To date, the cellular mechanisms involved in the progression of diseases characterized by chronic inflammation, such as rheumatoid arthritis (RA), remain largely unknown. However, cell-to-cell contact interactions between CD4+ helper T (Th) cells and monocytes have been implicated in the induction and maintenance of pro-inflammatory cytokine synthesis that is characteristic to the pathogenesis of RA. One such cytokine produced during monocyte-Th cell contact is interleukin (IL)-1 β, a mediator directly involved in the characteristic tissue destruction that occurs in the synovia of individuals with RA. Previous studies in our laboratories have shown that ligation of CD40 on monocytes with CD40 ligand (CD40L) present on activated Th cells induces monocyte IL-1β synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the exacerbation and maintenance of chronic inflammatory responses. This dissertation represents efforts to elucidate components of the CD40 signaling pathway critical to monocyte activation and how CD40-mediated signaling events are modulated by the anti-inflammatory cytokines IL-4 and IL-10. Using either monocytes isolated from human peripheral blood or a monocytic cell line (THP-1), cellular kinases and transcription factors activated upon CD40 ligation were examined by western blot analyses and electrophoretic mobility shift assays (EMSA), respectively. CD40-dependent interleukin-1β synthesis in monocytes was abrogated by inhibitors of protein tyrosine kinase (PTK) activity but not by inhibitors of protein kinase C (PKC). The extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) mitogen-activated protein kinases (MAPK's) were specifically activated upon CD40 ligation, and specific inhibition of Erk1/Erk2 activation diminished IL-10 production in a dose-dependent manner. Both IL-4 and IL-10 reduced Erk1/Erk2 activation and synergized in this effect. Finally, STAT3, a member of the family of transcription factors involved in cytokine signaling, was highly phosphorylated in monocytes treated with IL-10 or with IL-10 and IL-4 in combination but not with IL-4 alone. Together these results suggest that in monocytes (1) CD40-mediated IL-1β synthesis and NF-κB activation require PTK activity, (2) CD40-mediated IL-1β production is critically dependent upon Erk1/Erk2 activity, (3) both IL-4 and IL-10 target the Erk1/Erk2 signaling cascade in the downregulation of IL-1β synthesis, (4) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway in that these cytokines are synergistic with respect to their ability to inhibit CD40-mediated Erk1/Erk2 activation and IL-1β synthesis, and differ in their ability to block CD40-mediated rescue from apoptosis, and (5) STAT3 activation may be directly involved in the downregulatory effects of IL-10 on CD40 signaling. (Abstract shortened by UMI.)

<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.

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. 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. 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.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 177-206). Also available on the Internet.

The aim of cancer immunotherapy is to eradicate tumours by inducing a tumour-specific immune response. This thesis focuses on how antibodies and peptides can improve antigen presentation and the subsequent tumour-specific T cell response. Tumour recognition by the immune system can be promoted through delivery of antigen in the form of a vaccine. One example is the development of a therapeutic peptide vaccine containing both CD4+ and CD8+ T cell epitopes. So far, peptide vaccinations have shown limited success in clinical trials and further improvements are needed, such as choice of adjuvant and T cell epitopes, as well as targeted delivery of peptides and adjuvants to the same DC. In paper I, we describe the development of a peptide-peptide conjugate (with a tumour T cell epitope) that, via immune complex formation and FcγR binding, enhance antigen uptake and activation of DCs. The conjugate consists of three tetanus toxin-derived linear B cell epitopes (MTTE) that were identified based on specific IgG antibodies in human serum. Three MTTE peptide sequences were conjugated to a synthetic long peptide (SLP) that consists of a T cell epitope derived from the desired target tumour. In paper II, the conjugate was evaluated in a modified Chandler loop model containing human blood, mimicking blood in circulation. The conjugate was internalised by human monocytes in an antibody-dependent manner. A conjugate containing the model CMV-derived T cell epitope pp65NLV generated recall T cell responses dependent on MTTE-specific antibodies and the covalent conjugation of the three MTTE with the SLP. In paper III, a CD40-specific antibody was characterised for local treatment of solid tumours. The antibody eradicated bladder tumours in mice and induced T cell-mediated immunological memory against the tumour. In paper IV, we characterised the Chandler loop model (used in paper II) for its potential use in predicting cytokine release syndrome (CRS) in response to monoclonal antibodies (mAbs). Superagonistic antibodies (e.g., OKT3) induced rapid cytokine release whereas no cytokine release was induced by antibodies (e.g., cetuximab) associated with low incidence of CRS in the clinic. In conclusion, this thesis work demonstrates proof-of-concept of improved strategies for antibody- and peptides-based cancer immunotherapies and their potential use in multiple cancer indications.

Le CD40 ligand (CD40L) est une molécule thrombo-inflammatoire qui prédit des événements cardiovasculaires. Les plaquettes constituent la principale source du CD40L soluble (sCD40L) dans la circulation, avec la capacité d’influencer la fonction des plaquettes par l'intermédiaire de sa liaison à ses récepteurs plaquettaires : CD40, αIIbβ3 et α5β1. Nous avons précédemment démontré que la stimulation des plaquettes humaines avec le sCD40L induit une activation du nuclear factor kappa B (NF-κB), qui pourra jouer un rôle non génomique en amorçant les plaquettes. En effet, l’amorçage des plaquettes par le sCD40L augmente fortement l’activation et l’agrégation plaquettaire en réponse à des doses sous-optimales d’agonistes. Cependant, l’implication des différents récepteurs dans l’amorçage des plaquettes suite à l’activation du NF-κB par le sCD40L demeure inconnue. De plus, le transforming growth factor-B (TGF-B)-activated Kinase (TAK1), un régulateur majeur de l’inflammation, est activé par le sCD40L dans les cellules nucléées, et pourra ainsi être impliqué dans la signalisation sCD40L/NF-κB plaquettaire. D’autre part, le sCD40L se trouve en quantité élevée dans la circulation sanguine des patients coronariens. De plus, le traitement antiplaquettaire à l’Aspirine (ASA) est inefficace chez certains patients et son efficacité est réduite chez les patients à hauts risques d’évènements coronariens, ce qui augmente leur risque de sur-activation plaquettaire et de développer une thrombose. Des taux sanguins élevés de médiateurs thrombo-inflammatoires, tels que le sCD40L, peuvent expliquer de telles variabilités. Ainsi, ce projet a été entrepris dans le but d’élucider l’impact de l’axe sCD40L/NF-κB sur la fonction plaquettaire et sa modulation par l’ASA. Dans le premier volet de mon projet, nous avons identifié les récepteurs de sCD40L impliqués dans l'activation du NF-κB plaquettaire, leur signalisation en aval et leur implication dans l'agrégation plaquettaire. Nous avons montré que les plaquettes exprimaient les récepteurs du CD40L : CD40, αIIbβ3 et α5β1 et libéraient du sCD40L. Le sCD40L seul a induit une activation de NF-κB plaquettaire. Cet effet était absent des plaquettes de souris déficientes en CD40 (CD40-/-) et inhibé par le blocage de CD40, mais n'était pas affecté par le blocage de αIIbβ3 ou α5β1. L’axe sCD40L/CD40 a aussi activé TAK1 en amont de NF-κB. Dans les études fonctionnelles, le sCD40L seul n’a pas eu d’effet sur l’agrégation plaquettaire, mais a potentialisé l’agrégation en présence de doses sous-optimales de thrombine; cet effet a été aboli par des inhibiteurs de CD40, TAK1 et NF-κB. Cette première étude nous a permis de conclure que le sCD40L amorce les plaquettes via des voies de signalisation impliquant CD40/TAK1/NF-κB, ce qui prédispose les plaquettes à une activation et agrégation accrues en réponse à des stimuli thrombotiques. Dans le deuxième volet de mon projet, nous avons émis l’hypothèse qu’en présence de taux élevés de sCD40L, l’efficacité de l’ASA peut varier et avons visé à déterminer les effets de l’ASA sur la signalisation et l’agrégation des plaquettes en présence de sCD40L. Les effets de l'ASA sur les plaquettes humaines traitées au sCD40L, en réponse à des concentrations sous-optimales de collagène ou de thrombine, ont été évalués sur l'agrégation, la sécrétion de thromboxane A2 (TxA2) et la phosphorylation de p38 MAPK, NF-κB, TAK1 et la chaîne légère de la myosine (MLC). Le sCD40L a significativement augmenté la sécrétion de TxA2 par les plaquettes, en réponse à des doses sous-optimales de collagène et de thrombine; cet effet a été inversé par l'ASA. L'ASA n'a pas inhibé la phosphorylation de p38 MAPK, NF-κB, TAK1, que ce soit avec une stimulation plaquettaire par le sCD40L seul ou en présence des agonistes. Le sCD40L a potentialisé l'agrégation plaquettaire, un effet complètement inversé et partiellement réduit par l'ASA en réponse à une dose sous-optimale de collagène et de thrombine, respectivement. Les effets de l'ASA sur les plaquettes traitées au sCD40L avec du collagène étaient liés à l'inhibition du changement de forme des plaquettes et à la phosphorylation de la MLC. En résumé, l'ASA n'affecte pas la signalisation plaquettaire du sCD40L, mais empêche son effet sur la sécrétion de TxA2 et l'agrégation plaquettaire en réponse au collagène, via un mécanisme impliquant l'inhibition de la MLC. En conclusion, ce projet nous a permis de déterminer que l’amorçage des plaquettes par le CD40L via l’activation de NF-κB dépend du récepteur CD40 et la signalisation via TAK1. Ainsi, l’axe sCD40L/CD40/TAK1/NF-κB potentialise l’activation et l’agrégation en réponse à des stimuli thrombotiques, ce qui peut favoriser l’occurrence d’événements athéro-thrombotiques chez les patients coronariens. De plus, étant donné que l’ASA n’a pas d’effet sur la signalisation via l’axe sCD40L, le ciblage de cet axe dans les plaquettes peut avoir un potentiel thérapeutique chez les patients coronariens présentant des taux élevés de sCD40L et ne réagissant pas ou moins à l'ASA. / CD40 ligand (CD40L) is a thrombo-inflammatory molecule that predicts cardiovascular events. Platelets are the main source of soluble CD40L (sCD40L) in the circulation, with the ability to influence platelet function through its binding to platelet receptors: CD40, αIIbβ3, and α5β1. We have previously demonstrated that stimulation of human platelets with sCD40L induces activation of nuclear factor kappa B (NF-κB), which may play a non-genomic role in priming platelets. Indeed, platelet priming by sCD40L strongly enhances platelet activation and aggregation in response to suboptimal doses of agonists. However, the involvement of the different receptors in platelet priming following activation of NF-κB by sCD40L remains unknown. In addition, the transforming growth factor-B (TGF-B) -activated Kinase (TAK1), a major regulator of inflammation, is activated by sCD40L in nucleated cells, and may be involved in sCD40L/NF-κB signaling. On the other hand, sCD40L is high in the bloodstream of coronary patients, who receive Aspirin (ASA) for secondary prevention. However, antiplatelet therapy with ASA is ineffective in some patients and its efficacy is reduced in patients at high risk for coronary events, increasing their risk of platelet over-activation and developing thrombosis. High blood levels of thrombo-inflammatory mediators, such as sCD40L, may explain such variability. Thus, we undertake this project to elucidate the impact of the sCD40L/NF-κB axis on platelet function and its modulation by ASA. In the first part of our project, we aimed to identify the sCD40L receptors involved in the activation of platelet NF-κB, their downstream signaling and their involvement in platelet aggregation. We have shown that platelets express CD40L receptors: CD40, αIIbβ3 and α5β1 and release sCD40L. sCD40L alone induced activation of platelet NF-κB. This effect was absent in CD40-/- mouse platelets and inhibited by CD40 blockade, but was not affected by αIIbβ3 or α5β1 blockade. The sCD40L/CD40 axis has also activated TAK1 upstream of NF-kB. In functional studies, sCD40L alone had no effect on platelet aggregation but potentiated aggregation in the presence of suboptimal thrombin doses. The inhibitors of CD40, TAK1 and NF-κB abolished this effect. This first study concluded that sCD40L primes platelets via CD40/TAK1/NF-κB signaling pathways, which predisposes platelets to increased activation and aggregation in response to thrombotic stimuli. In the second part of our project, we hypothesized that in the presence of high levels of sCD40L; the effectiveness of ASA may vary. Accordingly, we aimed at determining the effects of ASA on signaling and aggregation of platelets in the presence of sCD40L. The effects of ASA on human platelets treated with sCD40L, in response to suboptimal concentrations of collagen or thrombin, were evaluated on aggregation, thromboxane A2 (TxA2) secretion and phosphorylation p38 MAPK, NF-κB, TAK1 and myosin light chain (MLC). sCD40L significantly increased TxA2 secretion by platelets in response to suboptimal doses of collagen and thrombin; this effect has been reversed by the ASA. ASA did not inhibit the phosphorylation of p38 MAPK, NF-κB, TAK-1, with platelet stimulation by sCD40L either alone or in the presence of agonists. sCD40L potentiated platelet aggregation, an effect completely reversed and partially reduced by ASA in response to a suboptimal dose of collagen and thrombin, respectively. In addition, the effects of ASA on sCD40L-treated platelets with collagen related to inhibition of platelet shape change and phosphorylation of MLC. In summary, ASA does not affect platelet signaling via sCD40L, but prevents its effect on TXA2 secretion and platelet aggregation in response to collagen, via a mechanism involving inhibition of MLC. In conclusion, this project allowed us to determine that platelet priming by sCD40L via activation of NF-κB depends on the CD40 receptor and signaling via TAK1. Thus, the sCD40L/CD40/TAK1/NF-κB axis potentiates activation and aggregation in response to thrombotic stimuli, which may promote occurrence of atherothrombotic events in coronary patients. In addition, since ASA has no effect on platelet signaling via the sCD40L axis, targeting of this axis in platelets may have therapeutic potential in coronary patients with high levels of sCD40L that are none or less responding to ASA.

No description available.

Homocysteine-Methionine (HM) cycle produces a universal methyl group donor S-adenosylmethionine (SAM), a competitive methylation inhibitor S-adenosylhomocysteine (SAH), and an intermediate amino acid product homocysteine (Hcy). Elevated plasma levels of Hcy is termed as hyperhomocycteinemia (HHcy) which is an established risk factor for cardiovascular disease (CVD) and neural degenerative disease. We were the first to describe methylation inhibition as a mediating biochemical mechanism for endothelial injury and inflammatory monocyte differentiation in HHcy-related CVD and diabetes. We proposed metabolism-associated danger signal (MADS) recognition as a novel mechanism for metabolic risk factor-induced inflammatory responses, independent from pattern recognition receptor (PRR)-mediated pathogen-associated molecular pattern (PAMP)/danger-associated molecular pattern (DAMP) recognition. In this study, we examined the relationship of HM cycle gene expression with methylation regulation in human disease. We selected 115 genes in the extended HM cycle, including 31 metabolic enzymes and 84 methyltransferases (MT), examined their mRNA levels in 35 human disease conditions using a set of public databases. We discovered that: 1) HM cycle senses metabolic risk factor and controls SAM/SAH-dependent methylation. 2) Most of metabolic enzymes in HM cycle (8/11) are located in cytosol, while most of the SAM-dependent MTs (61/84) are located in the nucleus, and Hcy metabolism is absent in the nucleus. 3) 11 up-regulated, 3 down-regulated and 24 differentially regulated SAM/SAH-responsive signal pathways are involved in 7 human disease categories. 4) 8 SAM/SAH-responsive H3/H4 hypomethylation sites are identified in 8 disease conditions. We conclude that HM cycle is a key metabolic sensor system which mediates receptor-independent MADS recognition and modulates SAM/SAH-dependent methylation in human disease. We propose that HM metabolism takes place in cytosol and that nuclear methylation equilibration requires nuclear-cytosol transfer of SAM, SAH and Hcy. CD40 is a cell surface molecule which is expressed on antigen presenting cells such as monocyte, macrophage, dendritic cells and neutrophils. The costimulatory pair, CD40 and CD40L, enhances T cell activation and induce chronic inflammatory disease. Also, DNA hypomethylation on CD40 promotor induces inflammatory monocyte differentiation in chronic kidney disease. In order to figure out if CD40 is a prototypic HM cycle regulated master gene, RNA-seq analysis were performed for CD40+ and CD40- monocytes from mouse peripheral blood and 1,093 differentially expressed genes (DEGs) were selected from those two groups. All the DEGs modulate as much as 15 functional gene groups such as cytokines, enzymes and transcriptional factors. Furthermore, CD40+ monocytes activated trained immunity pathways especially in Acetyl-CoA generation and mevalonate pathway. In HM cycle, CD40 is a prototypic HM cycle regulated master gene to induce the most of the Hcy metabolic enzymes as well as MT, which can further modulate the methylation-regulated pathological signaling. / Biomedical Sciences