Quentin Baca

The maintenance of pregnancy relies on finely tuned immune adaptations. We demonstrate that these adaptations are precisely timed, reflecting an immune clock of pregnancy in women delivering at term. Using mass cytometry, the abundance and functional responses of all major immune cell subsets were quantified in serial blood samples collected throughout pregnancy. Cell signaling-based Elastic Net, a regularized regression method adapted from the elastic net algorithm, was developed to infer and prospectively validate a predictive model of interrelated immune events that accurately captures the chronology of pregnancy. Model components highlighted existing knowledge and revealed previously unreported biology, including a critical role for the interleukin-2-dependent STAT5ab signaling pathway in modulating T cell function during pregnancy. These findings unravel the precise timing of immunological events occurring during a term pregnancy and provide the analytical framework to identify immunological deviations implicated in pregnancy-related pathologies.

Stroke is a leading cause of cognitive impairment and dementia, but the mechanisms that underlie post-stroke cognitive decline are not well understood. Stroke produces profound local and systemic immune responses that engage all major innate and adaptive immune compartments. However, whether the systemic immune response to stroke contributes to long-term disability remains ill-defined. We used a single-cell mass cytometry approach to comprehensively and functionally characterize the systemic immune response to stroke in longitudinal blood samples from 24 patients over the course of 1 year and correlated the immune response with changes in cognitive functioning between 90 and 365 days post-stroke. Using elastic net regularized regression modelling, we identified key elements of a robust and prolonged systemic immune response to ischaemic stroke that occurs in three phases: an acute phase (Day 2) characterized by increased signal transducer and activator of transcription 3 (STAT3) signalling responses in innate immune cell types, an intermediate phase (Day 5) characterized by increased cAMP response element-binding protein (CREB) signalling responses in adaptive immune cell types, and a late phase (Day 90) by persistent elevation of neutrophils, and immunoglobulin M+ (IgM+) B cells. By Day 365 there was no detectable difference between these samples and those from an age- and gender-matched patient cohort without stroke. When regressed against the change in the Montreal Cognitive Assessment scores between Days 90 and 365 after stroke, the acute inflammatory phase Elastic Net model correlated with post-stroke cognitive trajectories (r = -0.692, Bonferroni-corrected P = 0.039). The results demonstrate the utility of a deep immune profiling approach with mass cytometry for the identification of clinically relevant immune correlates of long-term cognitive trajectories.

Trypsin is covalently linked to a photopolymerized sol-gel monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of N(alpha)-benzoyl-l-arginine ethyl ester (BAEE) and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith is via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group links to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N alpha-benzoyl-L-arginine (BA), the digestion product of BAEE. The BA is separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, we determined that 97 ng of trypsin is bound to the 1-cm microreactor located at the entrance of capillary column. The bioactivity of the trypsin-PSG-PEG microreactor at 20 degrees C for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20 degrees C after 16 h of incubation time.