Positron Emission Tomography (PET) permits regional myocardial perfusion, fibrosis and oxidative metabolism to be non-invasively quantified with radioactive tracers such as [15O]-water and [1-11C]-acetate. PET is an established research tool in congestive heart failure (CHF), a major cause of morbidity and mortality. However, as CHF is a syndrome that eventually affects all aspects of cardiac and systemic hemodynamic function, more clinically relevant information from a single PET scan is desirable. The aim of this thesis therefore was to develop and implement some new concepts in cardiac PET. A new method for the measurement of cardiac output with any tracer was validated in animal experiments and CHF patients. The early pulmonary retention of [1-11C]-acetate was inversely related to left ventricular (LV) function in animals and was directly proportional to lung water content and severity of LV diastolic dysfunction in patients. Eight patients with acute myocardial infarction were followed with serial PET from 3 hours to 3 weeks after trombolytic treatment. PET revealed that myocardial perfusion and the extraction and utilization of fuel substrates all decreased closer to the infarct centre. The rate of oxygen utilization within the infarct at 3 h predicted degree of myocardial fibrosis, pulmonary oedema and tissue viability at 3 weeks. Seventeen patients with CHF due to chronic ischemic cardiomyopathy and severely reduced LV function were evaluated with [1-11C]-acetate PET before and after coronary artery bypass surgery. There was a dramatic improvement in physical performance and symptoms, which was not correlated to the standard LV ejection indices. PET revealed that functional improvement was associated with improved LV loading conditions, reversed remodeling and homogenization of oxidative metabolism rather than increased output.

Reverse transcriptase (RT) is a crucial enzyme for retrovirus replication, and its presence in the virion is indispensable for infectivity. This thesis illustrates the use of RT activity assays as tools for quantitation and characterization of different retroviruses, particularly HIV. A non radioactive assay, using microtiter plates, for the RT of Moloney murine leukemia virus (MMuLV) was developed. Assay conditions for MMuLV and HIV-1 RT, together with isozyme specific RT activity blocking antibodies, were shown useful for discrimination between RTs from different retrovirus genera. RT activity assay for HIV-1 was found to quantitate different subtypes more equally efficient than p24 antigen assays did. Viral load (VL), the amount of HIV particles in the blood, is an important marker of the clinical status of an infected person. A method for VL determination based on RT activity (ExaVir Load) was developed. After plasma pretreatment, to inactivate cellular DNA polymerases, virions in patient plasma were immobilized on a gel, which was washed to remove disturbing factors. The virions were lysed with a detergent containing buffer and the lysate eluted. Finally, the RT activity in the lysate was determined and found to correlate strongly to VL by RNA according to a PCR based standard method (Roche Amplicor 1.5). The second version of the method was able to measure VL down to approximately 400 HIV-1 RNA copies/ml. The usefulness of RT from the VL procedure for determination of susceptibility towards anti-HIV drugs was demonstrated, and the results were in agreement with genotypic data. Due to its technical simplicity, and ability to detect a broad range of HIV-1 subtypes, ExaVir Load and the drug susceptibility application are interesting for clinical use, particularly but not only in resource limited settings. The concept is also potentially useful for research purposes, e.g. in combination with specific RT assay conditions.

Neurofibromatosis type-2 (NF2) is an autosomal dominant disorder with the clinical hallmark of bilateral eighth cranial nerve schwannomas. However, the diagnostic criterion is complicated by the presence of a variable phenotype, with the severe form presenting with additional tumors such as peripheral schwannoma, meningioma and ependymoma. We constructed a microarray spanning 11Mb of 22q, encompassing the NF2 gene, to detect deletions in schwannoma. Forty seven patients were analyzed and heterozygous deletions were detected in 45% of tumors. Using this array-based approach, we also detected genetic heterogeneity in a number of samples studied. Despite the high sensitivity and the comprehensive series of studied schwannomas, no homozygous deletions affecting the NF2 gene were detected <b>(paper I)</b>. In order to detect more subtle deletions within the NF2 locus, a higher-resolution gene-specific array was developed, for the detection of disease-causing<b> </b>deletions using a PCR-based non-redundant strategy. This novel approach for array construction significantly increased the reliability and resolution of deletion-detection within the NF2 locus <b>(paper II)</b>. To further expand the coverage of the 11 Mb microarray, we constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number. This 22q array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb <b>(paper III)</b>. Using this array, we analyzed sporadic and familial schwannomatosis samples, which revealed two commonly deleted regions within the immunoglobulin lambda locus and the GSTT1/CABIN1 locus. These regions were further characterized using higher-resolution non-redundant arrays, bioinformatic tools, positional cloning and mutational screening. Missense mutations were detected in the CABIN1 gene, which may contribute to the pathogenesis of schwannomatosis and therefore requires further study <b>(paper IV)</b>. Meningioma is the second most common NF2-associated tumor and loss of 1p has been previously established as a major genetic factor for disease initiation/progression and also correlates with increased morbidity. We analyzed 82 meningiomas using a chromosome 1 tiling-path genomic microarray. The distribution of aberrations detected supports the existence of at least four regions on chromosome 1, which are important for meningioma tumorigenesis <b>(paper V)</b>.

Microarray-based comparative genomic hybridization (array-CGH) has emerged as a versatile platform with a wide range of applications in molecular genetics. This thesis focuses on the development of array-CGH with a specific aim to approach disease-related questions through improved strategies in array construction and enhanced resolution of analysis. In <b>paper I</b>, we applied an array covering 11 Mb of 22q, encompassing the NF2 locus, for deletion detection in sporadic schwannoma. Hemizygous deletions and tumor heterogeneity were identified. Array-CGH was established as a reliable platform for detection of DNA dosage alterations. <b>Paper II</b> described the construction of the NF2 gene-specific microarray for high-resolution scanning of deletions in the NF2 locus. We report a novel PCR-based non-redundant strategy for microarray fabrication, which considerably improved the sensitivity and reliability of deletion detection. <b>Paper III</b> reported the first tiling-path array comprehensively covering a human chromosome. The usefulness of the 22q-array was demonstrated by applying it to detect DNA dosage-alterations in 22q-associated disorders. In <b>paper IV</b>, we optimized array-CGH protocols for deletion detection in 22q11 deletion-syndrome. We showed that genomic and cDNA clones are not optimal for analysis of 22q11 locus and that PCR-based non-redundant strategy is reliable for deletion detection in such regions. In <b>paper V</b>, we utilized the 22q-array for understanding the genetic basis of schwannomatosis. Two commonly deleted regions were identified within the IGL and the GSTT1/CABIN1 loci. Further investigations using high-resolution arrays, bioinformatic analysis and mutational screening were performed. Missense mutations, specific to the schwannomatosis- and NF2 samples, were identified in the CABIN1 gene. <b>Paper VI</b> described the first array-CGH study for comprehensive and high-resolution profiling of deletions spanning the 17q11 locus. Both typical and atypical deletions were identified in NF1 samples. Bioinformatic analysis revealed novel segmental duplications, which can potentially mediate 17q11 deletions.

Intra-articular glucocorticoid injection therapy is frequently used to relieve symptoms of arthritis, but there is considerable variation in injection routines among physicians. One issue of debate concerns the importance of synovial fluid aspiration during the injection procedure. In the present randomised controlled study of patients with rheumatoid arthritis (RA), a significantly reduced risk for arthritis relapse was observed when arthrocentesis was included in the intra-articular injection procedure of the knee. Furthermore, there is no consensus about the post-injection regimes. Previous studies have shown beneficial effects of post-injection rest of the knee, but also injection routines for other joints often include such recommendations. The present randomised controlled trial showed that 48-hour rest in elastic orthosis after intra-articular injection in the wrist did not improve the outcome. Thus, the effect of post-injection rest varies between different joints. The improved treatment result of post-injection rest of the knee is supposed to be caused by retarded steroid resorption from the joint. In order examine the metabolic effects in cartilage, bone and the hypothalamic-piuitary-adrenal (HPA)-axis, resting and mobile RA patients were studied after intra-articular knee injections. Serum levels of the injected glucocorticoid, triamcinolone hexacetonide (THA), were analysed, as well as cartilage oligomeric matrix protein (COMP) as a marker of cartilage turnover, osteocalcin for bone formation and deoxypyridinoline for bone resorption. The HPA-axis was assessed using serum levels of cortisol and adrenocorticotropine hormone. The result showed a short term and reversible suppression of the HPA-axis and bone formation, whereas bone resorption was unaffected. No differences between mobile and resting patients were observed. In both groups reduction of COMP levels were seen, but these were significantly more pronounced in resting patients, suggesting a cartilage-protective effect. The THA levels increased similarly in both groups, indicating that rest did not affect glucocorticoid resorption. Consequently, another explanation for the beneficial effects of postinjection rest of knee synovitis should be considered. In the present material the incidence of infectious complications of intra-articular treatment was less than 1/12,000 injections. The findings in this thesis can be applied in the clinical practice and should be considered when new guidelines for intra-articular glucocorticoid therapy are created.

Nitric oxide (NO) is expressed in inflammatory tissues. However, NO effects are controversial in inflammation; NO is described as acting in a dose dependent manner and possess both pro-inflammatory and anti-inflammatory properties. The present thesis explored the role of NO in relation to white blood cell (WBC) and protein system activation by foreign surfaces in simulated extracorporeal circulation (SECC) using human whole blood from volunteer donors. Three doses of NO, 40 ppm, 80 ppm and 500 ppm, were administered and an array of markers of WBC and protein activation were studied. Neutrophil degranulation was detected with myeloperoxidase (MPO), human neutrophil lipocalin (HNL) and lactoferrin (LF); eosinophil degranulation with eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO); and basophil degranulation with histamine. Furthermore, whole blood and WBC capacity to produce reactive oxygen species (ROS) were studied and cytokine release was measured with IL-1 and IL-10. Complement activation was measured with C3a and C5b-9 complex and contact system activation with FXIIa-C1INH, FXIIa-AT, FXIa-C1INH and FXIa-AT. NO increased neutrophil degranulation at all dose levels and 80 ppm NO increased basophil degranulation; whereas, NO exerted no effect on eosinophil degranulation, WBC subset counts, cytokine release or capacity to produce ROS. In addition, while increasing both specific and azurophil degranulation with 40 ppm, 80 ppm and 500 ppm, NO reversed the classical degranulation hierarchy with 500 ppm and azurophil degranulation became predominant. Furthermore, NO effect was greater with 500 ppm than with 80 ppm, indicating a dose response effect. The lack of iNOS mRNA expression in WBC and lack of L-NAME effect on degranulation and nitrite/nitrate production, together with absent increase in nitrite/nitrate in controls, excluded autocrine or paracrine regulation of degranulation. FXIIa-AT and FXIa-AT complexes increased and became predominant during early recirculation, whereas FXIIa-C1INH and FXIa-C1INH complexes were predominant at baseline but remained unaltered, suggesting contact system inhibition predominantly via AT. C3a and C5b-C9 increased. NO had no effect on either contact or complement system activation; however, 500 ppm NO shortened active clotting time. In conclusion, the present data suggest that NO has a direct effect on neutrophil and basophil degranulation. Recognition of NO as an enhancer of degranulation may give access to new therapeutic tools for local and systemic inflammatory therapies; whereas, the identification of increased AT mediated inhibition of FXIIa and unchanged C1INH complexes presents new possibilities for therapeutic intervention in conditions such as hereditary angioedema and heart surgery.

In patients with unstable coronary artery disease (CAD) biochemical markers are emerging as useful tools in clinical management. In this thesis we studied the use of markers of low-density lipoprotein (LDL) oxidation and renal function. Our study populations consisted of unstable CAD patients included in the Fast Revascularisation during Instability in Coronary artery disease (FRISC)-II trial and healthy controls. Patients were followed for 2 years regarding death and myocardial infarction (MI). Using receiver operating characteristic curve analysis, we found that oxidized low-density lipoprotein (OxLDL), especially when combined with high-density lipoprotein, compared to traditionally measured lipids/lipoproteins, and a new lipoprotein marker, lipoprotein associated-phospholipase A2, was better at discriminating between healthy controls and CAD patients. In patients, OxLDL was found to be an independent prognostic marker associated with an increased risk of MI, of particular use in patients with no evidence of myocardial necrosis. In our study on the effects of an early invasive treatment strategy in unstable CAD patients with mild to moderate renal dysfunction (i.e. creatinine clearance &lt;90mL/min) we found that in patients randomized to invasive treatment, the rates of death/MI and MI alone were significantly lower than in patients randomized to non-invasive treatment. In patients treated invasively, no detrimental effects were seen on renal function at follow-up at 6 months. In healthy controls, we investigated new markers of renal (cystatin C) and cardio-renal function (N-terminal probrain natriuretic peptide, [NT-proBNP]) regarding reference levels and physiological determinants. We found that cystatin C is influenced by age whereas NT-proBNP is influenced by age and gender. Our studies suggest that OxLDL and renal dysfunction are associated with a poor prognosis in unstable CAD patients and that these markers demonstrate potential for clinical use. In the search for new markers related to renal function we have contributed with reference levels of cystatin C and NT-proBNP.

This thesis focuses on the involvement of apoptosis and proliferation in the mechanisms of menstruation and hormonal replacement therapy, HRT, as well as in the mechanisms of progesterone therapy in endometrial carcinoma. The aim of the first study was to investigate endometrium for 4 days before and for 2 days during menstruation. In the epithelium, rapid increase in the apoptotic index, decreasing expression of estrogen receptor α (ER) and progesterone receptor (PR), and minimal proliferation were observed prior to menstruation. In the stroma, an increase in the expression of ER and PR and proliferation was seen before the final decrease, and increased apoptosis was seen during menstruation. Thus, apoptosis is involved in the remodeling of the endometrium during menstruation. Postmenopausal endometrium showed unaffected homeostasis, i.e. unchanged ratio between apoptotic index and Ki-67 index during substitution therapy. ER expression was decreased both in the epithelium and stroma, while PR showed some increase in receptor expression. The unchanged homeostasis contributes to endometrial safety during combined continuous HRT. Unchanged apoptosis and increasing proliferation were observed with increasing tumor grade in 29 patients with endometrioid endometrial carcinoma, which may contribute to greater aggression as tumor grade increases. Decreased proliferation was observed after medroxy-progesterone at 20 mg per day particularly in the foci of maximal proliferation in G1 and G2 tumors. The expression of ER was unchanged, while PR was decreased in the foci of maximal expression for PR in G1 and G2 tumors. Since high proliferation and PR expression also coexisted in the same foci, evaluated in G1 and G2 tumors, the effect of progesterone could be facilitated in these tumor groups. High expression of sex steroid receptors was also a predicting factor for good response to progesterone (= decrease in proliferation), while the amount of stroma could not predict that effect.

To diagnose rheumatoid arthritis (RA) during the early stages of the disease is often difficult. The disease course shows great inter-individual variation from mild, self-limiting to very severe destruc-tive disease with extra-articular manifestations. Early aggressive treatment with potentially toxic drugs has been shown to improve the long-term outcome. Thus, it is desirable to make an early reliable di-agnosis and to identify those patients who would benefit from being treated most aggressively. The aim of this thesis was to evaluate laboratory and clinically markers of inflammation as predic-tors of disease course, to compare dual-energy X-ray absorptiometry (DXA) and conventional radiog-raphy (CR) as measures of joint destruction and to investigate the significance of antibodies against cyclic citrullinated peptide (anti-CCP antibodies), rheumatoid factors (RFs) and HLA shared epitope (SE) alleles for the relative risk of future development of RA and as predictors of disease severity in patients with early RA. Patients with RA of recent onset are included in the early RA programme at the Department of Rheumatology, University Hospital, Umeå and are followed longitudinally. The prediction of markers of inflammation for bone loss and radiological outcome was analyzed in the first 43 patients recruited. Radiographs of hands and feet (Larsen score) and bone mineral density (BMD) in hands (DXA), were assessed at baseline, after 1 and 2 years. The disease activity was evaluated clinically and by labora-tory tests. Radiological damage increased significantly during the study and was particularly corre-lated with Larsen score at baseline. BMD in hands decreased significantly in postmenopausal women and the decrease was greater than in healthy matched controls. Radiological progression and bone loss in hands was retarded by an early response to therapy. In a case-control study within the Medical Biobank and the Maternity cohort of Northern Sweden, patients from the early RA programme were identified among blood donors from whom samples had been collected years before onset of symptoms. The prevalence of anti-CCP antibodies and RFs (IgA-RF, IgG-RF and IgM-RF) was investigated in samples from 83 individuals (pre-patients) and com-pared with matched controls. SE alleles were assessed in a sub-group of 59 individuals. Anti-CCP antibodies and RFs preceded onset of RA by several years and increased in prevalence closer to dis-ease onset. Anti-CCP antibodies and IgA-RF significantly predicted the onset of RA. The combination of anti-CCP antibodies and SE alleles was associated with a high relative risk for future development of RA. In a later co-analysis between the register of patients in the early RA programme (n=138) and the Medical Biobank and the Maternity cohort, 93 pre-patient samples were identified. The significance of SE alleles and of the presence of anti-CCP antibodies and RFs before and at disease onset for disease activity and severity was studied. Radiographs of hands and feet were assessed at baseline and after 2 years (Larsen score). The presence of anti-CCP antibodies in pre-patient samples and at baseline was associated with radiological damage, as was presence of all RFs at baseline. A higher titre of anti-CCP antibodies was associated with greater radiological progression. The titre was lowered by a therapeutic response. In multiple logistic regression analyses anti-CCP antibodies, IgA-RF, ESR and swollen joint count predicted greater radiological progression, whilst a therapeutic response predicted a lesser pro-gression. In conclusion, anti-CCP antibodies and IgA-RF are predictors for future onset of RA and for radio-logical destruction and progression. The combination of anti-CCP antibodies and SE alleles is associ-ated with a high relative risk for future RA. Therapeutic response decreases the radiological progres-sion and the bone loss in hands and lowers the titre of anti-CCP antibodies. Conventional radiography is a better measure of joint destruction than DXA.

Lynch syndrome (Hereditary Nonpolyposis Colorectal Cancer, HNPCC) is the most common hereditary syndrome predisposing to colorectal cancer, accounting for 1-3% of all colorectal cancer. This multi-organ cancer predisposition syndrome is caused by mutations in the mismatch repair (MMR) genes, especially MLH1 and MSH2, and to lesser extents MSH6 and PMS2, which lead to widespread genetic instability and thus microsatellite instability (MSI). Hereditary cancer often manifests in two or more tumours in a single individual; 35-40% of Lynch syndrome patients have synchronous or metachronous tumours of the two major Lynch syndrome-related cancers: colorectal and endometrial. The main purposes of the work underlying this thesis were to identify persons at risk of Lynch syndrome or other types of hereditary colorectal cancer, to estimate the cancer risks associated with these predispositions and to identify the underlying genetic causes. A population-based cohort of 78 persons with double primary colorectal or colorectal and endometrial cancer was identified. Cancer risks in their 649 first-degree relatives were estimated in relation to tumour MSI status (positive or negative) and age at diagnosis (before or after 50 years of age) in the probands. The overall standardised incidence ratio was 1.69 (95% CI; 1.39-2.03). The highest risks for Lynch syndrome-associated cancers: (colorectal, endometrial, ovarian and gastric) were found in families with young MSI-positive probands, likely representing Lynch syndrome families. Importantly, no overall risk was found in families with old probands, irrespective of MSI status. Blood samples were available from 24 MSI-positive patients for mutation screening of MLH1, MSH2 and MSH6. Sequence variants or rearrangements predicted to affect protein function were found in 16 patients. Six novel variants were found: two large rearrangements, two truncating and two missense mutations. The missense mutations were found to segregate in the families. Studies of allele frequencies, MSI and loss of immunostaning in tumours from family members further supports the hypothesis that these missense changes play a role in Lynch syndrome, as do the non-conservative nature and evolutionary conservation of the amino acid exchanges. Five families had mutations in MLH1, five in MSH2, and six in MSH6. The unexpectedly large impact of MSH6 was in genealogical studies shown to be due to a founder effect. Cumulative risk studies showed that the MSH6 families, despite their late age of onset, have a high lifetime risk for all Lynch syndrome-related cancers, significantly higher in women (89% by age 80 years) than in men (69%). The gender differences are in part due to high endometrial (70%) and ovarian cancer risk (33%) in addition to the high colorectal cancer risk (60%). These findings are of great importance for counselling and surveillance of families with MSH6 mutations. Finally, in a large family with MSI-negative hereditary colorectal cancer for which the MMR genes and APC had been excluded as possible causes, a genome-wide linkage analysis was performed, resulting in a suggested linkage to chromosome 7. Conclusions: Relatives of probands with MSI-positive, double primary colorectal and endometrial cancer diagnosed before the age of 50 years have significantly increased risks of Lynch syndrome-related cancers. MSH6 mutations, which have unusually high impact in this study population due to a founder effect, confer high cumulative risks of cancer despite the generally late age of onset.