Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae.

Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).

Mapping of a gene predisposing to early-onset Alzheimer's disease to chromosome 14q24.3.

Genetic linkage studies with chromosome 21 DNA markers and mutation analysis of the beta-amyloid protein precursor gene located in 21q21.3 have indicated that early-onset Alzheimer's disease (EOAD) is a heterogeneous disorder for which at least one other chromosomal locus exists. We examined two extended histopathologically confirmed EOAD pedigrees, AD/A and AD/B, with highly informative short tandem repeat (STR) polymorphisms and found complete linkage of the disease to a (CA)n dinucleotide repeat polymorphism at locus D14S43 in 14q24.3 (Zmax = 13.25 at theta = 0.0). Using additional chromosome 14 STR polymorphisms we were able to delineate the region containing the EOAD gene to an area of, at most, 8.9 centiMorgans between D14S42 and D14S53, flanking D14S43 on both sides.

A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q.

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

The immunogenicity of chimeric antibodies.

Mice were immunized with model xenogeneic (both the VH frameworks and the CH domains of human origin), chimeric (just VH frameworks human), or self antibodies, and the antiantibody responses were dissected. Only the self antibody did not elicit a response. A strong response was elicited by the most xenogeneic antibody with approximately 90% against the C and approximately 10% against the V. The anti-V response was not attenuated in the chimeric antibody, demonstrating that foreign VH frameworks can be sufficient to lead to a strong antiantibody response. The magnitude of this xenogeneic anti-VH response was similar to that of the allotypic response elicited by immunizing mice of the Igha allotype with an Ighb antibody. Thus, although chimerization can diminish antiantibody responses, attention should be paid both to V region immunogenicity and to polymorphism.

Transglutaminase: new insights into gelatin nanoparticle cross-linking.

Gelatin nanoparticles (GNPs) have demonstrated to be beneficial as a biodegradable and biocompatible delivery system. So far, nanoparticles prepared by the two-step desolvation technique were subsequently cross-linked by glutaraldehyde to guarantee storage stability. Although in vivo and in vitro toxicological studies have not revealed any glutaraldehyde related undesired effects, an alternative to chemical cross-linking could ease future clinical use in humans. Therefore, the recombinant enzyme microbial transglutaminase was used to examine its cross-linking abilities in nanoparticle production. Various process parameters, such as incubation time, temperature, medium, pH and the particle purification were evaluated regarding their impact on particle size and its distribution. Cross-linking reactions were best at 25°C using an ion-free solvent at a neutral pH and have been terminated after 12 h. Preliminary storage stability testing indicated adequate consistency of particle size and particle distribution making transglutaminase a potential candidate for glutaraldehyde substitution in future GNP production.

A novel technique for selective NF-kappaB inhibition in Kupffer cells: contrary effects in fulminant hepatitis and ischaemia-reperfusion.

BACKGROUND AND AIMS: The transcription factor nuclear factor kappa B (NF-kappaB) has risen as a promising target for anti-inflammatory therapeutics. In the liver, however, NF-kappaB inhibition mediates both damaging and protective effects. The outcome is deemed to depend on the liver cell type addressed. Recent gene knock-out studies focused on the role of NF-kappaB in hepatocytes, whereas the role of NF-kappaB in Kupffer cells has not yet been investigated in vivo. Here we present a novel approach, which may be suitable for clinical application, to selectively target NF-kappaB in Kupffer cells and analyse the effects in experimental models of liver injury. METHODS: NF-kappaB inhibiting decoy oligodeoxynucleotides were loaded upon gelatin nanoparticles (D-NPs) and their in vivo distribution was determined by confocal microscopy. Liver damage, NF-kappaB activity, cytokine levels and apoptotic protein expression were evaluated after lipopolysaccharide (LPS), d-galactosamine (GalN)/LPS, or concanavalin A (ConA) challenge and partial warm ischaemia and subsequent reperfusion, respectively. RESULTS: D-NPs were selectively taken up by Kupffer cells and inhibited NF-kappaB activation. Inhibition of NF-kappaB in Kupffer cells improved survival and reduced liver injury after GalN/LPS as well as after ConA challenge. While anti-apoptotic protein expression in liver tissue was not reduced, pro-apoptotic players such as cJun N-terminal kinase (JNK) were inhibited. In contrast, selective inhibition of NF-kappaB augmented reperfusion injury. CONCLUSIONS: NF-kappaB inhibiting decoy oligodeoxynucleotide-loaded gelatin nanoparticles is a novel tool to selectively inhibit NF-kappaB activation in Kupffer cells in vivo. Thus, liver injury can be reduced in experimental fulminant hepatitis, but increased at ischaemia-reperfusion.

Importance of using complementary process analyzers for the process monitoring, analysis, and understanding of freeze drying.

The aim of the present paper is to demonstrate the importance of using complementary process analyzers (PAT tools) for the process monitoring, analysis, and understanding of freeze drying. A mannitol solution was used as a model system. Raman spectroscopic, near-infrared (NIR) spectroscopic, plasma emission spectroscopic, and wireless temperature measurements (TEMPRIS) were simultaneously performed in-line and real-time during each freeze-drying experiment. The combination of these four process analyzers to monitor a freeze-drying process is unique. The Raman and NIR data were analyzed using principal component analysis (PCA) and multivariate curve resolution (MCR), while the plasma emission spectroscopic and wireless temperature measurement data were analyzed using univariate data analysis. It was shown that the considered process analyzers do not only complement but also mutually confirm each other with respect to process step end points, physical phenomena occurring during freeze drying (process understanding), and product characterization (solid state). Furthermore and most important, the combined use of the process analyzers helped to identify flaws in previous studies in which these process analyzers were studied individually. Process analyzers might wrongly indicate that some process steps are fulfilled. Finally, combining the studied process analyzers also showed that more information per process analyzer can be obtained than previously described. A combination of Raman and plasma emission spectroscopy seems favorable for the monitoring of nearly all critical freeze-drying process aspects.

Reshaping human antibodies: grafting an antilysozyme activity.

The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the "humanizing" of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

Rational design of a stable, freeze-dried virus-like particle-based vaccine formulation.

Virus-like particles (VLPs) have been extensively explored as vaccine candidates since the mid-1980s. Numerous VLPs have been designed as vaccines for prevention of virus-induced infectious diseases and for the therapeutical treatment of chronic diseases and drug addiction. Recently, a vaccine against nicotine addiction, which is based on VLPs of the RNA phage Qb to which nicotine haptens are covalently coupled via succinimate linkers (NicQb), has attracted a great deal of interest. Phase II clinical trials with this vaccine have shown that it is efficacious for smoking cessation in humans when antinicotine antibody levels are sufficiently high. For commercialization, the development of stable formulations enabling storage for prolonged periods is required. Hereby, lyophilization, a well-established method leading to stable and dry formulations, is often applied. In this study, we investigated the influence of different pH values and various excipients such as surfactants, polyols, sugars, and salts on the stability of NicQb in liquid formulations, during freeze thawing, freeze drying, and finally upon storage of the dried product. Lyophilized NicQb formulations were developed which were stable over 6 months at ambient temperature with fully retained biological activity. Hereby, it was found that a combination of the surfactant polysorbate 20 and the disaccharide trehalose was capable to prevent NicQb aggregation and to preserve its integrity (nicotine binding and integrity of VLP shell). Furthermore, asymmetrical flow field-flow fractionation (AF4), a new, promising analytical tool, was established for the investigation of VLP stability.

Protein engineering.

Ten years of protein engineering have seen the synthesis of novel therapeutic agents and the analysis of the structure, activity, specificity, stability and folding pathways of proteins. It is hoped that protein engineering will eventually lead to the design of novel catalytic sites on either novel or existing proteins.

Development of a sprayable hydrogel formulation for the skin application of therapeutic antibodies.

A formulation of an antibody with antibacterial properties for topical use on Staphylococcal skin infections was developed and characterized. The best formulation was obtained with 1.5% (w/v) sodium carboxymethylcellulose containing 10 mg/ml immunoglobulin. Spraying forces and rheological behavior were measured in order to characterize the hydrogel formulation. The percentage of antibody aggregates in gel as well as the antibody release, folding and target binding properties of the released antibody were analyzed to proof an acceptable shelf life and no significant changes in the activity of the antibody over time. No microbial contamination was observed in the chosen non-airless application container. Functional testing of the topical skin formulation was performed with an ex vivo biopsy culture model of dog skin. Histological analysis indicated efficacy in protection from Staphylococcus mediated skin damage and antibody delivery restricted to the epidermal surface. The results demonstrate that this hydrogel is suitable for cutaneous antibody applications in the medical field.

Correlation of in vivo and in vitro release data for rh-INFalpha lipid implants.

Previous in vitro experiments had shown that rh-INFalpha releasing tristearin implants feature promising properties making them an excellent tool for the delivery of therapeutic proteins. Sustained release for periods up to one month could be achieved, associated with high protein stabilization. The objective of this study was to investigate for the first time the in vivo release properties of these implants in rabbits and to establish an in vivo-in vitro correlation. Computer modeling was used to simulate rh-INFalpha serum levels based on pharmacokinetic data. Protein serum concentrations on therapeutically relevant nearly constant levels could be detected for 9 days. Modeling revealed that in vivo release correlated closely with the release monitored in vitro.

Structure determination and crystal chemistry of large repeat mixed-layer hexaferrites.

Hexaferrites are an important class of magnetic oxides with applications in data storage and electronics. Their crystal structures are highly modular, consisting of Fe- or Ba-rich close-packed blocks that can be stacked in different sequences to form a multitude of unique structures, producing large anisotropic unit cells with lattice parameters typically >100 Šalong the stacking axis. This has limited atomic-resolution structure solutions to relatively simple examples such as Ba2Zn2Fe12O22, whilst longer stacking sequences have been modelled only in terms of block sequences, with no refinement of individual atomic coordinates or occupancies. This paper describes the growth of a series of complex hexaferrite crystals, their atomic-level structure solution by high-resolution synchrotron X-ray diffraction, electron diffraction and imaging methods, and their physical characterization by magnetometry. The structures include a new hexaferrite stacking sequence, with the longest lattice parameter of any hexaferrite with a fully determined structure.

Engineered mutants of HGF/SF with reduced binding to heparan sulphate proteoglycans, decreased clearance and enhanced activity in vivo.

BACKGROUND: Although a number of growth factors bind cell-surface heparan sulphate proteoglycans (HSPGs), the role of this interaction is unclear except for fibroblast growth factor which requires HSPG binding for signalling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signalling. RESULTS: To map the binding sites for HSPG and the Met receptor in HGF/SF, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in the hairpin structure of the amino-terminal domain or in kringle 2 have been replaced. Two of the mutants (HP1 and HP2) showed greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Furthermore, when compared with wild-type HGF/SF, the HP1 mutant exhibited a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver. CONCLUSIONS: These results establish the following: the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; high-affinity binding of HGF/SF to HSPGs is not essential for signalling; one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor; and HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo.

Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift.

To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens.

Complement recruitment using bispecific diabodies.

We describe the engineering of antibody fragments produced in bacteria for recruitment of complement effector functions. From a phage display repertoire we isolated human antibody fragments directed against complement C1q, and linked these to lysozyme-specific antibody fragments, creating bispecific antibodies (diabodies). One diabody was able to recruit C1q, resulting in efficient lysis of lysozyme-coated sheep erythrocytes, and also induced rosette-formation of erythrocytes with human monocytes and phagocytosis after phorbol ester stimulation. These diabodies may have therapeutic applications requiring the activation of complement.

Retargeting serum immunoglobulin with bispecific diabodies.

Monospecific antibody fragments produced in bacteria lack the Fc portion of antibodies, and are therefore unable to recruit natural effector functions. We describe the use of a bispecific antibody fragment (diabody) to recruit the whole spectrum of antibody effector functions by retargeting serum immunoglobulin (Ig). One arm of the diabody was directed against the target antigen, and the other against the serum Ig. The bispecific diabodies were able to recruit complement, induce mononuclear phagocyte respiratory burst and phagocytosis, and promote synergistic cytotoxicity towards colon carcinoma cells in conjunction with CD8+ T-cells. Further, by virtue of binding to serum Ig their half-life (beta-phase) was increased fivefold compared to a control diabody of the same molecular weight. Such bispecific diabodies may provide an attractive alternative to monoclonal antibodies for serotherapy.

Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and [gamma-32P]-ATP.

A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags. Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria. Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium. The tag was not subject to proteolysis either in the broth or in human plasma. After affinity purification the antibody fragments were phosphorylated with [gamma-32P]ATP and casein kinase II. The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities. In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity. Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.