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Current and novel markers for intraamniotic infection after preterm prelabor rupture of membranes
No full textBackgroundIntraamniotic infection (IAI) is one of the main complications associated with preterm prelabor rupture of membranes (PPROM), affecting up to 60% of such pregnancies. Neonates born following IAI are at high risk of early-onset neonatal sepsis (EONS), acquiring the infection in utero. Preterm neonates are particularly susceptible to multi-organ dysfunction secondary to sepsis, which is associated with high mortality rates. Antibiotic treatment for the mother is insufficient in cases of IAI; evacuation of the pregnancy is necessary to resolve the infection and reduce secondary morbidity and mortality for both the mother and the fetus/neonate. Vigilant monitoring of PPROM pregnancies for signs of IAI is crucial to identify those affected and at high risk of EONS, to initiate timely delivery and thereby reducing adverse outcomes. However, misdiagnosis can lead to unnecessary preterm deliveries. Accurate diagnostic tools for IAI are essential, yet no easily available noninvasive method exists for confirming IAI antenatally and current practices lack reliable markers, possibly resulting in both over- and underdiagnosis of IAI. There is an urgent need to enhance the diagnostic accuracy of IAI and identify those at increased risk of EONS.The aim of this thesis was to investigate current and novel antenatal markers of IAI following PPROM, with the goal of improving diagnostic accuracy.Material and methodsThe three studies included in this thesis were historical cohort studies derived from a single original study cohort, sharing common inclusion criteria: PPROM in singleton pregnancies, with delivery from 24+0 to 33+6 gestational weeks in Stockholm County, Sweden, from 2012 to 2019. While the studies share a common cohort, they each had slightly different exclusion criteria tailored to their specific research objectives. Data were extracted from existing medical records and cardiotocography traces.Study I and II examined the association between exposure to IAI and the outcome of changes in short-term variation of fetal heart rate, a potential novel marker of IAI. For the exposure, IAI, we used the diagnosis of EONS in the neonate as a proxy, due to the absence of a reliable method to confirm IAI antenatally and given the strong association between IAI and EONS. Cardiotocography traces were analyzed using a computerized algorithm to assess short-term variation (the primary outcome) and baseline frequency (a secondary outcome). We compared values in the last cardiotocography trace before the start of labor between the groups using linear regression and examined the difference in change over time prior to start of labor using locally estimated scatterplot smoothing curves and linear mixed models.Study III examined the association between antenatal characteristics and EONS to elucidate their role as markers of IAI. Logistic regression was employed, with EONS as the dependent variable. A risk-indicating model was developed based on complete case analysis, examining the proportion of cases within pairs of risk factors, and utilizing relative excess risk due to interaction tables. The model's performance was evaluated using diagnostic performance metrics.For all three studies, adjustments were made using meticulously constructed directed acyclic graphs to identify variables as confounders or mediators.ResultsShort-term variation declined significantly more steeply in those exposed to IAI compared to those not exposed, beginning approximately 20 hours before the start of labor. This resulted in a 21.5% (95% confidence interval [CI], -32.6 to -8.7) or 1.41 msec (95% CI, -2.52 to -0.31) lower mean short- term variation in the last cardiotocography trace before start of labor in the IAI-exposed group compared to the non-exposed group. Baseline frequency increased more in the IAI-exposed group than the non-exposed group. The decline in short-term variation was only partially explained by this observed rise in baseline frequency, and it exhibited a larger relative difference and began earlier than the rise in baseline frequency.A negative correlation was found between short-term variation and EONS, with a 37% increase in the risk of EONS for each 1 msec decrease in mean STV. A short-term variation below 4 msec was associated with a 2.62-fold increase in the risk for EONS. Baseline frequency was positively correlated with the risk of EONS, and a baseline frequency above 160 bpm was associated with a 3.75-fold increased risk of EONS. Maternal diabetes, maternal temperature >38℃, and positive urinary or vaginal/cervical culture were significantly associated with an increased risk of EONS, with odds ratios of 4.37 (95% CI, 1.41 to 13.56), 6.42 (95% CI, 2.94 to 14.02) and 2.62 (95% CI, 1.14 to 6.03), respectively.A risk-indicating model for EONS was developed which demonstrated an AUC of 0.7348 and surpassed the diagnostic performance of the clinicians' suspicion of IAI. The model indicates an elevated risk of EONS in pregnancies with positive urinary and/or vaginal/cervical culture in conjunction with at least one of the following markers: maternal temperature ≥38℃, maternal diabetes, fetal baseline frequency ≥160 bpm, or fetal short-term variation ≤4 msec. Maternal white blood cell (WBC) count was only marginally associated with EONS, and when categorizing the WBC count at 15,000 cells/mm3 the association was no longer significant. No association was identified with maternal C-reactive protein levels, maternal pulse, or maternal symptoms.ConclusionOur findings suggest that short-term variation of fetal heart rate may be a promising novel marker for IAI, warranting further investigation and development. Maternal diabetes also emerged as a novel marker. Among the current antenatal markers for IAI, maternal temperature, positive vaginal and/or cervical cultures, and fetal tachycardia are the most important to consider. A risk-indicating model for EONS, incorporating significant markers for EONS, outperformed clinicians' suspicion of IAI.List of scientific papersI. Short-term variation of the fetal heart rate as a marker of intraamniotic infection in pregnancies with preterm prelabor rupture of membranes: a historical cohort study. Birgisdottir BT, Hulthén Varli I, Saltvedt S, Lu K, Abtahi F, Ådén U, Holzmann M. J Matern Fetal Neonatal Med. 2024 Dec;37(1):2345855. https://doi.org/10.1080/14767058.2024.2345855II. Changes in short-term variation of antenatal cardiotocography to identify intraamniotic infection: a historical cohort study. Birgisdottir BT, Andersson T, Hulthén Varli I, Saltvedt S, Lu K, Abtahi F, Ådén U, Holzmann M. J Matern Fetal Neonatal Med. 2025 Dec;38(1):2434059. https://doi.org/10.1080/14767058.2024.2434059III. Risk factors and risk-indicating model for early-onset neonatal sepsis after preterm prelabor rupture of membranes: a historical cohort study. Birgisdottir BT, Andersson T, Hulthén Varli I, Saltvedt S, Abtahi F, Ådén U, Holzmann M. [Manuscript] </p
Computational approaches to study germline-encoded and expressed adaptive immune receptor repertoires
No full textThe somatic recombination of a couple variable (V), diversity (D), and joining (J) genes into millions of B and T cell receptors (BCRs and TCRs) is the key mechanism by which the adaptive immune system recognizes an endlessly diverse set of pathogens. The loci encoding V, D, and J genes are complex: nearly every individuals has a unique set, deletions and duplications are commonplace, and intergenic and interallelic variation is enormous. TCRs are made of either an alpha and beta chain or a gamma and delta chain. BCRs comprise two identical heavy chains and two identical light chains, the latter from either the kappa or lambda locus. My thesis is a study of TCR and BCR population diversity, response to antigenic stimuli, and the computational methods used to analyze them. Our lab developed a method called IgDiscover to determine personal genotypes from expressed TCR and BCR repertoire data. As a computational scientist, I applied our lab's method as well as population genetics methods, amplicon denoising approaches, and methods I developed during my thesis work to elucidate the genetics and functionality of TCRs and BCRs.In paper I, we sequenced all four types of TCR repertoires (alpha, beta, delta, gamma) in 45 individuals across four diverse superpopulations (sub-Saharan African, European, East Asian, South Asian) and applied IgDiscover to determine each person's TCR V, D, and J genotype. We discovered 175 previously undescribed V and J alleles, with a majority containing coding variants. We reported large inter-individual and inter-population diversity and many instances of population specific alleles. Non-African specific Neanderthal novel variants of the TRGV4, TRAJ24, TRAJ26, and TRAV12-2 genes were discovered. The TRGV4 variant was particularly divergent from previously described alleles and exhibited decreased binding to the TRGV4 butyrophilin-like (BTNL) ligand.In paper II, we longitudinally sequenced the immunoglobulin heavy (IGH) repertoires of the BCR and isolated monoclonal antibodies of two individuals first infected by SARS-CoV-2 then immunized with an mRNA spike vaccine. The sampling timepoints following infection and vaccination were timed to capture the peak of adaptive immune system activity, allowing us to trace numerous BCR lineages through time. We functionally characterized 31 SARS-CoV-2 neutralizing antibodies, with four neutralizing Omicron BA.1, despite the study occurring prior to the emergence of Omicron, demonstrating that affinity maturation can broaden the response. Combined IGH repertoire and monoclonal antibody lineage tracing revealed that vaccination expanded a broad range of affinity-matured infection- induced antibody lineages.Paper III describes a novel method for identifying and filtering PCR chimeras in adaptive immune receptor repertoire sequencing (AIRR-seq) data. We analyzed 319 published libraries and demonstrated the universal presence of chimeras in AIRR-seq data with different frequencies depending on the type of AIRR dataset. Further, we created new AIRR-seq libraries to determine which PCR conditions produced more chimerism. Evaluation on simulated TCR and IGH data revealed that our tool performed better than existing non-domain-specific software making it a useful tool for the AIRR community.List of scientific papersI. Martin Corcoran, Mark Chernyshev*, Marco Mandolesi*, Sanjana Narang, Mateusz Kaduk, Kewei Ye, Christopher Sundling, Anna Färnert, Taras Kreslavsky, Carolina Bernhardsson, Maximilian Larena, Mattias Jakobsson, Gunilla B Karlsson Hedestam"Archaic humans have contributed to large-scale variation in modern human T cell receptor genes"Immunity, 2023, 56, 635-652 *Equal Contribution.https://doi.org/10.1016/j.immuni.2023.01.026II. Mark Chernyshev*, Mrunal Sakharkar*, Ruth I. Connor, Haley L. Dugan, Daniel J. Sheward, C. G. Rappazzo, Aron Stålmarck, Mattias N. E. Forsell, Peter F. Wright, Martin Corcoran, Ben Murrell, Laura M. Walker, Gunilla B. Karlsson Hedestam"Vaccination of SARS-COV-2-infected individuals expands a broad range of clonally diverse affinity-matured B cell lineages"Nature Communications, 2023, 14, article 2249 *Equal Contribution.https://doi.org/10.1038/s41467-023-37972-1III. Mark Chernyshev*, Aron Stålmarck*, Martin Corcoran, Gunilla B. Karlsson Hedestam, Ben Murrell"Detection of PCR chimeras in adaptive immune receptor repertoire sequencing using hidden Markov models" * Equal Contribution. [Submitted]</p
Advancing neuromuscular electrical stimulation optimizing comfort and hemodynamic efficiency
No full textPhysical inactivity and immobilization are significant contributors to global health challenges, including venous thromboembolism (VTE), muscle atrophy, and chronic diseases. Neuromuscular electrical stimulation (NMES) has emerged as a promising intervention to counteract these effects by mimicking muscle contractions and enhancing venous circulation. However, traditional NMES systems face barriers such as discomfort, complex setups, and poor compliance.This thesis explores innovative approaches to improve NMES comfort and efficiency by integrating textile electrodes into wearable garments, such as socks, and optimizing stimulation parameters. The research comprised five interrelated studies investigating the design, efficacy, and usability of textile- based NMES systems.Study I examined the construction of textile electrodes, focusing on how hydration and pressure affected stimulation performance. Results demonstrated that hydrated electrodes under moderate compression (~20 mmHg) significantly improved comfort and reduced both current intensity requirements and performance variability for effective muscle activation.Study II mapped motor points on the calf muscles to identify areas optimal for electrode placement. A probability heatmap was developed to guide electrode positioning, thereby enhancing stimulation precision without requiring professional assistance.Study III compared newly developed transverse textile electrodes integrated into socks (TTE-socks) with standard gel electrodes placed on motor points (MPE) at different intensity levels. Both configurations effectively increased venous hemodynamics in key veins, such as the popliteal and femoral veins, compared to baseline. However, in most of the comparisons, NMES via TTE- socks produced significantly greater hemodynamic responses, though it required slightly higher current and caused marginally more discomfort.Study IV optimized NMES parameter settings for the TTE-sock by testing combinations of frequency (1 or 36 Hz) and phase duration ranging from 75-400 microseconds (us). Findings revealed that low-frequency stimulation at 1 Hz, combined with a phase duration between 150 us and 400 us, was the most energy-efficient while maintaining comfort, making it suitable for long-term use in wearable devices.Study V explored the hemodynamic effects of frequency (1 Hz vs. 36 Hz) and plateau time using TTE-socks. Low-frequency 1 Hz NMES produced distinct single twitches that enhanced venous flow with minimal discomfort. In contrast, high-frequency 36 Hz NMES induced sustained tetanic contractions associated with greater discomfort but also higher hemodynamic efficacy. These findings underscore the importance of tailoring NMES parameters to individual needs for optimal compliance and therapeutic outcomes.The thesis highlights the transformative potential of wearable NMES systems integrated into everyday garments to address physical inactivity-related health issues. By combining textile-based electrodes with optimized stimulation protocols, this research presents a user-friendly solution that may enhance comfort, mobility, and compliance. The integration of NMES into clothing has the potential to revolutionize preventive healthcare for populations at risk of VTE or unable to engage in regular physical activity due to aging or chronic conditions. Future research should focus on large-scale clinical trials to validate these findings and explore broader applications of wearable NMES technology across diverse medical contexts.List of scientific papersI. Euler L, Juthberg R, Flodin J, Guo L, Ackermann PW, Persson NK. Textile electrodes: influence of electrode construction and pressure on stimulation performance in neuromuscular electrical stimulation (NMES). Annu Int Conf IEEE Eng Med Biol Soc. 2021;2021:1305-1308. https://doi.org/10.1109/EMBC46164.2021.9630649II. Schriwer E, Juthberg R, Flodin J, Ackermann PW. Motor point heatmap of the calf. J Neuroeng Rehabil. 2023 Mar;20(1):28. https://doi.org/10.1186/s12984-023-01152-5III. Sundstrom C, Juthberg R, Flodin J, Guo L, Persson NK, Ackermann PW. Effects on hemodynamic enhancement and discomfort of a new textile electrode integrated in a sock during calf neuromuscular electrical stimulation. Eur J Appl Physiol. 2023 May;123(9):2013- 2022. https://doi.org/10.1007/s00421-023-05212-5IV. Juthberg R, Flodin J, Guo L, Rodriguez S, Persson NK, Ackermann PW. Neuromuscular electrical stimulation in garments optimized for compliance. Eur J Appl Physiol. 2023 Apr;123(8):1739-1748. https://doi.org/10.1007/s00421-023-05181-9 (+ Correction Eur J Appl Physiol. 2023 Aug;123(8):1749 https://doi.org/10.1007/s00421-023-05211-6). V. Juthberg R, Flodin J, Aliaga N, Guo L, Rodriguez S, Persson NK, Ackermann PW. Electrically Induced Hemodynamic Enhancement via Sock-Integrated Electrodes is More Comfortable and Efficient at 1 Hz as compared to 36 Hz. [Accepted]</p
Biomarker and pathogenic study of immune-mediated neuropathies
No full textThere is a lack of reliable biomarkers for diagnosis, prognosis assessment, and monitoring of treatment response in immune-mediated neuropathies. The diagnosis is predominantly based on clinical assessment and electrophysiological findings. Although immune-mediated neuropathies are treatable, existing therapies are nonspecific and fail to provide adequate benefit for a significant number of patients.In Study I, we investigated the diagnostic and prognostic value of axonal injury biomarkers, neurofilament light (NfL) chain and total tau (T-tau), in plasma and CSF of patients with Guillain-Barre syndrome (GBS), chronic inflammatory demyelinating polyradiculoneuropathy, multifocal motor neuropathy (MMN), and paraproteinemic demyelinating neuropathy (PDN). In GBS, CIDP, and PDN, levels of NfL were elevated in both plasma and CSF, whereas in patients with MMN the levels of NfL were elevated only in plasma. NfL in plasma and CSF was associated with prognosis in GBS and may also help distinguish MMN from amyotrophic lateral sclerosis (ALS). Additionally, T-tau in plasma emerged as a novel diagnostic biomarker for both acute and chronic immune-mediated neuropathies.In Study II, we analysed over 1100 biomarkers in plasma and CSF using proximity extension assay (PEA) technology in two independent cohorts (discovery and replication) of patients with GBS and CIDP. We identified several candidate biomarkers with potential diagnostic value for GBS and CIDP. However, the most consistent finding was upregulation of interleukin-8 (IL8) in CSF. To validate this finding, we further analysed levels of IL8 using a chemiluminescence assay in a third (validation) cohort, which included patients with GBS, CIDP, PDN, MMN, healthy individuals, and other disease controls. IL8 in CSF is validated as a diagnostic biomarker for GBS and CIDP, and shows potential as a prognostic biomarker for both short- and long-term outcomes in GBS.Beta-trace protein (BTP) is the second most abundant protein in CSF after albumin, yet it has not previously been studied in patients with immune-mediated neuropathies. In Study III, we investigated the levels of BTP in CSF and plasma of patients with GBS, CIDP, disease controls, and healthy controls. In CIDP, levels of BTP were elevated in both CSF and plasma, while in GBS only in CSF. Notably, in patients with GBS and CIDP, levels of BTP in CSF were elevated regardless of the Q.Alb, suggesting that BTP may serve as a more informative diagnostic biomarker than albumin. Furthermore, the levels of BTP in CSF may have predictive value for treatment response in CIDP.Numerous attempts to develop new therapies for GBS other than intravenous immunoglobulin and plasma exchange have been unsuccessful. Previous studies have shown that blocking the tyrosine kinase receptor platelet-derived growth factor receptor-alpha (PDGFRa) with imatinib, a small tyrosine kinase inhibitor, regulate the function of the brain-blood barrier. In Study IV, we found that imatinib reduced the severity of experimental autoimmune neuritis (EAN), an animal model of GBS, by improving blood-nerve barrier (BNB) integrity. This conclusion was supported by clinical scoring, functional electrophysiological testing, and histological assessment of the BNB. These findings demonstrate the efficacy of imatinib in treating EAN and provide evidence for its potential clinical application in patients with GBS.In summary, this thesis identifies candidate biomarkers for immune-mediated neuropathies and proposes a novel therapeutic strategy for GBS.List of scientific papersI. Ivan Kmezic, Kristin Samuelsson, Anja Finn, Zane Upate, Kaj Blennow, Henrik Zetterberg, Rayomand Press. Neurofilament light chain and total tau in the differential diagnosis and prognostic evaluation of acute and chronic inflammatory polyneuropathies. Eur J Neurol. 2022 Sep;29(9):2810-2822. https://doi.org/10.1111/ene.15428II. Ivan Kmezic, Rasmus Gustafsson, Katharina Fink, Anders Svenningsson, Kristin Samuelsson, Caroline Ingre, Tomas Olsson, Magnus Hansson, Ingrid Kockum, Milena Zeitelhofer Adzemovic, Rayomand Press. Validation of elevated levels of interleukin-8 in the cerebrospinal fluid, and discovery of new biomarkers in patients with GBS and CIDP using a proximity extension assay. Front Immunol. 2023 Nov 23:14:1241199. https://doi.org/10.3389/fimmu.2023.1241199III. Ivan Kmezic, Rasmus Gustafsson, Magnus Hansson, Rayomand Press. Beta-trace protein in chronic inflammatory demyelinating polyradiculoneuropathy and Guillain-Barré syndrome clinical utilization and a new insight into pathophysiological mechanisms. Online ahead of print. J Neurol Sci. 2025 Feb 28:472:123439. https://doi.org/10.1016/j.jns.2025.123439IV. Ivan Kmezic, Charles Dorris, Humberto Skott, Manuel Arauji, Rayomand Press, Manuel Zeitelhofer, Milena Zeitelhofer Adzemovic. Imatinib ameliorates experimental autoimmune neuritis, an animal model of Guillain-Barre syndrome. [Manuscript]</p
Nutrition and health-related quality of life after multimodality treatment of gastroesophageal cancer
No full textBackground: Gastroesophageal cancer (GEC) is notorious for causing extreme weight loss and symptoms contributing to weight loss are often the first sign of the disease. Malnutrition is not only associated with increased morbidity and mortality but does also impact on eligibility for multimodality treatment which might further deteriorate nutritional status and exacerbate weight loss. While nutrition is a key component in the standardized postoperative recovery protocol, nutritional route is not included due to lack of scientific evidence. At Karolinska University Hospital patients undergoing esophagectomy routinely receive a nutritional jejunostomy. Potential advantages of this approach were evaluated in study I. Quality of life measurements does not only assessing overall quality of life but also assess nutritional aspects. Study II assess effects of neoadjuvant treatment on Health-Related Quality of Life (HRQL) while study IV evaluates HRQL on time-to-surgery from neoadjuvant chemoradiotherapy (nCRT). Study III is a pilot-study for pharmacological treatment of nutritional issues in patients operated for GEC where HRQL serves as a secondary outcome.Aim: The overall aim of this thesis was to investigate measures involved in the multimodal treatment of GEC to improve nutritional aspects and HRQL, enhancing understanding and treatment and ultimately contributing to reduce morbidity and mortality.Method and results: Study I examined 1871 patients who underwent GEC surgery in Sweden between 2006 and 2017. Multivariable logistic regression showed that postoperative complications were more common in patients with a nutritional jejunostomy (39.0% vs. 44.4%, p=0.019). A sub-analysis demonstrated benefit of jejunostomy among patients with postoperative leakage (OR 0.19 for Clavien-Dindo score ≥ IIIb, range 0.04- 0.94). Study II evaluated the effects of neoadjuvant therapy vs. surgery alone in 361 patients undergoing curatively intended GEC surgery at Karolinska University Hospital between 2013 and 2020. Multivariable linear regression demonstrated less pain and fewer problems related to eating restrictions in patients assigned to neoadjuvant treatment (-11, p=0.004, - 12, p=0.005). Study III assessed safety and tolerability of Octreotide LAR depot, 10mg, administered intramuscularly at 7 days, 1 month and 2 months postoperatively. The study included 20 patients (10 gastrectomy, 10 esophagectomy) and was conducted from September 2021 to October 2023. Of the 20 participants, 19 (95%) completed the treatment. Mild to moderate adverse events were reported in 12 participants (60%). Two patients experienced serious adverse events, although none attributed to the exposure. Study IV was a secondary endpoint analysis within a multicenter randomized controlled trial, in which patients were randomized between standard time-to-surgery (4-6 weeks) and prolonged time-to-surgery (10-12 weeks). Among the 192 patients who completed at least one HRQL-questionnaire, 95 (49%) were assigned to the prolonged time-to-surgery. Preoperatively, after nCRT, patients in the prolonged time-to-surgery group reported better HRQL compared to those in the standard time-to-surgery group, but postoperatively no clinically significant differences were found.Conclusions: The studies included in this thesis demonstrates beneficial use of selective jejunostomy and improved preoperative HRQL after neoadjuvant treatment compared to surgery alone. It demonstrates tolerability of Sandostatin LAR depot, which might potentially mitigate the undesired postoperative weight-loss in patients with GEC. Finally, the results demonstrates that postoperative HRQL is unaffected by time-to-surgery after esophagectomy for cancer.List of scientific papersI. Holmén A, Hayami M, Szabo E, Rouvelas I, Agustsson T, Klevebro F. Nutritional jejunostomy in esophagectomy for cancer, a national register-based cohort study of associations with postoperative outcomes and survival. Langenbecks Arch Surg. 2021 Aug;406(5):1415-1423. https://doi.org/10.1007/s00423-020-02037-0Il. Holmen A, Jebril W, Ida S, Agustsson T, Lampi M, Rouvelas I, Sunde B, Klevebro F. Effects of neoadjuvant therapy on health-related quality of life for patients with gastroesophageal cancer. Eur J Surg Oncol. 2023 Nov;49(11):107008. https://doi.org/1016/j.ejso.2023.107008III. Holmén A, Lampi M, Rouvelas I, Sunde B, Agustsson T, Lindberg G, Klevebro F. Postoperative Treatment with Octreotide in Patients with Gastroesophageal Cancer: An Open-Label Phase 2 Study. [Submitted]IV. Holmen A, Murad F, Nilsson K, Rouvelas I, Lindblad M, Szabo E, Halldestam I, Smedh U, Wallner B, Johansson J, Johnsen G, Aahlin E.K, Johannsessen H-O, Alexandersson von Döbeln G, Hjortland G.O, Wang N, Shang Y, Borg D, Quaas A, Bertella I, Bruns C, Schröder W, Nilsson M, Klevebro F, Sunde B. Health-related quality of life comparing standard and prolonged time-to-surgery after neoadjuvant chemoradiotherapy for esophageal cancer - results from the multicenter, randomized, controlled NeoRes II trial. [Manuscript]</p
Advanced care in patients with out-of-hospital cardiac arrest
No full textOut-of-hospital cardiac arrest (OHCA) is one major cause of mortality and is responsible for up to one fifth of deaths globally. Despite intense efforts to improve survival only one out of ten patients survive and even in cases with favourable resuscitation characteristics, survival chances decline fast if initial treatment fails. These patients are often referred to as refractory OHCA and the overall aim of this thesis is to characterize and investigate whether survival in this group can be improved using advanced circulatory support, compared to standard treatment alone. Additionally, treatment of OHCA with hypothermia at the intensive care unit is investigated.Methods and resultsStudy I is a registry-based observational cohort study evaluating the change in use of target temperature management (TTM) in Sweden following the TTM-trial and whether the treatment shift from 33 ℃ to 36 ℃ had any impact on 6-month survival. A total of 2,899 patients with OHCA were included for analysis of which 1,402 were treated with TTM. Following the publication of the TTM-trial, treatment with TTM declined, against guideline recommendations, from 70.5% to 54.5% but without significant impact on 6-month survival between the groups TTM33 (47.2%) and TTM36 (47.3%) (adjusted OR 1.12, 95% CI 0.8 - 1.56).Study II is an observational study evaluating the implementation of an extracorporeal cardiopulmonary resuscitation (ECPR) program for refractory OHCA in Stockholm, Sweden. Data were prospectively collected and outcomes included feasibility, safety and clinical aspects. A total of 95 patients with refractory OHCA were included for analysis of which 22/95 (23%) achieved ROSC prior to ECPR, 39/95 (41%) were excluded for extracorporeal membrane oxygenation (ECMO), 34/95 (36%) had ECMO initiated and finally 23 patients on ECMO were admitted alive to the ICU. The target time interval of ECMO-initiation within 60 minutes was met in 9% of cases. The most reported safety aspect among those cannulated were complicated vascular access (n=6) and severe bleeding at access site requiring intervention (n=2). Survival to discharge among those admitted to ICU on ECMO was 23% (9/23) of which 78% (7/9) had good neurologic function. Overall survival was 25% (24/95) and most of the survivors treated with ECMO, 8 out of 9 patients, had more than the intended 60 minutes to ECMO-initiation.Study III is a registry-based observational study investigating the burden of coronary artery disease in relation to the number of defibrillations among patients with OHCA. Patients with initial shockable rhythm who underwent coronary angiography were included and grouped according to number of defibrillations: 1-3, 4-6, 7-9 and ≥ 10. Outcomes included coronary angiography findings described as acute occlusion, chronic occlusion, multivessel disease and percutaneous coronary intervention (PCI) as well as 1-year survival. In total 2,594 patients were included for analysis. Most patients had significant stenosis (84% - 69%) with the highest prevalence among those receiving 1-3 defibrillations. PCI was also more common among those receiving 1-3 defibrillations (74%) compared to the other groups of defibrillations (62-54%). Acute coronary occlusions were similar between the groups (44% - 38%). One- year survival declined with increased number of defibrillations regardless of adjustment of coronary angiography findings and percutaneous coronary intervention.Study IV is a registry-based observational study exploring whether a simple combination of patient and prehospital characteristics can identify patients with initial shockable rhythm with high risk of not achieving ROSC and who might benefit from early transport. Multiple variables were analysed for importance using random forest and the proportion of patients without ROSC based on resuscitation time were analysed using Kaplan Meier curves. Outcome was "no ROSC" at hospital arrival. A subgroup analysis was performed on patients fulfilling the ECPR-criteria from study II. In total 10,651 patients were included of which 5,390 patients (51%) did not achieve ROSC. The most important variables for predicting "no ROSC" was absence of ROSC ten minutes after EMS arrival and adrenaline administered, both for the overall cohort and the subgroup meeting ECPR-criteria. Combining these two variables identified 83% (n=2592/3108) without ROSC in the full cohort and 81% (n=464/572) in ECPR subgroup.ConclusionsFollowing the publication of the TTM-trial, the use of TTM in Sweden decreased, against guideline recommendations, but without any effect on 6-month survival.The implementation of an ECPR program in Stockholm, Sweden, was feasible without any major safety aspects but the intended time intervals were not met.Despite this survival rates were comparable to previous ECPR studies, and most survivors had more than 60 minutes to ECMO initiation.Among patients with shockable OHCA undergoing coronary angiography we did not observe an increase in burden of coronary artery disease with increased number of defibrillations.In cases of shockable OHCA, absence of ROSC 10 minutes after EMS arrival and adrenaline administered had the highest impact on predicting "no ROCS" and combined they identified 83% without ROSC in the full cohort and 81% in the ECPR subgroup.List of scientific papersI. Abazi L, Awad A, Nordberg P, Jonsson M, Taccone FS, Wickerts CJ, Svensson L, Hollenberg J, Ringh M, Forsberg S. Long-term survival in out-of-hospital cardiac arrest patients treated with targeted temperature control at 33 ℃ or 36 ℃: A national registry study. Resuscitation. 2019;143:142-147. https://doi.org/10.1016/j.resuscitation.2019.08.029II. Lis Frykler Abazi, Andreas Liliequist, Felix Böhm, Magnus Hedberg, Moa Simonsson, Anders Backman, Malin Ax, Frieder Braunschweig, Linda Mellbin, Rickard Linder, Leif Svensson, Juliane Jurga, Per Nordberg, Mattias Ringh, Sune Forsberg, Jacob Hollenberg Implementation of an extracorporeal resuscitation (ECPR) program for out-of-hospital cardiac arrest in Stockholm, Sweden: Feasibility, safety, and outcome Resusc Plus. 2024;18:100596. https://doi.org/10.1016/j.resplu.2024.100596III. Lis Frykler Abazi, Sune Forsberg, Felix Böhm, Martin Jonsson, Mattias Ringh, Gabriel Riva, Ludvig Elfwén, Per Nordberg, Akil Awad, Charlotte Miedel, Anette Nord, Andreas Claesson, Nils Witt, Jacob Hollenberg Burden of coronary artery disease in relation to the number of defibrillations in out-of-hospital cardiac arrest. [Submitted]IV. Lis Frykler Abazi, Mattias Ringh, Per Nordberg, Martin Jonsson, Araz Rawshani, Anette Nord, Andreas Claesson, Gabriel Riva, Jacob Hollenberg, Sune Forsberg On-scene identification of refractoriness in out-of-hospital cardiac arrest with shockable rhythm. [Manuscript]</p
Progression of chronic kidney disease : risk factors, sex differences and intervention
No full textBackgroundMortality and morbidity in pre-dialytic patients are substantial, including in people with mild to moderate chronic kidney disease (CKD). There are also unexplained sex-specific differences regarding prevalence, incidence, mortality and disease progression. Some studies suggest CKD is more prevalent in women, however the risk of kidney replacement therapy is higher for men. Explanations for this relationship includes unequal access to kidney replacement therapy, a faster progression in men, increased mortality in pre-dialytic stages among women and differences in monitoring and management. Diabetes is one of the leading causes of CKD, why adequate blood glucose control is a primary clinical goal for these patients. Broccoli sprout have a high content of glucoraphanin, a precursor of sulforaphane, which has been shown to decrease oxidative stress. Translational studies have shown that supplementation with broccoli sprout extract can improve metabolic control, but the effect in people with CKD remains uncertain. Hypoparathyroidism is a rare disease with unknown prevalence in Sweden and is a condition characterized by low level of parathyroid hormone. It can lead to severe and sometimes life-threatening hypocalcemia. Treatment therefore consists of calcium and vitamin D supplements, and introduced more recently, treatment with parathyroid hormone analogues. However, to what extent a regimen with high doses of calcium and vitamin D could cause kidney damage over time is not known.ObjectiveThe aim of study I was to investigate sex-specific differences in mortality and disease progression in patients with CKD stage 3b to 5, while study III explored differences between men and women in monitoring and management of people with CKD in a non-referred, general population. The aim of study II was to investigate the risk for CKD among people with hypoparathyroidism. Study IV investigated whether broccoli sprout extract could improve metabolic control in people with CKD and type 2 diabetes.Methods and resultsStudy I used the Swedish Renal Registry-Chronic Kidney Disease (SRR-CKD). Incident patients with non-dialysis CKD 3b-5 identified between 2010 and 2018 were included. The outcomes of interest were sex-specific differences in time to CKD progression, death and repeated assessment of estimated glomerular filtration rate (eGFR). Of 26 279 incident cases, women had a lower risk for progression of CKD stage (SHR 0.88, 95% CI 0.85-0.92), mortality by any cause (SHR 0.90, 95% CI 0.85-0.94) and less steep decline of eGFR, compared to men.Study II identified people with hypoparathyroidism through the Swedish National Patient Registry and Prescribed Drug Registry. A total of 1562 individuals with hypoparathyroidism and without prior CKD were included. The Total Population Registry was used to find matched controls. The outcomes of interest were time to a diagnosis with CKD and urolithiasis and incidence rates of hospitalization. Patient with hypoparathyroidism had an increased risk of CKD (HR 4.45, 95% CI 3.66-5.42) and were more likely to be hospitalized due to CKD (HR 3.49, 95% CI 2.84-4.30), compared to controls.Study III used the Stockholm Creatinine Measurements Project, a repository of laboratory data in Region Stockholm between 2009 and 2017. A total of 227 847 individuals with at least one outpatient eGFR Study IV was a double-blind, placebo-controlled trial including people with CKD 3b-4 and type 2 diabetes. A total of 99 individuals were randomized to either broccoli sprout extract or placebo for 12 weeks. Metabolic control measured by fasting glucose, HbA1c, insulin and oral glucose tolerance test did not differ between treatment groups at the end of follow-up.ConclusionsMen with CKD were more likely to progress to a more advanced CKD stage and had a steeper decline in eGFR, while women were less likely to receive a CKD diagnosis, have timely monitoring and to be referred to a nephrologist. Also, people with hypoparathyroidism had a markedly higher risk for developing CKD compared to controls. In the clinical trial, broccoli sprout extract did not improve metabolic control in people with CKD and type 2 diabetes.List of scientific papersI. Swartling O, Rydell H, Stendahl M, Segelmark M, Trolle Lagerros Y, Evans M. CKD Progression and Mortality Among Men and Women: A Nationwide Study in Sweden American Journal of Kidney Diseases, 2021;78(2):190-199https://doi.org/10.1053/j.ajkd.2020.11.026II. Swartling O, Evans M, Spelman T, Kamal W, Kämpe O, Mannstadt M, Trolle Lagerros Y, Björnsdottir S. Kidney Complications and Hospitalization in Patients with Chronic Hypoparathyroidism: A Cohort Study in Sweden Journal of Clinical Endocrinology and Metabolism, 2022;107(10):e4098-e4105https://doi.org/10.1210/clinem/dgac456III. Swartling O, Yang Y, Clase CM, Fu EL, Hecking M, Hodlmoser S, Trolle Lagerros Y, Evans M, Carrero JJ. Sex Differences in the Recognition, Monitoring, and Management of CKD in Health Care: An Observational Cohort Study Journal of the American Society of Nephrology, 2022;33(10):1903- 1914https://doi.org/10.1681/asn.2022030373IV. Avesani C, Swartling O, Rosengren AH, Heimbürger O, Svensson MK, Guron G, Segelmark M, Lundberg S, Jonsson A, Mörtberg J, Bratt O, Bonnevier U, Fernström A, Christensson A, Fontanive R, Evans M, Stenvinkel P, for the INITIATE Study Group No effect of broccoli sprout extract on glycemic control in patients with CKD and type 2 diabetes: A randomized clinical trial [Manuscript]</p
Characterization of novel genetic variants causing mitochondrial disease
No full textBackground. The motivation for this thesis is the desire to improve diagnostics, to increase knowledge about mitochondrial diseases and thus be able to help more patients. To give the patients an explanation for their illness, in some cases offering treatment and in other cases offering prenatal diagnostics to families who have lost a child is incredibly rewarding. Over the years, a lot of unclear variants have been detected in clinical genetic diagnostics at my workplace, Centre for Inherited Metabolic Diseases (CMMS) at Karolinska University Hospital. With the introduction of next-generation sequencing (NGS) about 12 years ago, the number of solved cases has increased exponentially but this is also true for the number of unclear variants detected. In order to find out whether a variant is disease-causing in the patient, the following questions must be addressed. Is the variant rare enough? Is the inheritance pattern as expected? What does the genetic variant lead to at the mRNA and/or protein level? What is the disease mechanism? Can the patient's clinical and biochemical picture be linked to the specific gene? In this thesis, we validate the pathogenicity of some of these variants and report the molecular, clinical, and biochemical findings in the patients.Methods. This work had not been possible without Clinical Genomics at Science for Life Laboratory at Karolinska Institutet that has the NGS platforms for research and diagnostics and that, in close collaboration with CMMS and Clinical Genetics and Genomics at the hospital, has developed all necessary bioinformatic tools needed for handling the enormous amount of data generated and the annotation and sorting of detected variants. The collaboration is called Genomic Medicine Center Karolinska (GMCK) and has the mission to enable the implementation of genomics-based diagnostics into Swedish healthcare. For validation we have used Drosophila melanogaster as an animal model, primary cells (fibroblasts and myoblasts) from the patients, different in-vitro and biochemical assays, immunostaining, Western and Southern blots, Blue Native Gel electrophoresis and RNA sequencing.Results. A novel variant, p.Tyr955His, in the mitochondrial DNA polymerase POLYA was shown to have a dominant negative effect on mtDNA synthesis and ligation and was found to cause an early-onset severe disease. The mtDNA variant m.10372G>A in the gene for mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 3 (MT-ND3) caused a severe complex I deficiency in skeletal muscle and led to an adult-onset polyneuropathy. In addition we describe a new disease gene, NDUFB7, in this thesis. The causative variant was an intronic variant that resulted in abnormal splicing of mRNA and absence of the protein in cells from the patient. Rescue experiments were performed to connect the complex I deficiency to NDUFB7. In the last study several synonymous and intronic variants in the genes PDHA1, PDHX and TPK1 were shown to lead to exon skipping or intronic inclusions and to cause primary or secondary pyruvate dehydrogenase deficiency in the patients.Significance. For the patients and the families included in the studies the importance is to get a genetic diagnosis, to obtain a better prognosis for the course of the disease and specific genetic counseling. The genetic diagnosis can guide the treatment, e.g. ketogenic diet in patients with pyruvate dehydrogenase deficiency, thiamine supplementation in thiamine pyrophosphokinase deficiency but also the choice of antiepileptic drugs and to enable prenatal diagnostics in future pregnancies. In a larger context the thesis contributes to increase knowledge about novel variants, disease genes, disease mechanisms, and phenotypes associated with mitochondrial disorders.List of scientific papersI. A multi-systemic mitochondrial disorder due to a dominant p.Y955H disease variant in DNA polymerase gamma. Siibak T, Clemente P, Bratic A, Bruhn H, Kauppila TES, Macao B, Schober FA, Lesko N, Wibom R, Naess K, Nennesmo I, Wedell A, Peter B, Freyer C, Falkenberg M, Wredenberg A Hum Mol Genet. 2017 Jul 1;26(13):2515-2525https://doi.org/10.1093/hmg/ddx146II. Novel Mutation m.10372A>G in MT-ND3 Causing Sensorimotor Axonal Polyneuropathy Bruhn H, Samuelsson K, Schober FA, Engvall M, Lesko N, Wibom R, Nennesmo I, Calvo-Garrido J, Press R, Stranneheim H, Freyer C, Wedell A, Wredenberg A Neurol Genet. 2021 Mar 15;7(2):e566.https://doi.org/10.1212/nxg.0000000000000566III. Severe congenital lactic acidosis and hypertrophic cardiomyopathy caused by an intronic variant in NDUFB7. Correia SP, Moedas MF, Naess K, Bruhn H, Maffezzini C, Calvo- Garrido J, Lesko N, Wibom R, Schober FA, Jemt A, Stranneheim H, Freyer C, Wedell A, Wredenberg A Hum Mutat. 2021 Apr;42(4):378-384.https://doi.org/10.1002/humu.24173IV. Novel Synonymous and Deep Intronic Variants Causing Primary and Secondary Pyruvate Dehydrogenase Complex Deficiency. Bruhn H, Naess K, Ygberg S, Peña-Pérez L, Lesko N, Wibom R, Freyer C, Stranneheim H, Wedell A, Wredenberg A Hum Mutat. 2024 https://doi.org/10.1155/2024/1611838</p
Whole genome sequencing in pediatric ALL - a tool for understanding chromosomal aberrations and improving diagnostic procedures
No full textWhole genome sequencing is a revolutionary technology that has changed the field of genomics. By providing unprecedented base pair resolution, WGS allows for precise identification of disease causing genetic abnormalities. While cytogenetic and molecular methods are well established in clinical practice, the application of WGS in cancer diagnostics remains at the developmental stage. Additionally, detecting complex genomic events currently requires a multi- modal approach, as each method is targeted and has limited resolution. This reliance on multiple techniques often leads to cumbersome workflows and requires input from various experts with specialized knowledge.One of the diseases that requires genetic characterization is acute lymphoblastic leukemia (ALL). This disease is known to be genetically heterogeneous, with structural genomic changes playing a central role in leukemogenesis. Currently, ALL is divided into genetic subgroups, each associated with different outcomes. Detecting and identifying the genetic aberrations driving disease progression often requires the use of multiple methods. Therefore, having a single method capable of detecting all genetic aberrations would be highly beneficial.In this thesis, the efforts to assess the value of WGS as a stand-alone method in clinical diagnostics are described. The aim is not only to detect the genetic aberrations described in the treatment protocols, but also to identify emerging genetic subgroups and novel gene fusions that have diagnostic significance or can be used for targeted therapy.In Paper I WGS was applied to a pediatric B-cell ALL (B-ALL) cohort to investigate its feasibility to detect clinically relevant genetic aberrations. Two sequencing depths (30x versus 90x coverages) and two analysis approaches (leukemia-only vs leukemia/normal) were tested, and the results were comprehensively compared to findings from standard of care (SoC) methods. Based on our analysis, sequencing leukemia samples with 30x coverage was sufficient to detect all obligatory aberrations. Additionally, almost all patients without stratifying genetic markers could be allocated to one of the emerging subgroups. The paper demonstrates the potential of WGS in the diagnostic setting of ALL .In Paper II we applied WGS to genetically characterize a case of pediatric T-cell ALL (T-ALL) that did not respond to standard therapy. WGS detected a novel JAK2:CCDC88C gene fusion, resulting in a chimeric transcript. This novel transcript, containing the tyrosine kinase domain of JAK2, produces a chimeric protein expected to respond to a JAK2 inhibitor. This case report shows how WGS can be used to find personalized treatment options for leukemia patients.In Paper III we present a proof-of-concept study describing the use of patient- specific genetic markers detected by WGS to monitor disease. This is performed by designing sensitive quantitative assays targeting these genetic markers. WGS was applied to detect structural variants (SVs) and other relevant leukemic genetic alterations resulting in unique sequences in six pediatric ALL cases. We then used this data to identify patient-specific targets and designed droplet digital PCR (ddPCR) assays to enable the absolute quantification of these targets. Subsequently, the technical feasibility of these patient-specific targets was assessed for monitoring measurable residual disease (MRD) in genomic DNA and cell-free tumor DNA. The results showed that the sequences provided by WGS enabled us to identify patient-specific targets that could be used for sensitive and specific detection of leukemic cells.Manuscript IV explores the complexity of a specific ALL subtype. We applied WGS and whole transcriptome sequencing (WTS) to perform a detailed characterization of the genomic and transcriptomic profiles of Philadelphia- positive ALL (Ph+ALL). We have integrated the findings from SoC diagnostics and MRD information to comprehensively investigate the features of Ph+ALL and potential for refining diagnostics for these patients.Together, these studies explored the potential of WGS in clinical diagnostics and enhanced the understanding of genetic aberrations in pediatric ALL.List of scientific papersI. Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia. F. Rezayee, J. Eisfeldt, A. Skaftason, I. Öfverholm, S. Sayyab, A. C. Syvänen, K. Maqbool, H. Lilljebjörn, B. Johansson, L. Olsson- Arvidsson, C. Orsmark Pietras, A. Staffas, L. Palmqvist, T. Fioretos, L. Cavelier, L. Fogelstrand, J. Nordlund, V. Wirta, R. Rosenquist, and G. Barbany. Front Oncol 2023, 13:1217712. https://doi.org/10.3389/fonc.2023.1217712II. Case Report: Whole genome sequencing identifies CCDC88C as a novel JAK2 fusion partner in pediatric T-cell acute lymphoblastic leukemia. A. Krstic, F. Rezayee, L. Saft, A. Hammarsjö, P. Svenberg, and G. Barbany. Front. Pediatr. 2023, 10:1082986. https://doi.org/10.3389/fped.2022.1082986III. Patient-specific assays based on whole-genome sequencing data to measure residual disease in children with acute lymphoblastic leukemia: a proof of concept study. C. Arthurt; F. Rezayee; N. Mogensen; L. Saft; R. Rosenquist; M. Nordenskjold; A. Harila-Saari; E. Tham; G. Barbany Front Oncol 2022, 12, 899325. https://doi.org/10.3389/fonc.2022.899325IV. Unveiling the complexity of Philadelphia-positive acute lymphoblastic leukemia through the integration of genomic, phenotypic, and measurable residual disease data. F. Rezayee, J. Eisfeldt, A. Krstic, P. Svenberg, J. Joelsson, K. Belander Strålin, H. Vogt, S. Deneberg, S. Volanthen, L. Saft, R. Rosenquist, and G. Barbany. [Manuscript]</p
Prevention and treatment of cognitive decline in a mouse model of cranial radiotherapy
No full textPediatric brain cancers remain the leading cause of cancer-related mortality in children. With advancements in medical technology, survival rates have significantly improved, with more children reaching the five-year survival milestone. However, for many, survival comes at a high cost. Cranial radiotherapy, a cornerstone in tumor management and metastasis control, leaves lasting imprints on the developing brain. Among its most devastating consequences are late-emerging cognitive complications that persist throughout life, profoundly affecting the quality of life for survivors. Despite growing recognition of these impairments, the underlying mechanisms remain elusive, and no effective treatments exist.Furthermore, sex has emerged as a potential risk factor influencing the severity of these deficits, adding another layer of complexity to an already pressing clinical challenge.One of the leading hypotheses attributes these cognitive deficits to the depletion of neurogenesis, the birth of new neurons, in the hippocampus, a central hub for learning and memory. The failure of hippocampal neurogenesis to recover after radiation is thought to stem from chronic microglial activation. As the primary immune cells of the central nervous system (CNS), microglia are essential for normal brain maturation, synaptic refinement, and immune surveillance. Hence, understanding how microglia respond to irradiation over time, could provide critical insights into the mechanisms driving radiation-induced cognitive impairments.This thesis investigates the long-term alterations in microglia across the mouse lifespan, both in normal aging and following cranial irradiation. A particular focus is also placed on the sex-dependent neuroinflammatory response. Beyond identifying key molecular events after irradiation, we sought to identify key components of the inflammatory cascade that could be therapeutically targeted to mitigate radiation-induced brain injury.To enable this investigation, we developed a rapid and robust single-cell isolation protocol for brain subregions, facilitating the enrichment of traditionally hard-to- isolate microglia and vascular cells. Importantly, our method eliminates the need for additional enrichment steps, making it highly reproducible and well-suited for unbiased longitudinal single-cell RNA sequencing studies across different brain regions, time points, and cell types.Leveraging this novel approach, we conducted a comprehensive longitudinal analysis of the hippocampal response to cranial irradiation, spanning up to one year post-exposure. Microglia emerged as the most distinct and dynamically affected population, prompting an in-depth examination of their radiation- induced states. Our findings revealed the presence of temporally specific radiation-associated microglia (RAM) subtypes, characterized by a delayed yet sustained inflammatory response in the hippocampus. This persistent activation, spearheaded by microglia, appeared to play a crucial role in shaping long-term microglial dynamics following irradiation. We observed a progressive loss of microglia, which failed to compensate for their depletion through self-renewal. This deficit ultimately led to the infiltration of monocyte-derived macrophages, which gradually adopted a microglial-like phenotype. Functional analysis revealed a correlation between these molecular events and episodes of neuronal asynchrony, suggesting a potential link between radiation-induced microglial dysfunction and impaired network activity.Building upon these findings, we explored sex-dependent differences in the inflammatory response to cranial irradiation. Transcriptomic profiling revealed a more pronounced inflammatory signature in female hippocampi compared to males, driven primarily by microglia and likely mediated by distinct molecular mechanisms. Expanding our analysis beyond the hippocampus to cognition- related areas such as the cortex and cerebellum, we uncovered differential susceptibilities across brain regions. While females exhibited the strongest inflammatory response in the hippocampus, they also showed the greatest microglial loss in the cortex-a pattern mirrored in males, albeit to a lesser extent.To translate these insights into potential therapeutic strategies, we employed a combination of computational and in vivo approaches to identify key regulators of the inflammatory pathway. By modulating the expression of these targets at critical time points using clinically approved antisense oligonucleotides, we demonstrated that dampening their activity could effectively reduce irradiation- induced neuroinflammation in the hippocampus. These findings highlight a promising avenue for mitigating cognitive decline in pediatric brain cancer survivors.Finally, to contextualize our findings within the broader landscape of neurodegenerative processes, we examined microglial dynamics in an AppNL-G-F knock-in mouse model, resembling amyloid pathology and Alzheimer's disease. Our analysis revealed dynamic transcriptional changes in microglia before and after plaque accumulation, coinciding with disruptions in hippocampal network activity, impaired synchronization of fast-spiking interneurons and plaque formation.Overall, this thesis provides novel and critical insights into microglial responses during normal brain aging and in the aftermath of cranial irradiation. By elucidating sex-specific vulnerabilities and identifying key inflammatory mediators, our work lays the foundation for potential therapeutic interventions aimed at preserving cognitive function. Ultimately, these findings bring us one step closer to improving the quality of life for pediatric brain tumor survivors and their families.List of scientific papersI. Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses Efthalia Preka#, Alejandro Lastra Romero#, Ying Sun, Yara Onetti Vilalta, Thea Seitz, Adamantia Fragkopoulou, Christer Betsholtz, Ahmed M Osman, Klas Blomgren #Shared first authorship Heliyon, volume 10, issue 16 (2024); e35838https://doi.org/10.1016/j.heliyon.2024.e35838II. Microglia Adopt Temporally Specific States after Irradiation, Correlating with Neuronal AsynchronyAlejandro Lastra Romero#, Efthalia Preka#, Giusy Pizzirusso, Luis Enrique Arroyo-García, Georgios Alkis Zisiadis, Nuria Oliva-Vilarnau, Yana Ruchiy, Thea Seitz, Kai Zhou, Arturo Gonzalez Isla, Lara Friess, Ying Sun, Maria Querol Canut, Alia Shamik, Yiran Xu, Changlian Zhu, Carlos F. D. Rodrigues, André Fisahn, Bertrand Joseph, Lena-Maria Carlson, Adamantia Fragkopoulou, Volker M Lauschke, Christer Betsholtz, Ahmed M Osman*, Klas Blomgren* #Shared first authorship, *Shared senior authorship [Manuscript]III. Sex- and brain region-dependent inflammatory responses to cranial irradiationEfthalia Preka, Maria Querol Canut, Sophie Sandner, Allanah Greer, Kalliopi-Maria Tamvaki, Xeni Androni, Yara Onetti Vilalta, Ahmed M. Osman, Klas Blomgren, Adamantia Fragkopoulou [Manuscript]IV. Antisense Therapy Targeting the cGAS-STING Pathway to Alleviate Neuroinflammation after Cranial RadiotherapyEfthalia Preka, Maria Querol Canut, Sophie Sandner, Allanah Greer, Apostolia-Ioli Diamanti, Niki Throumpari, Renato Esposito, Lei Huang, Zunpeng Liu, Yara Onetti Vilalta, Manolis Kellis, Ahmed M. Osman, Fredrik Kamme, Klas Blomgren, Adamantia Fragkopoulou [Manuscript]V. Dynamic microglia alterations associate with hippocampal network impairments: A turning point in amyloid pathology progression Giusy Pizzirusso, Efthalia Preka#, Julen Goikolea#, Celia Aguilar-Ruiz, Patricia Rodriguez-Rodriguez, Guillermo Vazquez-Cabrera, Simona Laterza, Maria Latorre-Leal, Francesca Eroli, Klas Blomgren, Silvia Maioli, Per Nilsson, Adamantia Fragkopoulou, André Fisahn, Luis Enrique Arroyo-García #Equal authorshipBrain, Behavior and Immunity, July 2024, Volume 119, Pages 286-300https://doi.org/10.1016/j.bbi.2024.04.009</p