Label-free in vitro assays predict the potency of anti-disialoganglioside chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against solid tumor cells. METHODS: We used real-time, label-free cellular impedance sensing to evaluate the potency of anti-disialoganglioside (GD2) targeting CAR T-cell products against GD2+ patient-derived GBM stem cells over a period of 2 days and 7 days in vitro. We compared CAR T products using two different modes of gene transfer: retroviral transduction and virus-free CRISPR-editing. Endpoint flow cytometry, cytokine analysis and metabolomics data were acquired and integrated to create a predictive model of CAR T-cell potency. RESULTS: Results indicated faster cytolysis by virus-free CRISPR-edited CAR T cells compared with retrovirally transduced CAR T cells, accompanied by increased inflammatory cytokine release, CD8+ CAR T-cell presence in co-culture conditions and CAR T-cell infiltration into three-dimensional GBM spheroids. Computational modeling identified increased tumor necrosis factor α concentrations with decreased glutamine, lactate and formate as being most predictive of short-term (2 days) and long-term (7 days) CAR T cell potency against GBM stem cells. CONCLUSIONS: These studies establish impedance sensing as a high-throughput, label-free assay for preclinical potency testing of CAR T cells against solid tumors.

Daily acute intermittent hypoxia to improve walking function in persons with subacute spinal cord injury: a randomized clinical trial study protocol.

BACKGROUND: Restoring community walking remains a highly valued goal for persons recovering from traumatic incomplete spinal cord injury (SCI). Recently, studies report that brief episodes of low-oxygen breathing (acute intermittent hypoxia, AIH) may serve as an effective plasticity-inducing primer that enhances the effects of walking therapy in persons with chronic (> 1 year) SCI. More persistent walking recovery may occur following repetitive (weeks) AIH treatment involving persons with more acute SCI, but this possibility remains unknown. Here we present our clinical trial protocol, designed to examine the distinct influences of repetitive AIH, with and without walking practice, on walking recovery in persons with sub-acute SCI (< 12 months) SCI. Our overarching hypothesis is that daily exposure (10 sessions, 2 weeks) to AIH will enhance walking recovery in ambulatory and non-ambulatory persons with subacute (< 12 months) SCI, presumably by harnessing endogenous mechanisms of plasticity that occur soon after injury. METHODS: To test our hypothesis, we are conducting a randomized, placebo-controlled clinical trial on 85 study participants who we stratify into two groups according to walking ability; those unable to walk (non-ambulatory group) and those able to walk (ambulatory group). The non-ambulatory group receives either daily AIH (15, 90s episodes at 10.0% O2 with 60s intervals at 20.9% O2) or daily SHAM (15, 90s episodes at 20.9% O2 with 60s intervals at 20.9% O2) intervention. The ambulatory group receives either 60-min walking practice (WALK), daily AIH + WALK, or daily SHAM+WALK intervention. Our primary outcome measures assess overground walking speed (10-Meter Walk Test), endurance (6-Minute Walk Test), and balance (Timed Up & Go Test). For safety, we also measure levels of pain, spasticity, systemic hypertension, and autonomic dysreflexia. We record outcome measures at baseline, days 5 and 10, and follow-ups at 1 week, 1 month, 6 months, and 12 months post-treatment. DISCUSSION: The goal of this clinical trial is to reveal the extent to which daily AIH, alone or in combination with task-specific walking practice, safely promotes persistent recovery of walking in persons with traumatic, subacute SCI. Outcomes from this study may provide new insight into ways to enhance walking recovery in persons with SCI. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02632422 . Registered 16 December 2015.

Novel method for action potential measurements from intact cardiac monolayers with multiwell microelectrode array technology.

The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.

Variability of Leg Kinematics during Overground Walking in Persons with Chronic Incomplete Spinal Cord Injury.

Incomplete spinal cord injury (iSCI) often leads to partial disruption of spinal pathways that are important for motor control of walking. Persons with iSCI present with deficits in walking ability in part because of inconsistent leg kinematics during stepping. Although kinematic variability is important for normal walking, growing evidence indicates that excessive variability may limit walking ability and increase reliance on assistive devices (AD) after iSCI. The purpose of this study was to assess the effects of iSCI-induced impairments on kinematic variability during overground walking. We hypothesized that iSCI results in greater variability of foot and joint displacement during overground walking compared with controls. We further hypothesized that variability is larger in persons with limited walking speed and greater reliance on ADs. To test these hypotheses, iSCI and control subjects walked overground. Kinematic variability was quantified as step-to-step foot placement variability (end-point), and variability in hip-knee, hip-ankle, and knee-ankle joint space (angular coefficient of correspondence [ACC]). We characterized sensitivity of kinematic variability to cadence, auditory cue, and AD. Supporting our hypothesis, persons with iSCI exhibited greater kinematic variability than controls, which scaled with deficits in overground walking speed (p < 0.01). Significant correlation between ACC and end-point variability, and with walking speed, indicates that both are markers of walking performance. Moreover, hip-knee and hip-ankle ACC discriminated AD use, indicating that ACC may capture AD-specific control strategies. We conclude that increased variability of foot and joint displacement are indicative of motor impairment severity and may serve as therapeutic targets to restore walking after iSCI.

Constraints on Stance-Phase Force Production during Overground Walking in Persons with Chronic Incomplete Spinal Cord Injury.

Persons with incomplete spinal cord injury (iSCI) face ongoing struggles with walking, including reduced speed and increased reliance on assistive devices (ADs). The forces underlying body weight support and gait, as measured by ground reaction forces (GRFs), are likely altered after iSCI because of weakness and AD dependence but have not been studied. The purpose of this study was to examine GRF production during overground walking after iSCI, because greater insight into GRF constraints is important for refining therapeutic interventions. Because of reduced and discoordinated motor output after iSCI, we hypothesized that persons with iSCI would exert smaller GRFs and altered GRF modifications to increased cadence compared with able-bodied (AB) persons, especially when using an AD. Fifteen persons with chronic iSCI, stratified into no AD (n = 7) and AD (n = 8) groups, walked across an instrumented walkway at self-selected and fast (115% self-selected) cadences. Fifteen age-matched AB controls walked at their own cadences and iSCI-matched conditions (cadence and AD). Results showed fore-aft GRFs are reduced in persons with iSCI compared with AB controls, with reductions greatest in persons dependent on an AD. When controlling for cadence and AD, propulsive forces were still lower in persons with iSCI. Compared with AB controls, persons with iSCI demonstrated altered GRF modifications to increased cadence. Persons with iSCI exhibit different stance-phase forces compared with AB controls, which are impacted further by AD use and slower walking speed. Minimizing AD use and/or providing propulsive biofeedback during walking could enhance GRF production after iSCI.

Effects of acute intermittent hypoxia on hand use after spinal cord trauma: A preliminary study.

OBJECTIVE: To test the hypothesis that daily acute intermittent hypoxia (AIH) combined with hand opening practice improves hand dexterity, function, and maximum hand opening in persons with chronic, motor-incomplete, cervical spinal cord injury. METHODS: Six participants completed the double-blind, crossover study. Participants received daily (5 consecutive days) AIH (15 episodes per day: 1.5 minutes of fraction of inspired oxygen [FIo2] = 0.09, 1-minute normoxic intervals) followed by 20 repetitions of hand opening practice and normoxia (sham, FIo2 = 0.21) + hand opening practice. Hand dexterity and function were quantified with Box and Block and Jebsen-Taylor hand function tests. We also recorded maximum hand opening using motion analyses and coactivity of extensor digitorum and flexor digitorum superficialis muscles using surface EMG. RESULTS: Daily AIH + hand opening practice improved hand dexterity, function, and maximum hand opening in all participants. AIH + hand opening practice improved Box and Block Test scores vs baseline in 5 participants (p = 0.057) and vs sham + hand opening practice in all 6 participants (p = 0.016). All participants reduced Jebsen-Taylor Hand Function Test (JTHF) time after daily AIH + hand opening practice (-7.2 ± 1.4 seconds) vs baseline; 4 of 6 reduced JTHF time vs sham + hand opening practice (p = 0.078). AIH + hand opening practice improved maximum hand aperture in 5 of 6 participants (8.1 ± 2.7 mm) vs baseline (p = 0.018) and sham + hand opening practice (p = 0.030). In 5 participants, daily AIH-induced changes in hand opening were accompanied by improved EMG coactivity (p = 0.029). CONCLUSIONS: This report suggests the need for further study of AIH as a plasticity "primer" for task-specific training in spinal cord injury rehabilitation. Important clinical questions remain concerning optimal AIH dosage, patient screening, safety, and effect persistence. CLINICALTRIALSGOV IDENTIFIER: NCT01272336.

Modulation of hand aperture during reaching in persons with incomplete cervical spinal cord injury.

The intact neuromotor system prepares for object grasp by first opening the hand to an aperture that is scaled according to object size and then closing the hand around the object. After cervical spinal cord injury (SCI), hand function is significantly impaired, but the degree to which object-specific hand aperture scaling is affected remains unknown. Here, we hypothesized that persons with incomplete cervical SCI have a reduced maximum hand opening capacity but exhibit novel neuromuscular coordination strategies that permit object-specific hand aperture scaling during reaching. To test this hypothesis, we measured hand kinematics and surface electromyography from seven muscles of the hand and wrist during attempts at maximum hand opening as well as reaching for four balls of different diameters. Our results showed that persons with SCI exhibited significantly reduced maximum hand aperture compared to able-bodied (AB) controls. However, persons with SCI preserved the ability to scale peak hand aperture with ball size during reaching. Persons with SCI also used distinct muscle coordination patterns that included increased co-activity of flexors and extensors at the wrist and hand compared to AB controls. These results suggest that motor planning for aperture modulation is preserved even though execution is limited by constraints on hand opening capacity and altered muscle co-activity. Thus, persons with incomplete cervical SCI may benefit from rehabilitation aimed at increasing hand opening capacity and reducing flexor-extensor co-activity at the wrist and hand.

Neuromuscular constraints on muscle coordination during overground walking in persons with chronic incomplete spinal cord injury.

OBJECTIVE: Incomplete spinal cord injury (iSCI) disrupts motor control and limits the ability to coordinate muscles for overground walking. Inappropriate muscle activity has been proposed as a source of clinically observed walking deficits after iSCI. We hypothesized that persons with iSCI exhibit lower locomotor complexity compared to able-body (AB) controls as reflected by fewer motor modules, as well as, altered module composition and activation. METHODS: Eight persons with iSCI and eight age-matched AB controls walked overground at prescribed cadences. Electromyograms of fourteen single leg muscles were recorded. Non-negative matrix factorization was used to identify the composition and activation of motor modules, which represent groups of consistently co-activated muscles that accounted for 90% of variability in muscle activity. RESULTS: Motor module number, composition, and activation were significantly altered in persons with iSCI as compared to AB controls during overground walking at self-selected cadences. However, there was no significant difference in module number between persons with iSCI and AB controls when cadence and assistive device were matched. CONCLUSIONS: Muscle coordination during overground walking is impaired after chronic iSCI. SIGNIFICANCE: Our results are indicative of neuromuscular constraints on muscle coordination after iSCI. Altered muscle coordination contributes to person-specific gait deficits during overground walking.

Daily intermittent hypoxia enhances walking after chronic spinal cord injury: a randomized trial.

OBJECTIVES: To test the hypothesis that daily acute intermittent hypoxia (dAIH) and dAIH combined with overground walking improve walking speed and endurance in persons with chronic incomplete spinal cord injury (iSCI). METHODS: Nineteen subjects completed the randomized, double-blind, placebo-controlled, crossover study. Participants received 15, 90-second hypoxic exposures (dAIH, fraction of inspired oxygen [Fio2] = 0.09) or daily normoxia (dSHAM, Fio2 = 0.21) at 60-second normoxic intervals on 5 consecutive days; dAIH was given alone or combined with 30 minutes of overground walking 1 hour later. Walking speed and endurance were quantified using 10-Meter and 6-Minute Walk Tests. The trial is registered at ClinicalTrials.gov (NCT01272349). RESULTS: dAIH improved walking speed and endurance. Ten-Meter Walk time improved with dAIH vs dSHAM after 1 day (mean difference [MD] 3.8 seconds, 95% confidence interval [CI] 1.1-6.5 seconds, p = 0.006) and 2 weeks (MD 3.8 seconds, 95% CI 0.9-6.7 seconds, p = 0.010). Six-Minute Walk distance increased with combined dAIH + walking vs dSHAM + walking after 5 days (MD 94.4 m, 95% CI 17.5-171.3 m, p = 0.017) and 1-week follow-up (MD 97.0 m, 95% CI 20.1-173.9 m, p = 0.014). dAIH + walking increased walking distance more than dAIH after 1 day (MD 67.7 m, 95% CI 1.3-134.1 m, p = 0.046), 5 days (MD 107.0 m, 95% CI 40.6-173.4 m, p = 0.002), and 1-week follow-up (MD 136.0 m, 95% CI 65.3-206.6 m, p < 0.001). CONCLUSIONS: dAIH ± walking improved walking speed and distance in persons with chronic iSCI. The impact of dAIH is enhanced by combination with walking, demonstrating that combinatorial therapies may promote greater functional benefits in persons with iSCI. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that transient hypoxia (through measured breathing treatments), along with overground walking training, improves walking speed and endurance after iSCI.

Enabling techniques for in vitro studies on mammalian spinal locomotor mechanisms.

The neonatal rodent spinal cord maintained in vitro is a powerful model system to understand the central properties of spinal circuits generating mammalian locomotion. We describe three enabling approaches that incorporate afferent input and attached hindlimbs. (i) Sacral dorsal column stimulation recruits and strengthens ongoing locomotor-like activity, and implementation of a closed positive-feedback paradigm is shown to support its stimulation as an untapped therapeutic site for locomotor modulation. (ii) The spinal cord hindlimbs-restrained preparation allows suction electrode electromyographic recordings from many muscles. Inducible complex motor patterns resemble natural locomotion, and insights into circuit organization are demonstrated during spontaneous motor burst 'deletions', or following sensory stimuli such as tail and paw pinch. (iii) The spinal cord hindlimbs-pendant preparation produces unrestrained hindlimb stepping. It incorporates mechanical limb perturbations, kinematic analyses, ground reaction force monitoring, and the use of treadmills to study spinal circuit operation with movement-related patterns of sensory feedback while providing for stable whole-cell recordings from spinal neurons. Such techniques promise to provide important additional insights into locomotor circuit organization.