KI Open Archive Karolinska Institutet
Not a member yet
10598 research outputs found
Sort by
Molecular identities and connections of neuronal classes within the myenteric and submucosal plexuses of the mouse small intestine
No full textThe gastrointestinal (GI) tract contains its own intrinsic nervous system - the Enteric Nervous System (ENS) - which regulates essential gut functions, including intestinal motility, fluid exchange across the epithelial lining, local blood flow, and interactions with immune-endocrine system. These activities are mediated by independent neuronal circuits composed of motor neurons (MNs), intrinsic primary afferent neurons (IPANs) and interneurons (INs), organized into two concentric layers: the myenteric and submucosal plexuses. Previous studies have applied pharmacology, nerve lesion, tracing and antibody staining to characterize enteric neurons, and have found a variety of neuron subtypes with different morphological, neurochemical and electrophysiological features. However, little was known about the molecular diversity of enteric neurons largely due to the technical challenge of isolating sparsely distributed neurons in the GI tract.In this thesis, I aim to characterize the molecular neuron subtypes of ENS in mouse small intestine and further develop a method to investigate the neuron-neuron connectivity using single-cell RNA sequencing, genetic mouse lines and viral tracing tools.In study I, we focused on the neurons in myenteric plexus. Single cell RNA-seq of myenteric neurons identified 12 neuron classes (ENCs). Based on the marker genes revealed for ENCs, we acquired Cre driven mouse lines to further characterize the morphology, projection and neurochemical codes of three (ENC6, ENC7 and ENC12) putative IPANs. We found that only ENC6 corresponds to classic IPAN features. Single cell RNA sequencing of small intestine from embryonic 15.5 and 18,5 stages identified a unique developmental principle that all neurons differentiate within two trajectories (denoted Branch A and B).In study II, we focused our attention to another major plexus - submucosal plexus. Single cell RNA-seq revealed three neuron classes (smENCs), with one previously unidentified class that exhibits IPAN feature, which was validated functionally. Based on the marker genes of each class, we acquired three transgenic mouse line, and in combination of AAV vectors, we characterized the morphology and connectivity of these neurons, revealing homotypic and heterotypic connection between the three neuron classes and the close contact between all three neuron classes and 5-HT+ enterochromaffin cells. Subsequent transcriptomic analysis of ENS cells from post-natal (P7) mouse small intestine revealed a binary fate split (branch A and B) through which the identity of submucosal neurons is made, which is similar to the developmental process of myenteric plexus.In study III, we demonstrated a new method to investigate neuronal connectivity in the ENS through AAV-mediated expression of a transsynaptic tracer: wheat germ aggluttinin (WGA) fused with mCherry (mWmC). We first showed that the mWmC can target enteric neurons efficiently. Transsynaptic labelling to non-ENS cells including enteric glia cells was not observed, also not when WGA was expressed in motor neurons, known to directly interact with the smooth muscle. As examples of selective neuron classes, we assessed the neuronal connectivity of myENC5, myENC7 and myENC10 with mWmC. We find that these three subtypes of myenteric neurons have their own preferential connection to other types of neurons. In the submucosal plexus, transmission was also apparent.In conclusion, this thesis presented a comprehensive molecular map of enteric neurons, with previously undiscovered neuron classes. We further incorporated a newly developed transsynaptic tracer to study the neuronal connectivity in the ENS. With the three studies included in this thesis, we aim to enhance the understanding of enteric neurobiology and provide new tools and biological insights for future research on gastrointestinal physiology and pathophysiology.List of scientific papersI. Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing. Morarach, K*., Mikhailova, A*., Knoflach, V., Memic, F., Kumar, R., Li, W., Ernfors, P., Marklund, U. Nature Neuroscience 24, 34-46, 2021. https://doi.org/10.1038/s41593-020-00736-xII. The transcriptomes, connections and development of submucosal neuron classes in the mouse small intestine. Li, W*., Morarach, K*., Liu, Z., Banerjee, S., Chen, Y., Harb, A.L., Kosareff, J.M., Hall, C.R., López-Redondo, F., Jalalvand, E., Mohamed, S.H., Mikhailova, A., Linden, D.R., Marklund, U. Nature Neuroscience 28, 1146-1159, 2025. https://doi.org/10.1038/s41593-025-01962-xIII. Assessing connectivity in the enteric nervous system using WGA-mediated anterograde tracing. Wei Li, Ulrika Marklund. [Manuscript]</p
Studies of innate regulation of sterile inflammation in cancer and autoimmunity
No full textThe Immune system is a complex system that is composed of specialized celltypes but also response mechanisms that reside in all cells of our bodies. The immune system also relies on barrier functions and the coordinated response to insult of these barriers and infection has evolved to restore a balance with our environment. This includes mechanisms related to fighting infection but also to take care of the cells in our bodies to remove damaged cells and cells that have turned into cancer cells and no longer follow the signals in their environment. Dysregulation of the immune system can lead to both cancer and inflammatory diseases. As we know more of the immune system research has focused on targeting the immune system both to enhance and steer the response to combat cancer but also to block detritus responses and prevent autoimmunity and that the immune system destroys our own tissues and organs. In this thesis the focus is to evaluate how to overcome the immune suppression in cancer as well as to determine how the immune system is regulated during inflammatory pressure that can lead to autoimmunity.Paper I. Here we identify Neutrophils as a cell type that responds to immunotherapy for cancer and blocks the effect of the treatment. We find that IFNγ produced as a result of activation of cytotoxic cells upregulate the negative costimulatory receptor PDL1 on neutrophils. Depletion of this negative loop increases efficacy of immunotherapy and Neutrophils change their phenotype to an aging-reversed and anti-tumor state.Paper II. In this study we investigate the involvement of endosomal pattern recognition receptors in immunotherapy for cancer targeting myeloid cells. We find that myeloid cells need to sense the tumor microenvironment using these receptors to be targetable for treatment. Mechanistically, we find that in absence of endosomal TLRs myeloid cells do not upregulate the inflammasome machinery needed to respond to myeloid targeting.Paper III. Unconventional T cells have the capacity to both regulate adaptive immunity as well as exert cytotoxicity. Of these iNKT cells are quick responders and are activated by glycolipids presented on the MHC Class I like molecule CD1d. Here we find that cancer associated fibroblasts express CD1d within the tumor and during stress responses they present glycolipids to iNKT cells and activate them. Thus, we define a new pathway for regulating innate lymphocyte within the tumor microenvironment.Paper IV. In this study we investigate how B cells are regulated during autoinflammatory responses resulting in production of autoantibodies in connection to the systemic autoimmune disease SLE. We find that there is a balance between conventional and unconventional T follicular helper cells in the regulation of B cells. Thus, when we activate iNKT cells to differentiate into iNKTfh cells they influence the numbers of Tfh cells and in effect the autoreactive B cell response.List of scientific papersI. Pei SD, Pan YY, Liang H, Ravetch JV, Soehnlein O, Karlsson MCI. Neutrophil regulation of immunotherapy for cancer is controlled by type II interferon. In revision. [Submitted]II. Pan YY, Pei SD, Liang H, Westeberg K, Yu YD, He N, Ravetch JV, Mak TW, McGaha TL, Karlsson MCI. Myeloid targeting for immunotherapy of cancer requires endosomal pattern recognition. In revision. [Submitted]III. Pei S, Sjölund J, Pan Y, Pietras K, Karlsson MCI. Cancer-associated fibroblasts express CD1d and activate invariant natural killer T cells under cellular stress. Cell Mol Immunol. 2024 Jan;21(1):91-94. https://doi.org/10.1038/s41423-023-01082-1IV. He C, Wang S, Moreews M, Pei S, Chen G, Li QZ, Del Monte Monge A, Ramiro AR, Cai C, Gaya M, Barral P, Buggert M, Batista FD, Karlsson MCI. The balance between conventional and unconventional T follicular helper cells influences autoreactive B cell responses. Cell Rep. 2025 May 27;44(5):115602. https://doi.org/10.1016/j.celrep.2025.115602</p
Development and validation of prognostic factors and a diagnostic deep learning algorithm in uveal melanoma
No full textThe uveal tract comprises three anatomical structures: the iris, the ciliary body, and the choroid. Uveal melanoma (UM) is the most common primary malignant intraocular tumor in adults and has a high propensity for metastasis. Unfortunately, once radiologically detectable metastases develop, the median patient survival is only about 1.5 years. No major improvements in patient survival have been achieved during the last several decades, making it essential to identify factors that can help improve outcomes. Detecting lesions at the earliest possible stage-before they grow large-and accurately distinguishing the few truly malignant lesions from the larger number of benign counterparts is likely an effective way to enhance survival.Recognizing the factors that influence disease progression is critical for improving prognosis. Without this understanding, meaningful progress remains limited. Therefore, the main focus of this dissertation is to investigate clinical factors that help us better understand the disease, as well as to identify technological tools that can aid in earlier diagnosis. By gaining deeper insight, we aim to improve patient outcomes and make a meaningful impact on the course of the disease.In paper I, we compared the long-term prognosis for patients with iris melanomas with small choroidal melanomas. It has previously been described that iris melanomas have a more favorable prognosis compared to choroidal melanomas. However, iris melanomas are typically relatively small at diagnosis, which could contribute to their favorable prognosis. Tumor diameter, which is one of the strongest predictors of uveal melanoma prognosis, has not been taken into full account in previous studies. We have therefore compared the two tumor types and adjusted for tumor size. Our findings did not show any survival differences between iris and choroidal melanomas.In paper II, we investigated the association between vasculogenic mimicry (VM), presenting symptoms, patient outcome and the area density of periodic acid-Schiff positive histological patterns (PAS density). PAS stains a range of tissues including VM, which is a fluid conducting extracellular matrix pattern that has in many cancers been linked to worse prognosis. One of our previous studies observed that patients with uveal melanoma having a shadow in the visual field as a symptom were more likely to have retinal detachment and worse prognosis. In this study, we demonstrated that the likely reason a shadow in the visual field is associated with a worse prognosis is that these tumors are more prone to have higher PAS density and display VM.In paper III, we wanted to investigate the relationship between obesity and metabolic factors with uveal melanoma prognosis. High body mass index (BMI) is generally believed to be linked to a less favorable prognostic factor. However, our results showed that obesity and high serum levels of leptin were associated with a favorable prognosis, described as an obesity paradox.In paper IV, we developed a deep learning algorithm to distinguish small choroidal melanomas from nevi. Early detection in melanomas is crucial, as even small melanomas can metastasize and become life threatening but detecting them at an early stage is often challenging. Additionally, the task of distinguishing these lesions often falls on non-experts, making decision supporting tools even more valuable. Our algorithm not only matched but, in some cases, outperformed human ophthalmologists, making it a potential tool for clinicians in determining tumor monitoring frequencies and deciding when to refer for further evaluation or treatment.List of scientific papersI. Sabazade S, Herrspiegel C, Gill V, Stålhammar G. No differences in the long-term prognosis of iris and choroidal melanomas when adjusting for tumor thickness and diameter. BMC Cancer. 2021 Nov 24;21(1):1270. Erratum in: BMC Cancer. 2022 Jan 4;22(1):32. doi: 10.1186/s12885-021-09167- 8.https://doi.org/10.1186/s12885-021-09002-0II. Sabazade S, Gill V, Herrspiegel C, Stålhammar G. Vasculogenic mimicry correlates to presenting symptoms and mortality in uveal melanoma. J Cancer Res Clin Oncol. 2022 Mar;148(3):587-597.https://doi.org/10.1007/s00432-021-03851-9III. Sabazade S, Opalko A, Herrspiegel C, Gill VT, Plastino F, André H, Stålhammar G. Obesity paradox in uveal melanoma: high body mass index is associated with low metastatic risk. Br J Ophthalmol. 2024 Mar 20;108(4):578-587. https://doi.org/10.1136/bjo-2022-322877IV. Sabazade S, Lumia Michalski MA, Bartoszek J, Fili M, Holmström M, Stålhammar G. Development and Validation of a Deep Learning Algorithm for Differentiation of Choroidal Nevi from Small Melanoma in Fundus Photographs. Ophthalmol Sci. 2024 Aug 30;5(1):100613. https://doi.org/10.1016/j.xops.2024.100613</p
Stigma, reproductive and mental health among transgender and gender diverse individuals
No full textBackgroundTransgender and gender diverse (TGD) individuals are exposed to widespread stigma since they break gender norms dictating that all individuals should identify, look and behave according to binary gender expectations determined by the sex they were assigned at birth. The positioning of TGD individuals as abnormal and inferior is used to marginalize and control them, diminishing their access to resources and autonomy. The stress that stigma entails harms their mental health, causing important health discrepancies. On a structural level stigma against TGD individuals is present in discriminatory laws, policies and negative population attitudes that systematically disadvantage them or fail to protect their rights. This includes legislation that enforces mandatory sterilization before legal gender recognition can be approved. It remains unclear how legislation and population attitudes affect European TGD individuals health care encounters and ability to build a family. Moreso, the health care needs of gestational TGD individuals require further exploration.AimThe broader aim of this thesis was to explore how structural stigma affects health care seeking behaviors, health care encounters and the ability of TGD individuals to build a family and sustain their mental health as parents. A secondary aim was to deepen the understanding of gender dysphoria, stigma exposure, mental health concerns and health care needs among gestational TGD individuals to support law reforms and improve the mental health and quality of care for TGD individuals.MethodsThis thesis includes four studies involving quantitative and qualitative methodologies. Studies I and II are qualitative. They explored the experiences of TGD individuals who have undergone pregnancy and childbirth in Sweden after mandatory sterilization was removed from the law on legal gender recognition. The studies used inductive thematic content analysis based on face-to-face interviews.Studies III and IV are cross sectional. They mainly used multilevel logistic regression to assess the association between country level structural stigma and individual outcomes among TGD individuals living in Europe. These studies relied on a composite index, measuring structural stigma at the country level based on legislation and population attitudes. Two large scale cross-European surveys on discrimination provided the individual level data.ResultsStudy I: Health care providers in gender-affirming, antenatal and obstetrical care were perceived to regard pregnancy and masculinity as incompatible. Participants were systematically marginalized in reproductive health care, experienced microaggressions, and a lack of knowledge. They took the lead to ensure that their health needs were met. Former sterilization regulations compromised trust and expectations on health care providers, limiting disclosure. The quality of care was inconsistent.Study II: The ability to sustain gender congruence during pregnancy was central to participants. Gender norms, gender dysphoria and the coming out process affected family planning. Pregnancy and masculinity could be reconciled by renegotiating the feminine connotations of pregnancy, accessing gender- affirming treatment, dissociating from the pregnant body and hiding the pregnancy. Peer interactions contributed to resilience. Body changes and interpersonal stigma strained participants mental health, making it harder to handle microaggressions and claim their gender identity.Study III: Country-level structural stigma varied greatly across countries in Europe and was negatively associated with access to gender-affirming care and positively associated with gender identity concealment in health care. Gender identity concealment was associated with a lower exposure to discrimination in health care in lower and higher structural stigma countries.Study IV: Parenthood prevalence among TGD individuals in Europe was 8.3%. There was no statistically significant association between country-level structural stigma and parenthood. Biological parenthood was twice as common among participants AMAB than AFAB. The odds ratio of biological parenthood differed according to gender identity. Parenthood status and structural stigma did not predict depressive symptoms.ConclusionsStructural and interpersonal stigma affect trust in health care providers, limiting disclosure of clinically relevant information and access to gender-affirming care. Structural stigma marginalize gestational TGD individuals in reproductive health care, exposing them to health risks. Health professionals are at risk of conflating legal and medical boundaries when making decisions. To support health equity policy makers should eliminate structural stigma against TGD individuals. Meanwhile clinicians can empower TGD individuals who wish to undergo pregnancy by supporting their gender congruence and mental health. Further research is needed to assess the influence of structural stigma on TGD family building and parental health.List of scientific papersI. Falck, F., Frisén, L., Dhejne, C., & Armuand, G. (2021). Undergoing pregnancy and childbirth as trans masculine in Sweden: experiencing and dealing with structural discrimination, gender norms and microaggressions in antenatal care, delivery and gender clinics. International journal of transgender health. 22(1-2), 42-53. https://doi.org/10.1080/26895269.2020.1845905II. Falck, F. A. O. K., Dhejne, C. M. U., Frisén, L. M. M., & Armuand, G. M. (2024). Subjective Experiences of Pregnancy, Delivery, and Nursing in Transgender Men and Non-Binary Individuals: A Qualitative Analysis of Gender and Mental Health Concerns. Archives of sexual behavior. 53(5), 1981-2002. https://doi.org/10.1007/s10508-023-02787-0III. Falck, F., & Bränstrom, R. (2023). The significance of structural stigma towards transgender people in health care encounters across Europe: Health care access, gender identity disclosure, and discrimination in health care as a function of national legislation and public attitudes. BMC public health. 23(1), 1031. https://doi.org/10.1186/s12889-023-15856-9IV. Falck, F., Smart, B. D., Frisen, L., & Bränstrom, R. (2025). Structural stigma, parenthood patterns and depressive symptoms among transgender and gender diverse individuals across 30 countries in Europe. International journal of transgender health. 1-17. https://doi.org/10.1080/26895269.2025.2537879</p
Non-coding RNAs in psoriasis : insights into epidermal dysregulation and inflammation
No full textPlaque psoriasis is a common chronic inflammatory skin disorder characterised by pruritic and scaly focal skin lesions. Characteristic skin changes include abnormal keratinocyte proliferation, dysregulated epidermal differentiation, activation of inflammatory pathways, and infiltration of immune cells into the skin. The inflammatory axis of the cytokines interleukin-23 (IL-23) and IL-17A is recognised as a central signalling pathway in the development of psoriasis and serves as an effective therapeutic target.Non-coding RNAs have received increased attention over the past three decades as essential regulators of gene expression and translation in development, homeostasis, and disease. While some previous research has characterised the roles of various non-coding RNAs in psoriasis, their functions remain poorly understood. Therefore, this thesis investigates long non-coding RNAs and microRNAs and their role in psoriasis inflammatory signalling and keratinocyte dysfunction. Our work utilised bulk and single-cell RNA sequencing to profile the non-coding transcriptome, and we validated our findings through in vitro keratinocyte culture in monolayer and 3D epidermal models, in vivo mouse models of psoriasis skin inflammation, RT-qPCR and RNA in situ hybridisation.Study I characterised miR-378a, a miRNA upregulated in psoriasis and regulated by IL17A that activates NF-KB signalling by repressing NFKBIA. Delivery of miR-378a lead to exacerbated skin inflammation in the imiquimod mouse model and potentiated the inflammatory effects of IL-17A in cell culture through a positive feedback mechanism, emphasising its pathogenic role in psoriasis.Study II investigated the evolutionarily conserved role of miR-149 as an intrinsic suppressor of skin inflammation by repressing the TWEAK receptor. In psoriasis, miR-149 is downregulated, and epidermal deletion of miR-149 in mice leads to increased production of inflammatory mediators by keratinocytes, even in the absence of inflammatory stimuli, with no apparent phenotype resulting from its deletion in mice. In the imiquimod and Il-23 models of skin inflammation, epidermal miR-149 deletion exacerbated skin inflammation, increased immune cell infiltration, and production of inflammatory mediators. Single-cell RNA sequencing of the Il-23 model of skin inflammation identified mast cell infiltration as a notable cellular change caused by miR-149 epidermal depletion; pharmaceutical induction of mast cell apoptosis reduced imiquimod-induced skin inflammation in mice. These findings emphasise the importance of miR-149 in suppressing TWEAK/TWEAKR signalling to prevent skin inflammation, suggesting that restoring epidermal miR-149 could be a potential future treatment option for psoriasis.Study III systematically describes the landscape of long non-coding RNAs in epidermal keratinocytes within psoriasis lesions and healthy skin. The skin-enriched lncRNA RP11-295G20.2 is found to be upregulated in psoriasis, regulated by IL-17A and early epidermal differentiation, and is demonstrated to suppress late-stage keratinocyte differentiation. Based on our findings, we renamed it cytoplasmic differentiation-associated epidermal RNA CYDAER.Study IV created a single cell atlas of long non-coding RNAs in keratinocytes and immune cells in psoriasis and healthy epidermis, identifying distinct non-coding signatures of psoriasis cell states. LINC01137 is identified as a long non-coding RNA specifically overexpressed in psoriasis activated keratinocyte subtypes and regulated by IL-17A. LINC00892 is specifically expressed in Th1 and Th17 helper T cell subsets and proliferative cytotoxic T cells, possibly indicating T cell activation in psoriasis.Collectively, the four constituent studies of this thesis elucidate the critical roles of differentially expressed non-coding RNAs in the pathogenesis of psoriasis, including amplifying inflammatory signalling, disrupting keratinocyte differentiation, and modulating immune cells. The findings contribute to an improved understanding of psoriasis pathogenesis and provide potential therapeutic targets and disease biomarkers.List of scientific papersI. miR-378a regulates keratinocyte responsiveness to interleukin-17A in psoriasis. Ping Xia, Lorenzo Pasquali, Chenying Gao, Ankit Srivastava, Nupur Khera, Jan Cedric Freisenhausen, Longlong Luo, Einar Rosen, Anke van Lierop, Bernhard Homey, Andor Pivarcsi, Eniko Sonkoly. British Journal of Dermatology, 2022 Aug;187(2):211-222. https://doi.org/10.1111/bjd.21232II. Loss of epidermal microRNA-149 sensitizes to skin inflammation. Longlong Luo, Hao Yuan, Ankit Srivastava, Karl Annusver, Nupur Khera, Piyal Saha, Roxane Prieux, Kunal Das Mahapatra, Evelyn Kelemen, Menil Dholakia, Jan C Freisenhausen, Milena Petkova, Taija Mäkinen, Gunnar Pejler, Maria Kasper, Andor Pivarcsi, Eniko Sonkoly. [Manuscript]III. RNA Sequencing Reveals the Long Non-Coding RNA Signature in Psoriasis Keratinocytes and Identifies CYDAER as a Long Non-Coding RNA Regulating Epidermal Differentiation. Jan Cedric Freisenhausen*, Longlong Luo*, Evelyn Kelemen, Jonathan Elton, Viktor Skoog, Andor Pivarcsi, Eniko Sonkoly. Experimental Dermatology, 2025 Feb;34(2):e70054. https://doi.org/10.1111/exd.70054IV. Single-cell transcriptomic analysis uncovers keratinocyte- and immune-specific long non-coding RNAs in psoriasis epidermis. Jan Cedric Freisenhausen*, Longlong Luo*, Huaitao Cheng, Martin Enge, Andor Pivarcsi, Enikö Sonkoly. [Manuscript]* equal contribution.</p
Posttraumatic stress disorder and alcohol use disorder in women
No full textBackground: Most people experience traumatic events, for instance a natural disaster or the unexpected death of a loved one.Some people then develop posttraumatic stress disorder (PTSD), a debilitating disorder characterized by intrusion symptoms, e.g. intense distress and nightmares, avoidance, negative changes in cognitions and mood as well as changes in arousal and reactivity, e.g. startling easily and finding it difficult to concentrate. PTSD is approximately twice as common among women as among men.Some people find that alcohol provides short-term relief from their PTSD symptoms, for example by reducing intense distress or being able to sleep and wake without remembering one's nightmares. Over time alcohol use can lead to alcohol use disorder (AUD), which is characterized by problematic alcohol use. There are other trajectories to comorbid PTSD and AUD, but this is the most supported by research to date. PTSD and AUD often occur together.PTSD and AUD are associated with negative outcomes, e.g. other mental disorders, suicidality and ill physical health. Similarly, PTSD and alcohol use during pregnancy are associated with adverse outcomes for those pregnant as well as their expected children, including antepartum complications and fetal alcohol spectrum disorders (FASD).Comorbid PTSD and AUD tend to be more severe and more impairing than either disorder on its own. For instance, higher rates of comorbid mental disorders, suicidality and homelessness have been found among people with comorbid PTSD and AUD than among individuals with either PTSD or AUD.Comorbid PTSD and AUD are regarded as difficult to treat. Traditionally, sequential treatment, where AUD was treated first, then PTSD, was suggested. Patients were typically required to achieve and maintain abstinence before PTSD treatment was initiated, something which potentially is a great barrier to PTSD treatment for those with comorbid PTSD and AUD.Great strides have been made in developing treatment of comorbid PTSD and AUD, but the evidence on how to treat comorbid PTSD and AUD is not yet robust. Women are overrepresented among those with comorbid PTSD and AUD, yet, underrepresented in the extant treatment research. Trials of treatment of comorbid PTSD and AUD have included mainly men. Women and men may have different treatment needs and may also respond differently to treatment. So, we need to know more about treatment of comorbid PTSD and AUD in women.Objectives: The present thesis sought to estimate the current prevalence of PTSD and alcohol use during pregnancy in Stockholm, Sweden, and to investigate the safety, feasibility, and efficacy of concurrent treatment of comorbid PTSD and AUD, which does not require abstinence, in treatment- seeking women with comorbid PTSD and AUD in Swedish healthcare.Methods: Cross-sectional studies were conducted to estimate the current prevalence of PTSD and alcohol use during pregnancy. A pilot study was undertaken to investigate the safety and feasibility of concurrent treatment of PTSD and AUD in treatment-seeking women in Swedish healthcare. A randomized clinical trial was conducted to investigate whether concurrent treatment of PTSD and AUD reduces PTSD symptom severity and alcohol use more than AUD treatment in treatment-seeking women with comorbid PTSD and AUD in Swedish healthcare.Results: Approximately 4.1 percent of pregnant people are estimated to have current PTSD and approximately 4.2 percent estimated to use alcohol during pregnancy in Stockholm, Sweden. Concurrent treatment of PTSD and AUD in women was safe and feasible. In the randomized clinical trial, PTSD symptom severity and alcohol use decreased from baseline to 9-month follow-up for both treatments. There was a significantly greater reduction in PTSD symptom severity in the concurrent treatment arm than in the AUD treatment arm. There was no detectable difference in alcohol use between treatments.Conclusions: Further efforts to spread information about alcohol use during pregnancy may be needed, continued screening for alcohol use during pregnancy is warranted as well as treatment of risky alcohol use and AUD, when necessary, to reduce the risk of adverse outcomes for those pregnant as well as their expected children. It may be useful to investigate screening for PTSD in antenatal care further, to evaluate whether systematic screening for PTSD should be introduced in antenatal care. The present findings indicate that concurrent treatment of PTSD and AUD is feasible, safe, and efficacious for treatment-seeking women with comorbid PTSD and AUD in Swedish healthcare, and that abstinence is not required before or during treatment.List of scientific papersI. Persson, A; Lindmark, S; Petersson, K; Gabriel, E; Thorsell, M; Lindström, K; Göransson, M; Cardell, G; Magnusson, Å. Fear of childbirth, potentially traumatic events and posttraumatic stress disorder during pregnancy in Stockholm, Sweden: A cross-sectional study. Sexual & Reproductive Healthcare, 2020, Vol. 25, p. 100516. https://doi.org/10.1016/j.srhc.2020.100516II. Persson, A; Lindmark, S; Petersson, K; Gabriel, E; Thorsell, M; Lindström, K; Göransson, M; Cardell, G; Magnusson, Å. Alcohol and illicit and non-medical prescription drug use before and during pregnancy in Stockholm, Sweden: A cross-sectional study. Sexual & Reproductive Healthcare, 2021, Vol. 29, p. 100622. https://doi.org/10.1016/j.srhc.2021.100622III. Persson, A; Back, S E; Killeen, T K; Brady, K T; Schwandt, M L; Heilig, M; Magnusson, A. Concurrent Treatment of PTSD and Substance Use Disorders Using Prolonged Exposure (COPE): A pilot study in alcohol-dependent women. Journal of Addiction Medicine, 2017, Vol. 11(2), p. 119-125. https://doi.org/10.1097/ADM.0000000000000286IV. Persson, A; Axén, Å; Capusan, A J; Magnusson, Å; Heilig, M. Concurrent Treatment of Posttraumatic Stress Disorder and Alcohol Use Disorder in Women: A Randomized Clinical Trial. JAMA Network Open, 2025, Vol. 8(7), p. e2521087. https://doi.org/10.1001/jamanetworkopen.2025.21087</p
Towards clinically reliable artificial intelligence for prostate cancer pathology
No full textHistopathological assessment of prostate biopsies plays a central role in determining patient prognosis and guiding treatment decisions. This evaluation is typically performed by pathologists using the Gleason scoring system on hematoxylin and eosin (H&E) stained tissue sections. However, the process is subjective and prone to significant intra- and inter-pathologist variability, which can result in under- and over-treatment of patients. In cases where a definitive diagnosis cannot be made from H&E slides alone, additional immunohistochemical (IHC) staining is often used to confirm malignancy, though this introduces added cost, time, and workload. The digitisation of prostate biopsies into Whole Slide Images (WSIs) and the application of Artificial Intelligence (AI) have shown great potential to support pathologists by improving diagnostic consistency and reducing variability. Deep learning models, in particular, have demonstrated diagnostic performance comparable to that of expert pathologists. Yet, real-world deployment remains limited due to a lack of protocolised studies assessing model generalisability different laboratories, whole slide scanners, patient populations and clinically challenging cases. More recently, foundation models have shown promising pan-cancer detection capabilities, including prostate cancer, but evidence on their performance in disease-specific diagnostics is limited. This thesis addresses these gaps by developing and validating AI models for prostate cancer diagnosis with a focus on generalisability, clinical integration, and potential applications such as reducing reliance on IHC.In Study I, we developed software for accessing the .isyntax WSI format, which is the proprietary format of the Philips UFS scanner-the first scanner to obtain U.S. Food and Drug Administration clearance. Accessing these digital images allowed us to use them in subsequent studies to train AI models. In Study II, we evaluated the use of physical colour calibration for reducing scanner-induced colour variability in WSIs, with the goal of assessing the model's performance across cohorts digitised by different scanner vendors. Colour calibration showed superior performance over colour normalisation techniques, improving cancer detection and Gleason grading performance. In Study III, we have pre-registered a study protocol for the development and retrospective validation of an improved, weakly supervised AI model for diagnostic assessment of digitised prostate biopsies, and in Study IV, we have developed and validated the model rigorously following the predefined protocol. To our knowledge, this represents the largest retrospective validation of an AI model for such a purpose, including approximately 100,000 digitised core needle biopsies from 7,342 patients spanning across 15 laboratories in 11 countries. The model was trained end-to-end and showed excellent generalisation capabilities, achieving pathologist-level diagnosis and grading across international cohorts (i.e., external or internal validation with respect to the scanner used for digitisation and clinical laboratory). We compared the developed model, which we refer to as task-specific (TS), with the foundation models (FM), UNI and Virchow2. Given the potential of FMs as few- shot learners, we investigated whether their performance improves with increased prostate pathology training data. Results showed a consistent improvement in Gleason scoring across all validation cohorts for all models when more prostate pathology training data were available. We further assessed performance across different scanners and clinically challenging cases, observing improvements with increased task-specific training data. Notably, foundation models used up to 35 times more energy than the TS model during model prediction, raising concerns about their sustainability. In Study V, we used our in-house TS model for an important clinical challenge: reducing IHC staining in prostate cancer. Using a sensitivity-prioritised scenario, we retrospectively evaluated the model's performance on H&E-stained biopsies from international cohorts, selecting cases where pathologists had originally assessed the H&E-stained biopsy alongside the IHC. By lowering the malignancy classification threshold to prioritise sensitivity, the model identified all malignant slides without a single false negative, while reducing IHC usage up to 44.4% across international cohorts.To conclude, the constituent papers of this thesis address important and timely challenges in digital pathology, paving the way for future prospective clinical trials of AI models in real-world settings. These efforts support the broader integration of AI-assisted diagnostics into routine clinical practice, with the ultimate goal of reducing variability in histopathological assessment and enhancing patient care.List of scientific papersI. Mulliqi N*, Kartasalo K*, Olsson H, Ji X, Egevad L, Eklund M, Ruusuvuori P. OpenPhi: an interface to access Philips iSyntax whole slide images for computational pathology. Bioinformatics 2021 Aug 6;37(21):3995-3997.https://doi.org/10.1093/bioinformatics/btab578II. Ji X, Salmon R, Mulliqi N, Khan U, Wang Y, Blilie A, Olsson H, Pedersen BG, Sørensen KD, Ulhøi BP, Kjosavik SR, Janssen EAM, Rantalainen M, Egevad L, Ruusuvuori P, Eklund M, Kartasalo K. Physical Color Calibration of Digital Pathology Scanners for Robust Artificial Intelligence-Assisted Cancer Diagnosis. Modern Pathology 2025 Jan 16;38(5):100715.https://doi.org/10.1016/j.modpat.2025.100715III. Mulliqi N, Blilie A, Ji X, Szolnoky K, Olsson H, Titus M, Gonzalez GM, Boman SE, Valkonen M, Gudlaugsson E, Kjosavik SR, Asenjo J, Gambacorta M, Libretti P, Braun M, Kordek R, Łowicki R, Hotakainen K, Väre P, Pedersen BG, Sørensen KD, Ulhøi BP, Rantalainen M, Ruusuvuori P, Delahunt B, Samaratunga H, Tsuzuki T, Janssen EAM, Egevad L, Kartasalo K, Eklund M. Study Protocol: Development and Retrospective Validation of an Artificial Intelligence System for Diagnostic Assessment of Prostate Biopsies. medRxiv 2024.07.04.24309948. [Manuscript Preprint]https://doi.org/10.1101/2024.07.04.24309948IV. Mulliqi N, Blilie A, Ji X, Szolnoky K, Olsson H, Boman SE, Titus M, Gonzalez GM, Mielcarz JA, Valkonen M, Gudlaugsson E, Kjosavik SR, Asenjo J, Gambacorta M, Libretti P, Braun M, Kordek R, Łowicki R, Hotakainen K, Väre P, Pedersen BG, Sørensen KD, Ulhøi BP, Ruusuvuori P, Delahunt B, Samaratunga H, Tsuzuki T, Janssen EAM, Egevad L, Eklund M, Kartasalo K. Foundation Models - A Panacea for Artificial Intelligence in Pathology? arXiv:2502.21264v2 [cs.CV]. [Manuscript Preprint]https://doi.org/10.48550/arXiv.2502.21264V. Blilie A*, Mulliqi N*, Ji X, Szolnoky K, Boman SE, Titus M, Gonzalez GM, Asenjo J, Gambacorta M, Libretti P, Gudlaugsson E, Kjosavik SR, Egevad L, Janssen EAM, Eklund M, Kartasalo K. Artificial Intelligence- Assisted Prostate Cancer Diagnosis for Reduced Use of Immunohistochemistry. arXiv:2504.00979v1 [cs.CV]. [Manuscript Preprint]https://doi.org/10.48550/arXiv.2504.00979* Authors contributed equally</p
The delivery challenge : synthetic solutions for next-generation gene editing tools
No full textGene therapy has progressed rapidly in recent years, with several treatments- such as CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based therapies for sickle cell disease and beta thalassemia- receiving FDA approval. Permanent gene editing generally follows two strategies: gene replacement, where a new copy of a gene is introduced, and gene repair, where the existing gene is precisely corrected. Gene repair is preferred when feasible, as it preserves the gene's natural regulatory environment.The field has seen an explosion of new gene editing tools. Innovations like base editing and prime editing now allow for the correction of nearly any type of genetic mutation with high precision. The CRISPR/Cas9 system, which revolutionized gene editing and earned a Nobel Prize, stands out for its simplicity: retargeting it only requires changing the RNA guide, unlike earlier systems such as TALENs (Transcription Activator-Like Effector Nuclease) or zinc finger nucleases. TALENs and zinc finger nucleases were the first programmable gene editors; however, because they rely solely on protein-DNA interactions, they require custom protein engineering for each new DNA target, significantly limiting their versatility and scalability Moreover, CRISPR/Cas9 can be engineered into versatile fusion proteins for applications beyond cutting DNA (DeoxyriboNucleic Acid), including epigenetic modulation and targeted base conversions.However, a major barrier remains: efficient delivery into target cells. Cells are inherently resistant to foreign macromolecules, and current delivery methods often face inefficiency, toxicity, cost limitations, etc.This PhD project focused on developing a synthetic delivery system for CRISPR/Cas9 RNPs (RiboNucleoProteins)-the active protein-RNA complexes- rather than delivering the system as DNA or mRNA (messenger RiboNucleic Acid). The aim was to create a cost-effective, scalable method capable of transfecting even challenging cell types such as T cells, iPSCs (induced Pluripotent Stem Cells), and primary human MuSCs (Muscle Stem Cells). By simplifying and expanding access to efficient RNP delivery, this work seeks to advance both research and therapeutic gene editing applications.Paper I - Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP.This study demonstrates a proof-of-concept for repurposing nucleotide- delivering CPPs (Cell-Penetrating Peptides) for Cas9 RNP delivery. We show that Cas9 RNP can form nanoparticles with the PF14 CPP, enabling efficient uptake across multiple cell lines. Gene editing efficiency was evaluated using both the Stoplight reporter system and endogenous targets via fluorescence- and genomic analysis. Our results highlight CPP-mediated delivery as a simple, effective strategy that outperforms nearly all non-viral methods available at the time in terms of efficiency.Paper II - Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotide in vitro This study builds on previous work from our lab using endosomolytic small molecules. In collaboration with AstraZeneca, we designed new variations of a previously identified EEE (Endosomal Escape Enhancers), resulting in the development of EEE4. This molecule efficiently delivers ONs (OligoNucleotides) to a broad range of cell types with minimal cellular toxicity. The findings establish a foundation for future studies, with EEE4 representing a significant improvement over earlier generations both in terms of efficiency and safety.Paper III - Nanoparticle-based delivery of RNP gene editors using modified cell-penetrating peptides This study expands on our previous work by introducing a new peptide family, hPep, for delivering a broader range of gene editing proteins to an expanded set of cell types. These peptides maintain the high efficiency of PF14 while being gentler on cells, demonstrating the ability to deliver cargo with diverse charges - an ability not previously seen in nanoparticle-forming CPPs. A key finding was the role of silica nanoparticles (sub-40 nm) in enhancing editing efficiency. These nanoparticles likely serve as a protein and peptide adsorption core, forming more effective nanoparticles than those produced in HBG (HEPES Buffered Glucose) buffer alone. Additionally, we achieved efficient delivery to hard-to-transfect cells, including iPSCs and MuSCs, highlighting the potential of this method for advanced gene editing applications ex vivo.Paper IV - A Small-Molecule-Based Universal Endosomal Escape Strategy for Protein DeliveryBuilding on Paper II, this study uses the recently developed EEE4 endosomolytic small molecule for protein delivery of gene editing tools. It introduces a novel concept: naked gene editors can remain stable in whole serum and undergo sufficient endocytosis to enable genome editing, as long as they are efficiently released from endosomes. The method is highly scalable, easy to implement, and effective in therapeutically relevant cell types, including T cells and iPSCs. Furthermore, the study reveals a strong correlation between protein endocytosis and editing efficiency, with increased uptake leading to a significant rise in editing. This insight opens a promising path for further optimization.List of scientific papersI. Oskar Gustafsson, Julia Rädler, Samantha Roudi, Tõnis Lehto, Mattias Hällbrink, Taavi Lehto, Dhanu Gupta, Samir El Andaloussi, Joel Z Nordin. Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP. https://doi.org/10.3390/pharmaceutics13060878II. H. Yesid Estupiñán, Tom Baladi, Samantha Roudi, Michael J. Munson, Jeremy Bost, Oskar Gustafsson, Daniel Velásquez-Ramírez, Deepak Kumar Bhatt, Daniel Hagey, Dennis Hekman, Shalini Andersson, Samir EL Andaloussi and Anders Dahlen. Design and screening of novel endosomal escape compounds that enhance functional delivery of oligonucleotide in vitro. https://doi.org/10.1016/j.omtn.2025.102522III. Oskar Gustafsson, Supriya Krishna, Sophia Borate, Marziyeh Ghaeidamini, Xiuming Liang, Osama Saher, Raul Cuellar, Björn K. Birdsong, Samantha Roudi, H. Yesid Estupiñán, Evren Alici, CI Edvard Smith, Elin K. Esbjörner, Olivier Gerrit de Jong, Simone Spuler, Helena Escobar, Joel Z. Nordin, Samir EL Andaloussi. Nanoparticle-based delivery of RNP gene editors using modified cell-penetrating peptides. [Manuscript]IV. Oskar Gustafsson, H. Yesid Estupiñán, Juliette Suermondt, Samantha Roudi, Julia Radler, Sophia Borate, Xiuming Liang, Evren Alici, Joel Z. Nordin, Samir EL Andaloussi. A Small-Molecule-Based Universal Endosomal Escape Strategy for Protein Delivery. [Manuscript]</p
Intrapartum-related adverse perinatal outcomes : burden, consequences, and models of care from studies in eastern Uganda
No full textIntroduction: Intrapartum-related complications are a major contributor to adverse perinatal outcomes, including stillbirths, neonatal deaths, and intrapartum-related neonatal encephalopathy (IP-NE). However, most existing research focuses on single outcomes, often missing the broader spectrum of birth-related complications. There is also limited cohort evidence on the long- term consequences for infants who survive with IP-NE, despite known risks of developmental disabilities. Furthermore, although parental involvement in the care of newborns correlates with improved outcomes, the practical implementation of strategies that promote parent-newborn closeness, especially in low-resource neonatal settings, remains insufficiently documented.The objective: This study aimed to determine the prevalence of intrapartum- related adverse perinatal outcomes, assess one-year outcomes for survivors, and explore the experiences and perceptions of mothers and healthcare providers regarding parental participation in the care of critically ill newborns.Methods: The study was conducted at two hospitals in Eastern-Central Uganda. Sub-study 1 was a cross-sectional study with a nested case-control design, examining the births of babies weighing over 2,000 grams between June and December 2022. Data from a prospective perinatal registry were used to estimate the prevalence of intrapartum-related adverse outcomes, including stillbirths, neonatal deaths within 24 hours, and IP-NE, which was defined using an Apgar score 5.5 mmol/L, and a Thompson score of >5. Sub-study 2 followed infants diagnosed with IP-NE for one year and compared their survival outcomes to healthy newborns using the life table method. Sub-studies 3 and 4 were exploratory qualitative studies involving 18 in- depth interviews with mothers and 16 interviews with healthcare providers in neonatal units. These interviews were audio-recorded, transcribed, and analysed using reflexive thematic analysis.Results: Among 6,550 births, 10.2% resulted in adverse perinatal outcomes, including 3.8% stillbirths, 0.6% neonatal deaths, and 5.7% instances of IP-NE. Emergency referrals and caesarean sections did not alter the relationship between IP-NE and obstetric risk, except in cases of prolonged or obstructed labour. At the 12-month follow-up, healthy and mild IP-NE neonates displayed relatively high cumulative survival rates, each exceeding 95%. Neonates with moderate IP-NE experienced a lower survival rate of 87%. Notably, those with severe IP-NE faced the poorest outcomes, with only a 27% chance of survival to 12 months.From the qualitative studies, the fear of losing their baby was a predominant concern influencing both healthcare providers' and parents' experiences and perceptions in the NCU. Healthcare providers are the authority in the NCU; they focus solely on delivering lifesaving medical care and use mothers as assistants to achieve this goal. On the other hand, mothers manifested conditional trust in healthcare contingent upon improving their babies. The mothers were eager to participate in caregiving activities to improve the survival chances of their infants, which was the priority.Conclusions: This thesis shows that intrapartum-related adverse outcomes affect over 10% of births in Eastern Uganda, including stillbirths, early neonatal deaths, and IP-NE. Emergency referral and caesarean section had no impact on IP-NE risk. Severe IP-NE had the poorest outcomes, with only 27% surviving to one and a half years without disability. Moderate cases had better survival (87%), but only 67% were disability-free. Mild and healthy neonates had over 95% survival. Qualitative findings indicated that healthcare providers guide parental involvement in the NCU. The parents were willing to participate and comply with the rules, driven by their desperation for their babies' survival.List of scientific papersI. Wanduru P, Straneo M, Sadoo S, Tann C, Kakooza-Mwesige A, Rolland M, Hanson C. Prevalence, and risk of adverse intrapartum-related outcomes in Uganda: a cross-sectional study with nested case- control. [Submitted]II. Wanduru P, Straneo M, Sun S, Waiswa P, Kakooza-Mwesige A, Hanson. Infant Outcomes Following Intrapartum-Related Neonatal Encephalopathy in Eastern Central Uganda: A Cohort Study. [Manuscript]III. Wanduru P, Hanson C, Waiswa P, Kakooza-Mwesige A, Alvesson HM. Mothers' perceptions and experiences of caring for sick newborns in Newborn Care Units in public hospitals in Eastern Uganda: a qualitative study. Reprod Health. 2023;20(1):106. Epub 20230720.https://doi.org/10.1186/s12978-023-01649-1IV. Wanduru P, Hanson C, Kwesiga D, Kakooza-Mwesige A, Mölsted Alvesson H, Waiswa P. Parental participation in newborn care in the view of health care providers in Uganda: a qualitative study. Reproductive Health. 2024;21(1):155. https://doi.org/10.1186/s12978-024-01896-w</p
Phenotypes and therapy in heart failure and cardiovascular disease
No full textBackground Phosphodiesterase 5 inhibitors (PDE5i) are the first line treatment for erectile dysfunction. However, both human and animal studies have shown that they also exert beneficial effects on endothelial function, inflammation and hemodynamics. In epidemiological studies their use has been associated with lower risk of incident cardiovascular (CV) disease and of death/heart failure hospitalisation (HFH) after a myocardial infarction.Unmet needs in heart failure (HF) vary according to ejection fraction (EF) subgroups. Patients with HF and reduced EF (HFrEF) have access to life- prolonging therapies, but the implementation of these treatments remains suboptimal. In contrast, patients with HF and preserved EF (HFpEF) have limited therapeutic options, largely due to the heterogeneity of this condition, the multitude of competing risk factors, and limited pathophysiological understanding. Whether an additional HF phenotype characterized by a supranormal EF could help improve prognosis in HFpEF remains unclear.Aims The aim of study I was to assess the prognosis of men with stable coronary artery disease (CAD) treated with PDE5i vs alprostadil. The overarching aim of the subsequent three studies was to improve the understanding of certain aspects of drug implementation in HFrEF and of prognosis in HFpEF. Specifically, these studies aimed to:In HFpEF: Investigate the frequency distribution of EF, identify patient characteristics associated with EF >60%, and evaluate prognosis across varying EF values (study II) . In HFrEF: Determine the probability of mineralocorticoid receptor antagonist (MRA) discontinuation and its predictors during the first year of treatment among new users, as well as the probability of MRA reinitiation in the subsequent year (study III) In HFrEF: Examine first-year adherence to and discontinuation of sodium- glucose cotransporter 2 inhibitors (SGLT2i), along with predictors of these outcomes among new users, and evaluate the probability of SGLT2i reinitiation in the following year (study IV) Materials All studies were register-based. For study I, the cohort was selected by linking data from the National Patient Register (NPR) and the National Prescribed Drug Register. For study II, the cohort was derived from the Swedish HF Registry (SwedeHF). For studies III and IV, the cohorts were selected by linking data from SwedeHF and the National Prescribed Drug Register. Linkage with Statistics Sweden was used to retrieve socioeconomic variables in all studies. The NPR was also used to obtain information on comorbidities (for all studies) and on hospitalizations (for studies I and II). Finally, the Cause of Death Register was utilized in all studies.Study I: Among 18,542 Swedish men with stable CAD 16,548 were subsequently treated with PDE5i and 1,994 with alprostadil, between 2006-2013. PDE5i users had fewer comorbidities, required less comedications, and had higher level of education. Treatment with PD5i was independently associated with lower risk of all-cause and CV death, revascularisation, myocardial infarction and HFH. The lower risk of death was dose dependent and stronger in younger men.Study II: Among 5,576 patients with HFpEF enrolled in SwedeHF between 2017- 2021 and with EF measured continuously, 21% had EF>60%. An EF>60% was associated with more severe symptoms, female sex, hypertrophic cardiomyopathy, hypertension and valvular disease. The risk of all-cause and non- CV death and all-cause hospitalisation was higher with EF>55% in crude but not in adjusted analyses.Study III: Among 11,474 patients with HFrEF enrolled in SwedeHF between 2006- 2021 and initiated on MRA 71% remained on therapy at one year. Advanced kidney disease, hyperkalaemia, lack of follow-up in specialty care, more severe heart failure, comorbidities, and markers of sociodemographic frailty were associated with discontinuation. Among those who discontinued, 46% reinitiated treatment the following year.Study IV: Among 6,216 patients with HFrEF, enrolled in SwedeHF, who were initiated on SGLT2i therapy between 2019-2022 first-year adherence was 84% and only 16% discontinued therapy at one year. Chronic kidney disease, hypertension, diabetes mellitus, ischemic heart disease, lower income and non- use of anticoagulants were associated with non-adherence and discontinuation. Among discontinuers, 42% reinitiated treatment the following year.Conclusions The studies in this thesis suggest that:1) In men with stable CAD treatment with PDE5i vs alprostadil is independently associated with better prognosis, but confirming any causal relationship requires a randomized controlled trial.2) Patients with higher EF in HFpEF may be underrepresented in SwedeHF, have a specific phenotype and tend to have a higher crude risk of death and hospitalization for non-CV causes.3) One-third of new MRA users with HFrEF discontinue treatment within the first year, although nearly half of them restart therapy in the following year.4) Treatment persistence and adherence among new SGLT2i users with HFrEF in SwedeHF is generally very high.List of scientific papersI. Daniel P. Andersson; Laura Landucci; Ylva Trolle Lagerros; Alessandra Grotta; Rino Bellocco; Mikael Lehtihet; Martin J. Holzmann. Association of Phosphodiesterase-5 Inhibitors Versus Alprostadil with Survival in Men with Coronary Artery Disease. J Am Coll Cardiol, 2021 Mar 30;77(12):1535-1550. https://doi.org/10.1016/j.jacc.2021.01.045II. Laura Landucci; Ulrika Ljung Faxén; Lina Benson; Giuseppe MC Rosano; Ulf Dahlström; Lars H. Lund; Gianluigi Savarese. Characterizing heart failure across the spectrum of the preserved ejection fraction: does heart failure with supranormal ejection fraction exist? Data from the Swedish Heart Failure Registry. J Am Heart Assoc, 2025 Mar 13. https://doi.org/10.1161/JAHA.124.037502III. Laura Landucci; Ulrika Ljung Faxén; Lina Benson; Ulf Dahlström; Juan J. Carrero; Gianluigi Savarese; Lars H. Lund. Discontinuation and reinitiation of mineralocorticoid receptor antagonists in patients with heart failure and reduced ejection fraction. Eur J Heart Fail, 2024 Nov 26. https://doi.org/10.1002/ejhf.3523IV. Laura Landucci; Ulrika Ljung Faxén; Lina Benson; Gianluigi Savarese; Lars Lund. Adherence, discontinuation and reinitiation of Sodium- Glucose cotransporter 2 Inhibitors in patients with heart failure and reduced ejection fraction. [Manuscript]</p