Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ～10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.     

Altered immunodominance hierarchies of CD8+ T cells in the spleen after infection at different sites is contingent on high virus inoculum

Activated epitope-specific CD8+ T cells after virus infection can be organized into hierarchies (immunodominance), based on their ability to focus the response on few viral determinants. The mechanisms responsible for immunodominance can be multifactorial, with CD8+ T cells precursor frequencies recently highlighted as a key regulator. Employing the LCMV infection model, we demonstrate that the hierarchies were altered when comparing different sites of infection but only at high viral doses. These findings have significant implications when investigating immunity to viruses with different replication abilities that may override the influence of T cell precursor frequencies.     

Migration Stimulating Factor (MSF) promotes fibroblast migration by inhibiting AKT

The protein kinase AKT is activated strongly by many motogenic growth factors, yet has recently been shown capable of inhibiting migration in several cell types. Here we report that treatment with Migration Stimulating Factor (MSF), a truncated form of fibronectin that promotes the migration of many cell types, inhibits AKT activity in human fibroblasts and endothelial cells. In fibroblasts, treatment with either MSF or the AKT inhibitor, Akti-1/2, stimulated migration into 3D collagen gels to a similar extent and the effects of Akti-1/2 on migration could be blocked by the expression of an inhibitor-resistant mutant, AKT1 W80A. These data indicate that MSF promotes fibroblast migration, at least in part, by inhibiting the activity of AKT.     

Tissue section AFM: In situ ultrastructural imaging of native biomolecules

Conventional approaches for ultrastructural high-resolution imaging of biological specimens induce profound changes in bio-molecular structures. By combining tissue cryo-sectioning with non-destructive atomic force microscopy (AFM) imaging we have developed a methodology that may be applied by the non-specialist to both preserve and visualize bio-molecular structures (in particular extracellular matrix assemblies) in situ. This tissue section AFM technique is capable of: i) resolving nm–µm scale features of intra- and extracellular structures in tissue cryo-sections; ii) imaging the same tissue region before and after experimental interventions; iii) combining ultrastructural imaging with complimentary microscopical and micromechanical methods. Here, we employ this technique to: i) visualize the macro-molecular structures of unstained and unfixed fibrillar collagens (in skin, cartilage and intervertebral disc), elastic fibres (in aorta and lung), desmosomes (in nasal epithelium) and mitochondria (in heart); ii) quantify the ultrastructural effects of sequential collagenase digestion on a single elastic fibre; iii) correlate optical (auto fluorescent) with ultrastructural (AFM) images of aortic elastic lamellae.     

Prophylactic and therapeutic effects of Mytilus edulis fatty acids on adjuvant-induced arthritis in male Wistar rats

Lipid-rich fractions from the flesh tissue of Mytilus edulis were obtained by solvent extraction and chromatographic separation, and tested for anti-inflammatory (AI) activity in vitro and in vivo. Inhibition of leukotriene production by isolated human neutrophils in response to calcium ionophore stimulation in the presence of exogenous arachidonic acid substrate was demonstrated for the hydrolysed triglyceride fraction of the crude lipid extract. This fraction was subsequently tested for in vivo AI activity using the mycobacterial adjuvant-induced polyarthritis rat model. The hydrolysed triglyceride fraction showed significant AI activity when dosed therapeutically (10 mg/kg BW/day, p.o., for 6 days from the onset of arthritis), decreasing body weight loss by 55% and hind paw swelling by 65% compared to the arthritic control. The (non-hydrolysed) crude lipid extract was effective when dosed prophylactically (30 mg/kg BW/day, p.o., for 16 days starting on day ?2 of arthritigen inoculation). Structural analysis by GC and GC–MS revealed in the extracts an abundance of EPA (20:5n-3) and DHA (22:6n-3) (37% of total fatty acids), along with a small quantity of a rare anti-inflammatory n-3 analogue of arachidonic acid, namely 7, 11, 14, 17-eicosatetraenoic acid (20:4n-3).