Access to modified geiparvarins using Pd(0)-mediated C-C bond forming reactions

Geiparvarin is a natural product which contains both a 3(2H)-furanone and a coumarin moiety in its structure. The aim of this project was to investigate the use of Pd(0)-mediated C–C bondforming reactions to produce structurally modified geiparvarins. Chapter 1 consists of a review of the relevant literature, including that pertaining to the syntheses of selected naturally occurring 3(2H)-furanones. The known syntheses of geiparvarin and closely related analogues are examined, along with the documented biological activity of these compounds. The synthetic routes which allow access to 4-substituted-3(2H)-furanones are also described. Chapter 2 describes in detail the synthesis of a variety of novel structurally modified geiparvarins by two complementary routes, both approaches utilising Pd(0)-mediated crosscoupling reactions, and discusses the characterisation of these compounds. The preparation of 5-ethyl-3(2H)-furanones is described, as is their incorporation into geiparvarin and the corresponding 5″-alkylgeiparvarin analogues via formation and dehydration of intermediate alcohols. Halogenation of 5-ethyl-3(2H)-furanones and the corresponding geiparvarin derivatives is discussed, along with further reactions of the resulting halides. Preparation of 3″-arylgeiparvarins involving both Suzuki–Miyura and Stille reactions, using the appropriate intermediate iodides and bromides, is described. The application of Stille and Heck conditions to give 3″-ethenylgeiparvarin analogues and Sonogashira conditions to produce 3″-ethynylgeiparvarin analogues, using the relevant intermediate iodides, is also extensively outlined. Chapter 3 contains all of the experimental data and details of the synthetic methods employed for the compounds prepared during the course of this research. All novel compounds prepared were fully characterised using NMR spectroscopy, IR spectroscopy, mass spectrometry and elemental analysis; the details of which are included.

Biased group decision-making and the effect of computer-mediated communication: separating the effects of anonymity, voting and blind picking

The influence of communication technology on group decision-making has been examined in many studies. But the findings are inconsistent. Some studies showed a positive effect on decision quality, other studies have shown that communication technology makes the decision even worse. One possible explanation for these different findings could be the use of different Group Decision Support Systems (GDSS) in these studies, with some GDSS better fitting to the given task than others and with different sets of functions. This paper outlines an approach with an information system solely designed to examine the effect of (1) anonymity, (2) voting and (3) blind picking on decision quality, discussion quality and perceived quality of information.

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC).

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-gamma) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-gamma did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-gamma antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-gamma. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-gamma was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-gamma antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-gamma and IL-10 modulation.

Equal IgG antibody response to pneumococcal vaccination in all stages of human immunodeficiency virus disease.

To evaluate the immunogenicity and safety of a 23-valent pneumococcal vaccine in human immunodeficiency virus (HIV)-seropositive patients, 80 men and 18 women received 1 dose of the vaccine (Pneumo 23; Pasteur Mérieux MSD, Brussels). The total IgG antibody response against all 23 Streptococcus pneumoniae capsular antigens was measured. Antibody levels were expressed in arbitrary units per microliter, referring to a standard curve. Geometric mean titers of the total IgG capsular antibodies on the day of vaccination and 30-45 days later were compared. The ratios of titers after and before vaccination in patients with > 500, 200-500, and < 200 CD4 lymphocytes/microL were 10, 10, and 12.6, respectively. Nonresponse (ratio < 4) occurred in 17% of patients and was unrelated to CD4 cell count. The vaccine was well tolerated; no serious side effects occurred. In 83% of the patients with HIV infection, the total antipneumococcal IgG level was higher after vaccination.

Particulate allergens potentiate allergic asthma in mice through sustained IgE-mediated mast cell activation.

Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and a cellular infiltrate dominated by eosinophils. Numerous epidemiological studies have related the exacerbation of allergic asthma with an increase in ambient inhalable particulate matter from air pollutants. This is because inhalable particles efficiently deliver airborne allergens deep into the airways, where they can aggravate allergic asthma symptoms. However, the cellular mechanisms by which inhalable particulate allergens (pAgs) potentiate asthmatic symptoms remain unknown, in part because most in vivo and in vitro studies exploring the pathogenesis of allergic asthma use soluble allergens (sAgs). Using a mouse model of allergic asthma, we found that, compared with their sAg counterparts, pAgs triggered markedly heightened airway hyperresponsiveness and pulmonary eosinophilia in allergen-sensitized mice. Mast cells (MCs) were implicated in this divergent response, as the differences in airway inflammatory responses provoked by the physical nature of the allergens were attenuated in MC-deficient mice. The pAgs were found to mediate MC-dependent responses by enhancing retention of pAg/IgE/FcεRI complexes within lipid raft–enriched, CD63(+) endocytic compartments, which prolonged IgE/FcεRI-initiated signaling and resulted in heightened cytokine responses. These results reveal how the physical attributes of allergens can co-opt MC endocytic circuitry and signaling responses to aggravate pathological responses of allergic asthma in mice.

B-lymphocyte effector functions in health and disease.

B-lymphocytes have traditionally been thought to contribute to immunity and autoimmune disease through terminal differentiation into plasma cells that secrete antibody. However, studies in mice and recent clinical studies have demonstrated that genetically altered B-cell function and B-cell-targeted therapies can significantly affect autoimmune diseases that were predominantly thought to be T-cell-mediated. B-cell depletion in mouse models of disease has also led to the identification of alternative B-cell effector functions that regulate normal immune responses and autoimmune disease. This review highlights multiple B-cell effector mechanisms, including the promotion of cellular immunity, the negative regulation of immune responses, and the production of pathogenic antibodies.

Gait and behavior in an IL1β-mediated model of rat knee arthritis and effects of an IL1 antagonist.

Interleukin-1 beta (IL1β) is a proinflammatory cytokine that mediates arthritic pathologies. Our objectives were to evaluate pain and limb dysfunction resulting from IL1β over-expression in the rat knee and to investigate the ability of local IL1 receptor antagonist (IL1Ra) delivery to reverse-associated pathology. IL1β over-expression was induced in the right knees of 30 Wistar rats via intra-articular injection of rat fibroblasts retrovirally infected with human IL1β cDNA. A subset of animals received a 30 µl intra-articular injection of saline or human IL1Ra on day 1 after cell delivery (0.65 µg/µl hIL1Ra, n = 7 per group). Joint swelling, gait, and sensitivity were investigated over 1 week. On day 8, animals were sacrificed and joints were collected for histological evaluation. Joint inflammation and elevated levels of endogenous IL1β were observed in knees receiving IL1β-infected fibroblasts. Asymmetric gaits favoring the affected limb and heightened mechanical sensitivity (allodynia) reflected a unilateral pathology. Histopathology revealed cartilage loss on the femoral groove and condyle of affected joints. Intra-articular IL1Ra injection failed to restore gait and sensitivity to preoperative levels and did not reduce cartilage degeneration observed in histopathology. Joint swelling and degeneration subsequent to IL1β over-expression is associated limb hypersensitivity and gait compensation. Intra-articular IL1Ra delivery did not result in marked improvement for this model; this may be driven by rapid clearance of administered IL1Ra from the joint space. These results motivate work to further investigate the behavioral consequences of monoarticular arthritis and sustained release drug delivery strategies for the joint space.

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism.

Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a "pseudosubstrate" mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1.

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.

Developmental cascades of peer rejection, social information processing biases, and aggression during middle childhood.

This study tested a developmental cascade model of peer rejection, social information processing (SIP), and aggression using data from 585 children assessed at 12 time points from kindergarten through Grade 3. Peer rejection had direct effects on subsequent SIP problems and aggression. SIP had direct effects on subsequent peer rejection and aggression. Aggression had direct effects on subsequent peer rejection. Each construct also had indirect effects on each of the other constructs. These findings advance the literature beyond a simple mediation approach by demonstrating how each construct effects changes in the others in a snowballing cycle over time. The progressions of SIP problems and aggression cascaded through lower liking, and both better SIP skills and lower aggression facilitated the progress of social preference. Findings are discussed in terms of the dynamic, developmental relations among social environments, cognitions, and behavioral adjustment.

Morphing low-affinity ligands into high-avidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer.

Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in nontarget tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy--dynamic affinity modulation (DAM)--using elastin-like polypeptide diblock copolymers (ELP(BC)s) that self-assemble from a low-affinity to high-avidity state by a tunable thermal "switch", thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELP(BC)s on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELP(BC) self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micrometer-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM and provide rational design parameters for future applications of this technology for targeted drug delivery.

Reductive ligation mediated one-step disulfide formation of S-nitrosothiols.

A one-step reductive ligation mediated disulfide formation of S-nitrosothiols was developed. This reaction involves the reaction of the S-nitroso group with phosphine-thioesters to form sulfenamide and thiolate intermediates, which then undergo a fast intermolecular disulfide formation to form stable conjugates. This reaction can be used to design new biosensors of S-nitrosated proteins.

Magnitude and breadth of a nonprotective neutralizing antibody response in an efficacy trial of a candidate HIV-1 gp120 vaccine.

BACKGROUND: A candidate vaccine consisting of human immunodeficiency virus type 1 (HIV-1) subunit gp120 protein was found previously to be nonprotective in an efficacy trial (Vax004) despite strong antibody responses against the vaccine antigens. Here we assessed the magnitude and breadth of neutralizing antibody responses in Vax004. METHODS: Neutralizing antibodies were measured against highly sensitive (tier 1) and moderately sensitive (tier 2) strains of HIV-1 subtype B in 2 independent assays. Vaccine recipients were stratified by sex, race, and high versus low behavioral risk of HIV-1 acquisition. RESULTS: Most vaccine recipients mounted potent neutralizing antibody responses against HIV-1(MN) and other tier 1 viruses. Occasional weak neutralizing activity was detected against tier 2 viruses. The response against tier 1 and tier 2 viruses was significantly stronger in women than in men. Race and behavioral risk of HIV-1 acquisition had no significant effect on the response. Prior vaccination had little effect on the neutralizing antibody response that arose after infection. CONCLUSIONS: Weak overall neutralizing antibody responses against tier 2 viruses is consistent with a lack of protection in this trial. The magnitude and breadth of neutralization reported here should be useful for identifying improved vaccines.