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.

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.

Immunopathogenesis of Asthma and Atopic Diseases – The specific Role of a selected Panel of Genes in human T helper Cell Differentiation

T helper cell (Th) functions are crucial for proper immune defence against various intra- and extracellular pathogens. According to the specific immune responses, Th cells can be classified into subtypes, Th1 and Th2 cells being the most frequently characterized classes. Th1 and Th2 cells interact with other immune cells by regulating their functions with specific cytokine production. IFN, IL-2 and TNF- are the cytokines predominantly produced by Th1 cells whereas Th2 cells produce Th2-type cytokines, such as IL-4, IL-5 and IL-13. Upon TCR activation and in the presence of polarizing cytokines, Th cells differentiate into effector subtypes from a common precursor cell. IFN and IL-12 are the predominant Th1 polarizing cytokines whereas IL-4 directs Th2 polarization. The cytokines mediate their effects through specific receptor signalling. The differentiation process is complex, involving various signalling molecules and routes, as well as functions of the specific transcription factors. The functions of the Th1/Th2 cells are tightly regulated; however, knowledge on human Th cell differentiation is, as yet, fairly poor. The susceptibility for many immune-mediated disorders often originates from disturbed Th cell responses. Thus, research is needed for defining the molecular mechanisms involved in the differentiation and balanced functions of the Th cells. Importantly, the new information obtained will be crucial for a better understanding of the pathogenesis of immune-mediated disorders, such as asthma or autoimmune diseases. In the first subproject of this thesis, the role of genetic polymorphisms in the human STAT6, GATA3 and STAT4 genes were investigated for asthma or atopy susceptibility in Finnish asthma families by association analysis. These genes code for key transcription factors regulating Th cell differentiation. The study resulted in the identification of a GATA3 haplotype that associated with asthma and related traits (high serum IgE level). In the second subproject, an optimized method for human primary T cell transfection and enrichment was established. The method can be utilized for functional studies for the selected genes of interest. The method was also utilized in the third subproject, which aimed at the identification of novel genes involved in early human Th cell polarization (0-48h) using genome-wide oligonucleotide arrays. As a result, numerous genes and ESTs with known or unknown functions were identified in the study. Using an shRNA knockdown approach, a panel of novel IL-4/STAT6 regulated genes were identified in the functional studies of the genes. Moreover, one of the genes, NDFIP2, with a previously uncharacterized role in the human Th differentiation, was observed to promote IFN production of the differentiated Th1 cells. Taken together, the results obtained have revealed potential new relevant candidate genes serving as a basis for further studies characterizing the detailed networks involved in the human Th cell differentiation as well as in the genetic susceptibility of Th-mediated immune disorders.

Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.

Pluripotency and genetic stability of human pluripotent stem cells

Pluripotent cells have the potential to differentiate into all somatic cell types. As the adult human body is unable to regenerate various tissues, pluripotent cells provide an attractive source for regenerative medicine. Human embryonic stem cells (hESCs) can be isolated from blastocyst stage embryos and cultured in the laboratory environment. However, their use in regenerative medicine is restricted due to problems with immunosuppression by the host and ethical legislation. Recently, a new source of pluripotent cells was established via the direct reprogramming of somatic cells. These human induced pluripotent stem cells (hiPSCs) enable the production of patient specific cell types. However, numerous challenges, such as efficient reprogramming, optimal culture, directed differentiation, genetic stability and tumor risk need to be solved before the launch of therapeutic applications. The main objective of this thesis was to understand the unique properties of human pluripotent stem cells. The specific aims were to identify novel factors involved in maintaining pluripotency, characterize the effects of low oxygen culture on hESCs, and determine the high resolution changes in hESCs and hiPSCs during culture and reprogramming. As a result, the previously uncharacterized protein L1TD1 was determined to be specific for pluripotent cells and essential for the maintenance of pluripotency. The low oxygen culture supported undifferentiated growth and affected expression of stem cell associated transcripts. High resolution screening of hESCs identified a number of culture induced copy number variations and loss of heterozygosity changes. Further, screening of hiPSCs revealed that reprogramming induces high resolution alterations. The results obtained in this thesis have important implications for stem cell and cancer biology and the therapeutic potential of pluripotent cells.

Gene Regulation in The Human Immune System. Gene Expression Signatures of Th2 Cell Differentiation, Type 1 Diabetes, and Intrauterine Immune Adaptation

The human immune system is constantly interacting with the surrounding stimuli and microorganisms. However, when directed against self or harmless antigens, these vital defense mechanisms can cause great damage. In addition, the understanding the underlying mechanism of several human diseases caused by aberrant immune cell functions, for instance type 1 diabetes and allergies, remains far from being complete. In this Ph.D. study these questions were addressed using genome-wide transcriptomic analyses. Asthma and allergies are characterized by a hyperactive response of the T helper 2 (Th2) immune cells. In this study, the target genes of the STAT6 transcription factor in naïve human T cells were identified with RNAi for the first time. STAT6 was shown to act as a central activator of the genes expression upon IL-4 signaling, with both direct and indirect effects on Th2 cell transcriptome. The core transcription factor network induced by IL-4 was identified from a kinetic analysis of the transcriptome. Type 1 diabetes is an autoimmune disease influenced by both the genetic susceptibility of an individual and the disease-triggering environmental factors. To improve understanding of the autoimmune processes driving pathogenesis in the prediabetic phase in humans, a unique series of prospective whole-blood RNA samples collected from HLA-susceptible children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study was studied. Changes in different timewindows of the pathogenesis process were identified, and especially the type 1 interferon response was activated early and throughout the preclinical T1D. The hygiene hypothesis states that allergic diseases, and lately also autoimmune diseases, could be prevented by infections and other microbial contacts acquired in early childhood, or even prenatally. To study the effects of the standard of hygiene on the development of neonatal immune system, cord blood samples from children born in Finland (high standard of living), Estonia (rapid economic growth) and Russian Karelia (low standard of living) were compared. Children born in Russian Karelia deviated from Finnish and Estonian children in many aspects of the neonatal immune system, which was developmentally more mature in Karelia, resembling that of older infants. The results of this thesis offer significant new information on the regulatory networks associated with immune-mediated diseases in human. The results will facilitate understanding and further research on the role of the identified target genes and mechanisms driving the allergic inflammation and type 1 diabetes, hopefully leading to a new era of drug development.

Clinical and genetic markers in disease progression – a study among subjects with obstructive lung diseases

Asthma, COPD, and asthma and COPD overlap syndrome (ACOS) are chronic pulmonary diseases with an obstructive component. In COPD, the obstruction is irreversible and the disease is progressive. The aim of the study was to define and analyze factors that affected disease progression and patients’ well-being, prognosis and mortality in Chronic Airway Disease (CAD) cohort. The main focus was on COPD and ACOS patients. Retrospective data from medical records was combined with genetic and prospective follow-up data. Smoking is the biggest risk factor for COPD and even after the diagnosis of the disease, smoking plays an important role in disease development and patient’s prognosis. Sixty percent of the COPD patients had succeeded in smoking cessation. Patients who had managed to quit smoking had lower mortality rates and less psychiatric diseases and alcohol abuse although they were older and had more cardiovascular diseases than patients who continued smoking. Genetic polymorphism rs1051730 in the nicotinic acethylcholine receptor gene (CHRNA3/5) associated with heavy smoking, cancer prevalence and mortality in two Finnish independent cohorts consisting of COPD patients and male smokers. Challenges in smoking cessation and higher mortality rates may be partly due to individual patient’s genetic composition. Approximately 50% of COPD patients are physically inactive and the proportion was higher among current smokers. Physically active and inactive patients didn’t differ from each other in regard to age, gender or comorbidities. Bronchial obstruction explained inactivity only in severe disease. Subjective sensation of dyspnea, however, had very strong association to inactivity and was also associated to low health related quality of life (HRQoL). ACOS patients had a significantly lower HRQoL than either the patients with asthma or with COPD even though they were younger than COPD patients, had better lung functions and smaller tobacco exposure.