Thèse (M.Sc.)--Université Laval, 2007. / Bibliogr.: f. 101-110. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Evidence linking periodontitis and cardiovascular disease points to systemic inflammation as the primary etiology. The investigation into this association has focused on periodontitis as a low grade chronic infection, and subsequently, systemic inflammation and the cascade of numerous markers and mediators integrally involved in the human inflammatory response. The aim of this study was to measure the differences in circulating levels of C-reactive protein and human soluble CD40 ligand as markers and mediators of systemic inflammation in the serum of patients with moderate to severe periodontitis before, during, and after non-surgical periodontal therapy.Twenty-one patients were treated with full mouth scaling and root planing performed on two separate appointments spaced approximately 2 weeks apart. The third visit, an evaluation of non-surgical therapy, occurred 8 weeks after completion of scaling and root planing. Blood samples were procured from each patient at the first and second appointment prior to scaling and root planing and again at a third visit. CRP and sCD40 ligand levels were measured using ELISA. CRP levels remained relatively constant throughout the study maintaining a mean level of 2.44 mg/L at visit 1, 2.53 mg/L at visit 2, and 2.63 mg/L at visit 3. These differences were not statistically significant. sCD40 ligand mean levels were reported to be 847.7 pg/mL, 637.3 pg/mL, and 859.5 pg/mL, respectively for visits 1, 2, and 3. These values were not statistically significant. Within the limitations of this study, no evidence was found to directly relate non-surgical periodontal therapy with the lowering of systemic markers CRP and human soluble CD40 ligand.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanisms involved in the induction of allograft survival, we determined the fate of tracer populations of alloreactive T cell receptor (TcR) transgenic CD8+ T cells circulating in a normal microenvironment. In the first portion of this thesis, we observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive TcR transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4 and B7-l/2 by CTLA4-Ig, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive TcR transgenic CD8+ T cells. Also in support of our hypothesis, depletion of CD4+ T cells, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive TcR transgenic CD8+ T cells. We continued by examining the effects of IFN-γ, IL-10 and IL-4. None of these cytokines had any significant effect on the deletion of alloreactive TcR transgenic CD8+ T cells induced by co-stimulation blockade. The last part of this thesis studied the behavior of alloreactive TcR transgenic CD8+ T cells during the maintenance phase of allograft survival induced by our two-element protocol. Using a hematopoietic TcR transgenic chimera system, our results demonstrated that levels of alloreactive CD8+ T cells remained low throughout the maintenance phase. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by co-stimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade by co-stimulation blockade.

Malgré la mise en oeuvre de la leucoréduction systématique, les transfusions plaquettaires restent génératrices de réactions post-transfusionnelles (encore appelées « Effets Indésirables Receveur, EIR »). Nous savons que les plaquettes sanguines relarguent des molécules proinflammatoires (e.g. CD40 ligand soluble ou sCD40L) durant leur préparation et stockage et leurs taux augmentés sont associées aux EIR. Le travail de cette thèse vise à mieux comprendre les mécanismes de survenue des EIR post transfusion plaquettaire, dans le contexte de l’inflammation. La première partie de nos travaux n’a pas retrouvé de polymorphismes génétiques du gène CD40LG qui pourraient modifier l’affinité du couple Récepteur/Ligand, en cas d’EIR. Dans un second temps, une caractérisation haplotypique de CD40LG n’a pas non plus permis de retrouver d’association avec l’apparition d’EIR. Nous avons ensuite tenté d’identifier des marqueurs génétiques de surexpression de sCD40L (et par conséquence, d’EIR). Pour cela, nous avons étendu l’investigation à douze polymorphismes de CD40LG mais aussi des marqueurs génétiques connus comme indépendamment liés à l’expression de sCD40L : au niveau de son récepteur CD40 et d’ITGA2. Ont été retrouvés associés à une modification significative de sécrétion de sCD40L, d’une part le polymorphisme rs126643 (ITGA2), un haplotype étendu de CD40LG et aussi un haplotype interchromosomique (CD40LG–CD40–ITGA2). Toutefois, ces haplotypes n’ont pas pu être retrouvés associés à l’apparition d’EIR. Nous avons également montré que lors du stockage de concentrés plaquettaires (CP) non leucoréduits, les « modificateurs de réponse biologique » (BRM) leucocytaires diffèrent d’une part, dominent et influencent d’autre part les BRM d’origine plaquettaire. Nous nous sommes ensuite intéressés à un autre BRM individualisé dans les produits transfusés : l’ADN mitochondrial, classé comme un signal de danger endogène (ou DAMP) et nous avons retrouvé une augmentation significative de ceux-ci dans les CP ayant induit un EIR. Nous avons alors entrepris une étude protéomique (LC-MS/MS) et transcriptomique (RNA-seq) sur des CP ayant induit ou non un EIR. L’enrichissement des protéines et gènes différentiellement exprimés présente principalement un rôle dans l’activation plaquettaire, la coagulation, l’apoptose et la dégranulation qui représentent des processus en liaison étroite. Le transcriptome plaquettaire a confirmé l’étiologie apoptique par la mise en évidence de la dérégulation calcique mitochondriale. De plus, l’obtention de l’enrichissement de sa voie intrinsèque permet d’expliquer l’abondance des ADNmt retrouvés précédemment dans les CP associés aux EIR. Par ailleurs, l’état d’activation et la dégranulation des plaquettes expliquent l’abondance des BRM retrouvés en cas d’EIR. Ainsi, nous avons identifié de nouveaux marqueurs inflammatoires hautement exprimés dans le groupe EIR. Après validation de ces résultats par des études complémentaires, ces marqueurs pourraient être proposés comme cibles de prévention d’EIR en modifiant, par exemple la préparation des CP / Despite the implementation of systematic leucoreduction, platelet transfusions are still generating transfusion reactions (also called "Adverse Events or AEs"). We know that platelets release proinflammatory molecules during preparation and storage of platelet components (PCs) notably the soluble CD40 ligand or sCD40L and high levels are associated with AEs. In this thesis, we aimed to understand the mechanisms of occurrence of inflammatory platelet transfusion reactions. In the first part, we investigated the genetic polymorphisms of CD40LG. We failed to identify a significant association with AEs but we identified genetic markers of sCD40L overexpression: a CD40LG haplotype and also an interchromosomal haplotype (CD40LG-CD40-ITGA2). However, these haplotypes have not been found to be associated with AEs. In the second part, we showed that, during storage of non leucoreduced PCs, leukocyte-derived "biological response modifiers" (BRMs) dominate and influence the platelet-derived BRMs. Then, we found increased levels of another individualized BRM in PCs involved in AEs: mitochondrial DNA, classified as an endogenous danger-associated molecular pattern (DAMP). In the last part, we performed an integrated proteomic and transcriptomic study by LC-MS/MS and RNA-seq, respectively, to better understand the pathophysiology of AEs in a case-control approach. The biological enrichment of differentially expressed genes revealed a significant association with platelet activation, apoptosis and inflammatory mechanisms which may be involved in platelet transfusion reactions. This study provides novel insights into the molecular mechanisms underlying the occurrence of AEs and could give paths to prevent them

<p>BACKGROUND: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. CD40 ligand is another membrane molecule, which ligates to cell types associated with atherosclerotic plaques thereby mediating intraplaque inflammation and weakening of the fibrous cap. Acute coronary syndrome (ACS) is a multi-factorial disease in which TF and CD40 ligand have prominent roles. Single nucleotide poly-morphisms (SNPs) in the TF and CD40 ligand genes may influence the development, pro-gression and outcome in ACS. AIM: The aim of this thesis was to investigate the genetic and molecular control of TF expression in healthy individuals and in patients with ACS. More-over, the aim was to investigate whether SNPs in the TF and CD40L genes respectively were associated with risk and outcome in ACS and / or with plasma concentrations of these pro-teins. RESULTS: A real-time PCR method that allowed sensitive and dynamic quantification of TF mRNA was established and used for the identification of a high and low response phe-nomenon of TF mRNA. The TF high and low response correlated with the expression of toll-like receptor 4 (TLR-4) thus linking TF to innate immunity in a novel fashion. Investigation of several SNPs in the TF and CD40L genes led to the identification of the 5466 A>G in the TF gene and the -3459 A>G SNP in the CD40L gene. The 5466 G allele was associated with cardiovascular death in patients with ACS and increased TF procoagulant activity in human monocytes, which explained the clinical association. The -3459 G allele regulated the produc-tion of soluble CD40L but was not related with patient outcome. Soluble CD40L levels above median were associated with the risk of MI in patients with ACS. A prolonged treatment with dalteparin was more efficient in patients presenting with high levels of sCD40L, which further supports sCD40L as a marker of a prothrombotic state. CONCLUSIONS: The results of this thesis adds to our current knowledge of factors influencing TF expression and activity by demonstrating the effects of TF gene variants, cell signalling molecules, CD40 ligand protein and gene variation. All of these effects have the potential to modify the risk of development, progression and outcome in the acute coronary syndrome and exemplify the interplay between coagulation and inflammation, in which both TF and CD40 ligand are active.</p>

<p>Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses.</p><p>We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy.</p><p>In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.</p>

BACKGROUND: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. CD40 ligand is another membrane molecule, which ligates to cell types associated with atherosclerotic plaques thereby mediating intraplaque inflammation and weakening of the fibrous cap. Acute coronary syndrome (ACS) is a multi-factorial disease in which TF and CD40 ligand have prominent roles. Single nucleotide poly-morphisms (SNPs) in the TF and CD40 ligand genes may influence the development, pro-gression and outcome in ACS. AIM: The aim of this thesis was to investigate the genetic and molecular control of TF expression in healthy individuals and in patients with ACS. More-over, the aim was to investigate whether SNPs in the TF and CD40L genes respectively were associated with risk and outcome in ACS and / or with plasma concentrations of these pro-teins. RESULTS: A real-time PCR method that allowed sensitive and dynamic quantification of TF mRNA was established and used for the identification of a high and low response phe-nomenon of TF mRNA. The TF high and low response correlated with the expression of toll-like receptor 4 (TLR-4) thus linking TF to innate immunity in a novel fashion. Investigation of several SNPs in the TF and CD40L genes led to the identification of the 5466 A&gt;G in the TF gene and the -3459 A&gt;G SNP in the CD40L gene. The 5466 G allele was associated with cardiovascular death in patients with ACS and increased TF procoagulant activity in human monocytes, which explained the clinical association. The -3459 G allele regulated the produc-tion of soluble CD40L but was not related with patient outcome. Soluble CD40L levels above median were associated with the risk of MI in patients with ACS. A prolonged treatment with dalteparin was more efficient in patients presenting with high levels of sCD40L, which further supports sCD40L as a marker of a prothrombotic state. CONCLUSIONS: The results of this thesis adds to our current knowledge of factors influencing TF expression and activity by demonstrating the effects of TF gene variants, cell signalling molecules, CD40 ligand protein and gene variation. All of these effects have the potential to modify the risk of development, progression and outcome in the acute coronary syndrome and exemplify the interplay between coagulation and inflammation, in which both TF and CD40 ligand are active.