Measurement of Bioactivity of Immunomodulatory Treatment of Relapsing Multiple Sclerosis. With Emphasis on MxA Protein

Background Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, which mainly affects young adults. In Finland, approximately 2500 out of 6000 MS patients have relapsing MS and are treated with disease modifying drugs (DMD): interferon- β (INF-β-1a or INF-β-1b) and glatiramer acetate (GA). Depending on the used IFN-β preparation, 2 % to 40 % of patients develop neutralizing antibodies (NAbs), which abolish the biological effects of IFN-β, leading to reduced clinical and MRI detected efficacy. According to the Finnish Current Care Guidelines and European Federation of Neurological Societis (EFNS) guidelines, it is suggested tomeasure the presence of NAbs during the first 24 months of IFN-β therapy. Aims The aim of this thesis was to measure the bioactivity of IFN-β therapy by focusing on the induction of MxA protein (myxovirus resistance protein A) and its correlation to neutralizing antibodies (NAb). A new MxA EIA assay was set up to offer an easier and rapid method for MxA protein detection in clinical practice. In addition, the tolerability and safety of GA were evaluated in patients who haddiscontinued IFN-β therapy due to side effects and lack of efficacy. Results NAbs developed towards the end of 12 months of treatment, and binding antibodies were detectable before or parallel with them. The titer of NAb correlated negatively with the amount of MxA protein and the mean values of preinjection MxA levels never returned to true baseline in NAb negative patients, but tended to drop in the NAb positive group. The test results between MxA EIA and flow cytometric analysis showed significant correlation. GA reduced the relapse rate and was a safe and well-tolerated therapy in IFN-β-intolerant MS patients. Conclusions NAbs inhibit the induction of MxA protein, which can be used as a surrogate marker of the bioactivity of IFN-β therapy. Compared to flow cytometricanalysis and NAb assay, MxA-EIA seemed to be a sensitive and more practical method in clinical use to measure the actual bioactivity of IFN-β treatment, which is of value also from a cost-effective perspective.

Ecological and Epidemiological Analyses of Multiple Sclerosis Relapse Rate

The underlying cause of many human autoimmune diseases is unknown, but several environmental factors are implicated in triggering the self-destructive immune reactions. Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system, potentially leading to persistent neurological deterioration. The cause of MS is not known, and apart from immunomodulatory treatments there is no cure. In the early phase of the disease, relapsing-remitting MS (RR-MS) is characterized by unpredictable exacerbations of the neurological symptoms called relapses, which can occur at different intervals ranging from 4 weeks to several years. Microbial infections are known to be able to trigger MS relapses, and the patients are instructed to avoid all factors that might increase the risk of infections and to properly use antibiotics as well as to take care of dental hygiene. Among those environmental factors which are known to increase susceptibility to infections, high ambient air inhalable particulate matter levels affect all people within a geographical region. During the period of interest in this thesis, the occurrence of MS relapses could be effectively reduced by injections of interferon, which has immunomodulatory and antiviral properties. In this thesis, ecological and epidemiological analyses were used to study the possible connection between MS relapse occurrence, population level viral infections and air quality factors, as well as the effects of interferon medication. Hospital archive data were collected retrospectively from 1986-2001, a period in time ranging from when interferon medication first became available until just before other disease-modifying MS therapies arrived on the market. The grouped data were studied with logistic regression and intervention analysis, and individual patient data with survival analysis. Interferons proved to be effective in the treatment of MS in this observational study, as the amount of MS exacerbations was lower during interferon use as compared to the time before interferon treatment. A statistically significant temporal relationship between MS relapses and inhalable particular matter (PM₁₀) concentrations was found in this study, which implies that MS patients are affected by the exposure to PM₁₀. Interferon probably protected against the effect of PM₁₀, because a significant increase in the risk of exacerbations was only observed in MS patients without interferon medication following environmental exposure to population level specific viral infections and PM₁₀. Apart from being antiviral, interferon could thus also attenuate the enhancement of immune reactions caused by ambient air PM₁₀. The retrospective approach utilizing carefully constructed hospital records proved to be an economical and reliable source of MS disease information for statistical analyses.

Emancipating and developing learning activity: Systemic intervention and re-instrumentation in higher education.

The central theme of this thesis is the emancipation and further development of learning activity in higher education in the context of the ongoing digital transformation of our societies. It was developed in response to the highly problematic mainstream approach to digital re-instrumentation of teaching and studying practises in contemporary higher education. The mainstream approach is largely based on centralisation, standardisation, commoditisation, and commercialisation, while re-producing the general patterns of control, responsibility, and dependence that are characteristic for activity systems of schooling. Whereas much of educational research and development focuses on the optimisation and fine-tuning of schooling, the overall inquiry that is underlying this thesis has been carried out from an explicitly critical position and within a framework of action science. It thus conceptualises learning activity in higher education not only as an object of inquiry but also as an object to engage with and to intervene into from a perspective of intentional change. The knowledge-constituting interest of this type of inquiry can be tentatively described as a combination of heuristic-instrumental (guidelines for contextualised action and intervention), practical-phronetic (deliberation of value-rational aspects of means and ends), and developmental-emancipatory (deliberation of issues of power, self-determination, and growth) aspects. Its goal is the production of orientation knowledge for educational practise. The thesis provides an analysis, argumentation, and normative claim on why the development of learning activity should be turned into an object of individual|collective inquiry and intentional change in higher education, and why the current state of affairs in higher education actually impedes such a development. It argues for a decisive shift of attention to the intentional emancipation and further development of learning activity as an important cultural instrument for human (self-)production within the digital transformation. The thesis also attempts an in-depth exploration of what type of methodological rationale can actually be applied to an object of inquiry (developing learning activity) that is at the same time conceptualised as an object of intentional change within the ongoing digital transformation. The result of this retrospective reflection is the formulation of “optimally incomplete” guidelines for educational R&D practise that shares the practicalphronetic (value related) and developmental-emancipatory (power related) orientations that had been driving the overall inquiry. In addition, the thesis formulates the instrumental-heuristic knowledge claim that the conceptual instruments that were adapted and validated in the context of a series of intervention studies provide means to effectively intervene into existing practise in higher education to support the necessary development of (increasingly emancipated) networked learning activity. It suggests that digital networked instruments (tools and services) generally should be considered and treated as transient elements within critical systemic intervention research in higher education. It further argues for the predominant use of loosely-coupled, digital networked instruments that allow for individual|collective ownership, control, (co-)production, and re-use in other contexts and for other purposes. Since the range of digital instrumentation options is continuously expanding and currently shows no signs of an imminent slow-down or consolidation, individual and collective exploration and experimentation of this realm needs to be systematically incorporated into higher education practise.

Magnetic Resocance Imaging Methods in the Follow-up of Multiple Sclerosis and in Farby Disease

Multiple sclerosis (MS) is a chronic immune-mediated inflammatory disorder of the central nervous system. MS is the most common disabling central nervous system (CNS) disease of young adults in the Western world. In Finland, the prevalence of MS ranges between 1/1000 and 2/1000 in different areas. Fabry disease (FD) is a rare hereditary metabolic disease due to mutation in a single gene coding α-galactosidase A (alpha-gal A) enzyme. It leads to multi-organ pathology, including cerebrovascular disease. Currently there are 44 patients with diagnosed FD in Finland. Magnetic resonance imaging (MRI) is commonly used in the diagnostics and follow-up of these diseases. The disease activity can be demonstrated by occurrence of new or Gadolinium (Gd)-enhancing lesions in routine studies. Diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI) are advanced MR sequences which can reveal pathologies in brain regions which appear normal on conventional MR images in several CNS diseases. The main focus in this study was to reveal whether whole brain apparent diffusion coefficient (ADC) analysis can be used to demonstrate MS disease activity. MS patients were investigated before and after delivery and before and after initiation of diseasemodifying treatment (DMT). In FD, DTI was used to reveal possible microstructural alterations at early timepoints when excessive signs of cerebrovascular disease are not yet visible in conventional MR sequences. Our clinical and MRI findings at 1.5T indicated that post-partum activation of the disease is an early and common phenomenon amongst mothers with MS. MRI seems to be a more sensitive method for assessing MS disease activity than the recording of relapses. However, whole brain ADC histogram analysis is of limited value in the follow-up of inflammatory conditions in a pregnancy-related setting because the pregnancy-related physiological effects on ADC overwhelm the alterations in ADC associated with MS pathology in brain tissue areas which appear normal on conventional MRI sequences. DTI reveals signs of microstructural damage in brain white matter of FD patients before excessive white matter lesion load can be observed on conventional MR scans. DTI could offer a valuable tool for monitoring the possible effects of enzyme replacement therapy in FD.

Multiple sclerosis and pregnancy: clinical and immunological aspects

Background: Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system that affects most commonly young women in their childbearing age. Previous studies have shown that MS relapse rate usually reduces during pregnancy and increases again after delivery. Patients with MS and their treating physicians are interested to know more about the risks the disease can cause to pregnancy and how pregnancy affects the disease. The reasons for increased relapse rate after delivery are not entirely clear, but loss of pregnancy related immune tolerance and changes in the hormonal status at the time of delivery seem to be of relevance. Aims and methods: The aims of this study were to follow the natural course of MS during and after pregnancy, evaluate pregnancy related risks among MS patients, follow the inflammatory response of MS patients during and after pregnancy and clarify the risk of relevant co-morbidities known to affect other autoimmune diseases after pregnancy and compare these results to healthy controls. This study was a part of a prospective nation-wide follow-up study of 60 Finnish MS patients. All eligible MS patients were enrolled in the study during the years 2003-2005. A prospective followup continued from early pregnancy until six months postpartum. MS relapses, EDSS scores and obstetric details were recorded. Blood samples were obtained from the patients at early, middle, and late pregnancy, after delivery and one month, three months and six months postpartum. Results: MS patients were no more likely to experience pregnancy or delivery complications than the Finnish mothers in general. The need of instrumental assistance, however, was higher among mothers with MS. Disease activity followed the course seen in previous studies. The majority of mothers (90.2%) breastfed their babies. Contrary to previous results, breastfeeding did not protect MS patients from disease worsening after delivery in present study. Mothers with active pre-pregnancy disease chose to breastfeed less frequently and started medication instead. MS patients presented with higher prevalence of elevated thyroid autoantibodies postpartum than healthy controls, but the rate of thyroid hormonal dysfunction was similar as that of healthy controls. The mode of delivery nor the higher rate of tissue damage assessed with C-reactive protein concentration were not predictive of postpartum relapses. The prevalence of gestational diabetes was slightly higher among mothers with MS compared to Finnish mothers in general, but postpartum depression was observed in similar rates. MS patients presented with significantly lower serum concentrations of vitamin D during pregnancy and postpartum than healthy controls. Conclusions: Childbearing can be regarded as safe for mothers with MS as it is for healthy mothers in general. Breastfeeding can be recommended, but it should be done only after careful evaluation of the individual risk for postpartum disease activation. Considering MS patients tend to develop thyroid antibody positivity after delivery more often than healthy controls and that certain treatments can predispose MS patients to thyroid hormonal dysfunction, we recommend MS mothers to be screened for thyroid abnormalities during pregnancy and after delivery. Increased risk for gestational diabetes should be kept in mind when following MS mothers and glucose tolerance test in early pregnancy should be considered. Adequate vitamin D supplementation is essential for MS mothers also during pregnancy and postpartum period.

ERBB4 mutations in cancer and amyotrophic lateral sclerosis

ErbB receptor tyrosine kinases, epidermal growth factor receptor (EGFR, also known as ErbB1), ErbB2 (HER2 or NEU), ErbB3 (HER3), and ErbB4 (HER4), transduce signals borne by extracellular ligands into central cellular responses such as proliferation, survival, differentiation, and apoptosis. Mutations in ERBB genes are frequently detected in human malignant diseases of epithelial and neural origin, making ErbB receptors important drug targets. Targeting EGFR and ErbB2 has been successful in eg. lung and breast cancer, respectively, and mutations in these genes can be used to select patients that are responsive to the targeted treatment. Although somatic ERBB4 mutations have been found in many high-incidence cancers such as melanoma, lung cancer, and colorectal cancer and germ-line ERBB4 mutations have been linked to neuronal disorders and cancer, ErbB4 has generally been neglected as a potential drug target. Thus, the consequences of ERBB4 mutations on ErbB4 biology are largely unknown. This thesis aimed to elucidate the functional consequences and assess the clinical significance of somatic and germ-line ERBB4 mutations in the context of cancer and amyotrophic lateral sclerosis. The results of this study indicated that cancer-associated ERBB4 mutations can promote aberrant ErbB4 function by activating the receptor or inducing qualitative changes in ErbB4 signaling. ERBB4 mutations increased survival or decreased differentiation in vitro, suggesting that ERBB4 mutations can be oncogenic. Importantly, the potentially oncogenic mutations were located in various subdomains in ErbB4, possibly providing explanation for the characteristic scattered pattern of mutations in ERBB4. This study also demonstrated that hereditary variation in ERBB4 gene can have a significant effect on the prognosis of breast cancer. In addition, it was shown that hereditary or de novo germ-line ERBB4 mutations that predispose to amyotrophic lateral sclerosis inhibit ErbB4 activity. Together, these results suggest that ErbB4 should be considered as a novel drug target in cancer and amyotrophic lateral sclerosis.

Organizing for Systemic Innovations – Research on Knowledge, Interaction and Organizational Interdependencies

Systemic innovation has emerged as an important topic due to the interconnected technological and sociotechnical change of our current complex world. This study approaches the phenomenon from an organizing perspective, by analyzing the various actors, collaborative activities and resources available in innovation systems. It presents knowledge production for innovation and discusses the organizational challenges of shared innovation activities from a dynamic perspective. Knowledge, interaction, and organizational interdependencies are seen as the core elements of organizing for systemic innovations. This dissertation is divided into two parts. The first part introduces the focus of the study and the relevant literature and summarizes conclusions. The second part includes seven publications, each reporting on an important aspect of the phenomenon studied. Each of the in-depth single-case studies takes a distinct and complementary systems approach to innovation activities – linking the refining of knowledge to the enabling of organizations to participate in shared innovation processes. These aspects are summarized as theoretical and practical implications for recognizing innovation opportunities and turning ideas into innovations by means of using information and organizing activities in an efficient manner. Through its investigation of the existing literature and empirical case studies, this study makes three main contributions. First, it describes the challenges inherent in utilizing information and transforming it into innovation knowledge. Secondly, it presents the role of interaction and organizational interdependencies in innovation activities from various novel perspectives. Third, it highlights the interconnection between innovations and organizations, and the related path dependency and anticipatory aspects in innovation activities. In general, the thesis adds to our knowledge of how different aspects of systems form innovations through interaction and organizational interdependencies. It highlights the continuous need to redefine information and adjust organizations and networks based on ongoing activities – stressing the emergent, systemic nature of innovation.

Imaging Neuroinflammation in Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system CNS), where inflammation and neurodegeneration lead to irreversible neuronal damage. In MS, a dysfunctional immune system causes auto‐reactive lymphocytes to migrate into CNS where they initiate an inflammatory cascade leading to focal demyelination, axonal degeneration and neuronal loss. One of the hallmarks of neuronal injury and neuroinflammation is the activation of microglia. Activated microglia are found not only in the focal inflammatory lesions, but also diffusely in the normal‐appearing white matter (NAWM), especially in progressive MS. The purine base, adenosine is a ubiquitous neuromodulator in the CNS and also participates in the regulation of inflammation. The effect of adenosine mediated via adenosine A2A receptors has been linked to microglial activation, whereas modulating A2A receptors may exert neuroprotective effects. In the majority of patients, MS presents with a relapsing disease course, later advancing to a progressive phase characterised by a worsening, irreversible disability. Disease modifying treatments can reduce the severity and progression in relapsing MS, but no efficient treatment exists for progressive MS. The aim of this research was to investigate the prevalence of adenosine A2A receptors and activated microglia in progressive MS by using in vivo positron emission tomography (PET) imaging and [11C]TMSX and [11C](R)‐PK11195 radioligands. Magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) was performed to evaluate structural brain damage. Non‐invasive input function methods were also developed for the analyses of [11C]TMSX PET data. Finally, histopathological correlates of [11C](R)‐PK11195 radioligand binding related to chronic MS lesions were investigated in post‐mortem samples of progressive MS brain using autoradiography and immunohistochemistry. [11C]TMSX binding to A2A receptors was increased in NAWM of secondary progressive MS (SPMS) patients when compared to healthy controls, and this correlated to more severe atrophy in MRI and white matter disintegration (reduced fractional anisotropy, FA) in DTI. The non‐invasive input function methods appeared as feasible options for brain [11C]TMSX images obviating arterial blood sampling. [11C](R)‐PK11195 uptake was increased in the NAWM of SPMS patients when compared to patients with relapsing MS and healthy controls. Higher [11C](R)‐PK11195 binding in NAWM and total perilesional area of T1 hypointense lesions was associated with more severe clinical disability, increased brain atrophy, higher lesion load and reduced FA in NAWM in the MS patients. In autoradiography, increased perilesional [11C](R)‐PK11195 uptake was associated with increased microglial activation identified using immunohistochemistry. In conclusion, brain [11C]TMSX PET imaging holds promise in the evaluation of diffuse neuroinflammation in progressive MS. Being a marker of microglial activation, [11C](R)‐ PK11195 PET imaging could possibly be used as a surrogate biomarker in the evaluation of the neuroinflammatory burden and clinical disease severity in progressive MS.

The effects of mutated cationic amino acid transporter y+LAT1 at the cellular and systemic level

y+LAT1 is a transmembrane protein that, together with the 4F2hc cell surface antigen, forms a transporter for cationic amino acids in the basolateral plasma membrane of epithelial cells. It is mainly expressed in the kidney and small intestine, and to a lesser extent in other tissues, such as the placenta and immunoactive cells. Mutations in y+LAT1 lead to a defect of the y+LAT1/4F2hc transporter, which impairs intestinal absorbance and renal reabsorbance of lysine, arginine and ornithine, causing lysinuric protein intolerance (LPI), a rare, recessively inherited aminoaciduria with severe multi-organ complications. This thesis examines the consequences of the LPI-causing mutations on two levels, the transporter structure and the Finnish patients’ gene expression profiles. Using fluorescence resonance energy transfer (FRET) confocal microscopy, optimised for this work, the subunit dimerisation was discovered to be a primary phenomenon occurring regardless of mutations in y+LAT1. In flow cytometric and confocal microscopic FRET analyses, the y+LAT1 molecules exhibit a strong tendency for homodimerisation both in the presence and absence of 4F2hc, suggesting a heterotetramer for the transporter’s functional form. Gene expression analysis of the Finnish patients, clinically variable but homogenic for the LPI-causing mutation in SLC7A7, revealed 926 differentially-expressed genes and a disturbance of the amino acid homeostasis affecting several transporters. However, despite the expression changes in individual patients, no overall compensatory effect of y+LAT2, the sister y+L transporter, was detected. The functional annotations of the altered genes included biological processes such as inflammatory response, immune system processes and apoptosis, indicating a strong immunological involvement for LPI.