On the induction of inflammatory reactions by Streptococcus pyogenes

Streptococcus pyogenes is an important human pathogen that causes significant morbidity and mortality worldwide. In order to successfully infect the human host, S. pyogenes is equipped with tools that modulate the human immune defence. The present thesis explores how different streptococcal proteins interfere with innate immunity, adaptive immunity, and blood coagulation. M protein is a surface-bound streptococcal virulence factor with antiphagocytic properties. M proteins can be released from the bacterial surface, and in the present thesis we report that soluble M1 protein is a potent activator of monocytes and T cells. M1 protein-induced monocyte activation involves Toll-like receptor (TLR) 2, and results in secretion of the pro-inflammatory cytokines IL-6, IL-1? and TNF?. In addition, monocytes treated with M1 protein up-regulate Tissue Factor (TF) expression on the cell surface, leading to activation of the extrinsic pathway of coagulation. T cell induction upon M1 protein stimulation results in a potent Th1 type response. Activation does not require normal antigen processing, it is MHC class II dependent, and V? restricted with preferential expansion of V?2 and 4 bearing T cells, suggesting that M1 protein functions as a superantigen. Thrombin activatable fibrinolysis inhibitor (TAFI) is a human procarboxypeptidase circulating in plasma. When activated, TAFI inhibits fibrinolysis and modulates the immune system by cleaving inflammatory mediators such as C5a, fibrinopeptides and bradykinin. Here we find that TAFI binds to Streptococcal collagen-like surface protein A and B on S. pyogenes. These bacteria can also recruit the two TAFI activators thrombin and plasmin to the bacterial surface, leading to the activation of surface-bound TAFI. Taken together, the findings may help explain how S. pyogenes modulates the immune response in order to successfully infect its human host

Personalized marketing - A qualitative study on tailored marketing online from a consumer's perspective

When consumers today navigate and shop online they meet different types of advertisements, both directly on the e-commerce website and through other channels. Many companies use personalized marketing as a way to create relationships with consumers. However, personalization today is a sensitive area and often twinned with privacy issues. Many consumers feel as they are being stalked. The purpose of this thesis is to investigate how consumers perceive personalized marketing in purchasing situations online when it is used as a customer relationship management-tool. The result should be of great use for practitioners that deal with retailing online in order to maximize the effect of their marketing efforts. Through qualitative interviews we intend to investigate consumers’ sensitivity concerning e-retailers data gathering on the Internet. Our main findings concern the degree of personalization in marketing messages, when personalization strengthens the consumer-company relationship and when it is seen as an intrusion on privacy

Heparin-binding protein: a diagnostic biomarker of urinary tract infection in adults.

Urinary tract infections (UTIs) are associated with significant morbidity and high frequency of antibiotic prescription. Diagnosing UTI is often difficult, particularly in the critically ill patient and in patients with unspecific and mild symptoms. The standard rapid tests have limited value, and there is a need for more reliable diagnostic tools. Heparin-binding protein (HBP) is released from neutrophils and has previously been studied as a diagnostic and predictive biomarker in different bacterial infections

PKCε, Via its Regulatory Domain and Independently of its Catalytic Domain, Induces Neurite-like Processes in Neuroblastoma Cells

To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCα, βII, δ, and ε fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCε–EGFP induced cell processes whereas the other isoforms did not. The effect of PKCε–EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCε regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCε and neurites induced during retinoic acid- and growth factor–induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCε– PSC1V3 protein contained α-tubulin, neurofilament-160, and F-actin, but the PKCε–PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y.  These data suggest that PKCε, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation

The activation loop tyrosine 823 is essential for the transforming capacity of the c-Kit oncogenic mutant D816V.

Oncogenic c-Kit mutations have been shown to display ligand-independent receptor activation and cell proliferation. A substitution of aspartate to valine at amino acid 816 (D816V) is one of the most commonly found oncogenic c-Kit mutations and is found in >90% of cases of mastocytosis and less commonly in germ-cell tumors, core-binding factor acute myeloid leukemia and mucosal melanomas. The mechanisms by which this mutation leads to constitutive activation and transformation are not fully understood. Previous studies have shown that the D816V mutation causes a structural change in the activation loop (A-loop), resulting in weaker binding of the A-loop to the juxtamembrane domain. In this paper, we have investigated the role of Y823, the only tyrosine residue in the A-loop, and its role in oncogenic transformation by c-Kit/D816V by introducing the Y823F mutation. Although dispensable for the kinase activity of c-Kit/D816V, the presence of Y823 was crucial for cell proliferation and survival. Furthermore, mutation of Y823 selectively downregulates the Ras/Erk and Akt pathways as well as the phosphorylation of STAT5 and reduces the transforming capacity of the D816V/c-Kit in vitro. We further show that mice injected with cells expressing c-Kit/D816V/Y823F display significantly reduced tumor size as well as tumor weight compared with controls. Finally, microarray analysis, comparing Y823F/D816V cells with cells expressing c-Kit/D816V, demonstrate that mutation of Y823 causes upregulation of proapoptotic genes, whereas genes of survival pathways are downregulated. Thus, phosphorylation of Y823 is not necessary for kinase activation, but essential for the transforming ability of the c-Kit/D816V mutant.Oncogene advance online publication, 1 December 2014; doi:10.1038/onc.2014.383

Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

AbstractHypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leads to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma

Activated Transcription of the Human Neuropeptide Y Gene in Differentiating SH-SY5Y Neuroblastoma Cells Is Dependent on Transcription Factors AP-1, AP-2Α, and NGFI

Activated transcription of the human neuropeptide Y gene ( NPY ) was investigated in SH-SY5Y neuroblastoma cells at the onset of sympathetic neuronal differentiation induced by 12- O -tetradecanoylphorbol 13-acetate (TPA) and serum or by nerve growth factor (NGF). As determined by transient expression, two NGF response elements (REs) were required for transcription induced by NGF in SH-SY5Y cells with stable expression of an exogenous NGF receptor TRK-A gene (SH-SY5Y/trk). TPA treatment in the presence of serum induced NPY transcription in both wild-type SH-SY5Y (SH-SY5Y/wt) and SH-SY5Y/trk cells. A TPA RE (TRE), overlapping the proximal NGF RE, was identified by expression of the v-Jun oncoprotein that enhanced NPY transcription. Suppression of TPA-induced NPY transcription was obtained by expression of a dominant negative Jun protein, selective protein kinase C inhibition, or introduction of a mutated TRE, whereas NGF-induced NPY transcription was inhibited to a lesser degree. The transcription factor AP-2Α was shown to bind cooperatively to the NPY promoter with either AP-1 or NGFI-A to the shared TRE and NGF RE and to the distal NGF RE, respectively. These results show that transcription factors AP-1, AP-2Α, and NGFI-A are involved in activated NPY transcription during the onset of neuronal differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65864/1/j.1471-4159.1998.70051887.x.pd

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling.

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in context of regulating Src family kinases. Here we present an addition role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling