological evaluation. In general, we found that high-dose exposure to DES and KTZ disrupted folliculogenesis in rats. When comparing endpoint sensitivity between different exposure periods, our results suggested that no profound differences can be observed, although pubertal exposure allowed the inclusion of vaginal opening as a sensitive endpoint to estrogenic chemicals. In addition, we showed that the quantification results obtained from the SPC method were significantly correlated with that of traditional histological assessment. Therefore, SPC could be used as a complementary method to prioritize groups for histology analysis.In summary, in vitro ovarian tissue culture can be used to study the impact of chemicals on follicle survival and growth, and underlying mechanisms. Utilizing this model, we found that exposure to the selected chemicals affected folliculogenesis, through disruption of their energy metabolism and increased oxidative stress. Similarly, we showed that high-dose DES and KTZ exposure disrupted follicle growth in rats, but not in low- and middle-dose groups. This suggests that the investigated endpoints in the in vivo study were not sensitive enough. It advocates the need for a sensitive and human-relevant assay for the screening of EDCs present in the global market. The identified common signatures and biomarkers might be used as a base for the future development of such screening assays after validation. Even though this is a small step, we are moving towards the future of a chemical-safe world.List of scientific papersI. Jie Hao1*, Tianyi Li1, Manuel Heinzelmann, Elisabeth Moussaud, Filipa Lebre, Kaarel Krjutškov, Anastasios Damdimopoulos, Catarina Arnelo, Karin Pettersson, Ernesto Alfaro-Moreno, Cecilia Lindskog, Majorie van Duursen, Pauliina Damdimopoulou. Effects of in vitro activation vs fragmentation on human ovarian tissue and follicle growth in culture. 1Equal contribution *Corresponding author [Submitted]II. Tianyi Li*, Paraskevi Vazakidou, Pim E G Leonards, Anastasios Damdimopoulos, Eleftheria Maria Panagiotou, Catarina Arnelo, Kerstin Jansson, Karin Pettersson, Kiriaki Papaikonomou, Majorie van Duursen, Pauliina Damdimopoulou. Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using In Vitro Models. Toxicology. 2023 Feb;485:153425. *Corresponding author https://doi.org/10.1016/j.tox.2023.153425 III. Tianyi Li, Richelle D Björvang, Jie Hao*, Valentina Di Nisio, Anastasios Damdimopoulos, Cecilia Lindskog, Kiriaki Papaikonomou, Pauliina Damdimopoulou. Persistent Organic Pollutants Dysregulate Energy Homeostasis in Human Ovaries In Vitro. *Corresponding author. [Submitted]IV. Julie Boberg, Tianyi Li, Sofie Christiansen, Monica Kam Draskau, Pauliina Damdimopoulou, Terje Svingen, and Hanna KL Johansson*. Comparison of female rat reproductive effects of pubertal versus adult exposure to known endocrine disruptors. Front Endocrinol (Lausanne). 2023 Oct 3:14:1126485. *Corresponding author https://doi.org/10.3389/fendo.2023.1126485 V. Tianyi Li*, Julie Boberg, Hanna K L Johansson, Valentina Di Nisio, Sofie Christiansen, Terje Svingen, Pauliina Damdimopoulou. Quantitative analysis of ovarian surface photographs as a tool for assessment of chemical effects on folliculogenesis and ovulation in rats. Reprod Toxicol. 2023 Aug 1;119:108416. *Corresponding author https://doi.org/10.1016/j.reprotox.2023.108416 </p