Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken (Gallus domesticus). IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary. Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms. The use of immunoglobulin therapy broadens the arsenal available to combat pathogens in medicine and IgY is a promising candidate, both as an alternative to antibiotics and as a useful tool in research and diagnostics.

Diabetes mellitus (DM) is one of the leading causes of end stage renal failure. An increased susceptibility to renal ischemia/reperfusion (I/R)-injury was found in DM rats. Unilateral renal ischemia for as short as 20 minutes led to an irreversible progressive injury in DM kidneys, whereas the injury in non-DM kidneys was almost reversible. The renal I/R injury was characterized by anuria, infiltration of inflammatory cells, tubular atrophy, dilation of the remaining tubuli and tubulointerstitial fibrosis. Necrotic areas were found in the inner parts of the outer medulla and in the papilla. The renal medulla was the most vulnerable part of the kidney. This was seen both by the extent of fibrosis four and eight weeks after I/R and by the presence of TUNEL-positive (apoptotic) cells 6h after ischemia. Increased accumulation of HA and enhanced CD44 expression was seen after I/R in DM kidneys. Treatment with long acting insulin 7-14 days before I/R, decreased the number of apoptotic cells in the renal medulla and protected renal function and morphology after the insult, while insulin treatment after the injury did not have any protective effect. Short acting insulin given 2-6 hours before I/R partially protected renal function but did not improve the morphological picture. Treatment with the angiotensin II receptor type 1 blocker candesartan, the PAF-antagonist UR-12670, the immunosuppressive agents tacrolimus and cyclosporin A, or prednisolone did not improve the outcome of the renal I/R injury in DM. Injection of cobalt protoporphyrin (CoPP) intraperitoneally in order to induce an over-expression of heme oxygenase-1 (HO-1) resulted in a trend towards a better function in DM kidneys after I/R. However, the induction of HO-1 by intraperitoneal CoPP injection was not achieved in all rats, when examined by western blot. In conclusion, unilateral renal I/R leads to a severe progressive injury in DM kidneys. Insulin treatment before ischemia, but not after, reduces the renal injury in DM rats. Studies using a more reliable administration of CoPP are required to decide if induction of HO-1 protects against renal I/R injury in DM.

The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted. The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C>T), 434(G>C) and 562(G>C). The two first were located to the mature peptide-coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterized by fibrosis and eosinophils and ECP has been suggested in fibrosis development.

In unstable coronary artery disease (CAD) it still is a matter of debate which patients should undergo early revascularisation. In the FRISC II study (n=2457) an early invasive strategy was, compared to a primarily non-invasive strategy, associated with reduced mortality and myocardial infarction (MI) rates. However, in this heterogeneous group of patients, tools for an appropriate selection to revascularisation are needed. From the FRISC II study we evaluated the prognosis, the angiographic extent of CAD and the effects of an early invasive strategy in relation to risk variables on admission. The occurrence of ST depression and/or elevated levels of Troponin T were associated with a higher risk for death and MI, more severe CAD and also with a reduction of death or MI by the early invasive strategy. Elevated levels of the inflammatory markers C-reactive protein (CRP) and interleukin-6 (Il-6) were associated with a higher mortality but an unchanged MI rate. Elevated levels of Il-6, but not CRP, identified patients with a large reduction of mortality by the invasive strategy. Age ≥ 70 years, male gender, diabetes, previous MI, ST depression and elevated levels of troponin and markers of inflammation were independently associated with an adverse outcome. The FRISC-score was constructed using these 7 variables. At FRISC-score ≥ 5 an early invasive strategy markedly reduced mortality and MI, at FRISC–score 3-4 death/MI was reduced, whereas in patients with a FRISC-score 0-2 neither mortality nor death/MI was influenced. In unstable CAD, a non-invasive strategy seems justified only for patients at low risk, i.e. FRISC score < 2. In patients with intermediate and high risk, i.e. FRISC-score ≥ 3, an early invasive strategy is recommended.

Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.

Reduced performance of the left ventricle to eject blood – left ventricular systolic dysfunction (LVSD) – is a common predecessor of the heart failure syndrome. With or without symptoms, LVSD is associated with a poor prognosis. However, with adequate treatment, the development or progression of symptoms, the need for hospitalisation and mortality can all be reduced. In the present work, the occurrence of LVSD was evaluated by echocardiography in a community-based sample of 75-year-old men and women (n = 433). LVSD was a common condition, with a prevalence rate of 6.8%. In nearly half the participants with LVSD, there was no clinical evidence of heart failure. Community-based screening for asymptomatic LVSD has been proposed as a strategy to reduce the incidence of heart failure. Because of the high costs and low availability, echocardiography is not a suitable screening tool. The plasma concentration of B-type natriuretic peptide (BNP) has been the most advocated screening tool. Another alternative is the standard 12-lead electrocardiogram (ECG). Both the ECG and BNP were effective in excluding LVSD in our 75-year-old community-based sample. However, compared with BNP, the ECG had considerably better specificity. In screening for LVSD, BNP had a diagnostic value in addition to the ECG, but only in individuals with abnormal ECGs. The left ventricular ejection fraction (LVEF) measured by echocardiography is a well-established index for describing left ventricular systolic function. The wall motion index (WMI) and the amplitude of mitral annulus motion (MAM) are suggested as alternative echocardiographic methods. Compared with MAM, the WMI had a more favourable agreement with the LVEF in our 75-year-old participants. Nonetheless, MAM was a strong predictor of mortality. MAM predicted the risk of all-cause and cardiac mortality independently of other risk factors. In addition, when it came to cardiac mortality, the predictive ability of MAM was independent of the LV function measured as the WMI.

This thesis studied environmental factors in relation to asthma and respiratory symptoms among schoolchildren in two countries. In Sweden, 1014 pupils (5-14 year) in 8 schools participated. Wheeze was reported by 7.8%, current asthma by 5.9%, doctor-diagnosed asthma by 7.7%, cat allergy by 6.8% and dog allergy by 4.8%. Current asthma was less common among those consuming more fresh milk and fish. Doctor-diagnosed asthma was less common among those consuming olive oil. Cat, dog and horse allergens were common in settled dust and related to respiratory symptoms. Pupils consuming butter and fresh milk had less respiratory symptoms in relation to allergen exposure. In schools with increased levels of microbial volatile organic compounds and selected plasticizers (Texanol and TXIB) asthma and respiratory symptoms were more common. In Korea, 2365 pupils (9-11 year) in 12 schools participated (96%). In total, wheeze was reported by 8.0%, current asthma by 5.7%, doctor-diagnosed asthma by 5.4%, cat allergy by 2.6% and dog allergy by 4.9%. Contamination of dog and mite (Dermatophagoides farinae) allergen was common while cat allergen was uncommon. Remodelling, changing floor and building dampness at home were positively associated with asthma and respiratory symptoms. The strongest associations were found for floor dampness. Indoor/outdoor concentration of NO2, formaldehyde and ultrafine particles (UFP) at schools were positively associated with asthma and respiratory symptoms. When comparing Sweden and Korea, Korean pupils had more breathlessness and asthma but reported less cat and pollen allergy. Swedish schools had CO2-levels below 1000 ppm, while most Korean schools exceeded this standard. Since both home and school environment may affect pupil’s asthma and respiratory symptoms, air quality should be an important health issue. Moreover, changes in dietary habits may be beneficial to decrease asthma and allergies. Furthermore, interaction between diet and environment needs to be further investigated.

Tissue factor (TF) is a transmembrane glycoprotein that initiates the blood coagulation cascade and is also involved in cell migration, tumour metastasis and angiogenesis. Pathologic expression of tissue factor by monocytes contributes to several thrombotic complications like acute coronary artery disease and disseminated intravascular coagulation. The aim of this thesis was to investigate the clinically important pathologic expression of TF in myelo-monocytic leukaemia cells and reveal the cellular signals leading to the suppression of TF expression. The studies in this thesis indicate that TF is a marker of immature myelo-monocytic cells. Markedly higher levels of TF were expressed in immature myelo-monocytic cell lines compared to mature monocyte-like cells. Induction of terminal differentiation in immature cells resulted in down-regulation of TF expression, irrespective of the specific phenotypes induced by retinoic acid (RA) or vitamin D3 in monoblastic U-937 cells. TF suppression was also found independent of differentiation pathways, i.e. monocytic or granulocytic. The nuclear receptor activation requirements for transcriptional suppression of TF by retinoic acid (RA) were shown to differ between acute promyelocytic leukaemia (APL) NB4 and U-937 cells. In NB4 cells the binding of the agonist to the RA receptor (RAR)α alone is needed for down-regulation of TF, whereas ligand binding to both RARαand retinoic X receptor was necessary for efficient suppression of TF expression in U-937 cells. To analyse the transcriptional regulation of TF, stable NB4 and U-937 clones expressing the luciferase gene under the control of various 5' flanking regions of the TF gene were selected. Different promoter regions were found to control the basal TF transcriptional activity. Analysis of protein binding to the 140 bp promoter region, responsible for basal TF activity in NB4 cells, revealed binding of RFX-1. RA suppressed the promoter activity in NB4, but not in U-937 cells. The ectopic expression of the APL fusion proteins PML/RARα or PLZF/RARα in U-937 reporter clones were shown to confer sensitivity to RA-induced suppression of TF promoter activity. These results provide a more detailed picture of TF regulation in leukaemic and haematopoietic cells and may have a bearing on new clinical treatment strategies in APL and other leukaemias.

Bakgrund: Långvarig nacksmärta är vanligt och drabbar ca 50% av världens befolkning. I Sverige drabbas kvinnor oftare än män. Ospecifik långvarig nacksmärta saknar känt ursprung. Behandlingen går oftast ut på att dämpa symtom och utbilda patienten i smärtan. Att istället utgå från att öka måluppfyllelse är ovanligare. Syftet med examensarbetet var att undersöka hur tilltro till behandling, katastroftankar samt smärtstillande medicinering påverkade graden av måluppfyllelse hos kvinnor i arbetsför ålder med ospecifik långvarig nacksmärta efter en rehabiliteringsperiod. Även sambandet mellan tilltro till behandling och katastroftankar undersöktes.  Metod: Detta är en retrospektiv longitudinell studie och sekundäranalys av data från en RCT studie med data tillhörande 65 kvinnor. Tilltro till behandling, katastroftankar och medicinering mättes innan rehabiliteringsperioden och måluppfyllelse mättes efter.  Resultat: Mann-whitney U-test visade ingen skillnad i måluppfyllelse efter rehabilitering mellan de med hög respektive neutral grad av tilltro till behandling (p=0, 395), låg respektive neutral grad av katastroftankar (p=0,59).  Sperman´s Rho test fann inget samband mellan tilltro och katastroftankar (p=0,751). De som tog medicin regelbundet hade en signifikant högre grad av måluppfyllelse mot de som ej tog medicin regelbundet (p=0,025).   Slutsats: Studien ger inte stöd för att tilltro till behandling och katastroftankar påverkar graden av måluppfyllelse. Inget samband mellan katastroftankar och graden av tilltro till behandling tycks finnas. Resultatet bör tolkas i ljuset av att data saknades för de med låg tilltro respektive höga katastroftankar. Smärtmedicinering tycks ha en positiv påverkan på måluppfyllelse. Mer kunskap om hur/vilken medicinering som påverkar måluppfyllelse behövs.

<p>Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement <i>in vivo</i> has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation <i>in vivo</i> that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation.</p><p>The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples.</p><p>A methodology for measurement of the total amount (sum of free and esterified) of an F₂-isoprostane, 8-iso-prostaglandin F_2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F_2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride.</p><p>Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F_2α and a major prostaglandin F_2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation.</p><p>In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F_2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet.</p><p>In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation <i>in vivo</i> under basal conditions and in the pathology of diseases.</p>