The effectiveness of most chemotherapeutic regimens is limited by the toxicity of the therapy to normal healthy cells. Therapies to selectively modulate abnormal T cells bearing the interleukin 2 receptor (IL-2R) have been developed to treat diseases associated with aberrant immune response. This study describes the development and optimization of a targeted gene or oligonucleotide therapy vehicle to IL-2R bearing T cells for selective elimination of these cells. In this work, a monoclonal antibody to the IL-2R was used to target the oligonucleotide delivery vehicle which consisted of a polyamidoamine dendrimer. Optimization of the delivery vehicle involves understanding the factors that govern its association with oligonucleotide, the pathway of IL-2R endocytic trafficking, and the stability of the oligonucleotide in the biological milieu. Oligonucleotide stability in a cellular environment was examined intra- and extracellularly. Results showed that the rate of intracellular degradation of oligonucleotides was much greater than extracellular degradation. Binding of oligonucleotides to dendrimers was demonstrated as a function of dendrimer generation. The total binding capacities for dendrimers differed depending upon dendrimer size and surface group, whereas equilibrium binding affinity was comparable for all dendrimers tested. Binding of oligonucleotide delivery vehicle to the cell surface and subsequent internalization was inversely related to dendrimer size, and in all cases, significantly less than binding and internalization of the natural ligand for the IL-2R. Based on experimental results, a kinetic model of the delivery vehicle was derived which includedthe dependence of binding and internalization on dendrimer size and surface charge and intracellular degradation of oligonucleotide. Based on model predictions, we show that larger dendrimers carry more oligonucleotide than the smaller dendrimer vehicles, and delivery is more effective with larger vehicles. This work establishes our ability to predict the effects of different delivery vehicle properties on oligonucleotide delivery and aids in the development of design criteria for new vehicles for delivery of antisense, siRNA, or genes to IL-2R bearing cells.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "August 2008" Includes bibliographical references.

Le rôle de l'interleukine 2 (IL-2) dans les cellules dendritiques (DC) humaines n’est pas bien défini. Dans ce travail, nous avons exploré les effets de l'IL-2 sur des DC dérivées de monocytes. Nous démontrons une expression constitutive des chaines des β et γ du récepteur de l’IL-2 sur les DC ainsi qu’une une expression inductible de la chaîne α en réponse à des agents ‘maturants’ tels le LPS et le TNFα. L’IL-2 induit la phosphorylation du facteur de transcription STAT5 provoquant l’augmentation de la synthèse d'IFN-γ par les DC sans modifier leur phénotype ou leur survie. En revanche, nous n'avons pas mis en évidence d’activation des autres voies de signalisation du récepteur de l’IL-2. Nous avons également démontré que l'IL-2 augmente les capacités des DC à activer les lymphocytes T CD4+ allogéniques et les lymphocytes T CD8+ indépendamment des lymphocytes T auxilliaires. Nous n’avons pas pu mettre en évidence la sécrétion endogène d'IL-2 par les DC cependant les anticorps anti-CD25 diminuent les capacités allostimulatrices de DC en absence d’IL-2 exogène. Ainsi nos travaux indiquent que l’expression de CD25 par les DC matures est un événement clé menant la DC à un nouvel état d’activation. / Human dendritic cells (DC) express interleukin 2 (IL-2) receptor α-chain (CD25), but the role of IL-2 in DC is poorly understood. In this work, we explored the effects of IL-2 on monocyte-derived DC. First, we demonstrated the constitutional expression of β and γ chain of IL-2R on DC, while the α-chain was inducible by LPS and TNFα. Then we found that IL-2 does not affect DC phenotype and apoptosis but increases IFN-γ synthesis in DC through activation of transcription factor STAT5. Moreover we reported that IL-2 increases the ability of DC to activate allogeneic CD4+ T cells and helpless CD8+ T lymphocytes, most likely because of IL-2–triggered IFN-γ synthesis. We have not been able to demonstrate the endogenous secretion of IL-2 by DC, however anti-CD25 decreased allostimulatory capacity of DC in the absence of exogenous IL-2.In summary, we disclose that IL-2 induces DC functional maturation and activation. Interestingly, our study suggests a direct effect of anti-CD25 monoclonal antibodies on DC and that CD25 expression regulation on human DC could be used to control immune response in vivo.

Interleukin-2-Rezeptor-Antikorper (IL-2RA) sind monoklonale Anti-IgG-Antikörper, die den Interleukin-2-Rezeptor aktivierter T-Zellen blockieren und so zu einer signifikanten Reduktion von akuten Abstoßungsreaktionen (AR) nach Nierentransplantation führen. Ziel dieser Arbeit ist die Durchführung einer Meta-Analyse aller verfügbaren kontrollierten Studien, die Erwachsene und Kinder nach primarer Lebertransplantation mit einer Induktionstherapie mit IL-2RA behandelten und diese mit Placebo oder keiner Heilbehandlung verglichen. Die Meta-Analyse soll zeigen, dass durch IL-2RA-Gabe die AR, SRAR (steroidresistente AR), Tod des Patienten (D), Transplantatverlust (GL) sowie CNI- und Steroid-bedingte Nebenwirkungen durch Dosisreduktion signifikant reduziert werden. Sechs Studien mit pädiatrischen und 20 Studien mit adulten Transplantatempfängern erfüllten die Einschlusskriterien. Die Analyse der Endpunkte wurde mit dem Modell mit zufälligen Effekten durchgeführt. Die IL-2RA-Induktionstherapie kann als sicher und ohne signifikante Nebenwirkungen für mindestens zwölf Monate bei Patienten nach Lebertransplantation beurteilt werden. IL-2RA reduziert signifikant die AR bei pädiatrischen (RR 0,38 CI [0,22-0,66]; p=0,0126; NNT=4) und adulten Patienten (RR 0,83 CI [0,74-0,93]; p=0,0007; NNT=18) nach Lebertransplantation und SRAR bei adulten Patienten (RR 0,66 CI [0,48-0,91]; p=0,011; NNT=29). D und GL werden reduziert. In adulten Studien der Vergleichsgruppe delayed/reduced CNI wurden eine signifikante Reduktion der gepoolten renalen Dysfunktionen (RR 0,46; CI [0,27-0,78]; p=0,004; 5 Kohorten), des Serum-Kreatinin (MD -0,05; [0,11-0,01]; p=0,03; 3 Kohorten) sowie eine signifikante Steigerung der eGFR beobachtet. Bei adulten Patienten der Vergleichsgruppe no/low steroids zeigte sich eine signifikante Reduktion des PTDM (RR 0,42; CI [0,31-0,55]; p<0,0001; NNT=12) und des HTN (RR 0,49; CI [0,25-0,96]; p=0,04; NNT=11).

O Diabetes Mellitus tipo 1 (DM1A) é uma doença autoimune caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas, levando à perda progressiva da capacidade de secreção de insulina pelas células beta pancreáticas. A homeostase das células T, ou seja, o desenvolvimento e manutenção apropriados dos números e funções das células T são essenciais para a integridade do sistema imune. Classicamente acreditava-se que as células T CD4+ poderiam se subdividir em duas populações efetoras distintas, T helper 1 e T helper 2. Recentemente, foram descritas duas novas vias de ativação de linfócitos T CD4+: a via Th17, que tem papel fundamental na autoimunidade; a via T regulatória, onde células T CD4+CD25+ high são essenciais na tolerância periférica e proteção contra autoimunidade. As Interleucinas (IL) de cadeia gama comum agem em várias etapas desta diferenciação linfocítica. A IL-21 é o membro mais recente desta família de citocinas, que inclui também: IL-2, IL-4, IL-7 , IL-9 e IL-15. A IL-21 atua através da interação com seu receptor, o IL-21R, apresentando ações pleiotrópicas e, como regra, pró-inflamatórias. Em estudos com modelos animais de diabetes autoimune verificou-se que a IL-21 e seu receptor são essenciais para o desenvolvimento da doença, porém ainda não há estudos sobre a ação desta interleucina no DM1 em humanos. O objetivo de nosso estudo foi avaliar o papel dos receptores das interleucinas de cadeia gama comum na patogênese do DM1A através da determinação da expressão da proteína de superfície e do RNA mensageiro destes receptores em pacientes com DM1A de início recente, em comparação com indivíduos controles normais, e da correlação destes valores com títulos de autoanticorpos pancreáticos. Estudamos a expressão da proteína de superfície do IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) e IL-7R (CD127) em linfócitos T periféricos de 35 pacientes com DM1 e 25 controles sadios utilizando citometria de fluxo. O tempo médio de diagnóstico do DM1 foi de 3 meses, e todos os pacientes estavam em uso de insulina no momento da coleta de sangue. Auto-anticorpos pancreáticos (anti-GAD65 e anti-IA2) foram dosados através de radioimunoensaio. A expressão do RNAm de IL-21R, IL-2R e IL-2R foi quantificada por PCR em tempo real em 23 dos pacientes portadores de DM1A. Detectamos, pela primeira vez, diminuição significativa na expressão proporcional de IL-21R, CD25 e CD122 em linfócitos TCD3+ e TCD4+, além de diminuição na expressão de CD124 em linfócitos T CD4+ e CD127 em linfócitos T CD3+. Verificamos também redução significativa na quantidade de células TCD4+CD25+high (T regulatórias) nos pacientes DM1A. Não houve correlação entre expressão dos receptores de superfície das interleucinas de cadeia gama comum e títulos de autoanticorpos pancreáticos. Realizamos o PCR em tempo real para quantificar a expressão do RNA mensageiro (RNAm) dos receptores de interleucinas de cadeia gama comum, e avaliar se esta correspondia à expressão das proteínas de superfície obtida através de citometria de fluxo. Comparamos a expressão do RNAm de IL-21R, IL-2R e IL-2R nos pacientes DM1A dividindo-os em tercis de acordo com os valores de expressão de proteína de superfície obtidos por citometria de fluxo em linfócitos T CD3+, e verificamos que não houve diferença entre os 3 grupos na expressão relativa dos genes estudados. Portanto, em nossa casuística a redução da expressão da proteína de superfície dos receptores de interleucinas de cadeia gama comum possivelmente decorreu de alterações posteriores à transcrição do RNA mensageiro / Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by pancreatic infiltration of T and B lymphocytes, macrophages and dendritic cells, leading to a progressive destruction of the insulin-producing -cells. Homeostasis of T cells can be defined as the ability of the immune system to maintain normal T-cell counts and to restore T-cell numbers following T-cell depletion or expansion. It was classically believed that the CD4+ T cells could be activated into two distinct effector populations, T helper1 and T helper2. It was recently described two new pathways of CD4+ T lymphocytes activation: the Th17 pathway, that plays a fundamental role in autoimmunity and the regulatory pathway (Treg), where CD4+CD25+high T cells are essential to maintain peripheral tolerance and therefore protect against autoimmunity. The common gamma chain cytokines interfere with several steps of the CD4+ T lymphocytes differentiation. Interleukin-21 (IL-21) is the most recent member of this family, that also includes IL-2, IL-4, IL-7, IL-9 and IL-15, and has pleiotropic effects on the immune system. Interleukin-21 acts through interaction with its receptor, the IL-21R, which is expressed in a great variety of immune cells. Various studies with animal models of autoimmune diabetes demonstrated that IL-21 and its receptor are essential for the development of the disease, but there are no studies evaluating the role of this interleukin and its receptor in T1DM in humans. The aim of our study was to assess the role of common gamma chain-dependent cytokine receptors in the pathogenesis of T1D, by determining the expression of the surface protein and mRNA of these receptors in recent-onset T1D patients and correlating these values with titles of pancreatic autoantibodies. We studied the surface protein expression of IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) and IL-7R (CD127) in peripheral T lymphocytes of 35 patients with T1D and 25 healthy controls using flow cytometry. Mean T1D duration was 3 months and all patients were using insulin at the time of blood withdraw. Pancreatic autoantibodies (anti-GAD65 and anti-IA2) were assessed by radioimmunoassay. The mRNA expression of IL-21R, IL-2R and IL-2R was quantified by real time PCR in 23 of the T1D patients. We detected for the first time a statistically significant decrease in the proportional expression of IL-21R, CD25 and CD122 on CD3+ and CD4+ T lymphocytes, a decrease in the expression of CD124 on CD4+ T cells and CD127 on CD3+ T lymphocytes. We also observed a significant reduction in the amount of CD4+ CD25+high (T regulatory cells) in T1D patients. There was no correlation between the expression of the surface receptors of common gamma chain cytokines and titles of pancreatic autoantibodies. We performed real-time PCR to quantify RNA expression of common gamma-chain interleukin receptors, and evaluate if these values corresponded to those of surface proteins obtained using flow cytometry. We compared the mRNA expression of IL-21R, IL-2R and IL-2R in T1D patients by dividing them into tertiles according to the expression values of surface protein obtained by flow cytometry in CD3+T lymphocytes. We observed that there was no difference in the relative expression of mRNA among the 3 groups of patients. Therefore, in our study, the reduction of surface protein expression of common gamma chain cytokines receptors was possibly due to alterations that occurred after the transcription of mRNA

O Diabetes Mellitus tipo 1 (DM1A) é uma doença autoimune caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas, levando à perda progressiva da capacidade de secreção de insulina pelas células beta pancreáticas. A homeostase das células T, ou seja, o desenvolvimento e manutenção apropriados dos números e funções das células T são essenciais para a integridade do sistema imune. Classicamente acreditava-se que as células T CD4+ poderiam se subdividir em duas populações efetoras distintas, T helper 1 e T helper 2. Recentemente, foram descritas duas novas vias de ativação de linfócitos T CD4+: a via Th17, que tem papel fundamental na autoimunidade; a via T regulatória, onde células T CD4+CD25+ high são essenciais na tolerância periférica e proteção contra autoimunidade. As Interleucinas (IL) de cadeia gama comum agem em várias etapas desta diferenciação linfocítica. A IL-21 é o membro mais recente desta família de citocinas, que inclui também: IL-2, IL-4, IL-7 , IL-9 e IL-15. A IL-21 atua através da interação com seu receptor, o IL-21R, apresentando ações pleiotrópicas e, como regra, pró-inflamatórias. Em estudos com modelos animais de diabetes autoimune verificou-se que a IL-21 e seu receptor são essenciais para o desenvolvimento da doença, porém ainda não há estudos sobre a ação desta interleucina no DM1 em humanos. O objetivo de nosso estudo foi avaliar o papel dos receptores das interleucinas de cadeia gama comum na patogênese do DM1A através da determinação da expressão da proteína de superfície e do RNA mensageiro destes receptores em pacientes com DM1A de início recente, em comparação com indivíduos controles normais, e da correlação destes valores com títulos de autoanticorpos pancreáticos. Estudamos a expressão da proteína de superfície do IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) e IL-7R (CD127) em linfócitos T periféricos de 35 pacientes com DM1 e 25 controles sadios utilizando citometria de fluxo. O tempo médio de diagnóstico do DM1 foi de 3 meses, e todos os pacientes estavam em uso de insulina no momento da coleta de sangue. Auto-anticorpos pancreáticos (anti-GAD65 e anti-IA2) foram dosados através de radioimunoensaio. A expressão do RNAm de IL-21R, IL-2R e IL-2R foi quantificada por PCR em tempo real em 23 dos pacientes portadores de DM1A. Detectamos, pela primeira vez, diminuição significativa na expressão proporcional de IL-21R, CD25 e CD122 em linfócitos TCD3+ e TCD4+, além de diminuição na expressão de CD124 em linfócitos T CD4+ e CD127 em linfócitos T CD3+. Verificamos também redução significativa na quantidade de células TCD4+CD25+high (T regulatórias) nos pacientes DM1A. Não houve correlação entre expressão dos receptores de superfície das interleucinas de cadeia gama comum e títulos de autoanticorpos pancreáticos. Realizamos o PCR em tempo real para quantificar a expressão do RNA mensageiro (RNAm) dos receptores de interleucinas de cadeia gama comum, e avaliar se esta correspondia à expressão das proteínas de superfície obtida através de citometria de fluxo. Comparamos a expressão do RNAm de IL-21R, IL-2R e IL-2R nos pacientes DM1A dividindo-os em tercis de acordo com os valores de expressão de proteína de superfície obtidos por citometria de fluxo em linfócitos T CD3+, e verificamos que não houve diferença entre os 3 grupos na expressão relativa dos genes estudados. Portanto, em nossa casuística a redução da expressão da proteína de superfície dos receptores de interleucinas de cadeia gama comum possivelmente decorreu de alterações posteriores à transcrição do RNA mensageiro / Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by pancreatic infiltration of T and B lymphocytes, macrophages and dendritic cells, leading to a progressive destruction of the insulin-producing -cells. Homeostasis of T cells can be defined as the ability of the immune system to maintain normal T-cell counts and to restore T-cell numbers following T-cell depletion or expansion. It was classically believed that the CD4+ T cells could be activated into two distinct effector populations, T helper1 and T helper2. It was recently described two new pathways of CD4+ T lymphocytes activation: the Th17 pathway, that plays a fundamental role in autoimmunity and the regulatory pathway (Treg), where CD4+CD25+high T cells are essential to maintain peripheral tolerance and therefore protect against autoimmunity. The common gamma chain cytokines interfere with several steps of the CD4+ T lymphocytes differentiation. Interleukin-21 (IL-21) is the most recent member of this family, that also includes IL-2, IL-4, IL-7, IL-9 and IL-15, and has pleiotropic effects on the immune system. Interleukin-21 acts through interaction with its receptor, the IL-21R, which is expressed in a great variety of immune cells. Various studies with animal models of autoimmune diabetes demonstrated that IL-21 and its receptor are essential for the development of the disease, but there are no studies evaluating the role of this interleukin and its receptor in T1DM in humans. The aim of our study was to assess the role of common gamma chain-dependent cytokine receptors in the pathogenesis of T1D, by determining the expression of the surface protein and mRNA of these receptors in recent-onset T1D patients and correlating these values with titles of pancreatic autoantibodies. We studied the surface protein expression of IL-21R, IL-2R (CD25), IL-2R (CD122), IL-4R (CD124) and IL-7R (CD127) in peripheral T lymphocytes of 35 patients with T1D and 25 healthy controls using flow cytometry. Mean T1D duration was 3 months and all patients were using insulin at the time of blood withdraw. Pancreatic autoantibodies (anti-GAD65 and anti-IA2) were assessed by radioimmunoassay. The mRNA expression of IL-21R, IL-2R and IL-2R was quantified by real time PCR in 23 of the T1D patients. We detected for the first time a statistically significant decrease in the proportional expression of IL-21R, CD25 and CD122 on CD3+ and CD4+ T lymphocytes, a decrease in the expression of CD124 on CD4+ T cells and CD127 on CD3+ T lymphocytes. We also observed a significant reduction in the amount of CD4+ CD25+high (T regulatory cells) in T1D patients. There was no correlation between the expression of the surface receptors of common gamma chain cytokines and titles of pancreatic autoantibodies. We performed real-time PCR to quantify RNA expression of common gamma-chain interleukin receptors, and evaluate if these values corresponded to those of surface proteins obtained using flow cytometry. We compared the mRNA expression of IL-21R, IL-2R and IL-2R in T1D patients by dividing them into tertiles according to the expression values of surface protein obtained by flow cytometry in CD3+T lymphocytes. We observed that there was no difference in the relative expression of mRNA among the 3 groups of patients. Therefore, in our study, the reduction of surface protein expression of common gamma chain cytokines receptors was possibly due to alterations that occurred after the transcription of mRNA

Objectives: To assess the expression of the symptoms of jaw muscle pain and its association with alterations in biochemistry, other symptoms and the carriage of staphylococci. Methods: Three different study populations were assessed. The first was selected and examined by the author and consisted of 43 pain and 41 age and sex matched controls. The second was a study of CFS patients who were blinded to the author and the author subsequently examined the associations between jaw muscle symptom reporting and the standardised biochemistry measures. The third study was also blinded to the author but included an investigation of staphylococci and certain cytokine and biochemistry measures. Results: The three studies clearly establish an association between the carriage of toxicogenic coagulase negative staphylococci and the expression of jaw muscle pain in both males and females. These associations were homogeneous and were found whether the patients were selected on the basis of having jaw muscle pain or selected from within a population of patients selected on the basis of having Chronic Fatigue Syndrome. The studies associated the changes with variations in biochemistry and these were in turn associated with symptom expression within the jaw muscle pain patients. These biochemical alterations included the dysregulation of immune cell counts, cytokines, electrolyte and protein metabolism. These symptoms and biochemical changes were associated with pain severity and illness duration and staphylococcal toxin production. From the data a model was developed which shows the mechanisms involved in the development of chronic pain in the jaw muscles. Conclusions: The carriage of toxicogenic coagulase-negative staphylococci were found to be associated with the expression of jaw muscle pain and the alterations in biochemistry associated with these symptoms.

Objectives: To assess the expression of the symptoms of jaw muscle pain and its association with alterations in biochemistry, other symptoms and the carriage of staphylococci. Methods: Three different study populations were assessed. The first was selected and examined by the author and consisted of 43 pain and 41 age and sex matched controls. The second was a study of CFS patients who were blinded to the author and the author subsequently examined the associations between jaw muscle symptom reporting and the standardised biochemistry measures. The third study was also blinded to the author but included an investigation of staphylococci and certain cytokine and biochemistry measures. Results: The three studies clearly establish an association between the carriage of toxicogenic coagulase negative staphylococci and the expression of jaw muscle pain in both males and females. These associations were homogeneous and were found whether the patients were selected on the basis of having jaw muscle pain or selected from within a population of patients selected on the basis of having Chronic Fatigue Syndrome. The studies associated the changes with variations in biochemistry and these were in turn associated with symptom expression within the jaw muscle pain patients. These biochemical alterations included the dysregulation of immune cell counts, cytokines, electrolyte and protein metabolism. These symptoms and biochemical changes were associated with pain severity and illness duration and staphylococcal toxin production. From the data a model was developed which shows the mechanisms involved in the development of chronic pain in the jaw muscles. Conclusions: The carriage of toxicogenic coagulase-negative staphylococci were found to be associated with the expression of jaw muscle pain and the alterations in biochemistry associated with these symptoms.