From cytologic features to serum markers: advancing prognostication of uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. As surveys show, almost all patients diagnosed with UM want prognostic information. However, the tools available today for early prognostication in UM are limited; beyond what information can be obtained by tumor size and location, current methods typically rely on tumor tissue obtained from enucleated eyes or invasive biopsies, or on repeated sampling of peripheral plasma or serum. Therefore, improving the toolbox for prognostication of aggressive vs. less aggressive disease is the focus of this dissertation. In paper I, we explored the scope of digital image analysis of cytologic features in uveal melanoma. We know that cytologic features such as shape and size of tumor cells can predict metastatic death in uveal melanoma. Currently, however, cytologic analysis is conducted manually by visual inspection of pathologists, which is time-consuming and impacts its reproducibility. In this study, we analyzed twelve morphometric variables in a large number of uveal melanoma cells. We then correlated the results with BRCA associated protein-1 (BAP-1) expression and BAP-1 gene mutation status, monosomy 3, gene expression classification as well as patient survival. We demonstrated that digital image analysis of variables describing the shape and size of tumor nuclei correlated to BAP-1 status, to monosomy 3, and to gene expression class. Mean time consumption per tumor was less than 2.5 min. Thus, digital image analysis is a fast and highly reproducible technique, that for the first time allows to objectively quantify cytologic tumor features in large tumors and gives prognostic information on survival in UM. In paper II, we explored if biopsies from posterior UM hold the same prognostic information as tissue from enucleated specimens. We classified BAP-1 expression in transvitreal biopsies of posterior UM and correlated our results with BAP-1 expression in subsequent biopsies. We found that BAP-1 expression in transvitreal biopsies was concordant with BAP-1 in enucleated specimens. Moreover, BAP-1 expression in transvitreal biopsies identified patients with poor metastasis-free survival. Thus, transvitreal biopsies can be a good prognostic tool for patients that do not undergo enucleation. The results also indicate the need to further study BAP-1 expression in even smaller UMs, to infer the tumor size at which loss of BAP-1 starts to occur, and to correlate it with the early seeding of micrometastases. In paper III, we explored prognostic testing on serum samples obtained from patients at diagnosis of uveal melanoma. We first screened for cancer-related proteins by protein profiling in order to find potential biomarkers. Second, we performed ELISA to evaluate the serum levels of the best candidates. Third, receiver operating characteristics were used to define thresholds for metastatic risk. This led to a prognostic test (serUM-Px) that stratifies patients into low, intermediate, and high-risk categories which we tested in a training cohort and validation cohort. We found that serUM-Px is a prognostic test, based on a single peripheral venous blood sample at the time of UM diagnosis, which can be used to stratify patients into low, intermediate and high metastatic risk categories. Thus, this test predicts metastases many years in advance without the need for biopsy or repeated sampling. In paper IV, we wanted to assess the prognostic utility of Tenascin C (TNC) in uveal melanoma. Therefore, we collected peripheral blood samples from 82 patients with small posterior uveal melanomas between 1996 and 1999 as described above for paper III. TNC concentrations were examined 2021. RNA sequencing data of TNC from an additional 80 larger tumors were collected. These levels were subsequently linked with the cumulative incidence of metastasis-related death through competing risk data analysis. In our analysis we observed no significant disparities in tumor size, age at diagnosis, visual acuity, serum protein levels, or treatment modality between patients with above or below median serum or tumor TNC levels. However, above median TNC RNA was associated with BAP1 mutation, monosomy 3, and epitheloid cytomorphology. The competing risk analysis indicated increased metastatic mortality in patients with above median TNC and primary tumor TNC RNA compared to their below median counterparts. We conclude that TNC is a prognostic biomarker in uveal melanoma that exhibits elevated levels in peripheral blood and tumors at diagnosis in patients destined for metastatic death

Monitoring of the oral anticoagulants apixaban and rivaroxaban to optimize individual safety and efficacy

The oral anticoagulants apixaban and rivaroxaban are administered at fixed doses without requiring routine monitoring. However, there is a dose and exposure dependency in efficacy and safety, and monitoring the anticoagulants may improve treatment outcomes. The aim of this thesis was to evaluate different laboratory methods for monitoring apixaban and rivaroxaban and to show the typical exposure levels in patients. Additionally, the study aimed to study the exposure in certain specific populations (i.e. those with obesity and renal impairment), to elucidate whether monitoring would be especially valuable for these patients. In studies I and II, we evaluated various coagulation assays for the monitoring of apixaban and rivaroxaban. While PT-INR (venous) and aPTT were not sensitive enough to be used for this purpose, the anti-Factor Xa assay showed a strong correlation with LC/MS-MS. However, for precise estimations of apixaban/rivaroxaban concentrations in the lower range, LC-MS/MS is the preferred method to use. A large interindividual variability in trough concentrations was observed, 12-fold for apixaban (6-fold within the same dosage group) and 17-fold for rivaroxaban (17-fold for the standard dose and 3- fold for the reduced dose). In study III, we explored the influence of obesity on apixaban trough and peak concentrations. A specific dose recommendation for obese patients may not be necessary given that the concentrations were largely similar to those in matched normal weight patients. However, obese patients exhibited an extensive 18-fold interindividual variability in trough concentrations. Consequently, monitoring apixaban concentrations could be valuable for specific obese patients to ensure optimal treatment outcomes In study IV, we examined the impact of renal function on apixaban exposure in AF patients. Patients with moderate renal impairment had twice as high apixaban trough concentrations compared to patients with normal renal function. This indicates that the recommended 5 mg twice daily dose might be excessive for some patients with moderate renal impairment, and that monitoring ought to be recommended

Randomised clinical trials with hyperbaric oxygen in COVID-19 and Long COVID : transcriptomic insights into benefits and harms

The flow from transcription of genes through translation and processing of proteins is a common basis for all life. Redox homeostasis is crucial for the defence against oxidative stress. We adapt through hormesis; non-lethal stress regulates redox-sensitive systems to maintain homeostasis. If the stress is chronic or acutely overwhelming, the cells can either go into apoptosis or into senescence to maintain homeostasis. Similar effects have been seen with HBOT as with intermittent oxygen deprivation. Hyperbaric oxygen therapy (HBOT) is delivered in a pressure chamber by breathing 100% oxygen intermittently, several times a week, in an ambient pressure equivalent to 10-20 meters of seawater. The aim of this thesis was to evaluate potential harms of HBOT for novel indications and to explore biomarkers in experimental and clinical trials in order to enable future precision medicine. We used methods evaluated on healthy volunteers in randomised clinical trials (RCTs) conducted in compliance with good clinical practice (ICH-GCP). In Paper I, we evaluated Electron paramagnetic resonance (EPR) spectroscopy for measuring reactive oxygen species (ROS) in blood and RNA sequencing (RNAseq) of monocytes in peripheral blood (PBMC), and compared HBOT and HIIT in ten healthy volunteers. We could measure ROS in blood in the same physiological range in both interventions. We also discovered pathways involved in adaption to hypoxia and inflammation that were similar in both interventions. In Papers II and III, we evaluated harms and explored RNAseq in PBMC in an open label RCT where 31 patients with severe COVID- 19 were randomised to HBOT or best practice. We observed similar frequencies of adverse events (AEs) in the two groups and could not see any negative effect on vital signs or oxygenation. We discovered a unique transcriptomic signature in the subjects that had received HBOT. The differentially expressed genes were associated with the unfolded protein response, apoptosis, and immune response. In Paper IV, we evaluated harms and described health related quality of life (HRQoL)in an interim analysis of the first 20 subjects froma placebo controlled RCT where 80 patients with Long COIVD were randomised to HBOT or sham treatment. We reported more AEs than expected and severe physical and mental disabilities with a very poor HRQoL. Most AEs were mild, and all were transient. We have shown that HBOT shares similarities in immune response with HIIT in healthy volunteers. HBOT has a favourable profile of harms and has a potent immunomodulatory effect that is associated with fast recovery for critical COVID-19 patients. HBOT has a favourable profile of harms for patients with post COVID-19 condition. The results provide a base for future clinical trials with HBOT

Prenatal risk factors for severe cardiovascular diseases up to middle-age : a Nordic collaborative study

Background and objectives: Cardiovascular diseases (CVDs) are major causes of death and disability. However, the established traditional risk factors cannot explain a substantial proportion of CVD cases, prompting investigations into novel risk factors. A growing body of evidence underscores the potential role of suboptimal intrauterine conditions on the development of CVD. Nonetheless, our knowledge about the associations between factors contributing to an adverse intrauterine environment and the risk of developing CVD remains limited. The overall objective of this thesis is to enhance our comprehension of the potential role of prenatal risk factors in developing CVD later in life. More specifically, the thesis aims to study the following research questions: (1) Are negative birth outcomes such as preterm birth, being small (SGA) or large (LGA) for gestational age related to the atrial fibrillation risk later in life (Study I)? (2) Is maternal preeclampsia and its subtypes linked to increased risks of stroke and ischemic heart disease in the offspring (Study II)? (3) Is maternal polycystic ovary syndrome (PCOS) associated with the risks of overall CVD and its major subtypes in her offspring (Study III)? and (4) Is prenatal exposure to maternal severe stress related to the risk of heart failure later in life (Study IV)? Methods: We performed four register-based prospective cohort studies, including all live singletons from Denmark (Study I: 1978-2016, Studies II-IV: 1973-2016) and Sweden (Studies I-IV: 1973-2014), and live births from a randomly selected 90% of all births in Finland Studies I and II; 1987-2014). The size of the study population was 8,012,433 in Study I, 8,475,819 in Study II, 6,839,703 in Study III, and 6,758,560 in Study IV. Information on birth outcomes, maternal and offspring's health and covariates were obtained through linkage to population-based socioeconomic and health registers. Each study participant was followed up until the earliest diagnosis of the CVD of interest, emigration, death, or end of follow-up (Denmark: December 31, 2016; Finland: December 31, 2014; Sweden: December 31, 2020), whichever occurred first. We examined the association between prenatal exposures (including preterm birth, SGA, LGA, maternal preeclampsia, PCOS, and severe stress) and CVD outcomes in offspring using multivariable Cox regression models. Furthermore, we used family- based study designs, i.e. sibling and cousin comparison analyses, to account for unmeasured familial genetic and environmental confounders. Additionally, we investigated the mediating roles of abnormal birth outcomes and congenital heart disease in case of some of the observed associations. Results: In Study I, we found that being born preterm or LGA was linked to an increased risk of atrial fibrillation in both childhood and adulthood. The associations persisted in the sibling comparison analyses. In contrast, SGA was related to an increased atrial fibrillation risk in childhood but not in adulthood. In Study II, we found that individuals prenatally exposed to maternal preeclampsia had higher risks of stroke and ischemic heart disease than those unexposed, and that the associations were more pronounced in cases of severe than milder forms of preeclampsia. The associations of the severe forms of maternal preeclampsia with the offspring's risk of stroke remained in the sibling comparison analyses. In Study III, maternal PCOS was associated with elevated risks of overall CVD, hypertensive disease, stroke, and ischemic heart disease in the population analysis; most of these associations, except that observed in case of stroke, remained in the cousin comparison analysis. When investigating the interaction between maternal PCOS and its prevalent comorbidities, we found that individuals born to mothers with both PCOS and its common comorbid conditions, i.e. diabetes, hypertensive disease, or psychiatric disorders, had higher CVD risks than those born to mothers with only PCOS. In Study IV, we found that offspring exposed to maternal loss of a close family member the year prior to or during pregnancy did not have a higher risk of heart failure than those unexposed. However, the severe forms of maternal bereavement, specifically loss due to unnatural causes and loss of a child or partner, were linked to an increased risk of heart failure in the offspring. When splitting follow-up for Studies I-IV at the age of 18, we found that the association between preterm birth and the risk of atrial fibrillation, maternal preeclampsia and the risk of stroke, and severe maternal stress and the risk of heart failure were stronger during childhood than during adulthood. In the mediation analyses, there was some evidence that the association between maternal PCOS and the risk of CVD in offspring was to a modest extent mediated by preterm birth, LGA, and congenital heart disease. In the case of the link between severe maternal stress and the risk of heart failure, we observed considerable contributions from congenital heart disease and preterm birth. Conclusions: This thesis revealed associations of prenatal risk factors with increased risks of CVD up to early middle-age. Specifically, our findings suggest that preterm birth, SGA, and LGA were related to elevated risks of atrial fibrillation. Additionally, maternal preeclampsia, especially its severe types, was associated with elevated risks of stroke and ischemic heart disease in offspring, while maternal PCOS was associated with increased risks of overall CVD and its major types. Moreover, severe maternal stress was associated with an elevated risk of heart failure in offspring. If subsequent studies confirm our findings, early-life prevention and targeted intervention programs may be developed to inform health policies, eventually resulting in a reduced burden of CVD

Modifiable risk factors, blood proteins, and venous thromboembolism

Venous thromboembolism (VTE) refers to blood clots in the veins, which is an underappreciated vascular disease that can cause disability and mortality. Although some triggers for VTE (e.g., surgery, fracture, infection, hospitalization, and cancer) have been established, the associations of modifiable risk factors and blood proteins with the risk of VTE remain uncertain. This PhD project aimed to 1) investigate the associations of obesity and lifestyle factors with VTE risk; 2) explore the associations of blood proteins with VTE risk; and 3) establish protein pathways linking modifiable risk factors to VTE development. In Paper I, we explored the associations of overall and central obesity with the risk of VTE using both cohort and Mendelian randomization analyses. We found a potentially causal association between obesity and VTE risk. Waist circumference might be a preferable indicator linking obesity to VTE. Around 12.4% and 23.7% of VTE cases could be prevented if the population maintained a healthy body mass index and waist circumference, respectively. In Paper II, using the prospective cohort design, we investigated the associations of cigarette smoking, alcohol and coffee intake, physical activity, and diet with the risk of incident VTE. We found that high levels of physical activity and a healthy diet were associated with lower VTE risk in women and men. Cigarette smoking showed a positive association with VTE only in women. Alcohol and coffee intake was not associated with VTE. In Paper III, we explored the association between ultra-processed food intake and the risk of VTE using the prospective cohort design. A higher ultra-processed food intake was associated with a moderately increased risk of VTE. This association was not modified by age, sex, or body mass index. In Paper IV, we conducted a prospective cohort and Mendelian randomization study to estimate the associations of 257 blood proteins with VTE risk. The cohort analysis identified 21 blood proteins associated with incident VTE. Machine-learning analysis found that body mass index and von Willebrand factor shared an identical highest ranking concerning the contribution to the prediction model. Mendelian randomization analysis confirmed 7 protein-VTE associations. In Paper V, we performed a two-stage network Mendelian randomization analysis to decipher proteomic pathways underlying the associations of 15 modifiable risk factors with VTE. We found that several proteins, in particular annexin II and coagulation factor XI, mediated the associations of obesity, smoking, and insomnia with VTE. Proteome-wide Mendelian randomization analysis identified many VTE-associated proteins with druggable potentials. In summary, the above five studies identified modifiable risk factors and blood proteins for VTE development and further revealed protein pathways underlying the associations between modifiable risk factors and VTE. These findings may deepen understanding of VTE pathogenesis and facilitate precision prevention and drug development for VTE

Exploring signaling pathways in endothelial mechanotransduction

Mechanotransduction, the process by which cells sense and convert mechanical forces from their surroundings, plays a pivotal role in cellular function. For vascular endothelial cells, coping with the continuous and significant shear stress caused by blood flow is essential for the stability of the circulatory system. Dysfunction in this mechanism can lead to various vascular pathologies, including atherosclerosis and aneurysms. Therefore, understanding how endothelial cells form mechanosensory complexes and thereby respond to external forces is crucial. This thesis aims to study the involvement of the Angiomotin protein family in endothelial mechanotransduction. The Amot protein family, including Angiomotin (Amot), Angiomotin-Like 1 (AmotL1), and Angiomotin-like 2 (AmotL2), shares common structures as well as protein interaction motifs. However, they exhibit significantly different roles in vascular functions. In Paper I, we elucidate how Amot binds to Talin within the integrin adhesome and regulates force transmission between fibronectin and the cytoskeleton in migrating endothelial cells. Additionally, we demonstrate that deletion of Amot impairs both physiological and pathological angiogenesis. In Paper II, AmotL2 isshown to bind VE cadherin through p120 catenin and connect to the nuclear membrane via actin filaments in aortic endothelial cells, thereby transmitting junctional mechanical signals. Depletion of AmotL2 resulted in a pro-inflammatory response and, in severe cases, leads to the spontaneous formation abdominal aortic aneurysms (AAAs) in male adult mice. In Paper III, it is demonstrated that AmotL1 not only binds to N-cadherin but is also associated with focal adhesion proteins. This suggests that AmotL1 functions may extend beyond endothelial cell junctions to include interactions with the extracellular matrix. Additionally, we provide a comprehensive summary of the protein binding profiles of all Amot proteins, as obtained from BioID-MS analysis, thus offering a global perspective on this protein family. In conclusion, Amot family proteins, despite their involvement in separate cellular processes, share common connections with a set of junction-related proteins. Furthermore, they exhibit unique, specific binding partners, offering mechanistic insights into the distinct activities of individual Amot proteins

Skeletal muscle responses to physical activity in health and metabolic disease

Sedentary lifestyles, characterised by a lack of physical activity and prolonged periods of sitting, have been linked to reductions in whole-body metabolic flexibility and the increased risk of metabolic diseases, including type 2 diabetes. This can be attributed, at least partially, to the direct negative effects of physical inactivity on skeletal muscle insulin sensitivity, oxidative capacity, and overall metabolic health. In addition, sedentary behaviour can lead to anabolic resistance, resulting in losses of skeletal muscle mass and strength, which can further contribute to conditions like sarcopenic obesity, impairing physical performance and overall quality of life. Conversely, physical activity plays a crucial role in maintaining and improving skeletal muscle health. Exercise is associated with various adaptations in skeletal muscle that enhance tissue oxidative capacity, substrate handling, insulin sensitivity, as well as skeletal muscle mass and strength. These positive changes in skeletal muscle contribute to improvements in systemic metabolic wellbeing. The molecular mechanisms underlying skeletal muscle adaptation to the perturbations caused by physical activity are complex and involve intrinsic processes within the muscle fibre itself, as well as communication between different cell populations in composite skeletal muscle tissue. However, our understanding of the intricate details of these mechanisms remains incomplete. Gaining deeper insights into the regulation of skeletal muscle adaptation could not only facilitate personalised exercise recommendations but also uncover novel opportunities for drug discovery, ultimately leading to improvements in human health. Despite the well-known benefits of exercise, physical activity guidelines are often not met by the general population. Therefore, there is a pressing need for low-level entry paradigms that can promote physical activity and reduce sedentary behaviour for the betterment of individual and public health. One such approach is the incorporation of frequent activity breaks or ‘exercise snacks’ into daily routines, which involves short-duration physical activity breaks throughout the day to disrupt prolonged periods of sitting. These interventions have demonstrated efficacy for cardiometabolic health in controlled settings, such as laboratory-based clinical trials. However, it is essential to evaluate the benefits of breaking sedentary time using strategies that better mimic real-world scenarios to inform practical public health guidelines. In this thesis, the following objectives were pursued: (1) To assess the translational efficacy of interrupting sedentary time in improving cardiometabolic health. (2) To investigate the skeletal muscle transcriptome following exercise or physical inactivity in the context of health and metabolic diseases. (3) To determine the metabolic effects of physical activity- responsive transcription factors and signalling molecules in skeletal muscle. Study I revealed that even a minor addition of ≈750 steps dispersed throughout the day, equivalent to ≈10 minutes of extra walking time, improved dynamic glucose control in individuals with obesity and insulin resistance. Notably, those who engaged in higher levels of physical activity while interrupting sedentary time experienced greater benefits, indicating that more breaks from sedentary behaviour lead to better metabolic health outcomes. Nevertheless, adherence to the intervention, which involved 3-min activity bouts every 30 min between 08:00-18:00, was lower than anticipated. This raises questions about the long- term feasibility of such approaches when considered in isolation from other modifiable lifestyle factors, including changes in dietary habits or structured exercise routines. Study II employed a comprehensive meta-analytical approach to compare the skeletal muscle transcriptomic response to acute aerobic or resistance exercise, exercise training, and physical inactivity. This analysis revealed distinct gene signatures in skeletal muscle after a single bout of exercise in the naïve state, which differed from those observed after training of the same exercise modality. Interestingly, there was greater overlap in the skeletal muscle transcriptome between acute aerobic and resistance exercise than there was between acute exercise and exercise training. These findings highlight the refinement of the adaptive response in skeletal muscle over time through dedicated training to a specific exercise modality. Study II identified the transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3) as a gene that is upregulated in skeletal muscle after exercise but downregulated in response to physical inactivity. Study III delved deeper into the role of NR4A3 in the context of physical inactivity and revealed its regulatory role in translation within skeletal muscle. Depletion of NR4A3 resulted in skeletal muscle atrophy and compromised glucose oxidation, instead favouring increased lactate production. Therefore, decreased levels of NR4A3 during physical inactivity may directly contribute to muscle disuse atrophy and impaired skeletal muscle metabolism. Furthermore, study II identified that individuals with obesity and/or type 2 diabetes exhibit an altered skeletal muscle transcriptional response to exercise training compared to healthy individuals. Study IV uncovered a heightened inflammatory response during the recovery period after exercise in individuals with type 2 diabetes. This response was attributed to an increased influx of immune cells into skeletal muscle tissue, potentially facilitating crosstalk between different cell types within the skeletal muscle interstitial space. Notably, the cytokine stromal cell-derived factor 1 (CXCL12/SDF-1) was found to be expressed by macrophages or endothelial cells in response to factors released by skeletal muscle fibres or hypoxia, respectively. CXCL12 activation, in turn, promoted the proliferation of skeletal muscle satellite cells, which could be integral for adaptive remodelling following exercise. In conclusion, the research presented in this thesis emphasises the central role of physical activity in improving human health, with a specific focus on the ability of exercise to induce adaptations in skeletal muscle. The findings herein shed light on the intricate molecular mechanisms underlying skeletal muscle responses to physical activity, which contribute to the metabolic fitness of this tissue and of the human body as a whole

Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.Knut and Alice Wallenberg Foundation (2019-0059)Swedish Research Council (2019-1209, 2017-06014)Swedish Association against RheumatismErling Persson Foundation (2017-10-09)German Federal Ministry of Education and Research (GO-Bio-project aidCURE; 031A385)Federal State of Hesse (LOEWE-project 13, IME Fraunhofer Project Group TMP at Goethe University)Fraunhofer Cluster of Excellence for Immune-Mediated Diseases CIMDSpanish Ministry of Universities through the European Union (NextGeneration EU)Publishe

Cellular and molecular mechanisms of inflammatory arthritis and fibromyalgia

In Study I, we examined the impact of the hR100E-NGF mutation on inflammatory pain and bone erosion in both female and male mice. Our findings indicate that the hR100E-NGF mutation did not affect the development of the peripheral sensory nervous system at the lumbar DRG, sciatic nerve, ankle joint, or glabrous skin. Moreover, hR100E-NGF mice displayed sensory thresholds similar to those of the hWT-NGF mice in response to mechanical, heat, or cold stimulation under normal conditions. The hR100E-NGF and hWT-NGF mice developed comparable mechanical and heat sensitivity impairments after the intra-articular injection of complete Freund’s adjuvant. Notably, the hR100E-NGF mice were insensitive to nociceptive stimulation in the deeper tissues assessed by weight bearing and gait analysis. Furthermore, mRNA analysis from the inflamed joint showed a differential sex-dependent gene expression profile between hR100E-NGF female and male mice. Finally, the hR100E-NGF female but not the male mice were protected against the CFA-bone erosion. These data collectively demonstrate that the R100E NGF mutation effectively protects against joint pain-like behaviors in both male and female mice while providing bone protection exclusively to female mice in a monoarthritis model. We propose that manipulating the signaling of NGF and its receptors in a manner similar to the R100E mutation could be a promising approach to treating chronic pain and maintaining bone health, particularly in women. Study II investigated the effects of injecting purified IgG from fibromyalgia (FM) patients and healthy controls (HC) in mice. We found that the injection of FM IgG but not IgG from healthy controls (HC) induces pressure, mechanical, and cold hypersensitivity in mice that were coupled to enhanced nociceptor responsiveness to mechanical and cold stimulation. The FM IgG-injected mice also developed impaired muscular strength and decreased locomotor activity. Moreover, FM IgG bound and stimulated satellite glial cells (SGCs) in vivo and in vitro. No FM or HC IgG accumulation was found in the brain or spinal cord of the injected mice. Our study also demonstrated that FM IgG can bind to satellite glial cells and neurons in the human DRG. In addition, we observed a significant reduction in the intraepidermal nerve fiber density in the mice 14 days after the FM IgG injection. Our results suggest that transferring FM IgG into mice can replicate some peripheral FM symptoms. This study can provide a valuable animal model for studying the peripheral physiology of FM. Our discovery could significantly advance the understanding and treatment of fibromyalgia and other related conditions. However, more research is needed to understand the cellular and molecular mechanisms involved in FM-IgG-mediated changes in mice. Study III aimed to investigate the frequency of anti-satellite glial cell (SGC) antibodies and the antibody association with the disease severity in FM patients. We used serum (Karolinska Institutet, Sweden; n=30/group) and plasma (McGill University, Canada; n=35/group) samples collected from FM patients and HCs. Our results showed a higher binding intensity of the FM IgG to SGC in vitro. Furthermore, the frequency of SGC bound to FM IgG was significantly higher than HC IgG-treated cells. These findings correlated with pain intensity and fibromyalgia impact questionnaire scores (FIQ, questionnaire was only assessed in the Karolinska cohort). Further cluster analysis separated the FM group into severe and mild groups. Additionally, we found that serum from FM patients contains IgG that binds in greater proportion to SGC in the human DRG, measured by higher signal intensity. There were no differences in the binding intensity to neuronal cell bodies or axons between FM and HC serum samples. Finally, the previous results were confirmed using an FM serum sample with high levels of anti-SGC antibodies in 5 more human DRGs. To summarize, our report indicates that levels of anti-human SGC and anti-mouse SGC antibodies are elevated in patients with FM, which are linked to a more severe form of the disease. Patient stratification based on their profile of anti-SGC antibodies might benefit from therapies aiming to decrease circulating IgG or prevent IgG binding. Our results point to the possible involvement of anti-SGC antibodies and SGCs in the severity of FM; however, more in-depth studies are necessary to elucidate the antigen or antigens expressed in the SGC that bind to the circulating anti-SGC antibodies. In Study IV, we aimed to explore the neuroimmune signature of the FM skin. We processed 16 FM and 16 HC sex-matched skin biopsies by immunohistochemistry. Using a pan-neuronal marker, we found lower intraepidermal nerve fiber density (IENFD) in the FM compared with HC skin. Moreover, the length and volume of dermal NF200+ nerve profiles were significantly elevated, but we found no changes in the length of dermal or epidermal Gap43+ nerve profiles in the FM group. Similarly, we found no changes in the total volume of CD31+ blood vessels between FM and HC skin. Our results showed that the density of non-nerve associated S100b+, CD68+, and CD163+ cells was significantly lower in the FM skin. Furthermore, the dermal CD117+FcERI+ mast cells in the dermis of FM patients were significantly increased compared with the HCs. Additionally, we found similar densities of CD207+, CD3+, or Neutrophil elastase+ cells between FM and HC skin biopsies. mRNA analysis of FM skin showed no changes in Cd68, Cd163, Cx3cr1, or FceR1 mRNA levels between FM and HC skin. In summary, this study reveals crucial dermal and epidermal changes in FM skin, particularly regarding nerve fibers and certain immune cell populations. These findings are highly relevant as they provide deeper insights into the complex interactions between the nervous and immune systems in FM. Understanding these changes could be key to developing more effective treatments for FM, focusing on both the neuropathic and immune components of the disease

The single leg squat in clinical testing : aspects of reliability, validity, and associated factors

Background: The Single Leg Squat (SLS) test is a functional test widely used in clinical settings to examine and evaluate rehabilitation goals. Research indicates that the SLS is reliable when the knee relative to the foot is dichotomously assessed. However, the assessment of functional movements often comprises more complex analyses of the whole kinetic chain with several body segments which highlights the need to develop and test a standardised multi-segmental SLS. Movement quality is an important aspect when using prevention programs in the clinical context, and the SLS can be used for this purpose. As knee injuries are common among athletes and especially among female soccer players, further investigation of the SLS in this population is warranted. Overall, the development of both quantitative and qualitative measurements needs to be studied to improve clinical testing. Clinically, portable marker-less motion capture (MMC) systems are suggested to be an adequate substitute for a three-dimensional analysis system, and one such novel MMC system is the QinematicTM. Before a test such as the SLS, or any other new measurement instrument, can be used in clinical settings, it is important to explore its measurement properties. Aim: The overall aim of this thesis was to develop and assess aspects of reliability and validity of the SLS among physically active people, and from a biopsychosocial perspective investigate factors associated with the SLS in a sample of female soccer players. Methods: Study I was a systematic review and meta-analysis that investigated the current literature regarding the intra- and inter-rater reliability of visually assessed SLS, including the Forward Step-Down (FSD) and Lateral Step-Down (LSD) tests. Study II was a laboratory-based test-retest reliability and validity study of a three-dimensional MMC system, the QinematicTM. Study III was an intra- and inter-rater reliability study of a standardised multi-segmental SLS developed from the findings in Study I. Study IV was a cross-sectional observational study using linear regression models to explore if demographic and biopsychosocial factors associated with the outcome of the SLS, assessed as a total score for all segments and as a separate knee segment in a sample of elite and sub-elite female soccer players. An additional analysis investigated the possibility of the SLS to discriminate injured soccer players from non-injured players. Results: In Study I, the pooled results of ICC/kappa showed a “moderate” agreement for inter-rater reliability and a “substantial” agreement for intra-rater reliability of the SLS, including the FSD and LSD. In Study II, the QinematicTM showed “substantial relative reliability” but “poor absolute reliability”. Regarding validity, a “moderate” agreement between the visual assessment and Qinematic™ data for various knee angles was shown and the best discriminative ability of the SLS was found at a knee angle of 6°. In Study III the proposed multi-segmental SLS showed a “moderate” inter-rater reliability and an “almost perfect” intra-rater reliability. In Study IV, the outcome of the SLS was associated with previous injuries and various demographic-, biomechanical- and psychosocial factors depending on the tested leg. The total score associated with hip strength for both the dominant and the non-dominant leg, and the knee segment associated with division inherency for both the dominant and non-dominant leg. The additional analysis showed that the SLS was not able to discriminate between players with and without previous or present injuries. Conclusion: The SLS seems to be a reliable and clinically useful multi-segmental test of movement quality in contrast to the QinematicTM system. The SLS was, in a sample of female elite and sub-elite soccer players, associated with a variety of biopsychosocial factors when assessed as a total score or as a separate knee segment. The results imply that several factors need to be considered when assessing the SLS among female soccer players such as leg dominance, division inherency, hip strength, and psychosocial factors