Enantiodivergent asymmetric catalysis with the tropos BIPHEP ligand and a proline derivative as chiral selector.

A catalytic system based on the tropos ligand BIPHEP and (S)-proline methyl ester as chiral selector was studied for Rh-catalysed asymmetric catalysis. By careful control of the catalyst preformation conditions, the enantioselectivity could be completely reversed in asymmetric hydrogenation of prochiral olefins maintaining the same absolute level in favorable cases. The enantiodivergent asymmetric catalysis could be rationalised by the interplay of the dynamic chirality (tropos) of the phosphine ligand and the coordination of the proline selector. Treating a suitable Rh-BIPHEP precursor with the (Sc)-proline-based ionic liquid led to an equimolar mixture of (RaSc)- and (SaSc)-diastereomers that is kinetically stable at 0 °C. At higher temperature, an irreversible diastereomerisation process was observed resulting in the diastereomerically pure (RaSc)-complex [Rh{(Ra)-BIPHEP}{(Sc)-ProlOMe}]. Whereas the use of the pure (RaSc)-complex led to 51% ee (R) in the hydrogenation of methyl 2-acetamidoacrylate, the S-product was formed with almost identical enantioselectivity when the (RaSc)/(SaSc)-mixture was applied under identical conditions. This inversion was associated with the relative stability of the diastereomers in the equilibria forming the catalytically active substrate complex. The possibility to use this different reactivity to control the direction of enantioselectivity was demonstrated for the hydrogenation of different substrates whereby ee's of up to 80% could be achieved. Moreover, the (RaSc)-complex led to high enantioselectivities of up 86% ee in the asymmetric hydroboration of styrene, approaching the performance of the atropos BINAP ligand for this reaction.

Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines.

Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

Inhibition of type I allergic responses with nanogram doses of replicon-based DNA vaccines.

BACKGROUND: Allergic diseases have become a major public health problem in developed countries; yet, no reliable, safe and consistently effective treatment is available. DNA immunization has been shown to prevent and balance established allergic responses, however, the high dose of conventional DNA vaccines necessary for the induction of anti-allergic reactions and their poor immunogenicity in primates require the development of new allergy DNA vaccines. We evaluated protective and therapeutic effects of a Semliki-Forest Virus replicase-based vs a conventional DNA vaccine in BALB/c mice using the model allergen beta-galactosidase. METHODS: Immunoglobulin (Ig)E suppression was determined by a basophil release assay as an in vitro correlate for allergen-specific crosslinking capacity of IgE reflecting the in vivo situation in an allergic individual. Th1 memory responses were measured by cytokine detection via enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT). RESULTS: Nanogram amounts of a replicase-based vector triggered a Th1 response comparable with that achieved with the injection of 20,000-times more copies of a conventional DNA plasmid, and induced IgE suppression in both a protective and a therapeutic setting. CONCLUSIONS: Replicase-based DNA vaccines fulfill the stringent criteria for an allergy DNA vaccine, i.e. low dose, strong Th1 immunogenicity and memory, lack of 'therapy-induced' IgE production and anaphylactic side effects. Moreover, by triggering apoptosis in transfected cells, their unique 'immunize and disappear' feature minimizes the hypothetical risks of genomic integration or induction of autoimmunity.

The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.

In healthy cells, fusion and fission events participate in regulating mitochondrial morphology. Disintegration of the mitochondrial reticulum into multiple punctiform organelles during apoptosis led us to examine the role of Drp1, a dynamin-related protein that mediates outer mitochondrial membrane fission. Upon induction of apoptosis, Drp1 translocates from the cytosol to mitochondria, where it preferentially localizes to potential sites of organelle division. Inhibition of Drp1 by overexpression of a dominant-negative mutant counteracts the conversion to a punctiform mitochondrial phenotype, prevents the loss of the mitochondrial membrane potential and the release of cytochrome c, and reveals a reproducible swelling of the organelles. Remarkably, inhibition of Drp1 blocks cell death, implicating mitochondrial fission as an important step in apoptosis.

Mining the melanosome for tumor vaccine targets: P.polypeptide is a novel tumor-associated antigen.

To identify novel, tumor-specific target antigens for vaccine development, we studied immune responses to P.polypeptide, an M(r) 110,000 integral melanosomal membrane protein associated with the Prader-Willi syndrome. Together with expressed sequence tag (EST) and serial analyses of gene expression (SAGE) library analyses, reverse transcription-PCR and Northern blotting verified that P.polypeptide expression was limited to melanoma and melanocytes. A single dominant epitope corresponding to positions 427-435 (IMLCLIAAV) was identified using allele-specific epitope forecasting combined with work in HLA-A*0201/K(b) transgenic mice. This epitope was then used to generate de novo human P.polypeptide-specific CD8+ T cells capable of recognizing P.polypeptide expressing human tumor cell lines in an HLA-A*0201-restricted fashion. Thus, P.polypeptide may be valuable in the creation of novel therapeutic anticancer vaccines.

Designing immune responses with genetic immunization and immunostimulatory DNA sequences.

Genetic immunization or DNA vaccination represents a rapidly developing technology with new perspectives for the prevention and therapy of infectious diseases and it offers new approaches for the treatment of autoimmunity, tumors and even allergy. DNA vaccines are comprised of plasmid DNA which encodes antigen molecules directly in the transfected cells of a target organism. In contrast to protein-induced immune responses, DNA vaccines stimulate both humoral and cell-mediated immune reactions. In the present review we present a palette of unique features of genetic immunization like the effect of CpG motifs, the influence of mode and site of gene delivery and the modulation of immune responses by co-delivery of cytokines, colony stimulating factors, adhesion molecules and other stimulatory molecules. In addition, modulation of the immune response via translation, processing and presentation will be discussed, which in sum demonstrate the elegant possibilities of genetic immunization to induce tailor-made immune responses.

Olefin metathesis in supercritical carbon dioxide.

Liquid or supercritical carbon dioxide (scCO(2)) is a versatile reaction medium for ring-opening metathesis polymerization (ROMP) and ring-closing olefin metathesis (RCM) reactions using well-defined metal catalysts. The molybdenum alkylidene complex 1 and ruthenium carbenes 2 and 3 bearing PCy(3) or N-heterocyclic carbene ligands, respectively, can be used and are found to exhibit efficiency similar to that in chlorinated organic solvents. While compound 1 is readily soluble in scCO(2), complexes 2 and 3 behave like heterogeneous catalysts in this reaction medium. Importantly, however, the unique properties of scCO(2) provide significant advantages beyond simple solvent replacement. This pertains to highly convenient workup procedures both for polymeric and low molecular weight products, to catalyst immobilization, to reaction tuning by density control (RCM versus acyclic diene metathesis polymerization), and to applications of scCO(2) as a protective medium for basic amine functions. The latter phenomenon is explained by the reversible formation of the corresponding carbamic acid as evidenced by (1)H NMR data obtained in compressed CO(2). Together with its environmentally and toxicologically benign character, these unique physicochemical features sum up to a very attractive solvent profile of carbon dioxide for sustainable synthesis and production.

Nucleic acid for the treatment of cancer: genetic vaccines and DNA adjuvants.

Despite some interesting pilot experiments more than a century ago, nucleic acid has only recently been added to the list of agents used for the prevention and therapy of cancer. Two distinct features of nucleic acids are used for this purpose: in DNA and RNA vaccines, genetic information for pathogen- or tumor-derived antigens is delivered to the host who then produces the encoded antigen and initiates an immune response. In DNA adjuvants, immunostimulatory sequences (CpG motifs) present in DNA of bacterial origin are used. Such sequences are delivered in the form of oligonucleotides or within the sequence of DNA vaccine. In addition, CpG oligonucleotides by themselves have successfully been used to stimulate the immune system in an antigen-independent manner for the treatment of experimental tumors. DNA and RNA vaccines for the treatment and prevention of cancer and other diseases suffer from two some shortcomings: insufficient immunogenicity and--in the case of RNA--low stability. A variety of strategies are being explored to improve the efficacy of nucleic acid vaccines (genetic vaccines) especially for self-antigens in the case of cancer. Among the most recent improvements are self-replicating RNA vaccines and replicase-based DNA-vaccines in which antigen expression is under the control of an alphaviral replicase. Despite highly promising results in many animal tumor models the efficacy of nucleic acid vaccines and adjuvants in the clinic remains to be seen.

Removal of the circumsporozoite protein (CSP) glycosylphosphatidylinositol signal sequence from a CSP DNA vaccine enhances induction of CSP-specific Th2 type immune responses and improvesprotection against malaria infection.

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.

Supercritical carbon dioxide as solvent and temporary protecting group for rhodium-catalyzed hydroaminomethylation.

Supercritical carbon dioxide (scCO2) acts simultaneously as solvent and temporary protecting group during homogeneously rhodium-catalyzed hydroaminomethylation of ethyl methallylic amine. Cyclic amines are formed as the major products in scCO,, whereas the cyclic amide is formed preferentially in conventional solvents. Multinuclear high-pressure NMR spectroscopy revealed that this selectivity switch is mainly due to reversible formation of the carbamic acid in the solvent CO2, which reduces the tendency for intramolecular ring closure at the Rh-acyl intermediate. These results substantiate the general concept of using scCO2 as a protective medium for amines in homogeneous catalysis and demonstrate for the first time its application for selectivity control.

Complement activation and inflammatory processes in Drusen formation and age related macular degeneration.

Recent studies implicate inflammation and complement mediated attack as early events in drusen biogenesis. The investigations described here sought to determine whether primary sites of complement activation could be identified within drusen substructure, and whether known inhibitors of the terminal pathway of complement are present in drusen and/or retinal pigmented epithelial (RPE) cells that lie in close proximity to drusen. Immunohistochemical examination shows two fluid phase regulators of the terminal pathway, vitronectin (Vn, S-protein) and clusterin (apolipoprotein J), to be present in drusen; Vn also accumulates in the cytoplasm of RPE cells that are closely associated with drusen. The membrane associated complement inhibitor, complement receptor 1, is also localized in drusen, but it is not detected in RPE cells immunohistochemically. In contrast, a second membrane associated complement inhibitor, membrane cofactor protein, is present in drusen associated RPE cells, as well as in small, spherical substructural elements within drusen. These previously unidentified elements also show strong immunoreactivity for proteolytic fragments of complement component C3 that are characteristically deposited at sites of complement activation. It is proposed that these structures represent residual debris from degenerating RPE cells that are the targets of complement attack. It is likely that RPE cell debris entrapped between the RPE monolayer and Bruch's membrane serves as a chronic inflammatory stimulus and a potential nucleation site for drusen formation. Thus, the process of drusen biogenesis may be envisaged as a secondary manifestation of primary RPE pathology that is exacerbated by consequences of local inflammatory processes.

Genetic vaccination against malaria infection by intradermal and epidermal injections of a plasmid containing the gene encoding the Plasmodium berghei circumsporozoite protein.

The circumsporozoite protein (CSP) from the surface of sporozoite stage Plasmodium sp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encoding Plasmodium berghei CSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced by P. berghei CSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.

Steel-promoted oxidation of olefins in supercritical carbon dioxide using dioxygen in the presence of aldehydes

Oxidation of olefins occurs effectively in supercritical carbon dioxide as the reaction medium with dioxygen as the primary oxidant and aldehydes as sacrificial co-oxidants. No catalyst is required, but the reaction is promoted by the stainless steel of the reactor walls. Depending on the substrate, vinylic oxidation or epoxidation can be the prevailing pathway. Epoxidation is particularly effective for substrates with internal double bonds and for long-chain terminal olefins.

Mechanistic aspects of dihydrogen activation and transfer during asymmetric hydrogenation in supercritical carbon dioxide.

A new "CO2-philic" chiral rhodium diphosphinite complex was synthesized and applied as catalyst precursor in the asymmetric hydrogenation of dimethyl itaconate in scCO2, scC2H6 and various liquid organic solvents. Deuterium labeling studies and parahydrogen-induced polarization (PHIP) NMR experiments were used to provide the first detailed mechanistic insight into the activation and transfer of the dihydrogen molecule during hydrogenation in scCO2. Chemical interactions between CO2 and reactive intermediates of the catalytic pathway could be excluded as possible explanations for the experimentally verified difference in the catalytic behavior in scCO2 and hexane.

Identification of a MHC class II-restricted human gp100 epitope using DR4-IE transgenic mice.

CD4+ T cells play a central role in the induction and persistence of CD8+ T cells in several models of autoimmune and infectious disease. To improve the efficacy of a synthetic peptide vaccine based on the self-Ag, gp100, we sought to provide Ag-specific T cell help. To identify a gp100 epitope restricted by the MHC class II allele with the highest prevalence in patients with malignant melanoma (HLA-DRB1*0401), we immunized mice transgenic for a chimeric human-mouse class II molecule (DR4-IE) with recombinant human gp100 protein. We then searched for the induction of CD4+ T cell reactivity using candidate epitopes predicted to bind to DRB1*0401 by a computer-assisted algorithm. Of the 21 peptides forecasted to bind most avidly, murine CD4+ T cells recognized the epitope (human gp10044-59, WNRQLYPEWTEAQRLD) that was predicted to bind best. Interestingly, the mouse helper T cells also recognized human melanoma cells expressing DRB1*0401. To evaluate whether human CD4+ T cells could be generated from the peripheral blood of patients with melanoma, we used the synthetic peptide h-gp10044-59 to sensitize lymphocytes ex vivo. Resultant human CD4+ T cells specifically recognized melanoma, as measured by tumor cytolysis and the specific release of cytokines and chemokines. HLA class II transgenic mice may be useful in the identification of helper epitopes derived from Ags of potentially great clinical utility.

The promise of nucleic acid vaccines.

Establishing the effective use of 'naked' nucleic acids as vaccines would undoubtedly be one of the most important advances in the history of vaccinology. While nucleic acids show much promise for use as vaccine vectors in experimental animals, not a single naked nucleic acid vector has been approved for use in humans. Indeed, data from human clinical trials is scant: nucleic acid vaccines have not been clearly demonstrated to have any convincing efficacy in the prevention or treatment of infectious disease or cancer. Here we illustrate possible mechanisms underlying effective nucleic acid vaccination. We focus on progress that has been made in the improvement of their function. Additionally, we identify promising new strategies and try to forecast future developments that could lead to the real success of nucleic acid vaccines in the prevention and treatment of human disease.

Enhancement of tumor-specific immune response with plasmid DNA replicon vectors.

To enhance the immunogenicity of nucleic acid vaccines, we used plasmid DNA vectors that contained replicons derived from the prototype alphavirus, Sindbis, and another alphavirus, Semliki Forest virus. When transfected into cells or injected directly into animal muscle, these plasmids launch a self-replicating RNA vector (replicon) which in turn directs the expression of a model tumor antigen. Immunization with plasmid DNA replicons elicited immune responses at doses 100 to 1000-fold lower than conventional DNA plasmids and effectively treated mice bearing an experimental tumor expressing the model antigen. Significantly, replicon-based DNA plasmids did not produce a greater quantity of antigen; instead, antigen production differed qualitatively. Plasmid DNA replicons mediated antigen production that was homogeneous in all transfected cells and associated with the apoptotic death of the host cells. Because of their safety and efficacy, plasmid DNA replicons may be useful in the development of recombinant vaccines for infectious diseases and cancer.