SDF-1 and PDGF enhance [alpha]v[beta]5-mediated ERK activation and adhesion-independent growth of human pre-B cell lines

CD23 acts through the [alpha]v[beta]5 integrin to promote growth of human pre-B cell lines in an adhesion-independent manner. [alpha]v[beta]5 is expressed on normal B-cell precursors in the bone marrow. Soluble CD23 (sCD23), short CD23-derived peptides containing the arg-lys-cys (RKC) motif recognized by [alpha]v[beta]5 and anti-[alpha]v[beta]5 monoclonal antibodies (MAbs) all sustain growth of pre-B cell lines. The chemokine stromal cell-derived factor-1 (SDF-1) regulates key processes during B-cell development. SDF-1 enhanced the growth-sustaining effect driven by ligation of [alpha]v[beta]5 with anti-[alpha]v[beta]5 MAb 15F-11, sCD23 or CD23-derived RKC-containing peptides. This effect was restricted to B-cell precursors and was specific to SDF-1. The enhancement in growth was associated with the activation of extracellular signal-regulated kinase (ERK) and both these responses were attenuated by the MEK inhibitor U0126. Finally, platelet-derived growth factor also enhanced both [alpha]v[beta]5-mediated cell growth and ERK activation. The data suggest that adhesion-independent growth-promoting signals delivered to B-cell precursors through the [alpha]v[beta]5 integrin can be modulated by cross-talk with receptors linked to both G-protein and tyrosine kinase-coupled signalling pathways

Characterisation of human B-lymphocyte specific antigens and receptor immunoglobulins

Considerable attention has recently been focused on the mechanisms which allow B-lymphocytes to effect simultaneous biosynthesis and expression of receptor and effector forms of the immunoglobulin molecule as mandated by the Clonal Selection Hypothesis. The genetic mechanisms responsible for the above phenomenon have now been established for the biosynthesis of IgM in murine lymphocytes. Also of great interest is the possible regulatory influence exerted by carbohydrate moieties on the secretion and membrane deposition of immunoglobulin molecules. The objectives of the work described in this thesis were to attempt to ascertain whether or not human B-lymphocytes expressing IgG, IgA and IgE adopted similar genetic strategies to allow simultaneous expression of structurally distinct membrane and secretory heavy chain polypeptides. The role of N-linked oligosaccharides in secretion and membrane deposition of immunoglobulin molecules was also studied and compared with data from similar investigations on the biosynthesis of human transplantation antigens in the hope of revealing possible carbohydrate-influenced structural signals necessary for transport and expression of immunoglobulins. The antibiotic tunicamycin was used to inhibit N-glycosy1ation of proteins in cells actively engaged in protein biosynthesis. SDS-PAGE analysis of specifically immunoprecipitated immunoglobulins of different classes synthesised in cells treated with tunicamycin revealed that four-chain immunoglobulin molecules were readily formed in all cells. It is concluded that immunoglobulin assembly occurs independently of N-glycosylation of the newly-synthesised immunoglobulin chains. HLA-A,-B,-C/beta2 microglobulin complexes were also efficiently assembled in the presence of tunicamycin. Evidence is presented to support the hypothesis that 19S IgM and 11S IgA polymers are formed in cells treated with tunicamycin. No conclusive evidence was obtained with respect to incorporation of J-chain into nonglycosylated polymeric immunoglobulins. The presence of N-linked oligosaccharides on the immunoglobulin heavy chain appeared to be mandatory for the efficient secretion of IgM and IgA from human cells synthesising these classes of immunoglobulin. By contrast non-N-glycosylated IgG and IgE molecules were efficiently secreted from IgG and IgE producing human B-lymphocytes. Three experimental approaches were adopted to elucidate the functional role of N-linked carbohydrate side chains of immunoglobulin molecules in the efficient membrane deposition of these molecules. In one case it was found that non-N-glycosylated IgM could be demonstrated at the cell membrane of tunicamycin-treated cells: this data was shown to be incorrect by more rigorous experimental analyses. It is concluded that N-glycosylation of immunoglobulins is necessary for efficient membrane deposition of IgM and IgA. In contrast, HLA-A,-B and -C alloantigens and also HLA-DR alloantigens were detected at the membrane of tunicamycin-treated cells and, in the case of HLA-A2 and HLA-B7, the non-glycosylated molecules were alloantigenic as judged by their serological reactivity with alloantisera. Biosynthesis of membrane and secretory forms ofy -heavy chain polypeptides was studied in the B-lymphoblastoid cell line Bec-11. Normal Bec-11 cells synthesised three intracellular forms of Y-Chain of AMWs 64K, 55K and 52K and two of these chains are secreted into the culture supernatant (52K and 55K). The 64K species was presumed to be the membrane form of y-heavy chain and this hypothesis was supported by the finding that IgG isolated from aliquots of radioiodinated Bec-11 membrane proteins contained y-heavy chains of AMW 64K. (Abstract shortened by ProQuest.)

The Quixotism of a Relationally Constituted Contract Law

Relational contract theory is often seen as a rival to classical contract. Despite its inception in the early 1970s, the full theory does not seem to have made much headway into the substantive law of contract within England and Wales outside of doctrinal rhetoric in good faith. Part of this stagnation is due to the convolution that surrounds relational contract theory, both in its descriptive and normative claims. This thesis will attempt to rectify the situation and elucidate relational contract theory as the neutral analytic tool that it was designed to be. It will be shown that relational theory is not inherently opposed to the continuation of the classical law and that a change to a relationally constituted law of contract would be damaging. While the classical law is founded on false premises, the effect is not as dire as academics posit, and in a world that is better off with this flawed doctrine relational theory will encounter the awkward question of its utility. The fact that the theory would not do well in substantive law does not make its norms and narrative any less accurate, and this thesis maintains it still has a place. In determining an adequate place the thesis will reference an obscure jurisprudential theory developed by Niklas Luhmann known as Autopoietic Systems Theory. With reference to this, the thesis determines that relational contract theory is a description of the interplay between psychic and social systems. The theory is both separate and distinct from the legal subsystem of society, which observes the theory's noises and generates its own internal communications. On this abstract theoretical level, the thesis will deduce that further distortion of the theory by the legal system is highly likely, and therefore integration is unlikely to find any real success

Secondary structure of human apolipoprotein A-I(1–186) in lipid-mimetic solution

AbstractThe solution structure of an apoA-I deletion mutant, apoA-I(1–186) was determined by the chemical shift index (CSI) method and the torsion angle likelihood obtained from shift and sequence similarity (TALOS) method, using heteronuclear multidimensional NMR spectra of [u-13C, u-15N, u-50% 2H]apoA-I(1–186) in the presence of sodium dodecyl sulfate (SDS). The backbone resonances were assigned from a combination of triple-resonance data (HNCO, HNCA, HN(CO)CA, HN(CA)CO and HN(COCA)HA), and intraresidue and sequential NOEs (three-dimensional (3D) and four-dimensional (4D) 13C- and 15N-edited NOESY). Analysis of the NOEs, Hα, Cα and C′ chemical shifts shows that apoA-I(1–186) in lipid-mimetic solution is composed of α-helices (which include the residues 8–32, 45–64, 67–77, 83–87, 90–97, 100–140, 146–162, and 166–181), interrupted by short irregular segments. There is one relatively long, irregular and mostly flexible region (residues 33–44), that separates the N-terminal domain (residues 1–32) from the main body of protein. In addition, we report, for the first time, the structure of the N-terminal domain of apoA-I in a lipid-mimetic environment. Its structure (α-helix 8–32 and flexible linker 33–44) would suggest that this domain is structurally, and possibly functionally, separated from the other part of the molecule

Respirable antisense oligonucleotides: a new drug class for respiratory disease

Respirable antisense oligonucleotides (RASONs), which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery

Layered Silicate-Alginate Composite Particles for the pH-Mediated Release of Theophylline

Numerous natural and synthetic clay minerals have proven to be excellent drug carriers for high drug-loaded and sustained release formulations due to their considerable ion exchange, adsorption, and swelling capacities. Moreover, the synthetic smectite clays have added advantages in terms of compositional purity and controlled cation exchange capacity in comparison to natural clays. This study involves the intercalation of theophylline (TP) in a synthetic clay, Laponite® (LP), followed by the inclusion of the resulting intercalates into sodium alginate (SA) beads to achieve pH-controlled drug release. Maximum intercalated drug incorporation of 68 mg/g was obtained by ion exchange at pH 1.2 and confirmed by an increase in basal spacing of the clay from 12.9 to 15.5 Å. TP release from the binary LP-TP intercalates in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was found to be 40% and 70%, respectively. LP-TP particles were also incorporated in an SA matrix via polymer crosslinking using CaCl2(aq) to improve the pH selective release. The ternary polymer-clay-drug composite particles effectively prevented the release of TP at low pH in SGF and resulted in sustained release in SIF, with 40% dissolution within 120 min

Pollen-derived adenosine is a necessary cofactor for ragweed allergy

BACKGROUND: Ragweed (Ambrosia artemisiifolia) is a strong elicitor of allergic airway inflammation with worldwide increasing prevalence. Various components of ragweed pollen are thought to play a role in the development of allergic responses. Aim of the study was to identify critical factors for allergenicity of ragweed pollen in a physiologic model of allergic airway inflammation METHODS: Aqueous ragweed pollen extract, the low molecular weight fraction or the major allergen Amb a 1 were instilled intranasally on 1 - 11 consecutive days and allergic airway inflammation was evaluated by bronchoalveolar lavage, lung histology, serology, gene-expression in lung tissue and measurement of lung function. Pollen-derived adenosine was removed from the extract enzymatically in order to analyze its role in ragweed-induced allergy. Migration of human neutrophils and eosinophils towards supernatants of ragweed-stimulated bronchial epithelial cells was analyzed RESULTS: Instillation of ragweed pollen extract, but not of the major allergen or the low molecular weight fraction, induced specific IgG1 , pulmonary infiltration with inflammatory cells, a Th2-associated cytokine signature in pulmonary tissue and impaired lung function. Adenosine aggravated ragweed-induced allergic lung inflammation. In vitro, human neutrophils and eosinophils migrated towards supernatants of bronchial epithelial cells stimulated with ragweed extract only if adenosine was present CONCLUSIONS: Pollen-derived adenosine is a critical factor in ragweed-pollen induced allergic airway inflammation. Future studies aim at therapeutic strategies to control these allergen-independent pathways