It is important to account for all sources of uncertainty when evaluating the clinical or cost-effectiveness of health care technologies. Therefore, this thesis takes as its basis a cost-effectiveness study in liver transplantation and identifies two previously unexplored issues that can arise in clinical and cost-effectiveness studies. A literature review of studies evaluating the effectiveness, costs or cost-effectiveness of solid organ transplantation confirmed that these issues were important and relevant to other transplantation studies. The first issue concerns the selection of an appropriate method for estimating mean study costs in the presence of incomplete (censored) data. Twelve techniques were identified and their accuracy was compared across artificially created mechanisms and levels of censoring. Lin's method with known cost histories and short interval lengths is recommended for accurately estimating mean costs and their uncertainty. It is assumed that these findings are generalisable to any solid organ transplant study where censoring is an issue. The second issue explored in this thesis relates to methods for measuring uncertainty around survival, HRQL and cost estimates derived from prognostic models in the absence of observed data. Probabilistic sensitivity analysis is recommended for measuring prognostic model parameter uncertainty and estimating individual patient outcomes and their uncertainties, as it is able to incorporate the additional uncertainty from using prognostic models to estimate control group outcomes. This thesis shows the quantitative importance of these issues and the methodological guidance offered should enable decision makers to have more confidence in clinical and cost-effectiveness estimates. Providing decision makers with a fuller estimate of the uncertainty around clinical and cost effectiveness estimates will aid them in decisions about the necessity of conducting further research in to the clinical or cost-effectiveness of health care technologies.

No description available.

HLA-G is a protein normally expressed during pregnancy, protecting the fetus from the maternal immune system. Previous studies have shown an association between HLA-G expression post-transplantation and lower incidences of organ rejection. To further examine this beneficial role, we conducted a prospective study following a cohort of heart transplant recipients for one year and measuring their plasma HLA-G levels at various time points. Preliminary analyses failed to reveal an association between HLA-G and various parameters of rejection and vasculopathy. However, we decided to examine the in vitro effects of HLA-G in a smooth muscle cell (SMC) migration assay and whether HLA-G can be modulated pharmacologically. We made the novel observations that purified HLA-G was capable of inhibiting migration of SMCs, a key event in the development of cardiac allograft vasculopathy. IL-10, an anti-inflammatory cytokine, was capable of upregulating HLA-G in a Jeg-3 cell line. The modulation of HLA-G may represent a strategy to protect again vasculopathy, which is a leading cause of morbidity and mortality in heart transplant recipients.

HLA-G is a protein normally expressed during pregnancy, protecting the fetus from the maternal immune system. Previous studies have shown an association between HLA-G expression post-transplantation and lower incidences of organ rejection. To further examine this beneficial role, we conducted a prospective study following a cohort of heart transplant recipients for one year and measuring their plasma HLA-G levels at various time points. Preliminary analyses failed to reveal an association between HLA-G and various parameters of rejection and vasculopathy. However, we decided to examine the in vitro effects of HLA-G in a smooth muscle cell (SMC) migration assay and whether HLA-G can be modulated pharmacologically. We made the novel observations that purified HLA-G was capable of inhibiting migration of SMCs, a key event in the development of cardiac allograft vasculopathy. IL-10, an anti-inflammatory cytokine, was capable of upregulating HLA-G in a Jeg-3 cell line. The modulation of HLA-G may represent a strategy to protect again vasculopathy, which is a leading cause of morbidity and mortality in heart transplant recipients.

Background: Management of a transplant recipient involves the use of multiple immunosuppressant drugs. Currently there is no test that reflects the overall immune status of the patient. This results in under or over suppression of the immune system and consequently increases in morbidity and mortality rates. Evaluation of the proliferative response of PBMC's to a mitogen PHA by measurement of intracellular ATP was evaluated as a tool to assess the immune response in kidney transplant patients. Method: PBMC's were separated from the blood samples of healthy controls and kidney transplant patients on cyclosporine, sirolimus, and tacrolimus based regimens by density gradient centrifugation, cells were counted and incubated overnight with and without PHA. The luciferin-Iuciferase enzyme reaction which induces bioluminescence and the Turner Biosystem luminometer were used to measure intracellular ATP levels in relative light units (RLU). An A TP standard curve was generated for each test. Results: The ATP (nglml) levels measured in the transplant recipients were lower and statistically significantly different (p< 0.0001) than the healthy controls. No statistically significant difference was measured between the cycIosporine and sirolimus drug groups. Patients on tacrolimus gave a statistically significant (p<O.0001) stronger immune response than those receiving cyclosporine and sirolimus. Overall, the immune response results of kidney transplant patients were statistically significantly lower than the healthy control by 981 nglml. Linear regression analysis revealed no correlation between patient A TP (nglml) levels and therapeutic drug blood levels, immunosuppressant drug dosages, creatinine levels and white cell counts. The immune responses of patients who were diagnosed with infection or were clinically stable were characterised as low or moderate, of interest, one patient who was diagnosed with rejection was found to have a strong immune response (>501 nglml ATP). Conclusion: Future studies to determine the predictive value of the A TP assay in directing immunosuppressive therapy are required. The assay described in this study is simple, sensitive and rapid and has possible application in immunological monitoring in a variety of conditions that affects the immune system. Keywords: kidney transplantation, immunosuppression, bioluminescence, lymphocyte, Adenosine Triphosphate (A TP), Phytohemmagglutinin (PHA) / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.

Islet transplantation is a more physiological way to treat type 1 diabetes. However, shortage of donor tissue and chronic administration of immune suppressive drugs has limited the widespread application of this therapy for all patients with type 1 diabetes, particularly children suffering from this disease. Xenogeneic islet transplantation particularly neonatal porcine islets (NPI) holds promise for clinical transplantation because of the potentially unlimited supply of islets. New evidence suggests that monoclonal antibodies (mAbs) specific for immune cell surface molecules could be employed in the prevention of islet graft rejection as well as induction of immunological tolerance to the transplanted grafts without the need for continuous administration of harmful immune suppressive drugs. It was shown by our group that short-term administrations of a combination of anti-LFA-1 and anti-CD154 mAbs which targets both adhesion and costimulatory pathways of T cell activation, is highly effective in preventing NPI xenograft rejection. In this thesis, we determined whether short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs could induce tolerance to NPI xenografts. Our data show that this combination of mAbs can induce dominant, species and tissue specific tolerance to NPI xenografts which is mediated by regulatory T cells in non-autoimmune prone B6 mice. We also found that T cell subsets such as CD4+ and CD8+ T cells as well as antigen presenting cells (APC) play an important role in the induction and maintenance of tolerance to NPI xenografts. In addition we found that PD-1/PDL interaction is important for induction and maintenance of tolerance to NPI xenografts. Finally, we found that this combined mAb therapy was effective in preventing NPI xenografts rejection in autoimmune prone NOD mice when it was combined with anti-CD4 mAb. It is may hope that the research presented in this thesis will provide insight into the nature of the immune responses to xenogeneic islet transplantation in humans and aid in the development of effective, tolerance inducing therapies, so that patients with T1DM will once again know a life free from their disease. / Experimental Surgery

Thesis (M Nursing)--University of South Australia, 1997

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes a life-long presence in the infected host. The adaptive immune response is indispensable in controlling HCMV infection. Consequently, healthy individuals show no or mild symptoms following primary infection. In contrast, immunocompromised individuals who develop primary infection or recrudescence of HCMV can experience severe morbidity, and sometimes mortality. HCMV-specific T cell populations undergo changes in the architecture of their T cell receptor (TCR) repertoire following each episode of viral reactivation. A diverse TCR repertoire is thought to be required to provide the most efficient protection against virus infection. Perturbation to this repertoire, as can occur in immunocompromised individuals following transplantation, can lead to an increase risk of developing virus-associated clinical disease. Therefore, the study of factors influencing TCR selection is critically important in both healthy and immunocompromised individuals. To further understand the factors governing TCR selection in a persistent virus infection, the current thesis examined this process in different settings. CD8+ T cell responses to persistent viral infections are characterised by the accumulation of T cells exhibiting an oligoclonal T cell repertoire, with a parallel reduction in the naïve T cell pool. However, the precise mechanism for this phenomenon remains elusive. Here, we showed that HCMV-specific CD8+ T cells recognising distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T cell repertoire, or a private, diverse T cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public TCR was co-incident with an atypical peptide-MHC (pMHC) structure, whereby the epitope adopted a helical conformation that bulged from the peptide-binding groove, whilst a diverse TCR profile was observed in response to the epitope that formed a flatter, more ‘featureless’ landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared to the T cells expressing diverse TCR. These findings provide new insights into our understanding of how the biology of antigen presentation, in addition to the structural features of the pMHC, might shape the T cell phenotype and its corresponding repertoire architecture. Next, the role of HCMV in shaping the global and antigen-specific TCR repertoire in healthy donors was examined. First, exposure to HCMV led to an inflation of terminally differentiated CD57-expressing T cells. This effect was not seen in HCMV seronegative individuals who showed evidence of exposure to another persistent herpesvirus, Epstein-Barr virus (EBV). More importantly, these terminally differentiated CD8+ T cells in HCMV-exposed individuals displayed a highly skewed architecture of their peripheral blood T cell repertoire, with large monoclonal/oligoclonal expansions. However, ex vivo analyses of HCMV-specific T cells revealed a heterogeneous pattern of CD57 expression that showed no correlation to the antigenic source of its cognate epitope. Based on these observations, we proposed that exposure to HCMV drives the differentiation of not only the global T cell population, but select HCMV-specific T cell populations as well, and that expression of CD57 by these cells was co-incident with an oligoclonal T cell repertoire. Finally, the TCR repertoire was examined in a cohort of solid organ transplant (SOT) recipients, where primary infection or recrudescence of latent virus infection can be manifested either as asymptomatic or symptomatic disease. We examined 18 SOT recipients, and observed that symptomatic HCMV or EBV infection or recrudescence following solid organ transplantation was co-incident with a dramatic skewing of the TCR repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no HCMV/EBV infection or recrudescence showed minimal or no skewing of the TCR repertoire, and maintained TCR repertoire diversity. Interestingly, this disparate repertoire showed no correlation with levels of viral load in the peripheral blood. More importantly, we showed that large monoclonal/oligoclonal repertoire expansions was linked to the loss of antigen-specific T cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, while SOT recipients who maintained peripheral blood TCR repertoire diversity and functional antigen-specific T cell responses could resist clinical symptomatic disease in spite of high levels of viral load. Therefore, the work presented in this thesis provides additional evidence on the factors governing TCR selection in HCMV-exposed healthy individuals, as well as the consequences that perturbation to the TCR repertoire has on the functionality of the T cell compartment in immunocompromised individuals.

Fetal pig islet-like cell clusters (ICCs) were transplanted into the thymus or omentum of STZ-induced diabetic pigs immunosuppressed with cyclosporine (CsA) and deoxyspergualin (DSG), as a potential treatment for type 1 diabetes. C-peptide levels in response to glucagon and arginine significantly increased over time using 1 litter of ICCs with highest levels obtained at 100 days post-transplantation. Increasing the number of ICCs to 2 litters was not advantageous. Histology of the graft showed all 4 pancreatic endocrine cells. Normoglycaemia was achieved for transient periods without insulin administration in 4 out of 12 pigs. These results suggest sub-optimal insulin production, possibly due to the adverse effects of CsA on the grafted β cells. The effect of CsA on adult porcine β cells was investigated and adverse effects were shown. Renal toxicity and adverse changes to the haematological parameters did not occur despite high CsA levels although minimal toxicity to the liver was observed. The results indicate that the use of CsA monotherapy in the maintenance phase to prevent rejection of allografted pancreatic β cells may become a subsequent problem over time. As an alternative to chronic immunossuppression, anti-CD3 monoclonal antibody was administered for 10 days in pigs. Using anti-CD3 alone, only 1 out 4 pigs showed cells positive for insulin. The addition of a 5-day CsA course administered the day before transplantation did not promote allograft survival. The use of DSG for 10 days with anti-CD3 promoted graft survival with the histology showing the 4 endocrine cells 3 weeks post-transplantation. An attempt was made to replace any form of immunossuppression by encapsulating fetal pig ICCs in barium alginate, which were able to remain viable when transplanted in NOD/SCID mice. Fibrosis was detected in BALB/c mice transplanted with encapsulated fetal ICCs suggesting that fetal pig ICCs shed antigens that elicit an immune response. Results from this study show that although fetal pig ICCs may be a viable source of insulin-producing cells, the use of CsA to prevent rejection has adverse effects on graft function. Encapsulation as well as transient immunosuppression is worthy of further investigation to prevent rejection of fetal pig ICCs.

Transplants of olfactory ensheathing cells (OECs) from olfactory bulbs have recently been shown to support regrowth and reinnervation of damaged spinal cord, which has led to improved functional recovery. Using complete transection in adult rat, the studies presented in this thesis examine the role of peripherally derived olfactory tissue in promoting axonal regeneration and functional recovery. Chapter One and Two provide the background to the area of spinal cord regeneration and the methods used in this thesis. Chapter Three shows that transplants of OECs from rat olfactory lamina propria (OLP) are able to support axon regrowth in the lesioned spinal cord. The BBB score was significantly higher in experimental rats (5.4???0.84) compared with control animals (1.9???0.33) (P&lt0.001). These dissociated OECs from OLP can promote axonal regrowth through the lesion. Histological assessment showed that: 1) axons labelled with Fluororuby grew into the injury site in OECs-transplanted rats, with occasional fibres extending into the rostral cord; 2) brainstem neurons in the raphe nucleus were retrogradely labeled with Fluororuby; and 3) serotonergic axons were detectable distal to the lesion in OECs-transplanted rats. No fibres grew into the injured region and no retrograde labeling or serotonergic fibres were seen in control animals. The role of regenerated serotonergic fibres in OECs-transplanted rats is discussed. Chapter Four demonstrates that solid pieces of OLP dissected from the nose can re-establish the continuity of the transected cord and supply the OECs that can migrate to the cord stumps to support the axon regeneration. Experimental rats which received OLP from olfactory mucosa showed significantly greater locomotive recovery (BBB scores: OLP, 5.0???1.9; control, 1.5???0.5, p&lt0.0001). In animals with OLP transplants, histological analysis indicated that nerve fibres, expressing neurofilament and serotonin were present at the transection site. Locomotive recovery of the hindlimbs occurred, similar to that seen after OECs transplantation. Retrograde labeling of medullary raphe neurons and gigantocellular reticular nucleus occurred following Fluororuby injection in the cord distal to the lesion, further supporting the supraspinal origin of the 5-HT innervation in the present studies. These results indicate that OLP is effective in promoting partial spinal cord repair. Chapter Five examines functional recovery of spinal reflex circuitry, ie., H-reflex excitability using paired stimuli, in OLP-transplanted rats compared with normal and respiratory lamina propria (RLP) transplanted animals. H-reflex amplitude of the conditioned response was significantly reduced in OLP transplanted rats compared to RLP transplanted animals (p&lt 0.05). Therefore, hindlimb reflex excitability can be modulated by OLP transplants after transection of the spinal cord in adult rats. Chapter Six examines whether functional recovery can occur if transplantation of OLP tissue is delayed by 1 month after the spinal cord transection. The BBB score was significantly higher in experimental rats (4.3???0.8 for OLP) compared with control animals (1.0???0.3, P&lt 0.001), but recovery was less than after acute transplantation. Asx before, histological assessment of OLP animals showed: a) serotonergic axons were present in the cord below the transection site; b) brainstem raphe nuclei was retrogradely labeled; c) bisbenzimide pre-labeled cells from OLP transplants migrated in host spinal cord. These changes were not seen in control animals. These results indicate that OLP has the ability to promote axonal regeneration in chronically injured cord of adult rats. Chapter Seven compares the results from these three types of intervention. In conclusion, these studies show that peripherally derived OECs or solid pieces of OLP can promote partial spinal cord repair in acute or chronic transection injuries. Such tissue might provide a potential source for autologous grafting in human paraplegia.