<p>Transplantation of islets of Langerhans is a potential treatment of type 1 diabetes that aims to restore normal glucose homeostasis. Microencapsulation of islets could enable transplantation in the absence of immunosuppression, which would be beneficial as the side effects associated with immunosuppression outweigh the potential benefits of islet transplantation. Alginate is a polysaccharide that can be harvested from brown algae and is often used for microencapsulation of cells.</p><p>The aim of this study was to evaluate alginate/poly-L-lysine/alginate capsules with regard to their biocompatibility and permeability to cytokines. Moreover, the function of microencapsulated islets was studied <i>in vitro</i> as well as their ability to reverse hyperglycaemia in diabetic mice.</p><p>Microencapsulated rodent islets functioned well <i>in vitro</i>, with similar insulin release rates and glucose oxidation rates as naked islets. However, when cultured with interleukin-1β and tumour necrosis factor-α, microencapsulated islets were functionally suppressed, showing that the capsules are permeable to these cytokines. The biocompatibility of capsules varied depending on their composition. The presence of poly-L-lysine in the capsule decreased the biocompatibility. However, the biocompatibility of the capsules was improved when the coating alginate had been epimerised, i.e. enyzmatically tailored. Transplantation of microencapsulated allogeneic islets to immune competent mice lowered blood glucose concentrations up to 1 month after implantation. The success of the microencapsulated islet graft depended on the composition of the alginate/poly-L-lysine/alginate capsule used, as capsules that had poor biocompatibility failed to reverse hyperglycaemia more than transiently in athymic nude mice.</p><p>In conclusion, alginate/poly-L-lysine/alginate capsules can protect islets of Langerhans from allogeneic rejection in mice. However, the composition of the capsule is of critical importance in the success of transplantation. Epimerised alginates may provide a novel capsule with ideal properties for microencapsulation of islets of Langerhans.</p>

<p>The airway epithelium plays a crucial role in protecting the underlying connective tissue (CT) from noxious agents. Damage and shedding of the epithelium are observed in the airways of asthma, cystic fibrosis and rhinitis patients. The aim of this study was to investigate the mechanisms by which epithelial damage occurs, and the consequences of such damage for the inflammatory process in the airways. In this study, cultured normal human bronchial epithelial cells, excised rat tracheae, and cultured murine mast cells were used as model systems. Metabolic alterations, morphological changes and cell-cell contact stabilities were investigated.</p><p>The T-helper (Th)-1 cytokines, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin (IL)-1β were found to be pro-inflammatory, leading to major morphological changes, inhibitions in desmosome formation, and accelerated cell death. The Th2 cytokines, IL-4, IL-5, and IL-13 were found to cause no changes in cell death, nitric oxide levels and desmosome formation but instead an increase in proliferation, therefore were anti-inflammatory in this respect.</p><p>Increasing the osmolarity of the airway surface liquid (ASL) altered the integrity of the tight junction (TJ) and allowed a 4-kDa compound to penetrate the epithelial layer and access the CT. This effect was reversible if the ASL was returned to 295 mOsm. Intentionally breaking the TJ with EGTA and subsequent osmolar changes in ASL demonstrated the importance of TJ and the fragility of the CT under hyperosmolar stress, leading to a disrupted CT with larger capillaries and altered elemental ion content and epithelial denudation. </p><p>Hydrocortisone was shown to downregulate IL-4-induced IL-6 upregulation in murine mast cells. Furthermore, incubating mast cells with hydrocortisone lead to a new subpopulation that was morphologically unique, that displayed new cell surface markers (CD44 and CD61) and that lacked CD54. These changes modify the interactions of mast cells with surrounding cells in the CT and epithelium.</p><p>In conclusion, the balance between pro- and anti-inflammatory cytokines and ASL osmolarity may influence the role of the airway epithelium as a barrier. The pharmacological use of hyperosmolarity to disrupt TJ reversibly may help facilitate the delivery of drugs through the airway epithelium.</p>

<p>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.</p><p>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.</p><p>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.</p><p>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.</p>

<p>Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. </p><p>The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. </p><p>We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. </p><p>Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. </p><p>In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.</p>

<p>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.</p><p>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.</p><p>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.</p>

<p>Diabetes mellitus is a bihormonal disorder with hyperglycemia due to deficiency of insulin and hypersecretion of glucagon. To improve diabetes treatment it is important to clarify the signal transduction of glucagon secretion. The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), an important determinant of hormone secretion, and the membrane potential were recorded in individual mouse α-cells. Glucagon and insulin secretion were measured from mouse islets and glucagon secretion from hamster glucagonoma cells. Glucose inhibited glucagon secretion from islets and glucagonoma cells with maximal effect at 7 mM, indicating a direct action on the α-cells. High concentrations of glucose paradoxically stimulated glucagon secretion. Whereas glucose inhibition of glucagon release was associated with lowering of [Ca²⁺]_i, stimulation of secretion at high glucose concentrations was Ca²⁺-independent. Adrenaline, which is a potent stimulator of glucagon secretion, increased [Ca²⁺]_i by α₁- and β-adrenergic mechanisms involving mobilization of intracellular Ca²⁺ from the endoplasmic reticulum (ER) and influx of the ion across the plasma membrane. Ca²⁺ mobilization could be attributed to generation of inositol 1,4,5-trisphosphate and cAMP, and influx occurred through voltage-dependent L-type channels activated by a depolarizing store-operated current. Glucose hyperpolarized the α-cells and inhibited adrenaline-induced [Ca²⁺]_i signalling. At 3 mM, glucose had a pronounced stimulatory effect on Ca²⁺ sequestration in the ER, shutting off store-operated Ca²⁺ influx. The α-cells express ATP-regulated K⁺ channels but pharmacological blockade of these channels neither interfered with the hyperpolarizing and [Ca²⁺]_ilowering effects of glucose nor with the inhibition of glucagon secretion. In contrast, activation of the depolarizing store-operated mechanism prevented glucose-induced, hyperpolarization, lowering of [Ca²⁺]_i and inhibition of glucagon secretion. It is proposed that adrenaline stimulation and glucose inhibition of glucagon release involve modulation of a store-operated depolarizing current. The U-shaped dose response relationship for glucose-regulated glucagon secretion may explain the hyperglucagonemia in diabetes.</p>

Transplantation of islets of Langerhans is a potential treatment of type 1 diabetes that aims to restore normal glucose homeostasis. Microencapsulation of islets could enable transplantation in the absence of immunosuppression, which would be beneficial as the side effects associated with immunosuppression outweigh the potential benefits of islet transplantation. Alginate is a polysaccharide that can be harvested from brown algae and is often used for microencapsulation of cells. The aim of this study was to evaluate alginate/poly-L-lysine/alginate capsules with regard to their biocompatibility and permeability to cytokines. Moreover, the function of microencapsulated islets was studied in vitro as well as their ability to reverse hyperglycaemia in diabetic mice. Microencapsulated rodent islets functioned well in vitro, with similar insulin release rates and glucose oxidation rates as naked islets. However, when cultured with interleukin-1β and tumour necrosis factor-α, microencapsulated islets were functionally suppressed, showing that the capsules are permeable to these cytokines. The biocompatibility of capsules varied depending on their composition. The presence of poly-L-lysine in the capsule decreased the biocompatibility. However, the biocompatibility of the capsules was improved when the coating alginate had been epimerised, i.e. enyzmatically tailored. Transplantation of microencapsulated allogeneic islets to immune competent mice lowered blood glucose concentrations up to 1 month after implantation. The success of the microencapsulated islet graft depended on the composition of the alginate/poly-L-lysine/alginate capsule used, as capsules that had poor biocompatibility failed to reverse hyperglycaemia more than transiently in athymic nude mice. In conclusion, alginate/poly-L-lysine/alginate capsules can protect islets of Langerhans from allogeneic rejection in mice. However, the composition of the capsule is of critical importance in the success of transplantation. Epimerised alginates may provide a novel capsule with ideal properties for microencapsulation of islets of Langerhans.

The airway epithelium plays a crucial role in protecting the underlying connective tissue (CT) from noxious agents. Damage and shedding of the epithelium are observed in the airways of asthma, cystic fibrosis and rhinitis patients. The aim of this study was to investigate the mechanisms by which epithelial damage occurs, and the consequences of such damage for the inflammatory process in the airways. In this study, cultured normal human bronchial epithelial cells, excised rat tracheae, and cultured murine mast cells were used as model systems. Metabolic alterations, morphological changes and cell-cell contact stabilities were investigated. The T-helper (Th)-1 cytokines, tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin (IL)-1β were found to be pro-inflammatory, leading to major morphological changes, inhibitions in desmosome formation, and accelerated cell death. The Th2 cytokines, IL-4, IL-5, and IL-13 were found to cause no changes in cell death, nitric oxide levels and desmosome formation but instead an increase in proliferation, therefore were anti-inflammatory in this respect. Increasing the osmolarity of the airway surface liquid (ASL) altered the integrity of the tight junction (TJ) and allowed a 4-kDa compound to penetrate the epithelial layer and access the CT. This effect was reversible if the ASL was returned to 295 mOsm. Intentionally breaking the TJ with EGTA and subsequent osmolar changes in ASL demonstrated the importance of TJ and the fragility of the CT under hyperosmolar stress, leading to a disrupted CT with larger capillaries and altered elemental ion content and epithelial denudation. Hydrocortisone was shown to downregulate IL-4-induced IL-6 upregulation in murine mast cells. Furthermore, incubating mast cells with hydrocortisone lead to a new subpopulation that was morphologically unique, that displayed new cell surface markers (CD44 and CD61) and that lacked CD54. These changes modify the interactions of mast cells with surrounding cells in the CT and epithelium. In conclusion, the balance between pro- and anti-inflammatory cytokines and ASL osmolarity may influence the role of the airway epithelium as a barrier. The pharmacological use of hyperosmolarity to disrupt TJ reversibly may help facilitate the delivery of drugs through the airway epithelium.

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.

Neuroblastoma is one of the most common solid cancers of early childhood. In Sweden, approximately 10-15 cases occur annually. The overall five-year neuroblastoma survival in Europe is approximately 45%. Since cancer treatment involves drugs with risks of side effects in the growing child, there is a need for more effective and less toxic drugs. One new approach in cancer treatment is inhibition of tumor angiogenesis, i.e., of new blood vessel growth into the tumor. An angiogenesis inhibitor may be combined with cytostatic drugs to enhance the efficacy. The aim of this study was to investigate how drugs could be used to inhibit angiogenesis and tumor growth in a xenograft model of human neuroblastoma in nude mice. The tumors express the angiogenesis stimulator vascular endothelial growth factor (VEGF) on both protein and mRNA levels. The angiogenesis inhibitors SU5416 (an inhibitor of VEGF signalling) and TNP-470 (an inhibitor of endothelial cell proliferation) inhibited angiogenesis in our model. TNP-470, however, inhibited angiogenesis without significant reduction of the tumor growth, in contrast to SU5416. We also discovered that the cytostatic drug CHS 828 could cause regression of neuroblastoma tumors in the model when given orally at a low daily dose, alone or in combination with the angiogenesis inhibitor SU5416 or TNP-470. Furthermore, a new use of the cardiac glycoside digoxin was found. Digoxin inhibited FGF-2 -stimulated bovine capillary endothelial cell growth in vitro, and inhibited angiogenesis in vivo in the chick chorioallantoic membrane assay (CAM). It also inhibited neuroblastoma growth by approximately 50% in our neuroblastoma model. In conclusion, CHS 828 and digoxin represent two classes of drugs with potent antitumor effects that may be valuable in treatment of neuroblastoma, either alone or in combination with angiogenesis inhibitors.