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The diversity of MAIT cells across the human body and in COVID-19
No full textMAIT cells guard the immune homeostasis of barrier sites across the human body and circulate through peripheral blood, constantly patrolling for signals of infection and inflammation. Despite their presence in virtually every organ, our knowledge of the immunobiology of MAIT cells in the human immune system is largely limited to peripheral blood. The aim of this thesis is to advance our knowledge on the role of MAIT cells in human tissue immunology. To this end, we explored the distribution, activation, and functional adaptation of MAIT cells across different human tissues, particularly in response to viral disease such as COVID-19.In Study I, we characterized human MAIT cells in donor-matched organs or blood and detected compartmentalization patterns regarding population size and site- specific adaptations. We demonstrate heterogeneity of MAIT cells within the same individual, featuring a CD39hi CD27low barrier-protective phenotype in the gut and a dynamically adoptable, liver-dominant CD56-associated enhanced effector program.In Study II, we monitor the dynamics of MAIT cell activity in patients suffering from acute COVID-19. We detected a decline of MAIT cells in circulation and a recruitment to the inflamed lungs of COVID-19 patients. MAIT cells were highly inflammatory in the infected airways, and circulating MAIT cells showed signs of residual activation which was associated with disease severity.In Study III, we followed the survivors of severe COVID-19 in longitudinal-paired analysis and detected a transient inconsistent recovery of MAIT cells. In the early resolution phase of the disease, MAIT cells normalized numerically in the circulation, but remained residually activated. In later convalescence, MAIT cell numbers declined, and were functionally impaired in a subset of patients exhibiting an exhausted PD-1high phenotype.Together, this thesis explores the role of MAIT cells in human mucosal immunology to shed light on aspects of MAIT cell diversity in health and viral disease.List of scientific papersI. Kammann T, Cai C, Sekine T, Mouchtaridi E, Boulouis C, Nilsén V, Rivera-Ballesteros O, Müller TR, Gao Y, Raineri EJM, Akhirunnesa M, Adamo S, Constantz C, Niessl J, Weigel W, Kokkinou E, Stamper C, Marchalot A, Bassett J, Ferreira S, Rødahl I, Wild N, Brownlie D, Tibbitt C, Mak JYW, Fairlie DP, Leeansyah E, Michaelsson J, Marquardt N, Mjösberg J, Jorns C, Buggert M, Sandberg JK. MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles. Sci Immunol. 2024; 9(99):eadn2362. https://doi.org/10.1126/sciimmunol.adn2362II. Parrot T*, Gorin JB*, Ponzetta A, Maleki KT, Kammann T, Emgård J, Perez-Potti A, Sekine T, Rivera-Ballesteros O, Karolinska COVID-19 Study Group, Gredmark-Russ S, Rooyackers O, Folkesson E, Eriksson LI, Norrby-Teglund A, Ljunggren HG, Björkström NK, Aleman S, Buggert M, Klingström J, Strålin K, Sandberg JK. MAIT cell activation and dynamics associated with COVID-19 disease severity. Sci Immunol. 2020; 5(51):eabe1670. https://doi.org/10.1126/sciimmunol.abe1670III. Kammann T*, Gorin JB*, Parrot T*, Gao Y, Ponzetta A, Emgård J, Maleki KT, Sekine T, Rivera-Ballesteros O, Karolinska COVID-19 Study Group, Gredmark-Russ S, Rooyackers O, Skagerberg M, Eriksson LI, Norrby-Teglund A, Mak JYW, Fairlie DP, Björkström NK, Klingström J, Ljunggren HG, Aleman S, Buggert M, Strålin K, Sandberg JK. Dynamic MAIT Cell Recovery after Severe COVID-19 Is Transient with Signs of Heterogeneous Functional Anomalies. J Immunol. 2024; 212(3):389- 396. https://doi.org/10.4049/jimmunol.2300639* contributed equally</p
T cell subset composition differs between blood and cerebrospinal fluid in amyotrophic lateral sclerosis.
No full textInflammation is a hallmark of amyotrophic lateral sclerosis (ALS) and is often assessed through biological samples. Due to the easier access, peripheral blood is more commonly phenotyped instead of cerebrospinal fluid (CSF) or affected tissues in ALS. Here, using flow cytometry, we compared the composition of T cell subsets in blood and CSF in ALS patients. We found consistent but weak correlations between blood and CSF for all T cell subsets examined. This finding implies that blood and CSF offer complementary information when characterizing T cell immunity in ALS and blood may not be used as a surrogate for CSF
Discrete approaches for leveraging g protein-coupled receptors as therapeutic tools
No full textG protein-coupled receptors (GPCRs) represent the largest and most therapeutically valuable family of human membrane proteins, serving as targets for over a third of FDA- approved drugs. Despite their clinical success, detailed mechanistic, structural, and interactome-level knowledge of GPCRs remains limited. This thesis uses multiple approaches to explore these aspects of GPCR biology, with a focus on brain-expressed aminergic GPCRs.First, G protein recruitment and G protein inwardly rectifying K+ (GIRK) channel activation assays were employed to investigate Ulotaront, a novel antipsychotic, at three aminergic receptors: the trace amine-associated receptor 1 (TAAR1), the dopamine D2 receptor (D2R), and the serotonin receptor 1A (5-HT1AR). Ulotaront was found to be a full agonist at TAAR1, with modest, a partial, weaker agonist at 5-HT1AR and a weak partial agonist with low potency at D2R. Using TAAR1- knockout (KO) mice, we demonstrated that TAAR1 is required for Ulotaront's effects on lowering core body temperature (CBT), baseline locomotion, and reversing MK-801-induced pre-pulse inhibition (PPI) disruption.Virtual screening is a powerful method to discover GPCR ligands and typically requires high-resolution protein structures. Recent advances in artificial intelligence (AI)-based protein structure prediction prompted us to evaluate the performance of AlphaFold2 and homology modelling in virtual screening to discover new TAAR1 ligands. After enriching TAAR1 models generated by both methods, docking screens identified 25 TAAR1 agonists, which were evaluated using a TAAR1-Gas recruitment assay. The hit- rate achieved with the AlphaFold screen was more than double that of the homology model screen, and the most potent agonist discovered was further tested for selectivity. The lead compound from the AlphaFold screen showed favourable pharmacokinetics and TAAR1-dependent antipsychotic-like effects in mouse behavioural assays.While virtual docking has proven successful in the identification of GPCR ligands, computational approaches to predict quantitative ligand parameters are less established. To address this, molecular dynamics (MD) simulations, G protein recruitment, and radioligand competition assays were conducted to improve our understanding of D2R activation mechanics. This led to the development of a computational pipeline that accurately predicted D2R ligand efficacy and affinity (R2: 0.82 and 0.7 goodness-of-fit with experimental results). These studies also provided insights into specific residues involved in ligand specificity at D2R and B2AR.Finally, we systematically explored the interactomes of human GPCRs with accessory proteins using state-of-the-art suspension-bead arrays (SBAs), focusing on receptor-activity modifying proteins (RAMPs) and p11. The RAMP screen revealed over 50 new GPCR-RAMP interactions and validated endogenous GPCR-RAMP complexes in SK-N- MC cells using quantitative in situ proximity assays. Similarly, the p11 screen identified over 20 GPCR-p11 interactions, showing that these complexes increased p11 protein levels and that p11 inhibited TAAR1 expression and signalling.In conclusion, this thesis uses a variety of strategies to improve our understanding of GPCR biology. The findings clarify how existing GPCR-targeted drugs work, present methods for identifying and optimizing GPCR ligands, and highlight the role of accessory proteins in modulating GPCR responses, potentially leading to new directions in research and drug discovery.List of scientific papersI. Saarinen M#, Mantas I, Flais I, Ågren R, Sahlholm K, Millan MJ, Svenningsson P#. TAAR1 dependent and independent actions of the potential antipsychotic and dual TAAR1/5-HT1A receptor agonist SEP- 363856. Neuropsychopharmacology. 2022 Dec;47(13):2319-2329. https://doi.org/10.1038/s41386-022-01421-2II. Díaz-Holguín A*, Saarinen M*, Vo DD, Sturchio A, Branzell N, Cabeza de Vaca I, Hu H, Mitjavila-Domenech N, Lindqvist A, Baranczewski P, Millan MJ, Yang Y, Carlsson J#, Svenningsson P#. AlphaFold accelerated discovery of psychotropic agonists targeting the trace amine-associated receptor 1. Sci Adv. 2024 Aug 9. https://doi.org/10.1126/sciadv.adn1524III. Chen Y*, Kahlous NA*, Saarinen M, Pérez-Conesa S, Svenningsson P, Delemotte L#, Carlsson J#. Revealing molecular determinants of ligand efficacy and affinity at the D2 dopamine receptor through molecular dynamics simulations. [Manuscript]IV. Kotliar IB*, Bendes A*, Dahl L*, Chen Y, Saarinen M, Ceraudo E, Dodig- Crnković T, Uhlen M, Svenningsson P, Schwenk JM#, Sakmar TP#. Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins. Sci Adv. 2024 Aug 2. https://doi.org/10.1126/sciadv.ado9959V. Saarinen M#, Kotliar IB, Hoffkes I, Bowin CF, Bendes A, Dahl L, Glaros V, Abney A, Bendes A, Kreslavsky T, Schwenk JM, Sakmar TP, Svenningsson P#. Receptor activity drives the GPCR-p11 interactome. [Manuscript]*Equal contribution # Corresponding author</p
Impaired lung function and the development of cardiovascular disease
No full textImpaired lung function is a prevalent condition in the general population, posing a significant risk for morbidity and mortality. There is a well-documented association between compromised lung function and increased susceptibility to cardiovascular disease (CVD). However, further research is needed to fully elucidate the underlying mechanisms and implications of this relationship. Notably, atherosclerosis, which is characterized by endothelial dysfunction in its early stages, underpins most CVD cases.To expand our knowledge on endothelial dysfunction and lung function, Study I investigated three distinct measurements of endothelial function alongside three different measurements of lung function in two community-based cohorts. The results indicated a modest association between reduced endothelial-dependent vasodilation (EDV), as measured invasively, and forced expiratory volume in one second percent of predicted value (FEV1 %pred). This novel finding suggests that endothelial dysfunction in the pulmonary vasculature may play a role in the deterioration of lung function early in the disease process.There are many biomarkers associated with CVD, but their relationship with impaired lung function remains less clear. Study II delve into specific protein biomarkers associated with CVD, seeking to determine their relevance in the context of diminished lung function. Three different study designs were utilized. Firstly, a cross-sectional analysis found that 22 out of 79 CVD protein biomarkers were associated with lower FEV1 %pred. Secondly, of these 22 proteins, only two proteins were linked to a more rapid decline of FEV1 %pred over a five-year period using a prospective cohort design in one of the study cohorts. Thirdly, causal inference was explored using Mendelian randomization in the UK Biobank, testing these two proteins. This analysis provided evidence supporting a potential causal role for growth-differentiation 15 (GDF-15) in the reduction of FEV1.Study III involved an additional investigation of protein biomarkers and lung function, examining 242 proteins linked to CVD or metabolism and its association to FEV1 %pred, forced vital capacity (FVC) %pred, and FEV1/FVC ratio. The results indicated that five proteins were significantly associated with either higher or lower FEV1%pred. In contrast, nine proteins were associated with either higher or lower FVC %pred. Notably, no proteins showed associations with the FEV1/FVC ratio in either cohort. These results suggest that the protein biomarkers were mainly associated with lower lung volume rather than airway obstruction.Study IV examined the impact of lower lung function and adverse outcomes in UK Biobank, a large-scale community database comprising approximately half a million participants. This study uncovered a progressive rise in both overall and cardiovascular mortality, myocardial infarction, ischemic stroke, and heart failure, with lower FEV1 or FVC %pred. This trend was consistent regardless of whether the individuals exhibited normal spirometry at the outset or not. Lastly, we compared the population attributable fraction (PAF) and reported that lower FEV1 %pred and FVC %pred were at least as influential as established risk factors like diabetes, chronic kidney disease, and hypertension in determining the risk of adverse outcomes.The findings derived from these studies not only illuminate the pivotal role of lung function as a predictor of adverse outcomes but also emphasize its significance within the established landscape of CVD risk factors. Moreover, it underscores the importance of maintaining healthy blood vessels for overall health.However, despite an extensive exploration of protein biomarkers associated with cardiovascular disease, no potential biomarker candidate suitable for clinical use for lung function was identified. This outcome raises the possibility that pursuing individual biomarkers in this context may not be the most viable path forward.List of scientific papersThis thesis is based on the four following papers:I. Endothelial dysfunction is associated with impaired lung function in two independent community cohorts. Rydell A, Janson C, Lisspers K, Ställberg B, Nowak C, Carlsson AC, Feldreich T, Iggman D, Lind L, Ärnlöv J. Respir Med. 2018 Oct;143:123-128.https://doi.org/10.1016/j.rmed.2018.09.009II. Plasma proteomics and lung function in four community-based cohorts. Rydell A, Nowak C, Janson C, Lisspers K, Ställberg B, Iggman D, Leppert J, Hedberg P, Sundström J, Ingelsson E, Lind L, Ärnlöv J. Respir Med. 2021 Jan;176:106282.https://doi.org/10.1016/j.rmed.2020.106282III. Cardiovascular disease-linked plasma proteins are mainly associated with lung volume. Rydell A*, Nerpin E*, Zhou X, Lind L, Lindberg E, Theorell Haglow J, Fall T, Janson C, Lisspers K, Elmståhl S, Zaigham S, Melander O, Nilsson PM, Ärnlöv J, Malinovschi A. ERJ Open Res. 2023 Mar 27;9(2):00321-2022.https://doi.org/10.1183/23120541.00321-2022IV. FEV1 and FVC as Robust Risk Factors for Cardiovascular Disease and Mortality: Insights from a Large Population Study. Rydell A, Janson C, Lisspers K, Yi-Ting Lin, Ärnlöv J. Respir Med. 2024 Jun;227:107614.https://doi.org/10.1016/j.rmed.2024.107614*Equal contribution</p
Prevention of endophthalmitis after cataract surgery
No full textPostoperative endophthalmitis (PE) is a rare but potentially vision threatening condition that needs prompt diagnosis and treatment. To minimize, or preferably, eradicate the event of postoperative endophthalmitis, we analyzed PE cases regarding their bacteriology and susceptibility to given intracameral (IC) prophylactic antibiotics (Abs) over time.In Paper 1, 95 culture-positive cases of PE occurring after cataract operations in a large surgical unit 1990 - 2009 were analyzed in a retrospective chart study. We found that the use of cefuroxime prophylaxis intracamerally effectively reduced the rate of PE from 0.18% (with no IC cefuroxime) to 0.04% (with IC cefuroxime), p In Paper 2, the incidence of PE in unilateral compared to immediate sequential bilateral cataract surgery (ISBCS) was analyzed from data of the Swedish National Cataract Register (NCR) from 2002 - 2017. The rate of PE in ISBCS was found to be lower than that of unilateral surgeries, p = 0.01. We believe that the reassuring results can be explained by the selection of patients being less prone to develop PE, but possibly also by the preferred use of the combination of ampicillin + cefuroxime as intracameral prophylaxis in ISCBS patients.In Paper 3, the 2011 - 2017 database of the NCR was investigated to compare the PE rates and bacteriology of the three most used intracameral antibiotic regimes: cefuroxime, moxifloxacin and the combination of ampicillin + cefuroxime. There were no significant differences in the incidence of PE between the three antibiotic groups, but enterococcal infections were statistically significantly reduced by the two alternative treatments regimes in comparison to cefuroxime alone, p List of scientific papersI. Friling, E. & Montan, P. (2018). Bacteriology and cefuroxime- resistance in endophthalmitis following cataract surgery before and after the introduction of prophylactic intracameral cefuroxime: a retrospective single-centre study. Journal of Hospital Infection. https://doi.org/10.1016/j.jhin.2018.02.005Il. Friling, Emma & Johansson, Björn & Lundstrom, Mats & Montan, Per. (2021). Postoperative Endophthalmitis in Immediate Sequential Bilateral Cataract Surgery: A Nationwide Registry Study. Ophthalmology. 129. https://doi.org/10.1016/j.ophtha.2021.07.007III. Friling, Emma & Bro, Tomas & Lundstrom, Mats & Montan, Per. (2024). Endophthalmitis following cataract surgery and effect of different intracameral antibiotic regimes in Sweden 2011 - 2017. A national registry study. Journal of Cataract and Refractive Surgery. https://doi.org/10.1097/j.jcrs.0000000000001464</p
Shedding light on the blood-brain barrier in ischemic stroke
No full textBlood-brain barrier (BBB) breakdown is a hallmark of ischemic stroke and has been associated with a poor clinical prognosis for ischemic stroke patients. Tissue- plasminogen activator (tPA), the only approved drug for treatment of ischemic stroke, can cause intracerebral hemorrhage, a severe form of BBB breakdown, and it has been suggested that this is caused by tPA-mediated activation of platelet- derived growth factor (PDGF)-CC/PDGF receptor alpha (PDGFRα) signaling in the neurovascular unit.In this thesis, an in vivo two-photon imaging pipeline was established which enables studies on ischemic stroke progression in real time. With this technique, BBB breakdown can be assessed and quantified longitudinally in the same animal. We could demonstrate that during the first 7 days after ischemic stroke induction, BBB leakage displays distinct peaks during the acute and the subacute phase, respectively, which could be effectively reduced with the tyrosine kinase inhibitor imatinib, an inhibitor of PDGFRα signaling.We could further show that phosphorylation of the tight junction protein occludin causes a loss of tight junction integrity in endothelial cells which leads to an increase in BBB permeability. The phosphorylation of occludin was mediated by tPA-induced activation of the PDGF-CC/PDGFRα signaling pathway in perivascular PDGFRα+ cells and inhibiting phosphorylation of occludin reduced BBB permeability.In addition, our studies established the role of PDGF-CC/PDGFRα signaling in myofibroblast transdifferentiation in the fibrotic scar after experimental ischemic stroke. We showed that inhibiting PDGF-CC/PDGFRα signaling, by pharmacologic or genetic means, preserved cerebrovascular health, reduced the reactive gliosis response and decreased myofibroblast expansion within the fibrotic scar in the early chronic phase after ischemic stroke. Importantly we demonstrate that targeting myofibroblast expansion in the post-acute phase after ischemic stroke onset was associated with improved functional outcome, thus suggesting a novel post-acute treatment opportunity for ischemic stroke patients.Collectively, this thesis provides further insight into how tPA-mediated PDGF- CC/PDGFRα signaling regulates BBB breakdown and establishes the involvement of PDGF-CC/PDGFRα signaling in myofibroblast scar formation after ischemic stroke.List of scientific papersI. Jil Protzmann, Felix Jung, Lars Jakobsson and Linda Fredriksson. Analysis of ischemic stroke-mediated effects on blood-brain barrier properties along the arteriovenous axis assessed by intravital two- photon imaging. Fluids and Barriers of the CNS, 2024, 21, 35 https://doi.org/10.1186/s12987-024-00537-5II. Jil Protzmann, Manuel Zeitelhofer, Christina Stefanitsch, Daniel Torrente, Milena Z. Adzemovic, Kirils Matjunins, Stella J.I. Randel, Sebastian A. Lewandowski, Lars Muhl, Ulf Eriksson, Ingrid Nilsson, Enming J. Su, Daniel A. Lawrence and Linda Fredriksson. Reduced myofibroblast expansion in the fibrotic scar enhances recovery after ischemic stroke. [Manuscript] III. Andreia Goncalves, Enming J. Su, Arivalagan Muthusamy, Manuel Zeitelhofer, Daniel Torrente, Ingrid Nilsson, Jil Protzmann, Linda Fredriksson, Ulf Eriksson, David A. Antonetti and Daniel A. Lawrence. Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCß phosphorylation of occludin. Blood, 2022, 140(4):388-400 https://doi.org/10.1182/blood.2021014958</p
Prevention of cognitive decline after radiation therapy
No full textCentral nervous system tumours are the leading cause of cancer-related deaths in children and the second-most-common malignancy diagnosed after leukaemia. Advances in treatment regimens and multimodal strategies, from early surgical resection, to radiotherapy, complete craniospinal radiation, and chemotherapeutic treatments have greatly improved the chances of survival. However, they are usually accompanied by long-term, late neurocognitive deficits, which, in totality, contribute to reduced quality of life. The aim of this thesis was to illuminate the mechanisms behind the cognitive decline that arises after cranial radiotherapy in childhood cancer survivors and to explore potential treatment strategies to prevent it.Despite the effectiveness of radiotherapy in treating brain cancer, a biomarker that can estimate the extent of IR-induced brain injury and can be correlated with the clinical outcomes has yet to be established. Discovering a measurable molecule, that can be easily quantified in clinical samples that can be obtained through minimally invasive procedures would prove instrumental in predicting the severity of the neurocognitive complications and stratifying patients to the best treatment scheme to prevent or reduce the debilitating decline. In the first study of this thesis, we identified a very promising molecule, EDA2R (ectodysplasin A2 receptor), and we propose it as a potential biomarker. Ectodysplasin A2 receptor is a protein that belongs to the tumour necrosis factor family and is involved a variety of biological processes. After irradiation, we show that EDA2R is highly elevated in the cerebrospinal fluid as well as the blood of mice and is the only marker that remains at high concentrations even at two weeks after the treatment completion. Fulfilling the criteria of a biomarker, it remains to be correlated with cognitive performance in a clinical setting.Among the most dominant hypotheses on the explanation of radiotherapy-induced cognitive decline is the depletion of hippocampal neurogenesis, a physiological process through which new neurons are constantly being born in the from neural stem and progenitor cells (NSPCs), which reside deep in the hippocampus, the centre of memory and learning. The depletion of neurogenesis has also been theorized to derive from the neuroinflammatory microenvironment that radiotherapy induces, which hinders the abilities of NSPCs to differentiate into neurons, and is governed by aberrantly activated microglia. As the resident immune cells of the brain, microglia react immediately to irradiation and undergo a series of changes to adapt to the needs of their niche, and their irradiation-induced diminishing numbers. Through unbiased, longitudinal in vivo studies, we showed that microglia reactivate long after irradiation and adopt unique morphologies and inflammatory profiles in a temporal manner. We observed a biphasic response in the hippocampal tissue, characterized by interferon signalling and proliferation of microglia that leads to neuronal asynchrony, which improves over time, due to the arrival of macrophage-derived microglia. In addition, our investigation revealed subsets of microglia that try to divide even in the presence of DNA damage, leading to senescence.Besides raising and maintaining a neuroinflammatory alarm, microglia are also responsible for clearing dead neural progenitors and cell debris. Irradiation has also been shown to increase the levels of expression of genes that are related to phagocytosis. Several genetic and pharmacological tools have been developed to deplete microglial populations over the years. The usage of these approaches could prove instrumental in understanding the role of microglia in the depletion of neurogenesis. In addition, a study by Willis et al. in 2020 showed that depleting microglia and their subsequent repopulation attenuated cognitive decline in a traumatic brain injury model. With these in mind, we utilized the Cx3cr1CreERt2- YFP/+Rosa26DTA/+ mouse model to deplete microglia before subjecting the animals to cranial radiotherapy. Contrary to what we expected, the absence of microglia increased the production of pro-inflammatory factors and neither the depletion nor the subsequent repopulation had any impact on the loss of immature or proliferating neurons. These findings challenge the proposed role for a pro-inflammatory microenvironment in the dysregulation of hippocampal neurogenesis and suggest that the observed reduction of neurogenesis was solely due to IR..Cranial radiotherapy has been documented to lead to decreases in dendritic density and changes in the spine morphology of the hippocampal neurons. As the main sites of the excitatory synapses, changes in dendrites and spines will often lead to abnormal signaling and dysregulation of the circuit, functions that are essential for cognition, and might also lead to glutamatergic excitotoxicity and neuronal death. Since newborn neurons rely on external inputs from the hippocampal circuit to pass survival checkpoints, strengthening synaptic connectivity might increase the chances of survival of the damaged immature neurons and improve cognitive functions in patients.Memantine is an uncompetitive antagonist of the N-methyl-D-aspartate receptor (NMDAR), which has been shown to exert neuroprotective properties and increases synaptic plasticity and is currently used in the treatment of AlzheimerŐs disease patients. Due to these factors, memantine was considered as a great candidate for ameliorating radiation-induced cognitive decline, and several clinical trials have been completed and are underway since 2013, with very promising results. However, the exact mechanisms of how memantine exerts its neuroprotective effects are still unknown. For the final study of this thesis, we investigated the potential of memantine in preventing loss of neurogenesis. Memantine was administered in mice intraperitonially 30 minutes after cranial irradiation, and then it was supplied in their drinking water to achieve a steady state plasma concentration that recapitulates the clinical practice. Even though our results suggest no change in the numbers of surviving neurons in the hippocampal neurogenic niche, we showed that memantine led to an increase in the arborization of the dendritic processes of the young neurons. These data highlight memantineŐs potential in improving synaptic plasticity, the incorporation of immature neurons in the hippocampal circuitry and, by extension, in attenuating cognitive decline.List of scientific papersI. EDA2R reflects the acute brain response to cranial irradiation in liquid biopsies. Alejandro Lastra Romero, Thea Seitz, Georgios Alkis Zisiadis, Holli Jeffery, Ahmed M Osman*. Neuro Oncol. 2024 Apr 29:noae077. *: Corresponding author(s). https://doi.org/10.1093/neuonc/noae077 II. Microglia Adopt Temporally Specific Subtypes after Irradiation, Correlating with Neuronal Asynchrony. Alejandro Lastra Romero#, Efthalia Preka#, Giusy Pizzirusso, Luis Enrique Arroyo-Garcia, Georgios Alkis Zisiadis, Nuria Oliva-Vilarnau, Thea Seitz, Kai Zhou, Arturo Gonzalez Isla, Lara Friess, Ying Sun, Alia Shamik, Changlian Zhu, Carlos F. D. Rodrigues, AndrŽ Fisahn, Bertrand Joseph, Lena-Maria Carlson, Adamantia Fragkopoulou, Volker M Lauschke, Christer Betsholtz, Ahmed M Osman*, Klas Blomgren*. #These authors contributed equally to this study. *Corresponding author(s). [Manuscript]III. Microglia depletion and repopulation do not alter the effects of cranial irradiation on hippocampal neurogenesis. Kai Zhou#, Georgios Alkis Zisiadis#, Monique Havermans, Adamantia Fragkopoulou, Cecilia Dominguez, Makiko Ohshima, Ahmed M Osman, Carlos F. D. Rodrigues, Klas Blomgren*. #These authors contributed equally to this study. *Corresponding author(s). [Manuscript]IV. Memantine increases the dendritic complexity of hippocampal young neurons in the juvenile brain after cranial irradiation. Georgios Alkis Zisiadis, Androniki Alevyzaki, Elene Nicola, Carlos F. D. Rodrigues, Klas Blomgren, Ahmed M Osman*. Front Oncol. 2023 Oct 4;13:1202200. *Corresponding author(s). https://doi.org/10.3389/fonc.2023.1202200 </p
Exploring cannabinoids for indolent B-cell lymphomas
No full textThe overexpression of cannabinoid receptors is well described in several indolent lymphomas but the relevance of this finding is more uncertain. Our group has previously shown that mantle cell lymphoma (MCL)-derived cell lines known to overexpress the cannabinoid receptor 1 entered apoptosis after exposure to cannabinoids and that lymphomas xenografted onto mice shrank in size after exposure to cannabinoids. To further understand the potential effects of cannabinoids on indolent lymphomas, we therefore undertook a clinical trial and subsequent correlational studies which are described in this thesis.In the trial, we recruited 23 patients with leukemic indolent B-cell lymphomas from our hematologic out-patient clinic. The patients were given cannabinoids in the form of a mouth-spray (Sativex®) with doses escalating between the patients to identify the maximal tolerated dose. The patients were blood sampled at regular intervals, both during a control day and on the day on the cannabinoid study drug administration. After a week, a final sample was taken. This longitudinal sampling allowed us to investigate what, if any, effects the cannabinoids had on the indolent B-cell lymphoma cells. Our analysis told us that the absolute number of circulating lymphocytes decreased after administration of cannabinoids but there was no evidence of apoptosis, neither was there any evidence of decreased proliferation, in the lymphoma cells. Instead, we surmised that a migration of lymphocytes away from the blood stream was the most likely explanation to our findings.The second study focused on the pharmacological perspectives of the abovedescribed clinical trial. All 23 patients underwent testing of captured breath, saliva, urine and blood during their participation in the study. From this we learned that the newer method of captured breath to detect cannabinoids was very sensitive and gave false positives if not very carefully handled. We also found that the urine analysis was surprisingly sensitive for a long period of time and in some cases cannabinoids were detected a week after administration in patients that received very small doses of the study drug. Both these findings have practical implications, for example when testing is conducted in workplaces and in traffic monitoring.Our third study investigated the possible interplay between the two types of cannabinoid receptors and how they affect cell migration. We used primary cells from patients and several different MCL-cell lines to discern what type of cannabinoid receptor was important in cell migration. We also investigated how the receptors communicate with each other and interact with the known potent chemokine receptor C-X-C Chemokine Receptor type 4 (CXCR-4).Paper IV is a manuscript detailing our ongoing work to identify the effects cannabinoids have on lymphoma cells. In this paper we take frozen lymphoma cells from the clinical trial and analyse them using RNA-sequencing (RNA-seq). Since we had cells saved from all the various timepoint of the study, we could produce a longitudinal analysis of how mRNA-levels changed after administration of cannabinoids.List of scientific papersI. Christopher M. Melén*, Magali Merrien*, Agata M. Wasik, Georgios Panagiotidis, Olof Beck, Kristina Sonnevi, Henna-Riikka Junlén, Birger Christensson, Birgitta Sander** & Björn Engelbrekt Wahlin**. Clinical effects of a single dose of cannabinoids to patients with chronic lymphocytic leukemia. Leuk Lymphoma. 2022;63(6):1387- 1397. *First authors equal contribrution, **Last authors equal contribution. https://doi.org/10.1080/10428194.2021.2020776 II. Christopher M. Melén, Magali Merrien, Agata M Wasik, Birgitta Sander, Björn Engelbrekt Wahlin, Georgios Panagiotidis, Olof Beck. Δ9-THC and CBD in Plasma, Oral Fluid, Exhaled Breath, and Urine from 23 Patients Administered Sativex. Cannabis Cannabinoid Res. 2023 Apr 19. https://doi.org/10.1089/can.2022.0179 III. Magali Merrien, Agata M. Wasik , Christopher M. Melén, Mohammad Hamdy Abdelrazak Morsy, Kristina Sonnevi, Henna-Riikka Junlén, Birger Christensson, Björn E. Wahlin and Birgitta Sander. 2-Arachidonoylglycerol Modulates CXCL12-Mediated Chemotaxis in Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia. Cancers. 2023 Mar; 15(5): 1585. https://doi.org/10.3390/cancers15051585 IV. Christopher M. Melén, Magali Merrien, Agata M. Wasik, Birger Christensson, Birgitta Sander & Björn Engelbrekt Wahlin. Longitudinal RNA-seq on indolent lymphoma cells before and after exposure of cannabinoids in vivo. [Manuscript]</p
Understanding the roles of avidity in biological systems
No full textUnderstanding avidity is essential for elucidating complex molecular interactions and designing effective therapeutic strategies. This thesis aims to explore strategies and tools to understand how avidity influences receptor-ligand binding kinetics, cellular uptake mechanisms, intracellular trafficking pathways, and modulation of immune responses.In Paper I, we developed a method called NanoDeep that utilizes DNA sequencing to decipher the nanoscale spatial distribution of membrane proteins. NanoDeep can characterize differences in protein nanoenvironments in different cellular contexts and provides insights into the roles of the composition and spatial organization of protein nanoenvironments. NanoDeep has the potential to be a tool for simultaneous analysis of the spatial distribution of many proteins at the membrane without microscopy measurements, providing a means to understand the importance of membrane protein assemblies in cellular function.In Paper II, we used DNA origami nanotechnology to precisely control insulin multivalency and spatial arrangement at the nanoscale. This methodology enabled us to study the binding and signaling characteristics of insulin nanoclusters, revealing that the arrangement and number of insulin molecules significantly influence their interaction with insulin receptors and subsequent activation of the insulin receptor signaling cascade. These insights offer guidance for designing and refining insulin-based nanotherapeutics aimed at treating diabetes.In Paper III, we constructed and characterized a novel platform that consists of self-assembling virus-like particles displaying single-chain Fc molecules on their surface. This engineered platform facilitated the investigation of Fc valency on Fcγ receptor clustering and activation. The findings revealed that Fc valency influences the binding of single-chain Fc virus-like particles to various FcγR-expressing immune cells. Interestingly, single-chain Fc virus-like particles showed limited internalization and receptor clustering, hindering phagocytosis and FcγR activation. These insights shed light on the potential of engineered Fc multimers for developing innovative therapies targeting autoimmune diseases by suppressing FcγR activation and immune complex-mediated functions such as antibody-dependent cellular phagocytosis.In Paper IV, we used DNA origami nanostructures presenting aptamers that bind to transferrin receptors to study the relationship between avidity and receptor-mediated endocytosis. We found that the binding, uptake, and sorting of the DNA nanostructures were specific to the transferrin receptor, aptamer-mediated, and modulated by aptamer valency. These findings provide insights into the role of avidity in cellular uptake and suggest potential strategies for modulating the intracellular behaviour of DNA nanosheets for drug delivery.List of scientific papersI. Elena Ambrosetti, Giulio Bernardinelli, Ian Hoffecker, Leonard Hartmanis, Georges Kiriako, Ario de Marco, Rickard Sandberg, Björn Högberg, Ana I. Teixeira. A DNA-nanoassembly-based approach to map membrane protein nanoenvironments. Nat Nanotechnol. 16, 85–95 (2021). https://doi.org/10.1038/s41565-020-00785-0 II. Joel Spratt*, José M. Dias*, Christina Kolonelou, Georges Kiriako, Enya Engström, Ekaterina Petrova, Christos Karampelias, Igor Cervenka, Natali Papanicolaou, Antonio Lentini, Björn Reinius, Olov Andersson, Elena Ambrosetti, Jorge L. Ruas, Ana I. Teixeira. Multivalent insulin receptor activation using insulin–DNA origami nanostructures. Nat Nanotechnol. 19, 237–245 (2024). *These authors contributed equally to this work. https://doi.org/10.1038/s41565-023-01507-y III. Ekaterina Petrova, Georges Kiriako, Johan Rebetz, Karl Johansson, Stefan Wennmalm, Niels E.J. Meijer, B. Martin Hällberg, Ingemar André, Elena Ambrosetti, John W. Semple, Ana I. Teixeira. Engineering multivalent Fc display for FcγR blockade. [Submitted]IV. Georges Kiriako, Joel Spratt, José Dias, Sinisa Bjelic, Elena Ambrosetti, Ana I. Teixeira. Effects of avidity in transferrin receptor targeting. [Manuscript]</p
Exploring lipoprotein characteristics in human cardiometabolic disease
No full textBackground: Atherosclerosis underlies most cases of cardiovascular disease (CVD) which remains the principal cause of global mortality. CVD is significantly linked to cardiometabolic diseases such as diabetes and obesity. These disorders precipitate a cascade of metabolic dysregulations, including insulin resistance and altered lipid metabolism, culminating in modified lipoprotein profiles.Aim: Dissecting the characteristics of lipoproteins in the context of cardiometabolic diseases and their contribution to atherogenesis.Methods: The thesis is based upon five papers. I. Assessment of the impact of a novel potential therapeutic agent on hepatic lipid metabolism using modified mRNA molecules in human hepatoma cell lines. II. Characterization of HDL in systemic arterial and coronary blood from patients with Non-ST-Elevation Myocardial Infarction (NSTEMI). III. Evaluation of the influence of clustered cardiometabolic risk factors on the functional properties of HDL. IV. Investigation into mechanisms of lipoprotein retention in type 2 diabetes through analyses of lipoproteins in serum and interstitial fluid. V. Characterization of postprandial lipoproteins in serum and interstitial fluid among healthy controls and individuals with type 2 diabetes.Results: Key findings from the thesis papers were I. The overexpression of TGIF1 protein in hepatoma cells induced a favorable lipid profile alteration. II. Notable differences in HDL properties were found between coronary and systemic circulation in the immediate aftermath of NSTEMI, unrelated to HDL levels. III. Cardiometabolic risk factors exert a progressive negative impact on HDL function, with a major contribution of abdominal obesity to alterations in reverse cholesterol transport. IV. An apparent increase in vascular retention of atherogenic lipoproteins was seen in type 2 diabetes, manifested by reduced ApoB-containing lipoproteins in interstitial fluid concomittant with increased LDL affinity for arterial proteoglycans. Similar changes were observed with increasing age in controls. V. Distinct postprandial alterations in serum and interstitial fluid revealed atherogenic postprandial lipoprotein modifications in Type 2 diabetes.Conclusion: This thesis accentuates the relationship between lipoprotein properties and atherosclerosis in the context of cardiometabolic disorders. The insights highlight the importance of qualitative lipoprotein evaluation beyond conventional quantitative lipoprotein analysis. Such analyses should improve preventive strategies, including cardiovascular risk assessment and new therapeutic interventions.List of scientific papersI. Härdfeldt J, Hodson L, Larsson L, Pedrelli M, Pramfalk C. Effects on hepatic lipid metabolism in human hepatoma cells following overexpression of TGFβ induced factor homeobox 1 or 2. Biochim Biophys Acta Mol Cell Biol Lipids. 2019 May;1864(5):756-762. https://doi.org/10.1016/j.bbalip.2019.02.009 II. Cariello M, Salvia R, Härdfeldt J, Piglionica M, Rutigliano D, Caldarola P, Ossoli A, Vacca M, Graziano G, Battaglia S, Zerlotin R, Arconzo M, Crudele L, Sabbà C, Calabresi L, Moschetta A. Intracoronary monocyte expression pattern and HDL subfractions after non ST elevation myocardial infarction. Biochim Biophys Acta Mol Basis Dis. 2021 Jun 1;1867(6):166116. https://doi.org/10.1016/j.bbadis.2021.166116 III. Härdfeldt J, Cariello M, Simonelli S, Ossoli A, Scialpi N, Piglionica M, Pasculli E, Noia A, Berardi E, Suppressa P, Piazzolla G, Sabbà C, Calabresi L, Moschetta A. Abdominal obesity negatively influences key metrics of reverse cholesterol transport. Biochim Biophys Acta Mol Cell Biol Lipids. 2022 Feb;1867(2):159087. https://doi.org/10.1016/j.bbalip.2021.159087 IV. Björklund P*, Härdfeldt J*, Straniero S, Äikäs L, Öörni K, Rudling M, Angelin B. Transvascular retention of atherogenic lipoproteins in type 2 diabetes relates to their enhanced proteoglycan-binding susceptibility. *Co-First Authors. [Manuscript]V. Härdfeldt J*, Björklund P*, Straniero S, Rudling M, Angelin B. Fasting and postprandial lipoproteins in Interstitial Fluid in Type 2 Diabetes. *Co-First Authors. [Manuscript]</p