The determination of concentrations of large therapeutic molecules, like monoclonal antibodies (mAbs), in the interstitial brain fluid (ISF) is one of the cornerstones for the translation from preclinical species to humans of treatments for neurodegenerative diseases. Microdialysis (MD) and cerebral open flow microperfusion (cOFM) are the only currently available methods for extracting ISF, and their use and characterization for the collection of large molecules in rodents have barely started. For the first time, we compared both methods at a technical and performance level for measuring ISF concentrations of a non-targetbinding mAb, trastuzumab, in awake and freely moving mice. Without correction of the data for recovery, concentrations of samples are over 10-fold higher through cOFM compared to MD. The overall similar pharmacokinetic profile and ISF exposure between MD (corrected for recovery) and cOFM indicate an underestimation of the absolute concentrations calculated with in vitro recovery. In vivo recovery (zeroflow rate method) revealed an increased extraction of trastuzumab at low flow rates and a 6-fold higher absolute concentration at steady state than initially calculated with the in vitro recovery. Technical optimizations have significantly increased the performance of both systems, resulting in the possibility of sampling up to 12 mice simultaneously. Moreover, strict aseptic conditions have played an important role in improving data quality. The standardization of these complex methods makes the unraveling of ISF concentrations attainable for various diseases and modalities, starting in this study with mAbs, but extending further in the future to RNA therapeutics, antibody-drug conjugates, and even cell therapies.

LEV decreased degranulation of CD8+ T lymphocytes which may contribute to the increased incidence of upper respiratory tract infections in LEV treated patients. Inhibition of SV2A may be responsible for this effect.

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The weakness in myasthenia gravis (MG) is mediated by T helper cell (Th)-dependent autoantibodies against neuromuscular epitopes. So far, analyzing Th phenotypes or antigen specificities has yielded very few clues to pathogenesis. Here we adopt an alternative antigen-independent approach, analyzing T cell receptor (TCR) Vb usage/ expansions in blood from 118 MG patients. We found major expansions (! five standard deviations above the mean of 118 healthy, individually age-and sex-matched controls) in diverse Vb in 21 patients (17.6%, p<0.001) among CD4 + T cells, and in 45 patients (38.1%, p<0.001) among CD8 + T cells. In informative probands, the expanded CD4+ cells consistently showed a Th cell phenotype (CD57 + CXCR5 + ) and expressed Th1 cytokines. Furthermore, their expression of markers for activation, lymphocyte trafficking and B cell-activating ability persisted for ! 3 years. Surprisingly, we noted a selective decline in the expansions/their CD57 positivity while the probands' MG was improving. CDR3 spectratyping suggested mono-or oligoclonal origins, which were confirmed by the prevalent TCR Vb CDR3 sequences of Th cells cloned from repeat bleeds. Thus, our data provide evidence for persistent clonally expanded CD4 + B helper T cell populations in the blood of MG patients. These unexpected CD4 + expansions might hold valuable clues to MG immunopathogenesis.

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