Das Klasse II Tumorsuppressor-Gen H-REV107-1, ist in normalen Geweben ubiquitär exprimiert, zeigt jedoch häufig Expressionsverluste, vorzugsweise in Tumoren des Brustgewebes, des Ovars und der Lunge. Das H-REV107-1 Protein wirkt in vitro und in vivo als Wachstumssuppressor. Um den Mechanismus der H-REV107-1 bedingten Wachstumshemmung zu verstehen, haben wir mit Hilfe des LexA-basierten Hefe-2-Hybrid Systems interagierende Proteine identifiziert. Diese Suche wurde mit einem H-REV107-1 Deletionskonstrukt durchgeführt, dem die Membran-bindende Domäne fehlte. Diese Analyse lieferte eine Vielzahl von potentiellen Interaktionspartnern, darunter der Retinsäure Rezeptor RARG, das Calcium-bindende Proteine S100A6, der Translations-Elongationsfaktor ETF und das weitgehend unbekannte Protein p14.5 Die Bindungen des H-REV107-1 Proteins an die beiden potentiellen Kandidaten, den Transkriptionsfactor PC4 und die regulatorische Untereinheit der Protein Phosphatase 2A (PR65), wurden genauer untersucht. Wir haben dabei einen Proteinkomplex aus H-REV107-1, PC4 und STAT1 (Signal Transducer and Activator of Transcription 1) identifiziert, der vermutlich eine Rolle in der IFNgamma - abhängigen Wachstumshemmung in Ovarialkarzinom Zellen spielt. Da sich die Expression des H-REV107-1 Gens durch IFNgamma über den Transkriptionsfaktor IRF-1 stimulieren läßt, und in verschiedenen Zelllinien sowohl zur Hemmung des Wachstums, als auch zur Apoptose führt, vermuteten wir verschiedene Mechanismen der Wachstumshemmung durch den IFNgamma-Signalweg und H-REV107-1. Weitere Analysen der H-REV107-1 - vermittelten Apoptose zeigten, daß die Interaktion zwischen H-REV107-1 und PR65 eine wichtige Rolle in diesem Prozeß spielt. Um die Proteindomäne zu identifizieren, welche für die direkte Wechselwirkung von H-REV107-1 mit PR65 verantwortlich ist, wurden H-REV107-1 Mutanten generiert und mittels Co-Immunpräzipitation getestet. Die Prolin-reiche Sequenz am N-Terminus des H-REV107-1 Proteins konnte als verantwortliche Domäne für die Interaktion bestimmt werden. Die funktionelle Analyse dieser Interaktion zeigte die Hemmung der Protein Phosphatase 2A (PP2A) Aktivität in Ovarialkarzinom Zellen durch H-REV107-1. Der Einsatz der Mutanten im Phosphatase-Aktivitätstest zeigte, daß die selbe Domäne, die die Interaktion vermittelt, auch für die Hemmung der Phosphatase 2A verantwortlich ist. Diese Fakten deuteten auf eine wichtige Rolle der Phosphatase 2A in Ovarialkarzinom Zellen hin, weil sowohl die Verwendung des PP2A Inhibitors (Okadainsäure), als auch die Transfektion der Zellen mit einem H-REV107-1 - Expressionsplasmid zur Apoptose führten. Damit konnten wir zeigen, daß PP2A für das Überleben der Ovarialkarzinomzellen notwendig ist, und die Reaktivierung des H-REV107-1 Proteins durch IFNgamma zur Hemmung der Phosphatase und damit zur Apoptose führt. / The H-REV107-1 class II tumor suppressor gene is ubiquitously expressed in normal tissues and down-regulated in human breast, ovarian and lung tumours. H-REV107-1 has the capacity to suppress growth of tumour cells in vitro and in vivo. To understand the mechanism of H-REV107-1 mediated growth suppression I performed a search for H-REV107-1 interacting proteins using a LexA-based yeast two-hybrid system. I screened a human kidney cDNA library with a truncated form of the H-REV107-1 as a bait. This analysis revealed numerous potential interaction partners. Among them the retinoic acid receptor gamma (RARG), the calcium-binding protein S100A6, the translation termination factor ETF1, and the potential translational inhibitor protein P14.5. The interaction of H-REV107-1 with the transcriptional co-activator PC4 and with the regulatory subunit A of protein phosphatase 2A (PR65) was analysed in detail. H-REV107-1 binds ectopically expressed and endogenous PC4. In addition, a multiprotein complex between H-REV107-1, PC4 and the signal transducer and activator of transcription 1 (STAT1) was demonstrated. This complex is likely to be involved in IFNgamma-mediated growth suppression of ovarian carcinoma cells. Endogenous H-REV107-1 can be induced after application of IFNgamma through the IRF-1 transcription factor. This up-regulation of H-REV107-1 leads either to growth suppression via a G1 arrest or to apoptosis depending on the cell line, suggesting different mechanisms of IFNgamma-, and H-REV107-1- mediated growth suppression. Further investigation of the mechanism of H-REV107-1-dependent apoptosis revealed an important role of the interaction between H-REV107-1 and the PR65 protein. The use of several H-REV107-1 mutant proteins generated after disruption of the highly conserved domains identified the proline-rich N-terminal domain responsible for the interaction with PR65 in Co-immunoprecipitation studies. Functional investigation of the H-REV107-1 - PR65 interaction demonstrated that wild-type H-REV107-1 is able to inhibit PP2A activity, however a mutant protein lacking the N-terminal domain was devoid of this function. We sought to identify the functional relevance of the PP2A activity in ovarian carcinoma cells with normally have suppressed the H-REV107-1 gene. Treatment of OVCAR-3 cells with the PP2A inhibitor Okadaic acid and transient transfection of the cells with wild-type H-REV107-1 resulted in the activation of caspase-9, suggesting a role for PP2A in the survival of ovarian carcinoma cells. We suggest, that the down-regulation of H-REV107-1 in ovarian carcinomas is a prerequisite for the PP2A-dependent activation of yet unknown signalling pathways mediating tumour cell survival. Reactivation of H-REV107-1 gene expression via IFNgamma leads to the inhibition of PP2A activity and tumour cell death.

The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Large clinical trials testing hydrocortisone therapy in septic shock have produced conflicting results. Subgroups may benefit of hydrocortisone treatment depending on their individual immune response. We performed an exploratory analysis of the database from the international randomized controlled clinical trial Corticosteroid Therapy of Septic Shock (CORTICUS) employing machine learning to a panel of 137 variables collected from the Berlin subcohort comprising 83 patients including demographic and clinical measures, organ failure scores, leukocyte counts and levels of circulating cytokines. The identified theranostic marker was validated against data from a cohort of the Hellenic Sepsis Study Group (HSSG) (n = 246), patients enrolled in the clinical trial of Sodium Selenite and Procalcitonin Guided Antimicrobial Therapy in Severe Sepsis (SISPCT, n = 118), and another, smaller clinical trial (Crossover study, n = 20). In addition, in vitro blood culture experiments and in vivo experiments in mouse models were performed to assess biological plausibility. A low serum IFNg/IL10 ratio predicted increased survival in the hydrocortisone group whereas a high ratio predicted better survival in the placebo group. Using this marker for a decision rule, we applied it to three validation sets and observed the same trend. Experimental studies in vitro revealed that IFNg/IL10 was negatively associated with the load of (heat inactivated) pathogens in spiked human blood and in septic mouse models. Accordingly, an in silico analysis of published IFNg and IL10 values in bacteremic and non-bacteremic patients with the Systemic Inflammatory Response Syndrome supported this association between the ratio and pathogen burden. We propose IFNg/IL10 as a molecular marker supporting the decision to administer hydrocortisone to patients in septic shock. Prospective clinical studies are necessary and standard operating procedures need to be implemented, particularly to define a generic threshold. If confirmed, IFNg/IL10 may become a suitable theranostic marker for an urging clinical need.

Das Proteasomensystem der Zelle ist für die Degradation von Proteinen verantwortlich und spielt eine zentrale Rolle bei der Generierung von Epitopen, die auf MHC-Klasse-I Molekülen den cytotoxischen T-Lymphozyten (CTLs) präsentiert werden. Die Stimulation von Zellen mit Interferon-gamma (IFNgamma) führt zu der Bildung von Immunoproteasomen, die im Vergleich zu den konstitutiven Proteasomen eine verbesserte Generierung vieler MHC-Klasse-I Epitope aufweisen. In gesunden Mäusen werden Immunoproteasomen vorwiegend in den lymphatischen Geweben exprimiert, wohingegen nicht-lymphatische Gewebe hauptsächlich konstitutive Proteasomen enthalten. In der vorliegenden Arbeit wurde der Einfluss der Listeria monocytogenes Infektion auf die aus der Leber, der Milz, dem Dünndarm und dem Colon stammenden murinen 20S Proteasomen untersucht. Die Struktur der isolierten 20S Proteasomen wurde mittels zweidimensionaler Gelelektrophorese und Westernblot ermittelt, während die Funktion durch in vitro Prozessierung von drei oligomeren Peptidsubstraten analysiert wurde. Die Prozessierungsprodukte wurden mittels HPLC-ESI-Ionenfalle massenspektrometrisch identifiziert sowie quantifiziert. Die vorliegende Arbeit zeigt zum ersten Mal, dass nach einer Infektion die aus den nicht-lymphatischen Organen und Zellen isolierten 20S Proteasomen eine strukturelle und funktionelle Plastizität aufweisen: Nach der Infektion wurde die Bildung von Immunoproteasomen induziert, was mit der gesteigerten Generierung der immunrelevanten Fragmente korreliert werden konnte. Dies verlief unabhängig von der direkten Präsenz von Listeria monocytogenes in den Organen und wurde ausschließlich durch das Cytokin IFNgamma reguliert. Es konnte außerdem eine Zunahme der posttranslationalen Modifikation von Leberproteasomen mit dem Monosaccharid N-Acetylglucosamin nach der Infektion nachgewiesen werden. Des Weiteren wurde eine detaillierte Analyse der massenspektrometrischen Daten hinsichtlich des Schnittverhaltens der konstitutiven und Immunoproteasomen etabliert. Die Auswertung ergab, dass die Immunoproteasomen nach der Infektion durch schnellere und veränderte Nutzung bestehender Spaltstellen an der verbesserten Epitoppräsentation beteiligt sind. / The proteasome system of the cell is responsible for the degradation of proteins and plays a central role in the generation of epitopes which are presented to cytotoxic T-lymphocytes (CTLs) on MHC-class-I molecules. The stimulation of cells by interferon-gamma (IFNgamma) leads to the formation of immunoproteasomes that show an improved generation of many MHC-class-I epitopes compared to constitutive proteasomes. In healthy mice, immunoproteasomes are mainly expressed in the lymphatic tissues, whereas the non-lymphatic organs predominantly contain constitutive proteasomes. In this project the effect of Listeria monocytogenes infection on murine 20S proteasomes derived from the liver, spleen, small intestine and colon were investigated. The structure of the isolated proteasomes was analyzed by two-dimensional gel electrophoresis and western blots while the function was studied by in vitro processing of three oligomeric peptide substrates. Identification and quantification of the processing products was performed by HPLC-ESI-ion trap mass spectrometry. The project showed for the first time, that after infection 20S proteasomes isolated from non-lymphatic organs as well as from non-lymphatic cells displayed structural and functional plasticity: immunoproteasomes were induced post infection which could be correlated with the enhanced generation of immuno-relevant fragments. This was independent of the direct presence of Listeria monocytogenes in the organs and solely controlled by the cytokine IFNgamma. In addition, an increased posttranslational modification with the monosaccharide N-acetylglucosamine could be detected in liver-derived proteasomes after infection. Furthermore, a detailed analysis of the mass spectrometry data was established according to the cleavage site usage of constitutive and immunoproteasomes. The result was that immunoproteasomes are involved in improved generation of the immuno-relevant fragments by the faster cleavage and the changed usage of existing cleavage sites after infection.