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BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes due to their favorable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the central nervous system (CNS) remains to be demonstrated. EXPERIMENTAL APROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei Variant-specific Surface Glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, Single Photon Emission Computed Tomography (SPECT), or a combination of both methodologies. This enabled the quantification of unlabeled and (99m) Tc-labeled nanobodies using respectively a sensitive VSG-based nanobody-detection ELISA, radioactivity measurement in collected microdialysates, and SPECT image analysis. KEY RESULTS: The combined read-out methodologies demonstrate that disposition of Nb_An33 can be detected in the brain of healthy rats following intravenous injection and that inflammation-induced damage to the blood-brain barrier significantly increases the nanobody perfusion efficiency. We also illustrate the advantage of complementing SPECT analyses with intracerebral microdialysis in brain disposition studies and suggest that it is of interest to evaluate the blood-brain barrier penetrating potential of monovalent nanobodies in models of CNS-inflammation. The presented data also suggest that fast blood clearance might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can perfuse into the brain parenchyma, especially in pathological conditions where the blood-brain barrier integrity is compromised.

BACKGROUND AND PURPOSE: Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes. EXPERIMENTAL APPROACH: After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H). KEY RESULTS: We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface. CONCLUSIONS AND IMPLICATIONS: Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.

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BACKGROUND AND PURPOSE: Patients resuscitated from cardiac arrest (CA) have a high mortality rate. Prognostic evaluation based on clinical observations is uncertain and would benefit from the use of biochemical markers of hypoxic brain damage. The purpose of the study was to validate the use of the serum astroglial protein S-100 levels at admission with regard to regaining consciousness after out of hospital CA. METHODS: Fifty-eight patients resuscitated from out-of-hospital CA were followed up until they regained consciousness or until their death or permanent vegetative state occurred. Serum samples for measurement of S-100, using an immunoradiometric assay, were obtained at admission. RESULTS: At admission, the mean value+/-standard error of the mean of serum S-100 protein was significantly higher in patients who did not regain consciousness compared with patients who regained consciousness, respectively 4.66+/-0.61 microg/l and 0.84+/-0.21 microg/l. A serum S-100 value of >0.7 microg/l at admission was found to be a predictor that consciousness would not be regained, with a specificity of 85%, a sensitivity of 66.6%, a positive predictive value of 84%, a negative predictive value of 78% and an accuracy of 77.6%. CONCLUSIONS: Serum S-100 protein at admission gives reliable and independent information concerning the short term neurological outcome after resuscitation; and could be a good marker of brain cell damage.