The islets of Langerhans are small and delicate spheroid organs scattered in the pancreas responsible for insulin production. Transplantation of isolated islets is a beneficial therapy for patients with a severe form of type 1 diabetes. The islets, which normally are richly vascularized in the pancreas, are completely disconnected from the vascular support by the enzymatic digestion during the isolation procedure. Islet viability is affected throughout all steps in this process, from donor death and isolation of islets to culturing and the transplantation process itself. In this thesis a novel strategy to promote islet survival and to re-establish islet vasculature is presented. We show endogenous expression of 51 different genes related to inflammation in cultured islets. Among these genes high expression of MCP-1, MIF, VEGF, thymosin b-10 and IL-8, IL-1β, IL-5R-a, IFN-γ antagonist were found in all donors during the 5- and the 2-day cultures, respectively. Protein expression of these genes can stimulate inflammatory immune responses but also promote tissue repair by attracting curative cells such as endothelial cells (EC) leading to re-establishment of the vasculature. We have established a novel technique by formation of composite islets using EC and mesenchymal stem cells (MSC). EC adhered on the surface of the islets and created a potential blood tolerant surface. The EC-islets showed a degree of protection from the detrimental effects of instant blood-mediated inflammatory reaction (IBMIR) with the major components of IBMIR being decreased in in vitro assays. We combined MSC to the EC-islets with success. The MSC were found to have proliferative effect on EC and the combination of these two cell types on the islets further increased the EC covered surface compared to EC-islets. The EC-MSC-islets in co-culture formed vessel-like structures both into the islets and out to the surrounding matrix. The MSC enhanced the exogenous EC to form vessel-like network in the EC-MSC-islets indicating vascular support by the MSC. The novel strategy and conditions presented herein could alleviate problems related to survival of the islets by promoting revascularization. This would open up a new era in islet transplantation and allow more patients to benefit from this therapy. / Clinical immunology, islet group

Neural stem and progenitor cells (NSPC) can differentiate to neurons and glial cells. NSPC are easily propagated in vitro and are therefore an attractive tool for tissue regeneration. Traumatic brain injury (TBI) is a common cause for death and disabilities. A fundamental problem following TBI is tissue loss. Animal studies aiming at cell replacement have encountered difficulties in achieving sufficient graft survival and differentiation. To improve outcome of grafted cells after experimental TBI (controlled cortical impact, CCI) in mice, we compared two transplantation settings. NSPC were transplanted either directly upon CCI to the injured parenchyma, or one week after injury to the contralateral ventricle. Enhanced survival of transplanted cells and differentiation were seen when cells were deposited in the ventricle. To further enhance cell survival, efforts were made to reduce the inflammatory response to TBI by administration of ibuprofen to mice that had been subjected to CCI. Inflammation was reduced, as monitored by a decrease in inflammatory markers. Cell survival as well as differentiation to early neuroblasts seemed to be improved. To device a 3D system for future transplantation studies, NSPC from different ages were cultured in a hydrogel consisting of hyaluronan and collagen. Cells survived and proliferated in this culturing condition and the greatest neuronal differentiating ability was seen in cells from the newborn mouse brain. NSPC were also used in a model of peripheral nervous system injury, and xeno-transplanted to rats where the dorsal root ganglion had been removed. Cells survived and differentiated to neurons and glia, furthermore demonstrating their usefulness as a tool for tissue regeneration.

During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization. Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process. The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.

Bakgrund: Antalet patienter i behov av njurtransplantation per år är cirka 400-500, varav endast cirka 350 kommer att bli transplanterade. Bristen på organ är tydligt framträdande. I väntan på transplantation behöver patienten dialysbehandling, vilket ofta upplevs vara tidskrävande och utmattande. Sjuksköterskor bör ha kunskap om patienters upplevelser i samband med väntan på transplantation för att kunna utvecklas i sin roll som sjuksköterska och få en djupare förståelse för deras situation. Syfte: Syftet var att beskriva kroniskt njursjuka patienters upplevelser av att leva med dialysbehandling och väntan på transplantation. Metod: Studien är en litteratursammanställning av tio kvalitativa och kvantitativa vetenskapliga artiklar inom området, vilka har granskats, analyserats och sammanställts. Resultat: I resultatet framkom flera olika upplevelser, så som längtan efter frihet, utmattning, höga förväntningar, frustration och rädsla inför framtiden i samband med dialysbehandlingen och väntan på transplantationen. Slutsats: Kroniskt njursjuka patienters upplever sin situation som psykiskt, fysiskt och socialt påfrestande. En av sjuksköterskans viktigaste uppgifter i samband med bemötandet av patienter under dialysbehandling är att vara tillgänglig. Med en tillgänglig och öppen inställning till patienterna blir sjuksköterskan mer mottaglig för patienternas individuella behov. / Background: The number in need of kidney transplants per year is approximately 400-500, of which only about 350 will be transplanted. The shortage of organs is clearly prominent. In anticipation of the transplant patient needs dialysis, which is perceived to be time consuming and exhausting. Nurses should have knowledge of patients' experiences in connection with awaiting transplantation to be able to evolve in her role as a nurse and get a deeper understanding of the patients’ situation. Aim: The aim of this study was to describe patients’ experiences of living with chronic kidney failure with dialysis and awaiting transplantation. Method: The study is a literature compilation of ten qualitative and quantitative scientific articles in the field. The articles have been reviewed, analyzed and compiled. Results: The results revealed several different experiences, such as yearning for freedom, fatigue, high expectations, frustration and fear for the future in connection with the dialysis treatment and awaiting transplantation. Conclusion: Chronic kidney disease patients perceive their situation as mentally, physically and socially stressful. One of the nurse's most important tasks in connection with the treatment of patients in dialysis is to be available. With an accessible and open approach to the patient in general, a nurse becomes more responsive to patient's individual needs.

Väntan på transplantation kan vara en komplex situation för patient och anhörig. Studiens syfte var att belysa vad patienter och deras anhöriga upplever när de står i kö för en organtransplantation. Metoden som användes var en kvalitativ ansats där fyra självbiografier lästes och analyserades. I resultatet framkom att upplevelserna startade med beskedet om att transplantation var nödvändig för den fortsatta överlevnaden, tills beskedet om transplantation kom och patienterna lades in för operation. Analysen av den insamlade datan resulterade i fem kategorier: Insikten om sjukdomens allvar, Mellan hopp och förtvivlan, Tankar kring döden, Samvetskval och Ett efterlängtat besked. Resultatet kan användas till att öka förståelsen hos vårdpersonal för vad patienterna och deras anhöriga genomgår känslomässigt under deras väntan på organtransplantation. / The waiting period for organ transplantation can be a complex situation for patients and their relatives. The purpose of the study was to enlighten the experiences of patients and their relatives when they stand in queue for organ transplantation. With a qualitative attempt four autobiographies were read and analyzed. The result showed that the experiences started with the information that transplantation was essential for the patients’ continuance of life, to when they were hospitalized for the operation. The analysis of the data resulted in five categories: The insight about the gravity of the disease, Between hope and despair, Thoughts surrounding death, Qualms of conscience and A call much longed for. The result can be used to increase the understanding for what patients and their relatives go through emotionally during their wait for organ transplantation among the nursing staff.

Type 1 diabetes affects one in every 400-600 children and adolescents in the US. Standard therapy with exogenous insulin is burdensome, associated with a significant risk of dangerous hypoglycemia, and only partially efficacious in preventing the long term complications of diabetes. Pancreatic islet transplantation has emerged as a promising therapy for type 1 diabetes. However, this cell-based therapy is significantly limited by inadequate islet supply (more than one donor pancreas is needed per recipient), instant blood-mediated inflammatory reaction, and loss of islet viability/function during isolation and following implantation. In particular, inadequate revascularization of transplanted islets results in reduced islet viability, function, and engraftment. Delivery of pro-vascularization factors has been shown to improve vascularization and islet function, but these strategies are hindered by insufficient and/or complex release pharmacokinetics and inadequate delivery matrices as well as technical and safety considerations. We hypothesized that controlled presentation of angiogenic cues within a bioartificial matrix could enhance the vascularization, viability, and function of transplanted islets. The primary objective of this dissertation was to enhance allogenic islet engraftment, survival and function by utilizing synthetic hydrogels as engineered delivery matrices. Polyethylene glycol (PEG)-maleimide hydrogels presenting cell adhesive motifs and vascular endothelial growth factor (VEGF) were designed to support islet activities and promote vascularization in vivo. We analyzed the material properties and cyto-compatibility of these engineered materials, islet engraftment in a transplantation model, and glycemic control in diabetic subjects. The rationale for this project is to establish novel biomaterial strategies for islet delivery that support islet viability and function via the induction of local vascularization.

Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting. To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize. We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) can be induced to express cardiac-specific markers by embryonic cardiomyocytes in vitro. To determine whether this phenomenon occurs in vivo, we have developed a cell transplantation system using zebrafish embryonic recipients. We were unable to isolate expandable zebrafish kidney stromal (ZKS) cells from the kidney, the human BM equivalent; hence, we analyzed the established ZKS1 cell line. We found that ZKS1 expresses stromal genes, but also expresses hematopoietic genes not normally expressed by MSCs. Furthermore, we were unable to differentiate ZKS1 cells into adipocytes, osteoblasts or cardiomyocytes in vitro. We created a transgenic ZKS1(CMV:eGFP) cell line which, after transplantation into zebrafish blastulae, was observed within the host heart, among other tissues. Finally, pT2/S2tnnt2-GM2 and pT2/S2tnnt2-DsRed transposons were generated to mark ZKS1 cardiac differentiation. The zebrafish model established here will be useful for studying the molecular mechanisms of exogenous MSC cardiac differentiation in vivo.

Little is known about the early recovery of functional outcomes in the lung transplant population. This thesis investigated skeletal muscle strength, functional exercise capacity, health-related quality of life and daily physical activity pre- and early post-lung transplantation in a cohort of fifty participants. Significant functional limitations were observed pre-transplant, however levels of physical activity were higher on rehabilitation days as compared to non-rehabilitation days. Post-transplant, improvements in functional exercise capacity and physical activity lagged behind the early improvements in pulmonary function and health-related quality of life. Muscle strength was reduced at hospital discharge compared to pre-transplant levels, but improved to pre-transplant levels by three months post-transplant. In summary, significant functional limitation exists pre-transplant, and lung transplantation leads to significant improvement of functional outcomes; however functional recovery occurs at different time periods and to varying degrees, and does not reach levels of a healthy reference population by three months post-lung transplant.

Ischemia-reperfusion (IR) injury is a severe complication in lung transplantation characterized by inflammation, alveolar damage, and hypoxemia. Alpha 1-antitrypsin (A1AT), a protease inhibitor, is currently used clinically for the treatment of A1AT deficiency emphysema. A1AT has been shown to have the potential to reduce IR injury through its anti-inflammatory and anti-apoptotic effects. We hypothesized that A1AT will ameliorate IR injury through these effects. We tested A1AT in two models of IR: a cell culture model of simulated lung transplantation and a rat in situ pulmonary ligation model. In cell culture, we found that A1AT exerts its protective effects by inhibiting cell death and inflammatory cytokine release in a dose-dependent manner. In the rat pulmonary ischemia-reperfusion model, we found that A1AT improved lung function by inhibiting apoptosis and inflammation. There is potential for future application of A1AT in the treatment of IR injury in lung transplantation.