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

No / 1 In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECS). 2 LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-¿. 3 LPS stimulated a rapid increase in nuclear factor kappa B (NFKB) DNA-binding activity. Preincubation with the NFKB pathway inhibitors, N-¿-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IKB¿, blocked both IRF-1 induction and NFKB DNA-binding activity. 4 LPS and TNF¿ also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-I induction but did not affect GAS/ GAF DNA-binding. 5 Preincubation with TLCK, PDTC or infection with I¿Ba adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6 Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NF¿B isoforms in the formation of the GAS/GAF complex. 7 These studies show that NF¿B plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NF¿B isoforms with the GAS/GAF complex either directly or via an intermediate protein.

Staphylococcus aureus is the causative agent of a diverse array of acute and chronic infections, and some these infections, including infective endocarditis, joint infections, and medical device-associated bloodstream infections, depend upon its capacity to form tenacious biofilms on surfaces. Inserted medical devices such as intravenous catheters, pacemakers, and artificial heart valves save lives, but unfortunately, they can also serve as a substrate on which S. aureus can form a biofilm, attributing S. aureus as a leading cause of medical device-related infections. The major aim of this work was take compounds to which S. aureus would be exposed during infection and to investigate their effects on its capacity to form a biofilm. More specifically, the project investigated the effects of serum, and thereafter of catheter lock solutions on biofilm formation by S. aureus. Pre-coating polystyrene with serum is frequently used as a method to augment biofilm formation. The effect of pre-coating with serum is due to the deposition of extracellular matrix components onto the polystyrene, which are then recognized by MSCRAMMs. We therefore hypothesized that the major component of blood, serum, would induce biofilm formation. Surprisingly, serum actually inhibited biofilm formation. The inhibitory activity was due to a small molecular weight, heat-stable, non-proteinaceous component/s of serum. Serum-mediated inhibition of biofilm formation may represent a previously uncharacterized aspect of host innate immunity that targets the expression of a key bacterial virulence factor: the ability to establish a resistant biofilm. Metal ion chelators like sodium citrate are frequently chosen to lock intravenous catheters because they are regarded as potent inhibitors of bacterial biofilm formation and viability. We found that, while chelating compounds abolished biofilm formation in most strains of S. aureus, they actually augmented the phenotype in a subset of strains. We investigated the molecular basis of this phenomenon. Deletion and complementation analysis and thereafter antibody based inhibition assays confirmed a functional role for the surface adhesin clumping factor B as the causative determinant associated with the increased biofilm phenotype. Finally, we investigated the regulation of clumping factor B-mediated biofilm formation and the basis for the strain dependence. Regulation was determined to occur via two novel post-translational networks- one affecting ClfB activity, mediated by Ca2+ binding to the EF-Hand domain, and the other affecting protein stability, mediated by the enzymatic activity of the metalloprotease-aureolysin. Polymorphisms within the aureolysin gene sequence, between strains, was identified as the basis for some strains forming robust biofilms within chelated media versus other than do not exhibit this phenotype.

The complement system plays an important role in innate and adaptive immunity. Central to all complement activities is the function of complement component 3 (C3). C3 contains a C-terminal extension of ~150 residues known as the NTR (or C345C) domain. To address the role of the NTR domain in binding and functional activities of C3, a C3/C5 chimera was engineered, in which the NTR domain of C3 was replaced by the homologous domain of the closely related protein C5. Functionally, the C3(C5NTR) was devoid of classical pathway-dependent hemolytic activity and deficient in factor H- and CR1-dependent factor I cleavability. Direct binding SPR assays, using chip bound methylamine treated His6-tagged C3(C5NTR), showed a complete loss of C5 binding while retaining wild type binding with CR1, factor H and factor B. These results present the first evidence for a major C5 binding site within C3 NTR.

The complement system plays an important role in innate and adaptive immunity. Central to all complement activities is the function of complement component 3 (C3). C3 contains a C-terminal extension of ~150 residues known as the NTR (or C345C) domain. To address the role of the NTR domain in binding and functional activities of C3, a C3/C5 chimera was engineered, in which the NTR domain of C3 was replaced by the homologous domain of the closely related protein C5. Functionally, the C3(C5NTR) was devoid of classical pathway-dependent hemolytic activity and deficient in factor H- and CR1-dependent factor I cleavability. Direct binding SPR assays, using chip bound methylamine treated His6-tagged C3(C5NTR), showed a complete loss of C5 binding while retaining wild type binding with CR1, factor H and factor B. These results present the first evidence for a major C5 binding site within C3 NTR.

No description available.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Genetic as well as environmental factors are believed to be of importance in the etiology of rheumatoid arthritis (RA). There are a number of previous studies of genetic markers in RA, but so far no genetic linkage and only a few associations have been found. Of the associations only one (with the HLA antigen DR4) appears to be well documented. In most previous association studies the patients have not been divided according to sex and family history of RA. In this investigation the HLA antigens A, B and DR and five serum protein systems (Bf, C3, Pi, Hp and Tf) were studied in patients with erosive rheumatoid arthritis (RA), from northern Sweden. Special attention was paid to variations in the strength of associations accord­ing to sex and family history of polyarthritis. The following results were found:  The frequency of the HLA antigen B27 was significantly increased in the North-Swedish population (16.6%) and among patients with a family history of polyarthritis (42.6%). In agree­ment with previous investigations a significantly increased frequency of the DR4 antigen was found in the RA patients.  In the properdin factor B (Bf) system the S phenotype was found to be significantly in­creased in male patients and in patients with a family history of polyarthritis, more severe form of RA and high titres of rheumatoid factor.  No significant differences with respect to phenotype or gene frequencies were found in the C3 complement system. Thus, the association between RA and C3 found in previous investiga­tions was not confirmed.  A significant increase of rare alpha-1-antitrypsin (Pi) types (MS, MZ, MF and SZ) was found among RA patients. However, the increase concerned mainly Z heterozygotes and was more strongly pronounced among male patients.  In the haptoglobin system a significant increase of the Hp^ gene and the Hp2-2 type was found among patients with a family history of polyarthritis, more pronounced among males.  A significant increase of the transferrin gene and of the 2 type was found among male RA patients, more pronounced among patients with a family history of polyarthritis. In 6 out of 8 gene loci studied significant associations were found, which is in agreement with a multifactorial etiology of RA. The results were largely in agreement with the hypothesis that associations would be expected to be stronger in males and in patients with a family history of polyarthritis. A notable finding was the high frequency of first degree relatives (around 40%) with symmetric peripheral polyarthritis of which more than 70% had a diagnosis of RA verified by hospital records. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1985, härtill 6 uppsatser.</p> / digitalisering@umu

Inflammation involves a complex interplay of immunological responses in reaction to an infection from invading pathogens and to tissue injury. However, if the inflammatory stimulus is not eliminated or if there is an aberrant response, chronic inflammation and disease can result. Most inflammatory diseases are targeted by drugs that provide only symptomatic relief by reducing the associated pain and swelling, without combating the underlying cause and progression of the disease. The purpose of this thesis was to investigate potential drug targets in different sub-cellular compartments to learn more about inflammatory pathways and the consequences of inhibiting them using different classes of small-molecule agents. Chapter 1 is a brief overview of inflammation, outlining differences between acute and chronic inflammation in relation to normal and aberrant immune responses. Major drug classes that are currently in use are briefly discussed, and some potential new inflammatory drug targets in the nucleus, cytoplasm, plasma membrane and serum that are to be investigated in the thesis are described. Chapter 2 addresses histone deacetylase (HDAC) enzymes that modulate gene transcription, and investigates the regulatory potential of HDACs and their inhibitors in macrophages. Mainly murine macrophages were investigated, but preliminary data on human macrophages is also presented. Four kinds of small-molecule inhibitors of HDAC enzymes are described in relation to their capacity to exhibit pro- or anti- inflammatory gene expression. Broad-spectrum inhibitors, those that do not discriminate between different isoforms of HDACs, were found to induce expression of a number of pro-inflammatory mediators. This was also the case for compounds that selectively inhibit class-I HDACs. On the other hand, compounds that are known to selectively inhibit class-II HDACs did not regulate pro-inflammatory genes and could be classified as anti-inflammatory based on their profiles of gene expression. An inhibitor of class-III HDACs, the sirtuins, was also briefly investigated for anti-inflammatory gene expression. This separation of the pro- and anti-inflammatory responses suggested that specific inhibitors of class II HDACs might be promising for treating inflammatory conditions. Chapters 3 and 4 discuss a group of extracellular serum proteins that are produced through the complement pathway. Complement proteins circulate in plasma while some are formed on membranes through serine protease action, and effect both innate and adaptive immune responses. Chapter 3 deals with the alternative pathway (AP) of complement activation, in particular with the protease Factor B which plays an important role as part of a protein complex (C3 convertase) in the initiation and propagation of complement. Factor B is an inactive zymogen at pH 7, but is catalytically active at alkaline pH. Substrate-based inhibitors were developed for Factor B using a chromogenic assay previously optimised in our lab. Over 60 peptide aldehydes were evaluated, with two potent inhibitors of Factor B studied further, and were found to inhibit C3 convertase by blocking cleavage of the native substrate C3 as measured by SDS-PAGE. Chapter 4 examined the properties of a homologue of Factor B found in the classical pathway (CP), the protease C2. C2 was found herein to be an inactive zymogen at pH 7. Under alkaline conditions C2 cleaved both C3 and short chromogenic peptide substrates. Substrates of 7- or 8-residues, corresponding to those in the native substrate C3, were optimally processed. C2 was inhibited by inhibitors identified in Chapter 3, which also inhibited formation of the different C3 convertase formed through the CP, as well as preventing formation of the terminal membrane attack complex (MAC) as measured by an immobilized ELISA and haemolysis assay. The selectivity of small-molecule peptidic inhibitors for different serine proteases was investigated. Chapter 5 introduces two other unrelated inflammatory proteins which have been incompletely studied herein. Protease-activated receptor 2 (PAR-2) is a cell surface protein that spans the membrane like other G-protein coupled receptors. PAR-2 has been implicated in inflammatory and proliferative diseased states, though its precise roles are still unclear. Here, PAR-2 mRNA expression was measured for different human cancer and immune cells. In macrophages, PAR-2 mRNA expression was inducible by HDACi, indicating that PAR-2 is negatively regulated by HDAC enzymes. The receptor was present and functional on the surface of HDACi-treated macrophages, permitting evaluation of PAR-2 agonists and antagonists. Caspase-1 is a cytosolic cysteine protease responsible for cleaving proIL-1β and proIL-18 into their active forms that are then secreted from cells as pro-inflammatory cytokines. Using monocytic cells, novel inhibitors of caspase-1 were developed and found to dose-dependently decrease levels of IL-1β released by the cell. Inhibition of caspase-1 may be useful for modulating circulating levels of the inflammatory cytokine IL-1β. The investigation of different prospective inflammatory targets in different locations of the cell has provided a lateral overview of inflammatory mechanisms and drug intervention strategies. Specific inhibitors have also been identified for these targets; HDACs in the nucleus, caspase-1 in the cytosol, PAR-2 and MAC on the cell surface, C2 and Factor B and C3 convertase in plasma. This unconventional study of the inflammatory network provides a different viewpoint of inflammatory proteins as drug targets.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.