Orientador: Valéria Marçal Felix de Lima / Resumo: A Leishmaniose Visceral (LV) é causada nas Américas, pelo protozoário intracelular obrigatório Leishmania infantum e os cães domésticos são os principais reservatórios urbanos do parasita e em áreas endêmicas, o aumento da LV em humanos tem sido associado ao aumento da infecção canina. Os atuais medicamentos disponíveis para a Leishmaniose Canina (CanL) não são completamente eficientes e meses após o tratamento a maioria dos cães apresentam recidiva, indicando a necessidade de buscar formas alternativas de tratamento. Na CanL, cães desenvolvem uma resposta imune celular (Th1) ineficiente para combater o parasita e a estimulação das vias de citocinas em células de defesa com proteínas recombinantes, tem o potencial de se tornar parte de métodos imunoterapêuticos eficazes. Neste estudo, as citocinas recombinantes caninas (IL-12, IL-2, IL-15 e IL-7) e o receptor solúvel de IL-10R1 (sIL-10R1), com atividade antagonista, foram avaliadas pela primeira vez em combinações (IL-12/IL- 2, IL-12/IL-15, IL-12/sIL-10R1, IL-15/IL-7) ou isoladamente (sIL-10R1) quanto à capacidade imunomodulatória em células mononucleares do sangue periférico (sigla em inglês PBMC) de cães com leishmaniose. Todas as combinações de proteínas recombinantes testadas mostraram melhorar a resposta linfoproliferativa. Além disso, as combinações de IL-12/IL-2 e IL-12/IL-15 promoveram a diminuição na expressão da proteína “Programed Cell Death 1” (PD-1) nos linfócitos. Estas mesmas combinações de citocinas e IL-12/sI... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral Leishmaniasis (LV) is caused in the Americas by the obligate intracellular protozoan Leishmania infantum and domestic dogs are the major urban reservoirs of the parasite and in endemic areas, and increase LV in humans has been associated with increased canine infection. The current medications available for Canine Leishmaniasis (CanL) are not completely effective and months after treatment most dogs present with relapse, indicating the necessity to looking for alternative forms of treatment. In CanL, dogs develop an ineffective cellular immune response (Th1) to combat the parasite. Then, the stimulation of cytokine pathways in defense cells with recombinant proteins, has the potential to become part of effective immunotherapeutic methods. In this study, the canine recombinant cytokines (IL-12, IL-2, IL-15 and IL-7) and the soluble receptor of IL-10R1 (sIL-10R1) with antagonistic activity, were evaluated for the first time in combinations IL-12/IL-2, IL-12/IL-15, IL12/sIL-10R1, IL-15/IL-7) or alone (sIL-10R1) for their immunomodulatory capacity in peripheral blood mononuclear cells (PBMC) from dogs with leishmaniasis. All combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, combinations of IL-12/IL-2 and IL-12/IL-15 promoted decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and IL-12/casIL-10R1 induced IFN-y production in PBMC. Furthermore, the combinat... (Complete abstract click electronic access below) / Mestre

Ko Kin Ming Jeffery. / Thesis submitted in: July 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 99-111). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Malignant Lymphoma --- p.1 / Chapter 1.2 --- Non-Hodgkin's Lymphoma --- p.1 / Chapter 1.3 --- Diffuse Large B-cell Lymphoma --- p.2 / Chapter 1.3.1 --- General Features of Diffuse Large B-cell Lymphoma --- p.2 / Chapter 1.3.2 --- Morphologic variants of Diffuse Large B-cell Lymphoma --- p.4 / Chapter 1.3.2.1 --- Centroblastic vairant --- p.5 / Chapter 1.3.2.2 --- Immunoblastic variant --- p.5 / Chapter 1.3.2.3 --- Anaplastic variant --- p.6 / Chapter 1.3.3 --- Immunophenotype of Diffuse Large B-cell Lymphoma --- p.6 / Chapter 1.3.3.1 --- Lineage-associated antigens --- p.6 / Chapter 1.3.3.1.1 --- B-cell lineage antigens --- p.6 / Chapter 1.3.3.1.2 --- T-cell lineage antigens --- p.7 / Chapter 1.3.3.2 --- Antigen involved in regulation of cell proliferation and apoptosis --- p.8 / Chapter 1.3.3.2.1 --- Proliferation markers --- p.8 / Chapter 1.3.3.2.2 --- Cell cycle regulators --- p.8 / Chapter 1.3.3.2.3 --- Protein controlling apoptosis --- p.10 / Chapter 1.3.4 --- Subtypes of Diffuse Large B-cell Lymphoma --- p.10 / Chapter 1.3.4.1 --- Classification method of DLBCL subtypes --- p.11 / Chapter 1.3.4.1.1 --- DNA microarray --- p.11 / Chapter 1.3.4.1.2 --- Immunohistochemistry pattern --- p.14 / Chapter 1.3.4.1.2.1 --- CD10 --- p.16 / Chapter 1.3.4.1.2.2 --- Bcl-6 --- p.16 / Chapter 1.3.4.1.2.3 --- CD138 --- p.17 / Chapter 1.3.4.1.2.4 --- MUM1/IRF4 --- p.17 / Chapter 1.3.4.2 --- Prognosis of 、DLBCL subtypes --- p.19 / Chapter 1.4 --- Interleukin 10 --- p.22 / Chapter 1.4.1 --- The IL-10 gene --- p.23 / Chapter 1.4.2 --- IL-10 promoter --- p.23 / Chapter 1.5 --- IL-10 receptor --- p.24 / Chapter 1.6 --- Cellular Signaling Pathways Regulated by IL-10 --- p.25 / Chapter 1.6.1 --- Jak/Stat Pathway --- p.25 / Chapter 1.6.2 --- Inhibition of NF B pathway --- p.26 / Chapter 1.7 --- Function of IL-10 --- p.27 / Chapter 1.7.1 --- Effects of IL-10 on immune cells in vitro --- p.27 / Chapter 1.7.2 --- Effects of IL-10 on B-cells --- p.28 / Chapter 1.8 --- IL-10 and IL-10 receptor in malignant diseases --- p.29 / Chapter 1.8.1 --- Melanoma --- p.29 / Chapter 1.8.2 --- Carcinoma --- p.30 / Chapter 1.8.3 --- Lymphoma --- p.30 / Chapter 1.9 --- Single Nucleotide Polymorphism (SNP) --- p.33 / Chapter 1.9.1 --- SNPs in cancer research --- p.34 / Chapter 1.9.1.1 --- Susceptibility to cancer and SNPs --- p.35 / Chapter 1.9.1.2 --- Outcome and SNPs --- p.35 / Chapter 1.10 --- SNP in the IL-10 promoter --- p.36 / Chapter 1.11 --- IL-10 promoter SNP in DLBCL --- p.37 / Chapter Chapter 2: --- Aims of Study --- p.39 / Chapter Chapter 3: --- Materials and Methods --- p.41 / Chapter 3.1 --- Sample Recruitment --- p.41 / Chapter 3.2 --- DNA preparation for Single Nucleotide Polymorphism (SNP) analysis --- p.41 / Chapter 3.2.1 --- Isolation of Peripheral Blood Mononuclear Cell (PBMC) from buffy coat from blood of normal control group --- p.41 / Chapter 3.2.2 --- Preparation for NHL and DLBCL samples from paraffin-embedded sections for DNA extraction --- p.42 / Chapter 3.2.3 --- DNA extraction for SNP analysis --- p.42 / Chapter 3.3 --- SNP analysis by Restriction Fragment Length Polymorphism (RFLP) --- p.43 / Chapter 3.3.1 --- Amplification of target site by PCR --- p.43 / Chapter 3.3.2 --- SNP analysis --- p.45 / Chapter 3.4 --- Determination of haplotypic frequency --- p.50 / Chapter 3.5 --- Classification of DLBCL by immunohistochemistry --- p.50 / Chapter 3.5.1 --- Staining pattern of CD10 --- p.53 / Chapter 3.5.2 --- Staining pattern of Bcl-6 --- p.54 / Chapter 3.5.3 --- Staining pattern of CD138 --- p.55 / Chapter 3.5.4 --- Staining pattern of MUM1/IRF4 --- p.56 / Chapter 3.6 --- Statistical Analysis --- p.57 / Chapter Chapter 4: --- Results --- p.58 / Chapter 4.1 --- SNPs of IL-10 promoter in normal controls --- p.58 / Chapter 4.1.1 --- Allelic Frequencies and genotype distributions --- p.58 / Chapter 4.1.2 --- Haplotypic Frequencies of normal controls --- p.58 / Chapter 4.2 --- SNP of the IL-10 promoter in non-Hodgkin's lymphomas --- p.59 / Chapter 4.2.1 --- Allelic frequencies and genotype distributions --- p.59 / Chapter 4.2.2 --- Haplolypic frequencies --- p.61 / Chapter 4.4 --- SNPs of the IL-10 promoter in DLBCL --- p.62 / Chapter 4.4.1 --- Allelic frequencies and genotype distributions --- p.62 / Chapter 4.4.2 --- Haplotypic frequencies --- p.64 / Chapter 4.5 --- SNP of the IL-10 promoter in different subtypes of DLBCL --- p.65 / Chapter 4.5.1 --- Classification of DLBCL by immunohistochemistry --- p.65 / Chapter 4.5.2 --- SNP of the IL-10 promoter in Germinal Center DLBCL (GC-DLBCL) --- p.67 / Chapter 4.5.2.1 --- Allelic frequencies and genotype distributions --- p.67 / Chapter 4.5.1.2 --- Haplotypic frequencies --- p.69 / Chapter 4.5.2 --- SNP of the IL-10 promoter in Activated Germinal Center DLBCL (AGC-DLBCL) --- p.70 / Chapter 4.5.2.1 --- Allelic frequencies and genotype distributions --- p.70 / Chapter 4.5.2.2 --- Haplotypic frequencies --- p.72 / Chapter 4.5.3 --- SNP of the IL-10 promoter in Activated non-Germinal Center DLBCL (ANGC-DLBCL) --- p.73 / Chapter 4.5.3.1 --- Allelic frequencies and genotype distributions --- p.73 / Chapter 4.5.3.2 --- Haplotypic frequencies --- p.75 / Chapter 4.5.4 --- SNP of the IL-10 promoter in Unclassified DLBCL (UC-DLBCL). --- p.76 / Chapter 4.5.4.1 --- Allelic frequencies and genotype distributions --- p.76 / Chapter 4.5.4.2 --- Haplotypic frequencies --- p.78 / Chapter 4.6 --- Summary of SNP of the IL-10 promoter in DLBCL subtypes --- p.79 / Chapter 4.7 --- Overall survival analysis --- p.80 / Chapter 4.7.1 --- Clinical data of DLBCL --- p.80 / Chapter 4.7.2 --- Cox Proportional Hazards Regression Analysis in DLBCL --- p.81 / Chapter Chapter 5: --- Discussion --- p.88 / Chapter 5.1 --- SNP for low IL-10 production in Hong Kong population --- p.88 / Chapter 5.2 --- NHL in low IL-10 production population --- p.90 / Chapter 5.2.1 --- The relationship between IL-10 and NHL --- p.90 / Chapter 5.2.2 --- Allelic frequencies and haplotype of the IL-10 promoter in NHL --- p.90 / Chapter 5.3 --- Classification of DLBCL --- p.91 / Chapter 5.3.1 --- Current prognostic analysis --- p.91 / Chapter 5.3.2 --- DLBCL subtypes distribution in Hong Kong is different from Caucasian --- p.92 / Chapter 5.4 --- IL-10 and DLBCL --- p.93 / Chapter 5.5 --- SNP of IL-10 promoter in DLBCL subtypes --- p.94 / Chapter 5.5.1 --- Allelic frequencies and haplotype of DLBCL subtypes --- p.94 / Chapter 5.5.2 --- Rare haplotypes were discovered in DLBCL --- p.94 / Chapter 5.6 --- Overall survival Analysis --- p.95 / Chapter 5.6.1 --- Univariate Cox Proportional Hazards Regression Analysis --- p.95 / Chapter 5.6.2 --- Bivariate Cox Proportional Hazards Regression Analysis --- p.96 / Chapter Chapter 6: --- Conclusion --- p.97 / References --- p.99

Tregs dienen zur Aufrechterhaltung der Balance im Immunsystem. Die Infektion, Aktivierung oder Induktion von Tregs durch Pathogene kann diese Balance empfindlich stören, eine Immunsuppression zur Folge haben und zur Ausbildung von Autoimmunerkrankungen oder Persistenzen beitragen. Das MV verfügt nicht nur über vielfältige Mechanismen der Immunsuppression, während einer MV-Infektion herrschen zudem Bedingungen vor, welche die Zahl und Aktivität von Tregs beeinflussen könnten. Aufgrund der Expression von Reifungsmarkern auf Trn ist zudem eine präferenzielle Infektion dieser Zellpopulation denkbar. MV-Infektionen können sowohl die akute MV-Enzephalitis, eine Autoimmunerkrankung, nach sich ziehen, als auch die Persistenz SSPE ausbilden. Ob diese Komplikationen mit spezifischen Aberrationen in der Menge und Aktivität von Tregs im Zusammenhang stehen, war bisher nicht bekannt. In dieser Arbeit konnte gezeigt werden, dass auf unstimulierten Trn der Reifungsmarker und MV-Rezeptor CD150 exprimiert wird und es in Folge dessen in vitro zu einer präferentiellen nicht produktiven Infektion und Depletion von Trn kommt. Ex vivo ließ sich ein deutlicher Depletionseffekt während der frühen akuten MV-Enzephalitis nachweisen, der nach Vaczinierung eines gesunden Probanden und Challenge eines immunisierten Affen nicht auftrat. Ob dieser Depletionseffekt ursächlich für die Enzephalitis ist, oder es sich um einen Begleiteffekt handelt ließe sich an Modellorganismen durch mitogene Manipulation der Trn während einer MV-Infektion untersuchen. Auch bei SSPE kann es zu einer Depletion von Trn kommen, dies scheint jedoch nicht mit der Progression dieser Erkrankung im Zusammenhang zu stehen. Wahrscheinlich ist dagegen ein Zusammenhang mit der Induktion von Tregs. In MV-stimulierten Proben von SSPE-Patienten wurde im Mittel signifikant mehr IL-10 exprimiert als in den Kontrollen. In Proben seropositiver gesunder Spender wurde IL-10 in den ersten Stunden nach MV-Stimulation fast ausschließlich von induzierten Tregs exprimiert. Weitere Versuche sind nötig, um die Evidenz zu steigern und zu ermitteln, ob auch in Patientenproben die frühe IL-10 Expression nach MV-Stimulation von induzierten Tregs dominiert wird. Zusammenfassend kann gesagt werden, dass es sowohl bei der MV-Enzephalitis als auch bei SSPE zu signifikanten Wechselwirkungen mit Tregs kommt. Ob sich eine MV-Enzephalitis auch ohne Depletion von Trn ausbilden kann und ob die Ausbildung von SSPE erhöhte IL-10 Expression voraussetzt, werden weitere Untersuchungen ergründen müssen. / Treg are supposed to keep the balance throughout the immune system. Infection, activation and induction of Treg by pathogens can interrupt with this balance. Immunosuppression autoimmunity and viral persistence can result from this interference. MV does not only cause immunosuppression during infection by multifaceted mechanisms, but also effects circumstances known to interfere with the frequency and activity of Treg. Due to the expression of maturity markers on the surface of Trn a preferential infection of this cell population by MV seems possible. MV-infection can involve both, acute MV-encephalitis, an autoimmune disease, and the virus persistence SSPE. It was not investigated until now, if thous complications are associated to specific aberrations in the frequency and activity of Treg. In this paper it was demonstrated that CD150, which is both a maturity marker and a MV-receptor, is expressed on unstimulated Trn. Due to this expression the Trn are infected preferentially and non productively in vitro leading to apoptosis. Ex vivo a noticeable depletion effect was detected during the early acute MV-encephalitis. This effect did not accure after vaccination of a healthy proband, and the challenge of an immunised monkey. Mitogenic manipulation of Trn in model organisms during MV-infection could give advice, if this depletion effect is causal to MV-Encephalitis or rather a concomitant effect. Depletion of Trn can also arise in the presence of SSPE, but this seems not to interfere with the progression of disease. By contrast there is presumable an interrelation of SSPE with the induction of Tregs. In MV-stimulated probes from SSPE-patients the median IL-10 expression was significantly higher than in controls. Probes from seropositive healthy donators were used to identify the IL-10 expressing cells. During the first hours after MV-stimulation IL-10 was mainly expressed by induced Treg. Subsequent investigations are necessary to enhance the evidence and to determine if induced Treg also dominate the early IL-10 expression after MV-stimulation in patient probes. To give a resume you could claim that there are significant interrelations between MV-encephalitis respectively SSPE and Treg. If a depletion of Trn is necessary for the development of MV-encephalitis and if increased levels of IL-10 expression are required for the formation of SSPE has to be examined in further experiments.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

<p>Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation.</p><p>The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated.</p><p>The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.</p>

Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation. The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated. The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.

Interleukin-1 (IL-1) is a prototypic multifunctional cytokine. IL-1 family include interleukin-1 a (IL-1 a), interleukin-1b (IL-1 b) and interleukin-1 receptor antagonist (IL-1 Ra). IL-1 b is the archetypeal pleiotropic cytokine which have been produced by many cells and exerting its biological effects on almost all cell types. IL-1 b is the most potent of known agents that are gastric cytoprotective, antiulcer, antisecretory and an inhibitor of gastric emptying. IL-1 Ra competes with IL-1 b for cell surface receptor occupancy. Host genetic factors that affect interleukin-1 (IL-1) have been reported to influence the susceptibility of Caucasians to gastric cancer. Whether Asians have the same genetic susceptibility remains unclear. In this study, the genetic associations of IL-1B and IL-1RN polymorphisms with gastric cancer and duodenal ulcer in Taiwan were evaluated. Genomic DNA from 140 unrelated Taiwanese patients with gastric adenocarcinoma, 94 with duodenal ulcer and 165 ethically matched healthy controls was typed for polymorphisms at positions ¡V31, -511, and +3954 in the IL-1B gene, and the variable number of tandem repeats polymorphisms in intron 2 of the IL-1RN gene. The allele frequencies of IL-1RN 2R in gastric cancer cases were much higher than those in healthy controls (9% vs. 3%, p = 0.781). The allele frequencies of IL-1B ¡V31, IL-1B ¡V511 and IL-1B +3954 did not differ. An increased risk of the development of intestinal type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 4.06 (95% confidence interval [CI]: 1.68 ¡V 9.79, p-value=0.085). And another increased risk of the development of diffuse type gastric carcinoma was found in IL-1RN 2R carriers with an odds ratio (OR) of 3.15 (95% confidence interval [CI]: 1.16 ¡V 8.56, p-value=0.061). A significant association was found in IL-1RN 2R/4R genotype and the risk of the development of duodenal ulcer, with an odds ratio (OR) of 2.57 (95% CI: 1.03 ¡V 6.38, p = 0.292). No significant relationship was noted in duodenal ulcer patients with IL-1B genotype examed in this study. Additionally, a synergistic interaction between blood type A and IL-1 RN 2R carriers existed in gastric cancer patients (OR= 4.51; 95% CI: 1.20 ¡V 16.88, p-value=0.516). The synergistic interaction was even stronger between blood type O and IL-1 RN 2R carriers of duodenal ulcer patients (OR= 10.3; 95% CI: 2.10 ¡V 50.61, p-value=0.160). In conclusion, the genetic polymorphisms of IL-1RN 2R and blood type A are associated with the development of gastric cancer. The genetic polymorphisms of IL-1RN 2R and blood type O are associated with the development of duodenal ulcer.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 180-198). Also available on the Internet.

Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7281. Adviser: Lu-Yu Hwang. Includes bibliographical references.

Viele Publikationen haben bereits den Einfluss verschiedener Zytokingenvariationen auf die Ensteheung einer Graft versus Host Disease untersucht. Ich habe den Einfluss verschiedener Zytokin- und Zytokinrezeptorgenvariationen des Interleukin 10-Gens auf die Ausprägung von GvHD und die Überlebensrate nach allogener Blutstammzelltranplantation bei Patienten mit einer hämatologischen Neoplasie untersucht. Die GvHD zählt zu einer gefürchteten Komplikation bei Patienten nach einer allogenen Stammzelltransplantation. Sie kann als akute Form innerhalb von 100 Tagen nach Transplantation, sowie als chronische Form im späteren Verlauf auftreten. Sowohl die akute wie auch die chronische GvHD können bis zum Tode des Patienten führen oder durch Organschäden und die langandauernde intensive Immunsuppression die Lebensqualität der Patienten sehr beeinträchtigen. Es fehlt bis heute das genaue Verständnis, warum einige Patienten eine schwerwiegende GvHD entwickeln, während andere Patienten von einer nur leichtgradig auftretenden GvHD auf Grund eines damit assoziierten GvL-Effektes sogar profitieren. Das Interleukin 10 spielt neben anderen Zytokinen aufgrund seiner antiinflammatorischen Funktion eine wichtige Rolle in der Pathophysiologie der GvHD. Durch IL 10 kommt es zu einer Herunterregulation von MHC Klasse 2 und Kostimulatorischen Molekülen auf der Zelloberfläche, womit die Fähigkeit antigenpräsentierender Zellen zur Antigenpräsentation supprimiert wird. Interleukin 10 bewirkt außerdem eine Hemmung der Produktion proinflammatorischer Faktoren wie z.B. Interferon a, oder TNF-a, sowie eine Suppression der Transkription von NF- kB. Für diese Analyse wurden die GvHD-relevanten klinischen Daten von 360 konsekutiven Patienten ausgewertet, die zwischen 2001 und 2011 in der Universitätsmedizin Göttingen eine allogene Transplantation erhielten und eine Einteilung in die Schweregrade der akuten und der chronischen GvHD vorgenommen. Es wurde eine Genotypisierung ausgewählter Single Nucleotide Polymorphisms des IL 10–Gens und des IL 10–Rezeptorgens mittels SNaPshot-Analyse und TaqMan® Genotyping Assays vorgenommen. Durch univariate, gefolgt von multivariaten logistischen Regressionsanalysen der Daten mit Bezug zum klinischen Verlauf wurden Genvarianten mit Einfluss auf das Auftreten und den Schweregrad der GvHD identifiziert. Für die Überlebenszeitanalysen wurde ein simpler Logrank–Test verwendet, um Variablen mit einem p-Wert < 0,20 herauszufiltern, mit denen anschließend eine Cox–Regressionsanalyse durchgeführt wurde. In Voruntersuchungen (v.a. Hansen et al) war der SNP IL-10 -597 Genotyp A (= IL-10 -592) im Patienten, in Kombination mit dem G-Allel im SNP IL-10Rβ K47E (=IL-10Rβ c238) im Spender, bereits als wichtiger protektiver Faktor gegen eine schwere aGvHD in Matched-related-Donor (MRD)-transplantierten Patienten beschrieben worden. Der protektive Effekt dieser Kombination war in unserem Datensatz über alle Patienten hinweg nur als Tendenz erkennbar, während sich der alleinige protektive Effekt des R K47E G-Allel im Spender bei Patienten mit malignen Lymphomen als signifikant erwies. Dieser Unterschied in unseren Analysen, kann an der inhomogenen Zusammenzusetzung des Kollektivs aus MRD- und MUD- transplantierten Patienten liegen. Der protektive Effekt der oben genannten Kombination wurde bisher nur in reinen MRD-Kollektiven nachgewiesen und war in reinen MUD-Kollektiven nicht vorhanden. Weiterhin erwies sich für die chronische GvHD der proximale SNP IL-10 -1087 Genotyp GG des Patienten als assoziiert mit einem erhöhten Auftreten von chronischer GvHD. Dieser Genotyp ist mit einer erhöhten IL 10 Produktion assoziiert. Laut Ergebnissen von Martin und Zhou et al., ist ein erhöhter IL 10–Spiegel mit einer cGvHD assoziiert. Unter den in dieser Untersuchung erstmalig analysierten Polymorphismen des IL 10-Rezeptorgens wurde ein signifikant erhöhtes Risiko für das Auftreten einer schweren akuten GvHD (Grad III und IV) für Patienten festgestellt, die mit einem Spender des Genotyps IL 10 Rα G351R AA transplantiert worden waren. Dieses Risiko war weiter erhöht für die Patienten festzustellen, die zur T-Zell-Depletion im Rahmen der Konditionierung ATG erhalten hatten. Der Einsatz des als prophylaktisch gegen das Auftreten einer schweren GvHD genutzen ATGs war bei dieser Patientengruppe also mit einer zusätzlichen Risikoerhöhung für das Auftreten einer schweren GvHD assoziiert. Der IL 10 Rezeptor besteht aus einer - und einer -Einheit. Analysen der -Einheit des IL 10-Rezeptors beziehen sich größtenteils auf den Rα G159S. Der Rα G351R wurde bisher nicht funktionell oder in genetischen Assoziationsstudien untersucht, weshalb die funktionelle Bedeutung dieses Polymorphismus bisher nicht bekannt ist. Eine funktionelle Rolle dieses Polymorphismus erscheint aber prinzipiell möglich, da er im codierenden Bereich des Gens liegt und mit einem Austausch der Aminosäure Glycin gegen Arginin verbunden ist. Die Bedeutung der in dieser Arbeit erstmalig untersuchten SNPs, insbesondere des Polymorphismus IL 10 Rα G351R AA, sollte in unabhängigen Patientenkollektiven hinsichtlich seiner klinischen Relevanz weiter überprüft werden, auch um die Assoziation mit einer höheren Inzidenz an schwerer akuter GvHD bei Patienten, die ATG erhielten, detaillierter zu untersuchen. Um den Effekt besser zu verstehen, sollte diese Genvariante auch in ihrer Funktionalität in vitro untersucht werden. Neben dem Interleukin 10 sind noch viele weitere Zytokine an der Entstehung bzw. der Verhinderung einer GvHD beteiligt und es ist wichtig, hier mit der Genotypisierung von Kandidatengenen fortzufahren, um weitere funktionell bedeutsame Genvarianten zu identifizieren. Im Erfolgsfalle wird durch die Identifizierung dieser Varianten die Erstellung eines genetischen Profils der Patienten vor der geplanten Stammzelltransplantation möglich. Mit Hilfe dieses Profils kann eine bessere Einschätzung des individuellen Risikos getroffen und gegebenenfalls die Medikation frühzeitig dementsprechend angepasst werden.