Herpesvirus saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor.

Herpesvirus Saimiri gene 13 (HVS13) exhibits 57% identity with the predicted sequence of a T cell-derived molecule termed CTLA8. Recombinant HVS13 and CTLA8 stimulate transcriptional factor NF-kappaB activity and Interleukin-6 (IL-6) secretion in fibroblasts, and costimulate T cell proliferation. An HVS13.

Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line.

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines.

Improved bicistronic mammalian expression vectors using expression augmenting sequence element (EASE).

We have recently reported on the isolation of a 5.7 kb segment of Chinese hamster ovary cell genomic DNA, Expression Augmenting Sequence Element (EASE), which when used in bicistronic expression vectors allows the development of stable Chinese hamster ovary cell pools in a five to seven week time period that express high levels of recombinant protein (6-25 mug 10-6 cells/day depending on the protein). In the present study, we have mapped the activity of the EASE to a 2.

Preparative protein purification on underivatized silica.

This article describes the use of underivatized silica gel as a preparative stationary phase for process purification of proteins. Although silica has been frequently used as a stationary phase backbone matrix, direct adsorption of proteins on underivatized silica has not been widely exploited for industrial applications. In this study an effort was made to fundamentally understand the interaction mechanisms between a protein and silica surface by using several proteins with a wide range of isoelectric points (pIs) and surface hydrophobicity.

Identification of potent human anti-IL-1RI antagonist antibodies.

Interleukin-1 (IL-1) blockade by IL-1 receptor antagonist benefits some arthritis patients by reducing joint damage. This fact inspired us to develop antagonist human therapeutic antibodies against IL-1R(I) using phage libraries that display single-chain variable fragment (scFv) antibody fragments. Panning libraries against human IL-1R(I) generated 39 unique scFv-phage whose binding to IL-1R(I) was competed by IL-1 ligands.

Substrate specificity and inducibility of TACE (tumour necrosis factor alpha-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity.

Tumour necrosis factor alpha (TNF alpha)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor alpha, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions.

High-level expression of full-length antibodies using trans-complementing expression vectors.

Recombinant antibodies are increasingly used as therapeutics for a wide variety of diseases. Generation of cell lines expressing high levels of recombinant antibody typically requires labor-intensive cloning and screening steps. We describe a mammalian expression system for the high-level production of full-length antibody molecules.

EASE vectors for rapid stable expression of recombinant antibodies.

Over the past 10 years, monoclonal antibodies and antibody fragments have become an increasingly important source of therapeutic molecules in the biotechnology industry. Drug development strategies rely on screening large numbers of candidate molecules in search of an optimized drug candidate. This strategy requires efficient production of ten to a few hundred milligrams of candidate molecules for screening in bioassays and animal models.

The soluble form of IL-1 receptor accessory protein enhances the ability of soluble type II IL-1 receptor to inhibit IL-1 action.

Regulation of the activity of the proinflammatory cytokine IL-1 is complex, involving transcriptional and translational control, precursor processing, a receptor antagonist (IL-1ra), and a decoy receptor. Here we report that the soluble form of the IL-1 receptor accessory protein (AcP) increases the affinity of binding of human IL-1alpha and IL-1beta to the soluble human type II IL-1 receptor by approximately 100-fold, while leaving unaltered the low binding affinity of IL-1ra. Soluble AcP is present in normal human serum at an average concentration greater than 300 ng/ml.

GM-CSF treatment for Crohn's disease: a stimulating new therapy?

Crohn's disease (CD) has been classically viewed as an overactive intestinal immune response to the normal constituents of the gut flora. Most therapeutic strategies to date have tried to suppress this overactive adaptive immune response. Recently, a novel, rather alternative therapeutic strategy has been proposed, wherein the approach is to stimulate the innate immune system with growth factors.

Vaccines for parasitic diseases.

Significant effort and progress has occurred over the last several years in the development of vaccines against three main tropical parasitic diseases (malaria, leishmaniases and schistosomiasis). However, an effective vaccine is not yet available. The difficulties in developing a vaccine against parasitic disease are complicated not only by the necessity to identify (and produce) appropriate, protective antigens but also a lack of complete understanding of the types of immune responses needed for protection.

Interleukin-15 interactions with interleukin-15 receptor complexes: characterization and species specificity.

Interleukin (IL-) 2 and IL-15 share the IL-2 receptor betagamma c subunits (IL-2Rbetagamma c) but have specific, unique alpha receptor subunits. We studied species specificity of human (hu), simian (si), and mouse (mu) IL-15 and found that hu and si IL-15 behaved similarly in all systems investigated. Hu and mu IL-15 bound hu or mu IL-15Ralpha with equal high affinity in the presence or absence of IL-2Rbetagamma c and exhibited similar proliferative activities on cells containing all three subunits.

Searching sequence databases via de novo peptide sequencing by tandem mass spectrometry.

There are many computer programs that can match tandem mass spectra of peptides to database-derived sequences; however, situations can arise where mass spectral data cannot be correlated with any database sequence. In such cases, sequences can be automatically deduced de novo, without recourse to sequence databases, and the resulting peptide sequences can be used to perform homologous nonexact searches of sequence databases. This article describes details on how to implement both a de novo sequencing program called "Lutefisk," and a version of FASTA that has been modified to account for sequence ambiguities inherent in tandem mass spectrometry data.

Regulation of nuclear translocation of nuclear factor-kappaB relA: evidence for complex dynamics at the single-cell level.

We have analysed activation of nuclear factor-kappaB (NF-kappaB) in response to interleukin-1 (IL-1) in human fibroblasts by tracking intracellular distribution and levels of endogenous relA, NF-kappaB1 and inhibitor of kappaB (I-kappaB) alpha using semi-quantitative confocal microscopy. Nuclear translocation of endogenous relA correlated with I-kappaBalpha degradation during stimulation with IL-1, whereas no effects were seen on levels or localization of NF-kappaB1. During pathway activation, relA was transported up a concentration gradient, resulting in a 3-4-fold increase in nuclear levels, but without any significant decrease in cytoplasmic concentration.

RIP4 is an ankyrin repeat-containing kinase essential for keratinocyte differentiation.

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood.

pH Transitions in ion-exchange systems: role in the development of a cation-exchange process for a recombinant protein.

Unexpected transient changes in effluent pH can occur during ion-exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation-exchange resin.

Interleukin 18 is a primary mediator of the inflammation associated with dextran sulphate sodium induced colitis: blocking interleukin 18 attenuates intestinal damage.

Background And Aims: Persistent inflammation observed in inflammatory bowel disease may be the consequence of an increased or aberrant immune response to normal gut constituents or an overall immune dysregulation and imbalance. Cytokines play an important role in immune regulation and interleukin 18 (IL-18) is one such cytokine that has emerged as being instrumental in driving CD4+ T cell responses towards a Th1-type. IL-18 can also directly mediate inflammation, moderate interleukin 1 activity, and can act on cell types other than T cells.

Genomic organization of the interleukin-1 locus.

Recent additions have expanded the interleukin (IL)-1 gene family to 10 members. We have determined the order, orientation, and intergenic distance of the nine IL-1 family genes that lie on human chromosome 2. We report cDNA sequences for the mouse orthologs of three of these genes.

IL-1 and IL-18 receptors, and their extended family.

The cytokines IL-1 and IL-18 are key molecules in both the innate and the adaptive immune response. Recently, important insights have been gained into the regulation of their functions. Moreover, it has become apparent that they are members of a larger family of related receptors, some of which can also be shown to contribute to host defense.

Tumor necrosis factor-alpha converting enzyme.

Tumor necrosis factor-alpha converting enzyme (TACE/ADAM17/CD156q) is a member of the 'A Disintegrin And Metalloprotease', or ADAM, family. It is a multi-domain, type I transmembrane protein that includes an extracellular zinc-dependent protease domain. TACE expression is largely constitutive, but the surface pool is downregulated following cell activation.

A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis.

TWEAK is a member of the TNF ligand family that induces angiogenesis in vivo. We report cloning of a receptor for TWEAK (TweakR) from a human umbilical vein endothelial cell (HUVEC) library. The mature form of TweakR has only one hundred and two amino acids and six cysteine residues in its extracellular region.

Plasmacytoid monocytes/T cells: a dendritic cell lineage?

Plasmacytoid monocytes/T cells were first described in 1958, yet their origin and function have remained enigmatic. Recently a series of publications brought these cells to the forefront of immunological research. Indeed, plasmacytoid monocytes/T-cells contain natural type-I interferon producing cells and can differentiate in vitro into dendritic cells (DC).

Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule.

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation.

Dendritic cells in models of tumor immunity. Role of Flt3 ligand.

Cancer immunotherapy might be based on the administration to cancer patients of dendritic cells (DC) 'pulsed' with tumor-specific antigens. An alternative approach is to directly expand and/or activate DC in vivo using the cytokine Flt3 Ligand (FL). In mice, FL can drive large expansion of both lymphoid-related DC that appears to selectively enhance Th1-like immune responses and myeloid-related DC that enhances a more mixed Th phenotype.