Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors

Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation

Agreement Between Transesophageal Echocardiographic Tricuspid Annular Plane Systolic Excursion Measurement Methods in Cardiac Surgery Patients.

OBJECTIVE: To assess the agreement between 2-dimensional tricuspid annular plane systolic excursion (2D-TAPSE), 2D-TAPSE-apex, and 2D speckle tracking echocardiography (STE-TAPSE) in a cross-section of routine cardiac surgery patients. DESIGN: Retrospective, observational study. SETTING: Tertiary, academic referral hospital. PARTICIPANTS: Patients undergoing elective cardiac surgery with intraoperative transesophageal echocardiography (TEE) imaging. INTERVENTIONS: TEE imaging was reviewed and evaluated for the following three different measurements of transthoracic echocardiography-TAPSE surrogates: 2D-TAPSE, 2D-TAPSE-apex, and STE-TAPSE. Statistical analyses, including 2-sample t tests, linear regression, and agreement using the Bland-Altman methods, were performed. MEASUREMENTS AND MAIN RESULTS: Modest correlation was demonstrated between STE-TAPSE and 2D-TAPSE (R CONCLUSIONS: Correlation and minimal bias were found between the several proposed TEE surrogates of transthoracic echocardiography-TAPSE; however, there was poor agreement. Therefore, these surrogates are not interchangeable, and each method needs to be separately validated for clinical use to relevant perioperative outcomes

Imaging the dynamics and microstructure of fibrin clot polymerization in cardiac surgical patients using spectrally encoded confocal microscopy

During cardiac surgery with cardiopulmonary bypass (CPB), altered hemostatic balance may disrupt fibrin assembly, predisposing patients to perioperative hemorrhage. We investigated the utility of a novel device termed spectrally-encoded confocal microscopy (SECM) for assessing fibrin clot polymerization following heparin and protamine administration in CPB patients. SECM is a novel, high-speed optical approach to visualize and quantify fibrin clot formation in three dimensions with high spatial resolution (1.0 μm) over a volumetric field-of-view (165 × 4000 × 36 μm). The measurement sensitivity of SECM was first determined using plasma samples from normal subjects spiked with heparin and protamine. Next, SECM was performed in plasma samples from patients on CPB to quantify the extent to which fibrin clot dynamics and microstructure were altered by CPB exposure. In spiked samples, prolonged fibrin time (4.4 ± 1.8 to 49.3 ± 16.8 min, p < 0.001) and diminished fibrin network density (0.079 ± 0.010 to 0.001 ± 0.002 A.U, p < 0.001) with increasing heparin concentration were reported by SECM. Furthermore, fibrin network density was not restored to baseline levels in protamine-treated samples. In CPB patients, SECM reported lower fibrin network density in protaminized samples (0.055 ± 0.01 A.U. [Arbitrary units]) vs baseline values (0.066 ± 0.009 A.U.) (p = 0.03) despite comparable fibrin time (baseline = 6.0 ± 1.3, protamine = 6.4 ± 1.6 min, p = 0.5). In these patients, additional metrics including fibrin heterogeneity, length and straightness were quantified. Note, SECM revealed that following protamine administration with CPB exposure, fibrin clots were more heterogeneous (baseline = 0.11 ± 0.02 A.U, protamine = 0.08 ± 0.01 A.U, p = 0.008) with straighter fibers (baseline = 0.918 ± 0.003A.U, protamine = 0.928 ± 0.0006A.U. p < 0.001). By providing the capability to rapidly visualize and quantify fibrin clot microstructure, SECM could furnish a new approach for assessing clot stability and hemostasis in cardiac surgical patients. © 2021 Wiley Periodicals LLC.12 month embargo; first published: 10 May 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Imaging the dynamics and microstructure of fibrin clot polymerization in cardiac surgical patients using spectrally encoded confocal microscopy

During cardiac surgery with cardiopulmonary bypass (CPB), altered hemostatic balance may disrupt fibrin assembly, predisposing patients to perioperative hemorrhage. We investigated the utility of a novel device termed spectrally-encoded confocal microscopy (SECM) for assessing fibrin clot polymerization following heparin and protamine administration in CPB patients. SECM is a novel, high-speed optical approach to visualize and quantify fibrin clot formation in three dimensions with high spatial resolution (1.0 μm) over a volumetric field-of-view (165 × 4000 × 36 μm). The measurement sensitivity of SECM was first determined using plasma samples from normal subjects spiked with heparin and protamine. Next, SECM was performed in plasma samples from patients on CPB to quantify the extent to which fibrin clot dynamics and microstructure were altered by CPB exposure. In spiked samples, prolonged fibrin time (4.4 ± 1.8 to 49.3 ± 16.8 min, p < 0.001) and diminished fibrin network density (0.079 ± 0.010 to 0.001 ± 0.002 A.U, p < 0.001) with increasing heparin concentration were reported by SECM. Furthermore, fibrin network density was not restored to baseline levels in protamine-treated samples. In CPB patients, SECM reported lower fibrin network density in protaminized samples (0.055 ± 0.01 A.U. [Arbitrary units]) vs baseline values (0.066 ± 0.009 A.U.) (p = 0.03) despite comparable fibrin time (baseline = 6.0 ± 1.3, protamine = 6.4 ± 1.6 min, p = 0.5). In these patients, additional metrics including fibrin heterogeneity, length and straightness were quantified. Note, SECM revealed that following protamine administration with CPB exposure, fibrin clots were more heterogeneous (baseline = 0.11 ± 0.02 A.U, protamine = 0.08 ± 0.01 A.U, p = 0.008) with straighter fibers (baseline = 0.918 ± 0.003A.U, protamine = 0.928 ± 0.0006A.U. p < 0.001). By providing the capability to rapidly visualize and quantify fibrin clot microstructure, SECM could furnish a new approach for assessing clot stability and hemostasis in cardiac surgical patients. © 2021 Wiley Periodicals LLC.12 month embargo; first published: 10 May 2021This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]