A elucidação dos mecanismos moleculares pelos quais tratamentos hormonais alteram o início da puberdade é de fundamental importância para o desenvolvimento de estratégias que reduzam a idade ao primeiro parto, e consequentemente a taxa de desfrute do rebanho Nelore. Foram investigados os efeitos do uso de dispositivos de progesterona, e do estradiol endógeno, sobre mecanismos moleculares controlando a obtenção da puberdade de novilhas Nelore peripúberes. Especificamente, como as diferenças na expressão de genes relativos à reprodução em duas áreas do hipotálamo. Trinta e cinco novilhas Nelores não púberes, e com idade entre 13 e 14 meses, foram divididas em quatro tratamentos experimentais (nove ou oito por tratamento): dispositivo de P4 sem estradiol (SP); dispositivo de P4 com estradiol (PE); sem dispositivo de P4 e sem estradiol (SS); e sem dispositivo de P4 e com estradiol (SE). As novilhas foram alimentadas no cocho pós desmame até atingirem 295 ± 11 kg, com fornecimento de água à vontade. Ao término do tratamento hormonal as novilhas foram abatidas e as porções de hipotálamo colhidas para processamento e armazenagem a -80 ºC. O RNA total do tecido hipotalâmico foi extraído, tratado com DNAse I e submetido à síntese de cDNA para estudo da expressão gênica por PCR em tempo real (qRT-PCR). Foram formados pools de RNA para a realização de um estudo abrangente da administração de progesterona e do efeito do estradiol endógeno e das diferenças entre áreas do hipotálamo, realizado por sequenciamento de nova geração (RNA-Seq), de forma a identificar possíveis genes candidatos no hipotálamo. Foram encontrados genes diferencialmente expressos alterados pelos tratamentos e entre as áreas do hipotálamo relativos à obtenção da puberdade. / The understanding of the molecular mechanisms by which nutrition, genetics and hormonal treatments affect the beginning of puberty is of great importance for developing strategies aiming to reduce the age at first calving, and therefore increase the slaughter rate in Nellore cattle. The effects of progesterone device and of endogenous estradiol on the molecular mechanisms controlling the attainment of puberty in Nellore heifers were investigated. Specifically, the molecular pathways of progesterone and estradiol were studied in the hypothalamus. Thirty five non-pubertal heifers, between 13 and 14 months of age, were divided into four treatment (nine or eight per treatment): P4 device without estradiol (SP), P4 device with estradiol (PE), without P4 device and without estradiol (SS), and without P4 device and with estradiol (SE). The heifers were fed after weaning until reach 295 ± 11 Kg, with water access. At the end of the hormonal treatments all heifers were slaughtered and the hypothalamus areas were harvested, processed and then also stored at -80°C. Total RNA of hypothalamus were extracted, treated with DNase I and submitted to cDNA synthesis for gene expression quantification by real time PCR (qRT-PCR). RNA samples were pooling to realize a comprehensive study of the effects of progesterone administration and endogenous estrogen on attainment of puberty by next-generation sequencing (RNA-Seq), in order to identify possible candidate genes in the hypothalamus. Genes diffentially expressed between hypothalamic areas and affected by treatments were found.

Investigou-se o efeito da leptina exógena e do maior consumo de energia, sobre o padrão da expressão de genes no hipotálamo de novilhas zebuínas; de modo a elucidar o mecanismo de sinalização da leptina no hipotálamo e os genes responsáveis pela obtenção da puberdade. Trinta e seis novilhas não púberes, e com idade entre 18 e 20 meses, foram divididas em três grupos experimentais: baixa energia (BAIXA), alta energia (ALTA), baixa energia com administração de leptina recombinante ovina (BAIXA+LEP), totalizando 56 dias de tratamento. Vinte e quatro novilhas foram abatidas ao apresentar sinais de puberdade, sendo eles: concentração de progesterona no soro superior a 1 ng/mL por duas amostras seguidas e presença de corpo lúteo detectável por ultra-sonografia. O hipotálamo foi colhido e armazenado a -80ºC. As amostras foram submetidas à extração do RNA total, tratadas com DNAse I e submetidas à síntese de cDNA. A quantificação relativa de quatro genes candidatos reconhecidamente envolvidos com a sinalização hipotalâmica da leptina em bovinos: NPY, NPY-Y1, NPY-Y4 e SOCS-3, foi feita através de PCR quantitativo (tempo real). Não houve efeito da administração de leptina sobre a expressão do NPY (P=0,70), ou de seus receptores: NPY-Y1 (P=0,27) e NPY-Y4 (P=0,92) no início da puberdade. A expressão de SOCS-3 foi reduzida (P=0,05) no hipotálamo de novilhas tratadas com leptina, o que sugere menor ação inibitória sobre a leptina. Em novilhas alimentadas com dieta de alta energia, a expressão do NPY-Y1 foi reduzida (P=0,04), o que indica que o hipotálamo estaria menos sensível à ação do NPY, permitindo a entrada precoce em puberdade. Nos demais genes estudados, NPY (P=0,75), NPY-Y4 (P=0,92) e SOCS-3 (P=0,24), a dieta não alterou significativamente suas expressões hipotalâmicas. Estudo mais abrangente do efeito da nutrição e da administração de leptina foi realizado através de hibridização em microarranjos de DNA, objetivando a identificação de possíveis genes candidatos, expressos no hipotálamo, que influenciam na obtenção da puberdade em novilhas Nelore tratadas com leptina ou submetidas à dieta de alta energia. Foram encontrados 78 genes cuja expressão foi alterada pela densidade energética da dieta (P=0,05) no hipotálamo das novilhas Nelore, destes foram selecionados os que apresentaram razões de expressão da ordem de 1,4 ou mais, totalizando 20 genes. Entre esses se destaca o gene da &beta;-arrestina 1 (ARRB1), que foi 1,40 vezes mais expresso (P=0,04) em novilhas submetidas à dieta de alta energia, pois atua na mediação da dessensibilização dos receptores acoplados à proteína-G-(GPCRs)1, como os receptores de NPY. Foram encontrados 134 genes diferencialmente expressos (P=0,05) devido a aplicação de leptina. Dentre os 80 genes que apresentaram razões superiores a 1,4, 18 genes tiveram a expressão reduzida, e 62 tiveram a expressão aumentada pela aplicação de leptina. Destes, alguns estão envolvidos na regulação da sinalização da leptina. O gene SRC foi menos expresso (1,64 vezes; P=0,04) em novilhas tratadas com leptina, o que sugere menor ação inibitória pela SHP-2. A proteína SOCS-2 foi 1,43 vezes (P=0,01) mais expressa no hipotálamo de novilhas tratadas com leptina. Sabe-se que, ao contrário de SOCS-1 e SOCS-3, CIS e SOCS-2 não se ligam, ou inibem, as janus kinases. O STAT-3 foi 2,14 vezes (P=0,03) mais expresso em novilhas tratadas com leptina, e sua ativação possibilita a ligação hipotalâmica da leptina com seu receptor (Ob-Rb). As IGFPB-1 e -2 foram mais expressas no hipotálamo de novilhas tratadas com leptina que em novilhas não tratadas, sendo IGFPB-1 1,78 vezes (P=0,04) mais expressa e IGFPB-2 1,89 vezes (P=0,05). As IGFPBs podem desempenhar função de potencialização da ação do IGF-1, ou exercer ação inibitória. Conclui-se que tanto o consumo de energia quanto a aplicação com leptina influenciaram o padrão de expressão gênica no hipotálamo de novilhas Nelore. A modulação da quantidade do receptor do NPY, NPY-Y1, no hipotálamo pode ser uma via importante pela qual a nutrição afeta o início da puberdade em novilhas. E ainda que estudos mais aprofundados de expressão dos genes encontrados nas hibridizações por microarranjo poderão revelar interações mais concisas entre os genes, a nutrição e a leptina na obtenção da puberdade. / It was investigated the effect of exogenous leptin and the high energy intake on gene expression pattern in the hypothalamus of zebuine heifers; in a way to elucidate the mechanism of leptin signaling in hypothalamus and the responsible genes for puberty. Thirty six heifers not in puberty at 18 and 20 months of age were divided in three experimental groups: low energy diet (LOW), high energy diet (HIGH), low energy diet with administration of recombinant ovine leptin (LOW+LEP), totalizing 56 days of treatment. Twenty four heifers were slaughtered when presented the signals of puberty: progesterone serum concentration above 1 ng/mL for two followed weeks and the presence of detectable corpus luteum by ultrasonography. The hypothalamus was collected and stored at -80ºC. Samples were submitted to total RNA extraction, treated with DNAse I and submitted to cDNA synthesis. The relative quantification of four candidate genes admittedly involved with hypothalamic leptin signaling in bovine: NPY, NPY-Y1, NPY-Y4 and SOCS-3, was evaluated through quantitative PCR (real time). There was no effect of leptin administration on NPY expression (P=0.70), or on its receptors: NPY-Y1 (P=0.27) and NPY-Y4 (P=0.92) in the onset of puberty. The expression of SOCS-3 was reduced (P=0.05) in the hypothalamus of heifers treated with leptin, what suggests lower inhibitory action over leptin. In heifers fed high energy diets, the expression of NPY-Y1 was reduced (P=0.04), which indicates that the hypothalamus would be less sensitive to the action of NPY, allowing the precocious onset of puberty. In other studied genes, NPY (P=0.75), NPY-Y4 (P=0.92) and SOCS-3 (P=0.24), the diet did not significantly altered their hypothalamic expressions. A more comprehensive study regarding the effect of nutrition and leptin administration was performed through the hybridization in DNA microarrangements, aiming the identification of possible candidate genes, expressed in hypothalamus that influence in the onset of puberty in Nelore heifers treated with leptin or submitted to high energy diets. It was found 78 genes whose expression was altered by the energy density of the diet (P<0.05) in the hypothalamus of Nelore heifers. From them, it was selected those genes which presented rates of expression in the order of 1.4 or more, totalizing 20 genes. From them, the highlight gene was &beta;-arrestin 1 (ARRB1) which was 1.40 more expressed (P=0.04) in heifers fed high energy diet due to its action in the mediation of receptors desensibilization coupled to protein-G-(GPCRs)1, as the receptors of NPY. It was found 134 genes differently expressed (P<0.05) due to leptin administration. From the 80 genes that presented rates of expression higher than 1.4, 18 genes had their expression reduced and 62 had their expression increased by leptin administration. Some of these 62 genes are involved in the regulation of leptin signaling. The gene SRC was the less expressed (1.64 times; P=0.04) in heifers treated with leptin what suggests lower inhibitory action by SHP-2. The protein SOCS-2 was 1.43 times (P=0.01) more expressed in the hypothalamus of heifers treated with leptin. It is known that on the contrary of SOCS-1 and SOCS-3, CIS and SOCS-2 do not bind or inhibit, as janus kinases. The STAT-3 was 2.14 times (P=0.03) more expressed in heifers treated with leptin and its activation enables the hypothalamic binding of leptin and its receptor (Ob-Rb). The IGFPB-1 and -2 were more expressed in the hypothalamus of heifers treated with leptin than the animals not treated, being the IGFPB-1 1.78 times (P=0.04) more expressed and the IGFPB-2 1.89 times (P=0.05). The IGFPBs could play a function of IGF-1 action enhancer or exert an inhibitory action. It is concluded that both energy intake and leptin administration influenced gene expression pattern in the hypothalamus of Nelore heifers. The modulation of the receptor quantity of NPY, NPY-Y1 in hypothalamus could be an important route in which nutrition affects the onset of puberty in heifers. Moreover, more detailed studies regarding gene expression in hybridization by microarrangement could reveal more concise interactions between genes, nutrition and leptin in the onset of puberty.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22378号 / 医科博第108号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 羽賀 博典, 教授 松村 由美 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21667号 / 医博第4473号 / 新制||医||1035(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 中川 一路, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bisherige per Tissue Engineering hergestellte Testsysteme der Mundschleimhaut basieren in der Regel auf allogenen und teils dysplastischen Keratinozyten. Dies schmälert die Aussagekraft der gewonnenen Ergebnisse hinsichtlich des Anspruchs, Nativgewebe bestmöglich nachzubilden. In der vorliegenden Arbeit sollte daher ein am Lehrstuhl für Tissue Engineering und Regenerative Medizin entwickeltes Protokoll zur Herstellung dreidimensionaler epidermaler Oralmukosaäquivalente auf Basis autologer Keratinozyten auf seine Eigenschaften und Einsatzmöglichkeit als in-vitro Testsystem untersucht werden. Nach erfolgreicher Isolierung und Kultivierung im Monolayer konnten insgesamt 420 Modelle zu drei verschiedenen Zeitpunkten (Passagen) aufgebaut werden. Die Untersuchung von Histologie, Viabilität und Barrierefunktion mittels MTT, TEER und Natriumfluoresceinpermeabilität konnte einen suffizienten Aufbau von verhorntem, mehrschichtigen oralen Plattenepithel nachweisen. Gleichzeitig konnte eine Abnahme der Epithelqualität mit steigendem Keratinozytenalter festgestellt werden. Eine sich anschließende Untersuchung von 14 Cytokeratinen sowie Apoptosemarkern per effizienzkorrigierter und normalisierter RT-qPCR konnte die Überlegenheit der dreidimensionalen autologen Oralmukosaäquivalente gegenüber der zweidimensionalen Monolayerkultur auf Genebene zeigen. / Current tissue-engineered oral mucosa test systems are usually based on allogenic, mostly dysplastic keratinocytes. Their ability to mimic native tissue sufficiently is therefore limited. Hence the aim of the present work was to examine the characteristics and possible applications of an autologous oral mucosa equivalent based on a protocol developed at the Chair of Tissue Engineering and Regenerative Medicine. Following isolation and monolayer cultivation, 420 equivalents could successfully be built at three different time points. Histology as well as viability and barrier assays (MTT, TEER, sodium-fluoresceine-permeability) revealed sufficient stratification and cornification. Concomitantly, decreasing epithelium quality was associated with a prolongated previous monolayer cultivation. In addition, efficiency corrected and normalized RT-qPCR of 14 cytokeratins and apoptosis marker genes showed the superiority of three dimensional oral mucosa equivalents over two dimensional monolayers on gene level.

Aging of the brain is the leading risk factor for neurodegenerative diseases and brain cancers and has deleterious effects on brain functions. It follows that attempts to reverse the aging process may be therapeutically valuable. Neural stem cells (NSC) have been shown to play a critical role in maintaining brain functions, and their number is severely decreased with age. The development of senescence-like characteristics and declining functions in NSCs have been proposed to be responsible for brain aging and tumorigenesis. MS-818 is a pyrrolopyrimidine that has been shown to increase the NSC population and reverse the decline of behavioral function in aged rodent models. While MS-818 has demonstrated such benefits, the mechanism by which it affects particular pathways of biological age in NSCs is not yet known. Understanding how MS-818 relates to the molecular mechanisms underlying cellular aging may help accelerate the development of anti-aging therapies for neurodegenerative diseases and cancer. This study attempts to elucidate the mechanism of action of MS-818 on NSCs using an in vitro accelerated-aging model produced by Hydroxyurea (HU) treatment. Our analysis of NSC population size post-MS-818 exposure supports the idea that MS-818 treatment can increase NSC proliferation. qPCR analysis of aging-related genes revealed HU treatment produced a trend of increased p16 and Il-6 and decreased Lamin B1 relative expression, supporting the notion that HU treatment can induce senescence in NSCs. MS-818 treatment alone also produced notable trends for targets including BRCA1. In addition, MS-818 treatment post-HU exposure appeared to influence the relative expression of targets, including PGC1a and Lamin B1. Such MS-818 treatment produced similarly noteworthy trends for the expression of genes including PGC1a, Lamin B1, BRCA1, RPTOR, and Il-6, whether in media containing 2.5% or 7.5% serum. These results indicate that MS-818 may have influenced some aging-related pathways.

No description available.

Aufgrund ihres sehr hohen Metastasierungsrisikos weisen Patienten mit klarzelligem Nierenzellkarzinom (kzNZK) eine sehr hohe Sterblichkeit auf. Mit den zurzeit zur Verfügung stehenden klinischen Parametern kann der Krankheitsverlauf der Patienten nach der operativen Entfernung des Primärtumors nur unzureichend vorhergesagt werden. Um das Nachsorge- und Therapieregime der Patienten zu optimieren, muss die Vorhersagegenauigkeit der bestehenden Prognosemodelle durch molekulare Marker erhöht werden. Um geeignete Gene für eine Abschätzung von Metastasierungsrisiko und krankheitsfreiem Überleben (DFS) zu identifizieren, wurden genomweite Expressionsanalysen sowohl an Lungenmetastasen (n = 24) als auch an Primärtumoren (n = 24) des kzNZK vorgenommen. Durch Vergleich von Metastasensubgruppen, die sich nach unterschiedlich langen DFS entwickelt hatten bzw. Primärtumoren, die nach unterschiedlich langen DFS Metastasen bedingten, wurden tumorintrinsische DFS-assoziierte Expressionsmuster identifiziert. Weiterhin wurden Gene identifiziert, deren Expression sich zwischen Primärtumoren unterschied, die im Krankheitsverlauf manifeste Metastasen bedingten und solchen, die dies nicht taten. Die differenzielle Expression funktionell interessanter, teilweise auch in anderen publizierten Microarraystudien an kzNZK bestätigter Gene wurde im Folgenden mittels quantitativer Polymerasekettenreaktion (qPCR) validiert. Anschließend wurde die Assoziation ausgewählter Gene mit klinischen Parametern und dem Überleben der Patienten untersucht. Ein von klinischen Parametern unabhängiger Einfluss auf den Krankheitsverlauf der Patienten wurde dabei für EDNRB und PECAM1 auf Expressionsebene (qPCR; n = 86) sowie für TSPAN7 auf Proteinebene (Immunhistochemie an „Tissue Microarrays“; n = 106) belegt. EDNRB und PECAM1 waren signifikant höher exprimiert in Primärtumoren mit günstigen klinischen Parametern (TNMI/II, G1/2, V0, N0/M0). TSPAN7 war vorwiegend in den Gefäßen der primären kzNZK nachweisbar; eine signifikant höhere Zahl TSPAN7-positiver Gefäße war ebenfalls in Tumoren mit günstigen klinischen Parametern zu verzeichnen (pT1/2, TNMI/II, N0). Überlebensanalysen zeigten ein signifikant längeres DFS für Patienten mit einer hohen im Vergleich zu solchen mit einer geringen EDNRB-Expression und für Patienten, die in beiden untersuchten Gewebestanzen der „Tissue Microarrays“ TSPAN7-positive Gefäße aufwiesen im Vergleich zu Patienten mit nur einer oder keiner TSPAN7-gefäßpositiven Stanze. Für Patienten mit einer hohen im Vergleich zu solchen mit einer geringen EDNRB- bzw. PECAM1-Expression oder mit zwei im Vergleich zu keiner oder einer TSPAN7-gefäßpositiven Gewebestanze war zudem ein signifikant längeres tumorspezifisches Überleben (TSS) zu verzeichnen. Mit Hilfe multivariater Cox-Regressionsanalysen wurde eine unabhängige günstige prognostische Relevanz für EDNRB auf das DFS sowie für EDNRB, PECAM1 und TSPAN7 auf das TSS nachgewiesen. Somit sind diese molekularen Faktoren geeignet, um die Genauigkeit der bestehenden und ausschließlich auf klinischen Parametern basierenden Prognosemodelle zu erhöhen. Für eine Abschätzung von DFS und Metastasierungsrisiko erscheint dabei insbesondere EDNRB geeignet. / Patients with clear cell renal cell carcinoma (ccRCC) have an extremely poor prognosis due to their high risk of metastases. Currently used clinico-patological parameters are insufficient for reliable prediction of metastatic risk and disease free survival (DFS) after surgical resection of the primary tumor. Molecular markers are strongly needed to improve outcome prediction, and thus to optimize the follow up and treatment schedule for patients with ccRCC. To identify genes which are suitable for the prediction of metastatic risk and DFS, genome-wide expression analyses were performed on pulmonary metastases (n = 24) and primary tumors (n = 24) obtained from patients with ccRCC. Tumor-intrinsic DFS-associated expression patterns were observed by comparing subgroups of metastases, which had developed within different DFS as well as primary tumors, which had caused metastases after different DFS. Furthermore, genes differentially expressed in primary tumors, which caused macroscopic metastases and tumors, which did not were identified. The differential expression of genes with a potential function in metastatic spread, which has in part been identified in independent published microarray studies as well, were validated by quantitative polymerase chain reaction (qPCR). Moreover, an independent prognostic impact on the survival of ccRCC patients was observed for the EDNRB und the PECAM1 gene expression (qPCR; n = 86) as well as for the TSPAN7 protein level (immunohistochemistry on tissue microarrays; n = 106). Primary tumors of patients with favourable clinico-pathological parameters (TNMI/II, G1/2, V0, N0/M0) showed a significantly higher EDNRB und PECAM1 gene expression than those with unfavorable parameters. TSPAN7 was predominantly detected in blood vessels of ccRCC tissues. In patients with favourable clinico-pathological parameters (pT1/2, TNMI/II, N0) a significantly higher number of TSPAN7-positive vessels was observed. Using survival analyses, a significantly longer DFS was observed for patients with a high compared to those with a low EDNRB expression as well as for patients with TSPAN7-positive vessels in both cores compared to no or one of the both cores investigated on tissue microarrays. A significantly longer TSS was observed for patients with a high EDNRB or PECAM1 expression as well as for patients with TSPAN7-positive vessles in both tissue cores investigated. Furthermore, EDNRB was an independent prognostic factor for the DFS of the patients; EDNRB, PECAM and TSPAN7 had an independent prognostic impact on the TSS. Therefore, these molecular markers are suitable to improve the accuracy of outcome prediction based on clinico-pathological parameters in ccRCC. For the prediction of DFS and metastatic risk EDNRB is particularly interesting.

Das Kolonkarzinom besitzt aufgrund seiner hohen Inzidenz und der zahlreichen Möglichkeiten, mittels moderner Therapiestrategien auf den Krankheitsverlauf Einfluss zu nehmen, eine hohe Relevanz für den klinischen Alltag. Etwa die Hälfte der an einem kolorektalen Karzinom erkrankten Patienten weisen Lebermetastasen auf, welche entweder bereits bei der primären Diagnosestellung vorliegen oder sich im weiteren Krankheitsverlauf ausbilden. Bei der Mehrzahl der Patienten mit Lebermetastasen ist derzeit nur eine palliative Therapie möglich. Die genaue Analyse kolorektaler Lebermetastasen ist somit unerlässlich, um eine gezielte Entwicklung neuer, effizienter Therapiestrategien für dieses große Patientenkollektiv zu ermöglichen. Ziel dieser Arbeit war es, möglichst genau Zytokinprofil, lymphozytäres Infiltrationsmuster und Verhältnis von pro- zu antiangiogenetischen Faktoren im Lebermetastasengewebe über einen biologisch maßgeblichen Wachstumszeitraum zu charakterisieren, um zu einem besseren Verständnis von Tumorimmunologie und Wachstumsverhalten kolorektaler Lebermetastasen beizutragen. Zu diesem Zweck wurde in einem Mausmodell die Ausbildung kolorektaler Lebermetastasen nach intraportaler Injektion von Tumorzellen (CT26.WT) untersucht. / Because of its high incidence and the numerous therapeutic strategies which allow the clinician to influence the course of the disease colorectal carcinoma figures prominently in the clinical daily routine. About half the patients with colorectal carcinoma suffer from hepatic metastasis, which is either already present at the primary diagnosis or develops later in the course of the disease. At present the majority of patients with hepatic metastasis can only be offered palliative therapy. A detailed analysis of hepatic metastasis is therefore essential for the specific development of new and efficient therapeutic strategies for this group of patients. The objective of this work was to characterize the cytokine profile, the lymphocytic infiltration patterns and the ratio of angiogenic and antangiogenic factors in the tissue of colorectal hepatic metastases as precisely as possible over a biologically significant timespan, thus contributing to a better understanding of tumor immunology and tumor growth. With this aim in view we analyzed the development of colorectal hepatic metastases in a mouse model after the intraportal injection of tumor cells (CT26.WT).

Bislang ist ungeklärt, warum PNPs teils schmerzhaft und teils schmerzlos verlaufen. Die in der vorliegenden Arbeit untersuchte Hypothese lautete, dass ein Ungleichgewicht zwischen pro- und anti-inflammatorischen Zytokinen der unterschiedlichen Schmerzausprägung zugrunde liegt.Es wurden 32 Patienten mit schmerzhafter PNP, 20 Patienten mit schmerzloser PNP und 44 Kontrollpersonen auf die Expression und Produktion ausgewählter pro- und anti-inflammatorischer Zytokine untersucht. Zur Messung der Schmerzhaftigkeit wurden etablierte Schmerzfragebögen verwendet. Zusätzlich wurden nahezu alle Patienten mit der Allgemeinen Depressionsskala befragt. Die Diagnose, Ätiologie, Dauer, klinische Manifestation der PNP sowie die Medikation der Patienten wurde auf standardisierten Erhebungsbögen dokumentiert. Zur Messung der Zytokine wurde morgens Blut in EDTA- und Serummonovetten asserviert und entsprechend der Messmethodik weiterverarbeitet. Die relative Genexpression wurde aus Gesamt-RNA mittels reverser Transkription und quantitativer real-time PCR, die Serumproteine mittels enzyme-linked immunosorbant assay gemessen. Die Patienten mit schmerzhafter PNP hatten in der Mehrzahl Neuropathie-typische Plussymptome und mittelstarke Schmerzen, die eine starke bis sehr starke Behinderung darstellten. Die hier untersuchten Zytokinmuster bei Patienten mit schmerzhafter und schmerzloser PNP zeigten eine Verschiebung zu pro-inflammatorischen Zytokinen bei Patienten mit schmerzhafter PNP. Die Zytokinexpression der Patienten mit schmerzhafter PNP war im Vergleich zu Patienten mit schmerzloser PNP und Kontrollen bezüglich der IL-2 und TNF Expression und Produktion signifikant erhöht. Umgekehrt lagen bei Patienten mit schmerzloser PNP die Produktion und die Expression des IL-4 im Vergleich zu Patienten mit schmerzhafter PNP und Kontrollen höher. Die Expression des IL-10 lag bei Patienten mit schmerzloser PNP ebenfalls höher als bei Patienten mit schmerzloser PNP und Kontrollen, unterschied sich aber auf Proteinebene nicht in den drei Gruppen. Die einleitend gestellte Hypothese, dass der schmerzhafte oder schmerzlose Verlauf einer PNP durch unterschiedliche Zytokinprofile bedingt ist, kann durch die vorliegenden Ergebnisse gestützt werden. In Zusammenschau mit den Daten aus der Grundlagenforschung scheint einem pro-inflammatorischen Zytokinmuster eine entscheidende Rolle an der Entstehung und Aufrechterhaltung neuropathischer Schmerzen zuzukommen. Für TNF sind entsprechende pathophysiologische Wirkungen bekannt. Anti-inflammatorische Zytokine, wie IL-4 und IL-10 zeigten analgetische Wirkungen im Tierversuch. Die Mitwirkung des IL-2 an peripheren Opioid-Rezeptoren lässt eine endogene periphere Analgesie vermuten. Hieraus lassen sich Folgerungen für zukünftige Diagnostik und Therapie neuropathischer Schmerzen ziehen. Durch Erkennung von Zytokin-Imbalancen wären schmerzhafte PNPs früher einer adäquaten Therapie zuzuführen. Durch die Modulation von Zytokinprofilen im Rahmen schmerzhafter PNPs könnten sich zusätzlich therapeutische Möglichkeiten eröffnen. / BACKGROUND: Pain is a common symptom in peripheral neuropathies. The factors determining why some peripheral neuropathies are painful and others are not are incompletely understood. Pro-inflammatory cytokines have been implicated to play a crucial role in the generation of pain. OBJECTIVE: To investigate whether cytokine profiles differ between patients with painful or painless neuropathy. METHODS: In this prospective study, we analyzed blood mRNA and protein levels of the pro-inflammatory cytokines interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4 and IL-10 in 32 patients with painful neuropathy, 20 patients with painless neuropathy, and 38 healthy control subjects, using quantitative real-time PCR and ELISA. RESULTS: Patients with a painful neuropathy had about twofold higher IL-2 mRNA (p = 0.001) and TNF mRNA (p < 0.0001) and protein levels (p = 0.009) than healthy control subjects and about twofold higher IL-2 and TNF mRNA (p = 0.03; p = 0.001) and protein levels (p = 0.04; p = 0.04) than patients with painless neuropathy. In contrast, mRNA levels of the anti-inflammatory cytokine IL-10 were about twofold higher in patients with painless neuropathy than in patients with painful neuropathy (p = 0.001) and controls (p = 0.004). IL-4 protein levels were 20-fold higher in patients with painless neuropathy (p < 0.0001) and 17-fold higher in patients with painful neuropathy (p < 0.0001) than in healthy control subjects. CONCLUSIONS: A pro-inflammatory cytokine profile seems to be associated with pain in the setting of a peripheral neuropathy, corroborating findings in animal models with experimental painful neuropathies. This may have implications for future treatment strategies.