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CD8<sup>+</sup> T cell subsets in acute infection and anti-tumor immunity
No full textCD8+ T cells are key immunotherapeutic targets due to their adaptability and functional diversity. This thesis investigates the utility of the chemokine receptor CX3CR1 in delineating CD8+ T cell functional potentials across various immunological contexts.In paper I, we introduce the CX3CR1 expression gradient as a predictive tool for assessing the functional potential of memory CD8+ T cells in both human and mouse models. Paper II explores differentiation-unrelated factors influencing CX3CR1 levels in CD8+ T cell cultures. We, therefore, could propose better practices to expand CX3CR1 gradient utility to adoptive cell therapy applications. Paper III examines the phenotypic characteristics of CX3CR1+ CD8+ T cells that arise in response to tumour antigens. I propose that unidentified spatial factors influence anti-tumour CX3CR1+ CD8+ T cell functional potentials and drive intratumoral cells toward dysfunction. Together, these studies contribute to a deeper understanding of CX3CR1 as a biomarker for CD8+ T cell differentiation, offering insights to enhance the clinical outcomes of CD8+ T cell- based therapies.List of scientific papersI. Graded expression of the chemokine receptor CX3CR1 marks differentiation states of human and murine T cells and enables cross-species interpretation. Anthonie Johan Zwijnenburg, Jyoti Pokharel, Renata Varnaitė, Wenning Zheng, Elena Hoffer, IMAN SHRYKI, Natalia Ramirez Comet, Marcus Ehrstrom, Sara Gredmark-Russ, Liv Eidsmo, Carmen Gerlach. Immunity. 2023, 56(8), 1955-1974.e10. https://doi.org/10.1016/j.immuni.2023.06.025II. The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells. Jyoti Pokharel*, IMAN SHRYKI*, Anthonie Johan Zwijnenburg, Ioana Sandu, Laura Krumm, Christina Bekiari, Rebecka Heinbäck, Victor Avramov, Josefin Lysell, Liv Eidsmo, Helena Erlandsson Harris and Carmen Gerlach. European Journal of Immunology. 2024, 54(1):e2350658. https://doi.org/10.1002/eji.202350658III. CD8+ T cell responses to cytoplasmic antigens in anti-tumor immunity. IMAN SHRYKI, Natalia Ramirez Comet, Carmen Gerlach. [Manuscript]* Equal contribution</p
Strategies to predict patient-specific outcomes after endovascular aneurysm repair
No full textAn abdominal aortic aneurysm (AAA) is an irreversible dilatation of the abdominal aorta marked by progressive vascular degeneration. AAAs are often asymptomatic and typically discovered incidentally. Swedish screening detected prevalence of AAA is approximately 1-2% in males over 65 years old and 0.5% in women over 70 years. Main risk factors include smoking, male sex, age and heredity. AAAs may rupture if not treated in time. Treatment is considered when the AAA diameter reaches 55 mm in men and 50 mm in women and performed by either open surgical repair (OSR) or endovascular aneurysm repair (EVAR). EVAR offers improved short-term results but is associated with more complications and inferior long-term outcome compared to OSR.The overall aim of this thesis was to explore and develop new insights regarding short and long-term outcomes in patients undergoing EVAR, through morphological, biomechanical and epidemiological analyses. The thesis studies focused on identifying the occurrence and factors related to endograft complications and device-specific failure. Furthermore, sac change after EVAR was examined as an indicator for clinical success. A final topic of this thesis was to investigate patients who exhibit optimal postoperative outcome and characterise predictive factors and sex-specific differences.Study I, a multicentre consecutive cohort of 924 patients treated with three contemporary EVAR devices were assessed for incidence of limb graft occlusion (LGO) and associated risk factors. The Zenith Alpha (ZA) device demonstrated the highest occlusion rate and was found to be an independent risk factor of LGO in addition to external iliac artery size and landing zone. Study II was a single centre analysis of 34 occluded Zenith Alpha patients from Study I with 95 consecutive control patients to investigate factors influencing LGO with the ZA. Morphological analysis of patient anatomy and limb features demonstrated a cumulative effect of oversizing, excessive limb overlap and a narrow aortic bifurcation as device-specific risks of LGO with the ZA. In Study III, 64 EVAR patients were stratified by presence of early sac regression within the first postoperative year. Patients with sac regression (n=39) exhibited less endograft- related complications, fewer reinterventions and improved long-term survival. Preoperatively measured ILT characteristics were associated with sac change in addition to aspirin use, AAA diameter and smoking. Study IV was a nationwide SWEDVASC investigation of 5411 EVAR patients to determine the proportion of patients presenting with optimal mid-term outcome defined as an absence of complications within the first postoperative year, no secondary aneurysm rupture and a minimum of three-year survival. This was true for 45% of all patients, a younger and less comorbid subgroup. These patients exhibited a more pronounced sac regression, irrespective of sex.In summary, device-type is a novel risk factor for LGO, and device-specific features of the ZA must be considered during EVAR to minimize risks of LGO. Early sac regression is a robust surrogate marker of clinical EVAR success and aneurysms have different prerequisites for sac change related to biomechanical, morphological and demographic features. Around half of patients demonstrates optimal mid-term outcome after EVAR and preoperative sex-specific considerations are vital, as they reveal differences in outcome and sac change.List of scientific papersI. Limb Graft Occlusion Following Endovascular Aneurysm Repair for Infrarenal Abdominal Aortic Aneurysm with the Zenith Alpha, Excluder, and Endurant Devices: A Multicentre Cohort Study. Bogdanovic M, Stackelberg O, Lindström D, Ersryd S, Andersson M, Roos H, Siika A, Jonsson M, Roy J. Eur J Vasc Endovasc Surg. 2021 Oct;62(4):532-539. https://doi.org/10.1016/j.ejvs.2021.05.015II. Predictors of Limb Graft Occlusion after Endovascular Aneurysm Repair with the Zenith Alpha Abdominal Endograft. Bogdanovic M, Huss M, Lindström D, Ersryd S, Andersson M, Roos H, Siika A, Jonsson M, Roy J. [Manuscript]III. Biomechanics and Early Sac Regression after Endovascular Aneurysm Repair of Abdominal Aortic Aneurysm. Bogdanovic M, Siika A, Lindquist Liljeqvist M, Gasser TC, Hultgren R, Roy J. JVS Vasc Sci. 2023 Mar 30;4:100104. https://doi.org/10.1016/j.jvssci.2023.100104IV. Identifying Patients with Optimal Mid-term Outcome after Elective Endovascular Aneurysm Repair - a Swedvasc Study. Bogdanovic M, Talvitie M, Siika A, Lindquist Liljeqvist M, Roy J, Hultgren R. [Manuscript]</p
The effect of eccentric exercise on decompression strain
No full textDuring high-altitude flight, extravehicular activity, and following diving, decompression sickness (DCS) can occur. Even mild symptoms of DCS (e.g., joint pain) may jeopardise a mission and necessitate treatment to resolve. The aetiology of DCS is thought to be the generation of gas bubbles within tissues and the vascular system, the latter known as venous gas emboli (VGE). These bubbles are believed to be formed from gas-saturated tissues and precursor bubbles, also known as micronuclei. There is limited knowledge regarding potential pre-decompression events that might provoke the development of DCS. Anecdotal observations made in reports suggest a relationship between strenuous exercise and musculoskeletal injury with an increased risk of DCS. However, no controlled studies in humans have been conducted to further explore this relationship. Given that aviators and divers frequently engage in strenuous physical activity, it is of interest, not least from a practical viewpoint, to determine whether and to what degree muscle damage induced by strenuous physical exercise may increase the risk of developing DCS. Eccentric contractions, where muscles lengthen under tension, cause greater exercise-induced muscle damage (EIMD) than concentric or isometric contractions. Although many eccentric exercises have been studied for their potential to cause EIMD, the effects of eccentric arm cycling remain unexplored.In this thesis, we examined the effect of eccentric exercise on the formation of VGE as a marker of decompression strain. The thesis is based on four separate studies with the collective aim of investigating the effects of eccentric exercise on muscle damage and the formation of high-altitude-induced VGE.The first study investigated eccentric arm cycling as a mode of exercise to induce muscle damage. The results show that 15 minutes of eccentric arm cycling is enough to induce EIMD, as evidenced by a reduction in isometric strength, delayed onset of muscle soreness, and elevation of markers indicative of muscle damage.The second study investigated the effect of eccentric upper-body exercise on the formation of VGE. The study included two conditions: (i) eccentric exercise performed 24 hours prior to decompression and (ii) no exercise (Control). The results show that performing eccentric exercise 24 hours prior to a continuous exposure to 24, 000 feet for 90 minutes led to an earlier onset and increased VGE load compared to the Control.The third study examined the effect of EIMD and its magnitude on high-altitude-induced VGE. The study included three conditions: (i) eccentric whole-body exercise, (ii) eccentric upper body exercise, and (iii) Control. The results show that the impact of eccentric exercise on high-altitude-induced VGE seems to vary depending on whether eccentric exercise has been performed in the upper body or lower body, rather than on the total muscle mass recruited.The fourth study investigated the effect of eccentric exercise on muscle damage and inflammation and explored their possible roles in hypobaric VGE formation. The study included VGE-data from studies II and III, along with blood samples collected in conjunction with Control/exercise interventions and altitude exposures. The findings suggest that eccentric EIMD and inflammation are associated with a higher decompression strain. Furthermore, VGE load seems to induce and exacerbate systemic inflammation in a dose-dependent manner.List of scientific papersI. The effect of eccentric arm cycling on muscle damage and injury-related biomarkers. Gottschalk F, Gennser M, Eiken O, Elia A. Clinical Physiology and Functional Imaging. 2024 Oct; Online ahead of print. https://doi.org/10.1111/cpf.12911II. Eccentric exercise 24 h prior to hypobaric decompression increases decompression strain. Gottschalk F, Eiken O, Elia A, Gennser M. European Journal of Applied Physiology. 2023 Sep; 123(9):2001-2011. https://doi.org/10.1007/s00421-023-05214-3III. Eccentric exercise before a 90-min exposure at 24,000 ft increases decompression strain depending on body region but not total muscle mass recruited. Gottschalk F, Gennser M, Günther M, Eiken O, Elia A. Experimental Physiology. 2024 Sep;109(9):1517-1528. https://doi.org/10.1113/ep091853IV. Eccentric exercise, muscle damage and inflammation in conjunction with high-altitude decompression. Gottschalk F, Gennser M, Günther M, Eiken O, Elia A. [Submitted]</p
Repeated cognitive assessments show stable function over time in patients with ALS.
No full textBACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multisystem disorder with not only motor symptoms but also extra-motor features including cognitive impairment. The most common cognitive profile observed in patients with ALS includes deficits in executive function, language, and social cognition. However, longitudinal studies on cognitive changes over time in ALS are sparse. We aimed to investigate the presence and nature of cognitive impairment at the time of ALS diagnosis and its association with survival as well as explore longitudinal cognitive change. METHOD: Patients (n = 216) were recruited at the Karolinska University Hospital in Stockholm, Sweden. Follow-up visits (n = 307 in total) were performed every 6 months. Cognitive impairment was assessed using the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) and/or Montreal Cognitive Assessment (MoCA). RESULTS: Cognitive impairment was observed in 38% of the patients at the time of ALS diagnosis, and the majority of these patients had deficits in executive function and/or language. Patients with cognitive impairment at the time of diagnosis had a more rapid decline in ALSFRS-R at 12- and 18-months follow-up, and a shorter survival. Cognitive function was stable during the first 2 years after diagnosis, and did not follow the trajectories of decline in motor functions. CONCLUSION: Cognitive impairment in ALS was associated with a faster decline of motor functions, and shorter survival. However, cognitive function did not deteriorate over time. Cognitive assessment is essential for the patients and caregivers to understand the phenotypic expression of ALS
Classification of asthma with cross-cohort clinical data and with enhancer mediated gene regulation
No full textInflammatory diseases such as IBD and asthma, along with metabolic disorders like obesity, are complex conditions shaped by a combination of genetic, environmental, and immunological factors. Understanding the molecular mechanisms driving these diseases is crucial for the development of targeted therapies and improving patient care. With the advent of bioinformatics and data- driven approaches, we have new opportunities to identify key biomarkers, regulatory mechanisms, and gene expression patterns associated with these conditions.In this study, we bring together different types of data, including proteomics, transcriptomics, and epigenomics, to investigate the roles of specific proteins, gene enhancers, and molecular markers in inflammatory and metabolic diseases. One of the key challenges we face is handling unstructured data from various sources, such as clinical trials and patient registries, especially when working with different cohort studies that have diverse data types and collection methods. By using robust computational tools and statistical methods, I addressed the complexity of organizing and analyzing these datasets, ensuring they are accurately combined across different cohorts.Paper-I investigates serum proteins encoded at known IBD risk loci. By profiling serum from Crohn's disease (CD) and ulcerative colitis (UC) patients, we identified differentially expressed proteins, including LACC1, linked to genetic variants associated with IBD. This targeted proteomic analysis provided insights into potential protein biomarkers that could distinguish between IBD subtypes, offering diagnostic and therapeutic potential.Paper-II applies biostatistical modeling and unsupervised clustering to explore asthma phenotypes by integrating clinical and molecular data from two large asthma cohorts (BIOAIR and U-BIOPRED). This data-driven approach revealed distinct asthma subgroups and associated biomarkers, emphasizing the heterogeneity of the disease. By combining bioinformatics and biostatistics, we contribute to personalized asthma management by identifying key phenotypic traits and inflammatory markers.Paper-III focuses on enhancer regulation in severe and mild childhood asthma. Using CAGE RNA sequencing, we identified asthma-specific enhancers and their interactions with transcription start sites (TSS) of genes linked to immune responses. Bioinformatics techniques were used to map enhancer activity and gene regulation, highlighting the role of non-coding regulatory elements in asthma severity. These findings enhance our understanding of gene regulation in asthma and provide novel targets for therapeutic interventions.Paper-IV explores enhancer activity in human white adipose tissue (WAT) and its role in insulin response. We identified active enhancers involved in regulating insulin-responsive genes in both obese and non-obese individuals, using chromatin conformation and CAGE sequencing data. This study uncovered key regulatory elements linked to obesity-related traits and insulin sensitivity, providing insights into metabolic regulation and potential intervention points for obesity and insulin resistance.Across all studies, bioinformatics and data-driven methodologies such as GWAS, CAGE-seq, and clustering algorithms were integral in identifying disease-relevant molecular signatures. The integration of multi-omics data allowed us to gain a comprehensive understanding of gene regulation, enhancer activity, and protein biomarkers in complex diseases, demonstrating the power of computational biology in advancing precision medicine.List of scientific papersI. Drobin K, Assadi G, Hong MG, Andersson E, Fredolini C, Forsström B, Reznichenko A, Akhter T, Ek WE, Bonfiglio F, Berner Hansen M, Sandberg K, Greco D, Repsilber D, Schwenk JM, D'Amato M, Halfvarson J. Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci. Inflammatory Bowel Diseases. 2019 Jan 10;25(2):306-316. https://doi.org/10.1093/ibd/izy326II. Akhter T, Kolmert J, James A, Andersson LI, Adcock IM, Wheelock CE, Dahlen SE, Kupczyk M, Daub CO. Explorative Biostatistical Modelling of Selected Clinical and Molecular Data from Two Patient Cohorts to Define Common Phenotypical Traits in Asthma. [Manuscript]III. Akhter T, Mileti E, Kere M, Kolmert J, Konradsen JR, Hedlin G, Melén E, Daub CO. Enhancers regulate genes linked to severe and mild childhood asthma. Heliyon. 2024 Jul 9;10(14). eCollection 2024 Jul 30.https://doi.org/10.1016/j.heliyon.2024.e34386IV. Mileti E, Kwok KH, Salvatore M, Raman A, Dias MS, Gustafsson C, Akhter T, Bonetti A, Andersson R, Månsson R, Mejhert N, Arner P, Ryden M, Daub CO. Enhancers Facilitate Acute Insulin Response in Human White Adipose Tissue. [Manuscript]</p
Understanding psychosocial and economic barriers to develop innovative people-centered models of tuberculosis care in Nepal
No full textBackgroundTuberculosis (TB) is the predominant cause of death from a single infectious disease in low- and middle-income countries (LMICs), causing 1.3 million deaths in 2022. Poverty, undernutrition, and poor living conditions drive TB. The majority of TB disease occurs in people who are poor, with little or no savings and marginal income to defray costs. Nepal is an LMIC in South Asia with a high TB prevalence of 416 people per 100,000 population. TB is predominantly diagnosed using a passive case-finding strategy (PCF), where people who feel ill, visit the government health centers of their own volition, and health providers evaluate symptoms. Despite the routine TB diagnosis and treatment is free of charge, the economic burden relative to household income is high, and psychosocial consequences include depression and high levels of stigma towards people with TB and their households in Nepal. To overcome these barriers, Nepal’s National TB Program strategies, as well as global TB strategies such as the END TB strategy, acknowledge that ending TB requires a comprehensive set of proven interventions rather than just a single biomedical approach. This warrants the development of effective strategies to address complex factors contributing to the development of TB, improving access to healthcare and reducing the negative impact of having TB. To do so, there is a need for a context-specific comprehensive understanding of the determinants of TB, the psychosocial characteristics of people with TB, and the burden of impact due to having TB. In addition, geographical barriers to accessing health care are a significant challenge in Nepal. Exploring the potential of innovative technologies such as drones for improving healthcare delivery provides a promising avenue to overcome such barriers and enhance access to services in remote areas. However, the existing evidence on effective and innovative TB care models in Nepal is scarce.AimThe thesis aims to provide context-specific evidence of barriers and facilitators to access TB diagnosis and care in Nepal and increase knowledge of the psychosocial characteristics and consequences of TB in Nepal.MethodsThe data for the thesis was collected between July 2017 and December 2019 from five districts of Nepal (Chitwan, Makwanpur, Dhanusha, Mahottari, and Pyuthan). The thesis contains four studies (I-IV). Study I involved quantitative, prospective longitudinal cohort studies that interviewed 221 people with TB and 119 people without active TB disease (controls). The study used an adapted and validated WHO Patient Cost questionnaire with additional structured questions on TB stigma, depression, and quality of life. The study evaluated the psychosocial factors of TB among people with TB who were at 8-12 weeks (baseline) and 22-26 weeks (follow-up) of treatment and compared them with single interviews taken with controls. Study II also included the longitudinal cohort study design and the same questionnaire and used the data from 221 people with TB (111 ACF and 110 PCF) to characterize the psychosocial consequences of TB and evaluate the role of ACF in mitigating the impact. Studies III and IV were qualitative studies that used semi-structured focus group discussions (FGD) guides. Study III involved seven FGDs with 54 TB stakeholders to understand the barriers and facilitators of accessing and engaging with TB diagnosis and care. Study IV included five FGDs with 40 community stakeholders and healthcare providers in Pyuthan district which explored their perceptions on using cargo drones to support TB diagnosis.ResultsStudy I: The determinants of TB were poverty and lack of education. People in the two lowest tertiles poorer (adjusted odds ratio (aOR):2.31; 95% confidence interval (CI) [1.2 – 4.45]), poorest (aOR:2.84; 95%CI [1.39-5.79]) and without education (aOR: 2.92; 95%CI [1.28-6.67]) were associated with being a person with TB. People with TB were more likely to have depression in comparison to the controls when measured at baseline (25/221, 11% versus 0/119, 0%; pStudy II: Among people who had TB, at baseline, one in three had mild or major depression (68/221, 31%). Compared to baseline (25/221, 11%), the proportion of people reporting major depression reduced at follow-up (11/221, 5%). There was no difference in stigma score, depression, and quality of life among people with TB identified by ACF or PCF.Study III: National multisectoral stakeholders perceived broader impediments to accessing and adhering to TB care, which encompassed individual, community, and health system levels. Such barriers included a lack of TB knowledge, psychosocial problems such as stigma, anxiety and depression, poor nutrition, low social support, and geographical impediments. However, the stakeholders perceived that these barriers could be addressed through basic health and TB education, mutual support, enhanced allowance from the NTP, and decentralized, community-based diagnostic services.Study IV: Stakeholders in Pyuthan expressed trust in drones, viewing them as an advantageous tool for transporting sputum samples, reducing distance and time barriers for TB care, and fostering community development opportunities. Nevertheless, perceived challenges in operating drones for TB included financial sustainability and the technical capacity of local people to independently operate drones in Nepal.ConclusionsThe findings in this thesis underscore the multifaceted challenges that people with TB in Nepal face during TB treatment. Poverty and lack of education were key determinants of TB. The results show that ACF has no role in mitigating stigma, depression, or improving quality of life. TB has profound effects on people because of stigma and depression during their treatment. These factors severely limit their ability to access and complete TB care pathways. This highlights the urgent need to integrate screening for depression into routine TB care. The studies also emphasize the need to enhance health and TB education, provide locally appropriate social protection interventions, and strengthen the health system by investing in the use of innovations such as drones to develop people-centered TB diagnosis and care in Nepal. Addressing the social, economic, and psychological dimensions of TB, alongside innovative healthcare solutions, will be key to achieving the goal of ending TB in Nepal.List of scientific papersI. Kritika Dixit, Bhola Rai, Noemia Teixeira de Siqueira-Filha, Raghu Dhital, Tara Prasad Aryal, Manoj Kumar Sah, Ram Narayan Pandit, Puskar Raj Paudel, Jens W. Levy, Job van Rest, Suman Chandra Gurung, Gokul Mishra, Knut Lönnroth, Stephen Bertel Squire, Laura Bonnett, Kristi Sidney Annerstedt, Maxine Caws, Tom Wingfield Poverty, food insecurity, stigma, depression and quality of life among people with and without tuberculosis in Nepal: a prospective cohort study with nested cross-sectional comparator arm. Infectious Diseases of Poverty. [Manuscript]II. Kritika Dixit, Bhola Rai, Noemia Teixeira de Siqueira-Filha, Raghu Dhital, Tara Prasad Aryal, Manoj Kumar Sah, Ram Narayan Pandit,Puskar Raj Paudel, Jens W. Levy, Job van Rest, Suman Chandra Gurung, Gokul Mishra, Knut Lönnroth, Stephen Bertel Squire, Kristi Sidney Annerstedt, Laura Bonnett, Ahmad Fuady, Maxine Caws, Tom Wingfield. Stigma, depression, and quality of life among people with pulmonary tuberculosis diagnosed through active and passive case finding in Nepal: a prospective cohort study. BMC Global and Public Health. 2024; 2 (1). https://doi.org/10.1186/s44263-024-00049-2III. Kritika Dixit, Olivia Biermann, Bhola Rai, Tara Prasad Aryal, Gokul Mishra, Noemia Teixeira de Siqueira-Filha, Puskar Raj Paudel, Ram Narayan Pandit, Manoj Kumar Sah, Govinda Majhi, Jens W. Levy, Job van Rest, Suman Chandra Gurung, Raghu Dhital, Knut Lönnroth, Stephen Bertel Squire, Maxine Caws, Kristi Sidney Annerstedt, Tom Wingfield. Barriers and facilitators to accessing tuberculosis care in Nepal: a qualitative study to inform the design of a socioeconomic support intervention. BMJ Open; 11(10):e049900. https://doi.org/10.1136/bmjopen-2021-049900IV. Kritika Dixit, Bhola Rai, Govind Majhi, Rajan Paudel, Raghu Dhital, Shraddha Acharya, Ganga Ram Budhathoki, Puskar Raj Paudel, Suman Chandra Gurung, Bishal Subedi, Pravin Lamsal, Uttam Pudasaini, Peter Small, Patrick Meier, Kristi Sidney Annerstedt, Maxine Caws. Healthcare providers' and community stakeholders’ perception of using drones for tuberculosis diagnosis in Nepal: An exploratory qualitative study. BMC Rural Health Services. [Manuscript]</p
Autoantibodies and lipids - novel contributors to pain in fibromyalgia?
No full textFibromyalgia (FM) is a chronic primary pain condition that affects 2-8% of the population, predominantly women (80%). It is characterized by pain that arises from musculoskeletal tissue, and skin. Other concomitant symptoms are usually fatigue, anxiety, depression, cognitive and sleep impairment. Although FM is non- destructive, it can significantly diminish the quality of life and contribute to high socio-economic costs. Albeit promising findings regarding changes in FM, its pathophysiology remains poorly understood, and efficient treatments are lacking. This thesis sought to investigate cellular and molecular contributors to pain in FM.Study I explored whether autoimmunity could be a key player in FM pathogenesis. Immunoglobulins G (IgG) isolated from the serum of FM patients were injected intraperitoneally into mice, resulting in polymodal hypersensitivities, reduced skin innervation, decreased locomotion, and reduced grip strength. Conversely, IgG isolated from healthy controls (HC). IgG was sequestered into the dorsal root ganglia (DRG), and consequently bound and activated satellite glial cells (SGC) in vitro and in vivo. Anti-SGC IgG also recognized epitopes present in postmortem human dorsal root ganglia (hDRG).Study II assessed anti-SGC IgG levels in individual serum or plasma samples from 2 FM cohorts (Sweden and Canada, n=30/35), and HC. Additionally, correlations of anti-SGC IgG and phenotypic characteristics were investigated. Our screening assay, consisting of primary cell cultures enriched for SGCs that were "live" incubated with serum or plasma, showed that FM IgG bound more frequently and strongly to in vitro murine SGCs, and postmortem human DRGs; predominantly in SGCs but also to some extent neuronal soma, compared to HC. The percentage of anti-SGC IgG from FM patients was significantly correlated with disease severity, in the form of VAS and FIQ scores, and to a lesser extent PPT. K-means clustering analysis based on VASaverage, VASmax and FIQ, split the patients into severe and mild FM groups. Importantly, the FM severe group presented increased frequency and binding intensity of FM IgG to SGCs, indicating a distinct subset of FM patients with increased IgG reactivity.Study III used lipidomic analysis to investigate lipid changes, associated to disease severity, and anti-SGC IgG levels in serum from FM normal-weight patients (n=35), compared to HC (n=33). High-performance liquid chromatography (HPLC) coupled to high-resolution mass spectrometry (LC- HRMS) was performed. Patients were divided into mild and severe groups, according to K-means clustering based on VASaverage, VASmax and FIQ scores. IgG binding to SGCs was assessed as described in Study II. Lipid levels were adjusted for independent covariates, such as BMI, age, and week of MS acquisition using linear regression (LR). Additionally, the relationship between anti-SGC IgG levels and lipid levels was analyzed with LR, and lipid levels and pain scores, such as VAS, FIQ, PPT and CPM were analyzed with Spearman rank's correlation. Decreased levels of lysophosphatidylcholine (LPC) (n=10), lysophsphatidylethanolamine (LPE) (n=7), phosphatidylcholine (PC) (n=4) and triglycerides (TGs) (n=5) were observed in FM. In contrast, diglycerides (DGs) (n=3) were found to be upregulated. A number of LPC species, such as 19:0, 22:0, and 24:1 were upregulated in the severe FM group, compared to the mild, and the latter two positively correlated with anti-SGC IgG levels. TGs correlated positively with pain ratings and to a lesser extent anti-SGC IgG levels. Sphingomyelins (SMs) (n=6) displayed increased concentrations in the mild FM group, compared to HC, but decreased in the severe FM group. Additionally, SMs (n=8) negatively correlated with clinical scores, with only one SM species showing a significant correlation with anti-SGC IgG levels. Not only SMs, but also one DG, PCs (n=3) and TGs (n=5) were negatively associated with phenotypic characteristics.Study IV measured Lysophosphatidic acid (LPA) in FM serum and cerebrospinal fluid (CSF). Autotaxin (ATX) levels from FM serum samples were also evaluated. For comparison, serum LPA levels were measured also in other painful conditions, such as osteoarthritis (OA), disc degeneration disc (DDD), and lumbar disc herniation (LDH). To investigate LPA and ATX levels, we employed LPA and ATX ELISA assays. We found a significant increase of LPA in the serum of FM patients, compared to HC. LPA levels correlated positively with conditioned pain modulation (CPM), and to a less extent VASmin. In CSF, no change of LPA levels nor ATX in the serum of FM patients was found. However, we observed a significant correlation between LPA and ATX levels in one FM cohort, substantiating the hypothesis of ATX being the responsible enzyme for the generation of LPA in FM. Interestingly, we found an increase of LPA in the serum of OA female patients, that correlated with VASpain and KOOS. Conversely, LPA levels were decreased in DDD, compared to HC. Our study highlights the presence of LPA in painful conditions, however in FM, a dual role is suggested, that requires further investigation.Taken together, this thesis highlights the presence of important peripheral contributors to pain in FM, such as autoimmunity, and changes in lipid metabolism, as seen in the following lipid classes: TG, LPC, SM as well as LPA, which can contribute to the symptomatology present in FM patients' subsets. Therapies that can reduce IgG titers or address the altered lipid metabolism might provide relief to these patients. Therefore, further mechanistic and longitudinal studies are required to fully elucidate pain mechanisms of FM.List of scientific papersI. Passive transfer of fibromyalgia symptoms from patients to mice. Andreas Goebel*, Emerson Krock*, Clive Gentry, Mathilde R. Israel, Alexandra Jurczak, Carlos Morado Urbina, Katalin Sandor, Nisha Vastani, Margot Maurer, Ulku Cuhadar, Serena Sensi, Yuki Nomura, Joana Menezes, Azar Baharpoor, Louisa Brieskorn, Angelica Sandström, Jeanette Tour, Diana Kadetoff, Lisbet Haglund, Eva Kosek, Stuart Bevan, Camilla I. Svensson#, and David A. Andersson#. J Clin Invest. 2021 Jul 1;131(13):e144201. https://doi.org/10.1172/JCI144201II. Fibromyalgia patients with elevated levels of anti-satellite glia cell immunoglobulin G antibodies present with more severe symptoms. Emerson Krock, Carlos E. Morado-Urbina, Joana Menezes, Matthew A. Hunt, Angelica Sandström, Diana Kadetoff, Jeanette Tour, Vivek Verma, Kim Kultima, Lisbet Haglund, Carolina B. Meloto, Luda Diatchenko, Eva Kosek, Camilla I. Svensson. Pain. 2023 Aug 1;164(8):1828-1840. https://doi.org/10.1097/j.pain.0000000000002881III. Fibromyalgia patients have altered lipid concentrations associated with disease symptom severity and anti-satellite glial cell IgG antibodies. Jenny E. Jakobsson* , Joana Menezes* , Emerson Krock, Matthew A. Hunt, Henrik Carlsson, Aina Vaidade, Payam Emami Khoonsari, Nilesh M. Agalave, Angelica Sandström, Diana Kadetoff, Jeanette Tour, Ida Erngren, Asma Al-Grety, Eva Freyhult, Katalin Sandor, Eva Kosek, Camilla I. Svensson, Kim Kultima. [Manuscript]IV. Comparative analysis of lysophosphatidic acid levels in fibromyalgia and other painful conditions. Joana Menezes, Jenny E. Jackobsson, Alex Bersellini, Farinotti, Emerson Krock, Matthew Hunt, Nils Simon, Sigita Venckute Larsson, Kim Kultima#, Eva Kosek#, Camilla I. Svensson#. [Manuscript]*, # Contributed equally</p
Advancements in testicular organoid research : insights for fertility preservation
No full textMale childhood cancer survivors who undergo highly gonadotoxic treatments and have not yet produced mature sperm can only maintain their fertility potential by testicular tissue cryopreservation. However, no clinically proven methods currently exist to restore fertility from these cryopreserved tissues. Fertility preservation for these patients is dependent on experimental techniques, including testicular tissue or cell transplantation, as well as in vitro methods, such as testicular organoids and testicular explant tissue culture.Firstly, we used a Matrigel-based 3D culture system to create prepubertal patient-derived testicular organoids and assess the impact of chemotherapy on organoid formation. Testicular cells from 11 boys were used for organoid formation and were compared with samples from 28 NORDFERTIL patients and 10 controls. Four of eleven prepubertal samples formed organoids with distinct cord-like structures and increased levels of testosterone and AMH. SOX9 expression was a key marker for successful organoid formation, and prior chemotherapy negatively affected its levels. This suggests SOX9 as a potential indicator for organoid formation and highlights the importance of Sertoli cells in maintaining testicular function and integrity.Further, we collected testicular samples from a paediatric cancer patient with B- cell acute lymphoblastic leukaemia to assess the effects of leukaemia contamination on prepubertal testicular tissue. Our results showed that primary cells from the enlarged leukemic testis formed organoids with seminiferous cord- like structures similar to the testicular tissue structure observed in explant tissue culture. In addition, no signs of leukemic infiltration were observed in the cultured explant tissues or organoids.Additionally, we evaluated testicular basement membrane protein composition during gonadal development and its role in germ cell maintenance. Our findings revealed that LAMA 1 is present in the testis during prenatal, prepubertal, and peripubertal stages, while LAMA 5 is exclusively found in the vasculature. Interestingly, the loss of LAMA 1 correlated with germ cell loss, underscoring its importance for testicular niche.Finally, we tested a synthetic biomimetic polyisocyanopeptides (PIC) based hydrogel (Noviogel) to generate murine testicular organoids. We showed that testicular cells self-aggregated in LN111 and LN121 cultures but not in LN521.Notably, only LN111-supplemented cultures produced organoids with distinct cord structures. These results demonstrate that primary mouse testicular cells can form organoids in a Matrigel-independent system and underscore the importance of specific laminin isoforms in organoid formation.In summary, we demonstrated that patient-derived testicular cells can form organoids with compartmentalized cord structures. Additionally, we established a Matrigel-independent culture system using a synthetic hydrogel, paving the way for advancements in tissue engineering, drug screening, toxicology, and regenerative medicine.List of scientific papersI. Prior exposure to alkylating agents negatively impacts testicular organoid formation in childhood cancer patients. Yanhua Cui, Femke Harteveld, Hajar Ali Mohammed Ba Omar, Yifan Yang, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Per Frisk, Kaisa Vepsäläinen, Cecilia Petersen, Rod T Mitchell, Jingtao Guo, João Pedro Alves-Lopes, Kirsi Jahnukainen, Jan-Bernd Stukenborg.Human Reproduction open. 2024; Aug 13;2024(3): hoae049.https://doi.org/10.1093/hropen/hoae049II. Organoid formation and explant-tissue culture of leukaemia-infiltrated prepubertal testicular tissue.Yanhua Cui, Jouko Lohi, Cecilia Lindskog, Kirsi Jahnukainen, Jan-Bernd Stukenborg. [Manuscript]III. Spermatogonia loss correlates with LAMA 1 expression in human prepubertal testes stored for fertility preservation.Magdalena Kurek, Elisabet Åkesson, Masahito Yoshihara, Elizabeth Oliver, Yanhua Cui, Martin Becker, João Pedro Alves-Lopes, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Cecilia Petersen, Olle Söder, Jingtao Guo, Rod T Mitchell, Kirsi Jahnukainen, Jan-Bernd Stukenborg Cells. 2021 Jan 27;10(2):241.https://doi.org/10.3390/cells10020241IV. Generation of testicular organoids in synthetic defined hydrogel.Yanhua Cui, Susana M. Chuva de Sousa Lopes, Jan-Bernd Stukenborg. [Manuscript]</p
Spatial immune dynamics in mucosal HIV infection
No full textSexual transmission is the most common mode of HIV infection, making the female genital and rectal tracts critical mucosal sites for preventing HIV transmission. Various factors, such as local inflammation and disruption of the epithelial barrier, have been suggested to increase the risk of sexual HIV transmission. Despite the significance of the female genital tract in this context, it remains less studied compared to other key sites like the gastrointestinal tract. Additionally, the long-term impact of HIV on the female genital tract is still not fully understood. Therefore, this thesis aims to investigate factors influencing HIV susceptibility and the long-term effects of chronic HIV infection on the female genital tract, utilizing a multi-omics approach that integrates conventional in situ bioimaging, spatial proteomics, and transcriptomics.Topical application of microbicides presents a promising strategy for preventing HIV in at-risk individuals. However, the failure of earlier microbicides underscores the need for more thorough safety evaluations. In Paper I, we performed an exploratory safety assessment following the topical rectal application of the promising microbicide Q-Griffithsin. We developed in situ bioimaging analysis workflows to evaluate its impact on the sensitive rectal epithelium and the CD4+ HIV target cell population. No adverse effects were observed on the rectal epithelium or on the frequency, density, and spatial distribution of CD4+ cells, indicating that Q-Griffithsin is safe based on these criteria.Depot medroxyprogesterone acetate (DMPA), a widely used hormonal contraceptive in HIV-endemic regions, has been linked to an increased risk of HIV acquisition, possibly due to induced inflammation, epithelial disruption and an altered genital microbiome, though the evidence remains conflicting. In Paper II, we used a multi-omics approach to thoroughly investigate the effects of DMPA on the cervical mucosa and its potential impact on HIV susceptibility. No differences in the cervical microbiome were detected at either the clinical or molecular level. Our results did however demonstrate a significant epithelial disruption, and an enhanced immunological profile associated with long-term DMPA use, both of which are factors linked to increased HIV susceptibility.Chronic systemic immune activation is a hallmark of HIV infection, but its effects on the female genital tract remain poorly understood. In Paper III, we performed a comprehensive analysis of the cervical transcriptional landscape in the context of chronic HIV infection and its impact on the cervical epithelium. Our transcriptional analysis revealed significant immune activation associated with HIV infection, primarily driven by interferon signaling and T cell activity. Additionally, we observed notable epithelial disruption, which was further validated through in situ bioimaging. These findings suggest a potential mechanistic link between cervical immune activation and epithelial instability, with important implications for HIV-related comorbidities in the female genital tract.In Paper IV, we extended our investigation of the T cell-related immune activation and epithelial disruption observed in previous studies, using the cutting-edge techniques, spatial transcriptomics and multi-epitope ligand cartography. Spatial transcriptomics revealed a tissue-wide induction of antibody-related genes, suggesting the recruitment of B cells or plasma cells into the cervical mucosa in association with HIV infection. Consistent with the findings from Paper III, we also observed significant induction of interferonrelated genes, alongside more sporadic activation of T cell-related genes. In situ bioimaging showed a marked expansion of tissue-resident memory CD8+ T cells and recruitment of peripheral CD8+ T cells. Interestingly, no changes were observed in the CD4+ T cell population, indicating that the systemic and gastrointestinal depletion of CD4+ T cells in HIV-infected individuals is not mirrored in the cervix of HIV-infected women.In conclusion, this thesis has explored factors associated with HIV infection and susceptibility in the rectal and female genital tracts using a multi-omics approach. We developed bioimage analysis workflows to assess epithelial integrity in both the single-layered rectal mucosa and the multi-layered stratified epithelium of the ectocervical mucosa. The findings provide new insights into immune activation, immune cell localization, and epithelial disruption linked to HIV susceptibility and infection. Notably, this is the first study to apply both bulk RNA sequencing and spatial transcriptomics to the female genital tract of HIV-infected women. Together, these results have important implications for HIV infection and prevention, potentially contributing to the development of more effective prevention strategies and targeted interventions for women at risk of, or living with, HIV.List of scientific papersI. A topical rectal douche product containing Q-Griffithsin does not disrupt the epithelial border or alter CD4+ cell distribution in the human rectal mucosa. Franzén Boger M, Benhach N, Hasselrot T, Brand RM, Rohan LC, Wang L, McGowan I, Edick S, Ho K, Meyn L, Matoba N, Palmer KE, Broliden K, Tjernlund A. Scientific Reports. 2023 May 9;13(1):7547. https://doi.org/10.1038/s41598-023-34107-wII. Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate. Bradley F, Franzén Boger M, Kaldhusdal V, Åhlberg A, Edfeldt G, Lajoie J, Bergström S, Omollo K, Damdimopoulos A, Czarnewski P, Månberg A, Oyugi J, Kimani J, Nilsson P, Fowke K, Tjernlund A, Broliden K. PLoS Pathogens. 2022 May 9;18(5):e1010494https://doi.org/10.1371/journal.ppat.1010494III. Sustained immune activation and impaired epithelial barrier integrity in the ectocervix of women with chronic HIV infection. Franzén Boger M, Hasselrot T, Kaldhusdal V, Miranda G, Czarnewski P, Edfeldt G, Bradley F, Rexaj G, Lajoie J, Omollo K, Kimani J, Fowke K, Broliden K, and Tjernlund A. [Submitted]IV. Spatial transcriptomics and in situ immune cell profiling of the host ectocervical landscape of HIV infected Kenyan sex working women. Franzén Boger M, Kaldhusdal V, Pascual-Reguant A, Kroh S, Uecker R, D. Burgener A, Lajoie J, Omollo K, Kimani J, Fowke K, E. Hauser A, Tjernlund A, and Broliden K. [Submitted]</p
Biomarkers for early detection and prognostic prediction of hepatocellular carcinoma
No full textHepatocellular carcinoma (HCC) is a highly lethal cancer, with over 40% of cases associated with chronic hepatitis B virus (HBV) infection. Early detection of HCC in high-risk HBV- infected populations can greatly improve patient survival; however, effective biomarkers for early detection are currently unavailable. Moreover, reliable biomarkers are needed to accurately predict patient prognosis due to the high heterogeneity of HCC. Liquid biopsy, through the analysis of circulating cell-free DNA (ccfDNA) released by tumor cells, offers a promising non-invasive approach for identifying HCC biomarkers.This thesis aimed to identify effective ccfDNA biomarkers for HCC, based on a population- based liver cancer screening cohort with a 7-year follow-up of 2,893 HBV-infected individuals. Over 400 samples were analysed, including 270 repeated samples collected before clinical diagnosis (pre-HCC) and non-HCC control samples randomly selected from the screening cohort, as well as 67 samples collected at the time of diagnosis from hospital HCC patients. We developed a novel bilateral single-strand sequencing method to analyse ccfDNA characteristics, including telomere and end sequences (Study I), genome-wide fragmentation (Study II), and tumor content (Study III).In Study I, we found that the short (25-60 nucleotides) single-stranded telomere G-tail DNA, but not double-stranded telomere DNA, was nearly 20-fold higher in HCC patients compared to non-HCC controls. We developed the telomere and end sequence in circulation (Telecon) model using samples from hospital HCC patients. The Telecon model perfectly distinguished HCC cases from controls, with an area under the curve (AUC) of 1.000 and 95% confidence interval (CI) of 0.998-1.000. Its performance in detecting pre-HCC samples improved as the time of diagnosis approached, with sensitivities increasing from 4.2% (> 4 years before diagnosis) to 59.1% (within 1 year before diagnosis) at 98.0% specificity. In the HBV-infected population, the model had an estimated positive predictive value of 10.2%. A high Telecon score was associated with shorter survival among HCC patients, with a hazard ratio (HR) of 3.22 (95% CI: 1.49-7.00).In Study II, we observed that HCC patients exhibited greater variation in ccfDNA fragmentation patterns across the genome, particularly on chromosomes 1q and 8q. A fragmentation model was developed using data from hospital HCC patients. This model excellently distinguished HCC patients from controls (AUC: 0.999; 95% CI: 0.997-1.000). The ccfDNA fragmentation score showed increasing performance in detecting pre-HCC samples, with sensitivities rising from 8.3% (> 4 years before diagnosis) to 36.4% (within 1 year before diagnosis) at 88.0% specificity. The positive predictive value was estimated at 1.1% in the HBV-infected population. A high ccfDNA fragmentation score was positively correlated with the Barcelona Clinic Liver Cancer (BCLC) stage and poorer survival (HR: 2.41; 95% CI: 1.13- 5.20).In Study III, the copy number-based ichorCNA algorithm was used to estimate tumor content in ccfDNA. We observed that tumor content decreased significantly after surgery. In non-HCC control samples, the specificity was 97.8%, with a mean tumor content of 0.011. In pre-HCC samples, the tumor content showed increasing sensitivities from 4.0% (≥ 4 years before diagnosis) to 22.7% (within 1 year before diagnosis), as mean tumor content increased from 0.014 to 0.026. In HCC samples, the sensitivity further increased to 30.4%, 81.8%, and 95.5% for patients at BCLC stages A, B, and C, respectively. HCC patients with high tumor content had poorer survival, particularly those at BCLC stage C (HR: 12.35; 95% CI: 1.42-107.90).In summary, all three methods-Telecon, genome-wide fragmentation, and tumor content-were effective in detecting HCC at diagnosis and predicting patient prognosis. Telecon, which measures short telomere G-tail, demonstrated the best performance in detecting samples before HCC diagnosis. However, detecting HCC prior to diagnosis remains challenging, and future studies that combine multiple features may improve accuracy.List of scientific papersI. Shifeng Lian*, Chenyu Lu*, Fugui Li*, Xia Yu, Limei Ai, Miao Yu, Biaohua Wu, Kuangrong Wei, Wenjing Zhou, Yulong Xie, Yun Du, Wen Quan, Panpan Wang, Li Deng, Zhiheng Liang, Xuejun Liang, Jiyun Zhan, Yong Yuan, Ellen T. Chang, Fang Fang, Zhiwei Liu, Mingfang Ji, Zongli Zheng. Early Detection and Disease Monitoring of Hepatocellular Carcinoma Using Circulating Telomere DNA. (*co-first authors) [Manuscript] II. Shifeng Lian, Chenyu Lu, Fugui Li, Xia Yu, Limei Ai, Biaohua Wu, Xueyi Gong, Wenjing Zhou, Yulong Xie, Yun Du, Wen Quan, Panpan Wang, Li Deng, Xuejun Liang, Jiyun Zhan, Yong Yuan, Fang Fang, Zhiwei Liu, Mingfang Ji, Zongli Zheng. Circulating DNA genome-wide fragmentation in early detection and disease monitoring of hepatocellular carcinoma. iScience. 2024;27:109701. https://doi.org/10.1016/j.isci.2024.109701III. Shifeng Lian, Chenyu Lu, Fugui Li, Xia Yu, Limei Ai, Biaohua Wu, Xueyi Gong, Wenjing Zhou, Xuejun Liang, Jiyun Zhan, Yong Yuan, Fang Fang, Zhiwei Liu, Mingfang Ji, Zongli Zheng. Monitoring hepatocellular carcinoma using tumor content in circulating cell-free DNA. Clinical Cancer Research. 2024;30:2772- 9. https://doi.org/10.1158/1078-0432.CCR-23-3449</p