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- Cunha Goncalves, Doris
(author)
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Levosimendan in early experimental sepsis: effects on the heart and hepatosplanchnic circulation
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Sepsis-related cardiovascular dysfunction associated with fluid-unresponsive tissue hypoperfusion might require inotropic treatment. This cardiovascular dysfunction seems to involve calcium desensitization and adrenergic unresponsiveness. We investigated the effects of clinically relevant plasma concentrations of the calcium sensitizer levosimendan during the first 6 h of endotoxemia in a model of experimental sepsis in pigs (21.8-44.0 kg). Levosimendan given to endotoxemic pigs receiving moderate volume resuscitation elicited tachycardia, hypotension and myocardial ischemia, as evidenced by a negative myocardial lactate flux (study I). Likewise, compared to non-treated controls, levosimendan did not improve systemic or hepatosplanchnic perfusion during endotoxin shock: the animals developed signs of tissue hypoperfusion with elevated blood lactate and low oxygen venous saturations (study II). Aggressive volume resuscitation before levosimendan treatment induced a hyperdynamic state that was sustained by levosimendan and norepinephrine treatment, whereas control animals gradually developed shock. Nevertheless, splanchnic blood flow was redistributed and the superior mesenteric artery, the hepatic artery and the portal vein blood flows (PVF) decreased. Similarly to study II, there were signs of systemic and hepatosplanchnic tissue hypoperfusion. In contrast, administration of dobutamine and norepinephrine, increased cardiac output (CO) and oxygen delivery, maintained PVF and improved tissue perfusion (study III). Load independent measurements of cardiac function showed that systolic function was actually enhanced during the first 2 h of sepsis, whereas diastolic function was depressed in both ventricles. The initial decrease seen in CO was a result of volume depletion, and recovered with aggressive volume resuscitation. Although endotoxin-induced lung injury caused early right ventriculovascular uncoupling and increased right ventricular (RV) myocardial oxygen demand, right coronary artery blood flow improved markedly with resuscitation, maintaining adequate myocardial perfusion (study IV). In resuscitated septic pigs, levosimendan supported RV function by increasing RV contractility at a low energy cost. CO and left ventricular ejection fraction increased, and right ventriculovascular coupling and mechanical efficiency tended to improve (study V). In conclusion, early treatment with levosimendan during resuscitated sepsis can increase CO and improves RV contractility at a low energy cost, but it does not improve hepatosplanchnic perfusion significantly, which is better achieved with dobutamine-norepinephrine. In addition, because levosimendan is an inodilator, its use in sepsis should be restricted to thoroughly fluid-resuscitated subjects.
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| 2. |
- Andersson, David
(author)
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Experimental Studies of Ursolic Acid and Alkaline Sphingomyelinase in Colon Cancer and Colitis
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Sphingomyelin (SM) is a sphingolipid present in all eukaryotic cell membranes. Hydrolysis of SM by sphingomyelinase (SMase) generates ceramide, which has effects on cell-cycle regulation and apoptosis. Alkaline sphingomyelinase (Alk-SMase) is the main SM-hydrolysing enzyme in the human intestine, and earlier studies have shown that it has anti-proliferative properties on colon cancer cells, and that its activity is decreased in colon tumors as well as in ulcerative colitis. Ursolic acid (UA) is a plant-derived pentacyclic triterpenoid with anti-cancer and anti-inflammatory properties, whose effects on instestinal disorders has not previously been studied. The aim of the studies in this thesis was to examine the effects of UA on experimental colon cancer and its relation to gut sphingolipid metabolism, and finally to generate recombinant human Alk-SMase and use it as a topical treatment in a rat colitis model. When treating human colon cancer cells (HT-29) with UA, we found that UA dose-dependently decreased cell proliferation and induced apoptosis, accompanied by activation of caspase 3, 8 and 9. UA also selectively increased the activity of intestinal alkaline sphingomyelinase before the activation of caspases, suggesting this to be an early event in UA-mediated apoptosis. In an in vitro study, we saw that UA as well as four other pentacyclic triterpenoids specifically enhanced the activity of purified rat intestinal Alk-SMase in a dose-dependent manner. In the third study, using an Azoxymethane (AOM) rat cancer model, we examined the effect of orally administered UA on the formation of aberrant crypt foci (ACF) and intestinal SMase activity. UA significantly reduced the incidence of ACF containing three or more crypts when administered during the initiation phase. AOM reduced mucosal Alk-SMase activity, and the inhibitory effects could not be prevented by UA. In both AOM-treated and normal rats, UA increased the activity of colonic neutral SMase markedly and that of acid SMase activity mildly, all of which indicates that UA has chemopreventive effects in the initiation phase of colon cancer associated with changes in SM metabolism. In the fourth and final study, we, for the first time, report a method to express a biologically active human Alk-SMase from Pichia pastoris yeast cells. We then used the expressed recombinant enzyme to treat a dextran sulfate sodium rat acute colitis model, and found that intrarectal instillation of Alk-SMase once daily for 1 week significantly reduced the inflammation score and protected the colonic epithelium from inflammatory destruction. We also found a tendency towards decreased tumor necrosis factor (TNF)-alpha expression in the Alk-SMase-treated group. To summarize, these studies show evidence that UA and alk-SMase have chemopreventive effects against colon cancer and inflammation, and provide evidence indicating a link between the effects of UA and sphingolipid metabolism. The last study also demostrates an effective method to generate recombinant alk-SMase for potential applications of the enzyme against colonic tumor and inflammation.
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| 3. |
- Asgeirsson, Daniel
(author)
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Permeability of the glomerular filtration barrier in relation to the size, shape, charge and deformability of the permeating molecules
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Characterization of the glomerular filtration barrier is of fundamental importance for understanding the pathophysiology of proteinuric kidney diseases. Furthermore, it can be of key value in the understanding of microalbuminuria, which is often associated with endothelial dysfunction in conjunction with cardiovascular disease. In the four studies of this doctoral thesis, macromolecular probes having different frictional ratios (f/f0) and charge were investigated in order to give deepened insights into the usefulness of polysaccharides as probes of the permselectivity of the glomerular filter for proteins. In Study I, the extensively cross-linked polysaccharide Ficoll, was shown to overestimate the true permeability of the glomerular filtration barrier to neutral globular proteins, indicating that size-selectivity is even more important than previously recognized. However, Ficolls in the SE-radius range 55-75Å, were not hyperpermeable across the large pores of the glomerular filter, indicating their usefulness for studying proteinuric diseases. In study II, the results of recent publications, indicating the lack of charge-selectivity in the glomerular filter, were shown to be artifactual. Thus, polysaccharides, when negatively charged, seemed to become expanded and to increase their f/f0, thereby becoming more hyperpermeable. Thus earlier findings, showing that the glomerular filter discriminates proteins based on charge, are still valid. In study III, we showed that the main size-restrictive barrier of the glomerular filter lies, not at the podocyte level, but closer to the plasma compartment, or may be evenly distributed along the thickness of the filter. This was indicated by the lack of concentration-polarization upon increasing GFR. Study IV demonstrated that the differential permeabilities of polysaccharides and proteins across the glomerular barrier were not observed across the continuous capillary walls of the peritoneal membrane, an enigma that warrants further study.
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| 4. |
- Bonde, Sara
(author)
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Death, survival, and morphological development of hippocampal granule cells born in an inflammatory environment
- 2009
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Doctoral thesis (other academic/artistic)abstract
- The brain continues to form new neurons throughout life. This process of adult neurogenesis has been thoroughly documented in several species including birds, rodents and humans. Adult neurogenesis is not a global process, but is confined to two subcompartments of the brain; the subventricular zone lining the lateral ventricles, and the subgranular zone (SGZ) in the hippocampal formation. A variety of stimuli such as voluntary exercise, epileptic seizure activity and inflammation can affect the basal level of neurogenesis. In the course of pathological conditions such as Alzheimer’s disease, multiple sclerosis, epilepsy and stroke, an inflammatory response is initiated in the brain. Prolonged epileptic seizure activity, status epilepticus (SE), strongly imposes on the integrity of the delicate brain structure and cell communication. SE not only induces inflammation, but also neuronal death and a transient increase of basal adult neurogenesis in the hippocampal formation. What role inflammation plays in a disease such as epilepsy, and how it affects the neurons born in the aftermath of seizure activity, is largely unknown. The specific aim of the four studies included in this thesis was to investigate the effect inflammation has on the amount of basal and seizure-induced neurogenesis, and if the morphological development or functional characteristics of new neurons is affected when the neuron is born into an inflammatory environment. In brief, the purpose was to investigate the quantity and quality of the neurogenic outcome in inflammation. To comprehend the interplay between neurogenesis and inflammation would provide a valuable insight into disease progression, and could ultimately be part of the treatment or even a cure for pathological conditions involving seizure activity and inflammation.
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| 7. |
- Nezirevic Dernroth, Dzeneta, 1969-
(author)
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Pheomelanin markers in melanoma with reference to their excretion into urine
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Skin pigmentation is an important issue in most cultures. Until recently we have not understood the most important elements of pigmentation regarding detailed chemical structure. The synthesis of melanin is very complex, and although core enzymes, other important proteins, and parts of the melanin structure have been identified much information in this context awaits disclosure.The function of the melanocyte and the deposition of melanin pigments into the keratinocytes are very important in the protection against UV light. Melanin pigments consist of high-molecular structures often described as brown to black eumelanin and yellow to red pheomelanin. Eumelanin is photoprotective, whereas pheomelanin is believed to be carcinogenic after UV radiation. There is strong evidence that people of fair complexion with freckles who tan poorly are at higher risk of developing melanoma. These people have a higher pheomelanin to eumelanin ratio in their skin.Melanoma, one of the most widely spread cancers, is derived from melanocytes. There is accumulating evidence that pigment constitution is highly involved in the development of melanoma. We found that patients with advanced melanoma secrete substantial amounts of pigment structures into the urine, in particular those with diffuse melanosis. In subsequently performed experiments we purified these pigments and subjected the product to chemical degradation by either hydrogen peroxide oxidation or hydriodic hydrolysis. Several new chromatographic methods were developed for the structural analysis of these products. Structural analysis of new chromatographic peaks was performed. In conclusion, complex pheomelanin structures as well as low molecular weight pigments and free benzothiazoles have been identified in the urine of patients with melanoma and diffuse melanosis.The present thesis provides new insight into melanogenesis and melanoma progression. This opens the doorway to further approaches to the investigation of melanins and can help to understand fundamental problems about the structure and biosynthesis of natural melanins.
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| 8. |
- Nilsson, Elin, 1979-
(author)
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Characterization of IgY for Oral Immunotherapy and Prevention of Pseudomonas aeruginosa Infections in Cystic Fibrosis Patients
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Chicken antibodies, commonly referred to as IgY, have several properties that make them suitable for oral treatment of infections and there is essentially no risk for development of resistance. The overall aims of this thesis were to investigate Anti-Pseudomonas IgY as prophylaxis against infections with Pseudomonas aeruginosa for cystic fibrosis (CF) patients and to characterize the antibody treatment. We found that Anti-Pseudomonas IgY has affinity for P. aeruginosa flagellin, the major component of the flagellum. This is important since the flagellum is required for host invasion and establishment of infection. Flagellin induces inflammation. The main cause of morbidity and mortality among CF patients is chronic colonization of the airways with P. aeruginosa. We have studied prophylactic treatment of 17 Swedish CF patients with Anti-Pseudomonas IgY for up to twelve years. The results were compared with a control group of 23 Danish CF patients. Patients treated with IgY had 2.3 P. aeruginosa positive cultures/100 treatment months vs. 7.0 cultures/100 treatment months in the control group (p=0.028), and the time from inclusion to the first recolonization was significantly longer in the IgY-treated group (p=0.012). Lung function was preserved and patients treated with IgY had good nutritional status at the end of the study. Furthermore, other bacteria have not emerged instead of P. aeruginosa. Freeze-drying of IgY and the content of IgY preparations for oral use was investigated. Besides IgY, 26 egg yolk proteins were identified. Some of the proteins are known to have antimicrobial and immunostimulatory effects, and could have a positive additive effect to IgY treatment. Cholesterol levels were low. Conclusion: Anti-Pseudomonas IgY is a promising complement in the prevention of Pseudomonas aeruginosa infections in CF patients, partly explained by the fact that IgY binds to flagellin.
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| 9. |
- Petzina, Rainer
(author)
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Negative pressure wound therapy in cardiac surgery
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Negative pressure wound therapy in cardiac surgery Rainer Petzina, M.D. Clinical Sciences, Lund, Lund University Poststernotomy mediastinitis is a devastating complication for patients undergoing cardiac surgery. Conventional treatment includes surgical revision, continuous irrigation with drainage and wound closure with the use of the greater omentum and muscle flaps. Widespread adoption of negative pressure wound therapy (NPWT) has been driven through favorable clinical experience and excellent healing effects. The aims of the research in this thesis were: I. To quantify cardiac output and left ventricular chamber volumes after NPWT, using magnetic resonance imaging (MRI). II. To examine the effects of NPWT on peristernal soft tissue blood flow after internal mammary artery harvesting, using laser Doppler velocimetry. III. To study the effect of NPWT on blood and fluid content of the sternal wound edge and bone marrow, using MRI (T2-STIR). IV. To identify the effects of NPWT on the position of the heart in relation to the thoracic wall, using MRI. An uninfected porcine sternotomy wound model was used for all studies. The hemodynamic effects of NPWT in cardiac surgery are debated. MRI measurements show that NPWT results in an immediate decrease in cardiac output, although to a lesser extent than shown in previous studies. MRI is known to be the most accurate method for quantifying cardiac output. Patients with poor blood perfusion of the sternotomy wound edge tissue have a higher risk of developing post-sternotomy mediastinitis. We show that the peristernal wound edge microvascular blood flow is decreased when the left internal mammary artery is harvested. NPWT therapy stimulates blood flow in the in the wound edge both before and after the mammary artery is removed. Stimulating blood flow to the wound edge in patients with impaired microcirculation may be crucial to ensure healing. MRI measurements show that NPWT increases sternotomy wound edge tissue fluid and/or blood content. Presumably, NPWT creates a pressure gradient that draws fluid from the surrounding tissue into the sternal wound edge and into the vacuum source. This “endogenous drainage” may be one possible mechanism by which osteitis is resolved. Heart rupture is a devastating complication to NPWT of sternotomy wounds. MR imaging shows that NPWT causes the heart to be sucked up towards the thoracic wall and, in some cases, the right ventricular free wall to bulge into the space between the sternal edges and the sharp edges of the sternum to poke into and deform the anterior surface of the heart. These can be effectively hindered by the placement of a rigid barrier over the anterior portion of the heart. Taken together, the studies of the present thesis demonstrate the effects of NPWT on the thorax and intrathoracic organs. NPWT alters wound edge microvascular blood flow and fluid content and affects heart pumping and heart position in relation to the thoracic wall. Rainer Petzina, MD Lund, January 15, 2009
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| 10. |
- Ronquist, Göran, 1971-
(author)
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Some Characteristics of Human Prostasomes and Their Relationship to Prostate Cancer
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.
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