Immune mechanisms in atherosclerosis; Focus on the role of the complement system

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the inner layer of the arterial wall. During atherogenesis, various structures that are recognized as non-self by the immune system, such as modified lipoproteins, are deposited in the arterial wall. Accordingly, atherosclerotic lesions and blood of humans and animals with atherosclerotic lesions show signs of activation of both innate and adaptive immune responses. Although immune attack is initially a self-protective reaction, which is meant to destroy or remove harmful agents, a chronic inflammatory state in the arterial wall accelerates atherosclerosis. Indeed, various modulations of the immune system of atherosclerosis-prone animals have provided us with convincing evidence that immunological mechanisms play an important role in the pathogenesis of atherosclerosis. This thesis focuses on the role of complement system, a player of the innate immunity, in atherosclerosis. Complement activation via any of the three different pathways (classical, alternative, lectin) proceeds as a self-amplifying cascade, which leads to the generation of opsonins, anaphylatoxins C3a and C5a, and terminal membrane-attack complex (MAC, C5b-9), all of which regulate the inflammatory response and act in concert to destroy their target structures. To prevent uncontrolled complement activation or its attack against normal host cells, complement needs to be under strict control by regulatory proteins. The complement system has been shown to be activated in atherosclerotic lesions, modified lipoproteins and immune complexes containing oxLDL, for instance, being its activators. First, we investigated the presence and role of complement regulators in human atherosclerotic lesions. We found that inhibitors of the classical and alternative pathways, C4b-binding protein and factor H, respectively, were present in atherosclerotic lesions, where they localized in the superficial proteoglycan-rich layer. In addition, both inhibitors were found to bind to arterial proteoglycans in vitro. Immunohistochemical stainings revealed that, in the superficial layer of the intima, complement activation had been limited to the C3 level, whereas in the deeper intimal layers, complement activation had proceeded to the terminal C5b-9 level. We were also able to show that arterial proteoglycans inhibit complement activation in vitro. These findings suggested to us that the proteoglycan-rich layer of the arterial intima contains matrix-bound complement inhibitors and forms a protective zone, in which complement activation is restricted to the C3 level. Thus, complement activation is regulated in atherosclerotic lesions, and the extracellular matrix is involved in this process. Next, we studied whether the receptors for the two complement derived effectors, anaphylatoxins C3a and C5a, are expressed in human coronary atherosclerotic lesions. Our results of immunohistochemistry and RT-PCR analysis showed that, in contrast to normal intima, C3aR and C5aR were highly expressed in atherosclerotic lesions. In atherosclerotic plaques, the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR, and a small fraction of them also expressed C3aR, mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. These results suggested that intimal cells can respond to and become activated by complement-derived anaphylatoxins. Finally, we wanted to learn, whether oxLDL-IgG immune complexes, activators of the classical complement pathway, could have direct cellular effects in atherogenesis. Thus, we tested whether oxLDL-IgG immune complexes affect the survival of human monocytes, the precursors of macrophages, which are the most abundant inflammatory cell type in atherosclerotic lesions. We found that OxLDL-IgG immune complexes, in addition to transforming monocytes into foam cells, promoted their survival by decreasing their spontaneous apoptosis. This effect was mediated by cross-linking Fc receptors with ensuing activation of Akt-dependent survival signaling. Our finding revealed a novel mechanism by which oxLDL-IgG immune complexes can directly affect the accumulation of monocyte-macrophages in human atherosclerotic lesions and thus play a role in atherogenesis.

Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.

Inflammatory and coagulation disturbances in acute pancreatitis

Acute pancreatitis (AP), a common cause of acute abdominal pain, is usually a mild, self-limited disease. However, some 20-30% of patients develop a severe disease manifested by pancreatic necrosis, abscesses or pseudocysts, and/or extrapancreatic complications, such as vital organ failure (OF). Patients with AP develop systemic inflammation, which is considered to play a role in the pathogenesis of multiple organ failure (MOF). OF mimics the condition seen in patients with sepsis, which is characterized by an overwhelming production of inflammatory mediators, activation of the complement system and systemic activation of coagulation, as well as the development of disseminated intravascular coagulation (DIC) syndrome. Vital OF is the major cause of mortality in AP, along with infectious complications. About half of the deaths occur within the first week of hospitalization and thus, early identification of patients likely to develop OF is important. The aim of the present study was to investigate inflammatory and coagulation disturbances in AP and to find inflammatory and coagulation markers for predicting severe AP, and development of OF and fatal outcome. This clinical study consists of four parts. All of patients studied had AP when admitted to Helsinki University Central Hospital. In the first study, 31 patients with severe AP were investigated. Their plasma levels of protein C (PC) and activated protein C (APC), and monocyte HLA-DR expression were studied during the treatment period in the intensive care unit; 13 of these patients developed OF. In the second study, the serum levels of complement regulator protein CD59 were studied in 39 patients during the first week of hospitalization; 12 of them developed OF. In the third study, 165 patients were investigated; their plasma levels of soluble form of the receptor for advanced glycation end products (sRAGE) and high mobility group box 1 (HMGB1) protein were studied during the first 12 days of hos-pitalization; 38 developed OF. In the fourth study, 33 patients were studied on admission to hospital for plasma levels of prothrombin fragment F1+2 and tissue factor pathway inhibitor (TFPI), and thrombin formation capacity by calibrated automated thrombogram (CAT); 9 of them developed OF. Our results showed significant PC deficiency and decreased APC generation in patients with severe AP. The PC pathway defects seemed to be associated with the development of OF. In patients who developed OF, the levels of serum CD59 and plasma sRAGE, but not of HMGB1, were significantly higher than in patients who recovered without OF. The high CD59 levels on admission to the hospital seemed to be predictive for severe AP and OF. The median of the highest sRAGE levels was significantly higher in non-survivors than in survivors. No significant difference between the patient groups was found in the F1+2 levels. The thrombograms of all patients were disturbed in their shape, and in 11 patients the exogenous tissue factor did not trigger thrombin generation at all ( flat curve ). All of the patients that died displayed a flat curve. Free TFPI levels and free/total TFPI ratios were significantly higher in patients with a flat curve than in the others, and these levels were also significantly higher in non-survivors than in survivors. The flat curve in combination with free TFPI seemed to be predictive for a fatal outcome in AP.

Health-Related Quality of Life, Costs and Treatment of Inflammatory Rheumatic Diseases in Routine Practice : With special emphasis on biological drugs

Rheumatoid arthritis (RA) and other chronic inflammatory joint diseases already begin to affect patients health-related quality of life (HRQoL) in the earliest phases of these diseases. In treatment of inflammatory joint diseases, the last two decades have seen new strategies and treatment options introduced. Treatment is started at an earlier phase; combinations of disease-modifying anti-rheumatic drugs (DMARDs) and corticosteroids are used; and in refractory cases new drugs such as tumour necrosis factor (TNF) inhibitors or other biologicals can be started. In patients with new referrals to the Department of Rheumatology of the Helsinki University Central Hospital, we evaluated the 15D and the Stanford Health Assessment Questionnaire (HAQ) results at baseline and approximately 8 months after their first visit. Altogether the analysis included 295 patients with various rheumatic diseases. The mean baseline 15D score (0.822, SD 0.114) was significantly lower than for the age-matched general population (0.903, SD 0.098). Patients with osteoarthritis (OA) and spondyloarthropathies (SPA) reported the poorest HRQoL. In patients with RA and reactive arthritis (ReA) the HRQoL improved in a statistically significant manner during the 8-month follow-up. In addition, a clinically important change appeared in patients with systemic rheumatic diseases. HAQ score improved significantly in patients with RA, arthralgia and fibromyalgia, and ReA. In a study of 97 RA patients treated either with etanercept or adalimumab, we assessed their HRQoL with the RAND 36-Item Health Survey 1.0 (RAND-36) questionnaire. We also analysed changes in clinical parameters and the HAQ. With etanercept and adalimumab, the values of all domains in the RAND-36 questionnaire increased during the first 3 months. The efficacy of each in improving HRQoL was statistically significant, and the drug effects were comparable. Compared to Finnish age- and sex-matched general population values, the HRQoL of the RA patients was significantly lower at baseline and, despite the improvement, remained lower also at follow-up. Our RA patients had long-standing and severe disease that can explain the low HRQoL also at follow-up. In a pharmacoeconomic study of patients treated with infliximab we evaluated medical and work disability costs for patients with chronic inflammatory joint disease during one year before and one year after institution of infliximab treatment. Clinical and economic data for 96 patients with different arthritis diagnoses showed, in all patients, significantly improved clinical and laboratory variables. However, the medical costs increased significantly during the second period by 12 015 (95% confidence interval, 6 496 to 18 076). Only a minimal decrease in work disability costs occurred mean decrease 130 (-1 268 to 1 072). In a study involving a switch from infliximab to etanercept, we investigated the clinical outcome in 49 patients with RA. Reasons for switching were in 42% failure to respond by American College of Rheumatology (ACR) 50% criteria; in 12% adverse event; and in 46% non-medical reasons although the patients had responded to infliximab. The Disease Activity Score with 28 joints examined (DAS28) allowed us to measure patients disease activity and compare outcome between groups based on the reason for switching. In the patients in whom infliximab was switched to etanercept for nonmedical reasons, etanercept continued to suppress disease activity effectively, and 1-year drug survival for etanercept was 77% (95% CI, 62 to 97). In patients in the infliximab failure and adverse event groups, DAS28 values improved significantly during etanercept therapy. However, the 1-year drug survival of etanercept was only 43% (95% CI, 26 to 70) and 50% (95% CI, 33 to 100), respectively. Although the HRQoL of patients with inflammatory joint diseases is significantly lower than that of the general population, use of early and aggressive treatment strategies including TNF-inhibitors can improve patients HRQoL effectively. Further research is needed in finding new treatment strategies for those patients who fail to respond or lose their response to TNF-inhibitors.

Spirometric studies on the adult general population of Helsinki : bronchodilation responses, determinants, and intrasession repeatability of FEV1, FEV6, FVC, and forced expiratory time

Spirometry is the most widely used lung function test in the world. It is fundamental in diagnostic and functional evaluation of various pulmonary diseases. In the studies described in this thesis, the spirometric assessment of reversibility of bronchial obstruction, its determinants, and variation features are described in a general population sample from Helsinki, Finland. This study is a part of the FinEsS study, which is a collaborative study of clinical epidemiology of respiratory health between Finland (Fin), Estonia (Es), and Sweden (S). Asthma and chronic obstructive pulmonary disease (COPD) constitute the two major obstructive airways diseases. The prevalence of asthma has increased, with around 6% of the population in Helsinki reporting physician-diagnosed asthma. The main cause of COPD is smoking with changes in smoking habits in the population affecting its prevalence with a delay. Whereas airway obstruction in asthma is by definition reversible, COPD is characterized by fixed obstruction. Cough and sputum production, the first symptoms of COPD, are often misinterpreted for smokers cough and not recognized as first signs of a chronic illness. Therefore COPD is widely underdiagnosed. More extensive use of spirometry in primary care is advocated to focus smoking cessation interventions on populations at risk. The use of forced expiratory volume in six seconds (FEV6) instead of forced vital capacity (FVC) has been suggested to enable office spirometry to be used in earlier detection of airflow limitation. Despite being a widely accepted standard method of assessment of lung function, the methodology and interpretation of spirometry are constantly developing. In 2005, the ATS/ERS Task Force issued a joint statement which endorsed the 12% and 200 ml thresholds for significant change in forced expiratory volume in one second (FEV1) or FVC during bronchodilation testing, but included the notion that in cases where only FVC improves it should be verified that this is not caused by a longer exhalation time in post-bronchodilator spirometry. This elicited new interest in the assessment of forced expiratory time (FET), a spirometric variable not usually reported or used in assessment. In this population sample, we examined FET and found it to be on average 10.7 (SD 4.3) s and to increase with ageing and airflow limitation in spirometry. The intrasession repeatability of FET was the poorest of the spirometric variables assessed. Based on the intrasession repeatability, a limit for significant change of 3 s was suggested for FET during bronchodilation testing. FEV6 was found to perform equally well as FVC in the population and in a subgroup of subjects with airways obstruction. In the bronchodilation test, decreases were frequently observed in FEV1 and particularly in FVC. The limit of significant increase based on the 95th percentile of the population sample was 9% for FEV1 and 6% for FEV6 and FVC; these are slightly lower than the current limits for single bronchodilation tests (ATS/ERS guidelines). FEV6 was proven as a valid alternative to FVC also in the bronchodilation test and would remove the need to control duration of exhalation during the spirometric bronchodilation test.

Common and rare variants of the renin-angiotensin system and their relation to antihypertensive drug responses

Most of the diseases affecting public health, like hypertension, are multifactorial by etiology. Hypertension is influenced by genetic, life style and environmental factors. Estimation of the influence of genes to the risk of essential hypertension varies from 30 to 50%. It is plausible that in most of the cases susceptibility to hypertension is determined by the action of more than one gene. Although the exact molecular mechanism underlying essential hypertension remains obscure, several monogenic forms of hypertension have been identified. Since common genetic variations may predict, not only to susceptibility to hypertension, but also response to antihypertensive drug therapy, pharmacogenetic approaches may provide useful markers in finding relations between candidate genes and phenotypes of hypertension. The aim of this study was to identify genetic mutations and polymorphisms contributing to human hypertension, and examine their relationships to intermediate phenotypes of hypertension, such as blood pressure (BP) responses to antihypertensive drugs or biochemical laboratory values. Two groups of patients were investigated in the present study. The first group was collected from the database of patients investigated in the Hypertension Outpatient Ward, Helsinki University Central Hospital, and consisted of 399 subjects considered to have essential hypertension. Frequncies of the mutant or variant alleles were compared with those in two reference groups, healthy blood donors (n = 301) and normotensive males (n = 175). The second group of subjects with hypertension was collected prospectively. The study subjects (n=313) underwent a protocol lasting eight months, including four one-month drug treatment periods with antihypertensive medications (thiazide diuretic, β-blocker, calcium channel antagonist, and an angiotensin II receptor antagonist). BP responses and laboratory values were related to polymorphims of several candidate genes of the renin-angiotensin system (RAS). In addition, two patients with typical features of Liddle’s syndrome were screened for mutations in kidney epithelial sodium channel (ENaC) subunits. Two novel mutations causing Liddle’s syndrome were identified. The first mutation identified located in the beta-subunit of ENaC and the second mutation found located in the gamma-subunit, constituting the first identified Liddle mutation locating in the extracellular domain. This mutation showed 2-fold increase in channel activity in vitro. Three gene variants, of which two are novel, were identified in ENaC subunits. The prevalence of the variants was three times higher in hypertensive patients (9%) than in reference groups (3%). The variant carriers had increased daily urinary potassium excretion rate in relation to their renin levels compared with controls suggesting increased ENaC activity, although in vitro they did not show increased channel activity. Of the common polymorphisms of the RAS studied, angiotensin II receptor type I (AGTR1) 1166 A/C polymorphism was associated with modest changes in RAS activity. Thus, patients homozygous for the C allele tended to have increased aldosterone and decreased renin levels. In vitro functional studies using transfected HEK293 cells provided additional evidence that the AGTR1 1166 C allele may be associated with increased expression of the AGTR1. Common polymorphisms of the alpha-adducin and the RAS genes did not significantly predict BP responses to one-month monotherapies with hydroclorothiazide, bisoprolol, amlodipin, or losartan. In conclusion, two novel mutations of ENaC subunits causing Liddle’s syndrome were identified. In addition, three common ENaC polymorphisms were shown to be associated with occurrence of essential hypertension, but their exact functional and clinical consequences remain to be explored. The AGTR1 1166 C allele may modify the endocrine phenotype of hypertensive patients, when present in homozygous form. Certain widely studied polymorphisms of the ACE, angiotensinogen, AGTR1 and alpha-adducin genes did not significantly affect responses to a thiazide, β-blocker, calcium channel antagonist, and angiotensin II receptor antagonist.

The effects of entacapone on cardiorespiratory, autonomic, and clinical responses in L-dopa-treated patients with Parkinson's disease

Rest tremor, rigidity, and slowness of movements-considered to be mainly due to markedly reduced levels of dopamine (DA) in the basal ganglia-are characteristic motor symptoms of Parkinson's disease (PD). Although there is yet no cure for this illness, several drugs can alleviate the motor symptoms. Among these symptomatic therapies, L-dopa is the most effective. As a precursor to DA, it is able to replace the loss of DA in the basal ganglia. In the long run L-dopa has, however, disadvantages. Motor response complications, such as shortening of the duration of drug effect ("wearing-off"), develop in many patients. In addition, extensive peripheral metabolism of L-dopa by aromatic amino acid decarboxylase and catechol-O-methyltransferase (COMT) results in its short half-life, low bioavailability, and reduced efficacy. Entacapone, a nitrocatechol-structured compound, is a highly selective, reversible, and orally active inhibitor of COMT. It increases the bioavailability of L-dopa by reducing its peripheral elimination rate. Entacapone extends the duration of clinical response to each L-dopa dose in PD patients with wearing-off fluctuations. COMT is important in the metabolism of catecholamines. Its inhibition could, therefore, theoretically lead to adverse cardiovascular reactions, especially in circumstances of enhanced sympathetic activity (physical exercise). PD patients may be particularly vulnerable to such effects due to high prevalence of cardiovascular autonomic dysfunction, and the common use of monoamine oxidase B inhibitor selegiline, another drug with effects on catecholamine metabolism. Both entacapone and selegiline enhance L-dopa's clinical effect. Their co-administration may therefore lead to pharmacodynamic interactions, either beneficial (improved L-dopa efficacy) or harmful (increased dyskinesia). We investigated the effects of repeated dosing (3-5 daily doses for 1-2 weeks) of entacapone 200 mg administered either with or without selegiline (10 mg once daily), on several safety and efficacy parameters in 39 L-dopa-treated patients with mild to moderate PD in three double-blind placebo-controlled, crossover studies. In the first two, the cardiovascular, clinical, and biochemical responses were assessed repeatedly for 6 hours after drug intake, first with L-dopa only (control), and then after a 2 weeks on study drugs (entacapone vs. entacapone plus selegiline in one; entacapone vs. selegiline vs. entacapone plus selegiline in the other). The third study included cardiovascular reflex and spiroergometric exercise testing, first after overnight L-dopa withdrawal (control), and then after 1 week on entacapone plus selegiline as adjuncts to L-dopa. Ambulatory ECG was recorded in two of the studies. Blood pressure, heart rate, ECG, cardiovascular autonomic function, cardiorespiratory exercise responses, and the resting/exercise levels of circulating catecholamines remained unaffected by entacapone, irrespective of selegiline. Entacapone significantly enhanced both L-dopa bioavailability and its clinical response, the latter being more pronounced with the co-administration of selegiline. Dyskinesias were also increased during simultaneous use of both entacapone and selegiline as L-dopa adjuncts. Entacapone had no effect on either work capacity or work efficiency. The drug was well tolerated, both with and without selegiline. Conclusions: the use of entacapone-either alone or combined with selegiline-seems to be hemodynamically safe in L-dopa-treated PD patients, also during maximal physical effort. This is in line with the safety experience from larger phase III studies. Entacapone had no effect on cardiovascular autonomic function. Concomitant administration of entacapone and selegiline may enhance L-dopa's clinical efficacy but may also lead to increased dyskinesia.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.