Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

In experimentally exposed mice, the environmental contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) produces significant suppression of adaptive immune responses at low doses. However, the underlying biochemical and cellular mechanisms of TCDD-induced immunotoxicity have remained elusive since the identification of these effects nearly 30 years ago. Antigen-presenting cells (APC) constitute various populations of cells essential for the initiation and maintenance of adaptive immune responses, and represent a potential target of TCDD toxicity. Thus, the studies presented here address the ability of TCDD to directly affect APC. The underlying objectives of these studies focus on the investigation of molecular signaling pathways and cellular processes potentially affected by TCDD. In order to eliminate conflicting variables found in vivo, we used ex vivo and in vitro models to address these objectives. Initial studies investigated the status and behavior of the aryl hydrocarbon receptor (AhR), a transcription factor recognized as the principal mediator of TCDD-induced immunotoxic effects, in the two main APC populations, macrophages and dendritic cells (DC). The results demonstrated that both APC populations expressed AhR. However, TCDD induced binding of AhR to dioxin response elements only in macrophages, and not DC. Because TCDD has been shown to alter DC function and survival in vivo, the possibility that TCDD altered other signaling pathways was addressed. Specifically, activation of the transcription factor NF-kB/Rel, integral in DC generation and function, was found to be suppressed by TCDD. This suppression was apparently mediated by a physical association between the AhR and proteins of NF-kB/Rel. Additional studies demonstrated that TCDD enhances the maturation of DC and appears to sensitize DC to apoptosis. These data establish that TCDD directly affects DC on the molecular and cellular levels and support several potential mechanisms of TCDD-induced immunotoxicity. / Graduation date: 2002

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 1999. / Includes bibliographical references. Also available on the Internet.

Dendrimers are hyperbranched macromolecules, with branches-upon-branches architectures, precise constitutions and molecular weights of several kiloDaltons (Figure 1). The dendritic structure remains to be an influential feature in the developments of dendrimer chemistry at large. Organometallic catalysis forms an active area, wherein the dendrimers find a defined importance. A number of dendrimer types have been utilized to study organometallic catalysis that combine the dendritic architectural principles. Chapter 1 of the Thesis summarizes the advances in the dendrimer-mediated catalyses, apart from an overview of the methods adopted to synthesize dendrimers. Chapter 2 describes the synthesis of newer types of larger generation poly(propyl ether imine) (PETIM) dendrimers. The molecular structure of a sixth generation PETIM dendrimer is shown in Figure 2. The PETIM series of dendrimers are synthesized by iterative synthetic cycles of two reductions and two Michael addition reactions. Modifications of the synthetic methods were identified, so as to facilitate the synthesis and purification of the higher generation dendrimers. Formation of the PETIM dendrimers, possessing a tertiary amine as the branch juncture and ether as the linker component, is assessed systematically by routine analytical techniques. The peripheries of these dendrimers possess either alcohols or amines or carboxylic acids or esters or nitriles, thereby opening up possibilities for varied studies. Architecturally-driven effects are searched constantly while integrating dendrimers in wide ranging studies. With knowledge that un-functionalized PAMAM and PPI dendrimers show fluorescence properties, we tested the PETIM dendrimers for their luminescence property. The photophysical properties of PETIM dendrimers presenting esters, alcohols, acid salts, nitriles and amines at their peripheries were studied. The anomalous fluorescence arising from alcohol terminated PETIM dendrimers (Figure 3) was established through a series of experiments. Various experimental parameters including pH, viscosity of the solvents, aging, temperature and concentration were used to assess the photochemical properties of the PETIM dendrimers. It was observed that generations 1 to 5 absorbed in the region of 260-340 nm, in MeOH and in aqueous solutions. Excitation of the OH-terminated dendrimer solutions at 330 nm led to an emission at ~390 nm (Figure 4). Dendrimers presenting esters, acid salts and amines at their peripheries also exhibited a similar excitation and emission wavelengths. An increase in the fluorescence intensity was observed at low pH and with more viscous solvents. Lifetime measurements showed at least two species (~2.5 and ~7.0 ns) were responsible for the emission. The quenching of the fluorescence originating from the PETIM dendrimers by inorganic anions was also established in the present study. The periodate, persulfate, perchlorate and nitrite anions quenched the fluorescence efficiently among several anions tested. An ‘oxygen-interacted moiety’, in addition to altered hydrogen bonding properties of the dendrimers, was presumed contribute to the anomalous fluorescence behavior. Chapter 3 of the Thesis elaborates photophysical studies of several PETIM dendrimers. Incorporation of catalytically active moieties at the peripheries of dendrimers was identified as an important avenue, in order to explore the effect of the dendritic architectures on the catalytic activities of chosen catalytic moieties. In order to assess the effect of the dendritic scaffold, in relation to both numbers and locations of the catalytic units, an effort was undertaken to study the catalytic activities of catalytic units, that are present in varying numbers within one generation. Partial and full phosphine-metal complex substituted three generations of dendritic catalysts were synthesized, by using a selective alkylation as a key step. The number of the primary amine groups led to define the number of phosphine groups at the peripheries. The primary amine groups were, in turn, prepared by a Michael addition of acrylonitrile and hydroxyl groups, followed by a reduction of the nitrile moieties to the corresponding amines. The first and the second generation PETIM dendrimers utilized in this study present up to four and eight hydroxyl groups at their peripheries. A partial etherification was exercised in order to mask few hydroxyl groups, useful to prepare the partially substituted phosphine groups. Subsequent Michael addition of acrylonitrile with remaining hydroxyl groups, to afford the nitrile terminated dendrimers, and a metal-mediated reduction of the nitrile to amine led to the required number of amine functionalized dendrimers. Functionalization of the peripheries with alkyldiphenyl phosphine moieties was conducted through a Mannich reaction of the amines with formaldehyde and diphenyl phosphine. The subsequent metal complexation with Pd(COD)Cl2 afforded a series of phosphine-Pd(II) complexes, for the zero, first and second generation PETIM dendrimers. Figure 5 shows the molecular structures of a partially and a fully substituted second generation dendrimer. Catalytic activities of the dendrimer-Pd(II) complexes were assessed in both Heck and Suzuki coupling reactions. A C-C bond forming reactions were studied, with the series of dendritic-Pd(II) catalysts, using Cs2CO3 as a base and at 40 oC. In an overall observation, it was found that an individual catalytic site showed a considerable increase in the catalytic activity when it was present in multiple numbers than as a single unit within the same generation (Figure 6). Figure 6. Bar diagrams of (a) Heck reaction and (b) Suzuki reaction, employing the dendritic catalysts 1 - 11. The Heck coupling reaction involved tert-butyl acrylate and iodobenzene, and the Suzuki coupling reaction involved phenyl boronic acid and iodobenzene. The observations revealed that: (i) the higher generation dendritic catalysts exhibited higher catalytic activities per catalytic site and (ii) the dendritic scaffold has a role in enhancing the activities of the individual catalytic sites. The catalysis study identified the catalytic activities that occurred when a series of catalysts within a given dendrimer generation was used. Such a study is hitherto unknown and the observations of this study address some of the pertinent queries relating to the efficiencies of multivalent dendritic catalysts. Chapter 4 of the Thesis describes the synthesis and characterization of series of organometallic PETIM dendrimer and studies of their catalytic activities. Studies on solid-supported catalysis present a significant importance in heterogeneous organometallic catalysis. Silica is a prominently utilized heterogeneous metal catalyst support. Functionalization of the solid supports with suitable chelating ligands is emerging as a viable strategy to circumvent not only the pertinent metal catalyst deterioration and leaching limitations, but also to stabilize the metal particles and to adjust their catalytic efficiencies. In exploring heterogeneous organometallic catalysis, functionalization of silica with a first generation phosphinated dendritic amine was undertaken. The synthetic scheme adopted to synthesize dendrimer functionalized silica is shown in Scheme 1. The reaction of the chloropropylated silica 4 with amine 3 was conducted in CHCl3. Complexation of the functionalized silica 5 with Pd(COD)Cl2 led to isolation of Pd(II)- impregnated silica. Scheme 1. Preparation of Pd nanoparticles stabilized by functionalized silica. It was anticipated that the ratio of phosphine to Pd(II) would be 1:0.5, resulting from a bidendate binding of the phosphine ligand to Pd metal. The observed ICP-OES result indicated that all phosphine ligands did not chelate the metal. With the desire to obtain the metal nanoparticles, the metal complex was subjected to a reduction, which was performed by conditioning 5-Pd(II) complex in EtOH. The Pd metal nanoparticle thus formed was characterized by physical methods, and the spherical nanoparticles were found to have >85 % size distribution between 2-4 nm (Figure 7). Analyses of the Pd(0) impregnated in dendrimer functionalized silica were performed using NMR, XPS spectroscopies, elemental analysis and microscopies. Figure 7. Transmission electron micrograph and histogram of 6, obtained after treatment with EtOH. The Pd-nanoparticle stabilized silica was used in the hydrogenation of several α, β-unsaturated olefins. The catalyst recycling experiments were conducted more than 10 times, and no loss in the catalytic activities were observed. Chapter 5 describes the functionalization of the silica support with diphenylphosphinomethyl-derivatized dendritic amine, palladium nanoparticle formation and the catalysis studies. Overall, the Thesis establishes the synthesis of larger generation PETIM dendrimers, studies of their anomalous fluorescence behavior, organometallic catalysis in solution, as well as, in heterogeneous conditions, pertaining to the C-C bond forming reactions and hydrogenation reactions. (For figure, graph and structural formula pl see the pdf file)

Numerous studies have demonstrated that tumour cells have the ability to alter immune function to create an immune suppressed environment. This allows tumour cells to escape immune surveillance and consequently the tumour can progress. Dendritic and T cells have critical roles in immune activation and tolerance and are thus major targets of tumour-mediated immune suppression. Understanding the mechanism(s) by which tumour cells modulate the immune system will facilitate the development of immune system-based therapies for cancer treatments. In this study we sought to determine the nature of, and cellular and molecular mechanisms underlying, changes in immune status during tumour progression using mouse models of prostate cancer. Detailed analysis of the immunological status in a mouse prostate dysplasia model (12T-7slow) revealed that immune suppression accompanied tumour progression. We found that T cells isolated from tumour-bearing hosts were hypo-responsive to antigen stimulation. Furthermore, we demonstrated that CD4+CD25+ regulatory T cells were responsible, at least in part, for this alteration. Anti-CD25 antibody treatment reduced, but did not prevent, tumour growth in either a transplanted prostate tumour model or a spontaneously developing prostate tumour model. In addition, an altered dendritic cell phenotype and an elevated frequency of CD4+CD25+ regulatory T cells were observed within the tumour mass. Similar alterations were observed in the prostate-specific Pten knockout mice which develop advanced prostate adenocarcinoma. Interestingly, evidence of immune activation, such as an increased frequency of activated T cells, was detected in the tumour microenvironment in both mouse prostate tumour models. To identify factors that may play critical roles in the altered immune cell phenotype observed in the tumour microenvironment, a global gene expression profiling analysis was carried out to evaluate the changes in immune-related gene expression patterns. This analysis provided additional evidence for the co-existence of immune suppression and immune activation. Moreover, subsequent analyses suggested that one differentially expressed transcript, interferon regulatory factor 7, and its target genes might be involved in modulating immune cells and/or tumour progression. Taken together, these studies have important implications for designing specific and effective anti-tumour immune therapy strategies that involve manipulation of tumour cells, dendritic cells and regulatory T cells.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Multiple sclerosis is a chronic autoimmune neurological disease characterized by inflammatory cell infiltration and demyelination in the central nervous system (CNS). It is considered to be mediated by Th1 and Th17 immune responses. Experimental autoimmune encephalomyelitis (EAE) is widely used as a mouse model to study MS as it has features and histopathology similar to that of MS. Tolerogenic dendritic cells (DC) are reported to efficiently prevent sensitization for EAE. In this research, we induced tolerogenic DC (DC10) by differentiating them with IL-10. Compared to immature DC, DC10 did not show increased expression of MHC II or the co-stimulatory molecules CD40, CD80 and CD86, and produced low levels of pro-inflammatory cytokines IL-1â, IL-6, and IL-12 but higher levels of IL-10. This is consistent with their possessing a tolerogenic phenotype. We found that three intraperitoneal (i.p.) injections of DC10 successfully inhibited the signs of established, ongoing EAE: DC10 significantly reduced the clinical scores, demyelination and cell infiltration in the spinal cord, as well as the production of IL-4, IL-6, IL-10, IL-17 and IFN-ã by spleen and lymph node (LN) lymphocytes. DC10 treatments did not significantly affect inflammatory cytokine mRNA levels in the CNS. We found that there was higher FoxP3 expression in the CNS in response to DC10 treatments relative to PBS-treated animals. We also found that DC10 treatments significantly enhanced IgG1, IgG2a and IgG2b production and total spleen and LN lymphocyte proliferation following challenge with myelin oligodendrocyte glycoprotein (MOG) antigen. As far as we know, this is the first report showing the successful therapeutic treatment with tolerogenic DC10 of established EAE in mice.

The molecular mechanisms that coordinate postnatal brain enlargement, synaptic properties and cognition remain an enigma. This study demonstrates that neuronal complexity controlled by p21-activated kinases (PAKs) is a key determinant for postnatal brain enlargement and synaptic properties. Double knockout (DK) mice lacking both PAK1 and PAK3 were severely impaired in postnatal brain growth, resulting in a dramatic reduction in brain volume at maturity. Remarkably, the reduced brain was accompanied by minimal changes in total cell count, due to a significant increase in cell density. However, the DK neurons have smaller soma, markedly simplified dendritic arbors/axons and reduced synapse density. Surprisingly, the DK mice were elevated in basal synaptic responses due to enhanced individual synaptic potency, but severely impaired in bi-directional synaptic plasticity. The PAK1/3 action is likely mediated by cofilin-dependent actin regulation because the activity of cofilin and the properties of actin filaments were specifically altered in the DK mice.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.