Aim: Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques, and this may correlate with serum markers of plaque instability and inflammation. Method: Circulating EMPs and inflammatory markers were measured in twenty healthy controls and seventy patients undergoing carotid endarterectomy. EMPs were quantified using flow cytometry. Bioplex assays profiled systemic inflammatory and bone-related proteins. Immunohistological analysis detailed the contribution of differentially-regulated systemic markers to plaque pathology. Alizarin red staining showed calcification. Results: EMPs were significantly higher in patients with carotid stenosis (greater than or equal to 70%) compared to controls, with no differences between asymptomatic vs symptomatic patients. Asymptomatic patients with unstable plaques exhibited higher levels of EMPs compared to those with stable plaques, with a similar trend observed in symptomatic patients. CXCL9 and SCGF-β were significantly elevated in asymptomatic patients with unstable plaques, with IL-16 and macrophage inhibitory factor significantly elevated in the stable plaque group. CXCL9, CTACK and SCGF-β were detected within all plaques, suggesting a contribution to both localised and systemic inflammation. Osteopontin and osteoprotegerin were significantly elevated in the symptomatic vs asymptomatic group, while osteocalcin was higher in asymptomatic patients with stable plaque. All plaques exhibited calcification, which was significantly greater in asymptomatic patients. This may impact on plaque stability. Conclusions: Circulatory EMP, CXCL9 and SCGF-β levels are raised in asymp-tomatic patients with unstable plaques, which could be important in identifying patients at most benefit from intervention.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1284103 / Thesis (PhD)-- School of Medicine, 2005

The ability of a cell to respond to extrinsic stimuli critically depends on its ability to regulate specific intracellular protein - protein interactions in a reversible manner and allow the temporal - spatial characteristics of the signal to be accurately transduced to downstream targets. Growth factor and cytokine receptors provide a link by which extracellular stimuli are propagated within the cell to accomplish specific cellular functions. Ligand - stimulation of these receptors activates cascades of intracellular events that transduce the signals that lead to a variety of cellular responses. The specificity of these signals and the fidelity with which they are communicated within the cell are critical for the fate of an organism as deregulation or misbalance of signalling networks is commonly associated with a wide range of pathologies and diseases. Cytokines are important regulatory proteins that regulate diverse cellular functions through their ability to bind to specific cell surface receptors. Most cytokines are pleiotropic effectors that regulate multiple cellular functions. For example, many cytokines can regulate diverse biological activities such as cell survival, proliferation and differentiation and in many cases these different biological activities can be independently regulated. The regulation of pleiotropic biological responses is mediated through the modulation of multiple intracellular signalling pathways. These pathways often present a high level of redundancy in terms of the biological functions that they control. However, pleiotropic cytokines have the ability to independently activate signalling pathways that lead to the regulation of specific biological functions such as survival, proliferation, differentiation or activation. The molecular mechanisms by which cytokines can regulate pleiotropic biological responses from the activation of a limited number, often redundant, of intracellular signalling pathways have not been fully resolved and remains one of the most important unanswered questions in cell biology. In particular, proteins and molecular mechanisms responsible for specifying different biological responses remain largely unidentified. In many cases, activation of multiple signalling pathways and integration of the signals they transduce is needed in order to modulate a biological function. One important mechanism by which signalling pathways are assembled within the cell is through the action of protein scaffolds that contain phosphotyrosine ( e.g. SH2, PTB ) or phosphoserine / threonine ( e.g. 14 - 3 - 3, WW, FHA, PBD, BCRT ) binding modules. Interestingly, although phosphotyrosine and phosphoserine / threonine - dependent signalling pathways are highly integrated within the cell, scaffold proteins containing both phosphotyrosine and phosphoserine or phosphothreonine - binding domains ( i.e. SH2 / PTB and WW / FHA / PBD / BCRT ) have not been identified. The broad aim of this thesis is to study the fundamental molecular mechanisms by which cytokines, through the binding of cell surface receptors, are able to activate and integrate signalling pathways that regulate and specify cellular responses. In particular, these studies examine the role of the 14 - 3 - 3 family of adaptor proteins in the assembly of signalling networks that couple the activated receptors of the haematopoietic cytokines IL - 3, IL - 5, and GM - CSF to downstream signalling targets and specific cellular functions such as survival and proliferation. The specific aims of this thesis are to examine the composition, molecular mechanisms of assembly and functional roles of signalling complexes that use the adaptor or scaffold protein 14 - 3 - 3 and are important for signal transduction in response to GM - CSF. This work shows that the phosphoserine / threonine - binding scaffold protein 14 - 3 - 3 ζ, previously reported to bind to Ser585 of the GM - CSF receptor, undergoes tyrosine phosphorylation. Using a panel of 14 - 3 - 3 ζ mutants a particular tyrosine residue, Tyr179, was found to be critical for the binding of the SH2 domain of Shc, the assembly of a PI3K signalling complex, the activation of the Akt / PKB signalling pathway and the control of cell survival in response to GM - CSF stimulation. Tyr179 of 14 - 3 - 3 ζ was also found to be important for specifying GM - CSF - mediated biological responses as it was found to play an important role in the control of cell survival versus cell proliferation. Furthermore, it was found that 14 - 3 - 3 ζ is able to simultaneously bind to Ser585 of the GM - CSF receptor and recruit Shc and PI3K through Tyr179, thus integrating phosphoserine / threonine and phosphotyrosine / dependent signalling pathways. The findings described in this thesis helped to identify a novel mechanism by which cytokine receptors achieve both integration in signalling and specificity in biological outcomes. The discovery that phosphoserine / threonine - binding proteins ( i.e. 14 - 3 - 3 ) are themselves tyrosine phosphorylated and able to recruit phosphotyrosine - binding molecules provides a new insight into how intracellular signal integration is achieved. Understanding how signal transduction is carried out within the cell is paramount to successful drug development in many therapeutic areas. The new insights in GM - CSF signalling provided by this work may help to successfully develop treatments to target diseases such as asthma, rheumatoid arthritis and leukaemia, where GM - CSF appears to play a pathogenic role. / Thesis (Ph.D.)--School of Medicine, 2006.

Preterm birth, defined as birth prior to 37 completed weeks gestation, is a serious health concern. Despite advances in health care screening and interventions, the rate of preterm birth in the United States has risen more than 30 percent since 1981. In 2006, Menon and associates reported the first multilocus genetic interaction in three single nucleotide polymorphisms predictive of spontaneous preterm birth in a population of White mothers. A gene association study using a case-control design was conducted to determine whether the results of the Menon study were also true for Black women and their infants. The case group included 22 preterm mothers and 22 preterm infants. The control group included 106 term mothers and 108 term infants. Study variables included high-risk and low-risk genetic combinations of the three single nucleotide polymorphisms. Birth certificate applications were reviewed to determine subject eligibility. Blood samples obtained by hospital personnel were sent to a laboratory for genotyping. It was predicted that there would be no statistically significant difference in the occurrence of the high-risk genetic patterns in the preterm Black mothers or their infants. Data analysis suggested that the multilocus genetic combinations reported by Menon were not predictors for preterm birth in Black mothers or their infants. Further study is recommended to identify genetic variations predictive of preterm birth across ethnic groups.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1a, IL-1b, IL-6, IL-8 and TNF-a were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the aforementioned equine genes.

Severe bloody diarrhea and subsequent serious post-diarrheal illnesses, including the hemolytic uremic syndrome and central nervous system complications, may develop following infections with Shiga toxin (Stx)-producing bacteria. The cytotoxic actions of Stxs destroy the microvasculature of organs, preventing function. A role for the cytokines tumor necrosis factor-alpha (TNF-[alpha]) and interleukin-1 beta (IL-1[beta]) in exacerbating disease may lie in their ability to up-regulate the Stx receptor, Gb3, on endothelial cell surfaces. A main source of proinflammatory cytokines is the macrophage, thus leading us to utilize the monocytic/macrophage-like cell line, THP-1, as a model for cytokine production in Stx pathogenesis. In addition to treating THP-1 cells with purified Stx1, cells were also treated with lipopolysaccharides (LPS), since bacterial LPS are known to be potent inducers of cytokines, and may be present during infection. Undifferentiated THP-1 cells are sensitive to Stx1 and do not produce TNF-[alpha] or IL-1[beta], while differentiated THP-1 cells, a better model for resident tissue macrophages, are less sensitive to Stx1 and produce TNF-[alpha] and IL-1[beta]. Prolonged expression of TNF-[alpha] mRNA over a 12 h time course experiment led us to inquire whether the extended elevation of transcripts involved Stx1induced mRNA stability. Our data suggest that the presence of Stx1 increases the stabilities of TNF-[alpha] and IL-1[beta] transcripts. In contrast to TNF-[alpha], the level of secreted IL-1[beta] protein does not correlate with the level IL-1[beta] mRNA, suggesting an alteration of post-translational processing and/or secretion of IL-1[beta]. Differentiated THP-1 cells produce chemokines in response to Stx1 and/or LPS treatments. Chemokines may enhance the destruction of tissue cells during an infection by mediating an inflammatory cell influx. Comparison of cytokine and chemokine mRNA and protein kinetics suggests that the regulation of expression may differ between individual cytokines and chemokines. Extension of experimental time courses demonstrated THP-1 cell sensitivity to killing by Stx1, especially in the presence of LPS. Further experiments revealed that undifferentiated and differentiated THP-1 cells were induced to undergo apoptosis following treatment with Stx1, LPS, and Stx1+LPS, and that caspase activation was involved. Collectively, these results allowed us to propose a model of the role of macrophages in Stx1 pathogenesis.

The replication of equine infectious anemia virus (EIAV) in macrophages not only leads to cell death, but also to the induction of a variety of cytokines that may affect immune function. Cytokine production may be responsible for the fever, anorexia, hemorrhages, lethargy or thrombocytopenia seen in the acute and chronic phases of equine infectious anemia (EIA). The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. We have extended studies of virulent and avirulent EIAV clones by examining the effects of Env proteins on cytokine expression in equine monocyte-derived macrophages (EMDM) using EIAV17, EIAV19, EIAV17SU, and EIAV17TM viruses. In the current studies a set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1a, IL-1b, IL-6, IL-8 and TNF-a were validated using QPCR primers and probes which were generated for the aforementioned equine genes.