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 <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.

Background: It is well known that performance of the right ventricle (RV) determines exercise capacity and may confer prognostic information in different cardiopulmonary diseases. To allow optimal patient management, ideal methods to assess right heart function are therefore important. Echocardiography is an attractive investigation for that purpose, although limited by the anatomical and functional complexities of the RV. The aim of the present thesis was to present applicable methods useful in clinical practice by traditional 2D/Doppler echocardiography and Doppler tissue imaging (DTI) in the assessment of global and regional RV function in both health and disease. Methods: The studies were performed on 4 different groups; (1) 255 healthy subjects (125 females), (2) 92 consecutive patients with different cardiac diseases (36 females), (3) 26 patients with systemic sclerosis, (SSc) (21 females) and (4) 26 consecutive patients with heart failure (8 females) undergoing cardiac catheterisation. Results: RV outflow tract fractional shortening (RVOT fs), which is a new method in the assessment of RV function, correlated significantly with RV systolic long axis motion (r= 0.66, p< 0.001), pulmonary artery acceleration time (r= 0.80, p< 0.001) and RV-right atrial peak systolic pressure drop gradient (r= -0.53, p< 0.001). Furthermore, RVOT fs was reduced in patients with pulmonary hypertension whereas RV systolic long axis motion was not in difference. This finding was confirmed after comparing RV function with invasive pressures. In healthy subjects, while the systolic myocardial velocities were preserved over age, the peak isovolumic contraction velocity (IVCv) was weakly increased with advanced age (r= 0.34, p< 0.01). Furthermore, both global and regional E/A ratios were reduced (r= -0.57, r= -0.67, p< 0.001 for both) with age whereas no alteration was found in the myocardial isovolumic relaxation time (IVRt). In patients with systemic sclerosis (SSc) both global (64± 23 vs. 39± 12 ms, p< 0.001) as well as regional (83± 40 vs. 46± 24 ms, p< 0.001) IVRt were prolonged. After evaluating echocardiographic parameters with invasive pressures we found a significant correlation between DTI derived IVRt and pulmonary artery systolic pressures (r= 0.83, p< 0.01) while the IVCv was related to the state of contractility (r= 0.77, p< 0.001). Furthermore, an IVCv below 6 cm/s was shown to be an accurate marker of increased right atrial pressure (>6 mm Hg). In conclusion, RVOT fs can be used as a complementary measurement of RV systolic function, being more sensitive to elevated pulmonary artery systolic pressures than the systolic longitudinal RV motion. Right heart function, mainly the diastolic function, is relatively weakly influenced by age compared to the left heart function. In patients with SSc, we found diastolic disturbances, including a prolonged IVRt and proposed the findings to be early markers related to intermittent pulmonary hypertension. This observation was strengthened after evaluating IVRt against invasive pulmonary artery systolic pressures. IVCv can be used to determine the state of RV contractility and also be used to identify patients with elevated filling pressures. The presented methods can be used to detect early signs of RV dysfunction which might prohibit right heart failure. All presented methods are non-invasive, reproducible, easy obtainable, and thus useful in clinical practice.

Cobalamin (vitamin B12) is one of several essential micronutrients needed by the human organism. Other important micronutritients, which interplay with vitamin B12, are folate and iron. During the last ten years, the attention has been drawn to different forms of neurological disorders supposed to be caused by vitamin B12 deficiency. Vitamin B12 deficiency states are common among elderly patients in primary health care and sometimes in hospital care, especially in geriatric practice. This is a study to define the cobalamin treatment traditions, among Swedish physicians in the period 1990 – 2000. The period was distinguished by an intense debate on the issue by the physicians, an increase of cobalamin consumption, and a shift from parenteral therapy towards oral high-dose therapy. It had been known that symptoms of cobalamin deficiency could start in the nervous system. This knowledge was reinforced by the application of homocysteine and methyl-malonic acid (MMA) in deficiency diagnosis. Introduction of homocysteine and MMA in deficiency diagnosis changed the view on deficiency prevalence, by identifying persons at risk to develop B12 deficiency prior to established symptoms. In this study, Swedish physicians are regarded mainly as receivers of communication about the markers homocysteine and MMA, and deficiency states of cobalamin and folate. The main senders were scientists from North America, Norway, Denmark, and Sweden. This study sets the senders and the receivers of cobalamin communication on a collegial level and quantifies and evaluates the feed-back from the receivers. The receivers, gen¬eral practitioners and geriatricians, appeared to be familiar with old knowledge and frontier concepts in the field. Thus, it is suggested that the increase of B12 prescriptions in Sweden 1990 – 2000 reflected an increased awareness of B12-associated clinical problems among the physicians managing the majority of deficiency patients, although a possible overconsumption of pharmaceutical drugs must be kept in mind.

Gene expression differences between patients and controls can be used to find susceptibility genes and drug targets for a disease. High-resolution strategies are required because the differences between the investigated groups may be small and numerous factors may affect the mRNA quantity. This thesis is based on the use of real-time RT-PCR combined with a new statistical approach, developed to detect small differences between patients and controls and differences due to patient subgroups. Comparisons between human brain biopsy and autopsy samples showed that post-mortem tissue can be used to make conclusions on the relative mRNA levels in the living brain. Power analysis based on human brain mRNA expression from 14 genes adjusted with two reference genes, revealed that a sample size of 50 patients and 50 controls was required to detect a 2-fold difference with a power and a confidence of 95%. A similar study in rats revealed that approximately the same sample size was required for rat brain mRNA expression studies. The mRNA levels of several genes were studied in 55 schizophrenia and 55 control prefrontal brain autopsies, using a novel and more powerful statistical analysis. The serotonin receptor 2C gene (HTR2C) showed a significant 1.5-fold decrease in the patients as compared to controls, and the monoamine oxidase B gene (MAOB) a 1.2-fold increase. The mechanism behind the decrease of HTR2C mRNA levels was investigated by studying the correlation of drug treatment and HTR2C promoter polymorphisms to the HTR2C expression levels. The observed decrease was present in untreated patients, suggesting that the HTR2C mRNA decrease is correlated with the disease and not the treatment. There was no association between promoter polymorphisms and HTR2C expression levels. Thus, the molecular mechanism for the decreased expression remains unclear. Nevertheless, the results support a role for monoaminergic synapses in schizophrenia.

Musculoskeletal disorders are common among professional driver groups. Ergonomic risk factors at work are often suggested as causative, aggravating or preserving. The general aim with this thesis is to investigate the association between musculoskeletal disorders and physical exposure with special with special focus on whole-body vibration (WBV), among professional drivers of all-terrain vehicles (ATVs). Drivers of ATVs are expsosed to high magnitudes of WBV and shock. This thesis included drivers of forest machines, snowgroomers and snowmobiles. A cross-sectional study revealed that ATV drivers had an increased risk of musculoskeletal symptoms in the neck-shoulder and thoracic regions, even after adjusting for age, smoking habits and psychosocial stress. Prevalence rates were in the range of 1.5-2.9 (CI:1.2-5.2) compared to an age-matched group from the general population. No group of ATV drivers had a significantly increased risk of low back pain. Trend analysis showed no association between symptoms and exposure time. A clinical investigation of a subgroup found that it was for ATV drivers with neck pain to have assymetrical and focal neuropathies, pure or in mix with a nociceptive disorder, in the neck and upper extremities (47-79%), which was in contrast to referents with neck pain who had more nociceptive disorders (27% prevalence of neuropathy). Two studies measured characteristics of seated WBV exposure in forest machines (forwarders), snowgroomers and snowmobiles. The magnitudes of WBV in ATVs, measured and analyzed according to ISO 2631-1, were between 0.5-3.5 m/s2 (frequency weighted vector sum), which was considered high compared to limits suggested by the international standard ISO 2631-1 and the physical agent directive from the Euoropean Union (0.5 m/s2, rms). Drivers of ATVs were exposed to horizontally directed WBV and shocks. Non-neutral neck postures are ergonomic risk factors that occured infrequently and with short duration. The magnitude of seated WBV in forwarder vehicles varied substantiálly depending on model, terrain condition and driver. This may result in different conclusions regarding health risk assessments. The main conclusion from this thesis is that musculoskeletal symptoms and disorders in the neck and upper extremities, among drivers of ATVs, may be a result of long-time exposure to shock-type and horisozontally oriented seated WBV.

Abstract Weak and osteoporotic bones in old age are an increasing cause of mortality and painful physical impairment of the elderly, especially in the western world. Bone mineral accrual during childhood and adolescence is thought to play a vital role in preventing osteoporosis. Identifying and optimizing the factors influencing peak bone mass is thus important for the prevention of osteoporosis and related fractures. A main aim of this thesis was to investigate the potential effects of various types of weight-bearing physical activity on bone accretion in young males just out of puberty. The results from our subgroups of athletes consisting of badminton, ice hockey, and soccer players suggest that weight-bearing physical activity gives rise to regional specific bone response that is determined by the degree of impact of the activity in areas subject to mechanical loading (papers I–IV). In summary, the bone is sensitive to loading after puberty in males, and important bone mass gains can be achieved by proper amount and type of exercise. Another aim of this thesis was to studythe effect of detraining on weight-bearing and non-weight-bearing bone in a cohort of adolescent males who participated in ice hockey and soccer training. Our results indicate that exercise-induced bone mineral density benefits decline, predominantly in weight-bearing bones, after retirement from an active sports career (papers II–IV). High bone density stemming from physical loading might be at least partly preserved even by reduced physical activity at nonweight-bearing sites after about three years of reduced activity (III, IV). A final aim was to follow prospectively the development of BMD during years of reduced activity in former male athletes, and evaluate whether exercise during adolescence could be associated with fewer fractures in old age. We found fewer fragility fractures in a cohort of 400 former athletes compared to in 800 age-matched controls. Thus, high bone density stemming from previous weight-bearing physical activity may reduce the risk of sustaining fragility fractures in the elderly. Key words: physical activity, peak bone mineral density, males.