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.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

Molecular patterns behind immunological and metabolic alterations in lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is a recessively inherited disorder characterised by reduced plasma and increased urinary levels of cationic amino acids (CAAs), protein malnutrition, growth failure and hyperlipidemia. Some patients develop severe immunological, renal and pulmonary complications. All Finnish patients share the same LPIFin mutation in the SLC7A7 gene that encodes CAA transporter y+LAT1. The aim of this study was to examine molecular factors contributing to the various symptoms, systemic metabolic and lipid profiles, and innate immune responses in LPI. The transcriptomes, metabolomes and lipidomes were analysed in whole-blood cells and plasma using RNA microarrays and gas or liquid chromatography-mass spectrometry techniques, respectively. Toll-like receptor (TLR) signalling in monocyte-derived macrophages exposed to pathogens was scrutinised using qRT-PCR and the Luminex technology. Altered levels of transcripts participating in amino acid transport, immune responses, apoptosis and pathways of hepatic and renal metabolism were identified in the LPI whole-blood cells. The patients had increased non-essential amino acid, triacylglycerol and fatty acid levels, and decreased plasma levels of phosphatidylcholines and practically all essential amino acids. In addition, elevated plasma levels of eight metabolites, long-chain triacylglycerols, two chemoattractant chemokines and nitric oxide correlated with the reduced glomerular function in the patients with kidney disease. Accordingly, it can be hypothesised that the patients have increased autophagy, inflammation, oxidative stress and apoptosis, leading to hepatic steatosis, uremic toxicity and altered intestinal microbe metabolism. Furthermore, the LPI macrophages showed disruption in the TLR2/1, TLR4 and TLR9 pathways, suggesting innate immune dysfunctions with an excessive response to bacterial infections but a deficient viral DNA response.

Nanoparticle Labels in Bioaffinity Assays

Nanoparticles offer adjustable and expandable reactive surface area compared to the more traditional solid phase forms utilized in bioaffinity assays due to the high surface to-volume ratio. The versatility of nanoparticles is further improved by the ability to incorporate various molecular complexes such as luminophores into the core. Nanoparticle labels composed of polystyrene, silica, inorganic crystals doped with high number of luminophores, preferably lanthanide(III) complexes, are employed in bioaffinity assays. Other label species such as semiconductor crystals (quantum dots) or colloidal gold clusters are also utilized. The surface derivatization of such particles with biomolecules is crucial for the applicability to bioaffinity assays. The effectiveness of a coating is reliant on the biomolecule and particle surface characteristics and the selected coupling technique. The most critical aspects of the particle labels in bioaffinity assays are their size-dependent features. For polystyrene, silica and inorganic phosphor particles, these include the kinetics, specific activity and colloidal stability. For quantum dots and gold colloids, the spectral properties are also dependent on particle size. This study reports the utilization of europium(III)-chelate-embedded nanoparticle labels in the development of bioaffinity assays. The experimental covers both the heterogeneous and homogeneous assay formats elucidating the wide applicability of the nanoparticles. It was revealed that the employment of europium(III) nanoparticles in heterogeneous assays for viral antigens, adenovirus hexon and hepatitis B surface antigen (HBsAg), resulted in sensitivity improvement of 10-1000 fold compared to the reference methods. This improvement was attributed to the extreme specific activity and enhanced monovalent affinity of the nanoparticles conjugates. The applicability of europium(III)-chelate-doped nanoparticles to homogeneous assay formats were proved in two completely different experimental settings; assays based on immunological recognition or proteolytic activity. It was shown that in addition to small molecule acceptors, particulate acceptors may also be employed due to the high specific activity of the particles promoting proximity-induced reabsorptive energy transfer in addition to non-radiative energy transfer. The principle of proteolytic activity assay relied on a novel dual-step FRET concept, wherein the streptavidin-derivatized europium(III)-chelate-doped nanoparticles were used as donors for peptide substrates modified with biotin and terminal europium emission compliant primary acceptor and a secondary quencher acceptor. The recorded sensitized emission was proportional to the enzyme activity, and the assay response to various inhibitor doses was in agreement with those found in literature showing the feasibility of the technique. Experiments regarding the impact of donor particle size on the extent of direct donor fluorescence and reabsorptive excitation interference in a FRET-based application was conducted with differently sized europium(III)-chelate-doped nanoparticles. It was shown that the size effect was minimal

Immunomodulation of allergic immune response during specific immunotherapy

Atopic, IgE-mediated allergies are one of the major public health problems in Finland and other Western countries. These diseases are characterized by type 2 T helper (Th2) cell predominated immune responses (interleukin-4 (IL-4), IL-5) against ubiquitous environmental allergens. Despite of adequate pharmacological treatment, more than 20% of the patients with allergic rhinitis develop asthma. Allergen specific immunotherapy (SIT) is the only treatment currently available to affect to the natural course of allergic diseases. This treatment involves repeated administration of allergens to the patients either via sublingual route (sublingual immunotherapy, SLIT) or by subcutaneous injections (subcutaneous immunotherapy, SCIT). Successful treatment with SCIT or SLIT has been shown to provide long-term remission in symptoms, and prevent disease progression to asthma, but the immunological mechanisms behind these beneficial effects are not yet completely understood. Increased knowledge of such mechanisms could not only help to improve SIT efficacy, but also provide tools to monitor the development of clinical response to SIT in individual patients, and possibly also, predict the ultimate therapeutic outcome. The aim of this work was to clarify the immunological mechanisms associated with SIT by investigating the specific allergen-induced immune responses in peripheral blood mononuclear cells (PBMC) of allergic rhinitis patients during the course of SLIT and SCIT. The results of this work demonstrate that both therapies induced increases in the protective, Th2-balancing Th1 type immune responses in PBMC, e.g. by up-regulating signaling lymphocytic activation molecule (SLAM) and interferon gamma (IFN-γ) expression, and augmented tolerogenic T regulatory (Treg) cell type responses against the specific allergens, e.g. by increasing IL-10 or Forkhead box P3 (FOXP3) expression. The induction of allergen-specific Th1 and Treg type responses during SLIT were dependent on the treatment dose, favoring high allergen dose SLIT. During SCIT, the early decrease in Th2 type cytokine production - in particular of IL-4 mRNA and IL-4/IFN-γ expression ratio - was associated with the development of good therapeutic outcome. Conversely, increases in both Th2 (IL-5) and Th1 (IFN-γ, SLAM) type responses and IL-10 mRNA production were seen in the patients with less effective outcome. In addition, increase in Th17 type cytokine (IL-17) mRNA production was found in the PBMC of patients with less effective outcome during both SLIT and SCIT. These data strengthen the current hypothesis that immunomodulation of allergen-specific immune responses from the prevailing Th2-biased responses towards a more Th1 type, and induction of tolerogenic Treg cells producing IL-10 represent the two key mechanisms behind the beneficial effects of SIT. The data also give novel insight into the mechanisms why SIT may fail to be effective in some patients by demonstrating a positive correlation between the proinflammatory IL-17 responses, Th2 type IL-5 production and clinical symptoms. Taken together, these data indicate that the analysis of Th1, Th2, Treg ja Th17-associated immune markers such as IL-10, SLAM, IL-4, IL-5 and IL-17 could provide tools to monitor the development of clinical response to SIT, and thereby, predict the ultimate clinical outcome already in the early course of the treatment.

Interleukin-4 induced leukocyte differentiation

Monocytes, macrophages and dendritic cells (DCs) are important mediators of innate immune system, whereas T lymphocytes are the effector cells of adaptive immune responses. DCs play a crucial role in bridging innate and adaptive immunity. Naïve CD4+ Th progenitors (Thp) differentiate to functionally distinct effector T cell subsets including Th1, Th2 and Th17 cells, which while being responsible for specific immune functions have also been implicated in pathological responses, such as autoimmunity, asthma and allergy. The main objective of this thesis is to dissect the signalling networks involved in the IL-4 induced differentiation of two important leukocyte subtypes, Th2 cells and DCs. Gene expression profiling lead to identification of over 200 genes which are differentially expressed during cytokine induced differentiation of human monocytes to DCs or macrophages and which are likely to be essential for the proper biological functions of these cell types. Transcriptome analysis demonstrated the dynamic regulation of gene expression by IL-12 and IL-4 during the initiation of Th cell differentiation, which was partly counteracted by an immunosuppressive cytokine, TGFβ, present in the culture media. Results from RNAi mediated gene knockdown experiments and global gene expression analysis elucidated that SATB1 regulates multiple genes important for Th cell polarization or function as well as may compete with GATA3 for the reciprocal regulation of IL-5 transcription. In conclusion, the results obtained have extended our system-level understanding of the immune cell differentiation processes and provide an excellent basis for the further functional studies which could lead to development of improved therapeutic approaches for a range of immunological conditions.

Blackcurrant seed oil for prevention of atopic dermatitis

One hypothesis for the increased incidence of atopic diseases has been that it is associated with changing dietary habits, especially the changed intake of essential fatty acids (EFAs). The metabolism of EFAs produces eiconasoids, prostaglandins and leukotrienes, which are essential to organs and play a major role in regulation of inflammation and immune response. In some studies persons with atopic dermatitis have been found to have reduced levels of EFAs. The first year of infancy as well as the foetal period are crucial for the development of atopic immune response. The composition of blackcurrant seed oil (BCSO) corresponds to the recommended ratio of EFAs n-3 and n-6 in the diet (1/3-1/4) and as a dietary supplement could, even in small doses, modify the unbalance of EFAs in an efficient way. The purpose of this study was to find out whether atopic allergies can be prevented by supplementing the diet of pregnant mothers with blackcurrant seed oil and whether it could affect the immunological balance of a child. We also sought to find out whether a blackcurrant seed oil supplementation can affect the composition of breast milk to suppress the T helper 2 lymphocyte (Th2) responses in infants. 313 pregnant mothers were randomly assigned to receive BCSO (n=151) or olive oil as placebo (n=162). Supplementation was started at the 8th to 16th weeks of pregnancy, 6 capsules per day (dose of 3 g), and continued until the cessation of breastfeeding. It was thereafter followed by direct supplementation to infants of 1 ml (1 g) of oil per day until the age of two years. Atopic dermatitis and its severity (SCORAD index) were evaluated, serum total IgE was measured and skin prick tests were performed at the age of 3, 12 and 24 months. Peripheral blood mononuclear cell (PBMC) samples were taken at the age of 3 and 12 months and breast milk samples were collected during the first 3 months of breastfeeding. Parental atopy was common (81.7%) in the studied infants, making them infants with increased atopy risk. There was a significantly lower prevalence of atopic dermatitis in the BCSO group (33%) than in the olive oil group (47%) at the age of 12 months. Also, SCORAD was lower in the BCSO group than in the olive oil group. Dietary intervention with BCSO had immunomodulatory effects on breast milk, inducing cytokine production from Th2 to Th1 immunodeviation. It decreased the level of IL-4 and elevated the level of IFN-γ. BCSO intervention did not affect cytokines in the children’s PBMC. However, children of smoking parents in the combined BCSO and olive oil group had significantly elevated levels of Th2 type cytokines IL-4, IL-5 and the proinflammator cytokine TNF. Dietary supplementation with BCSO is safe. It is well tolerated and transiently reduces the prevalence of atopic dermatitis at the age of 12 months. It can possibly become a potential tool in prevention of atopic symptoms when used at the early stages of life.

Cardiac Troponin Specific Autoantibodies: Analytical Tools for Exploring Their Impact on Cardiac Troponin I Testing

Cardiac troponin (cTn) I and T are the recommended biomarkers for the diagnosis and risk stratification of patients with suspected acute coronary syndrome (ACS), a major cause of cardiovascular death and disability worldwide. It has recently been demonstrated that cTn-specific autoantibodies (cTnAAb) can negatively interfere with cTnI detection by immunoassays to the extent that cTnAAb-positive patients may be falsely designated as cTnI-negative. The aim of this thesis was to develop and optimize immunoassays for the detection of both cTnI and cTnAAb, which would eventually enable exploring the clinical impact of these autoantibodies on cTnI testing and subsequent patient management. The extent of cTnAAb interference in different cTnI assay configurations and the molecular characteristics of cTnAAbs were investigated in publications I and II, respectively. The findings showed that cTnI midfragment targeting immunoassays used predominantly in clinical practice are affected by cTnAAb interference which can be circumvented by using a novel 3+1-type assay design with three capture antibodies against the N-terminus, midfragment and C-terminus and one tracer antibody against the C-terminus. The use of this assay configuration was further supported by the epitope specificity study, which showed that although the midfragment is most commonly targeted by cTnAAbs, the interference basically encompasses the whole molecule, and there may be remarkable individual variation at the affected sites. In publications III and IV, all the data obtained in previous studies were utilized to develop an improved version of an existing cTnAAb assay and a sensitive cTnI assay free of this specific analytical interference. The results of the thesis showed that approximately one in 10 patients with suspected ACS have detectable amounts of cTnAAbs in their circulation and that cTnAAbs can inhibit cTnI determination when targeted against the binding sites of assay antibodies used in its immunological detection. In the light of these observations, the risk of clinical misclassification caused by the presence of cTnAAbs remains a valid and reasonable concern. Because the titers, affinities and epitope specificities of cTnAAbs and the concentration of endogenous cTnI determine the final effect of circulating cTnAAbs, appropriately sized studies on their clinical significance are warranted. The new cTnI and cTnAAb assays could serve as analytical tools for establishing the impact of cTnAAbs on cTnI testing and also for unraveling the etiology of cTn-related autoimmune responses.

Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.

Bumblebees in a Changing Climate: Evaluating the Effects of Temperature on Queen Performance

Bumblebees are a very essential group of pollinating insects, but their populations have declined drastically during the past decades. We need to understand why their numbers are decreasing and what can be done to reverse this trend. Climate change-related phenomena, such as changes in the overwintering temperatures and spring conditions, are among the most prominent threats to bumblebees. Queens have a special role in the lifecycle of bumblebees because they overwinter and start new colonies the next year. Their successful performance: survival, overwintering ability, longevity, immune competence, and nest establishing capability in spring, is highly important for bumblebee populations. However, the effects of climate change on bumblebee queen performance remain unknown. The main objective of this thesis was to assess how temperature affects the performance of bumblebee queens during and after overwintering. The effects of warm temperature predicted by climate change scenarios on queen survival and stress-tolerance were studied by a four-month artificial diapause of bumblebee queens at two temperatures (9°C and 1.8°C). Bumblebee colonies were also reared in a laboratory and factors affecting colony characteristics were examined. In addition, queen performance during spring was studied in a starvation experiment using two temperatures (15°C as normal; 24°C as warmer than average) and queens collected from nature right after their emergence. My research revealed how temperature affects queen performance, and queen size was found to be an important factor determining the direction of some of these effects. We found a 0.4g weight threshold for bumblebee queens to be able to survive overwintering. In addition, during mild winters, larger queens have a higher chance than smaller ones to survive through winter and also to cope with immunological stresses after overwintering. During cold conditions, which are normal in the current climatic situation, this advantage disappears. In the spring starvation experiment, the starved queens survived approximately eight days longer in 15°C than in 24°C, which means that starvation risk rises significantly with increasing spring temperature, in a situation where food is scarce due to for example frost damage or asynchrony between bumblebees and their important food plants. These results could mean that in the future climate, larger queens are better able to survive the winter, initiate their nests and start rearing their offspring. This may be problematic, because I also detected two alternative strategies of colony development that differ between large and small queens; larger queens start to lay eggs earlier at nest initiation, their colonies mature later, they produce more workers, and they have a more strongly male biased sex allocation compared with smaller queens. If larger queens have a greater change of producing offspring after a mild winter, this could lead to a significant decline in the total production of new queens at a population level. Thus, it seems that queen size could act as one mechanism regulating the population level outcomes in different temperatures. The new information presented in my thesis reinforces that basic research, monitoring, and local species conservation of bumblebees both in Finland and globally must be increased to ensure that this highly important pollinator group survives in the face of climate change.

Players of Th-cell differentiation and immune dysregulation – focus on GIMAP family genes

The balance of T helper (Th) cell differentiation is the fundamental process that ensures that the immune system functions correctly and effectively. The differentiation is a fine tuned event, the outcome of which is driven by activation of the T-cell in response to recognition of the specific antigen presented. The co-stimulatory signals from the surrounding cytokine milieu help to determine the outcome. An impairment in the differentiation processes may lead to an imbalance in immune responses and lead to immune-mediated pathologies. An over-representation of Th1 type cytokine producing cells leads to tissue-specific inflammation and autoimmunity, and excessive Th2 response is causative for atopy, asthma and allergy. The major factors of Th-cell differentiation and in the related disease mechanisms have been extensively studied, but the fine tuning of these processes by the other factors cannot be discarded. In the work presented in this thesis, the association of T-cell receptor costimulatory molecules CTLA4 and ICOS with autoimmune diabetes were studied. The underlying aspect of the study was to explore the polymorphism in these genes with the different disease rates observed in two geographically close populations. The main focus of this thesis was set on a GTPase of the immunity associated protein (GIMAP) family of small GTPases. GIMAP genes and proteins are differentially regulated during human Th-cell differentiation and have been linked to immune-mediated disorders. GIMAP4 is believed to contribute to the immunological balance via its role in T-cell survival. To elucidate the function of GIMAP4 and GIMAP5 and their role in human immunity, a study combining genetic association in different immunological diseases and complementing functional analyses was conducted. The study revealed interesting connections with the high susceptibility risk genes. In addition, the role of GIMAP4 during Th1-cell differentiation was investigated. A novel function of GIMAP4 in relation to cytokine secretion was discovered. Further assessment of GIMAP4 and GIMAP5 effect for the transcriptomic profile of differentiating Th1-cells revealed new insights for GIMAP4 and GIMAP5 function.

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.