Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Microarrays in Lung Cancer Research: From Comparative Analyses to Verified Findings

Keuhkosyöpä on yleisimpiä syöpätauteja. Se jaetaan kahteen päätyyppiin: pienisoluiseen ja ei-pienisoluiseen keuhkosyöpään. Ei-pienisoluinen keuhkosyöpä jaetaan lisäksi alatyyppeihin, joista suurimmat ovat levyepiteeli-, adeno- ja suurisoluinen karsinooma. Keuhkosyövän tärkein riskitekijä on tupakointi, mutta muutkin työ- ja elinympäristön altisteet, kuten asbesti, voivat johtaa syöpään. Väitöstyössä tutkittiin kahdenlaisten keuhkosyöpäryhmien erityispiirteitä. Työssä kartoitettiin, onko löydettävissä muutoksia, jotka erottavat asbestikeuhkosyövät muista syövistä sekä luuytimeen varhaisessa vaiheessa leviävät keuhkosyövät leviämättömistä syövistä. Tutkimusten ensimmäisessä vaiheessa käytettiin mikrosirupohjaisia menetelmiä, jotka mahdollistavat jopa kaikkien geenien tarkastelun yhden kokeen avulla. Vertailevien mikrosirututkimusten avulla on mahdollista paikantaa geenejä tai kromosomialueita, joiden muutokset erottelevat ryhmät toisistaan. Asbestiin liittyvissä tutkimuksissa paikannettiin kuusi kromosomialuetta, joissa geenien kopiolukumäärän sekä ilmenemistason muutokset erottelivat potilaat altistushistorian mukaan. Riippumattomilla laboratoriomenetelmillä tehtyjen jatkoanalyysien avulla pystyttiin varmistamaan, että 19p-alueen häviämä oli yhteydessä asbestialtistukseen. Työssä osoitettiin myös, että 19p-alueen muutoksia voidaan indusoida altistamalla soluja asbestille in vitro. Tutkimuksessa saatiin lisäksi viitteitä asbestispesifisistä muutoksista signaalinvälitysreiteissä, sillä yhdessä toimivien geenien ilmentymisessä havaittiin eroja asbestille altistuneiden ja altistumattomien välillä. Vertailemalla luuytimeen syövän aikaisessa vaiheessa levinneiden ja leviämättömien keuhkoadenokarsinoomien muutosprofiileita toisiinsa, paikannettiin viisi aluetta, joilla geenien kopiolukumäärä- sekä ilmenemistason muutokset erottelivat ryhmät toisistaan. Jatkoanalyyseissä havaittiin, että 4q-alueen häviämää esiintyi adenokarsinoomien lisäksi levyepiteelikarsinoomiin, jotka olivat levinneet luuytimeen. Myös keuhkosyöpien aivometastaaseissa alue oli toistuvasti hävinnyt. Väitöstyön tutkimukset osoittavat, että vertailevien mikrosiruanalyysien avulla saadaan tietoa syöpäryhmien erityispiirteistä. Työssä saadut tulokset osoittavat, että 19p-alueen muutokset ovat tyypillisiä asbestikeuhkosyöville ja 4q-alueen muutokset luuytimeen aikaisessa vaiheessa leviäville keuhkosyöville.

The mast cell as a regulator of atherosclerotic plaque stability

Atherosclerosis is an inflammatory disease progressing over years via the accumulation of cholesterol in arterial intima with subsequent formation of atherosclerotic plaques. The stability of a plaque is determined by the size of its cholesterol-rich necrotic lipid core and the thickness of the fibrous cap covering it. The strength and thickness of the cap are maintained by smooth muscle cells and the extracellular matrix produced by them. A plaque with a large lipid core and a thin cap is vulnerable to rupture that may lead to acute atherothrombotic events, such as myocardial infarction and stroke. In addition, endothelial erosion, possibly induced by apoptosis of endothelial cells, may lead to such clinical events. One of the major causes of plaque destabilization is inflammation induced by accumulated and modified lipoproteins, and exacerbated by local aberrant shear stress conditions. Macrophages, T-lymphocytes and mast cells infiltrate particularly into the plaque’s shoulder regions prone to atherothrombotic events, and they are present at the actual sites of plaque rupture and erosion. Two major mechanisms of plaque destabilization induced by inflammation are extracellular matrix remodeling and apoptosis. Mast cells are bone marrow-derived inflammatory cells that as progenitors upon chemotactic stimuli infiltrate the target tissues, such as the arterial wall, differentiate in the target tissues and mediate their effects via the release of various mediators, typically in a process called degranulation. The released preformed mast cell granules contain proteases such as tryptase, chymase and cathepsin G bound to heparin and chondroitin sulfate proteoglycans. In addition, various soluble mediators such as histamine and TNF-alpha are released. Mast cells also synthesize many mediators such as cytokines and lipid mediators upon activation. Mast cells are capable of increasing the level of LDL cholesterol in the arterial intima by increasing accumulation and retention of LDL and by decreasing removal of cholesterol by HDL in vitro. In addition, by secreting proinflammatory mediators and proteases, mast cells may induce plaque destabilization by inducing apoptosis of smooth muscle and endothelial cells. Also in vivo data from apoE-/- and ldlr-/- mice suggest a role for mast cells in the progression of atherosclerosis. Furthermore, mast cell-deficient mice have become powerful tools to study the effects of mast cells in vivo. In this study, evidence suggesting a role for mast cells in the regulation of plaque stability is presented. In a mouse model genetically susceptible to atherosclerosis, mast cell deficiency (ldlr-/-/KitW-sh/W-sh mice) was associated with a less atherogenic lipid profile, a decreased level of lipid accumulation in the aortic arterial wall and a decreased level of vascular inflammation as compared to mast-cell competent littermates. In vitro, mast cell chymase-induced smooth muscle cell apoptosis was mediated by inhibition of NF-kappaB activity, followed by downregulation of bcl-2, release of cytochrome c, and activation of caspase-8, -9 and -3. Mast cell-induced endothelial cell apoptosis was mediated by chymase and TNF-alpha, and involved chymase-mediated degradation of fibronectin and vitronectin, and inactivation of FAK- and Akt-mediated survival signaling. Subsequently, mast cells induced inhibition of NF-kappaB activity and activation of caspase-8 and -9. In addition, possible mast cell protease-mediated mechanisms of endothelial erosion may include degradation of fibronectin and VE-cadherin. Thus, the present results suggest a role for mast cells in destabilization of atherosclerotic plaques.

Essential Thrombocythaemia : Diagnosis, Prognostic Aspects, and the Outcome of Finnish patients

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) characterized by thrombocytosis, i.e. a constant elevation of platelet count. Thrombocytosis may appear in MPDs (ET, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis) and as a reactive phenomenon. The differential diagnosis of thrombocytosis is important, because the clinical course, need of therapy, and prognosis are different in patients with MPDs and in those with reactive thrombocytosis. ET patients may remain asymptomatic for years, but serious thrombohaemorrhagic and pregnancy-related complications may occur. The complications are difficult to predict. The aims of the present study were to evaluate the diagnostic findings, clinical course, and prognostic factors of ET. The present retrospective study consists of 170 ET patients. Two thirds had a platelet count < 1000 x 109/l. The diagnosis was supported by an increased number of megakaryocytes with an abnormal morphology in a bone marrow aspirate, aggregation defects in platelet function studies, and the presence of spontaneous erythroid and/or megakaryocytic colony formation in in vitro cultures of haematopoietic progenitors. About 70 % of the patients had spontaneous colony formation, while about 30 % had a normal growth pattern. Only a fifth of the patients remained asymptomatic. Half had a major thrombohaemorrhagic complication. The proportion of the patients suffering from thrombosis was as high as 45 %. About a fifth had major bleedings. Half of the patients had microvascular symptoms. Age over 60 years increased the risk of major bleedings, but the occurrence of thrombotic complications was similar in all age groups. Male gender, smoking in female patients, the presence of any spontaneous colony formation, and the presence of spontaneous megakaryocytic colony formation in younger patients were identified as risk factors for thrombosis. Pregnant ET patients had an increased risk of complications. Forty-five per cent of the pregnancies were complicated and 38 % of them ended in stillbirth. Treatment with acetylsalicylic acid alone or in combination with platelet lowering drugs improved the outcome of the pregnancy. The present findings about risk factors in ET as well as treatment outcome in the pregnancies of ET patients should be taken into account when planning treatment strategies for Finnish patients.

The Pathobiology of the Saccular Cerebral Artery Aneurysm Rupture and Repair : A Clinicopathological and Experimental Approach

Backround and Purpose The often fatal (in 50-35%) subarachnoid hemorrhage (SAH) caused by saccular cerebral artery aneurysm (SCAA) rupture affects mainly the working aged population. The incidence of SAH is 10-11 / 100 000 in Western countries and twice as high in Finland and Japan. The estimated prevalence of SCAAs is around 2%. Many of those never rupture. Currently there are, however, no diagnostic methods to identify rupture-prone SCAAs from quiescent, (dormant) ones. Finding diagnostic markers for rupture-prone SCAAs is of primary importance since a SCAA rupture has such a sinister outcome, and all current treatment modalities are associated with morbidity and mortality. Also the therapies that prevent SCAA rupture need to be developed to as minimally invasive as possible. Although the clinical risk factors for SCAA rupture have been extensively studied and documented in large patient series, the cellular and molecular mechanisms how these risk factors lead to SCAA wall rupture remain incompletely known. Elucidation of the molecular and cellular pathobiology of the SCAA wall is needed in order to develop i) novel diagnostic tools that could identify rupture-prone SCAAs or patients at risk of SAH, and to ii) develop novel biological therapies that prevent SCAA wall rupture. Materials and Methods In this study, histological samples from unruptured and ruptured SCAAs and plasma samples from SCAA carriers were compared in order to identify structural changes, cell populations, growth factor receptors, or other molecular markers that would associate with SCAA wall rupture. In addition, experimental saccular aneurysm models and experimental models of mechanical vascular injury were used to study the cellular mechanisms of scar formation in the arterial wall, and the adaptation of the arterial wall to increased mechanical stress. Results and Interpretation Inflammation and degeneration of the SCAA wall, namely loss of mural cells and degradation of the wall matrix, were found to associate with rupture. Unruptured SCAA walls had structural resemblance with pads of myointimal hyperplasia or so called neointima that characterizes early atherosclerotic lesions, and is the repair and adaptation mechanism of the arterial wall after injury or increased mechanical stress. As in pads of myointimal hyperplasia elsewhere in the vasculature, oxidated LDL was found in the SCAA walls. Immunity against OxLDL was demonstrated in SAH patients with detection of circulating anti-oxidized LDL antibodies, which were significantly associated with the risk of rupture in patients with solitary SCAAs. Growth factor receptors associated with arterial wall remodeling and angiogenesis were more expressed in ruptured SCAA walls. In experimental saccular aneurysm models, capillary growth, arterial wall remodeling and neointima formation were found. The neointimal cells were shown to originate from the experimental aneurysm wall with minor contribution from the adjacent artery, and a negligible contribution of bone marrow-derived neointimal cells. Since loss of mural cells characterizes ruptured human SCAAs and likely impairs the adaptation and repair mechanism of ruptured or rupture-prone SCAAs, we investigated also the hypothesis that bone marrow-derived or circulating neointimal precursor cells could be used to enhance neointima formation and compensate the impaired repair capacity in ruptured SCAA walls. However, significant contribution of bone marrow cells or circulating mononuclear cells to neointima formation was not found.

Biological mechanisms behind clozapine-induced agranulocytosis

Clozapine is the most effective drug in treating therapy-resistant schizophrenia and may even be superior to all other antipsychotics. However, its use is limited by a high incidence (approximately 0.8%) of a severe hematological side effect, agranulocytosis. The exact molecular mechanism(s) of clozapine-induced agranulocytosis is still unknown. We investigated the mechanisms behind responsiveness to clozapine therapy and the risk of developing agranulocytosis by performing an HLA (human leukocyte antigens) association study in patients with schizophrenia. The first group comprised patients defined by responsiveness to first-generation antipsychotics (FGAs) (n= 19). The second group was defined by a lack of response to FGAs but responsiveness to clozapine (n=19). The third group of patients had a history of clozapine-induced granulocytopenia or agranulocytosis (n=26). Finnish healthy blood donors served as controls (n= 120). We found a significantly increased frequency of HLA-A1 among patients who were refractory to FGAs but responsive to clozapine. We also found that the frequency of HLA-A1 was low in patients with clozapine-induced neutropenia or agranulocytosis. These results suggest that HLA-A1 may predict a good therapeutic outcome and a low risk of agranulocytosis and therefore HLA typing may aid in the selection of patients for clozapine therapy. Furthermore, in a subgroup of schizophrenia, HLA-A1 may be in linkage disequilibrium with some vulnerability genes in the MHC (major histocompatibility complex) region on chromosome 6. These genes could be involved in antipsychotic drug response and clozapine-induced agranulocytosis. In addition, we investigated the effect of clozapine on gene expression in granulocytes by performing a microarray analysis on blood leukocytes of 8 schizophrenic patients who had started clozapine therapy for the first time. We identified an altered expression in 4 genes implicated in the maturation or apoptosis of granulocytes: MPO (myeloperoxidase precursor), MNDA (myeloid cell nuclear differentiation antigen), FLT3LG (Fms-related tyrosine kinase 3 ligand) and ITGAL (antigen CD11A, lymphocyte function-associated antigen 1). The altered expression of these genes following clozapine administration may suggest their involvement in clozapine-induced agranulocytosis. Finally, we investigated whether or not normal human bone marrow mesenchymal stromal cells (MSC) are sensitive to clozapine. We treated cultures of human MSCs and human skin fibroblasts with 10 µM of unmodified clozapine and with clozapine bioactivated by oxidation. We found that, independent of bioactivation, clozapine was cytotoxic to MSCs in primary culture, whereas clozapine at the same concentration stimulated the growth of human fibroblasts. This suggests that direct cytotoxicity to MSCs is one possible mechanism by which clozapine induces agranulocytosis.

Shwachman-Diamond syndrome : Clinical, genetic and radiological study

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder in which the cardinal symptoms arise from exocrine pancreatic insufficiency and bone marrow dysfunction. Previous studies have suggested increased risk of fatal complications among Finnish SDS infants. The genetic defect responsible for the disease was recently identified; the SBDS gene is located at chromosome 7q11 and encodes a protein that is involved in ribosome biosynthesis. The discovery of the SBDS gene has opened new insights into the pathogenesis of this multi-organ disease. This study aimed to assess phenotypic and genotypic features of Finnish patients with SDS. Seventeen Finnish patients with a clinical diagnosis of SDS were included in the study cohort. Extensive clinical, biochemical and imaging assessments were performed to elucidate the phenotypic features, and the findings were correlated with the SBDS genotype. Imaging studies included abdominal magnetic reso-nance imaging (MRI), brain MRI, cardiac echocardiography including tissue Doppler examination, and cardiac MRI. The skeletal phenotype was assessed by dual-energy X-ray absorptiometry and bone histomorphometry. Twelve patients had mutations in the SBDS gene. In MRI, a characteristic pattern of fat-replaced pancreas with occasional enhancement of scattered parenchymal foci and of pancreatic duct was noted in the SBDS mutation-positive patients while the mutation-negative patients did not have pancreatic fat accumulation. The patients with SBDS mutations had significantly reduced bone mineral density associated with low-energy peripheral fractures and vertebral compression fractures. Bone histomorphometry confirmed low-turnover osteoporosis. The patients with SBDS mutations had learning difficulties and smaller head size and brain volume than control subjects. Corpus callosum, cerebellar vermis, and pos-terior fossa structures were significantly smaller in SDS patients than in controls. Patients with SDS did not have evidence of clinical heart disease or myocardial fibrosis. However, subtle diastolic changes in the right ventricle and exercise-induced changes in the left ventricle contractile reserve were observed. This study expanded the phenotypic features of SDS to include primary low-turnover osteoporosis and structural alterations in the brain. Pancreatic MRI showed characteristic changes in the SBDS mutation-positive patients while these were absent in the mutation-negative patients, suggesting that MRI can be used to differentiate patients harbouring SBDS mutations from those without mutations. No evidence for clinical cardiac manifestations was found, but imaging studies revealed slightly altered myocardial function that may have clinical implications. These findings confirm the pleiotropic nature of SDS and underscore the importance of careful multidisciplinary follow-up of the affected individuals.

New approaches to improve the cord blood unit hematopoietic progenitor cell content

Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.

Affected sibling pairs with juvenile idiopathic arthritis : An immunogenetic study of the disease in multicase families

Genetic susceptibility to juvenile idiopathic arthritis (JIA) was studied in the genetically homogeneous Finnish population by collecting families with two or three patients affected by this disease from cases seen in the Rheumatism Foundation Hospital. The number of families ranged in different studies from 37 to 45 and the total number of patients with JIA, from among whom these cases were derived, was 2 000 to 2 300. Characteristics of the disease in affected siblings in Finland were compared with a population-based series and with a sibling series from the United States. A thorough clinical and ophthalmological examination was made of all affected patients belonging to sibpair series. Information on the occurrence of chronic rheumatic diseases in parents was collected by questionnaire and diagnoses were confirmed from hospital records. All patients, their parents and most of the healthy sibs were typed for human leukocyte antigen (HLA) alleles in loci A, C, B, DR and DQ. The HLA allele distribution of the cases was compared with corresponding data from Finnish bone marrow donors. The genetic component in JIA was found to be more significant than previously believed. A concordance rate of 25% for a disease with a population prevalence of 1 per 1000 implied a relative risk of 250 for a monozygotic (MZ) twin. An estimate for the sibling risk of an affected individual was about 15- to 20-fold. The disease was basically similar in familial and sporadic cases; the mean age at disease onset was however lower in familial cases, (4.8 years vs 7.4 years). Three sibpairs (3.4 expected) were concordant for the presence of asymptomatic uveitis. Uveitis would thus not appear to have any genetic component of its own, separate from the genetic basis of JIA. Four of the parents had JIA (0.2 cases expected), four had a type of rheumatoid factor-negative arthritis similar to that seen in juvenile patients but commencing in adulthood, and one had spondyloarthropathy (SPA). These findings provide additional support for the conception of a genetic predisposition to JIA and suggest the existence of a new disease entity, JIA of adult onset. Both the linkage analysis of the affected sibpairs and the association analysis of nuclear families provided overwhelming evidence of a major contribution of HLA to the genetic susceptibility to JIA. The association analysis in the Finnish population confirmed that the most significant associations prevailed for DRB1*0801, DQB1*0402, as expected from previous observations, and indicated the independent role of Cw*0401.

The role of phospholipid transfer protein and cholesteryl ester transfer protein in reverse cholesterol transport

In atherosclerosis, cholesterol accumulates in the vessel wall, mainly in the form of modified low-density lipoprotein (LDL). Macrophages of the vessel wall scavenge cholesterol, which leads to formation of lipid-laden foam cells. High plasma levels of high-density lipoprotein (HDL) protect against atherosclerosis, as HDL particles can remove peripheral cholesterol and transport it to the liver for excretion in a process called reverse cholesterol transport (RCT). Phospholipid transfer protein (PLTP) remodels HDL particles in the circulation, generating prebeta-HDL and large fused HDL particles. In addition, PLTP maintains plasma HDL levels by facilitating the transfer of post-lipolytic surface remnants of triglyceride-rich lipoproteins to HDL. Most of the cholesteryl ester transfer protein (CETP) in plasma is bound to HDL particles and CETP is also involved in the remodeling of HDL particles. CETP enhances the heteroexchange of cholesteryl esters in HDL particles for triglycerides in LDL and very low-density lipoprotein (VLDL). The aim of this thesis project was to study the importance of endogenous PLTP in the removal of cholesterol from macrophage foam cells by using macrophages derived from PLTP-deficient mice, determine the effect of macrophage-derived PLTP on the development of atherosclerosis by using bone marrow transplantation, and clarify the role of the two forms of PLTP, active and inactive, in the removal of cholesterol from the foam cells. In addition, the ability of CETP to protect HDL against the action of chymase was studied. Finally, cholesterol efflux potential of sera obtained from the study subjects was compared. The absence of PLTP in macrophages derived from PLTP-deficient mice decreased cholesterol efflux mediated by ATP-binding cassette transporter A1. The bone marrow transplantation studies showed that selective deficiency of PLTP in macrophages decreased the size of atherosclerotic lesions and caused major changes in serum lipoprotein levels. It was further demonstrated that the active form of PLTP can enhance cholesterol efflux from macrophage foam cells through generation of prebeta-HDL and large fused HDL particles enriched with apoE and phospholipids. Also CETP may enhance the RCT process, as association of CETP with reconstituted HDL particles prevented chymase-dependent proteolysis of these particles and preserved their cholesterol efflux potential. Finally, serum from high-HDL subjects promoted more efficient cholesterol efflux than did serum derived from low-HDL subjects which was most probably due to differences in the distribution of HDL subpopulations in low-HDL and high-HDL subjects. These studies described in this thesis contribute to the understanding of the PLTP/CETP-associated mechanisms underlying RCT.

Genomic alterations in Myeloproliferative Neoplasms and Myelodysplastic Syndromes

Myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic disorders whose etiology and molecular pathogenesis are poorly understood. During the past decade, enormous developments in microarray technology and bioinformatics methods have made it possible to mine novel molecular alterations in a large number of malignancies, including MPN and MDS, which has facilitated the detection of new prognostic, predictive and therapeutic biomarkers for disease stratification. By applying novel microarray techniques, we profiled copy number alterations and microRNA (miRNA) expression changes in bone marrow aspirate and blood samples. In addition, we set up and validated an miRNA expression test for bone marrow core biopsies in order to utilize the large archive material available in many laboratories. We also tested JAK2 mutation status and compare it with the in vitro growth pattern of hematologic progenitors cells. In the study focusing on 100 MPN cases, we detected a Janus kinase 2 (JAK2) mutation in 71 cases. We observed spontaneous erythroid colony growth in all mutation-positive cases in addition to nine mutation negative cases. Interestingly, seven JAK2V167F negative ET cases showed spontaneous megakaryocyte colony formation, one case of which also harbored a myeloproliferative leukemia virus oncogene (MPL) mutation. We studied copy number alterations in 35 MPN and 37 MDS cases by using oligonucleotide-based array comparative hybridization (array CGH). Only one essential thrombocythemia (ET) case presented copy number alterations in chromosomes 1q and 13q. In contrast, MDS cases were characterized by numerous novel cryptic chromosomal aberrations with the most common copy number losses at 5q21.3q33.1 and 7q22.1q33, while the most common copy number gain was trisomy 8. As for the study of the bone marrow core biopsy samples, we showed that even though these samples were embedded in paraffin and underwent decalcification, they were reliable sources of miRNA and suitable for array expression analysis. Further, when studying the miRNA expression profiles of the 19 MDS cases, we found that, compared to controls, two miRNAs (one human Epstein-Barr virus (miR-BART13) miRNA and one human (has-miR-671-5p) miRNA) were downregulated, whereas two other miRNAs (hsa-miR-720 and hsa-miR-21) were upregulated. However, we could find no correlation between copy number alterations and microRNA expression when integrating these two data. This thesis brings to light new information about genomic changes implicated in the development of MPN and MDS, and also underlines the power of applying genome-wide array screening techniques in neoplasias. Rapid advances in molecular techniques and the integration of different genomic data will enable the discovery of the biological contexts of many complex disorders, including myeloid neoplasias.

Basic and translational studies on species B adenoviruses

Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.