Wang Zhi-Jun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 54-63). / ABSTRACT --- p.VI / Chapter I. --- INTRODUCTION --- p.1 / Chapter A. --- Literature review --- p.1 / Chapter 1. --- Retinal iron toxicity --- p.2 / Clinical siderotic retinopathy --- p.2 / Experimental siderotic retinopathy --- p.4 / Free radical involvement in siderotic retinopathy --- p.5 / Chapter 2. --- Experimental photic retinopathy in rats --- p.8 / Morphologic features --- p.8 / Free radical involvement in photic retinopathy --- p.9 / Chapter 3. --- Mechanisms of cell death --- p.9 / Necrosis --- p.10 / Apoptosis --- p.10 / Chapter B. --- Statement of the problems --- p.15 / Chapter II. --- MATERIALS AND METHODS --- p.17 / Chapter A. --- Siderotic retinopathy model --- p.17 / Animals --- p.17 / Reagents and equipment --- p.18 / Surgical procedures --- p.18 / Chapter B. --- Histochemical methods --- p.18 / Reagents and equipment --- p.19 / Paraffin sections --- p.19 / H&E staining --- p.19 / TUNEL technique --- p.20 / Schmeltzer's iron staining --- p.21 / Chinoy's ascorbic acid staining --- p.21 / Chapter C. --- Biochemical methods --- p.21 / Reagents and equipment --- p.22 / DNA gel electrophoresis --- p.22 / Analysis of ascorbic acid and uric acid --- p.23 / Chapter III. --- RESULTS --- p.24 / Chapter A. --- Observations in rats --- p.24 / Morphologic changes after H&E staining --- p.24 / Visualization of apoptosis by TUNEL technique --- p.25 / Internucleosomal DNA fragmentation --- p.26 / Negative staining of iron in the ONL --- p.27 / Positive staining of ascorbic acid in the ONL --- p.27 / Chapter B. --- Observations in rabbits --- p.27 / Positive staining of ascorbic acid in all retinal layers --- p.27 / Apoptosis occurred in all retinal layers --- p.28 / Changes of ascorbic acid and uric acid after iron implantatio --- p.28 / Chapter IV. --- DISCUSSION --- p.48 / Chapter V. --- CONCLUSION --- p.53 / References --- p.54

Chlamydiales are obligate intracellular gram-negative bacteria that have gained high medical relevance. These important human pathogens cause diverse diseases including trachoma and wide spread sexually transmitted diseases. Chlamydia establishes membrane bound inclusions in the host cell and loots the host for nutritional requirements. Infections are usually recognized by the host immune system and eliminated systematically, by triggering apoptosis. However, the pathogen Chlamydia has evolved various strategies to prevent the detection as well as protect the invaded cell against apoptosis or any other form of cell death. The evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. The present study was aimed at investigating the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Caspases were differentially regulated upon Sn infection. Caspase-3 and -9 were not activated upon Sn infection and apoptosis induction; whereas caspases-8 was activated in Sn infected cells even without apoptosis induction. This indicates that, Sn utilizes death receptor association independent caspase activation for thriving in the host environment. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Proteins (IAP-1/2 and XIAP) and the Akt/PI3K pathway. Sn infection also 20 activated the pro-survival transcription factor NF-кB. Blocking any of these survival pathways sensitized the infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. The NF-кB mutant cells also showed reduced infectivity of Sn, which indicated an essential role of NF-кB in Sn infection. It was interesting to observe that, Acanthamoeba castellanii, a natural host of Sn, survived maintaining its trophozoite forms after infection with Sn upon starvation. The metacaspases, responsible for encystment could be regulated by Sn upon infection. This suggests an early level of gene regulation indicating how the pathogen evolved its ability to inhibit apoptosis in higher organisms. The resistance to apoptosis pathways subverted in Sn-infected cells was similar but not identical to those modulated by Chlamydia. Together, the data supports the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales. / Vertreter der Ordnung Chlamydiales sind obligat intrazelluläre gram-negative Bakterien mit einer zunehmenden medizinischen Relevanz. Diese Humanpathogene lösen verschiedene Krankheiten aus, unter anderem Blindheit und sexuell übertragbare Krankheiten. Chlamydia führt zur Ausbildung einer Membranumschlossenen Inklusion innerhalb der Wirtszelle und entzieht dem Wirt die nötigen Nährstoffe. Infektionen können vom Immunsystem des Wirts erkannt und systematisch mittels Apoptose bekämpft werden. Chlamydia hat jedoch verschiedene Strategien entwickelt um der Erkennung durch das Immunsystem zu entgehen und um ihre Wirtszelle vor dem Zelltod zu schützen. Ob die Fähigkeit den Zelltod der Wirtszelle zu regulieren evolutionär innerhalb der Ordnung der Chlamydiales, welche auch Chlamydia-ähnliche Organismen mit einem breiteren Wirtsspektrum beinhalten, konserviert ist wurde bislang noch nicht untersucht. In dieser Arbeit wurde das apoptotische Verhalten humaner mit dem Chlamydia-ähnlichen Organismus Simkania negevensis (Sn) infizierter Zellen untersucht. Mit Simkanien infizierte Zellen zeigen eine starke Apoptose-Resistenz, unabhängig ob diese über intrinsischen Stress oder durch die Aktivierung von Todesrezeptoren ausgelöst wurde. Die Apoptose-Signalkaskade wurde vor den Mitochondrien blockiert, da es weder zu einer Bax Translokation, Bax und Bak Oligomerisierung noch zur Cytochrom c Freisetzung in den infizierten Zellen kam. Auch Caspasen zeigten eine veränderte Regulation. Die Caspase-3 und -9 wurden während einer Sn-Infektion und Apoptose-Induktion nicht aktiviert. Die Caspase 8 jedoch ist während der Sn-Infektion unabhängig von einer Apoptose-Induktion aktiv. Diese Tatsache lässt vermuten, dass Sn sich eines von den Todesrezeptoren 22 unabhängigen Mechanismus der Caspaseaktivierung bedienen um in der Wirtzelle zu verbleiben. Infizierte Zellen zeigen eine Aktivierung verschiedener antiapoptotischer Signalwege wie der der Inhibitor of Apoptotis Proteins (IAP1/2 and XIAP)- und den Akt/PI3K Signalweg. Durch eine Sn-Infektion kommt es außerdem zur Aktivierung des Transkriptionsfaktors NF-kB. Wird einer dieser Überlebenssignalwege blockiert zeigen die infizierten Wirtszellen eine erhöhte Sensitivität gegenüber einer Apoptoseinduktion, was wiederum die zentrale Rolle der infektionsvermittelten Apoptoseresistenz aufzeigt. Eine Zelllinie mit mutiertem NF-kB zeigte eine verminderte Infektiosität von Sn, was die zentrale Rolle von NF-kB in der Sn-Infektion unterstreicht. Infektionsversuche mit Sn und dessen natürlichem Wirt Acanthamoeba castellanii zeigten, dass diese im Sn-infizierten Stadium die Aufrechterhaltung ihrer Trophozyten-Form nach Nährstoffmangel überlebten. Die Metacaspasen, welche für die Einkapselung verantwortlich sind konnten durch eine Sn-Infektion reguliert werden. Dies weist auf ein frühes Stadium der Genregulation hin und lässt vermuten wie die Pathogene die Fähigkeit zur Apoptose-Inhibition höherer Organismen erlangt haben könnten. Die durch eine Sn-Infektion vermittelte Resistenz der Wirtszelle gegenüber Apoptose-Signalwegen ist ähnlich, jedoch nicht identisch zu der durch Chlamydia vermittelten Resistenz. Diese Ergebnisse unterstützen die Hypothese eines evolutionär konservierten Signalwegs der Apoptoseresistenz als allgemeiner Nenner in der Ordnung der Chlamydiales.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates inside a vacuole, the so-called inclusion. During replication by a biphasic life-cycle Chlamydia secrete via their type 3 secretion system various effector proteins into the inclusion lumen, the inclusion membrane or the host cell cytosol to form their favored replication niche. Chlamydia-infected cells are highly resistant against apoptosis since the replicative form of Chlamydia is non-infectious and premature cell death would cause complete loss of one Chlamydia generation. The bacteria block apoptosis by preventing mitochondrial outer membrane permeabilization. Various proteins with anti-apoptotic function are enriched in Chlamydia-infected cells such as Mcl-1, cIAP2, Survivin or HIF1α. The accumulation of these proteins is a result of increased gene expression and direct protein stabilization. However, the molecular mechanisms and involved bacterial effector proteins are mostly unknown. With this work the molecular mechanisms of Mcl-1 stabilization and the participation of chlamydial factors were investigated. Mcl-1 is a member of the Bcl-2 protein family and has an extremely short half-life causing its permanent ubiquitination and subsequent degradation by the 26S proteasome under normal homeostasis whilst Mcl-1 accumulation results in apoptosis inhibition. It was shown that during C. trachomatis infection Mcl-1 ubiquitination is reduced causing its stabilization albeit no cellular ubiquitin-proteasome-system components are involved in this process. However, C. trachomatis express the two deubiquitinases ChlaDUB1 and ChlaDUB2 which are mostly uncharacterized. With this work the expression profile, subcellular localization, substrates and function of the deubiquitinases were investigated. It was shown that ChlaDUB1 is secreted to the surface of the inclusion where it interacts with Mcl-1 which is accumulated in the proximity of this compartment. By utilization of infection experiments, heterologous expression systems and in vitro experiments a direct interaction of ChlaDUB1 and Mcl-1 was demonstrated. Furthermore, it was shown that Mcl-1 is deubiquitinated by ChlaDUB1 causing its stabilization. During replicative phase of infection, ChlaDUB2 seems to be accumulated in the chlamydial particles. However, ChlaDUB2 substrates could not be identified which would give an indication for the physiological role of ChlaDUB2. Since 2011, a protocol to transform C. trachomatis with artificial plasmid DNA is available. As part of this work the transformation of C. trachomatis with plasmid DNA suitable for the permanent or inducible protein overexpression on a routinely basis was established. In addition, the first targeted homologous recombination into the chlamydial genome to replace the ChlaDUB1 gene by a modified one was performed and validated. The targeted homologous recombination was also used to create a ChlaDUB1 knock-out mutant; however deletion of ChlaDUB1 seems to be lethal for C. trachomatis. Due to the fact that ChlaDUB1-lacking Chlamydia could not be obtained an inhibitor screen was performed and identified CYN312 as a potential ChlaDUB1 inhibitor. Application of CYN312 during infection interfered with chlamydial growth and reduced Mcl-1 quantity in infected cells. Furthermore, CYN312 treated Ctr-infected cells were significantly sensitized for apoptosis. Taken together, C. trachomatis secretes the deubiquitinase ChlaDUB1 to the surface of the inclusion where it deubiquitinates Mcl-1 causing its accumulation in infected cells resulting in apoptosis resistance. Application of the ChlaDUB1 inhibitor CYN312 interferes with Mcl-1 stabilization sensitizing infected cells for apoptosis. / Chlamydia trachomatis ist ein obligat intrazelluläres Bakterium, welches sich in einer Vakuole, der sogenannten Inclusion vermehrt. Chlamydien durchlaufen einen zweiphasigen Entwicklungszyklus während welchem sie zu bestimmten Zeitpunkten der Infektion Effektorproteine mittels ihres Typ 3 Sekretionssystems in das Inclusionslumen, die Inclusionsmembran oder das Wirtszellzytoplasma sekretieren. Durch die Aktivität der Effektorproteine schaffen die Chlamydien die für sie favorisierten Bedingungen. Zusätzlich zeigen infizierte Zellen eine hohe Resistenz gegenüber Apoptose. Ein vorzeitiger Zelltod der Wirtszelle würde zum Verlust einer vollständigen Generation an Chlamydien führen, da die replizierende Form der Chlamydien nicht infektiös ist. Chlamydien hemmen die Wirtszellapoptose indem sie die Permeabilisierung der äußeren Mitochondrienmembran verhindern. Es ist bekannt, dass mehrere anti-apoptotische Proteine wie Mcl-1, cIAP2, Survivin oder HIF1α während der Infektion mit Chlamydien zu bestimmten Zeitpunkten angereichert werden und für die Apoptoseinhibition wichtig sind. Allerdings sind die molekularen Mechanismen sowie die beteiligten bakteriellen Proteine weitestgehend unbekannt. Mit dieser Arbeit wurden die molekularen Mechanismen der Mcl-1 Stabilisierung sowie die darin involvierten chlamydialen Proteine untersucht. Mcl-1, ein Mitglied der Bcl-2 Proteinfamilie, ist ein extrem instabiles Protein welches unter normalen Bedingungen permanent ubiquitiniert und vom 26S Proteasom abgebaut wird; eine Anreicherung von Mcl-1 hingegen führt zur Apoptoseinhibierung. In dieser Arbeit konnte gezeigt werden, dass während der Chlamydieninfektion Mcl-1 weniger ubiquitiniert wird was dessen Stabilisierung zur Folge hat. Es konnte jedoch keine Beteiligung von Komponenten des zellulären Ubiquitin-Proteasom-Systems festgestellt werden. C. trachomatis exprimiert zwei Deubiquitinasen welche weitestgehend uncharakterisiert sind. Ein weiteres Ziel dieser Arbeit war es das Expressionsprofil, die Lokalisierung, Substrate und die Funktion der Deubiquitinasen zu untersuchen. Es konnte gezeigt werden, dass ChlaDUB1 zur Oberfläche der Inclusion sekretiert wird und dort mit Mcl-1 interagiert, welches in diesem Kompartiment angereichert vorliegt. Unter Verwendung von Infektionsmodellen, heterologen Expressionssystemen sowie in vitro Experimenten konnte eine direkte Bindung beider Proteine sowie die spezifische Deubiquitinierung von Mcl-1 durch ChlaDUB1 gezeigt werden. Durch die permanente Deubiquitinierung mittels ChlaDUB1 wird Mcl-1 stabilisiert und im Bereich der Inclusionsoberfläche angereichert. Im Gegensatz zu ChlaDUB1 konnte ChlaDUB2 während der replikativen Phase der Infektion nicht im Zytoplasma sondern lediglich innerhalb der Bakterien detektiert werden. Außerdem konnten bislang keine Substrate für ChlaDUB2 identifiziert werden, welche auf die physiologische Funktion dieses Effektors schließen lassen könnten. Seit 2011 ist ein Protokoll für die Transformation von Chlamydien mit artifizieller Plasmid-DNA verfügbar. Als Teil dieser Arbeit wurde die routinemäßige Transformation von Chlamydien mit Plasmid-DNA zur permanenten und induzierbaren Proteinüberexpression etabliert. Außerdem konnte die erste gezielte homologe Rekombination ins chlamydiale Genom durchgeführt werden. Hierbei wurde das ChlaDUB1-Gen durch eine modifizierte Form ersetzt. Die Herstellung einer ChlaDUB1-Deletionsmutante mittels homologer Rekombination war jedoch nicht erfolgreich, da ChlaDUB1 vermutlich essentiell für C. trachomatis ist. Da ChlaDUB1-defiziente Chlamydien nicht generiert werden konnten, wurde ein Inhibitorscreen durchgeführt und CYN312 als ChlaDUB1-Inhibitor identifiziert. Die Anwendung von CYN312 während Infektionsversuchen zeigte eine deutliche Reduktion des Chlamydienwachstums sowie eine verminderte Mcl-1 Stabilisierung. Als Folge dessen waren Chlamydien-infizierte und mit CYN312 behandelte Zellen signifikant für die Apoptoseinduktion sensibilisiert. Mit der vorliegenden Arbeit konnte gezeigt werden, dass C. trachomatis die Deubiquitinase ChlaDUB1 während der Infektion an die Oberfläche der Inclusion sekretiert. Dort katalysiert ChlaDUB1 die Deubiquitinierung von Mcl-1 was dessen Anreicherung in infizierten Zellen und somit eine erhöhte Apoptoseresistenz zur Folge hat. Die Verwendung des ChlaDUB1-Inhibitors CYN312 verhindert die Mcl-1 Stabilisierung und sensibilisiert somit infizierte Zellen für Apoptose.

Die Expression der Hämatopoetischen Progenitor Kinase 1 (HPK1), einem Mitglied der Familie der „Germinal Centre“ Kinasen, ist im adulten Organismus auf die Zellen des hämatopoetischen Systems beschränkt. Die HPK1 wurde ursprünglich als ein Aktivator des JNK-Signalübertragungsweges beschrieben [Hu et al., 1996; Kiefer et al., 1996], und kürzlich wurde eine transiente Aktivierung der HPK1 nach TZR-Stimulation nachgewiesen. Auch wurde eine Assoziation der HPK1 mit dem Linker aktivierter T-Zellen (LAT) und den Adaptorproteinen Nck, Crk, Gads, Grb2, Grap, CrkL sowie SLP-76 gezeigt. Für die Aktivierung der nach TZR-Stimulation in den Lipid-Rafts lokalisierten HPK1 sind sowohl Lck als auch ZAP-70 notwendig [Liou et al., 2000; Liu et al., 2000a; Ling et al., 2001]. Diese Daten legen eine mögliche Funktion von HPK1 bei der TZR-vermittelten Signalübertragung nahe. Trotzdem konnte bisher eine physiologische Rolle der HPK1 im Rahmen der Immunrezeptor-Signalübertragung nicht nachgewiesen werden. In der vorliegenden Arbeit wird dargestellt, dass eine wichtige Funktion der HPK1 in T-Zellen nach TZR-Stimulation, die durch die Förderung des Aktivierungs-induzierten Zelltodes (AICD) vermittelt wird, in der Kontrolle der Termination der Immunantwort und damit Homöostase des Immunsystems besteht. Dies wurde durch retrovirale Überexpression der wildtypischen (wt) HPK1 in murinen CD4+ T-Zellen nachgewiesen, in denen die HPK1 zu einem Anstieg der spontanen und antiCD3-vermittelten Apoptose sowie zu einer gesteigerten Expression des Fas-Liganden (FasL oder auch CD95L) führte. Die Expression einer HPK1-„antisense“ (AS)-RNA in CD4+ T-Zellen bewirkte dagegen eine schwache, jedoch signifikant nachweisbare Hemmung der Apoptose und FasL-Expression. Die Apoptose-Hemmung durch die HPK1-AS-RNA war besonders stark in H2O2-stimulierten EL-4 T-Zellen ausgeprägt, in denen die Überexpression der wt HPK1 den durch reaktive Sauerstoffmetabolite (ROS) induzierten Zelltod verstärkte. Aus diesen Daten folgt, dass die HPK1 die T-Zell-Apoptose reguliert. In H2O2-stimulierten EL-4 T-Zellen führt die HPK1-Expression zu einer verstärkten und anhaltenden Aktivierung der c-Jun N-terminalen Kinase (JNK), die wahrscheinlich an der HPK1-vermittelten Apoptoseinduktion beteiligt ist. Unter den gleichen Bedingungen konnte eine schnelle Spaltung der HPK1 beobachtet werden. Die Überexpression der N- oder C-terminalen Spaltprodukte in CD4+ T-Zellen führte - wie die der Gesamt-HPK1 - zu einem Anstieg des AICD. In Übereinstimmung mit publizierten Daten konnten wir eine Hemmung der NFkB-Aktivität durch das C-terminale HPK1-Peptid nachweisen, das die IkBalpha-Degradation inhibiert. Die erzielten Ergebnisse führten uns in ihrer Gesamtheit zu folgendem Modell: während der Initiationsphase der T-Zell-Stimulierung werden nach schneller, transienter HPK1-Aktivierung pro- und anti-apoptotische Signale durch den JNK- und NFkB-Signalübertragungsweg vermittelt. Durch die Akkumulation der C-terminalen HPK1-Spaltprodukte kommt es später zur Inhibierung der NFkB-Aktivität und damit zu einer Verschiebung des Gleichgewichts zwischen den Überlebens- und Apoptose-stimulierenden Signalen zugunsten des AICD. Allerdings gibt es sicherlich weitere Faktoren und Signalwege, die an der HPK1-vermittelten Kontrolle der T-Zell-Apoptose beteiligt sind und von deren Untersuchung ein detaillierteres Verständnis der HPK1-Physiologie erwartet wird. Die Nukleären Faktoren aktivierter T-Zellen (NFAT´s) gehören zu einer Familie von Transkriptionsfaktoren, denen eine konservierte, ca. 300 Aminosäuren (aa) große DNA-Bindedomäne und eine Calcineurin-Bindedomäne gemeinsam ist. NFATc (auch NFATc1 oder NFAT2 genannt) und NFATp (NFATc2 oder NFAT1) werden in peripheren T-Zellen stark exprimiert und kontrollieren deren Effektorfunktionen u.a. über die Expression von IL-2, IL-3, IL-4, IL-5, IFNgamma und weiterer Lymphokine. Weitere von den NFAT´s kontrollierte Gene sind p21WAF1, der CD40L und der CD95L. Somit scheinen die NFAT´s bei der Zellzyklus-Kontrolle und beim AICD von T-Lymphozyten eine wichtige Rolle zu spielen. Daten unseres Labors zeigten, dass die T-Zell-Aktivierung zu einer massiven Induktion der kurzen Isoform A von NFATc innerhalb von 3-4 h führt [Chuvpilo et al., 1999b], noch vor dem Start des AICD. Dies ließ vermuten, dass sich die biologische Funktion von NFATc/A durch das Fehlen des C-terminalen Peptids von ca. 245 aa, das in allen anderen NFAT-Proteinen einschließlich der längeren Isoform NFATc/C vorhanden ist, unterscheidet. Deshalb sollte in der vorliegenden Arbeit die Induktion und Funktion von NFATc/A in murinen T-Lymphozyten untersucht werden. Es konnte gezeigt werden, dass die Infektion primärer CD4+ T-Lymphozyten mit NFATc/A-exprimierenden rekombinanten Retroviren, im Gegensatz zu der mit NFATc/C- oder NFATp-exprimierenden Retroviren, den AICD unbeeinflusst lässt. Dies deutet darauf hin, dass es durch die massive NFATc/A-Synthese nach Effektor-T-Zell-Aktivierung zur Induktion von Effektor-Funktionen kommt, ohne dass dabei die T-Zell-Apoptose beschleunigt wird. Im Gegensatz dazu üben die langen NFAT-Faktoren wie NFATc/C und NFATp eine pro-apoptotische Wirkung aus. / Hematopoietic progenitor kinase 1 (HPK1) is a member of germinal center kinases that is predominantly expressed in hematopoietic cells. The HPK1 has originally been described as an upstream Ser/Thr protein kinase of the JNK signaling cascade [Hu et al., 1996; Kiefer et al., 1996]. Recently, it was shown that T cell receptor (TCR) stimulation leads to a rapid but transient activation of HPK1 and its binding to the linker of activated T cells (LAT) and the adaptor proteins Nck, Crk and Gads. HPK1 which also binds to the signaling molecules SLP-76, Grb2, Grap and CrkL is localized in lipid rafts and needs lck and ZAP70 for its activation [Liou et al., 2000; Liu et al., 2000a; Ling et al., 2001]. These data suggested an involvement of HPK1 in the signaling transfer from the TCR to downstream signaling cascades. However, a role for HPK1 in T cell physiology remained to be shown. We show here that one of the functions of HPK1 activation upon T cell stimulation is to control the termination of immune response and, therefore, homeostasis of the immune system by supporting the ´Activation Induced Cell Death´ (AICD) of T cells. This is demonstrated by overexpressing wild type (wt) HPK1 which led to a substantial increase in spontaneous and antiCD3-mediated apoptosis as well as in Fas ligand (FasL or also CD95L) expression on murine CD4+ T cells. In contrast, expression of HPK1 antisense (AS)-RNA exerted a slight, albeit measurable suppressive effect on both apoptosis and FasL expression. Apoptosis suppression by overexpressing HPK1 AS-RNA was much more pronounced in H2O2-treated EL-4 T cells in which HPK1 overexpression stimulated cell death induced by reactive oxygen species (ROS) indicating that HPK1 indeed controls apoptosis in T cells. HPK1 expression also led to a sustained increase in c-Jun N-terminal kinase (JNK) activity suggesting that JNK activation contributes to the HPK1-mediated apoptosis in H202-treated EL-4 cells. Under the same conditions a rapid cleavage of HPK1 was observed, and overexpression of N- and C-terminal cleavage products in CD4+ T cells resulted, similar to full-length HPK1, in an increase in AICD. In agreement with published data we show that the C-terminal portion of HPK1 suppresses IkBalpha degradation thereby inhibiting NFkB activation. Taken together, these results led to a model in which, during the initial phase of T cell activation, the rapid and transient HPK1 induction provides both pro- and anti-apoptotic signals by activating JNK and NFkB signaling pathways, respectively. At a later stage, due to the accumulation of the C-terminal HPK1 cleavage product, NFkB activation is suppressed and the balance between survival and apototic signals is shifted favoring apoptosis. However, signalling events in addition to the stimulation of JNK and inhibition of NFkB signalling cascades might also be involved in the HPK1-mediated control of T cell apoptosis. The nature of these events remains to be elucidated. Nuclear Factor of activated T cells (NFAT) proteins belong to a family of transcription factors which share a common DNA binding domain of approximately 300 amino acids (aa) and a calcineurin binding domain. NFATc (also designated as NFATc1 or NFAT2) and NFATp (also designated as NFATc2 or NFAT1) are highly expressed in peripheral T cells and control their effector function, in particular the expression of lymphokines, such as IL-2, IL-3, IL-4, IL-5 and IFNgamma. Further target promoters for NFATs are the p21Waf1, the CD40 ligand and CD95 ligand promoters indicating that in addition to effector function NFATs are also involved in the control of the cell cycle and, in particular, AICD of T lymphocytes. We have shown previously that T cell activation leads to the massive induction of the short isoform A of NFATc within 3-4 h [Chuvpilo et al., 1999b], i.e. before the onset of AICD. This observation suggested that due to the lack of the C-terminal extension of approximately 245 aa which is present in all other NFAT proteins, NFATc/A might differ in biological function from other NFAT proteins, including its longer isoform NFATc/C. This view prompted us to investigate the induction and function of NFATc/A in murine T lymphocytes. Here we show that infection of primary CD4+ T lymphocytes with recombinant retroviruses expressing NFATc/A, in contrast to retroviruses expressing NFATc/C or NFATp, does not enhance AICD. These data suggest that the massive synthesis of NFATc/A upon activation of effector T cells ensures these cells to exert effector functions without inducing rapid apoptosis.

No description available.

Atopic eczema is a T-lymphocyte mediated chronic inflammatory skin disorder. The interaction of CD4+ T-lymphocytes with epidermal keratinocytes results in dysregulated, chronic inflammation and altered barrier function. T-lymphocyte induced keratinocyte apoptosis has been proposed as a mechanism by which epidermal integrity is impaired in eczema. Apoptosis of keratinocytes is thought to result from Tlymphocyte associated Fas ligand (FasL) binding to the death receptor Fas on keratinocytes. The primary aim of this project was to characterize the induction of keratinocyte apoptosis by T-lymphocytes and address the hypothesis that insulin-like growth factor-I (IGF-1), transforming growth factor [beta]1 (TGF[beta]1) and a milk derived growth factor extract containing TGF[beta] and IGF-I (whey growth factor extract; WGFE) protect keratinocytes from T-lymphocyte mediated apoptosis. To address the aims of this project, an in vitro co-culture model was developed combining T-lymphocytes with keratinocytes. Co-cultures were initially established using human Jurkat T-lymphocytes and human HaCaT keratinocytes with more extensive characterisation undertaken using primary CD4+ T-lymphocytes together with HaCaTs or normal human epidermal keratinocytes (NHEK). Annexin V and propidium iodide staining was established as the primary method for measuring keratinocyte apoptosis with this validated using sodium butyrate a known inducer of apoptosis. Changes in nuclear fragmentation and cell morphology were also examined as a key feature of apoptosis. The involvement of the Fas pathway was investigated by assessing T-lymphocyte FasL expression, keratinocyte Fas expression and downstream caspase activation. Inflammatory cytokines IFN[gamma] and TNF[alpha] were also examined due to their ability to induce Fas expression. Studies performed with T-lymphocytes demonstrated that keratinocyte apoptosis was induced, with this due primarily to direct T-lymphocytes and keratinocytes interactions, rather than soluble mediators in the co-culture milieu. Activated T-lymphocytes were found to have high levels of FasL and to upregulate keratinocyte Fas expression. The increased keratinocyte Fas was associated with increased IFN[gamma] levels in the co-culture media and activation of the caspase cascade. A Fas blocking antibody prevented T-lymphocyte induced keratinocyte apoptosis demonstrating that this was a Fas dependent event. As the primary function of keratinocytes is to terminally differentiate, the differentiation status of the cells induced to undergo apoptosis was examined. It was demonstrated that T-lymphocytes decrease the intensity of ?6 integrin expression by the keratinocytes. This marker identifies undifferentiated basal cells as high expressors of [alpha]6, with cells in the early stages of differentiation pathway found to be low expressors of [alpha]6. Co-staining with Annexin V demonstrated that the apoptotic keratinocytes were low expressors of [alpha]6 and thus cells committed to the early stages of differentiation. This suggested that the T-lymphocytes initiated the onset of keratinocyte terminal differentiation with this linked to the cells being more susceptible to death induced by T-lymphocyte by activation of the Fas pathway. The ability of TGF[beta]1, IGF-I and WGFE to inhibit T-lymphocyte induced keratinocyte apoptosis was examined. A combination of recombinant TGF[beta] (10ng) & IGF-I (100ng) was able to significantly inhibit keratinocyte apoptosis. A similar result was obtained with WGFE, and although these growth factor treatments were able to reduce the elevated IFN[gamma] levels in the co-culture media, they did not reduce T-lymphocyte induced Fas upregulation. The TGF?1 and IGF-I combination as well as WGFE did however prevent the T-lymphocyte induced shift from [alpha]6 bright to dim expressing keratinocytes. As such, the growth factor combinations appeared to protect the keratinocytes from T-lymphocyte mediated apoptosis by preventing them from committing to terminal differentiation. The studies in this thesis have characterised the Fas associated mechanisms by which T-lymphocytes induce keratinocyte apoptosis and suggest specific growth factor combinations may have the potential to ameliorate the reduced barrier function associated with inflammatory skin conditions such as atopic eczema.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Aging is characterized by a progressive decline in cardiac function. A critical contributor to the age-related impairment in heart function is the loss of cardiac myocytes through ??apoptosis??, or programmed cell death. A dramatic increase in the rate of apoptosis has been reported with aging in the rat left ventricle. In contrast, exercise training not only improves cardiac function, but also reduces the risk of heart disease. However, the ability of exercise training to modulate apoptotic signaling and apoptosis in the aging heart remains unknown. Therefore, the purpose of this study was to determine the effects of exercise training on apoptotic signaling and apoptosis in the aging heart. We hypothesized that (1) aging would increase pro-apoptotic signaling and apoptosis in the rat left ventricle, and (2) exercise training would ameliorate upregulation of Bcl-2 family-driven apoptosis in the heart. Four and 25 month old Fischer-344 rats were assigned to four groups: young control (YC), young trained (YT), old control (OC), and old trained (OT). Exercise training groups ran on a treadmill for 60 min/day at 15 m/min (15˚ incline), 5 d/wk for 12 wk. Protein expression of Bax, Bcl-2, caspase-9, and cleaved caspase-3 was measured using Western immunoblot analysis. Apoptosis (DNA fragmentation) was assessed using a cell death detection ELISA. Bax levels in OC were dramatically higher (+176.0%) compared to YC. In contrast, exercise training resulted in a significant decrease (-53.4%) in Bax in OT compared to OC. Bcl-2 levels in OC were lower (-26.3%) compared to YC. Conversely, exercise training significantly increased Bcl-2 levels by 117.8% in OT compared to OC. Caspase-9 levels were higher (+98.7%) in OC than YC, while exercise training significantly reduced caspase-9 levels in YT (-52.6%) and OT (-76.9%), respectively. Aging resulted in a dramatic increase (+122.8%) in cleaved caspase-3 levels and a significant decrease (-32.9%) with exercise training. Finally, apoptosis (DNA fragmentation) significantly increased (+163.8%) with aging and decreased (-43.9%) with exercise training. These novel data indicate that aging increases pro-apoptotic signaling and apoptosis in the left ventricle, while exercise training is effective in diminishing pro-apoptotic signaling and apoptosis in the aging heart.

Les granulocytes, neutrophiles et éosinophiles, sont des cellules effectrices de l'immunité innée. La régulation de la vie des granulocytes par le mécanisme de mort programmée cellulaire quest lapoptose procure un équilibre judicieux entre leur fonction de cellules effectrices dans la défense de lorganisme et le renouvellement sécurisé de ces cellules potentiellement dangereuses. Lapoptose spontanée constitue un processus nécessaire pour le maintien de lhoméostasie des cellules immunes, telles que les granulocytes. Mon travail de doctorat a permis didentifier de nouveaux mécanismes régulateurs de l'apoptose des granulocytes. La première étude démontre le rôle important des céramides de type C16 et C24 en tant que messagers secondaires dans la transmission du signal de mort dans les neutrophiles vieillissants. En effet, nous montrons que ces céramides, générées par la voie de synthèse de novo, s'accumulent spontanément dans les neutrophiles et précèdent l'apparition des signes de l'apoptose précoce ainsi que l'activation des caspases-8, -9 et -3. L'inhibition ou l'accélération pharmacologiques de cette accumulation ralenti ou accélère l'apoptose des neutrophiles, respectivement. Laccumulation des céramides C16 et C24 est également inhibée par le traitement des neutrophiles avec du GM-CSF, une cytokine proinflammatoire et anti-apoptotique. Par contre, nos résultats montrent que la mort induite par l'activation du récepteur Fas ne dépend pas de la synthèse de céramides par la voie de novo. Au cours de la seconde étude, nous avons identifié un mécanisme spécifique dans la régulation de l'apoptose des éosinophiles. Nous montrons que les éosinophiles expriment spontanément CD40 à leur surface et que lengagement de CD40 s'oppose à la mort des éosinophiles via l'induction de la protéine inhibitrice de l'apoptose cytoplasmique-2 (c-IAP2). En outre, nous apportons les évidences de l'existence d'un mécanisme similaire in vivo, en particulier dans l'inflammation allergique des voies respiratoires.

Background: Inflammation, if uncontrolled, can promote the formation of colon cancer. Intestinal lymphocytes (IL) are immune cells that can participate in inflammation through the generation of cytokines and by causing direct cellular injury. Apoptosis, or programmed cell death, regulates the population of lymphocytes and dysregulation of this process results in the prolonged activation of IL that occurs during inflammation. Therefore, inducing apoptosis of IL is a viable mechanism by which inflammation and possibly colon carcinogenesis could be prevented. Experimentally, inflammation may be induced in mice by: 1) the addition of the chemical irritant dextran sulfate sodium (DSS) to drinking water or 2) exposure to acute exercise (AE; intense treadmill running). Bovine lactoferrin (bLf) is a dietary whey protein with demonstrated ability to promote anti-inflammatory responses by reducing pro-inflammatory or increasing anti-inflammatory cytokine concentrations within the intestine. There are also reports that bLf can alter apoptotic proteins in the intestinal epithelium to favour cell death. Moreover, athletes have been reported to supplement their diets with whey protein; it is also known clinically that after heavy competition some athletes may experience gastrointestinal distress. The role, however, of bLf in reducing inflammation by initiation of apoptosis or alteration of cytokine levels in IL has not been examined. Objectives: The primary objective of this research was to determine whether dietary bLf affects mouse IL apoptosis and cytokine concentrations in: 1) a normal, non-inflamed state, 2) following AE challenge and 3) following DSS treatment. A second objective was to directly examine the potential protective effects of dietary bLf from inflammatory damage caused by DSS and AE challenge when administered alone or in combination with a carcinogen. Methods: A total of 252 female C57BL/6 mice were used in the experiments. Apoptotic proteins (Bcl-2, caspase 3, Bax, cytochrome c), inflammatory cytokine proteins (TNF-α and IL-10) and a transcription factor for pro-inflammatory cytokines (NFκB) were determined in isolated mouse IL by Western blot analysis. Flow cytometry was used to determine the extent of apoptosis in mouse IL subsets by measuring phosphatidylserine surface expression using Annexin V+ (ANN+) and propidium iodide staining. Tissue inflammation was determined by histology (H&E staining) on segments of mouse small and large intestine. Diets were prepared and pelletted to contain 20% total protein and contained either bLf or were the control formulation (no bLf). Mice were exposed to bLf containing diets for 4 d or 12 d prior to sacrifice. DSS was provided at 5% in the drinking water for 4 consecutive days prior to sacrifice. Animals were subjected to three repeated bouts of AE (each separated by 24 h rest) involving treadmill running and sacrificed either immediately or 24 h after the final exercise bout. In the experiment involving carcinogenesis, mice were given two subcutaneous injections of azoxymethane (AOM), followed by a two week incubation period, and subsequently exposed to bLf or the control diet. Results: Results from the first experiment determined that 2.0% bLf was effective at reducing mouse IL levels of TNF-α (p<0.05) (pro-inflammatory) and increasing the percentage of apoptotic CD4+ IL (CD4+/ANN+, p<0.05) in healthy mice. Thus, 2.0% bLf was used for the subsequent experiments. Dietary bLf administration in mice exposed to AE was associated with lower levels in mouse IL of the anti-apoptotic protein Bcl-2 (p<0.01) and of TNF-α (p<0.05) and NFκB (p<0.05), both pro-inflammatory proteins. Further, the exercise protocol resulted in oxidative stress, as measured by 8-iso prostaglandin F2α levels in plasma, but did not induce intestinal inflammation, evident by the absence of both tissue damage and infiltration of immune cells. Following DSS treatment, mice supplemented with bLf enriched diets had lower levels of both TNF-α (non-significant, 34% reduction) and NFκB (p<0.05) and increased concentrations (p<0.01) of cytochrome c, a mitochondrial protein associated with cell death. DSS exposure in mice resulted in gross morphological alterations and infiltration of immune cells in the small and large intestine; these changes in tissue histology were not affected by the addition of bLf. Mice injected with AOM and then subjected to DSS, but not AE, had increased numbers (p<0.001) of aberrant crypts, preneoplastic colonic lesions, compared to animals only receiving AOM injection. Dietary bLf did not affect any of these carcinogenic processes. Conclusions: Collectively, these results suggest that dietary bLf administration reduces pro-inflammatory cytokine levels and has limited effects on apoptosis of mouse IL. Moreover, these modifying effects of bLf did not result in mucosal protection, as evident in the inability of this protein to reduce DSS-induced tissue damage or formation of aberrant crypts. Physiological and Clinical Implications: Although the long term physiological consequences of bLf supplementation in the regulation of intestinal immune homeostasis require further study, the following clinical implications are tentatively suggested by the findings from this thesis research. First, dietary bLf supplementation does not provide direct protection of the intestine during inflammation either with or without exposure to the carcinogen (i.e., AOM); hence, bLf (at least in the dietary concentration and exposure used in these experiments) may not be useful in reducing the formation of aberrant crypts and carcinogenesis. Second, dietary bLf should not be recommended as a supplement at this time for athletes experiencing intestinal distress since it had no impact on tissue indicators of disease in a model (DSS) shown to produce extensive inflammation and tissue pathology. Nonetheless, the findings raise the possibility that bLf can modify both cytokines and apoptotic protein expression in IL and may influence some aspects of inflammatory processes in the gut.