Identification and in vivo characterization of a brain-penetrating nanobody.

BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.

Early weaning stress induces chronic functional diarrhea, intestinal barrier defects, and increased mast cell activity in a porcine model of early life adversity.

BACKGROUND: Early life adversity (ELA) is a risk factor for development of gastrointestinal disorders later in life. The underlying mechanisms through which ELA and sex interact to influence disease susceptibility remains poorly understood. METHODS: Utilizing a porcine early weaning stress (EWS) model to mimic ELA, we investigated the long-term effects of EWS on functional diarrhea, ileal permeability, mast cell activity and mast cell relationship with enteric ganglia. KEY RESULTS: Juvenile and adult EWS pigs exhibited chronic, functional diarrhea (EWS 43.6% vs late wean control(LWC) 4.8%, P<.0001), increased intestinal permeability (2 fold increase EWS vs LWC, P<.0001), and mast cell numbers (at 7 weeks and 20 weeks ~1.6 fold increase EWS vs LWC, P<.05). Compared with EWS male castrates (Male-C), females EWS pigs exhibited more frequent diarrhea (58.8% vs 29.9%, P=.0016), and increased intestinal permeability (1-2 fold higher in EWS females, P<.001). Increased mast cell numbers and their enhanced co-localization with neuronal ganglia were observed in both Male-C and female EWS pigs; however, female pigs exhibited greater release of mast cell tryptase upon activation with c48/80 (~1.5 fold increase, P<.05), compared with Male-C pigs. CONCLUSIONS AND INFERENCES: These data demonstrate that pigs exposed to ELA exhibit increased vulnerability to functional diarrhea, intestinal permeability and mast cell activity. Further, these studies also showed that EWS female and Male-C pigs exhibited dimorphic responses to EWS with female piglets exhibited greater susceptibility and severity of diarrhea, intestinal permeability and mast cell tryptase release. Together, these findings mimic some of the key pathophysiologic findings in human functional GI disorders functional gastrointestinal disorders (FGIDs) suggesting that the EWS porcine model could be a valuable preclinical translational model for FGID research associated with ELA.

Screening and Characterization Strategies for Nanobodies Targeting Membrane Proteins.

The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites). Notably, Nanobodies generally bind conformational epitopes making them very interesting tools to stabilize, dissect, and characterize specific protein conformations. In the clinic, several Nanobodies are under evaluation either as potential drug candidates or as diagnostic tools. In recent years, we have successfully generated high-affinity, conformation-sensitive anti-γ-secretase Nanobodies. γ-Secretase is a multimeric membrane protease involved in processing of the amyloid precursor protein with high clinical relevance as mutations in its catalytic subunit (Presenilin) cause early-onset Alzheimer's disease. Advancing our knowledge on the mechanisms governing γ-secretase intramembrane proteolysis through various strategies may lead to novel therapeutic avenues for Alzheimer's disease. In this chapter, we present the strategies we have developed and applied for the screening and characterization of anti-γ-secretase Nanobodies. These protocols could be of help in the generation of Nanobodies targeting other membrane proteins.

High quality structure of cleaved PAI-1-stab.

Here we report the crystal structure of a stablilized plasminogen activator inhibitor-1 variant (PAI-1-N150H-K154T-Q301P-Q319L-M354I (PAI-1-stab)) that shows a cleavage within the reactive centre loop. The new structure is of superior quality compared to the previously determined structure of the cleaved PAI-1-A335P mutant. We present a detailed comparison of the two structures and also compare them with the structure of the active PAI-1-stab. The structural data give important insights into the working mechanism of PAI-1 and also explain the role of various stabilizing mutations.

Multiphoton photochemical and collisional effects during oxygen-atom flame detection.

A Nd:YAG-pumped dye-laser system was used to two-photon excite oxygen atoms at 225.6 nm in an atmosphericpressure CH(4)-N(2)O-N(2) flame. Subsequent emission at 844.7 nm from the directly populated state as well as a stronger emission at 777.5 nm that was due to the O(3p(3)P ? 3p(5)P) collisional-energy transfer process was monitored. Two-photon-resonant oxygen-atom and hydrogen-atom (656.3-nm) emissions were also observed in the absence of a flame. Closer examination revealed that the tightly focused probe beam was producing these atoms by promoting multiphoton photolysis of the oxidizer as well as of the fuel molecules. Thus this type of laser-diagnostic probe is potentially quite intrusive, depending on the combustion region that is probed as well as on the laser energies used.