<p>S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated.</p><p>Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.</p><p>Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate <i>in vitro</i> evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease.</p><p>Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.</p>

<p>Apoptosis or programmed cell death is crucial for the resolution of inflammation, and phagocytosis of apoptotic cells initiates the release of actively anti-inflammatory responses from the phagocytes. Eosinophils are one of the most potent inflammatory cells in the body and is involved in a number of diseases, most commonly associated with parasitic infections and allergic diseases. Apoptosis in eosinophils is therefore one of the most important systems to avoid inflammation. This aim of the present investigation was to examine the mechanisms behind, and the consequences of this process in eosinophils. Apoptotic eosinophils have a unique surface receptor expression that indicates abilities to communicate with T-, B- and antigen presenting cells. They have a novel expression of CD49f, indicating an importance for binding to laminin or unknown functions of the VLA-6 receptor, possibly in the concept of phagocytosis of the apoptotic cell. </p><p>In apoptotic eosinophils the granules are translocated to the periphery of the cell, probably through a disruption of the cytoskeleton. This translocation makes the granules easily accessible and the apoptotic eosinophil can release considerable amounts of granule proteins in response to specific stimuli. The spontaneous release however, is decreased as compared with living cells. </p><p>Furthermore, the survival of eosinophils in response to an allergen challenge is increased in healthy subjects, but not in allergic patients. Mechanistically, this needs further investigation, but one theory is that it is due to the presence of specific IgE in patients in combination with differences in the response from the epithelial cells.</p>

<p>Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged <i>in vitro</i> culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon.</p><p>In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.</p>

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro. The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.

The kidney is the major organ responsible for the regulation of the composition and volume of the body fluids, which is essential for homeostasis. The glycosaminoglycan hyaluronan (HA), with extreme water-binding capacity, is present in the interstitium of the kidney with a heterogenous distribution. The importance of HA in renal water-handling is unknown and was the focus of the present investigation. Acute water-loading in rats caused the amount of papillary HA to increase and during water deprivation, the amount was reduced. Gerbils, with extreme urine concentrating capacity, have less HA in the renal papilla in normal conditions and responded diametrically different to water-loading (reduction in HA). Renomedullary interstitial cells (RMICs), which are probably the main producers of HA in the renal medulla, were cultured at different media osmolalities to mimic the milieu of the medulla during variations in the water balance. The amount of HA found in the media was decreased at high osmolalities and increased at low osmolalities, thereby strengthening the in vivo results. CD44, an HA-receptor involved in the uptake and degradation of HA, was expressed on RMICs in an osmolality dependent manner. During high media osmolality, the CD44 expression increased and at lower osmolalities, the opposite occurred, probably due to the need for uptake and degradation of HA. Renal ischemia-reperfusion injury causes a cortical accumulation of HA, up-regulation of CD44, and a depression of functional parameters. The time periods of ischemia correlated with the accumulation of HA which, in turn, was inversely correlated to GFR. Hyaluronidase injections in this setting failed to reduce HA levels and significantly improve renal function. In conclusion, the results from the present study suggest an important role for HA and RMICs in renal water-handling and that the intrarenal distribution of HA is altered after ischemia-reperfusion injury, which correlates with renal dysfunction.

The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important. In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect. The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of efficacy of agonists was UTP = ATP &gt;&gt; 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of 15 μM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y2, purinergic receptors on basolateral and that activation of these receptors results in the mobilization of Ca2+. 4) Increased luniinal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells.

S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated. Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated. Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate in vitro evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease. Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.

Apoptosis or programmed cell death is crucial for the resolution of inflammation, and phagocytosis of apoptotic cells initiates the release of actively anti-inflammatory responses from the phagocytes. Eosinophils are one of the most potent inflammatory cells in the body and is involved in a number of diseases, most commonly associated with parasitic infections and allergic diseases. Apoptosis in eosinophils is therefore one of the most important systems to avoid inflammation. This aim of the present investigation was to examine the mechanisms behind, and the consequences of this process in eosinophils. Apoptotic eosinophils have a unique surface receptor expression that indicates abilities to communicate with T-, B- and antigen presenting cells. They have a novel expression of CD49f, indicating an importance for binding to laminin or unknown functions of the VLA-6 receptor, possibly in the concept of phagocytosis of the apoptotic cell. In apoptotic eosinophils the granules are translocated to the periphery of the cell, probably through a disruption of the cytoskeleton. This translocation makes the granules easily accessible and the apoptotic eosinophil can release considerable amounts of granule proteins in response to specific stimuli. The spontaneous release however, is decreased as compared with living cells. Furthermore, the survival of eosinophils in response to an allergen challenge is increased in healthy subjects, but not in allergic patients. Mechanistically, this needs further investigation, but one theory is that it is due to the presence of specific IgE in patients in combination with differences in the response from the epithelial cells.

Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon. In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is an important signaling molecule as substrate for the phospholipase C (PLC)-catalyzed formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, and by directly regulating e.g. ion-channels, the cytoskeleton and vesicle trafficking in various types of cells. The present studies provide insights into the regulation of PLC activity and the plasma membrane concentration of PIP2 in individual insulin-secreting cells. Real-time monitoring of plasma membrane PIP2 was performed with evanescent wave microscopy and the PIP2/IP3-binding pleckstrin-homology-domain from PLC-δ1 fused to GFP. It was demonstrated that membrane depolarization and voltage-dependent Ca2+ influx are sufficient to activate PLC. Rise of the glucose concentration triggered Ca2+-dependent activation of PLC. Simultaneous measurements of the cytoplasmic Ca2+ concentration ([Ca2+]i) demonstrated that oscillations of [Ca2+]i resulting from periodic influx induced cyclic activation of PLC. Activation of muscarinic receptors caused a biphasic PLC response with an initial peak enhanced by positive feedback by Ca2+ mobilized from intracellular stores, followed by sustained activity depending on store-operated Ca2+-entry. Activation of PLC by Ca2+ mobilized from intracellular stores was part of the Ca2+-induced Ca2+ release mechanism by which glucagon stimulates primary mouse pancreatic β-cells. Experiments in permeabilized cells demonstrated rapid turnover of PIP2 with t1/2 ~ 16s. ATP stimulated concentration-dependent synthesis of plasma membrane PIP2, counteracted by the ADP analogue ADPβS. RT-PCR analysis identified transcripts of 10 different phosphoinositide-kinases. The ATP-stimulated PIP2 formation was mediated by type II and III PI4-kinases as well as by PIP5-kinase Iβ. It is concluded that the PIP2 concentration in the plasma membrane is regulated by the ATP/ADP ratio and that its hydrolysis by PLC is tightly controlled by [Ca2+]i in insulin-secreting cells.