Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

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The natural history for patients with de novo donor-specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pre-transplant donor-specific antibody (DSA), with a mean follow-up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA post-transplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years post-transplant. Independent predictors of dnDSA were HLA-DRβ1 MM > 0 (OR 5.66, p < 0.006); and non-adherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p=0.061). The median 10-year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody-mediated injury occurred and progressed in patients with dnDSA in the absence of graft dysfunction. Furthermore, non-adherence and cellular rejection contributed to both dnDSA development and the risk of progression to graft loss. (Human leukocyte antigen) HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for a subset of 286 donor–recipient pairs in which samples were available for high-resolution HLA-typing. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DR dnDSA alone (21.4 vs. 13.2, p<0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p<0.001). An optimal threshold for epitope mismatch (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA using a receiver operating characteristic analysis. Applying these thresholds, 0% and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years follow-up. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA when mismatched between the donor and recipient. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome.

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Medications that treat diseases such as Parkinson???s disease work by regulating dopamine transmission at synapses. Surprisingly, little is known about the mechanisms regulating dopamine release at synapses. In this thesis, we study mechanisms that regulate vesicle recycling in axons and dendrites of dopamine neurons. Key questions we addressed were: (1) Are vesicles in axons and dendrites associated with the same regulatory proteins, and thus by implication the same regulatory mechanisms, as in excitatory neurons; (2) Do vesicles undergo recycling, and (3) if so, are they characterised by a distinct pool size and rate of recycling. To study this, we cultured dopamine neurons and used immunocytochemistry to detect vesicular monoamine transporter 2 (VMAT2) and identify axons, dendrites and synaptic proteins, combined with labelling of recycling vesicles using FM 1-43. Vesicles in axons, but not in dendrites, were associated with presynaptic proteins such as Synaptophysin and Bassoon. We identified two kinds of presynaptic sites in axons: ???synaptic??? (located close to soma and dendrites??? and ???orphan???. The recycling vesicle pool size was smaller at orphan sites than at synaptic sites, and the initial rate of vesicle pool release was also lower at orphan sites. Both synaptic and orphan sites exhibited lower rates of vesicle pool release compared to hippocampal synapses, suggesting functional differences in presynaptic physiology between dopamine neurons and hippocampal neurons. In somatodendritic regions, VMAT2 was localised to the endoplasmic reticulum, Golgi, endosome, and large dense-core vesicles, suggesting that these vesicles might function as a part of the regulated secretory pathway in mediating dopamine release. None of the synaptic vesicle proteins we studied were detected in these regions, although some preliminary evidence of vesicle turnover was detected using FM 1-43 labelling. This thesis provides a detailed analysis of neurotransmitter release mechanisms in dopamine neurons. Our data suggests that presynaptic release of dopamine is mediated by mechanisms similar to those observed in excitatory neurons. In somatodendritic regions, our data suggests that VMAT2 is localised to organelles in secretory pathways, and that distinct mechanisms of release might be present at somatodendritic sites to those present in presynaptic sites. This thesis provides novel methods for analysing vesicle recycling in dopamine neurons, which provides the basis for further studies examining presynaptic function of dopamine neurons in normal brain function, disease, and therapeutic approaches.

Diabetes can be considered to be one of the main health epidemics of the 21st century. Studies conducted by the World Health Organisation (WHO) indicate that the number of people with diabetes in the year 2000 was 171 million and this is projected to increase to 366 million by 2030 (Wild et al. 2004). The increasing incidence of both Type 1 and Type 2 diabetes is due to population growth, aging, urbanisation, obesity and physical inactivity. The current treatment by insulin injections for individuals with Type 1 diabetes fails to overcome the long term microvascular and macrovascular complications associated with the disease. A major challenge in the treatment of diabetes is to provide patients with an insulin source that is capable of regulating blood glucose levels (BGL) on a minute to minute basis. Advances in medical research have enabled the investigation of a variety of potential alternative therapies that may provide Type 1 diabetic patients with a more superior control of BGL and consequently minimise complications. The utilisation of pancreases obtained from fetal pigs offers potential therapeutic value in the treatment of Type 1 diabetes. Islet-like cell clusters (ICCs) are obtained from such tissue following partial mechanical and enzymatic digestive procedures. ICCs are primarily composed of immature duct cells which, when transplanted, will mainly differentiate into insulin producing ?? cells. Such cells are able to normalise BGL in immunodeficient diabetic recipients and in immunocompetent recipients when anti-rejection drugs are administered. This study investigates microencapsulation as an immunoprotective strategy that has the potential to remove the need for immunosuppression when such cells are transplanted. A review of the literature related to current medical research in the field of diabetes is presented in Chapter 1. In order to achieve the aims of the study, an understanding of how fetal pig ICCs behave when placed within a barium alginate microcapsule both in vitro and in vivo is essential and this data is presented in Chapter 3. This chapter demonstrates that ICCs will survive and differentiate in their typical manner when enclosed within microcapsules and transplanted. Such encapsulated cells will function to normalise BGL when transplanted into diabetic immunodeficent mice for at least 25 weeks and the animals exhibit increased bodyweight. Microcapsules retrieved at this time point were observed to be intact with no breakages or evidence of cellular overgrowth. Transplantation of encapsulated insulin-producing cells into immunocompetent mice are described in Chapter 4. Allotransplantation of a microencapsulated mouse insulin-producing cell line into these diabetic mice also exhibited graft function, resulting in normal BGL in recipients. Large animal experiments are described in Chapter 5. Allotransplantation of microencapsulated fetal pig ICCs into diabetic pig recipients displayed evidence of transient graft function in terms of lower BGL and reduced exogenous insulin requirements. The xenotransplantion of encapsulated fetal pIg ICCs into diabetic immunocompetent mice described in Chapter 4 proved to be more challenging. The transplantation of such cells in this environment did not yield particularly positive results. BGL remained elevated in these recipients and the animals lost bodyweight post transplantation. This area of research warrants further investigation as it is likely that further measures such as transient immunosuppression in combination with microencapsulation will allow fetal pig ICCs to function in a xenograft setting.

Corneal transplantation is performed to restore vision or to relieve pain in patients with damaged or diseased corneas. However, approximately 40% of corneal allografts fail after 10 years. The most common cause of graft failure is irreversible immunological rejection, primarily mediated by CD4+ T cells, despite the topical application of glucocorticosteroids. The aim of this project was to investigate the anatomic site of antigen presentation during corneal transplantation in the rat, by using a lentiviral vector to express an anti-CD4 antibody fragment at potential sites of antigen presentation, including the donor corneal endothelium, the anterior segment of the eye and the cervical lymph nodes. Dual-gene lentiviral vectors were constructed by inserting the 2A self-processing sequence between two transgenes. This allowed expression of two transgenes within a single open reading frame. In vitro characterisation of the dual-gene vectors was performed in cell culture experiments, which showed that transgenic proteins were expressed at lower levels from dual-gene vectors compared to the expression from single-gene vectors and expression was lowest when the transgene was situated downstream of the 2A self-processing sequence. To locate the anatomic site of antigen presentation during corneal transplantation in rats, a lentiviral vector carrying an anti-CD4 antibody fragment was delivered to the corneal endothelium either immediately prior to corneal transplantation by ex vivo transduction of the donor corneas, or 5 days prior to corneal transplantation by anterior chamber injection into both the recipient and the donor rats. A separate group of recipient rats received intranodal injections of the lentiviral vector carrying an anti-CD4 antibody fragment into the cervical lymph nodes 2 days prior to corneal transplantation. Another group of rats underwent bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Corneal allografts were scored daily for opacity, inflammation and neovascularisation. Expression of the anti-CD4 antibody fragment from transduced tissues was detected using flow cytometry and polymerase chain reaction. Modest, but significant prolongation of corneal allograft survival was experienced by rats that received ex vivo transduction of the donor corneas with a lentiviral vector carrying an anti-CD4 antibody fragment immediately prior to corneal transplantation, but all grafts did eventually reject. Anterior chamber injection of the lentiviral vector carrying the anti-CD4 antibody fragment 5 days prior to corneal transplantation into both recipient and donor eyes did not prolong allograft survival. Intranodal injection of a lentiviral vector carrying an anti-CD4 antibody fragment did not prolong the survival of the corneal allografts, nor did bilateral lymphadenectomy of the cervical lymph nodes 7 days prior to corneal transplantation. Neither expression of the anti-CD4 antibody fragment in the cervical lymph nodes nor the removal of these nodes was able to prolong corneal allograft survival in rats, suggesting that T cell sensitisation could potentially occur elsewhere in the body. However, expression of the anti-CD4 antibody fragment from the donor corneal endothelium was able to prolong corneal allograft survival, suggesting that some antigen presentation might occur within the anterior segment of the eye. Based on the findings described in this thesis and those of others, I propose that antigen presentation in the rat occurs within anterior segment of the eye and within the secondary lymphoid tissues such as the cervical lymph nodes, and that inhibiting antigen presentation at one of these sites will delay graft rejection. However, to completely abolish antigen presentation during corneal transplantation in the rat, I hypothesise that antigen presentation within both the anterior segment of the eye and within the secondary lymphoid tissues must be inhibited.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1988. / Vita. Bibliography: leaves [352]-358.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 5 uppsatser.