Transplantation is the golden standard for the treatment of end-stage renal disease. During this process, the transplanted organ is often damaged. In this thesis, we investigated whether activation... Show moreTransplantation is the golden standard for the treatment of end-stage renal disease. During this process, the transplanted organ is often damaged. In this thesis, we investigated whether activation of the complement system, part of our innate immune system, plays a local role. We reviewed that properdin, the only known positive regulator of the complement system, was detected in serum, plasma and urine from patients with various complement-mediated renal diseases. In protocol biopsies obtained 10 days after transplantation, properdin was found deposited in addition to complement activation markers. Next, we showed that dendritic cells secrete properdin and a decrease in properdin levels during dendritic cell- T-cell interaction resulted in reduced T-cell proliferation and activation. We also showed that properdin is able to bind to surfaces of both viable and dead cells, contributing to complement activation. Macrophages can also produce properdin and negative regulators factor H and its splice variant FHL-1. Increasing knowledge on complement factor production by other cells than hepatocytes, including immune cells, hints towards a local role of the complement system in various processes. These findings contribute to a better understanding of the local role of the complement system and are important for the applications of (new) complement-inhibiting drugs. Show less
The aim of this thesis was to develop tools to detect and quantify HLA-specific memory B cells in peripheral blood of HLA-immunized individuals and to assess the applicability of the newly... Show moreThe aim of this thesis was to develop tools to detect and quantify HLA-specific memory B cells in peripheral blood of HLA-immunized individuals and to assess the applicability of the newly developed assays in the setting of clinical transplantation. Transplant patients who have already made a defense response to the foreign HLA on donor organ have an increased risk of developing antibody-mediated rejection, which may adversely affect the survival of the graft. In diagnostic HLA laboratories, serum of the patients is tested for the presence of HLA-specific antibodies before and after transplantation. Serum HLA antibodies are produced by plasma cells located in the bone marrow however, memory B cells can also play a role in antibody production against the donor organ. So far, the role of these cells has been neglected in the diagnostics. The research conducted in my PhD thesis enabled us to develop methods to detect HLA-specific memory B cells. By this means, patients who may potentially harbor HLA-specific memory B cells such as repeat transplant candidates, women receiving transplants from their partner or child and patients undergoing desensitization treatments may benefit from the assays that are described in this PhD thesis. Show less
This thesis emphasizes the presence of minor H antigen specific immune responses directly after birth, which will be present throughout life. The presence of minor H antigen mismatched... Show moreThis thesis emphasizes the presence of minor H antigen specific immune responses directly after birth, which will be present throughout life. The presence of minor H antigen mismatched microchimeric cells obtained through pregnancy from a mother or a child play a crucial role in this. Subsequent immunization against minor H antigens can lead to both cytotoxic and tolerogenic responses. Furthermore HA-1 specific T cells can share the same TCR Vbeta, yet being functionally different. The here performed studies enhances our understanding of immune reactions after HSCT and if applicable after renal transplantation, especially regarding the birth order effect and the assumed less favourable role of women as transplant donors. Show less
Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone... Show moreCurrently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic factors, enabling neuroprotection/tissue sparing in a rat model of spinal cord injury. In this model system, bone marrow stromal cell-mediated tissue sparing leads to motor and sensory function improvements. Moreover, we show that BMSCs__ neuroprotective ability can be enhanced by genetically modifying the cells to overexpress brain-derived neurotrophic factor. However, survival of BMSCs in the injured spinal cord is poor and limits their repair capacity. We investigated the effect of three immunosuppressig agents, Minocycline, Methylprednisolone and Cyclosporine, on macrophage suppression and BMSC survival. All three d rugs effectively reduced the macrophage response, without improving transplanted BMSC survival. Transplanting the cells within the reverse thermal gel ESHU, did significantly improve BMSC survival which was associated with increased tissue sparing and improved functional recovery. These effects are likely due to ESHU__s anti-oxidative properties. Future research will need to focus on combinations of neuroprotective BMSC transplants with axonal regenerating promoting therapies to further optimize BMSC-based therapy for spinal cord injury. Show less
The studies in this thesis describe the systematical search for factors involved in the pathophysiology of human renal I/R injury. Many of the processes assumed to be involved in renal I/R injury... Show moreThe studies in this thesis describe the systematical search for factors involved in the pathophysiology of human renal I/R injury. Many of the processes assumed to be involved in renal I/R injury based on animal studies could not be confirmed in our clinical study in humans. However, we found new evidence of complement activation and endothelial cell activation in human renal I/R injury. Moreover, there were large differences between deceased and living donor kidneys; brain dead donor kidneys have a unique proinflammatory cytokine release after reperfusion. Finally it appears that both brain dead and cardiac dead donor kidneys are not able to upregulate their metabolism-related genes upon reperfusion as living donor kidneys do, indicating that failure to restart metabolism may be a factor expanding I/R injury. All of these findings contribute to the understanding of renal I/R injury in humans and instigate the further search for therapeutical modalities to limit renal I/R injury. Show less
Transplant recipients generally require lifelong treatment with immunosuppressive medication to prevent rejection of the graft by their immune system. Inhibitors of the enzyme calcineurin,... Show moreTransplant recipients generally require lifelong treatment with immunosuppressive medication to prevent rejection of the graft by their immune system. Inhibitors of the enzyme calcineurin, including cyclosporin A and tacrolimus, constitute a very potent class of immunosuppressants that has revolutionized transplant medicine. However, their reputation has been showing cracks due to the severe side-effects associated with long-term use of these drugs, including an explosively increased risk of developing skin cancer. The pathophysiological mechanism of this phenomenon is not known, although a number of hypotheses have been put forward. In this dissertation, we show that oxidative stress, mainly derived from exposure to UVA radiation, may locally augment the effects of the calcineurin inhibitors; we propose that overly strong suppression of calcineurin activity may result in malignancy formation due to disruption of tumor-suppressive signaling pathways or disturbed immunosurveillance in skin. Show less
Pancreas transplantation and islet of Langerhans transplantation are potential solutions to treat patients with type 1 diabetes. However, pancreas grafts are scarce and there is a shortage of donor... Show morePancreas transplantation and islet of Langerhans transplantation are potential solutions to treat patients with type 1 diabetes. However, pancreas grafts are scarce and there is a shortage of donor pancreata relative to the number of patients needing a transplant. The aim of this thesis was to further optimize pancreas graft survival in pancreas transplantation and to optimize islet isolation outcomes in islet of Langerhans transplantation, leading to better use of available organs. The focus in pancreas transplantation should be on optimizing recipients to improve graft survival and on improving quality of pancreata procured by centers not performing pancreas transplantation (for example, by training procurement surgeons to optimize pancreas procurement, thereby increasing the number of transplantable organs. In islets transplantation, it is recommended that the reporting of donor, pancreas and isolation factors should become more standardized, which would enable us to determine more accurately which factors are important predictors for islet isolation outcome. Furthermore, if more biomedical factors (e.g. the presence of hyperemic islets) would be reported in addition to the other factors, we would be able to assess the independent effect of these biomedical factors for islet isolation outcome and eventually the effect on islet transplantation in the clinical setting. Show less
This thesis reports the isolation, characterization and allotransplantation in porcine retina of ciliary epithelium (CE)-derived cells, also known as retinal stem cells (RSCs). The self-renewal... Show moreThis thesis reports the isolation, characterization and allotransplantation in porcine retina of ciliary epithelium (CE)-derived cells, also known as retinal stem cells (RSCs). The self-renewal capacity and differentiation potential of these cells in vitro and in vivo makes them candidate donors in cell replacement approaches for treating retinal degenerative diseases. The use of pig as the animal model should facilitate translation of these findings to applications for human conditions. The work described in this thesis shows that cultures of self-renewing cells can be obtained in vitro from the newborn porcine CE and that these cells can differentiate into retinal and RPE phenotypes both in vitro and in vivo after transplantation in adult pigs. However, the nature and physiological role of these cells in potential endogenous repair mechanisms still remain to be explored. Notably, their remarkable plasticity in vitro makes them an interesting cell model to study this phenomenon of recent identification. Show less
This thesis describes the clinical significance of thebiomarker C4d, a split product of the complement system, in several manifestations of systemic autoimmunediseases such as SLE and... Show moreThis thesis describes the clinical significance of thebiomarker C4d, a split product of the complement system, in several manifestations of systemic autoimmunediseases such as SLE and antiphospholipid syndrome. The findings in this thesis suggest that this biomarker might be of use in unraveling disease mechanisms where complement activation is involved, as well as serving as a diagnostic tool to detect patients at risk of antibody mediated tissue injury Show less
Outline of the Thesis Part I Pneumocystis in kidney transplant recipients: transmission, risk factors , new diagnostic and chemo-prophylactic strategies. Chapter 2 describes the characteristics of... Show moreOutline of the Thesis Part I Pneumocystis in kidney transplant recipients: transmission, risk factors , new diagnostic and chemo-prophylactic strategies. Chapter 2 describes the characteristics of a large outbreak of Pneumocystis pneumonia among kidney transplant recipients. By performing a classical outbreak investigation and by application of new molecular genotyping techniques, the potential of the __interhuman transmission hypothesis__ is addressed and discussed. In Chapter 3 all currently available data on reported outbreaks of Pneumocystis pneumonia is systematically reviewed with the emphasis on mortality data, clinical risk factors and transmission analyses. In the case-control study described in Chapter 4, we performed a detailed risk factor analysis for development of PCP in kidney transplant recipients and used the multivariate output data to estimate the effects of several chemoprophylactic strategies by modeling the expected incidence and number-needed-to-treat to provide efficient PCP chemoprophylaxis over a 2-year period post transplantation. Chapter 5 reports the data of a prospective study on the serum markers S-adenosylmethionine and (1-->3)-_-D-glucan serum levels and correlation with clinical parameters in HIVnegative immunocompromised patients __ the majority kidney transplant recipients - with Pneumocystis pneumonia. Potential applicability for treatment monitoring and assessment of P. jirovecii pulmonary load is also discussed. Part II Genetic predisposition for development of invasive aspergillosis in stem cell transplant recipients Chapter 6 describes a multicenter study on the impact of the Y238X stop mutation in the human Dectin-1 receptor (which senses and attaches to glucan moieties of the fungal cell wall) on the risk of development of invasive aspergillosis in stem cell transplant recipients. In Chapter 7 a retrospective study of the influence of genetic variation in the macrophage activation route with respect to the relative additional risk for development of invasive aspergillosis is presented. Part III Experimental markers for detection of fungal infection: scintigraphic imaging. In Chapter 8 the clinical applicability of radiolabeled antimicrobial peptides and antifungal drugs for the diagnosis of invasive fungal infections is reviewed, together with a concise discussion about how promising agents should be further developed. The results of the thesis are summarized and discussed in Chapter 9. Show less
The research described in this thesis investigates the effect of viral infection on the risk of rejection. It was previously thought that a viral specific T-cell receptor could not crossreact... Show moreThe research described in this thesis investigates the effect of viral infection on the risk of rejection. It was previously thought that a viral specific T-cell receptor could not crossreact against allogeneic HLA molecules, however here it is proven that this is not only possible but is also common. The research performed in this thesis has important clinical implications for solid organ (kidney) and bone marrow transplantation. Viral infection commonly generates alloreactive T-cells, and may therefore be responsible for the failure of tolerance inducing regimens in primates and humans. It is shown that vaccination can also induce HLA-specific alloreactive T-cell responses and it is therefore recommended that vaccines be given to transplantation candidates at least three months prior to expected transplantation date. It is also shown that alloreactive T-cells can be tissue specific. In the final chapter of the thesis thesis it is shown that allogeneic cell therapy could conversely be useful to elicit a viral specific T-cell response in patients with infections, such as HIV. Show less
Over the last decades, restoration of renal function by renal allograft transplantation has evolved into the preferred treatment option for patients with end stage renal disease. The introduction... Show moreOver the last decades, restoration of renal function by renal allograft transplantation has evolved into the preferred treatment option for patients with end stage renal disease. The introduction of the calcineurin inhibitors (CNI) cyclosporine and tacrolimus have significantly contributed to this success. Adverse drug effects, together with the large inter-individual variation in pharmacokinetics of both drugs necessitates therapeutic drug monitoring (TDM). Nowadays, TDM is routinely performed by drug concentration measurement in blood. Unfortunately, the incidence of acute allograft rejection episodes is still 10-20% within first year after transplantation. A strategy to improve clinical immunesuppresion early after transplantation is improved monitoring. Next to advanced pharmacokinetic monitoring, such as estimated AUC monitoring, the development of pharmacodynamic markers could theoretically contribute to improve CNI therapy. Pharmacodynamic monitoring strategies, however, are still in an experimental phase and have not proven clinical benefit yet. They carry the theoretical advantage of monitoring the true effectiveness of immunosuppressive therapy. This led us to investigate pharmacodynamic monitoring as potential tool to guide drug dosing. We choose calcineurin activity as pharmacodynamic marker for monitoring and in this thesis, the analytical aspects, fundamental characteristics and insights in clinical usefulness of calcineurin activity measurement as a pharmacodynamic marker for CNI were investigated. Show less
Antibody-mediated rejection is increasingly recognised within the transplantation community as a cause or contributing factor in the rejection of transplanted organs. The humoral immune response... Show moreAntibody-mediated rejection is increasingly recognised within the transplantation community as a cause or contributing factor in the rejection of transplanted organs. The humoral immune response towards allografts involves B cells that, after T cell dependent activation, can differentiate into antibody producing cells. Whereas there are several therapeutic entities to treat cellular rejection, at present, no standard treatment for humoral rejection exists. The first part of this thesis describes the effects of different maintenance immunosuppressive drugs directly on B cells, as well as the effect on T cell help in vitro. The data presented in these chapters show differential effects of the drugs on B cells and T cell help. Furthermore, the effects of novel, experimental drugs on the in vitro activation of B cells are described. The second part of this thesis describes the development of a novel technique for monitoring the humoral alloimmune response. By using this technique, patients at risk for humoral rejection may be identified. The last part describes the difficulties and pitfalls of monitoring T cells that respond to alloantigen via the indirect pathway of allorecognition. In an alloimmune response, these are the T cells that provide help to B cells to become fully activated. Show less
Blood transfusions can lead to immunization or tolerance in the recipient. The latter is characterized by an improved transplant outcome after pretransplant blood transfusions. First observations... Show moreBlood transfusions can lead to immunization or tolerance in the recipient. The latter is characterized by an improved transplant outcome after pretransplant blood transfusions. First observations of improved kidney graft outcome after blood transfusion date 35 years back, however no exclusive immunological mechanism has been found yet. At the Leiden University Medical Center, patients on the waiting list for simultaneous pancreas-kidney transplantation (SPKT) received a non-leukocyte depleted, HLA-DR shared blood transfusion before transplantation. We showed that a pretransplant blood transfusion was able to diminish the severity of acute rejection episodes after SPKT. The need for HLA-DR sharing suggests a role for the indirect allorecognition. Our in vitro experiments focusing on the indirect allorecognition and on several subsets of recipient immune cells, showed that a pretransplant blood transfusion activates the recipient__s immune system. It is still not clear if and how this activation state can lead to transplantation tolerance. Additionally, there may be a role for microchimeric donor cells in the immunological mechanism leading to tolerance. Pretransplant blood transfusions may still be of clinical benefit in these times of potent immunosuppressive drug use, but requires the support of well-designed RCTs as well as more insight into the immunological mechanism. Show less
__Bones can break, muscles can atrophy, glands can loaf, even the brain can go to sleep, without immediately endangering our survival, but when the kidneys fail to manufacture the proper kind of... Show more__Bones can break, muscles can atrophy, glands can loaf, even the brain can go to sleep, without immediately endangering our survival, but when the kidneys fail to manufacture the proper kind of blood neither bone, muscle, gland nor brain can carry on__. This quote from Homer Smith's book 'From Fish to Philosopher' indicates the importance of the kidney for the maintenance of the harmonious composition of body fluids __ a function the kidney performs in a way that joins efficacy with elegance. Some of these processes are lined out in this thesis. The first chapter gives an introduction to kidney development, anatomy, and function, as well as a description of malfunction of the kidney, focusing on the development of proteinuria and failure of kidney transplants. The subsequent chapters describe studies that have been performed to investigate the biological mechanism that underly the development of proteinuria and long-term renal transplant failure. Show less
The term __chimerism__ originates from Greek mythology and refers to the creature Chimaera, whose body was in front a lion, the back a serpent and the midsection a goat. In medicine, the term... Show moreThe term __chimerism__ originates from Greek mythology and refers to the creature Chimaera, whose body was in front a lion, the back a serpent and the midsection a goat. In medicine, the term chimerism refers to an individual, organ or part consisting of tissues of diverse genetic constitution. Pregnancy, blood transfusion and organ transplantation are potential causes of chimerism. In this thesis the occurrence of chimerism is investigated in different organs of healthy women, of women with the autoimmune disease Systemic Lupus Erythematosus (SLE) and of women that received a renal allograft. To demonstrate chimerism, male cells were detected in female organs by using in situ hybridization of the Y chromosome. Chimerism was found in 18% of healthy organs, in about 50% of organs derived from women with SLE and in none of the skin tumors investigated from female renal allograft recipients. In various organ types and both in women with and without sons and women with and without a transfusion history, chimerism was present. In this thesis we describe these results and review data from the ancient and recent literature. With all these data in hand, we speculate about the sources of chimerism and its implications on immunity. Show less
The aim of this thesis was to unravel the role of the humoral immune system in rheumatoid arthritis patients by employing new immunosuppressive strategies, i.e. specific B-cell depletion with... Show moreThe aim of this thesis was to unravel the role of the humoral immune system in rheumatoid arthritis patients by employing new immunosuppressive strategies, i.e. specific B-cell depletion with Rituximab and non-specific lymfoablative treatment with high dose chemotherapy and hematopoeietic stem cell transplantation. This thesis evaluates the clinical benefit of these strategies as well as the immunological changes that coincide with clinical improvement. By combining clinical outcome with immunological parameters of the humoral immune system, these studies provide a unique approach to investigatepathologic mechanisms in rheumatoid arthritis. Show less
Successful tolerance induction therapies in rodents are for the most part unsuccessful in larger primates. Costimulation blockade by anti-CD40 or anti-CD40 + anti-CD86 in the life-supporting kidney... Show moreSuccessful tolerance induction therapies in rodents are for the most part unsuccessful in larger primates. Costimulation blockade by anti-CD40 or anti-CD40 + anti-CD86 in the life-supporting kidney allograft model in the rhesus monkey prevented graft rejection during treatment but did not induce tolerance. Costimulation blockade has to be integrated in conventional immunosuppressive therapies. However, conventional immunosuppressive drugs may antagonize the immunoregulatory effects of costimulation blockade. We describe how costimulation blockade prevented graft rejection in the immediate post transplantation period. CsA treatment was initiated only after 42 days post transplantation and this had a beneficial effect on graft survival, resulting in two of 4 monkeys surviving long-term without additional immunosuppressive treatment. Furthermore we found no beneficial effect of ATG induction therapy on costimulation blockade treatment. ATG induced rapid reappearance of CD8+ memory T-cells in the peripheral blood, possibly responsible for the observed accelerated rejection. Infiltrating cells in kidney graft biopsies and tissues revealed high expression of FOXP3 and other regulatory T-cell markers during rejection. We also have described the phenotypic and functional characteristics of naturally occurring regulatory T-cells in rhesus monkeys. Show less
The ultimate goal in the transplantation field is the induction and maintenance of donor specific tolerance. Treg cells that control immune responses to alloantigens give opportunities for... Show moreThe ultimate goal in the transplantation field is the induction and maintenance of donor specific tolerance. Treg cells that control immune responses to alloantigens give opportunities for tolerogenic therapies in transplantation. However, it is important to investigate the mechanisms of tolerance induction in order to use the optimal strategy. Therefore, we exploded both natural towards NIMA that can be induced during fetal life and induced tolerance by modulation of DC. Naturrally induced tolerance towards NIMA can have an influence on transplant outcome later in life. In this thesis we explored the influence of NIMA on the alloreactive T cells repetoire in healthy individuals and additionally we focused on the NIMA effect in patients transplanted with a NIMA haplotype mismatched kidney graft. In order to actively induce tolerance, we modulated DC to generate Treg cells, since this may be of clinical relevance in the future for patients that are on the waiting list for transplantation. In this thesis we explored the possibility of using modulated DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. Furthermore we describe an in vitro system for the use of human modulated DC to induce Treg cells. We show that two differentially modulated human DC can lead to different types of Treg cells. Finally, we examined the possibility to use in vitro tools to measure a possible tolerant state in patients. Monitoring of e.g. Treg cells and/or cytokines may give an indication which patients are at risk for rejection and which patients are more predisposed to tolerance. We describe the Elispot technique as a possible tool to monitor patients that received a renal allograft. In conclusion , this thesis contributes to the fundamental understanding of both natural and induced tolerance in transplantation and gives a handhold for future research. As donor-specific tolerance is still far away from the clinic, the in vitro monitoring tool described in this thesis may contribute to the optimalization of immunosuppressive therapies in transplant recipients. Show less
In this thesis, the possibilities and limitations of cell-based therapies after spinal cord injury are explored. Particularly, the potential of adult derived neural progenitor cell (NPC) grafts to... Show moreIn this thesis, the possibilities and limitations of cell-based therapies after spinal cord injury are explored. Particularly, the potential of adult derived neural progenitor cell (NPC) grafts to function as a permissive substrate for axonal regeneration was investigated. It was found that syngenic adult derived neural progenitor cells are able to survive transplantation in the acutely lesioned spinal cord and differentiate into glial phenotypes. When co-grafted with fibroblasts, glial fibrillary acidic protein expressing grafted NPC are able to replace the lesion defect and are able to induce contact mediated axon guidance and regenerative sprouting. NPC that are co-grafted with highly purified Schwann cells however migrate away from the lesion site, which is paralleled with a reduced axonal outgrowth. A close investigation of NPC that are transduced to express ectopic genes by using different viral vectors revealed that in vivo gene expression in genetically engineered neural progenitor cells is temporally limited and mostly restricted to undifferentiated NPC. Finally, MRI imaging at 17.6 Tesla was used to in vivo monitor structural changes following spinal cord injury in rats, enabling future longitudinal studies that allow direct correlation of structure with function. Show less
