La neuropiline-1 (Nrp-1) est une protéine transmembranaire agissant comme récepteur des sémaphorines de classe 3 (Sema3) et du facteur de croissance de l'endothélium vasculaire (VEGF). En plus de son rôle crucial dans le développement des systèmes nerveux et cardiovasculaires, Nrp-1 est impliquée dans des processus physiopathologiques impliquant certains de ses ligands classiques (Sema3 et VEGF) ou récemment caractérisés (TGF-β1 et PDGF) dans les tissus adultes. Dans le système immunitaire, Nrp-1 participe aux interactions entre les lymphocytes T et les cellules dendritiques, transmet les effets immunorégulateurs de Sema3A sur les lymphocytes T, et est impliqué dans le mécanisme suppresseur des lymphocytes T régulateurs (Treg) Foxp3+. Cependant, l'expression de Nrp-1 dans les lymphocytes T non-Treg humains et murins n'a été que peu étudiée. L'objectif de ce projet était de caractériser les populations de lymphocytes T Nrp-1+ non-Treg chez la souris, d'analyser leurs fonctions, d'identifier les mécanismes conduisant à l'expression de Nrp-1 dans ces cellules, et de comprendre le rôle joué par Nrp-1 dans les réponses immunitaires T. Mon travail s'est d'une part intéressé à une population de lymphocytes T non-conventionnels appelés lymphocytes « invariant natural killer T » (iNKT). Les lymphocytes iNKT sont des lymphocytes Tαβ dérivés du thymus aux propriétés immunomodulatrices reposant sur la sécrétion rapide de cytokines TH1 et TH2 après engagement de leur TCR semi-invariant par des complexes CD1d/glycolipide. Un sous-type distinct de cellules iNKT, dont l'origine et l'homéostasie sont encore mal connues, produit la cytokine pro-inflammatoire IL-17. Dans ce travail, j'ai montré que les lymphocytes iNKT émigrés thymiques récents sont identifiés spécifiquement par l'expression de Nrp-1. Les lymphocytes iNKT producteurs d'IL-17 expriment Nrp-1 et dépendent de l'export thymique. D'autre part, Nrp-1 est exprimé par les thymocytes immatures en division et certains lymphocytes Tαβ conventionnels mémoires en prolifération homéostatique rapide. In vitro, l'activation des lymphocytes T par le TCR induit l'expression de Nrp-1 de manière dépendante de la voie de signalisation Ca2+/calcineurine/NFAT. L'expression de Nrp-1 dans les lymphocytes T activés les sensibilise aux effets régulateurs de Sema3A et TGF-β1. En conclusion, ces résultats apportent de nouvelles données concernant l'expression et la fonction de Nrp-1 dans le système immunitaire. Plus généralement, cette étude permet d'envisager des stratégies thérapeutiques ciblant les processus dépendants de Nrp-1 dans les pathologies du système immunitaire et du système nerveux ou les cancers.

<p>Uppsatsens syfte har varit att försöka finna ”det effektivaste sättet att ta ut pengar från ett få-mansföretag”. För att kunna göra detta har vi granskat lagtexten i 56-57 kap IL, skatteverkets informationsbroschyrer, tagit fram ett antal beräkningsexempel och genomfört intervjuer med respondenter från de fem största revisionsbyråerna. När en företagare står inför valet hur den-ne ska ta ut pengar från sitt företag kan denne välja på att ta ut lön, göra pensionsavsättningar, ta ut utdelning eller låna in pengar till företaget med ränta.</p><p>Det mest effektiva sättet att ta ut pengar på varierar från företag till företag, men i de flesta fallen är det bäst att först och främst göra uttag genom löneutbetalningar till delägarna. An-ledningen till att delägarna först och främst ska ta ut lön är att den tillsammans med arbetsgi-varavgifterna är avdragsgilla i företaget och att lönen dessutom är pensions-, sjukpennings- och föräldrapenningsgrundande. Löneutbetalningen ska i första hand uppgå till någon av föl-jande gränser: sjukpenningsgrundande inkomst (7,5 prisbasbelopp = 307 500kr), undre skikt-gränsen för statlig inkomstskatt (328 800kr), pensionsgrundande inkomst (8,07 inkomstbasbe-lopp = 387 360kr) eller den inkomst som lönekravet kräver (275 400-688 500kr).</p><p>I andra hand ska företaget göra pensionsavsättningar på ca 7-10 procent av bruttolönerna till de anställda delägarna så de får samma skydd som en vanlig privatanställd tjänsteman. Efter att företaget har betalat ut lön och gjort pensionsavsättningar bör utdelning tas ut upp till gränsbeloppet. Gränsbeloppet bestäms utifrån det högsta av förenklingsregeln och huvudre-geln. Huvudregeln är vanligast förekommande då den ger ett högre gränsbelopp redan då företaget har bruttolöner på 360 000kr, förenklingsregeln ger alltid minsta gränsbelopp på två inkomstbasbelopp (91 800kr). Om utdelningsbara medel i företaget överstiger gränsbeloppet sparas detta belopp till senare år och eventuellt görs extra pensionsinsättningar till delägarna.</p>

Gene therapy has emerged as a field to modulate cell functions by introducing genes of interest to target cells. An emerging focus in this field is to employ non-viral vectors to deliver immunosuppressive cytokines to dendritic cells (DCs) to attenuate damaging immune responses. DCs serve as potential targets for suppression of T cell responses. In this work, we investigated the ability of polycation-modified polymeric particles complexed with interleukin-10 (IL-10) gene to modulate DCs. The delivery systems (designated as PSO10H6 and PLGAO10H6) were formed by coating cationic peptide O10H6 (O: ornithine; H: histidine) on the polystyrene (PS) and poly (lactic-co-glycolic acid) (PLGA) particulates. A mouse IL-10 encoding plasmid (pIL-10) was loaded on the surface of PSO10H6 and PLGAO10H6 via ionic interactions. Physical characterization of these particles revealed stable colloidal dispersions (diameters: 297.2&#177;14nm in PLGAO10H6-pIL-10 and 126.0&#177;8nm in PSO10H6-pIL-10). DNA molecules carried by PSO10H6 and PLGAO10H6 were protected from serum digestion. Results from in vitro gene transfection studies showed two-fold enhancement of IL-10 expression in bone marrow-derived DCs transfected with PSO10H6-pIL-10 and PLGAO10H6-pIL-10 compared to untransfected DCs. Their suppressive functions were evaluated in an in vitro mixed lymphocyte model. Results indicated that PSO10H6-pIL-10 and PLGAO10H6-pIL-10 modified DCs elicited weakest proliferation of allogeneic bulk T cells as well as CD4 and CD8 T cells among all the delivery modes. Using cell-embedded Matrigel as a surrogate graft, we showed that IL-10 gene-modified DCs suppressed host cell infiltration in vivo. These data suggested PSO10H6-pIL-10 and PLGAO10H6-pIL-10 deliver an overriding suppressive signal to T cells. Further studies revealed T cells stimulated by the IL-10 gene-modified DCs exhibited characteristics of regulatory T (Treg) cells, as evident by up-regulation of a Treg cell marker forkhead-type transcription factor 3 (Foxp3). This result was concomitant with an increase in of transforming growth factor beta (TGF-beta) production. &lt;br&gt;Taken together, this work demonstrated that PSO10H6 and PLGAO10H6 are effective in delivering pIL-10 to modulate DCs to suppress T cell responses. Collectively, the results raise the prospects of using PSO10H6 and PLGAO10H6 as vectors to deliver immunosuppressive genes to modulate T cell responses in vivo. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmaceutics / PhD / Dissertation

En riktad utdelning innebär att en eller flera aktieägare helt eller delvis avstår sin rätt till utdelning till förmån för en annan aktieägare. Beskattningen av utdelningar sker i allmänhet i enlighet med 42:12 Inkomstskattelagen (IL), som stadgar att den som har rätt till en utdelning när denna kan disponeras även skall beskattas för densamma. Paragrafen är tillämplig på riktade utdelningar och torde vid en första anblick innebära att det är aktieägaren som mottar den riktade utdelningen som är skattskyldig till utdelningsbeloppet. En av de grundläggande principerna inom aktiebolagsrätten som aktualiseras vid vinstutdelning i ett aktiebolag är likhetsprincipen, som innebär att alla aktier har samma rätt i bolaget. En riktad utdelning innebär att en eller flera aktieägare får en större utdelning än vad som betingas av dennes aktieinnehav. Således kan en riktad utdelning innebära ett avsteg från likhetsprincipen. Likhetsprincipen utgör ett skydd för aktieägarna och skall därför upprätthållas. Avsteg är dock möjliga men kräver samtliga aktieägares samtycke. Hur påverkar då likhetsprincipen och andra civilrättsliga regler beskattningen av en riktad utdelning? För att besvara denna fråga måste man först utreda vilket förhållande skatterätten har till civilrätten. Normalt sett följer skatterätten civilrätten, d v s rättshandlingar som accepteras inom civilrätten ligger även till grund för den skattemässiga bedömningen, vilket torde innebära att riktade utdelningar som genomförts på ett enligt civilrätten godtagbart sätt skall ligga till grund för beskattningen. Rättsläget är dock oklart och det är tänkbart att man skatterättsligt inte godtar transaktionen utan istället beskattar aktieägaren och därmed bortser från överlåtelsen. Detta synsätt skulle kunna motiveras av att den riktade utdelningen ses som en disposition av den aktieägare som avstår utdelning och att det därför är denna person och inte mottagaren som skall beskattas. Syftet med uppsatsen är att utreda vilka inkomstskatterättsliga konsekvenser en riktad utdelning ger upphov till, dels för den person som avstår utdelning, dels för den person som mottar den riktade utdelningen.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a bacterium mainly associated with aggressive forms of periodontitis. Among its virulence factors, a leukotoxin is suggested to play an important role in the pathogenicity. Periodontal infections with strains producing high levels of the leukotoxin are strongly associated with severe disease. Leukotoxin selectively kills human leukocytes and can disrupt the local defense mechanisms. Previous studies examining the role of the leukotoxin in host-parasite interactions have mainly focused on polymorphonuclear leukocytes (PMNs). In the inflamed periodontium, macrophages play a significant role in the regulation of the inflammatory reactions and the tissue breakdown and remodeling. Thus, the aim of this dissertation was to investigate death mechanisms of human macrophages exposed to leukotoxin. Human lymphocytes, PMNs, and monocytes/macrophages isolated from venous blood were exposed to purified leukotoxin or live A. actinomycetemcomitans strains producing variable levels or no leukotoxin. Different target cells were characterized by their expression of cell surface molecules. Cell death and viability were studied by examining cell membrane integrity and morphological alterations. Further, processes and cellular markers involved in apoptosis and necrosis were investigated. The expression and activation of pro-inflammatory cytokines of the leukotoxin-challenged leukocytes were examined at the mRNA and protein level. The biological activity of the secreted cytokines was investigated by testing the culture supernatants in a bone resorption assay. Additionally, different intracellular signaling pathways involved in the pro-inflammatory response from the macrophages were examined. Monocytes/macrophages were the most sensitive leukocytes for A. actinomycetemcomitans leukotoxin-induced lysis. This process in monocytes/ macrophages involved caspase-1 activation, and in addition, leukotoxin triggered abundant activation and secretion of IL-1β from these cells. The secreted IL-1β was mainly the 17 kDa bioactive protein and stimulated bone resorption. This activity could be blocked by an IL-1 receptor antagonist. When live bacteria were used, the A. actinomycetemcomitans-induced IL-1β secretion from human macrophages was mainly caused by the leukotoxin. Closer examination of the macrophages exposed to leukotoxin revealed that the induced cell death proceeded through a process that differed from classical apoptosis and necrosis. Interestingly, this process resembled a newly discovered death mechanism termed pyroptosis. The extensive leukotoxin induced IL-1β secretion did not correlate to increased levels of mRNA for IL-1β. It was mainly mediated by caspase-1 activation, since blocking it by a specific inhibitor also abolished the secretion of IL-1β. A similar pattern, but at much lower level, was seen for IL-18. In conclusion, these results show that A. actinomycetemcomitans leukotoxin induces a death process in human macrophages leading to a specific and excessive pro-inflammatory response. Our results indicate that this novel virulence mechanism of leukotoxin may play an important role in the pathogenic potential of A. actinomycetemcomitans.

Bone tissue is continuously degraded and rebuilt to respond to the needs of the body. Cells of the osteoblast lineage are responsible for the formation of bone, whereas the resorption of bone tissue is carried out by osteoclasts. To prevent imbalance between bone formation and resorption, these processes are delicately regulated by a complex network of both systemic factors and factors produced locally in the bone microenvironment, including members of the IL-6 family of cytokines. During the last decades, the presence of nerve fibers in skeletal tissue and presence of receptors for several neurotransmitters on both osteoblasts and osteoclasts, have suggested a possible role for neuropeptides in the regulation of skeletal metabolism. The overall aim of this study was to investigate the roles of cytokines in the IL-6 family and the neuropeptide VIP in regulation of osteotropic cytokine expression and bone metabolism in vitro. In Paper I, stimulation of bone resorption by the cytokine IL-6, in the presence of its soluble receptor sIL-6R, was demonstrated in mouse calvarial bones. OSM and LIF, other members of the IL-6 family of cytokines, were also shown to increase bone resorption. Furthermore, IL-6+sIL-6R, LIF, and OSM increased the expression of RANKL, which by binding to its receptor RANK functions as a crucial inducer of osteoclast formation and activation. In Paper II-IV, the effects of the neuropeptide VIP and related peptides on expression of osteotropic cytokines by osteoblasts and bone resorption in vitro have been studied. VIP and PACAP-38 both increased IL-6 production in osteoblasts in a time- and concentration-dependent manner. In contrast, no effect was seen with the related peptide secretin, indicating that the effects were mediated by the VPAC2 receptor. VIP and PACAP, in contrast to secretin, also induced IL-6 promoter activity in osteoblastic MC3T3-E1 cells transfected with an IL-6 promoter/luciferase construct. The effects of VIP on IL-6 were shown to be mediated by several intracellular pathways, including cAMP/PKA/CREB, AP-1, and C/EBP, but not NF-kB or the cAMP-activated Epac pathway. The release of IL-6 from osteoblasts was increased by several pro-inflammatory osteotropic cytokines, including interleukin-1b, an effect that was further potentiated by VIP, indicating a possible neuro-immunomodulatory interaction in the regulation of bone metabolism. VIP and PACAP-38 also increased the osteoblastic expression of RANKL and decreased the expression of OPG and M-CSF, factors crucial in regulation of differentiation and activation of osteoclasts. Although this indicated a possible bone resorptive effect, VIP was found to decrease osteoclast formation and bone resorption by directly targeting osteoclast progenitor cells through an inhibitory mechanism. In conclusion, the results in this thesis indicate that several cytokines in the IL-6 family stimulate bone resorption in calvarial bones in vitro, most likely through the RANKL-RANK interaction. Furthermore, expression of the osteotropic cytokine IL-6 in osteoblasts is stimulated by the neuropeptide VIP through VPAC2 receptors via several intracellular pathways, further strengthening the role of neuropeptides as local regulators of bone metabolism.

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology. In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis. Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro. In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells. / QC 20100729

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Wir untersuchten Liquor- und Serumproben von 40 an einem shuntversorgten Hydrocephalus erkrankten Kindern auf die Konzentration der Zytokine bFGF, TGF-β1, VEGF, IL-6, IGF-1 und Leptin sowie deren Korrelation mit dem Risiko von Shuntinsuffizienzen. Dabei konnten wir die Hypothese bestätigen, dass erhöhte Konzentration der fibrogenen Zytokine bFGF und TGF-β1 im Serum bzw. Liquor mit einem erhöhten Risiko für operationspflichtige Shuntinsuffizienzen durch Obstruktion des Schlauchsystems einhergehen, und dass diese Komplikationen mit steigenden Zytokinkonzentrationen umso eher eintreten. Außerdem war bFGF im Liquor von Kindern, die zum Abnahmezeitpunkt an einer Shuntdysfunktion durch Obstruktion oder Einwachsen des Shunts litten, signifikant höher als bei Kindern, die zum Zeitpunkt der Abnahme keine Shuntdysfunktion aus eben genannten Gründen hatten. Des Weiteren fanden wir Konzentrationsunterschiede für IL-6 im Liquor zwischen den einzelnen Ursachen der Erkrankung, wobei das Zytokin am höchsten bei Tumorpatienten war, gefolgt von posthämorrhagischem und postmeningitischem Hydrocephalus, und am niedrigsten bei Kindern mit kongenitaler ZNS-Fehlbildung.

In the present study our objectives were (1) to follow the temporal patterns of testicular LH and FSH receptor (LH-R and FSH-R) concentrations and affinity (Ka) during sexual maturation in bulls, to see if such patterns could explain the control of rapid testicular growth that occurs after 25 weeks of age, when serum gonadotropin concentrations are low; (2) to see if transformation growth factors (TGF- alpha and beta 1, 2 and 3) and interleukins (IL-1 and IL-6) are produced in the developing bovine testis and if their concentrations change during development; (3) to see if the onset of puberty could be hastened by treating bull calves subcutaneously (sc) with 3 mg of bLH (n=6) or 4 mg of bFSH (n=6) once every 2 days, from 4 to 8 weeks after birth. Mean LH-R concentrations decreased from 13 to 25 weeks of age and increased to 56 weeks of age (P<0.05). LH-RKa decreased from 9 to 17 weeks of age, increased to 29 weeks and declined to 33 weeks of age (P<0.05). FSH-R concentrations declined from 17 to 25 weeks of age then increased to 56 weeks of age (P<0.05). FSH-RKa increased from 17 to 25 weeks of age (P<0.05). Testicular TGF-alpha concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 33 to 56 weeks of age (P<0.05). Testicular TGF-beta 1 concentrations decreased from 17 to 21 weeks of age, increased to 25 weeks and decreased from 25 to 29 weeks of age (P<0.05). Testicular TGF-beta 2 concentrations increased from 5 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks and decreased at 29 weeks of age (P<0.05). Testicular TGF-beta 3 concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks of age and from 25 to 29 weeks of age (P<0.05). Mean testicular IL-1 alpha concentrations decreased from 5 to 9 weeks of age and 13 to 21 weeks of age (P<0.01) while mean testicular IL-1 beta concentrations decreased from 13 to 17 weeks and 29 to 33 weeks of age (P<0.01). Mean IL-1 bioactivity increased from 13 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks, decreased to 29 weeks and decreased from 33 to 56 weeks of age (P<0.05). Mean testicular IL-6 concentrations decreased (P<0.05) from 9 to 13 weeks of age, increased (P<0.05) to 21 weeks, decreased (P<0.05) to 25 weeks of age, increased (P<0.05) to 29 weeks and decreased (P<0.01) to 56 weeks of age. <p>We concluded that high concentrations of gonadotropin receptors might be critical to initiate postnatal testis growth and support it after 25 weeks of age in the face of low serum gonadotropin concentrations. Testicular TGF-alpha concentrations were higher in calves than adults while concentrations of TGF-beta and IL-1 were higher in the early postnatal period than the peripubertal period. The changes in testicular concentrations of TGFs and ILs led us to suggest a possible local regulatory role in development. Testicular IL-6 concentrations were higher in prepubertal calves than adults. Treatment of bull calves with bFSH from 4 to 8 weeks of age increased testicular growth (SC), hastened the onset of puberty (SC ≥ 28 cm), and enhanced spermatogenesis.