25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

Non-enterobacterial endotoxins stimulate human coronary artery but not venous endothelial cell activation via toll-like receptor 2

To determine whether non-enterobacterial endotoxins, which are likely to constitute the majority of the circulating endotoxin pool, may stimulate coronary artery endothelial cell activation. Interleukin-8 secretion, monocyte adhesion, and E-selectin expression were measured in human umbilical vein endothelial cells (HUVECs) and coronary artery endothelial cells (HCAECs) challenged in vitro with highly purified endotoxins of common host colonisers Escherichia coli, Porphyromonas gingivalis, Pseudomonas aeruginosa, and Bacteroides fragilis. HCAECs but not HUVECs expressed Toll-like receptor (TLR)-2 and were responsive to non-enterobacterial endotoxins. Transfection of TLR-deficient HEK-293 cells with TLR2 or TLR4/MD2 revealed that while E. coli endotoxin utilised solely TLR4 to signal, the endotoxins, deglycosylated endotoxins (lipid-A), and whole heat-killed bacteria of the other species stimulated TLR2-but not TLR4-dependent cell-signalling. Blockade of TLR2 with neutralizing antibody prevented HCAEC activation by non-enterobacterial endotoxins. Comparison of each endotoxin with E. coli endotoxin in limulus amoebocyte lysate assay revealed that the non-enterobacterial endotoxins are greatly underestimated by this assay, which has been used in all previous studies to estimate plasma endotoxin concentrations. Circulating non-enterobacterial endotoxins may be an underestimated contributor to endothelial activation and atherosclerosis in individuals at risk of increased plasma endotoxin burden.

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs β-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5–22 mmol/l)±leptin (0–10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of β-cell function in diabetes.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

Stable expression of a human-like sialylated recombinant thyrotropin in a chinese hamster ovary cell line expressing 'alfa'2,6-sialyltransferase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Role of HBD-2 and HBD-3 in Human T Cell Development

Human β-defensins (hBDs) are a family of cationic peptides able to directly kill a wide range of microorganisms including bacteria, fungi and viruses. In addition to their antimicrobial activities, defensins also contribute to the modulation of both the host innate and adaptive immunity. In this project, we demonstrate that the αCD3/28 co-stimulation of human CD4+ T cells in the presence of 10μg/ml hBD-2 or hBD-3 together causes an up-regulation in numbers of CD4+CD69+CD25+ and CD4+CD69-CD25+ T cell subsets, indicating that the treatment of hBD-2 and 3 enhances CD4+ T cell activation. Consistent with this finding, proliferation assay using CFSE suggests that hBD-2 and hBD-3 treatment in vitro induces the proliferation of CD4+ T cells following by 96hrs culture. Analysis of expression of the regulatory T cells (Tregs) specific marker, FoxP3, reveals a shift in the CD4+CD127-CD25+ Treg subset at 18hrs. However, at the later time point, we found that the percentage of FoxP3+cells decreased in the CD4+CD127-CD25+ Treg population, whereas the presence of the FoxP3+CTLA-4+ Treg subset increased. These data indicate that Treg suppressive function may be potentially defective following the co-incubation of purified T cells with either hBD-2 or hBD-3 for 42hrs in vitro due to the apparent loss of FoxP3 expression. We further characterise the role of hBD-2 and hBD-3 in driving human CD4+ T cells polarisation. Our in vitro data suggests that treatment with hBD-2 and hBD-3 can not only induces effector T cell (Teff) differentiation into RORγt+T-bet+ (Th17/Th1) cells, but can also trigger the differentiation of Treg expressing RORγt and T-bet rather than the master controller of Treg function, FoxP3. This apparent plasticity of T cell phenotype allows them to convert from Treg to Th1/17-like effector T cell phenotype following 18hrs in culture. By 42hrs in culture, treatment with hBD-2 and hBD-3 induced both Teff cell and Treg cell differentiation towards the Th17-like phenotype. Compared with the treatment with hBD-2, treatment with hBD-3 induced a more pronounced effect to increase levels of RORγt in CD4+ T cells. This elevated expression may, in turn, be responsible for the induction of higher IL-17A secretion. Consistent with this idea, it was found that treatment with hBD-3 but not hBD-2 was capable of inducing the higher level of secretion of IL-17A. Additionally, treatment with hBD-3 induced an increased expression of IL-6, which is capable of driving the differentiation of naïve T cells towards IL-17-producing Th17 cells. Functionally, using the Treg suppression assay, the data suggested that hBD-2 may dampen down Treg cell ability to induce suppression of Teff cell activity. Interestingly, co-culture with hBD-2 would also appear to increase Teff cell resistance to Treg immunoregulation in vitro. Further investigation using microarray gene analysis revealed chemokine C-C motif ligand 1 (CCL1) as potential genes responding to hBD-2 treatment. The blockade of CCL1 has been reported to inhibit Treg suppressive function. Thus, this study explored the function of these antimicrobial candidates in regulating CD4+ T cell plasticity which could result in hBD-2 and hBD-3 being able to regulate its own production, but also may regulate Treg and Teff cell development and function, thus strengthening the link between innate and adaptive immunity

Perturbative 2-body parent Hamiltonians for projected entangled pair states

We construct parent Hamiltonians involving only local 2-body interactions for a broad class of projected entangled pair states (PEPS). Making use of perturbation gadget techniques, we define a perturbative Hamiltonian acting on the virtual PEPS space with a finite order low energy effective Hamiltonian that is a gapped, frustration-free parent Hamiltonian for an encoded version of a desired PEPS. For topologically ordered PEPS, the ground space of the low energy effective Hamiltonian is shown to be in the same phase as the desired state to all orders of perturbation theory. An encoded parent Hamiltonian for the double semion string net ground state is explicitly constructed as a concrete example.

The frustrated accesion of the European Union to the European Convention of Human Rights: a brief analysis of opinion 2/13 of the Court of Justice of European Union

Trabajo redactado en inglés sobre la última sentencia 2/13, del Tribunal de Justicia de Europa sobre la adhesión de la Unión Europea al Convenio Europeo de Derechos Humanos fundamentales. Análisis de la opinión 2/13 y sus objeciones.

International human resource management in the introductory HRM course

This paper explores the extent to which students in the introductory HRM course in US institutions are likely to be exposed to information on international and cross-cultural aspects of HRM. Two methods are used: (1) an analysis of international content in fifteen popular introductory HRM textbooks and (2) a survey of professors teaching introductory HRM. The vast majority of responding instructors said their classes got some exposure to international issues in HRM, and most introductory texts included some relevant content. Critiques of international boxed features and dedicated IHRM chapters are provided, and suggestions for improving the quality and depth of IHRM content in introductory textbooks are made.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.