Dissertations / Theses on the topic 'TNF INDUCED'

Stress and stressful experiences are involved in the development and persistence of chronic physical and mental health disorders, and are an important concern for public health services. Previous studies have demonstrated acute stress can alter properties of neural circuits, specifically in the hippocampus. Tumour necrosis factor-alpha (TNF-alpha) is involved in AMPA-R trafficking, and evidence suggests it plays an important role in homeostatic, and possibly in Hebbian plasticity. Because TNF-alpha is regulated by stress, and is also important for plasticity; it is likely TNF-alpha is involved in stress-induced plasticity. Stress-induced changes in synaptic strength were recorded from 7 week old male wild-type, TNF-/- and DN-TNF (a dominant negative version of TNF) treated mice 24h after a 15 minute acute swim stress. DN-TNF, inhibits any soluble TNF-alpha while still permitting the action of membrane bound TNF-alpha. We recorded evoked excitatory postsynaptic currents from ventral hippocampal CA1 pyramidal cells in acute slices. We measured AMPA/NMDA ratios, as a measure of synaptic strength, along with rectification of the AMPA currents, to test for changes in AMPA receptor subunit composition. The present study demonstrates an increase in AMPA/NMDA ratio in stressed animals compared to unstressed controls, suggesting a stress-induced increase in synaptic strength in wild-type mice. Inhibition of TNF-alpha signalling prevents this stress-induced increase in synaptic strength, as DN-TNF treated mice had no change in AMPA/NMDA ratio following stress. Further, stress-induced synaptic changes were absent in TNF-/- mice. Stress does not affect the AMPA-R subunit composition at the 24h time point, as the rectification ratio was unaltered following stress. These results indicate stress induces an increase in synaptic strength within the CA1, in a manner dependent on TNF-alpha signalling.
Le stress et les expériences stressantes sont impliqués dans le développement et la persistance des troubles chroniques de santé physique et mentale, et elles représentent une préoccupation importante pour les services de santé publique. Des études antérieures ont démontré que le stress aigu peut modifier les propriétés des circuits neuronaux, en particulier dans l'hippocampe. Le facteur de nécrose tumorale-alpha (TNF-alpha) est impliqué dans le trafic des récepteurs AMPA (AMPA-R), et il semble qu'il joue un rôle important dans l'homéostasie, et peut-être dans la plasticité de Hebb. Puisque TNF-alpha est régulé par le stress et qu'il est également important pour la plasticité, il est probable que TNF-alpha soit impliqué dans la plasticité induite par le stress. Les changements de la force synaptique induits par le stress ont été enregistrées à partir de 7 semaines chez des souris (de type sauvage, TNF-alpha -/- et DN-TNF-alpha (une version dominante négative de TNF-alpha)) traitées 24h après 15 minutes de stress aigu dans l'eau. DN-TNF-alpha inhibe l'action de TNF-alpha soluble tout en permettant l'action de la membrane liée à TNF-alpha. Nous avons enregistré les courants postsynaptiques excitateurs évoqués à partir de cellules pyramidales dans des tranches aiguës de la région CA1 de l'hippocampe ventral. On a mesuré le ratio des récepteurs AMPA / NMDA pour quantifier la force synaptique en tenant compte de la correction des courants des récepteurs AMPA pour vérifier les changements dans la composition de leurs sous-unités. La présente étude démontre une augmentation du ratio des récepteurs AMPA / NMDA chez les animaux stressés par rapport aux animaux non stressés, ce qui suggère une augmentation de la force synaptique induite par le stress dans les souris de type sauvage. L'inhibition de la signalisation du TNF-alpha empêche cette augmentation de la force synaptique induite par le stress et les souris traitées avec DN-TNF-alpha n'ont pas modifié le rapport des récepteurs AMPA / NMDA en réponse au stress. En outre, les changements synaptiques induits par le stress étaient absents dans les souris TNF-alpha-/-. Le stress n'affecte ni la composition des sous-unités d'AMPA-R après 24h ni la correction des courants des récepteurs AMPA. Ces résultats indiquent que le stress induit une augmentation de la force synaptique dans la région CA1 via la signalisation de TNF-alpha.

Macrophages are the predominant cell type in the chronic inflammatory reaction associated with the development of mineral-dust-induced fibrosis. Macrophages are potent producers of cytokines, which have recently emerged as potentially important mediators in regulating inflammatory states. I investigated cytokine production by alveolar macrophages (AM), with a special emphasis on tumor necrosis factor-$\alpha$, in experimental lung fibrosis induced by mineral dusts. Exposure to fibrigenic dusts had a bimodal effect on TNF-$\alpha$ production by AM. First, suppressed TNF-$\alpha$ production after 1 and 3 weeks of exposure. Second, after 6 weeks of exposure TNF-$\alpha$ production was high. By contrast, interleukin-1 (IL-1) and interleukin-6 (IL-6) production was increased in animals with inflammation with and without fibrosis. Potentiations in IL-1 and IL-6 production were associated with the early stage of the inflammatory reaction and were inversely correlated with TNF-$\alpha$ changes. Interestingly, alterations in TNF-$\alpha$ production were associated with definite shifts in the distribution size of AM suggesting that the overall production of TNF-$\alpha$ may be regulated by the specific class of AM present at sites of inflammation. To our knowledge, our study brings the first evidence for a negative modulation of TNF-$\alpha$ during inflammatory reactions leading to lung fibrosis. Furthermore, experiments with neutralizing antibody to TGF-$\beta$ suggest a role for TGF-$\beta$ in down-regulating TNF-$\alpha$ production in our system.

Inflammatory bowel diseases (IBD) are chronic inflammatory processes that affect the gastrointestinal tract. IBD is characterized by an intestinal inflammation and epithelial cell injury leading to a poor psychosocial wellbeing and increased the risk for cancer. IBD pathogenesis is multifactorial, involving genetic predisposition, epithelial barrier defects, dysregulated immune responses, and environmental factors. The current gold standard therapy is TNF blockade, a key proinflammatory cytokine. TNF activates the NF-κB pathway inducing an anti-apoptotic cell response and it plays an important role in the regulation of the intestinal homeostasis. However, in certain situations, TNF can also trigger cell death through different signaling cascades, a typical feature seen in IBD. This work shows how apoptotic IEC areas in IBD human samples co-localize with NF-κB activation and A20 upregulation. Furthermore, using animal models as well as in vitro studies, we show how NF-κB activation and A20 upregulation are required events to trigger TNF- dependent cell death in intestinal epithelial cells. We also show how other cytokines that are usually upregulated in IBD interact with TNF to induce cell death. Specifically, lymphotoxin β receptor activation synergizes with TNF to trigger apoptosis. We have proven that intestinal epithelial cells undergo apoptotsis downstream of TNFR in a RIPK1 dependent manner, and that kinase inhibition of RIPK1 prevents cell death. Chronic NF-κB induces apoptosis downstream of TNF in a ROS dependent manner and A20 requires its linear ubiquitin binding domain, zinc finger seven, to enhance the formation of complex IIb or ripoptosome after TNF stimulation. Overall these results help to further understand the pathogenesis of IBD and suggests RIPK1 as a possible target for new drugs to treat IB.
Les malalties inflamatòries intestinals (MII) són patologies caracteritzades per una inflamació crònica del tracte gastrointestinal que indueix dany a l’epiteli intestinal incrementant la morbiditat del pacient i el risc de desenvolupar càncer. La patogènesis de les MII és multifactorial, i inclou susceptibilitat genètica, defectes en la barrera epitelial, una resposta immunitària descontrolada i factors ambientals. El tractament estàndard per les MII és el bloqueig del TNF, una citocina pro- inflamatòria. El TNF activa la via de senyalització del NF-κB induint una resposta anti-apoptòtica, al mateix temps que té una funció important en la regulació de l’homeòstasi intestinal. No obstant, en certs casos, el TNF es capaç d’induir mort cel·lular, una característica típica de les MII, a través de diferents vies de senyalització. Aquesta tesis mostra com les àrees apoptòtiques en l’epiteli de les MII correlacionen amb una activació de la via del NF-κB i un increment de A20. Usant models animals i experiments in vitro, demostrem com l’activació de NF-κB així com l’augment de A20 en les cèl·lules del epiteli intestinal, són events necessaris per induir la mort cel·lular depenent de TNF. També demostrem com altres citocines, que generalment estan augmentades en les MII, afavoreixen l’apoptosis secundaria a TNF. Específicament, l’activació de receptor de la limfotoxina beta incrementa la capacitat del TNF per induir mort cel·lular. Ensenyem com aquesta mort és secundaria a l’activitat quinasa de RIPK1 i com inhibint-la es pot prevenir la mort cel·lular. També mostrem com l’activació crònica de NF-κB indueix apoptosis secundaria al TNF degut a un increment de ROS mentre que A20 requereix el seu domini d’unió a cadenes d’ubiquitines lineals per afavorir la formació del complexe IIb o ripoptosoma posterior a l’estimulació per TNF. Així doncs, aquest treball aprofundeix més en el coneixement de la patogènesis de les MII i suggereix la inactivació de l’activitat quinasa de RIPK1 com una possible diana terapèutica.

The murine cytomegalovirus (MCMV) immediate early 1 (IE1) protein has been described as a trans-activator of viral and host gene expression. However, the precise role that IE1 plays in the viral life cycle, and in particular its effect on the host immune response is not known. This thesis investigates the functional relationship of the IE1 protein and the immune response induced after infection. By using an ie1-deletion mutant MCMV (MCMVdie1) it was demonstrated that, early after infection, tumor necrosis factor (tnf ) gene activation and protein production was significantly induced in infected-primary macrophages (M ) to a much greater extent than its wild type counterpart. In addition, preliminary studies on the signalling pathways activated upon infection were carried out in order to gain information about the pathways that might be involved in MCMVinduced modulation of tnf activation. Initial observations on the MAPK family members Erk1/2, p38 and JNK did not revealed any differential activation in the absence of IE1. However, due to a number of limitations, it was not possible to draw any firm conclusions from this study. Investigation of the role of IE1 in the in vivo production of TNF were also performed in both susceptible (BALB/c) and resistant (C57Bl/6) mice. These experiments confirmed the attenuated phenotype of MCMVdie1 in vivo, whereby the mutant strain grew to much lower titers than wild type. When cytokine production was assessed in relation to PFU levels a significant production of TNF after infection is observed in different organs of both mice strains. This raises the question whether IE1 contributes to MCMV modulation of TNF production in the natural host. Although, because it is still unclear whether the phenotype of MCMVdie1 in vivo is due to a defect in the virus or the result of a immune response, it was not possible to conclude unequivocally that IE1 is responsible for dampening this cytokine response. This thesis also tested whether the attenuated replication of MCMVdie1 in vivo was due to the increased TNF production induced after infection. An initial investigation in tnf depleted mice revealed that the MCMVdie1 growth phenotype is not due to TNF response. Overall, this study has provided insight into a potential immune modulatory function by MCMV associated with IE1 protein and the regulation of TNF in vivo and in vitro.

In type 1 diabetes (T1D) a combination of genetic predisposition and environmental factors triggers islet inflammation (insulitis) leading to a selective and gradual destruction of the pancreatic beta cells. Beta cells mainly die through apoptosis, triggered at least in part by pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ. Recent findings suggest that the mitochondrial pathway of cell death is involved in this death cascade. Array analysis indicated that TNF-α+IFN-γ induces transcription factors such as NF-ĸB, STAT1, and AP-1 in beta cells. We presently aimed to examine the pathway(s) of apoptosis triggered by TNF-α+IFN-γ in beta cells.

TNF-α+IFN-γ induces beta cell apoptosis through the intrinsic pathway of cell death. This involved activation of the BH3 only proteins DP5, PUMA and Bim. Knockdown (KD) of either DP5 or PUMA or both led to a partial protection of INS-1E cells (12-20%), while silencing Bim led to about 60% protection against cytokine-induced apoptosis. Bim is transcriptionally induced by activated STAT1. TNF-α+IFN-γ also induces downregulation of Bcl-XL, an anti-apoptotic Bcl-2 gene which inhibits Bim. Knocking down Bcl-XL alone led to increase in apoptosis, but this was prevented by the parallel KD of Bim.

The ultimate goal of our research is to protect beta cells from the autoimmune assault. Previous data revealed that JunB inhibits ER stress and apoptosis in beta cells treated with IL-β+IFN-γ. Here, TNF-α+IFN-γ up-regulated the expression of JunB which was downstream of activated NF-ĸB. JunB KD exacerbated TNF-α+IFN-γ induced beta cell death in primary rat beta cells and INS-1E cells. The gene networks affected by JunB were studied by microarray analysis. JunB regulates 20-25% of the cytokine-modified beta cell genes, including the transcription factor ATF3 and Bcl-XL. ATF3 expression was increased in cytokine-treated human islets and in vitro silencing of JunB led to >60% reduction in ATF3 overexpression. We confirmed direct JunB regulation of the ATF3 promoter by its binding to an ATF/CRE site. Silencing of ATF3 aggravated TNF-α+IFN-γ induced cell death in beta cells and led to the downregulation of Bcl-XL expression in INS-1E cells. Pharmacological upregulation of JunB using forskolin led to upregulation of ATF3 and consistent protection of these cells against cytokine-induced cell death, while genetic overexpression of JunB in mice increased ATF3 expression in the pancreatic islets and reversed the pro-apoptotic effects of cytokines on beta cells (±40 % protection).

As a whole, our findings indicate that TNF-α+IFN-γ triggers beta cell apoptosis by the upregulation of the pro-apoptotic protein Bim and downregulation of the Bcl-XL protein. These deleterious effects are at least in part antagonized by JunB via activation of ATF3.

Dans le diabète de type 1 (DT1), la combinaison de facteurs génétiques de prédisposition et de l'environnement déclenche l'inflammation des îlots de Langerhans (insulite) conduisant à une destruction sélective et progressive des cellules bêta du pancréas. Les cellules bêta meurent principalement d’apoptose, déclenchée au moins en partie par les cytokines pro-inflammatoires sécrétées par les cellules immunitaires comme l’IL-β, le TNF-α l’IFN-γ. De récentes découvertes suggèrent que la voie mitochondriale de la mort cellulaire jouerait un rôle dans la mort de ces cellules. L'analyse de réseaux de gène utilisant les biopuces d’ADN indique que l’association TNF-α+IFN-γ induit l’activation de facteurs de transcription tels que NF-ĸB, STAT1 et AP-1 dans la cellule bêta. Dans ce contexte, nous avons cherché à examiner les voies de l'apoptose déclenchées par le TNF-α+IFN-γ dans la cellule bêta.

En présence de TNF-α+IFN-γ les cellules bêta meurent par apoptose via la voie intrinsèque. L’activation des protéines pro-apoptotiques « BH3-seulement » dont DP5, PUMA et Bim étaient en cause de cette apoptose. Le « knockdown »1 (KD), de DP5 ou de PUMA, ou des deux en même temps conduit à une protection partielle des cellules INS-1E (12-20%), tandis que le KD de Bim conduit à environ 60% de protection contre l’apoptose induite par cette combinaison de cytokines. La transcription de Bim est induite par STAT1 activé. Parallèlement à la régulation positive de Bim, TNF-α+IFN-γ conduit à la régulation négative de la protéine Bcl-XL. Bcl-XL est une protèine anti-apoptotique de la famille de protèines Bcl-2 qui en general inhibe Bim. Réduire l’expression de Bcl-XL seul induit une augmention de l'apoptose, alors que le KD de Bim et Bcl-XL en parallèle empêche l'apoptose.

Le but ultime de notre recherche est de protéger les cellules bêta des agressions autoimmunitaires. Les données antérieures ont révélé que JunB inhibe le stress du réticulum endoplasmique et l'apoptose dans les cellules bêta traitées avec IL-β+IFN-γ. Nous avons observé que TNF-α+IFN-γ induit l'expression de JunB qui se produit en aval de NF-ĸB activé. Il est important de noter que l’inactivation de JunB par des agents interférants de l’ARN (siRNA) exacerbe la mort des cellules primaires bêta de rat et de cellules INS-1E induite par les cytokines. Les réseaux de gènes touchés par JunB ont été étudiés grâce a l'analyse en microréseaux. JunB règule 20-25% des gènes modifiés par des cytokines dans les cellules bêta, y compris le facteur de transcription ATF3 et Bcl-XL. L’expression d’ATF3 est augmenté dans les îlots humains traités avec les cytokines et la répression in vitro de JunB conduit à une réduction de >60% de l’expression d’ATF3. Nous avons confirmé la régulation d’ATF3 par JunB en montrant que JunB est directement lié au promoteur d’ATF3 via le site ATF/CRE. La diminution d’expression d’ATF3 en presence de TNF-α+IFN-γ a aggravé la mort cellulaire induite dans les cellules bêta et a conduit à la régulation négative de l'expression de Bcl-XL dans les cellules INS-1E. L’augmentation pharmacologique de JunB dans les cellules INS-1E par l’utilisation de forskolin a conduit à la régulation positive en aval d’ATF3 et par conséquente à la protection de cellules bêta vis-a-vis de effets indésirables des cytokines. Dans cette optique, la surexpression génétique de JunB dans le modèle Ubi-JunB de souris transgénique a conduit à une surexpression d’ATF3 dans les îlots pancréatiques et a permir d’inverser les effets pro-apoptotiques de cytokines sur la cellule bêta (protection ± 40%).

Globalement, ces résultats indiquent que TNF-α+IFN-γ déclenche l'apoptose des cellules bêta par la régulation positive du gène pro-apoptotique Bim et la régulation négative du gène anti-apoptotique Bcl-XL. Ces effets indésirables sont inhibé en partie par JunB via l’activation de ATF3.

1Pas d’équivalent en français. Signifie la réduction de l’expression d’un gène via utilisation d’un siRNA (agent interférant de l’ARN).

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Tumor necrosis factor (TNF) is a pleotropic cytokine that mediates many inflammatory and innate immune responses. TNF also causes apoptosis in certain transformed cell lines and cells that are infected with certain viruses or intracellular bacteria. Cytosolic phospholipase A2 (cPLA2) is an inflammatory enzyme that mediates its activities, by specifically catalyzing the release of arachidonic acid leading to the generation of eicosanoids. The activity of cPLA2 is necessary during TNF-induced apoptosis and the goal of this study was to identify signals that mediate the activation of cPLA2 during cell death. Intracellular calcium and phosphorylation are well documented to activate cPLA2 under many inflammatory conditions. Therefore, I examined the ability of these signals to regulate cPLA2 during TNF-induced apoptosis. I first examined calcium levels during the TNF-induced apoptosis of C3HA fibroblasts and determined that an influx of extracellular calcium occurs early during cell death. This influx, as well as the release [3H]arachidonic acid, was blocked by verapamil indicating that the calcium response is necessary for the activation of cPLA2 during this process. To analyze the effects that phosphorylation has during TNF-induced apoptosis, cPLA2 proteins, containing serine phosphorylation site mutations, were stably overexpressed in WM793 melanoma cells. Although PMA was able to enhance the release of [3H]arachidonic acid from cells that overexpressed cPLA2, the treatment of the same cells with TNF and cycloheximide had no effect. However, subsequent experiments using PMA demonstrated novel roles for the phosphorylation of Ser-437 and ?727. One was an activation role for Ser-437 as its phosphorylation enhanced [3H]arachidonic acid release. The other was an inhibitory role for the phosphorylation of Ser-727 as its mutation suppressed the release in response to PMA. In conclusion, though the activation of cPLA2, by calcium responses, during apoptotic and non-apoptotic systems is consistent, the regulation of cPLA2 by phosphorylation may involve both positive and negative regulatory signals.

Biomedical Neuroscience
Ph.D.
The use of highly active antiretroviral therapy (HAART) has significantly decreased the mortality rate of HIV-1 patients, however the increased survival has led to the development of complications associated with the persistence of the viral infection. Nearly half of HIV-1-infected individuals develop HIV-associated neurocognitive disorders (HAND) as the effects of the chronic infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxicity of HIV-1 has largely been attributed to the inflammation caused by viral replication and the altered signaling of astrocytes, microglia, and macrophages. Although HAART has improved the control of viral replication, the effects from inflammation remain a concern, particularly those of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α has been a therapeutic target for other diseases associated with chronic inflammation, such as rheumatoid arthritis, but emerging evidence has suggested that TNF-α signaling can have a dual role, especially in the CNS, proving the complexity in the modulation of the TNF-α pathway. Although the detrimental effects of TNF-α have been well-characterized, we lack a complete understanding of the beneficial role of TNF-α. TNF-α signaling has largely been considered to be neurotoxic but has been able to regulate neurite outgrowth in the context of neural development. Since TNF-α is upregulated in various neurodegenerative conditions, we considered potential outcomes of TNF-α on neurite outgrowth following injury. Initially, most would assume that TNF-α would prevent neurite outgrowth as apoptosis is a common outcome of TNF-α-induced signaling. If TNF-α signaling strictly prevents neurite outgrowth, anti-TNFα therapies could be considered to reverse this effect. However, upon induced injury, we observed an increase in neurite regrowth following induced injury in human primary fetal neurons, demonstrating a strong need for a deeper understanding of this dual role of TNF-α. Anti-TNF-α therapies have been considered for HIV-1-infected patients to reduce the chronic inflammation, however inhibiting TNF-α signaling could have side-effects that could prevent neuronal recovery from HIV-1 effects. Targeting pathways downstream of TNF-α signaling would be more advantageous to mediate the beneficial role of TNF-α in the CNS. We investigated the transcriptional effects of TNF-α treatment on neurons to uncover a potential pathway to promote neurite outgrowth. One pathway we have discovered to be beneficial in primary human fetal neurons is TNF-α-induced Ephrin B2 upregulation. Ephrin B2 (EphB2) receptors are important mediators of neuronal development and synaptic plasticity, however little has been established in regards to their role in HIV and inflammation, particularly in the CNS. EphB2 can mediate axonal development by providing retractive cues to assist the axon to reach the target, but EphB2 can also promote dendritic branching to improve learning and memory, which would be particularly beneficial for HAND patients that experience cognitive deficits. We observed a correlation between the upregulation of EphB2 in response to TNF-α and neurite outgrowth, which provides a potential pathway to repair damaged neurons and re-establish lost neuronal connections. Dendritic pruning and neuronal loss has been observed in HAND patients, so this ability to promote repair could prevent, improve, or recover the cognitive deficits experienced by HIV-patients with HAND. TNF-α, although primarily known to induce neurotoxicity, strongly activates the nuclear factor-kappaB (NF-κB) pathway, which can have a very wide range of transcriptional effects. Therefore, our hypothesis is that the TNF-α-induced neurite regrowth occurs through an upregulation EphB2 in an NF-κB-dependent pathway. TNF-α has been well established to induce NF-κB signaling, mostly by promoting the translocation of the NF-κB p65 DNA binding factor to the nucleus for transcriptional regulatory effects. NF-κB can regulate neuronal growth and process development of both dendrites and axons, which would correlate to the neurite regrowth observed following TNF-α upon induced injury. The regulation of EphB2 by NF-κB has not been extensively studied, but EphB2 can be negatively regulated by an NF-κB family member, c-Rel. We analyzed the EphB2 promoter and identified three NF-κB p65 binding sites upstream from the transcriptional start site, which provided insight to our hypothesis. We established that p65 directly binds to and can regulate EphB2 promoter activity in response to TNF-α. Since the dual role of TNF-α can be dependent on the receptor through which the signaling proceeds, either TNF-α receptor 1 (TNFR1) or TNF-α receptor 1 (TNFR2), we investigated if this upregulation of EphB2 is receptor dependent and determined EphB2 is induced primarily through activation of TNFR2. Neurons express both receptors, however, the effects of TNF-α to promote neuroprotection and repair primarily occur through the TNF-α/TNFR2 regulatory axis. Although we have been established the mechanism of TNF-α-induced EphB2 and there is a strong correlation with neurite outgrowth following induced injury, we considered the possibilities to modulate EphB2 in the absence of TNF-α to demonstrate the direct effects of EphB2 expression. Several approaches could be used to mediate EphB2 activation or inhibition in vitro. RNA interfering techniques, such as small interfering RNA (siRNA), are useful, but we were interested in a complete knockout strategy. Since our approach was to assess the effects of EphB2 knockout only on neurite outgrowth following induced injury, a knockout animal model would not be appropriate, as a lack of EphB2 would affect the development of the neurons, unless an inducible knockout model was established. This is a lengthy and elaborate process and, more importantly, would only be available in a non-human model. Other techniques, such as transcription activator like effector nucleases (TALENs), can generate knockout systems that are targeted to specific regions of a gene, but specific binding proteins must be created to recruit the endonucleases to the target. Clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) has emerged as a specific and relatively easy technique to knockout genes of interest and uses short RNA sequences to guide Cas9 endonucleases to target regions to create double stranded breaks in the DNA to silence the gene. Once concern with Cas9 is specificity to target only the desired region of the gene, as off-target effects can occur and may result in unwanted gene silencing. A Cas9 mutant, Cas9 nickase (Cas9n), has been created to have more specificity by requiring two guide RNAs to recruit two Cas9 nickases to generate a double stranded break as they function as nickases to only create a nick in one DNA strand. We developed this strategy to remove exon 1 of the EphB2 gene by using two pairs of Cas9 nickases, with four guide RNAs, to eliminate any chance for off-target effects but retaining the desired outcome of and EphB2 knockout. We validated the system by demonstrating that a knockout of EphB2 increases adhesion and prevents migration in human embryonic kidney 293T (HEK293T) cells. Although this cell model is not a neuronal cell model, the migration assay demonstrates the functional loss of EphB2. We also created an inducible Ephb2 system to overexpress EphB2. Together these provide essential tools to verify the direct involvement of EphB2 in neurite outgrowth. Taken together, our studies characterize a novel mechanism for neurite outgrowth following injury in neurons: TNF-α/TNFR2-induced EphB2 signaling in an NF-κB p65-dependent manner. In addition to the established mechanism, we developed a technique to assess the effects of EphB2 knockout and overexpression in the context of neurite outgrowth: EphB2-targeted-Cas9n and EphB2 inducible construct. This mechanism yields insight into a potential downstream pathway to be utilized to repair damaged regions in the brain and reverse cognitive deficits in neurodegenerative conditions, especially in a chronic inflammatory environment, such as HIV-1 infection. The strategies created provide a valuable toolset to demonstrate the direct effects of modulating EphB2 signaling, not only in neurons for effects on neuronal health and synaptic plasticity, but also in other disease models, such as glioblastoma, in which EphB2 was demonstrated to promote invasion and migration of tumor cells. These observations and the usefulness of the modulatory strategies likely extend to multiple neurodegenerative diseases that demonstrate cognitive deficits that correlate to neuroinflammation.
Temple University--Theses

The works presented in this dissertation provide insights into the mechanisms of chemotherapy-induced cognitive impairment (CICI or “ChemoBrain”) and take steps toward outlining a preventive strategy. CICI is now widely recognized as a complication of cancer chemotherapy experienced by a large percentage of cancer survivors. Approximately fifty percent of existing FDA-approved anti-cancer drugs generate reactive oxygen species (ROS). Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, produces the reactive superoxide radical anion (O2-•) in vivo. Dox treatment results in oxidation of plasma proteins, including ApoA-I, leading to TNF-α-mediated oxidative stress in plasma and brain. TNF-α elevation in brain leads to further central nervous system toxicity including mitochondrial dysfunction, neuronal death, and cognitive impairment. Co-administration of the antioxidant drug, 2-mercaptoethane sulfonate sodium (MESNA), prevents Dox-induced protein oxidation and subsequent TNF-α elevation in plasma without interfering with the cancer-killing ability of Dox. In studies presented in this dissertation, we measured oxidative stress in both brain and plasma of Dox-treated mice both with and without MESNA. MESNA ameliorated Dox-induced oxidative protein damage in plasma, confirming our prior studies, and in a new finding led to decreased oxidative stress in brain. Using novel object recognition (NOR), we demonstrated the Dox administration resulted in memory deficits. Using hydrogen magnetic resonance imaging spectroscopy (H1-MRS) techniques, we demonstrated that Dox administration led to a dramatic decrease in choline(phosphocholine)/creatine (Cho/Cr) ratios in mouse hippocampus. The activities of both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D(PLD) were severely diminished following Dox administration. The activity of PC-PLC was preserved when MESNA was co-administered with Dox. In the absence of TNF-α, MRS-indexed Cho/Cr ratio, PLD activity, and mitochondrial oxygen consumption are preserved in brain, and markers of oxidative stress are reduced. Together with results from our previous studies, these results provide strong evidence that TNF-α is strongly associated, if not responsible for CICI. We also tested the notion that O2-• is responsible for Dox-induced plasma protein oxidation and TNF-α release. O2-• resulted in increased oxidative damage to proteins when added to plasma and increased levels of TNF-α in macrophage culture, providing strong evidence that O2-• is responsible for these Dox-induced toxicities.

Matrix metalloproteinase (MMP) induced extra cellular matrix (ECM) degradation is a crucial process involved in the development of many chronic inflammatory diseases, including cardiac remodeling and cancer metastasis. In cardiac remodeling, the presence of pathological stimuli leads to elevated MMP-9 expression and impairment of cardiac performance, which subsequently develops into heart failure. While in tumorgenesis, MMP-9 has been found to play key roles in metastasis, as it can break physical barriers for the tumor. Therefore, searching for agents targeting MMP-9 is a new direction for the treatment of cardiac remodeling and cancer metastasis. Chinese herbal medicine is becoming increasingly used worldwide in recent decades. In the past twenty years, as many highly selective and sensitive bioassays were introduced into the bioactive compounds screening from herbal medicine, more than one hundred new drug candidates have been identified. Therefore, herbal medicine is a potential source of bioactive compounds. Panax notoginseng (PNG) is one of the most common traditional Chinese medicines to treat cardiovascular diseases, and it was also reported to have anti-cancer effect. We hypothesized that it contains bioactive compounds that could inhibit MMP-9 activity in cardiomyocytes and cancer cells. In order to examine the effect of PNG on cardiac remodeling and cancer metastasis, we employed TNF-α induced MMP-9 in H9c2 cell (a rat cardiomyocyte) and HepG-2 cell (a human hepatoma cell) as an in vitro assay, respectively. PNG was first extracted by four different extraction methods according to the polarity of the solvent. The most effective fraction in suppressing MMP-9 activity in TNF-α induced H9c2 cell was chosen for further separation by silica gel column chromatography and high performance liquid chromatography (HPLC) until a single compound was isolated. According to the result of spectroscopic analysis by NMR, the compound was identified as ginsenoside Rb1. For the bioactivity assays, real-time quantitative polymerase chain reaction (QPCR) and Enzyme-linked immunosorbent assay (ELISA) were used to measure the mRNA and protein expression of MMP-9, respectively. We also examined the MMP-9 activity by gelatin zymography. The results showed that both of the PNG extract obtained from 10% ethanol extraction method (PNG-3) and purified Compound P (ginsenoside Rb1) showed significant inhibitory effect on MMP-9 expression and activity in H9c2 cells and HepG-2 cells. We further examined the molecular mechanisms of the inhibitory effect of PNG-3. H9c2 and HepG-2 cells were pretreated with different kinase inhibitors followed by the activation by TNF-α. The results showed the protein kinase R (PKR) inhibitor could inhibit TNF-α induced MMP-9 in both of the two cell lines. Furthermore, the results of Western blot showed the PNG-3 suppressed the phosphorylation of eIF-2α which is a down-stream effector of PKR in TNF-α stimulated H9c2 and HepG-2 cells, respectively. Therefore, PNG-3 may act through PKR to regulate TNF-α induced MMP-9 activity. In summary, bioactivity guided fractionation is an effective way of isolating bioactive compounds from medicinal herbs. In addition, PNG containing ginsenoside Rb1 may be a potential candidate of MMP-9 inhibition for the treatment of cardiac remodeling and cancer metastasis.
published_or_final_version
Paediatrics and Adolescent Medicine
Master
Master of Philosophy

Thesis (MSc (Physiological Sciences))--Stellenbosch University, 2008.
Introduction: Skeletal muscle atrophy is a mitigating complication that is characterized by a reduction in muscle fibre cross-sectional area as well as protein content, reduced force, elevated fatigability and insulin resistance. It seems to be a highly ordered and regulated process and signs of this condition are often seen in inflammatory conditions such as cancer, AIDS, diabetes and chronic heart failure (CHF). It has long been understood that an imbalance between protein degradation (increase) and protein synthesis (decrease) both contribute to the overall loss of muscle protein. Although the triggers that cause atrophy are different, the loss of muscle mass in each case involves a common phenomenon that induces muscle proteolysis. It is becoming evident that interactions among known proteolytic systems (ubiquitin-proteosome) are actively involved in muscle proteolysis during atrophy. Factors such as TNF-α and ROS are elevated in a wide variety of chronic inflammatory diseases in which skeletal muscle proteolysis presents a lethal threat. There is an increasing body of evidence that implies TNF-α may play a critical role in skeletal muscle atrophy in a number of clinical settings but the mechanisms mediating its effects are not completely understood. It is also now apparent that the transcription factor NF-κB is a key intracellular signal transducer in muscle catabolic conditions. This study investigated the various proposed signalling pathways that are modulated by increasing levels of TNF-α in a skeletal muscle cell line, in order to synthesize our current understanding of the molecular regulation of muscle atrophy. Materials and Methods: L6 (rat skeletal muscle) cells were cultured under standard conditions where after reaching ± 60-65% confluency levels, differentiation was induced for a maximum of 8 days. During the last 2 days, myotubes were incubated with increasing concentrations of recombinant TNF-α (1, 3, 6 and 10 ng/ml) for a period of 40 minutes, 24 and 48 hours. The effects of TNF-α on proliferation and cell viability were measured by MTT assay and Trypan Blue exclusion technique. Morphological assessment of cell death was conducted using the Hoechst 33342 and Propidium Iodide staining method. Detection of apoptosis was assessed by DNA isolation and fragmentation assay. The HE stain was used for the measurement of cell size. In order to determine the source and amount of ROS production, MitoTracker Red CM-H2 X ROS was utilised. Ubiquitin expression was assessed by immunohistochemistry. PI3-K activity was calculated by using an ELISA assay and the expression of signalling proteins was analysed by Western Blotting using phospho-specific and total antibodies. Additionally, the antioxidant Oxiprovin was used to investigate the quantity of ROS production in TNF-α-induced muscle atrophy. Results and Discussion: Incubation of L6 myotubes with increasing concentrations of recombinant TNF-α revealed that the lower concentrations of TNF-α used were not toxic to the cells but data analysis of cell death showed that 10 ng/ml TNF-α induced apoptosis and necrosis. Long-term treatment with TNF-α resulted in an increase in the upregulation of TNF- α receptors, specifically TNF-R1. The transcription factors NF-κB and FKHR were rapidly activated thus resulting in the induction of the ubiquitin-proteosome pathway. Activation of this pathway produced significant increases in the expression of E3 ubiquitin ligases MuRF-1 and MAFbx. Muscle fibre diameter appeared to have decreased with increasing TNF-α concentrations in part due to the suppressed activity of the PI3-K/Akt pathway as well as significant reductions in differentiation markers. Western blot analysis also showed that certain MAPKs are activated in response to TNF-α. No profound changes were observed with ROS production. Finally, the use Oxiprovin significantly lowered cell viability and ROS production. These findings suggest that TNF-α may elicit strong catabolic effects on L6 myotubes in a dose and time dependent manner. Conclusion: These observations suggest that TNF-α might have beneficial effects in skeletal muscle in certain circumstances. This beneficial effect however is limited by several aspects which include the concentration of TNF-α, cell type, time of exposure, culture conditions, state of the cell (disturbed or normal) and the cells stage of differentiation. The effect of TNF-α can be positive or negative depending on the concentration and time points analysed. This action is mediated by various signal transduction pathways that are thought to cooperate with each other. More understanding of these pathways as well as their subsequent upstream and downstream constituents is obligatory to clarify the central mechanism/s that control physiological and pathophysiological effects of TNF-α in skeletal muscle.

Human cytomegalovirus (HCMV) infection induces a persistent reconfiguration of the NK cell compartment in some individuals, promoting the development of a mature NK cell subset, hallmarked by high expression levels of the CD94/NKG2C activating receptor (NKG2Cbright), together with additional phenotypic and functional features. The mechanisms underlying the adaptive differentiation and expansion of NKG2C+ NK cells remain unknown. In the present work we addressed the relationship between adaptive NKG2C+ NK cells and down-regulation of FcεRγ (FcRγ) expression, a phenotypic trait also associated with HCMV infection. Our results showed that expansions of adaptive NKG2Cbright and detection of FcRγ– NK cells largely coincided in a subgroup of HCMV+ individuals, presumably representing events related with a common virus-host interaction pattern. In addition, the existence of adaptive FcRγ-defective NK cells lacking NKG2C and activating KIRs supported the existence of alternative activation pathways promoting their differentiation. Finally, we disclose an association between NKG2C copy number and the distribution pattern of adaptive NK cell subsets. Several observations indirectly support the involvement in this process of a cognate interaction of CD94/NKG2C with ligand(s) displayed by HCMV-infected cells. To approach this issue, we developed a sensitive reporter system stably expressing CD94/NKG2C and DAP12 in the human Jurkat leukemia T cell line, segregated from other NK cell receptors involved in theinteraction with HCMV-infected cells. Signalling was detected by transfection of a reporter plasmid encoding Luciferase under NFAT/AP1-dependent promoter. Although Jurkat-NKG2C+ cells were activated by anti-CD94/NKG2C mAbs and HLA-E+ 721.221 cells, no response was detected to fibroblasts infected with AD169 or Towne HCMV strains, regardless of their ability to preserve surface HLA-E expression. On the other hand, infection with clinical isolates or with the endotheliotropic TB40/E strain triggered Jurkat-NKG2C+ activation; yet, this response was not inhibited by blocking mAbs and was independent of CD94/NKG2C expression. These results are discussed in the framework of previous observations supporting the hypothetical existence of specific ligand(s) for CD94/NKG2C in HCMV-infected cells.
La infección por el citomegalovirus humano (HCMV) promueve en algunos individuos el desarrollo persistente de una subpoblación de células Natural Killer (NK) maduras, que presentan una elevada expresión del receptor activador CD94/NKG2C (NKG2Cbright), junto a otras características fenotípicas y funcionales. Se desconocen los mecanismos que subyacen esta diferenciación y expansión adaptativa de las células NK. En este trabajo estudiamos la relación entre las células NK NKG2C+ y la perdida de expresión de FcR (FcR), otro rasgo fenotípico asociado a la infección por HCMV. Nuestros resultados indican que las expansiones de células adaptativas NKG2C+ y la detección de células NK FcR– coinciden en un subgrupo de individuos HCMV+, representando presumiblemente procesos relacionados con un patrón de interacción virus-huésped común. Además, la existencia de células NK FcR– que no expresan NKG2C ni KIRs activadores apuntan a la existencia de vías de diferenciación alternativas. Finalmente, hemos observado una asociación entre el número de copias del gen NKG2C y el patrón de distribución de las diferentes subpoblaciones de células NK adaptativas. Por otra parte, varias observaciones sugieren que la interacción del receptor CD94/NKG2C con un hipotético ligando en las células infectadas por HCMV contribuye al desarrollo de las células NK adaptativas. Para abordar la cuestión, desarrollamos un sistema sensor mediante la expresión estable de CD94/NKG2C y DAP12 en una línea leucémica humana T (Jurkat), que carece de otrosreceptores NK. La señalización se detecta transfectando un plásmido que codifica la luciferasa, con un promotor dependiente de NFAT/AP-1. Las células Jurkat-NKG2C+ se activan mediante la estimulación por anticuerpos monoclonales anti-CD94/NKG2C o células 721.221 HLA-E+, validando la sensibilidad del sistema. Sin embargo, no se detecta la respuesta a fibroblastos infectados por las cepas AD169 y Towne de HCMV, al margen de su expresión de HLA-E. Por el contrario, lad células infectadas por dos aislados clínicos de HCMV o por la cepa endoteliotrópica TB40/E activan a las células Jurkat-NKG2C+, si bien por un mecanismo independiente de CD94/NKG2C. Estos resultados se discuten en el marco de las observaciones que sustentan la hipotética existencia de ligando(s) específico(s) para CD94/NKG2C en las células infectadas por HCMV. xii

El neuroblastoma (NB) es un cáncer pediátrico que representa ~15% de muertes en cánceres infantiles. Los NBs de alto riesgo se caracterizan por una gran heterogeneidad e agresividad, que conlleva un mal pronóstico para el paciente. A pesar de las mejoras alcanzadas con las estrategias estándar en los últimos 20 años, la prevalencia de supervivencia después de cinco años continua por debajo del 50%. Esta situación pone de manifiesto la necesidad del desarrollo de nuevas estrategias para afrontar este problema. La activación de los receptores de muerte (DR) se ha propuesto como una alternativa a los tratamientos clásicos de quimio- y radio-terapia en distintos tipos de cáncer. En el caso de los NBs, esta aproximación fue descartada por el hecho de que entre un 50 y 70% de ellos no presentan expresión de caspasa-8. A pesar de ello, un grupo significativo de pacientes de NB se podrían beneficiar de un tratamiento que promoviera la muerte a través de la activación de los DR. Se conoce poco de la vía de señalización activada por los DR (especialmente Fas y TNFR1) y de su regulación en NBs, por ello consideramos básico su estudio antes de testar su posible relevancia terapéutica. Dado a que se ha descrito que la citoquina TNFα induce la expresión de Fas en diferentes tipos de cáncer, nosotros decidimos abordar el co-tratamiento de TNFα con FasL como estrategia terapéutica para el tratamiento de NB. Para llevar a cabo nuestro estudio, caracterizamos la señalización intracelular y la inducción de muerte a través de TNFR1 y Fas en ocho líneas celulares clínicamente representativas de NB. Observamos que el tratamiento con TNFα induce la expresión de Fas a través de la activación de la vía NF- κB e sensibiliza a la muerte inducida por FasL. A demás, el tratamiento con TNFα promueve la citotoxicidad de agentes genotóxicos, como cisplatino y etoposido, a través de la activación de la caspasa-8. La caracterización más en detalle que realizamos nos llevó a la conclusión que la heterogeneidad presente en neuroblastomas también se hace patente en los niveles de expresión de Fas y en su modulación por TNFα. La sensibilización a la muerte inducida por FasL, cisplatino o etoposido mediada por TNFα solo se podía observar en aquellos NBs donde TNFα era capaz de inducir la expresión de Fas. En conclusión, nuestros resultados evidencian que TNFα sensibiliza NBs a la muerte inducida por FasL a través de la inducción de la transcripción de FAS mediada por NF- κB. A demás, el pre-tratamiento con TNFα incrementa la muerte inducida por cisplatino y etoposido. Nuestros resultados revelan un nuevo mecanismo que pude mejorar los tratamientos que actualmente se utilizan para la erradicación de los NBs.
Neuroblastoma (NB) is a pediatric solid tumor that accounts for ~15% of all cancer-related deaths in infants. High-risk NBs are hallmarked by a high degree of heterogeneity and aggressiveness, which results in poor patient outcome. Despite the improvement of standard therapies in the last twenty years, five-year survival rates are still below 50%, which impels the development of new treatment strategies for this condition. Activation of death receptors (DRs) has been proposed as an alternative to standard chemo- and radio-therapies for various types of cancer. In NB, this approach has been largely disregarded, possibly due to the silencing of caspase-8 in 50-70% of the cases. Nevertheless, a significant group of NB patients could benefit from treatment that induces cell death through DR activation. Characterization of DR signaling (especially Fas and TNFR1) and their regulation in NB has been limitedly studied, but is a prerequisite for assessing their therapeutic relevance. Given that the cytokine TNFα has been described to induce Fas expression in various types of cancer, we addressed whether TNFα and FasL co-treatment could be a valid therapeutic strategy in NB. For the purpose of the study, TNFR1- and Fas-mediated signaling and cell death induction was characterized in a set of eight clinically representative NB cell lines. TNFα treatment was shown to induce Fas expression through NF-κB-mediated transcription of FAS and primed for FasL-induced cell death. Moreover, TNFα treatment enhanced the cytotoxic effects caused by DNA-damaging agents (i.e. cisplatin and etoposide) through caspase-8 activation. Further characterization revealed that the high degree of heterogeneity between NBs is also visible at the levels of Fas expression and modulation thereof by TNFα. TNFα-mediated priming for FasL-, cisplatin-, and etoposide-induced cell death was only observed for NBs that induced TNFα-mediated Fas expression. In conclusion, our findings reveal that TNFα primes NB for FasL-induced cell death through the NF-κB-mediated induction of Fas expression. Moreover, TNFα pre-treatment enhanced cisplatin- and etoposide-induced cell death. These findings unveil a novel mechanism that could improve the efficacy of treatment regimens currently used for the eradication of NB tumors.

Cell Biology
Ph.D.
Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance. Key clinical features of cumulative trauma disorders include pain, muscle weakness, and tissue fibrosis, although the etiology is still under investigation. Therefore, we first sought to characterize the temporal pattern of altered sensorimotor behaviors and inflammatory and fibrogenic processes occurring in forearm muscles and serum of young adult, female rats performing an operant, high repetition high force (HRHF) reaching and grasping task for 6, 12, or 18 weeks. Palmar mechanical sensitivity, cold temperature avoidance and spontaneous behavioral changes increased, while grip strength declined, in 18-week HRHF rats, compared to controls. Flexor digitorum muscles had increased MCP-1 levels after training and increased TNFα in 6-week HRHF rats. Serum had increased IL-1β, IL-10 and IP-10 after training. Yet both muscle and serum inflammation resolved by week 18. In contrast, IFNg increased at week 18 in both muscle and serum. Given the anti-fibrotic role of IFNg, and to identify a mechanism for the continued grip strength losses and behavioral sensitivities, we evaluated the fibrogenic proteins CCN2, collagen type I and TGFß-1, as well as the nociceptive/fibrogenic peptide substance P. Each increased in and around flexor digitorum muscles and extracellular matrix in the mid-forearm, and in nerves of the forepaw at 18 weeks. CCN2 was also increased in serum at week 18. At a time when inflammation had subsided, increases in fibrogenic proteins correlated with sensorimotor declines. Thus, muscle and nerve fibrosis may be critical components of chronic work-related musculoskeletal disorders. CCN2 and substance P may serve as potential targets for therapeutic intervention, and CCN2 as a serum biomarker of fibrosis progression. TGFß-1 and CCN2 are important mediators of tissue fibrosis by their stimulatory effect on extracellular matrix deposition, with CCN2 functions as a downstream mediator of TGFß-1. Substance P (SubP), a nociceptor-related neuropeptide, has also been linked to tissue fibrosis, although little work has been done to understand whether SubP directly causes fibrotic responses in tenocytes. Therefore, we sought to determine if SubP induces fibroblast proliferation and collagen production via CCN2 signaling directly or through the TGFß-1/CCN2 signaling pathway. We hypothesized that SubP may act directly through CCN2, independently from the TGFß-1/CCN2 signaling pathway, to increase fibroblast proliferation and fibrogenic and extracellular matrix protein production in vitro. To examine this question, we assayed cell proliferation and production of CCN2, TGFB1 and collagen type 1 in vitro using primary tendon fibroblasts (tenocytes) isolated from flexor digitorum tendons, and using rat dermal fibroblasts (RDF). We observed that cells isolated from flexor digitorum tendons that express proteins characteristic of tenocytes (vimentin and tenomodulin) underwent increased proliferation in a dose dependent manner after TGFß-1 treatment, but not SubP treatment, as did RDF cells. TGFß-1 treatment increased CCN2 production in both tenocytes and RDF cells, while SubP induced CCN2 production only in rat tenocytes. Expectedly, TGFß-1 treatment increased collagen expression in each cell type, as did SubP treatment alone using In-cell Western analysis. Interestingly, preliminary data that needs to be repeated showed that SubP treatment of each cell type enhanced TGFß-1 expression, assayed using In-cell Western and traditional western blot analyses. Our findings suggest that both SubP and TGFß-1 have distinct fibrogenic actions on tenocytes and dermal fibroblast and that both may be involved in tendinosis observed in animal models and patients with fibrosis. Inflammatory pain, muscle weakness, and tissue fibrosis are key clinical features of work-related musculoskeletal disorders. So, lastly, we evaluated the effects of therapeutic interventions on behavioral and cytokine changes in muscle, tendon and serum of HRHF rats that performed the reaching and grasping task for 11 weeks. We compared sensorimotor behavioral changes, and flexor digitorum tissue inflammation and fibrosis in rats receiving anti-TNFα therapy prophylactically during the initial training, or anti-TNFα therapy with or without rest as secondary interventions during the HRHF work task. Untreated or saline only treated animals at the end of the initial training period had decreased grip strength, increased mechanical sensitivity, and increased serum and tissue inflammatory cytokines (TNFα, IL-1ß, IL-6 and VEGF), changes prevented by prophylactic anti-TNFα treatment. Regarding the secondary interventions, four weeks of anti-TNFα therapy with or without rest, provided in HRHF task weeks 4-7, was more effective than rest alone for restoring grip strength; no treatments rescued forepaw mechanical sensitivity. Effectiveness of the 4-week anti-TNFα therapy extended to week 11, despite no further drug treatment after week 7, for maintenance of grip strength. Tissue cytokine analysis in week 11 showed that HRHF rats treated with saline had increased IL-18 in serum, muscle and tendon, and trends for increased muscle CCN2. Each treatment, particularly anti-TNF with or without rest, decreased serum and tendon IL-18 and IL-1alpha. Rats receiving combined rest and anti-TNFα therapy also had increased serum IL-10. Thus, similar short-term anti-TNFα therapy may be a potential intervention in WMSDs. These results demonstrate that both Substance P and CCN2 play important roles in the development of fibrosis in muscle and tendon in WMSDs based on our model of repetition reaching and grasping. Using in vitro methods, it was demonstrated that substance P is capable of inducing CCN2 in isolated tenocytes and rat dermal fibroblasts, independent of TGFß-1 signaling, a novel discovery that make suggest new treatments for fibrotic disorders. Finally, anti-TNFalpha treatment successfully prevented behavioral declines and increases in IL-18 in serum and tissues in our rat model when provided during the course of HRHF task performance.
Temple University--Theses

Pseudomonas aeruginosa has shown itself to be particularly adept at colonizing CF lungs and intractable to treatment even with the most aggressive therapy. Failure to control colonization with P. aeruginosa is a major cause of pulmonary debilitation that ultimately leads to almost all of the deaths in CF patients. During bacterial infections, macrophages produce many proinflammatory cytokines, among which TNF-a is believed to be the principal cytokine mediating many of the catastrophic host responses to bacterial infections. Little is known about the virulence factor(s) of P. aeruginosa in the pathogenesis of CF lung infections in terms of TNF-a production. The aim of this study was to determine the virulence factor(s) of P. aeruginosa with regard to TNF-a production. The kinetics of TNF-a production by live P. aeruginosa coincubated with RAW 264.7 macrophages was studied. RAW 264.7 macrophages were challenged with P. aeruginosa at concentrations of 104,106 and 108 CFU/ ml respectively. The production of TNF-a by macrophages increased in a time-dependent fashion with peak levels occurring at about 8 hours, after which the amount of TNF-a decreased, possibly related to TNF-a degradation by proteases from macrophages as well as P. aeruginosa itself. However, the data suggested a possible inverse relationship between the peak values of TNF-a produced and the number of bacteria added into macrophages, in that macrophages challenged by lower numbers of P. aeruginosa produced higher levels of TNF-a, accompanied by a slower rate of decline in TNF-a content. To understand what was occurring with both the P. aeruginosa and the macrophages during the production of TNF-a, the growth of P. aeruginosa and the viability of macrophages during their coincubation were studied in a time course. Depending on the starting bacterial number, the stationary phase of bacterial growth occurred between 8 and 12 hours. Eight hours after challenge with P. aeruginosa, macrophage viability started to decrease. The decrease of macrophage viability suggested P. aeruginosa had cytotoxic effect on macrophages. TNF-a production appeared to be related to both the growth stage of the P. aeruginosa culture and the presence of functioning macrophages. TNF-a production increased during the log growth phase of P. aeruginosa, after which the presence of TNF-a in the supernatants as well as macrophage viability decreased. These findings suggested the growth of P. aeruginosa and the viability of macrophages were important factors in P. aeruginosa induced TNF-a production. Macrophage-bacteria association is the initial step of macrophage phagocytosis. The relationship between the macrophage-bacteria association and TNF-a production by macrophages is not known. The effect of macrophage-P. aeruginosa association on TNF-a production was assessed by increasing their physical contact. The results showed that direct association of RAW 264.7 macrophages with P. aeruginosa significantly reduced TNF-a production, indicating the association of macrophages with P. aeruginosa may down-regulate the function of macrophage TNF-a production. This hypothesis was supported by further experiments which used transwell filter inserts. Transwell filter inserts keep the bacteria from direct contact with macrophages while allowing factors released from the bacteria to come into contact with the macrophages. These experiments demonstrated that the production of TNF-a was higher when P. aeruginosa were incubated in transwell filter inserts than when incubated directly in association with macrophages. These findings suggested that factors released from P. aeruginosa might play a major role in TNF-a production whereas the direct interaction of bacteria with macrophages may suppress TNF-a production. Presuming that released LPS might be the major virulence factor in the production of TNF-a by P. aeruginosa, the inhibition of P. aeruginosa induced TNFa production with different LPS antagonists was investigated. Eight P. aeruginosa LPS specific antibodies were acquired, and none of them were able to block TNF-a production by P. aeruginosa LPS even though they could bind specific epitopes of P. aeruginosa LPS by Western irnmunoblotting and ELISA. Polymyxin B (PMB) was shown to be cytotoxic to RAW 264.7 macrophages in this study and, therefore, is not a suitable antagonist for LPS-induced TNF-a production. However, Rhodopseudomonas sphaeroides diphosphoryl lipid A (RsDPLA) could inhibit P. aeruginosa induced TNF-a production by 70-75%. CEME, an cecropm-melittin hybrid, inhibited more than 90% of P. aeruginosa induced TNF-a production. In addition, inactivation of the released products of P. aeruginosa by heat-treatment resulted in 20% reduction of TNF-a production, indicating that heat stable products such as LPS must be responsible for 80% of TNF-a production by P. aeruginosa. Taken together the results suggest that LPS is the major virulence factor in the production of TNF-a by P. aeruginosa. The reasons behind the predilection and chronic persistence of mucoid P. aeruginosa in respiratory tract of CF patients are not completely understood. The ability of mucoid P. aeruginosa to induce TNF-a in murine alveolar macrophage was, therefore, compared with their nonmucoid counterparts. All the mucoid strains studied induced less TNF-a than their nonmucoid counterparts. The reduced TNF-a production seen with the mucoid form of P. aeruginosa may be attributed to the partial obstruction of LPS release by copious alginate coating around bacteria. This is consistent with the clinical findings that mucoid strains of P. aeruginosa are more suited to chronic rather than to acute respiratory infection in that reduced TNF-a as well as other virulence factors could temper the severity of infections.

碩士
長庚大學
天然藥物研究所
97
Rheumatoid arthritis (RA) is a complex systemic disease. Several factors have been implicated to trigger the mechanisms for the pathogenesis of RA. Although these factors concerning the increased inflammatory responses are well known, but the intracellular signaling pathways implicated in RA are not completely recognized. COX-2 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by TNF-alpha. However, the molecular mechanisms underlying TNF-alpha-induced cyclooxygenase-2 (COX-2) expression in human synoviocytes (HS) which contributes to inflammatory responses in RA were remain unclear. Here, we reported that TNF-alpha-induced COX-2 expression in human synoviocytes (HS), via TNF receptor-activated MAPKs, transactivation of Src, EGFR, PDGFR, PI3K/Akt, and protein kinase C (PKC), transcription factor NF-kappaB and AP-1 signaling pathways. Transfection with siRNA of p42, p38, JNK2, Src, EGFR, AKT and p65 abolished the COX-2 expression induced by TNF-alpha. Accordingly, TNF-alpha-induced activation of COX-2 promoter was also attenuated by pretreatment with inhibitors of MAPKs (U0126, SB202190, and SP600125), transactivation (PP1, AG1478, AG1296 and LY294002), PKCs (GF109203, Ro318220, Gö6976 and Rottlerin), NF-kappaB (Bay117082) and AP-1 (Tanshinone). Consistently, TNF-alpha-stimulated phosphorylation of p42/p44 MAPK, p38 MAPK, JNK, Src, EGFR and Akt was also reduced by pretreatment with U0126, SB202190, SP600125, PP1, AG1478 and LY294002, respectively. Furthermore, TNF-alpha-stimulated NF-kB translocation into nucleus in HS determined by immunofluorescence staining. Previous study shows that COX-2 promoter has an AP-1 binding region. We reported that TNF-alpha-induced mRNA of c-Jun and c-Fos (subunits of AP-1) expression in HS. In addition, TNF-alpha-induced phosphorylation of c-Jun was attenuated by the inhibitors of MAPKs, transactivation and PKCs. MAPKs, transactivation and PKCs pathways regulated AP-1 promoter activities were revealed by promoter assays. Moreover, TNF-alpha-induced PGE2 generation on HS via COX-2 expression was decreased by pretreatment with inhibitors of MAPKs, transactivation and PKCs, Bay11-7082, GR343 and Tanshinone. These results suggest that TNF-alpha induces COX-2 expression in HS via TNF receptor, Src, EGFR, PDGFR, PI3K/Akt, PKCs, MAPKs, transcription factor p65 and AP-1 pathways. Particularly, TNF-alpha-induced COX-2 expression via NF-kB pathway and was not mediated through MAPKs, RTKs and PKCs. These results will provide a new insight into the mechanisms of TNF-alpha in RA. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.

碩士
國立陽明大學
生理學研究所
96
Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine produced primarily by monocytes and macrophages. However, recent studies have indicated that adipose tissue also secreted TNF-α to regulate metabolism. Long-term treatment with TNF-α have been reported to induce lipolysis in 3T3-L1 adipocytes. However, the short-term effect of TNF-α on lipolysis is not well investigated. In addition, many studies demon- strated that TNF-α increased the expressions of inducible nitric oxide synthase (iNOS) and subsequent NO production in adipocytes. But the role of iNOS/NO in TNF-α-induced lipolysis is not clear. The aim of this study was to investigate the short-term regulatory mechanism of TNF-α-induced lipolysis in 3T3-L1 adipocytes. The lipolysis was determined by measuring glycerol release. The iNOS expression and subsequently NO production were detected by Western blot and Griess reagent. The iNOS inhibitor (S-ethyl-ITU), adenylyl cyclase inhibitor (SQ22536) and guanylyl cyclase inhibitor (LY83583) were used to clarify the involvement of iNOS, cAMP and cGMP in TNF-α-induced lipolysis. Transient transfection of iNOS shRNA was performed to further confirm the role of iNOS in TNF-α-induced lipolysis. The phosphorylation of hormone-sensitive lipase (HSL) was measured by immunoprecipitation and Western blot. Our results showed that short-term TNF-α treatment significantly increased lipolysis, iNOS expression, and NO production with the time- and dose-dependent manners. Furthermore, treatment with NO donor, S-nitroso-N- acetylpenicillamine (SNAP), also stimulated lipolysis in 3T3-L1 adipocytes. Moreover, pretreatment with iNOS and guanylyl cyclase inhibitor, but not adenylyl cyclase inhibitor, abolished TNF-α-induced lipolysis in 3T3-L1 adipocytes. Suppression of TNF-α-induced iNOS expression via shRNA significantly reduced TNF-α-induced lipolysis. Also, guanylyl cyclase inhibitor, but not adenylyl cyclase inhibitor decreased TNF-α-induced HSL phosphorylation. Based on these results, we concluded that short-term TNF-α treatment induced lipolysis in 3T3-L1 adipocytes by increasing expression of iNOS/NO, which elevated cGMP accumulation and subsequently induced phosphorylation of HSL.

碩士
國立成功大學
生物科技研究所
102
The cytokine, TNF-α, is an important pro-inflammatory cytokine released by several immune cells during infection or tissue damage and is involved in a diverse range of inflammatory, infectious, and malignant conditions. TNF-α also has many functions in mammal, such as induces inflammation, macrophage activation, cell proliferation, apoptosis, and so on. In the previous studies, the functions of teleost TNF-α of fish were simular as mammal, such as increased expression of inflammatory genes. But, more than one TNF-α isoforms were found in teleost fish. Some of this isoforms was different in their amino acid or DNA sequences. In our privious study, we had found 2 orange-spotted grouper TNF genes: gTNF-α1 and gTNF-α2, they were only 38% amino acid identical. And they expressed different mRNA pattern in peripheral blood leukocytes (PBLs) under LPS stimulated, the mechanism is still unknow. In this study, we try to understand the different function in vivo. We analysis the gene expression of immunostimulant stimulated fish by rea-ltime PCR. The results show (1) tnf-α1 has shown a much stronger expression relative to tnf-α2 after LPS, Poly I:C, vibrio and nervous necrosis virus infected. (2) In various recombinated grouper cytokines stimulated fish, tnf-α1 has shown stronger expression than tnf-α2 after injection rgIL-1β and rgIFN-γ, but tnf-α2 seems to be a more significant factor than tnf-α1 after rgCCL4 injection. However, tnf-α1 and tnf-α2 both down expression after rg-IL-6. (3)TNF-α1 and TNF-α2 have shown the cross activation in each other. Overall, the results indicate that maybe tnf-α1 has more important role on innate immunity, and tnf-α2 is a helper to induce tnf-α1expression.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily (TNFSF) and is as such initially expressed as type II class transmembrane glycoprotein from which a soluble ligand form can be released by proteolytic processing. While the expression of TWEAK has been detected at the mRNA level in various cell lines and cell types, its cell surface expression has so far only been documented for dendritic cells, monocytes and interferon-γ stimulated NK cells. The fibroblast growth factor-inducible-14 (Fn14) is a TRAF2-interacting receptor of the TNF receptor superfamily (TNFRSF) and is the only receptor for TWEAK. The expression of Fn14 is strongly induced in a variety of non-hematopoietic cell types after tissue injury. The TWEAK/Fn14 system induces pleiotropic cellular activities such as induction of proinflammatory genes, stimulation of cellular angiogenesis, proliferation, differentiation, migration and in rare cases induction of apoptosis. On the other side, Toll-like receptor3 (TLR3) is one of DNA- and RNA-sensing pattern recognition receptors (PRRs), plays a crucial role in the first line of defense against virus and invading foreign pathogens and cancer cells. Polyinosinic-polycytidylic acid poly(I:C) is a synthetic analog of dsRNA, binds to TLR3 which acts through the adapter TRIF/TICAM1, leading to cytokine secretion, NF-B activation, IRF3 nuclear translocation, inflammatory response and may also elicit the cell death. TWEAK sensitizes cells for TNFR1-induced apoptosis and necroptosis by limiting the availability of protective TRAF2-cIAP1 and TRAF2-cIAP2 complexes, which interact with the TNFR1-binding proteins TRADD and RIPK1. In accordance with the fact that poly(I:C)-induced signaling also involves these proteins, we found enhanced necroptosis-induction in HaCaT and HeLa-RIPK3 by poly(I:C) in the presence of TWEAK (Figure 24). Analysis of a panel of TRADD, FADD, RIPK1 and caspase-8 knockout cells revealed furthermore similarities and differences in the way how these molecules act in cell death signaling by poly(I:C)/TWEAK and TNF and TRAIL. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for these responses in TNF and TRAIL signaling. Lack of FADD protein abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis. Moreover, we observed that both long and short FLIP rescued HaCaT and HeLa-RIPK3 cells from poly(I:C)-induced apoptosis or necroptosis. To sum up, our results demonstrate that TWEAK, which is produced by interferon stimulated myeloid cells, controls the induction of apoptosis and necroptosis by the TLR3 ligand poly(I:C) and may thus contribute to cancer or anti-viral immunity treatment
Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) ist ein Mitglied der TNF-Superfamilie (TNFSF) und wird als solches anfänglich als Transmembranglykoprotein der Klasse II exprimiert, aus dem eine lösliche Ligandenform durch proteolytische Prozessierung freigesetzt werden kann. Während die Expression von TWEAK auf mRNA-Ebene in verschiedenen Zelllinien und Zelltypen nachgewiesen wurde, konnte ihre Zelloberflächenexpression bisher nur für dendritische Zellen, Monozyten und Interferon-γ-stimulierte NK-Zellen dokumentiert werden. Fibroblast growth factor-inducible-14 (Fn14) ist ein TRAF2-wechselwirkender Rezeptor der TNF-Rezeptor-Superfamilie (TNFRSF) und der einzige Rezeptor für TWEAK. Die Expression von Fn14 wird nach Gewebeverletzung in einer Vielzahl von nicht hämatopoetischen Zelltypen stark induziert. Das TWEAK / Fn14-System induziert pleiotrope zelluläre Aktivitäten, die von der proinflammatorischen Geninduktion über die Stimulierung der Angiogenese, Proliferation und Zelldifferenzierung bis hin zur Zellmigration und in seltenen Fällen zur Induktion von Apoptose reichen. Auf der anderen Seite spielt der Toll-like Rezeptor3 (TLR3), einer der DNA- and RNA-sensing pattern recognition receptors (PRRs), eine entscheidende Rolle in der ersten Verteidigungslinie gegen Viren und eindringende fremde Krankheitserreger und Krebszellen. Polyinosin-Polycytidylsäure-Poly (I: C) ist ein synthetisches Analogon von dsRNA, das an TLR3 bindet, das über den Adapter TRIF / TICAM1 wirkt und zu Zytokinsekretion, NF-B-Aktivierung, IRF3-Kerntranslokation und Entzündungsreaktion führt der Zelltod. TWEAK sensibilisiert Zellen für TNFR1-induzierte Apoptose und Nekroptose, indem es die Verfügbarkeit von schützenden TRAF2-cIAP1- und TRAF2-cIAP2-Komplexen begrenzt, die mit den TNFR1-bindenden Proteinen TRADD und RIPK1 interagieren. Entsprechend der Tatsache, dass diese Proteine auch von Poly (I: C) induziert werden, fanden wir eine verstärkte Nekroptose-Induktion in HaCaT und HeLa-RIPK3 durch Poly (I: C) in Gegenwart von TWEAK (Figure 24). Die Analyse eines Panels von TRADD-, FADD-, RIPK1- und Caspase-8-Knockout-Zellen ergab außerdem Ähnlichkeiten und Unterschiede in der Art und Weise, wie diese Moleküle bei der Zelltodsignalisierung durch Poly (I: C) / TWEAK und TNF und TRAIL wirken. RIPK1 erwies sich als essentiell für die Poly (I: C) / TWEAK-induzierte Caspase-8-vermittelte Apoptose, war jedoch für diese Reaktionen bei TNF- und TRAIL-Signalen entbehrlich. Das Fehlen von FADD-Protein hob TRAIL-, aber nicht TNF- und Poly (I: C) -induzierte Nekroptose auf. Darüber hinaus beobachteten wir, dass sowohl langes als auch kurzes FLIP HaCaT- und HeLa-RIPK3-Zellen vor Poly (I: C) -induzierter Apoptose oder Nekroptose retteten. Zusammenfassend zeigen unsere Ergebnisse, dass TWEAK, das von Interferon-stimulierten myeloischen Zellen produziert wird, die Induktion von Apoptose und Nekroptose durch den TLR3-Liganden Poly(I: C) steuert und somit zur Krebsbehandlung oder antiviralen Immunität beitragen kann

碩士
臺灣大學
微生物與生化學研究所
98
Autoimmune hepatitis is a chronic inflammation in the liver that occurs when the　body‘s immune system attacks the liver. Although the cause of autoimmune hepatitis is not entirely clear, some diseases, toxins and drugs may trigger autoimmune hepatitis insusceptible people, especially in women. Concanavalin A (Con A)-induced hepatitis in　mice is thought to mimic autoimmune hepatitis in human. In this study, we investigated　the role of TNF-α and IL-1 in Con A-induced hepatitis. We found that Con A-triggered　serum ALT and AST production and neutrophil infiltration in liver were markedly　reduced in TNF-α-/- mice, TNFR1-/- mice and IL-1R-/- mice. Furthermore, the number of　CD4+ T cells in liver was also reduced in mice deficient of TNF-α, and TNFR1. Overall　our findings imply that both TNF-α and IL-1 play important roles in mediating Con　A-induced hepatitis by regulating the recruitment of neutrophils into liver. Although the histopathology of early alcoholic liver disease, i.e., steatosis,inflammation, and necrosis, has been well documented, the exact mechanisms of pathogenesis of this devastating disease are still unknown. In this study, we established　the model of acute-alcohol hepatitis to investigate the role of TNF-α in ethanol-induced　acute liver injury. We found that serum ALT levels in TNFR1-deficient mice were　markedly reduced in alcohol-induced liver Injury. Neutrophil infiltration in alcohol-induced liver injury was also reduced in TNFR1-deficient mice. These results　demonstrated that TNF-α was involved in alcohol-induced hepatitis by mediating the recruitment of neutrophils into liver.

碩士
國立臺灣大學
免疫學研究所
85
Activated T lymphocytes undergo apoptosis when their CD3/TCR complex is re-cross-linked in the absence of costimulation, a process referred to as activation-induced cell death (AICD).This process is now believed to play a central role in the maintenance of immune tolerance as well as in the regulation of immune homeostasis.Therefore, it is important to understand how T cell undergoing AICD is controlled.So far it is suggested that AICD can mainly result from the interactions between Fas (APo-1/ CD95) and Fas-ligand (Fas-L).Furthermore, it is demostrated that TNF-alpha can mediate mature T-cell receptor-induced apoptosis through the TNF receptor-2 (TNFR2).However, the role of TNFR2 signaling in AICD has not been directly examined.In this study, the influences of TNFR2 siganling on activated mature T cells are examined.We firsts demostrate that recently activated T cells will undergo apoptosis when their surface TNFR2 molecules are crosslinked by specific antibodies. 48-72h activated CD8 Tcells are most sensitive to TNFR2-induced apoptosis, whereas activated CD4 T cells are resistant to TNFR2 signaling.In addition, the elevation of the expression of TNFR2 molecules is significantly different between CD4 and CD8 T cells after activation.We also find that CD28 costimulation can prevent anti-TNFR2-induced apoptosis of activated T cells.After the induction of TNFR2 signaling, level of IL-4 mRNA expression in survived CD8 T cells are higher than that in control group. Furthermore, we demostrate that soluble TNF can act as the natural ligand of TNFR2 and can cause apoptosis in activated CD8 T cells.These reluts have potential implications for understanding the role of TNFR2 signaling in the regulation of CD8 T cell responses.

碩士
國立陽明大學
微生物及免疫學研究所
102
TNF is a pleiotropic cytokine and considered as central mediators of broad biological activities. These include beneficial effects for the host in inflammation and in protective immune responses against infectious pathogens. On the other hand, TNF also exert host-damaging effects in apoptosis and in autoimmune diseases. Previously we have shown that TLR stimulation can mediate a novel inflammatory amplification through an intrinsic mechanism to augment subsequent TNF signaling in Tnf-deficient MEF cells. IL-1β is also an important inflammatory cytokine, exerts its biological effects by activating the transcription of various responsive genes. The IL-1 receptor type I (IL-1RI), receptor of IL-1β, shares the TIR domain and downstream pathways of TLR, so we would like to know whether IL-1β pretreatment also amplifies subsequent TNF-induced inflammatory response like TLR ligand. Here, we demonstrated acute IL-1β pretreatment sufficiently augment subsequent TNF-mediated response. This signal from IL-1β is MyD88 dependent. In further studies, we showed IL-1β augmented TNF-mediated IL-6 production is not through enhancing the TNF-downstream MAP kinase and NF-κB activities, but through differential NF-κB-regulated transcription. Furthermore, this differential NF-κB-regulated transcription happened on transcription elongation.