Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 105 PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

Therapeutic antibodies: Discovery and development using the ProteOn XPR36 biosensor interaction array system.

Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection.

Phage display-derived recombinant antibodies with TCR-like specificity against alpha-galactosylceramide and its analogues in complex with human CD1d molecules.

The glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of alpha-GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen-presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor-like (TCRL) specificity against the human CD1d (hCD1d)-alpha-GalCer complex. These Fab fragments were able to detect specifically hCD1d-alpha-GalCer complexes in cell-free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d(+) antigen-presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c-hCD1d-transgenic mice administered in vivo with alpha-GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of alpha-GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the alpha-GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of alpha-GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of alpha-GalCer and its analogues.

Antibody molecules discriminate between crystalline facets of a gallium arsenide semiconductor.

Seamless integration of biomolecules with manmade materials will most likely rely on molecular recognition and specific binding. In the following we show that combinatorial antibody libraries, based on the vast repertoire of the human immune system, can be harnessed to generate such binders. As a demonstration, we isolate antibody fragments that discriminate and bind selectively GaAs (111A) facets as opposed to GaAs (100). The isolated antibodies are utilized for exclusive localization of a fluorescent dye on (111A) surfaces in a structure comprising a mixture of (100) and (111A) surfaces. The potential importance of structure rigidity to facet recognition is suggested vis-a-vis published experiments with short and longer peptides.