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.

Circulating Glucocorticoid Bioactivity and Serum Glucocorticoid-Responsive Biomarkers during Steroid Therapy in Children and Adolescents with Inflammatory Bowel Disease

Objective: Glucocorticoid therapy is used worldwide to treat various inflammatory and immune conditions, including inflammatory bowel disease (IBD). In IBD, 80% of the patients obtain a positive response to the therapy; however the development of glucocorticoid-related side-effects is common. Our aim was therefore to study the possibility of optimizing glucocorticoid therapy in children and adolescents with IBD by measuring circulating glucocorticoid bioactivity (GBA) and serum glucocorticoid-responsive biomarkers in patients receiving steroid treatment for active disease. Methods: A total of sixty-nine paediatric IBD patients from the Paediatric Outpatient Clinics of the University Hospitals of Helsinki and Tampere participated in the studies. Control patients included 101 non-IBD patients and 41 disease controls in remission. In patients with active disease, blood samples were withdrawn before the glucocorticoid therapy was started, at 2-4 weeks after the initiation of the steroid and at 1-month intervals thereafter. Clinical response to glucocorticoid treatment and the development of steroid adverse events was carefully registered. GBA was analyzed with a COS-1 cell bioassay. The measured glucocorticoid therapy-responsive biomarkers included adipocyte-derived adiponectin and leptin, bone turnover-related collagen markers amino-terminal type I procollagen propeptide (PINP) and carboxyterminal telopeptide of type I collagen (ICTP) as well as insulin-like growth factor 1 (IGF-1) and sex hormone-binding globulin (SHBG), and inflammatory marker high-sensitivity C-reactive protein (hs-CRP). Results: The most promising marker for glucocorticoid sensitivity was serum adiponectin that associated with steroid therapy–related adverse events. Serum leptin indicated a similar trend. In contrast, circulating GBA rose in all subjects receiving glucocorticoid treatment but did not associate with the clinical response to steroids or with glucocorticoid therapy-related side-effects. Of notice, young patients (<10 years) showed similar GBA levels than older patients, despite receiving higher weight-adjusted doses of glucocorticoid. Markers of bone formation were lower in children with active IBD than in the control patients, probably reflecting the suppressive effect of the active inflammation. The onset of the glucocorticoid therapy further suppressed bone turnover. Inflammatory marker hs-CRP decreased readily after the initiation of the steroid, however the decrease did not associate with the clinical response to glucocorticoids. Conclusions: This is the first study to show that adipocyte-derived adiponectin associates with steroid therapy-induced side-effects. Further studies are needed, but it is possible that the adiponectin measurement could aid the recognition of glucocorticoid-sensitive patients in the future. GBA and the other markers reflecting glucocorticoid activity in different tissues changed during the treatment, however their change did not correlate with the therapeutic response to steroids or with the development of glucocorticoid-related side effects and therefore cannot guide the therapy in these patients. Studies such as as the present one that combine clinical data with newly developed biomolecular technology are needed to step-by-step build a general picture of the glucocorticoid actions in different tissues.

Health-related quality of life, symptoms and comorbidity in inflammatory bowel disease

Health-related quality of life (HRQoL) measurement has become an important outcome in treatment trials and in health policy decisions. HRQoL can be measured by using generic or disease-specific tools. Generic instruments can be used for comparing health status among patients in different health states and conditions but they do not focus specifically on the issues relevant in a particular disease. Disease-specific tools may be more responsive to changes within a specific condition. In earlier studies, impairment of HRQoL has been evident in patients with inflammatory bowel disease (IBD), especially when the disease is active. Data about the impact of comorbidity or demographic characteristics of the patients on HRQoL are partly controversial. This study, which comprised 2913 adult IBD patients, examined HRQoL using the disease-specific IBDQ and the general 15D instruments. The 15D scores of IBD patients were compared with scores of a gender and age matched general population sample. Frequency of IBD symptoms and arrangement of therapy were studied and compared with those of IBD patients in an earlier European study. Furthermore, data of other chronic diseases of the patients were obtained from the Social Insurance Institution s reimbursement register and comorbidity of IBD patients was compared with that of age and gender matched controls. --- Of the respondents, 37% reported that they suffered from disturbing IBD symptoms weekly. In 17% of the patients, the symptoms greatly affected the ability to enjoy leisure activities, and 14% stated that these symptoms greatly affected their capacity to work. Despite that, the great majority (93%) of patients expressed satisfaction with their current treatment, which exceeded the rate observed in the other European patients. The mean IBDQ score was 163, as the possible range is 32-224, and disease activity was strongly correlated with HRQoL. Older age, comorbid diseases, and female gender were also related to impairment of HRQoL. Lower HRQoL scores were seen also in newly-diagnosed patients and in those with a history of surgery, especially after stoma or ileal pouch-anal anastomosis (IPAA) operation. The range of 15D scores was 0.30-1.00, with mean of 0.87. As with the IBDQ, disease activity, older age and history of surgery were correlated with the score. Both the newly-diagnosed patients and patients with a long-lasting disease had lower scores than average even after adjusting for age. The 15D scores of IBD patients were significantly lower than those of the control group. A strong correlation was seen between the 15D and the IBDQ scores. Comorbidity with other chronic diseases was observed in 29% of IBD patients. Connective tissue diseases, chronic obstructive pulmonary diseases, pernicious anaemia, and coronary heart disease (CHD) were significantly increased in patients with IBD. Especially female IBD patients appeared to be at increased risk for CHD, and patients who reported weekly IBD symptoms had a higher risk for having other chronic diseases in addition to IBD. Comorbidity impaired HRQoL, as measured with both generic and disease-specific tools. In conclusion, HRQoL is impaired in IBD patients. An understanding of predictors of HRQoL will help to recognise patients who will need special support.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

Infliximabi-infuusioiden akuuttien haittavaikutusten esto asetyylisalisyylihapolla lapsipotilaiden IBD- ja reumasairauksien hoidossa

IBD- ja reumasairaudet ovat elämänlaatua merkittävästi heikentäviä, kroonisia tulehduksellisia sairauksia, joiden keskivaikeiden ja vakavien muotojen hoidossa suoneen annettava biologinen TNF-alfa -tulehdustekijän vasta-aine infliksimabi (Remicade®) on vankassa asemassa. Infliksimabin hyvän tehon kääntöpuolena esiintyy yleisesti haittoja, joista infuusion aikana tai pian sen jälkeen ilmenevät allergistyyppiset reaktiot ovat hoitoa hankaloittava ja jopa vaarallinen alaluokka. Infuusioreaktioiden estoon ei nykyisellään ole todistettavasti tehokkaita keinoja. Parasetamoli ja setiritsiini osoittautuivat tässä käyttötarkoituksessa tehottomiksi. HUS:n Lasten ja nuorten sairaalassa aloitettiin 11.3.2009 hoitokokeilu asetosalisyylihapolla (ASA, Disperin®), annosteltuna painonmukaisesti per os, päämääränä selvittää prospektiivisesti ASA:n käyttömahdollisuudet infuusioreaktioiden ehkäisyssä. Tämän tutkielman aineisto kerättiin esilääkekokeilun alun ja 24.6.2010 välisellä ajalla (yhteensä 67 viikkoa) infliksimabi-infuusiossa Lasten ja nuorten sairaalan osasto 2:lla käyneiden IBD- ja reumapotilaiden asiakirjoista. Vain 1 (0,2 %) ASA:n kanssa annetuista infuusioista johti infuusioreaktioon kun aiemmin parasetamolin ja setiritsiinin kanssa todettiin 11 (2,9 %) reaktiota. GraphPad Prism 5 -ohjelmistolla tehdyn tilastoanalyysin perusteella tulokset osoittavat ASA:n olevan erittäin lupaava infuusioreaktioiden estolääke.

Molekyylibiologisten menetelmien kehittäminen ihmisen suoliston sulfaattia pelkistävien bakteerien määrittämiseksi

Ihmisen ruuansulatuskanavan bakteeriston kehitys alkaa syntymästä, jolloin ensimmäiset bakteerit kansoittavat steriilin ruuansulatuskanavan. Bakteeristo kehittyy perimän, ympäristön ja varhaisen ruokavalion vaikutuksesta kohti monimuotoisempaa bakteeripopulaatiota. Aikuisen ruuansulatuskanavan normaalibakteeristo on varsin muuttumaton, mutta siihen vaikuttavat monet tekijät, kuten ikä, terveydentila, ruokavalio ja antibioottien käyttö. Bakteeriston koostumus vaihtelee ruuansulatuskanavan eri osissa ja bakteerimäärä kasvaa kohti paksusuolta, ollen paksusuolessa ja ulosteessa peräti 1010-1012 pmy/ml. Suurin osa ruuansulatuskanavan bakteereista on anaerobeja. Ruuansulatuskanavan bakteeristo vaikuttaa muun muassa suoliston kehittymiseen ja hiilihydraattien ja proteiinien hajotukseen sekä toimii osana immuunipuolustusta. Sulfaattia pelkistävät bakteerit (SRB) ovat monimuotoinen ryhmä pääosin anaerobisia bakteereita, jotka käyttävät aineenvaihdunnassaan elektronin vastaanottajana sulfaattia muuttaen sen lopulta sulfidiksi. SRB:t ovat sopeutuneet useisiin erilaisiin ympäristöihin. Niitä tavataan mm. vesistöjen sedimenteissä sekä ihmisen ruuansulatuskanavassa. Ihmisen ruuansulatuskanavassa on SRB:ta n. 105-108 pmy/g, ja niitä on löydetty erityisesti anaerobisista osista kuten suun ientaskuista ja paksusuolesta. SRB:t voivat olla haitaksi ruuansulatuskanavalle tuottamansa sulfidin vuoksi, joka esiintyy vesiliuoksessa vetysulfidina. Tämän on havaittu olevan toksista suoliston epiteelisoluille. Viimeaikoina on kiinnostuttu sulfaatinpelkistäjien yhteydestä suoliston sairaustiloihin, kuten tulehduksellisiin suolistosairauksiin (IBD). Pro gradu -tutkimukseni tavoitteena oli kehittää PCR-DGGE- ja qPCR-menetelmät ulosteen sulfaattia pelkistävien bakteerien määritykseen. Kohdegeeninä menetelmänkehityksessä käytettiin dsrAB-geeniä, joka koodaa dissimilatorista sulfiitinpelkistysentsyymiä. dsrAB-geeni on sulfaatinpelkistäjille ominainen konservoitunut geenialue, johon perustuvia tutkimuksia ei vielä ole paljon ihmispuolelta. qPCR-menetelmä saatiin optimoitua herkäksi ja spesifiseksi käyttäen dsrA-geenispesifisiä alukkeita, mutta PCR-DGGE-menetelmää ei saatu optimoitua käytössä olleilla alukkeilla, jotka monistivat PCR-DGGE:ssa myös negatiivikontrollikantoja. Tutkittaessa qPCR:lla IBD:tä (Crohn ja ulseratiivinen koliitti) sairastavien lasten ja terveiden kontrollihenkilöiden ulostenäytteistä eristettyä DNA:ta, merkittävää eroa SRB-määrissä ei havaittu eri ryhmien välillä. Crohnin tautia sairastavien aktiivisen vaiheen ja oireettoman vaiheen näytteiden välillä oli kuitenkin tilastollisesti merkitsevä ero (SRB-määrät; oireeton vaihe>oireellinen vaihe) (P <0,05).

Determination of intestinal permeability in healthy horses using the contrast medium iohexol

Tämä lisensiaatin tutkielma koostuu kolmesta osasta; kirjallisuuskatsauksesta, kokeellisesta osasta ja liitteistä. Iohexol on ionisoitumaton, trijodattu ja vesiliukoinen röntgenvarjoaine. Iohexolia on hyödynnetty lääketieteessä useita vuosia. Iohexolia on käytetty muun muassa angio- ja myelografiassa, lisäksi iohexolia on hyödynnetty arvioitaessa munuaiskerästen suodattumisnopeutta sekä suoliston läpäisevyyden muutoksia. Hevosen tulehduksellisessa suolistosairaudessa (Inflammatory bowel disease, IBD) suoliston rakenne ja sen läpäisevyys muuttuu; tyypillistä on tulehdussolujen kertyminen suoliston seinämään ja myös sidekudosmuodostusta saattaa esiintyä. Suolisto muutoksia saatetaan havaita sekä ohut- että paksusuolessa. IBD aiheuttaa hevoselle laihtumista, johtuen ravintoaineiden puutteellisesta imeytymisestä ja proteiinien menetyksestä suoleen suoliston häiriötilan yhteydessä. Tällä hetkellä IBD:n diagnostiikka perustuu tyypillisiin oireisiin, kliiniseen tutkimukseen, verinäytteisiin, glukoosin imeytymistestiin ja peräsuolesta otettuun koepalaan. IBD:n diagnostiikka on kuitenkin erittäin haastavaa ja tutkimusmenetelmiin liittyy lukuisia ongelmia, jotka vähentävät niiden luotettavuutta IBD:n diagnostiikassa. Tutkimuksemme tarkoituksena on kehittää hevosen IBD:n diagnostiikkaa entistä helpompaan, luotettavampaan ja turvallisempaan suuntaan. Tämän alustavan tutkimuksen tavoitteet olivat: (1) tutkia voidaanko iohexol havaita hevosen seerumissa oraalisen annostelun jälkeen ja (2) muodostaa iohexolin pitoisuuskuvaaja ajan funktiona terveillä hevosilla. Materiaalimme koostui kymmenestä terveestä hevosesta, joilla ei ollut havaittu laihtumista tai ripulia. Ennen iohexolin annostelua hevosille suoritettiin kliininen tutkimus ja verinäytteet otettiin maha-suolikanavan sairauden poissulkemiseksi. Hevosille suoritettiin myös mahalaukun tähystys. 16 tunnin paaston jälkeen 1 ml/kg Iohexolia annosteltiin 10 % -liuoksena nenämahaletkulla suoraan mahaan ja verinäytteet otettiin 0, 30, 60, 120, 180, 240, 300 ja 360 minuuttia annostelun jälkeen. Iohexolin pitoisuus määritettiin käyttämällä korkean erotuskyvyn nestekromatografiaa. Iohexolin pitoisuuksista tietyillä ajanhetkillä muodostettiin kuvaaja. Hevosilla ei havaittu maha-suolikanavan sairauksia. Kaikki hevoset olivat hyvässä kuntoluokassa ja mahalaukun tähystyksessä ei havaittu merkittäviä muutoksia. Verinäytteiden tulokset olivat viiterajoissa. Kaikki hevoset sietivät iohexolia hyvin ja haittavaikutuksia ei havaittu. Iohexol oli havaittavissa seerumissa 60 minuutin kuluttua annostelusta. Kuvaajassa voitiin havaita kaksi huippua. Statistiset menetelmät tukivat löydöksiä. Iohexol testi oli yksinkertainen suorittaa ja siihen ei liittynyt haittavaikutuksia. Annos 1ml/kg oli havaittavissa seerumissa. Iohexolin pitoisuuskuvaaja muodosti kaksi huippua, ja tämänkaltainen ilmiö on kuvattu kirjallisuudessa aikaisemmin useiden lääkkeiden tapauksessa. Hevosella ilmiö liittyy todennäköisesti maha-suolikanavan rakenteellisiin ja fysiologisiin eroavaisuuksiin ja lisätutkimuksia ilmiön varmistamiseksi tarvitaan. Iohexol näyttää olevan potentiaalinen merkkiaine suoliston läpäisevyyden arviointiin ja lisätutkimuksia IBD:tä sairastavien hevosten seerumin iohexolin pitoisuuksista verrattuna terveiden hevosten seerumin iohexolin pitoisuuksiin on suunnitteilla.

Matrix Metalloproteinases and their Tissue Inhibitors as Biomarkers in Ulcerative Colitis and Crohn s Disease

Matrix metalloproteinases (MMPs) represent a family of 23 metalloendopeptidases, collectively capable of degrading all components of the extracellular matrix. MMPs have been implicated in several inflammatory processes such as arthritis, atherosclerosis, and even carcinomas. They are also involved in several beneficial activities such as epithelial repair. MMPs are inhibited by endogenous tissue inhibitors of matrix metalloproteinases (TIMP). In this study, MMPs were investigated in intestinal mucosa of inflammatory bowel diseases (IBD), chronic intestinal disorders. The main focus was to characterize mucosal inflammation in the intestine, but also cutaneous pyoderma gangrenosum (PG), to assess similarites with IBD inflammation. MMPs and TIMPs were mainly examined in colonic mucosa, in adult Crohn s disease (CD), and paediatric CD, ulcerative colitis (UC), and indeterminate colitis (IC). Ileal pouch mucosa of proctocolectomized paediatric onset IBD patients was also investigated to characterize pouch mucosa. The focus was on finding specific MMPs that could act as markers to differentiate between different IBD disorders, and MMPs that could be implied as markers for tissue injury, potentially serving as targets for MMP-inhibitors. All examinations were performed using immunohistochemistry. The results show that immunosuppressive agents decrease stromal expression of MMP-9 and -26 that could serve as specific targets for MMP-inhibitors in treating CD. In paediatric colonic inflammation, MMP-10 and TIMP-3 present as molecular markers for IBD inflammation, and MMP-7 for CD. MMP expression in the the pouch mucosa could not be classified as strictly IBD- or non-IBD-like. For the first time, this study describes the expression of MMP-3, -7, -9, -12, and TIMP-2 and -3 in pouch mucosa. The MMP profile in PG bears resemblance to both intestinal IBD inflammation and cutaneous inflammation. Based on the results, MMPs and their inhibitors emerge as promising tools in the differential diagnosis of IBD and characterization of the disease subtype, although further research is necessary. Furthermore, the expression of several MMPs in pouch has been described for the first time. While further research is warranted, the findings contribute to a better understanding of events occurring in IBD mucosa, as well as pyoderma gangrenosum.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

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.

Basement membrane and provisional matrix components (collagen type IV, laminins and von Willebrand factor) in rheumatoid arthritis and Sjögren s syndrome

The aim of this study was twofold- Firstly, to determine the composition of the type IV collagen which are the major components of the basement membrane (BM), in the synovial lining of the rheumatoid arthritis (RA) patient and in the BM in the labial salivary gland of the Sjögrens syndrome (SS) patient. Secondly, this thesis aimed to investigate the role of the BM component laminin α4 and laminin α5 in the migration of neutrophils from the blood vessels thorough the synovial lining layer into synovial fluid and the presence of vWF in the microvasculature of labial salivary gland in SS. Our studies showed that certain α chains type IV collagen are low in RA compared to control synovial linings, while laminin α5 exhibited a pattern of low expression regions at the synovial lining interface towards the joint cavity and fluid. Also, high numbers of macrophage-like lining cells containing MMP-9 were found in the lining. MMP-9 was also found in the synovial fluid. Collagen α1/2 (IV) mRNA was found to be present in high amount compared to the other α(IV) chains and also showed intense labelling in immunohistochemical staining in normal and SS patients. In healthy glands α5(IV) and α6(IV) chains were found to be continuous around ducts but discontinuous around acini. The α5(IV) and α6(IV) mRNAs were present in LSG explants and HSG cell line, while in SS these chains seemed to be absent or appear only in patches around the ductal BM and tended to be absent around acini in immunohistochemical staining, indicating that their synthesis and/or degradation seemed to be locally regulated around acinar cells. The provisional matrix component vWF serves as a marker of vascular damage. Microvasculature in SS showed signs of focal damage which in turn might impair arteriolar feeding, capillary transudation and venular drainage of blood. However, capillary density was not decreased but rather increased, perhaps as a result of angiogenesis compensatory to microvascular damage. Microvascular involvement of LSG may contribute to the pathogenesis of this syndrome. This twofold approach allows us to understand the intricate relation between the ECM components and the immunopathological changes that occur during the pathogenesis of these inflammatory rheumatic disease processes. Also notably this study highlights the importance of maintaining a healthy ECM to prevent the progression or possibly allow reversal of the disease to a considerable level. Furthermore, it can be speculated that a healthy BM could quarantine the inflamed region or in case of cancer cells barricade the movement of malignant cells thereby preventing further spread to the surrounding areas. This understanding can be further applied to design appropriate drugs which act specifically to maintain a proper BM/BM like intercellular matrix composition.

Studies of the genetic epidemiology of cardiovascular disease: focus on inflammatory candidate genes

Cardiovascular disease (CVD) is a complex disease with multifactorial aetiology. Both genetic and environmental factors contribute to the disease risk. The lifetime risk for CVD differs markedly between men and women, men being at increased risk. Inflammatory reaction contributes to the development of the disease by promoting atherosclerosis in artery walls. In the first part of this thesis, we identified several inflammatory related CVD risk factors associating with the amount of DNA from whole blood samples, indicating a potential source of bias if a genetic study selects the participants based on the available amount of DNA. In the following studies, this observation was taken into account by applying whole genome amplification to samples otherwise subjected to exclusion due to very low DNA yield. We continued by investigating the contribution of inflammatory genes to the risk for CVD separately in men and women, and looked for sex-genotype interaction. In the second part, we explored a new candidate gene and its role in the risk for CVD. Selenoprotein S (SEPS1) is a membrane protein residing in the endoplasmic reticulum where it participates in retro-translocation of unfolded proteins to cytosolic protein degradation. Previous studies have indicated that SEPS1 protects cells from oxidative stress and that variations in the gene are associated with circulating levels of inflammatory cytokines. In our study, we identified two variants in the SEPS1 gene, which associated with coronary heart disease and ischemic stroke in women. This is, to our knowledge, the first study suggesting a role of SEPS1 in the risk for CVD after extensively examining the variation within the gene region. In the third part of this thesis, we focused on a set of seven genes (angiotensin converting enzyme, angiotensin II receptor type I, C-reactive protein (CRP), and fibrinogen alpha-, beta-, and gamma-chains (FGA, FGB, FGG)) related to inflammatory cytokine interleukin 6 (IL6) and their association with the risk for CVD. We identified one variant in the IL6 gene conferring risk for CVD in men and a variant pair from IL6 and FGA genes associated with decreased risk. Moreover, we identified and confirmed an association between a rare variant in the CRP gene and lower CRP levels, and found two variants in the FGA and FGG genes associating with fibrinogen. The results from this third study suggest a role for the interleukin 6 pathway genes in the pathogenesis of CVD and warrant further studies in other populations. In addition to the IL6 -related genes, we describe in this thesis several sex-specific associations in other genes included in this study. The majority of the findings were evident only in women encouraging other studies of cardiovascular disease to include and analyse women separately from men.

Pathophysiological factors of irritable bowel syndrome, and the effects of probiotic supplementation

Gastrointestinal symptoms and impaired quality of life caused by irritable bowel syndrome (IBS) affect up to 20% of the adult population worldwide. The exact aetiology and pathophysiology of IBS are incompletely understood. Clinical studies suggest that supplementation with certain probiotics may be beneficial in IBS, but there is not enough evidence to make general recommendations. The aim of this thesis was to investigate microbiota- and mucosa-associated pathophysiological factors of IBS, and to evaluate the long-term effects of multispecies probiotic supplementation on symptoms, quality of life, intestinal microbiota and systemic inflammatory markers in IBS. The intestinal microbiota composition in IBS patients and healthy control subjects was analysed by quantitative polymerase chain reaction (qPCR). Significantly lower counts for the Clostridium coccoides and the Bifidobacterium catenulatum groups were found in IBS compared to controls. Quantitative differences also appeared in subgroup analysis based on the predominant bowel habit: diarrhoea patients harboured significantly lower numbers of Lactobacillus spp. than the constipation-predominant patients, while higher counts for Veillonella spp. were detected in constipation-predominant patients compared to healthy controls. Analysis of mucosal biopsies by a metabolomic approach revealed multiple differences between patients and controls. The most prominent finding was an upregulation of specific lipid species, principally lysophosphatidylcholines and ceramides, in IBS. The effects of multispecies probiotic supplementation with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii JS, and Bifidobacterium breve Bb99 or Bifidobacterium animalis subsp. lactis Bb12 was evaluated in two, randomised, double-blind, placebo-controlled trials. Compared to placebo, the probiotic supplementation significantly reduced the total symptoms of IBS. No effects on bowel habit were seen. Health-related quality of life (HRQOL) is reduced in patients with IBS in comparison with the Finnish population on the whole. The probiotic supplementation improved one IBS-specific domain of quality of life (bowel symptoms), whereas no other effects on HRQOL were seen. The probiotics had no major effects on the predominant microbiota as measured by qPCR, but a microarray-based analysis suggested that the probiotic consumption stabilised the microbiota. No effects on serum sensitive-CRP or cytokines were detected. In conclusion, alterations in the microbiota composition and in the mucosal metabolite profile are potential pathophysiological factors of IBS. Multispecies probiotic supplementation alleviates the gastrointestinal symptoms of IBS, and improves the bowel symptoms domain of HRQOL. Probiotic supplementation in IBS is associated with a stabilisation of microbiota, but it does not influence systemic inflammatory markers.