Light and oxygen generate harmful reactive oxygen species (ROS) in the lens, causing biochemical changes that gradually disarrange the lens fibres resulting in light scattering and loss of transparency. In the healthy eye, this chronic exposure to oxidative stress may lead to age-related cataract. However, there are also some conditions that accelerate cataract formation, such as diabetes mellitus, in which increased glucose levels may contribute to increased generation of ROS. The superoxide dismutases (SOD) participate in the defence against ROS by catalysing the dismutation of superoxide radicals. The main SOD isoenzyme in the lens is copper-zinc superoxide dismutase (SOD1). The aim of this thesis was to explore if this antioxidant enzyme is important for the protection against age-related and diabetes-induced cataract development. Lenses from wild-type mice and mice lacking SOD1 were incubated in high levels of glucose in vitro and their transparency and damage evaluated daily. Also, the impact of nitric oxide was studied by adding a nitric oxide synthase inhibitor. Furthermore, in vivo cataract formation in relation to the oxidative status of the lens was evaluated in streptozotocin-induced diabetic mice as well as in non-diabetic mice of both genotypes. Finally, the spontaneous age-related cataract development was studied in both genotypes. In vitro, the SOD1 null lenses showed increased levels of superoxide radicals and developed dense nuclear lens opacities upon exposure to high levels of glucose. They also showed increased lens leakage of lactate dehydrogenase, reduced transport function across cell membranes, and increased water contents. However, the lens damage and cataract formation were eliminated when the synthesis of nitric oxide was inhibited. This indicates that both superoxide and nitric oxide have important roles in glucose-induced cataract development possibly through their reaction with each other which generates the highly reactive peroxynitrite. In vivo, both the SOD1 null and the wild-type mice showed cortical cataract changes after 8 weeks of diabetes, although the SOD1 null mice showed a more pronounced cataract formation than the wild-type mice in relation to the level of hyperglycaemia. As cataract formation was accentuated the lenses showed diminishing levels of glutathione but increasing amounts of protein carbonyls, suggesting a reduced lens antioxidant capacity as well as increased lens protein oxidation. Non-diabetic young (18 weeks of age) SOD1 null mice did not show any signs of cataract. At 1 year of age they had developed some cortical lens obscurity as compared to the wild-type mice which did not show equivalent changes until 2 years of age. The results presented in this thesis show that SOD1 null mice are more prone to develop diabetes-induced and age-related cataract than wild-type mice. The findings thus further endorse the importance of oxidative stress as a contributor to cataract development and indicate that both superoxide and nitric oxide may be damaging to the lens. I therefore conclude that the antioxidant enzyme SOD1 is important for the protection against cataract.

The primary aim of this thesis was to modify saphenous vein (SV) harvesting technique and evaluate its clinical importance. A new "no touch" (NT) technique of SV preparation was developed where the vein is harvested with a pedicle of surrounding tissue, which protects the vein from spasm therefore obviating the need for distension. Firstly, a prospective randomised study in 156 patients who underwent coronary artery bypass grafting was done to compare this new "no touch" technique to two others, the conventional (C) and the intermediate (I) techniques. A morphological study of the endothelium showed an endothelial integrity of 97% in NT vessels while about half of endothelial surface of veins harvested by the other two techniques was devoid of endothelium. At angiographic follow up, the patency for NT was 95.4%, 88.9% for grafts in group C and 86.2% for grafts in group I. A statistically significant difference in patency rate was found between the NT group and group C (p=0.025) and the poorest result was observed in group I. Secondly, the immunohistochemistry assessment using CD31-antibody confirmed a better-preserved endothelium for NT vessels. Putative NOS was identified by NADPH-diaphorase histochemistry and autoradiographic localization of [3H] L-nitroarginine (NOARG) binding. NADPH staining was almost continuous on the luminal aspect and was also present in the intact adventitia of NT vessels, which was markedly reduced in conventionally harvested veins. Autoradiographic analysis of specific NOARG binding showed greater binding in the no-touch vessels, confirming the histochemistry results. All three NOS isoforms were identified in the media of SV grafts. In NT, NOS I was abundant in adventitial nerves; NOS II was found in adventitial vasa vasorum and NOS III was associated with endothelial cells lining both the vessel lumen and microvessels within the adventitia. In conclusion, this study demonstrated that the endothelial integrity and NOS activity are better maintained when using the no-touch technique for vein graft harvesting. The vasorelaxant and thromboresistent activities of NO may be responsible for the reduced venospasm and improved early patency rates observed. Furthermore, the mechanical properties provided by the cushion of surrounding tissue in grafts harvested by NT technique may contribute to the observed high patency rate.

Nitric oxide (NO) is a mediator of vascular smooth muscle tension that metabolises rapidly in blood. NO delivered by inhalation can therefore be used as a selective pulmonary vasodilator to relieve pulmonary hypertension or to improve oxygenation with no systemic effects. In artificial ventilation nitric oxide has been administered in inspiration gas as a continuous gas flow or to form constant inspired concentration. Homogeneous inspired gas mixture has been regarded essential for successful therapy and the therapy has been characterized by the mixture NO concentration. The response in oxygenation on NO therapy has, however, been variable. Administration of NO as a short pulse synchronously with inspiration has been suggested to improve the response. In this study the NO administration was examined theoretically and experimentally with the aim to relieve pulmonary hypertension and improve oxygenation during artificial ventilation. For the experimental study a system for the synchronized administration was developed. The effect on oxygenation was studied during equine anaesthesia where hypoxemia develops regularly secondary to left-to-right shunt caused by atelectasis. By administering the NO as a short pulse in early inspiration to well ventilated lung areas the oxygenation could be effectively improved. Delayed administration to low ventilated lung areas was found possible for a negative contribution on oxygenation, which reduces the improvement gained in the well-ventilated lung areas. When NO is delivered into the whole inspiration, the net effect on oxygenation is the sum of these negative and positive contributions, whereas with pulsed delivery to the early inspiration the negative contribution can be avoided. This finding may be the main explanation for the varying response in oxygenation detected in patients as a response to NO inhalation. When the NO therapy aimed for the relief of induced pulmonary hypertension in pigs, no difference was observed between NO delivery as a short pulse or given to the whole inspiration. Maximum vasodilatation was observed with 105 nmol/min delivery rate. A larger delivery rate only contributed to an abrupt increase in pulmonary pressure at cessation of the delivery. The NO uptake from alveoli to tissue depends on the alveolar NO partial pressure. In a simulation this partial pressure was shown to be independent of the administration mode. Also the relationship between the NO uptake and delivery setting was not explicit. With pulsed delivery, expired NO can be reduced which was confirmed by the experimental results. This is important when the NO therapy is given in rebreathing circuit.

We have developed a methacholine provocation method, which detects bronchial responsiveness in more than 80% of healthy subjects. The method enables us to detect differences in bronchial responsiveness within the normal range. With this method FEV1 and Gaw had similar sensitivity in detecting small differences in bronchial responsiveness. Differences, between protocols when using doubling or fourfold concentration steps emphasize the importance to strictly adhere to a predefined protocol. Deep inhalation associated with the FEV1 manoeuvre decreases bronchial tone induced by methacholine for up to 6 minutes, which emphasizes the importance of exact timing between successive FEV1 measurements in bronchial provocation tests. There is a substantial overlap in bronchial responsiveness between healthy and asthmatic subjects and a deep inhalation at the end of the methacholine test challenge could not discriminate between asthmatic and non-asthmatic subjects. Inhalation of dust in a swine confinement building causes an intense airway inflammatory reaction with an extensive migration of inflammatory cells, predominantly neutrophils, into the upper and lower airways. Bronchial responsiveness to methacholine increased by about 3 doubling concentration steps and was normalized one week after exposure. However, exposure to dust in a swine confinement building did not yield increased bronchial responsiveness to eucapnic hyperventilation with dry air which is often observed in asthmatic subjects. Exhaled NO was approximately doubled five hours after exposure and in the present study we found no relationship between exhaled NO levels and bronchial responsiveness in healthy subjects. Protection with half-mask inhibited the dust induced increase of exhaled NO whereas the increase in bronchial responsiveness was influenced only to a minor extent. These findings, do not support the hypothesis that the increased bronchial responsiveness following organic dust exposure is directly caused by the inflammation. Instead, a possible direct effect on the smooth muscle and swelling of the airway mucosa and increased secretions due to the general inflammatory reaction probably leads to airway narrowing enhancing the post-exposure bronchial response to methacholine.

The development of diabetes mellitus depends on the balance between beta-cell proliferation and death. As mitogen-activated protein kinases (MAPK) may control this balance, the aim of this study was to investigate the events leading to MAPK activation in beta-cells and the consequences of these events. Overexpression of the SH2-domain containing adaptor protein Shb resulted in the assembly and activation of multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpression cells. This was paralleled by an attenuated activation of the MAP kinases ERK1/2. Thus, Shb-induced alterations in the IRS-1/PI3K/Akt/ERK pathway might explain the increased proliferation and apoptosis of beta-cells overexpressing Shb. The importance of the MAP kinase p38 in nitric oxide- and cytokine-induced beta-cell death was also investigated. Knock-down of p38 expression resulted in a lowered cell death rate in response to a nitric oxide donor. In transient transfections MKK3 over-expression resulted in increased p38 phosphorylation in RIN-5AH cells. In addition, a short-term MKK3 expression resulted in increased cytokine-induced cell death. A nitric oxide synthase inhibitor abolished the MKK3-potentiating effect on cytokine-induced cell death and inhibitors of phosphatases enhanced MKK3-stimulated p38 phosphorylation. Finally, as the dominant negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In further support for an MKK3/6-indepedent mechanism, the adaptor protein TAB1 significantly increased the cytokine- and nitric oxide-stimulated phosphorylation of p38. The TAB1-mediated activation of p38 was paralleled by a compensatory inhibition of ERK and JNK. In summary, p38 MAPK, activated mainly by TAB1, promotes, at least in part, beta-cell death in response to cytokines or nitric oxide.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which T-cells and macrophages invade the islets of Langerhans and selectively destroy the insulin producing β-cells, either directly or through the secretion of e.g. cytokines and nitric oxide (NO). This thesis has studied possible strategies to prevent T1DM. In β-cells and macrophages, NO is produced by inducible nitric oxide synthase (iNOS). In the first study, we found that 1400W, a highly selective inhibitor of iNOS could prevent interleukin (IL)-1β induced suppression of rat islet function in vitro, but not diabetes induced by multiple low dose streptozotocin (MLDS), a well established animal model for autoimmune diabetes, in vivo. Next, we wanted to test a new type of high affinity blocker of IL-1 action, called IL-1 trap, in vitro. Here we found that an IL-1 trap could prevent the suppressive effects by IL-1β on rat pancreatic islet function. Also, it was sufficient to block the action of IL-1β to prevent islet cell death induced by a combination of IL-1β, tumor necrosis factor-α and interferon-γ. In study III, a murine IL-1 trap was found to prolong islet graft survival in the recurrence of disease (ROD) model, a T1DM model that involves syngeneic transplantation of healthy pancreatic islets to diabetic nonobese diabetic mice. Mice treated with IL-1 trap displayed an increased mRNA level of the cytokine IL-4 in isolated spleen cells. This suggests a shift towards Th2-cytokine production, which in part could explain the results. Finally, simvastatin an anti-hypercholesterolemic drug that possesses anti-inflammatory properties e.g. by interfering with transendothelial migration of leukocytes to sites of inflammation was studied. We found that the administration of simvastatin could delay, and in some mice prevent, the onset of MLDS-diabetes, and prolong islet graft survival in the ROD model.

The activity of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) were regulated by kinase through phosphorylation. The cyclin-dependent kinase 5 (Cdk5) by associating with its neuron-specific activator p35 has been demonstrated to be essential for neurodegenerative neuronal death. This study focuses on the functional regulation of nNOS and eNOS by Cdk5/p35 complex in a phosphorylation dependent manner. We found that nNOS associated with Cdk5 by immunoprecipitation (IP) and in vitro phosphorylated by Cdk5 by autoradiograph. Nitrite (NO2-) production was significantly reduced in Cdk5 over-expressing N18 cells, suggested that Cdk5 down-regulated nNOS enzymatic activity. In addition, Cdk5 phosphorylated eNOS both in vitro and in vivo on Ser 113, and the Cdk5 inhibitor roscovitine suppressed the phosphorylation of eNOS. Interaction of wild-type eNOS and S113A mutant eNOS with Cdk5 was observed in co-immunoprecipitation experiments. Co-expression of S113A eNOS and Cdk5/p35 resulted in 2-fold enhancement nitrite (NO2-) generation than co-expression of eNOS and Cdk5/p35 in SH-SY5Y cells. These data indicate that Cdk5 phosphorylated nNOS and eNOS, as well as down regulated nNOS and eNOS activity. Our results supposed that Cdk5 associated with and regulated the activity of nNOS and eNOS through phosphorylation.

Background: Atopic disease is an increasing problem. Eczema affects 10-20% of young children, and 33-37% of children with eczema are food allergic. Among other factors, nitric oxide (NO) is thought to play a role in eczema and food allergy. Following the atopic march, pproximately 80% of children with atopic eczema will become sensitized to aeroallergens and develop asthma and/or allergic rhinitis. Skin prick test is used for investigating sensitization and is considered a safe method. However, systemic allergic reactions may appear when the test is performed. In diagnosing food allergy and for evaluating achievement of tolerance, the oral food challenge is the method of choice, and the double-blind placebocontrolled fashion is 'the gold standard'. Skin prick test: We examined six cases of generalized allergic reactions in connection with skin prick testing in order to identify risk factors, and thereby increase safety, and we investigated the necessity of performing skin prick tests in duplicate. We found that all six children with generalized reactions were <6 months of age. When analyzing skin prick tests in duplicate, we found only 1.3% that showed diverging results, and in infants <6 months even fewer, 0.9%. Food challenge: We developed recipes and a protocol for low-dose oral food challenge to milk and egg to be used in young children outgrowing their food allergy so as to facilitate early re-/introduction of small amounts of milk and egg. We performed 52 challenges, both open and double-blind placebo controlled. The recipes were validated for blinding. The lowdose challenge was tolerated well by the children and was easy to perform. Four children had a positive challenge outcome, all reacting to very small amounts of milk. All but two of the non-reacting children were able to introduce milk and egg into their diet. Nitric oxide and eczema: We investigated the effect of eczema treatment on the NO levels in urine. The sum of nitrite and nitrate was measured in urinary samples from 94 infants at two visits, with an interval of 6 weeks, and the results were compared with clinical data. The levels of NO products increased significantly when the eczema improved. The atopic march: The aim was to evaluate the atopic march in children with eczema, from referral at <2 years until 4½ years of age. We followed 123 children with eczema, 78 sensitized and 45 not sensitized to milk and/or egg, with respect to eczema severity, other allergic manifestations, development of airway sensitization, and achievement of food tolerance. The difference in severity of eczema at referral was significant when comparing food-sensitized with non-sensitized children. At follow-up, 62% were still affected by eczema, although 56% only mildly so. Tolerance was achieved in 81% of the children allergic to milk and 68% of those allergic to egg. Fifty-eight percent of the food-sensitized children and 26% of the non-sensitized children had become sensitized to aeroallergens, a significant difference. The difference in airway symptoms was not significant. Very few children were exposed to tobacco smoke in their homes. Conclusions: Increased precautions should be considered when performing skin prick tests in infants <6 months of age. The use of a single prick, to avoid the risk of summation of reactions, is justified when performing skin prick tests. We report recipes and a protocol for standardized open and double-blind placebo-controlled low-dose food challenge in young children, enabling the introduction of small amounts of egg and milk into the diet during tolerance development. NO products in urine increases when eczema improves. This might be due to a Th2/Th1 shift induced by the eczema treatment and skin healing, and the variation in NO response may be due to individual variations in NO-induced feedback downregulation of Th1 and Th2 proliferation. The prognosis for achieving clinical tolerance is very good in children early sensitized and allergic to milk and egg, but they will become significantly more often sensitized to aeroallergens.

Biofilms are communities of sessile microorganisms attached to a surface and imbeddedin a matrix of extracellular polysaccharide substances. These communities can be foundin diverse aquatic environments, such as in industrial pipes and in humans. By formingmicrocolony structures, which are highly resistant to adverse physical conditions as wellas antimicrobial agents, biofilms are very problematic when associated with e.g.persistent infections. In order to find new ways of controlling biofilm growth, theprocesses involved in biofilm development must be investigated further. The maininterest of this study is the occurrence of void formation inside biofilms. Thisphenomenon has been observed in several studies and has been correlated to cell deathinside the microcolonies. The occurrence of cell death has recently been associated withthe presence of nitric oxide in the biofilm. In this study, the implications of nitric oxideaccumulation on biofilm development were investigated using an individual-basedmodel. Specifically, the role of nitric oxide in void formation was considered. A largenumber of simulations were run using different parameter settings in order to determine ifnitric oxide could account for the occurrence of void formation observed experimentally.The general predictions made by the model system showed agreement to someexperimental data, but not to others. Sloughing, the detachment of chunks of cells fromthe biofilm, was observed in the majority of simulations. In some cases, the model alsopredicted the presence of live cells inside the voids, which has been observedexperimentally.

High salt intake contributes to the development of hypertension in salt-sensitive humans and animals and the mechanistic causes are poorly understood. In Dahl salt-sensitive (S) but not salt-resistant (R) rats, high salt diet increases cerebrospinal fluid (CSF) [Na+] and activates an aldosterone-mineralocorticoid receptor-epithelial sodium channel-endogenous ouabain (MR-ENaC-EO) neuromodulatory pathway in the brain that enhances the activity of sympatho-excitatory angiotensinergic and glutamatergic pathways, leading to an increase in sympathetic nerve activity (SNA) and blood pressure (BP). We hypothesize that high salt diet in Dahl S rats enhances Ang II release in the paraventricular nucleus (PVN), causing a decrease in local nitric oxide (NO) action and an increase in local glutamate release thereby elevating SNA, BP and heart rate (HR). The present study evaluated the effects of agonists or blockers of MR, ENaC, EO, nitric oxide synthase (NOS) or glutamate and AT1-receptors on the BP and HR responses to acute infusions of Na+ rich aCSF, intracerebroventricularly (icv), or in the PVN of Dahl S, R or Wistar rats or to high salt diet in Dahl S and R rats. In Wistar rats, aldosterone in the PVN enhanced the BP and HR responses to infusion of Na+ rich aCSF in the PVN, but not in the CSF, and only the enhancement was prevented by blockers of MR, ENaC and EO in the PVN. AT1-receptor blockers in the PVN fully blocked the enhancement by aldosterone and the responses to infusion of Na+ rich aCSF icv, or in the PVN. Na+ rich aCSF in the PVN caused larger increases in BP and HR in Dahl S vs. R rats and the responses to Na+ were fully blocked by an AT1-receptor blocker in the PVN. BP and HR responses to a NOS blocker in the PVN were the same, but L-NAME enhanced Na+ effects more in Dahl R than S rats. High salt diet attenuated increases in BP from L-NAME in the PVN of Dahl S but not R rats. AT1 and glutamate receptor blockers candesartan and kynurenate in the PVN decreased BP in Dahl S but not R rats on high salt diet. At the peak BP response to candesartan, kynurenate in the PVN further decreased BP whereas candesartan did not further decrease BP at the peak BP response to kynurenate. Our findings indicate that both an acute increase in CSF [Na+] and high salt intake in Dahl S rats increases AT1-receptor activation and decreases NO action in the PVN thereby contributing to the pressor responses to Na+ and presumably, to dietary salt-induced hypertension. The increased BP response to AT1-receptor activation in the PVN of Dahl S is mediated by enhanced local glutamate receptor activation. An MR-ENaC-EO pathway in the PVN can be functionally active and further studies need to assess its role in Dahl S rats on high salt intake.