Glu-Leu-Arg (ELR)-CXC chemokine-mediated neutrophil migration and activation plays a key role in many inflammatory diseases. Dysregulated neutrophil activation often leads to inflammatory responses such as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS).<p> Previously, we generated a bovine drug (i.e., bovine CXCL8(3-74)K11R/G31P, bG31P) by mutating the first two amino acids at the beginning of the N-terminus of bovine CXCL8/IL-8 and later substituting Arg for Lys11 and Pro for Gly31. Bovine G31P was shown to be a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle & guinea pigs, but a human equivalent thereof would be of significantly more use in human medicine. Published studies on the structure and function of human CXCL8 suggest that human CXCL8(3-72)K11R/G31P (i.e., hG31P) would not be a particularly effective chemokine antagonist. Thus, development of a humanized form of bG31P became a primary goal. I first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P and generated a human-bovine chimeric G31P (hbG31P; i.e., bCXCL8(3-44)K11R/G31P-hCXCL8(45-72)). I also made substitutions at each remaining human-discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T) within the 5 half of the hbG31P cDNA. The results showed that hbG31P and its analogues blocked CXCL8-induced human neutrophil chemotactic responses, reactive oxygen intermediate (ROI) release, and intracellular calcium flux. Humanized bovine G31P was also shown to significantly block pulmonary neutrophilic pathology in a guinea pig model of airway endotoxemia.<p> As bG31P, hbG31P and its further humanized forms showed essentially equivalent ELR-CXC chemokine antagonist activity, Dr. Fang Li, Ms Jennifer Town and I then generated a fully human form of bG31P, hG31P. <i>In vitro</i>, hG31P was shown to effectively inhibit CXCL-1-, -5-, and -8-induced neutrophil chemotactic responses, intracellular Ca2+ flux, and ROI release. Human G31P also desensitized heterologous G protein-coupled receptors (GPCR) including bacterial peptides (e.g., N-formyl-methionine-leucine-phenylalanine, fMLP), anaphylatoxin (e.g., complement 5a, C5a), lipid mediators (e.g., leukotriene B4, LTB4; platelet-activating factor, PAF) receptors. Moreover, hG31P, in a dose-dependent manner suppressed CXCL1 and CXCL8 expression by LPS-challenged airway epithelial cells and reversed the anti-apoptotic influence of ELR-CXC chemokines on neutrophils. <i>In vivo</i>, hG31P was significantly effective in blocking the pathology associated with airway endotoxemia, aspiration pneumonia, and intestinal ischemia and reperfusion injury, including neutrophil recruitment (70-95% reduction) into, and activation within, the airways or gut, chemokine or cytokine expression, and pulmonary vascular complications. The blockade of neutrophil recruitment by hG31P in aspiration pneumonia animals did not increase airway bacterial growth. The G31P treatment was protective in both mesenteric (i.e., local) and remote organ injury. These findings suggest that hG31P is not only a potent neutrophil antagonist, but an effective blocker of other inflammatory responses. These comprehensive anti-inflammatory effects indicate that hG31P could potentially provide a viable therapeutic approach for inflammatory diseases such as ALI /ARDS.

Sialyltransferases (STs), which catalyze the sialylation reaction by adding sialic acids to the terminal positions of oligosaccharide of glycoproteins and glycolipids, are over-expressed in cancer cells and associated with cancer metastasis. Until now, ST inhibitors are not applicable for clinical use because of poor cell permeability, although showing potent effect in vitro. In this study, we synthesize a lithocholic acid-based ST inhibitor AL10 and test its anti-metastatic effect. Overexpression of £\-2,3-ST is found in highly metastatic A549 and CL1-5 lung cancer cells. Confocal microscopy demonstrates that AL10 is cell permeable and may attenuate total sialylation on cell surface. AL10 has no cytotoxicity but inhibits adhesion, migration, actin polymerization and invasion of A549 and CL1-5 cells in vitro. Inhibition of adhesion and migration by AL10 is associated with reduced sialylation of beta1 integrin. In addition, activation of the beta1 integrin downstream signaling molecule focal adhesion kinase is also attenuated. More importantly, AL10 suppresses lung metastasis in vivo and this effect may be linked with reduced sialylation of the chemokine receptor CXCR4 which has been found to play a critical role in organ-specific metastasis. Serum biochemical assay indicates that AL10 does not affect liver and kidney functions of experimental animals. Taken together, we conclude that AL10 is an effective sialyltransferase inhibitor and exerts anti-metastatic effect in vivo via suppression of sialylation of beta1 integrin and CXCR4.

Würzburg, Univ., Diss., 2009

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. The role of cystein-x-cystein (CXC) chemokine ligand 10 (CXCL10, formerly interferon-y-inducible protein 10) in CVB3-induced myocarditis is unknown. To explore the contribution of CXCL10 to CVB3-induced myocarditis, we performed functional analyses using newly generated transgenic mice with cardiac-specific CXCL10 overexpression (Tg) and CXCL10 knock out (KO) mice. The mRNA levels of CXCL10 peaked in the myocardium at day 3 post-infection prior to immune infiltration, suggesting that mainly resident cells of the heart are the sources of CXCL10. Indeed, we showed that CXCL10 can be induced by IFN-y but not by CVB3 infection in cultured cardiomyocytes. Further, a transgenic mouse model with cardiac-specific overexpression of CXCL10 was generated. CXCL10 Tg mice had spontaneous infiltrations of mononuclear cells with limited mRNA upregulation of IFN-y and IL-10, which were not sufficient to cause the impairment of cardiomyocyte or cardiac function. Following CVB3 infection, the viral titre in the mouse hearts inversely correlated with the levels of CXCL10 at day 3 post-infection. Further, the decreased virus titers in the CXCL10 Tg mouse hearts led to reduced cardiac damage indicated by low serum cTnI levels and improved cardiac functional performance and vice versa in the KO mice. This antiviral ability of CXCL10 may be through increased recruitment of natural killer (NK) cells to the heart and increased IFN-y expression early post-infection. At days 7 and day 10 post-infection with massive influx of mononuclear cells, the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells as well as their associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines did not alter the viral clearance and mouse survival. Our data demonstrate for the first time that CXCL1 0 confers the protection to the heart during the early course of CVB3 infection, which may be primarily attributed to NK cell recruitment to the site of infection. This data suggest that CXCL10 is an important player in the orchestrated action of a group of cytokines and chemokines in combating against the CVB3-induced myocarditis in the early phase of infection.

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. The role of cystein-x-cystein (CXC) chemokine ligand 10 (CXCL10, formerly interferon-y-inducible protein 10) in CVB3-induced myocarditis is unknown. To explore the contribution of CXCL10 to CVB3-induced myocarditis, we performed functional analyses using newly generated transgenic mice with cardiac-specific CXCL10 overexpression (Tg) and CXCL10 knock out (KO) mice. The mRNA levels of CXCL10 peaked in the myocardium at day 3 post-infection prior to immune infiltration, suggesting that mainly resident cells of the heart are the sources of CXCL10. Indeed, we showed that CXCL10 can be induced by IFN-y but not by CVB3 infection in cultured cardiomyocytes. Further, a transgenic mouse model with cardiac-specific overexpression of CXCL10 was generated. CXCL10 Tg mice had spontaneous infiltrations of mononuclear cells with limited mRNA upregulation of IFN-y and IL-10, which were not sufficient to cause the impairment of cardiomyocyte or cardiac function. Following CVB3 infection, the viral titre in the mouse hearts inversely correlated with the levels of CXCL10 at day 3 post-infection. Further, the decreased virus titers in the CXCL10 Tg mouse hearts led to reduced cardiac damage indicated by low serum cTnI levels and improved cardiac functional performance and vice versa in the KO mice. This antiviral ability of CXCL10 may be through increased recruitment of natural killer (NK) cells to the heart and increased IFN-y expression early post-infection. At days 7 and day 10 post-infection with massive influx of mononuclear cells, the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells as well as their associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines did not alter the viral clearance and mouse survival. Our data demonstrate for the first time that CXCL1 0 confers the protection to the heart during the early course of CVB3 infection, which may be primarily attributed to NK cell recruitment to the site of infection. This data suggest that CXCL10 is an important player in the orchestrated action of a group of cytokines and chemokines in combating against the CVB3-induced myocarditis in the early phase of infection.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Streptococcus pneumoniae (the pneumococcus) remains a major cause of morbidity and mortality worldwide, particularly in young children and the elderly. It is responsible for a spectrum of diseases ranging from otitis media, to potentially fatal conditions such as pneumonia and meningitis, and is estimated to cost health services billions of dollars each year. The interaction of S. pneumoniae with the host generally begins in the nasopharynx, and invasive disease is almost invariably preceded by nasopharyngeal colonisation. In some circumstances, S. pneumoniae may translocate from the nasopharynx to the lungs where pneumonia can develop, and inflammation is believed to play a role in this process. The presence of pneumococci in the lungs also triggers an inflammatory response, which is important for clearance of the bacteria. However, a prolonged inflammatory response leads to tissue damage, and is linked with a poor prognosis of disease. It has been shown that respiratory epithelial cells are able to play an active part in the response to respiratory pathogens by releasing chemokines that are responsible for neutrophil recruitment, and it has recently been shown that infection of type II pneumocytes with S. pneumoniae leads to the release of interleukin (IL)-8. In order to determine the role of specific pneumococcal factors in eliciting a CXC chemokine response from type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562), monolayers of these cells were infected with wild type (WT) S. pneumoniae 039, or mutants deficient in choline binding protein A (CbpA), pneumococcal surface protein A (PspA), or pneumolysin (Ply), and the CXC chemokine mRNA response was measured by real-time RT-PCR. Release of IL-8 was also measured by ELISA. In response to WT D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA, and IL-8 protein. This response was increased 2-fold when a CbpA-negative (ACbpA) mutant was used to infect cells, suggesting that CbpA may have an inhibitory effect on the CXC chemokine response of these cells. Further investigatiDn demonstrated that this activity is dependent on the N-terminal region of CbpA and that all three N-terminal domains are required for this effect, as deletion of any one of these domains had the same effect on the CXC chemokine response as removing CbpA altogether. Infection with a PspA-negative mutant (APspA) led to a 2-fold decrease in the CXC chemokine response of A549 cells, compared to infection with WT D39 at 2 h, but no difference was seen in the response of Detroit-562 cells to this mutant compared to WT D39. Thus, PspA appears to have the ability to stimulate an early CXC chemokine release from A549 cells. Deletion of the first of 2 regions of the N-terminal a-helical domain of PspA reduced the ability of S pneumoniae to elicit a chemokine response to the same degree as removing PspA altogether, indicating that it is this region that is responsible for the chemokine inducing ability of PspA. Ply appeared to have no effect on the CXC chemokine response of A549 cells with no obvious difference seen in the response of these cells to APly compared to WT D39. However, infection of Detroit-562 cells with APly led to a 2-fold decrease in IL-8 mRNA and protein release compared to WT D39. Using D39 strains producing mutant forms of Ply with reduced cytotoxicity and/or complement activating abilities, the role of the cytotoxic activity of Ply was demonstrated to be important in generation of a chemokine response from both cell lines. Infection of A549 or Detroit-562 cells with mutants producing Ply with only 0.02% or 0.1% haemolytic activity led to a 2-fold decrease in IL-8 release compared to that elicited by WT D39. The complement activating ability of Ply also appeared to be important in the generation of a CXC chemokine response from A549 cells. Cells infected with a mutant that produced Ply with no complement activating ability released significantly less IL-8 than cells infected with WT D39. This activity of Ply did not appear to have an effect on the CXC chemokine release of Detroit-562 cells. Thus all three virulence factors investigated had some role in the ability of S. pneumoniae to generate a CXC chemokine response from respiratory epithehal cells, although their roles and the cell lines that were affected differed. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1225410 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2005

University, Diss., 2006--Frankfurt (Main). / Erscheinungsjahr an der Hauptitelstelle: 2005.

Universiẗat, Diss.--Jena, 2007.

Zugl.: Giessen, Universiẗat, Diss., 2007.

Zugl.: Giessen, Universiẗat, Diss., 2007.