CAR T cell therapy for pediatric central nervous system tumors: a review of the literature and current North American trials.

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children. Typical therapy for CNS tumors in children involves a combination of surgery, radiation, and chemotherapy. While upfront therapy is effective for many high-grade tumors, therapy at the time of relapse remains limited. Furthermore, for diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), there are currently no curative therapies. Chimeric antigen receptor T (CAR T) cell therapy is a promising novel treatment avenue for these tumors. Here, we review the preclinical evidence for CAR T cell use in pediatric brain tumors, the preliminary clinical experience of CNS CAR T cell trials, toxicity associated with systemic and locoregional CAR T cell therapy for CNS tumors, challenges in disease response evaluation with CAR T cell therapy, and the knowledge gained from correlative biologic studies from these trials in the pediatric and young adult population.

Pharmacology of Chimeric Antigen Receptor-Modified T Cells.

Cell-based immunotherapies using T cells that are engineered to express a chimeric antigen receptor (CAR-T cells) are an effective treatment option for several B cell malignancies. Compared with most drugs, CAR-T cell products are highly complex, as each cell product is composed of a heterogeneous mixture of millions of cells. The biodistribution and kinetics of CAR-T cells, following administration, are unique given the ability of T cells to actively migrate as well as replicate within the patient. CAR-T cell therapies also have multiple mechanisms of action that contribute to both their antitumor activity and their toxicity. This review provides an overview of the unique pharmacology of CAR-T cells, with a focus on CD19-targeting and B cell maturation antigen (BCMA)-targeting CAR-T cells.

Enhancing CAR T function with the engineered secretion of C. perfringens neuraminidase.

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Detection of an IL-6 Biomarker Using a GFET Platform Developed with a Facile Organic Solvent-Free Aptamer Immobilization Approach.

Aptamer-immobilized graphene field-effect transistors (GFETs) have become a well-known detection platform in the field of biosensing with various biomarkers such as proteins, bacteria, virus, as well as chemicals. A conventional aptamer immobilization technique on graphene involves a two-step crosslinking process. In the first step, a pyrene derivative is anchored onto the surface of graphene and, in the second step, an amine-terminated aptamer is crosslinked to the pyrene backbone with EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide) chemistry. However, this process often requires the use of organic solvents such as dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) which are typically polar aprotic solvents and hence dissolves both polar and nonpolar compounds. The use of such solvents can be especially problematic in the fabrication of lab-on-a-chip or point-of-care diagnostic platforms as they can attack vulnerable materials such as polymers, passivation layers and microfluidic tubing leading to device damage and fluid leakage. To remedy such challenges, in this work, we demonstrate the use of pyrene-tagged DNA aptamers (PTDA) for performing a one-step aptamer immobilization technique to implement a GFET-based biosensor for the detection of Interleukin-6 (IL-6) protein biomarker. In this approach, the aptamer terminal is pre-tagged with a pyrene group which becomes soluble in aqueous solution. This obviates the need for using organic solvents, thereby enhancing the device integrity. In addition, an external electric field is applied during the functionalization step to increase the efficiency of aptamer immobilization and hence improved coverage and density. The results from this work could potentially open up new avenues for the use of GFET-based BioMEMS platforms by broadening the choice of materials used for device fabrication and integration.

An integrated microfluidic platform for selective and real-time detection of thrombin biomarkers using a graphene FET.

Lab-on-a-chip technology offers an ideal platform for low-cost, reliable, and easy-to-use diagnostics of key biomarkers needed for early screening of diseases and other health concerns. In this work, a graphene field-effect transistor (GFET) functionalized with target-binding aptamers is used as a biosensor for the detection of thrombin protein biomarker. Furthermore, this GFET is integrated with a microfluidic device for enhanced sensing performances in terms of detection limit, sensitivity, and continuous monitoring. Under this platform, a picomolar limit of detection was achieved for measuring thrombin; in our experiment measured as low as 2.6 pM. FTIR, Raman and UV-Vis spectroscopy measurements were performed to confirm the device functionalization steps. Based on the concentration-dependent calibration curve, a dissociation constant of KD = 375.8 pM was obtained. Continuous real-time measurements were also conducted under a constant gate voltage (VGS) to observe the transient response of the sensor when analyte was introduced to the device. The target selectivity of the sensor platform was evaluated and confirmed by challenging the GFET biosensor with various concentrations of lysozyme protein. The results suggest that this device technology has the potential to be used as a general diagnostic platform for measuring clinically relevant biomarkers for point-of-care applications.

Hollow microcarriers for large-scale expansion of anchorage-dependent cells in a stirred bioreactor.

With recent advances in biotechnology, mammalian cells are used in biopharmaceutical industries to produce valuable protein therapeutics and investigated as effective therapeutic agents to permanently degenerative diseases in cell based therapy. In these exciting and actively expanding fields, a reliable, efficient, and affordable platform to culture mammalian cells on a large scale is one of the most vital necessities. To produce and maintain a very large population of anchorage-dependent cells, a microcarrier-based stirred tank bioreactor is commonly used. In this approach, the cells are exposed to harmful hydrodynamic shear stress in the bioreactor and the mass transfer rates of nutrients and gases in the bioreactor are often kept below an optimal level to prevent cellular damages from the shear stress. In this paper, a hollow microcarrier (HMC) is presented as a novel solution to protect cells from shear stress in stirred bioreactors, while ensuring sufficient and uniform mass transfer rate of gases and nutrients. HMC is a hollow microsphere and cells are cultured on its inner surface to be protected, while openings on the HMC provide sufficient exchange of media inside the HMC. As a proof of concept, we demonstrated the expansion of fibroblasts, NIH/3T3 and the expansion and cardiac differentiation of human induced pluripotent stem cells, along with detailed numerical analysis. We believe that the developed HMC can be a practical solution to enable large-scale expansion of shear-sensitive anchorage-dependent cells in an industrial scale with stirred bioreactors.

Continuous Real-Time Detection of Serotonin Using an Aptamer-Based Electrochemical Biosensor.

Serotonin (5-HT) is a critical neurotransmitter involved in many neuronal functions, and 5-HT depletion has been linked to several mental diseases. The fast release and clearance of serotonin in the extracellular space, low analyte concentrations, and a multitude of interfering species make the detection of serotonin challenging. This work presents an electrochemical aptamer-based biosensing platform that can monitor 5-HT continuously with high sensitivity and selectivity. Our electrochemical sensor showed a response time of approximately 1 min to a step change in the serotonin concentration in continuous monitoring using a single-frequency EIS (electrochemical impedance spectroscopy) technique. The developed sensing platform was able to detect 5-HT in the range of 25-150 nM in the continuous sample fluid flow with a detection limit (LOD) of 5.6 nM. The electrochemical sensor showed promising selectivity against other species with similar chemical structures and redox potentials, including dopamine (DA), norepinephrine (NE), L-tryptophan (L-TP), 5-hydroxyindoleacetic acid (5-HIAA), and 5-hydroxytryptophan (5-HTP). The proposed sensing platform is able to achieve high selectivity in the nanomolar range continuously in real-time, demonstrating the potential for monitoring serotonin from neurons in organ-on-a-chip or brain-on-a-chip-based platforms.

The IAP antagonist birinapant enhances chimeric antigen receptor T cell therapy for glioblastoma by overcoming antigen heterogeneity.

Antigen heterogeneity that results in tumor antigenic escape is one of the major obstacles to successful chimeric antigen receptor (CAR) T cell therapies in solid tumors including glioblastoma multiforme (GBM). To address this issue and improve the efficacy of CAR T cell therapy for GBM, we developed an approach that combines CAR T cells with inhibitor of apoptosis protein (IAP) antagonists, a new class of small molecules that mediate the degradation of IAPs, to treat GBM. Here, we demonstrated that the IAP antagonist birinapant could sensitize GBM cell lines and patient-derived primary GBM organoids to apoptosis induced by CAR T cell-derived cytokines, such as tumor necrosis factor. Therefore, birinapant could enhance CAR T cell-mediated bystander death of antigen-negative GBM cells, thus preventing tumor antigenic escape in antigen-heterogeneous tumor models in vitro and in vivo. In addition, birinapant could promote the activation of NF-κB signaling pathways in antigen-stimulated CAR T cells, and with a birinapant-resistant tumor model we showed that birinapant had no deleterious effect on CAR T cell functions in vitro and in vivo. Overall, we demonstrated the potential of combining the IAP antagonist birinapant with CAR T cells as a novel and feasible approach to overcoming tumor antigen heterogeneity and enhancing CAR T cell therapy for GBM.

Stimuli-Responsive Templated Polymer as a Target Receptor for a Conformation-based Electrochemical Sensing Platform.

The use of highly crosslinked molecularly imprinted polymers as a synthetic target receptor has the limitations of restricted accessibility to the binding sites resulting in slow response time. Moreover, such artificial receptors often require additional transduction mechanisms to translate target binding events into measurable signals. Here, we propose the development of a single-chain stimuli-responsive templated polymer, without using any covalent interchain crosslinkers, as a target recognition element. The synthesized polymer chain exhibits preferential binding with the target molecule with which the polymer is templated. Moreover, upon specific target recognition, the polymer undergoes conformation change induced by its particular stimuli responsiveness, namely the target binding event. Such templated single-chain polymers can be attached to the electrode surface to implement a label-free electrochemical sensing platform. A target analyte, 4-nitrophenol (4-NP), was used as a template to synthesize a poly-N-isopropylacrylamide (PNIPAM)-based copolymer chain which was anchored to the electrode to be used as a selective receptor for 4-NP. The electrode surface chemistry analysis and the electrochemical impedance study reveal that the polymer concentration, the interchain interactions, and the Hofmeister effect play a major role in influencing the rate of polymer grafting as well as the morphology of the polymers grafted to the electrode. We also show that the specific binding between 4-NP and the copolymer results in a substantial change in the charge transfer kinetics at the electrode signifying the polymer conformation change.

Lab-on-a-Chip Systems for Aptamer-Based Biosensing.

Aptamers are oligonucleotides or peptides that are selected from a pool of random sequences that exhibit high affinity toward a specific biomolecular species of interest. Therefore, they are ideal for use as recognition elements and ligands for binding to the target. In recent years, aptamers have gained a great deal of attention in the field of biosensing as the next-generation target receptors that could potentially replace the functions of antibodies. Consequently, it is increasingly becoming popular to integrate aptamers into a variety of sensing platforms to enhance specificity and selectivity in analyte detection. Simultaneously, as the fields of lab-on-a-chip (LOC) technology, point-of-care (POC) diagnostics, and personal medicine become topics of great interest, integration of such aptamer-based sensors with LOC devices are showing promising results as evidenced by the recent growth of literature in this area. The focus of this review article is to highlight the recent progress in aptamer-based biosensor development with emphasis on the integration between aptamers and the various forms of LOC devices including microfluidic chips and paper-based microfluidics. As aptamers are extremely versatile in terms of their utilization in different detection principles, a broad range of techniques are covered including electrochemical, optical, colorimetric, and gravimetric sensing as well as surface acoustics waves and transistor-based detection.

Editorial for the Special Issue on Printable and Flexible Electronics for Sensors.

Printable and flexible electronic materials have gained a tremendous amount of interest both in academia and in industry, due to their potential impact in many areas, including advanced manufacturing, healthcare, diagnostics, wearables, renewable energy, and defense, to name a few [...].

NON-ENZYMATIC ELECTROCHEMICAL DETECTION OF GLUTAMATE USING TEMPLATED POLYMER-BASED TARGET RECEPTORS.

We present a novel electrochemical biosensing platform for the detection of neurotransmitter glutamate using templated polymer-based target receptors. Our sensing approach demonstrates, for the first time, a non-enzymatic approach without the need of glutamate oxidase, leading to a more specific and rapid response. The proposed detection principle is based on the following two claims: (1) our templated polymer-based receptor results in specific molecular recognition of the target glutamate and, (2) upon target binding, the polymer undergoes a conformation change which can then be measured via electrochemical techniques. This sensing platform has the potential to provide direct monitoring of a variety of non-electroactive species and to eliminate the incorporation of enzymes thereby providing a simpler and more robust alternative to enzyme-based sensors.

A Single-chain Templated Polymer-based Target Receptor as a New Platform for Label-free Selective Electrochemical Sensing.

We report a novel single-chain polymer-based chemical receptor that can be used for the label-free electrochemical detection of an analyte with high selectivity. The polymer was developed using poly-N-isopropylacrylamide (pNIPAM) as a backbone structure in addition to other functional monomers that are used to imprint the template molecule 4-nitrophenol. The polymer also contains a redox reporting monomers (ferrocene) which create a change in the electrochemical signal upon molecular recognition. We hypothesize that the analyte binding to the receptor causes the polymer conformation change from the extended to the collapsed phase. After anchoring the polymer-based receptors onto the surfaces of the gold electrode, when exposed to the analyte, the changes in the electrochemical signals were observed which confirmed the selective target binding as well as the polymer conformation change as a result.

A Low-Cost Inkjet-Printed Aptamer-Based Electrochemical Biosensor for the Selective Detection of Lysozyme.

Recently, inkjet-printing has gained increased popularity in applications such as flexible electronics and disposable sensors, as well as in wearable sensors because of its multifarious advantages. This work presents a novel, low-cost immobilization technique using inkjet-printing for the development of an aptamer-based biosensor for the detection of lysozyme, an important biomarker in various disease diagnosis. The strong affinity between the carbon nanotube (CNT) and the single-stranded DNA is exploited to immobilize the aptamers onto the working electrode by printing the ink containing the dispersion of CNT-aptamer complex. The inkjet-printing method enables aptamer density control, as well as high resolution patternability. Our developed sensor shows a detection limit of 90 ng/mL with high target selectivity against other proteins. The sensor also demonstrates a shelf-life for a reasonable period. This technology has potential for applications in developing low-cost point-of-care diagnostic testing kits for home healthcare.

Selective Detection of Lysozyme Biomarker Utilizing Large Area Chemical Vapor Deposition-Grown Graphene-Based Field-Effect Transistor.

Selective and rapid detection of biomarkers is of utmost importance in modern day health care for early stage diagnosis to prevent fatal diseases and infections. Among several protein biomarkers, the role of lysozyme has been found to be especially important in human immune system to prevent several bacterial infections and other chronic disease such as bronchopulmonary dysplasia. Thus, real-time monitoring of lysozyme concentration in a human body can pave a facile route for early warning for potential bacterial infections. Here, we present for the first time a label-free lysozyme protein sensor that is rapid and selective based on a graphene field-effect transistor (GFET) functionalized with selectively designed single-stranded probe DNA (pDNA) with high binding affinity toward lysozyme molecules. When the target lysozyme molecules bind to the surface-immobilized pDNAs, the resulting shift of the charge neutrality points of the GFET device, also known as the Dirac voltage, varied systematically with the concentration of target lysozyme molecules. The experimental results show that the GFET-based biosensor is capable of detecting lysozyme molecules in the concentration range from 10 nM to 1 µM.

A biomechanical assessment of disc pressures in the lumbosacral spine in response to external unloading forces.

BACKGROUND CONTEXT: Axial back pain affects a large percentage of the population. Often aggravated by weight-bearing activity, these patients frequently have associated degenerative or post-traumatic lumbar disc disease. Aquatherapy is frequently used to transition patients from less activity limited by pain to greater activity by reducing weight-bearing load of the lumbar spine. Development of a means to permit patients similar spinal unloading while active during normal daily living would have the potential to promote similar effects. PURPOSE: The purpose of this study is to measure internal disc pressure at L4/L5 in response to forces exerted by an external vest. The study hypothesis anticipated an unloading of the lumbar spine during upright posture, as measured by intradicsal pressure at the L4/5 disc, correlating with external forces provided to the trunk by the device. STUDY DESIGN: A controlled experimental study of spine biomechanical loading was undertaken using isolated cadaver torsos obtained from an approved tissue source. Ages ranged at death with a mean of 65+/-6 years. METHODS: The distractive force created by inflating a set of pneumatic lifters within vests for treatment of low back pain were calibrated in a materials testing machine. Effects of inflation on the disc pressures within the lumbar spine then were tested. A microscopic pressure sensor (Samba, Gothenburg, Sweden) was placed into the nucleus of the L4/L5 disc of six isolated cadaver torsos (1 female, 5 male) using a 15-gauge spinal needle under direct fluoroscopic visualization. The pressure sensor was 0.42 mm in diameter, and had a calibrated response range of 0-7500 mm Hg. A pneumatically actuated lumbar vest was fit snugly to the torso. Each torso was supported in an upright, weight-bearing position for testing. The vest was inflated while the internal disc pressure was monitored and recorded. The data were analyzed to test for correlation between the amount of external unloading force provided by the vest and the intradiscal pressure measured in vitro. RESULTS: Application of external loads between the pelvis and ribcage by the vest demonstrated a maximum mean reduction of internal disc pressure at L4/L5 of 25% when the vest was inflated to a level producing approximately 400 N of effective load. The reduction in disc pressure was significantly different compared with baseline (upright, weight-bearing disc pressure without the vest) for all distraction settings (p<.01) except for the very lowest setting which was significant only at p=.025. CONCLUSIONS: Spinal unloading with an externally applied vest with adequate surface interface is effective in reducing intradiscal pressures. Ambulatory reduction of pressure would permit beneficial reduction of loads and permit patients with weight-bearing intolerance a better quality of life.

Extraosseous blood supply of the tibia and the effects of different plating techniques: a human cadaveric study.

OBJECTIVE: To describe the extraosseous blood supply of the tibia and how the blood supply of the distal tibia is influenced by different plating techniques. DESIGN: Microdissection of cadaveric adult hip disarticulation specimens following sequential arterial injections of india ink and Ward's Blue Latex was performed. Readily identifiable arterioles measured approximately 0.5 mm in diameter. Their artery of origin was identified, and their position along the medial, lateral, and posterior aspects of the tibia was documented relative to the tibial plafond. Additionally, six matched pairs of limbs were used to assess the effects of different plating techniques on the extraosseous blood supply along the medial aspect of the distal tibia. SETTING: University anatomy laboratory. PATIENTS/PARTICIPANTS: Nine matched pairs ( = 18) of randomly obtained, adult cadaveric hip disarticulation specimens. INTERVENTION: India ink followed by Ward's Blue Latex was injected into the superficial femoral artery at the level of the inguinal crease after cleansing of the arterial system. The skin, subcutaneous tissue, and muscles were dissected from the leg, exposing the arterial system and the extraosseous vessels of the tibia. MEAN OUTCOME MEASUREMENTS: The extraosseous blood supply of each aspect of the tibial diaphysis was determined. Each extraosseous arteriole was identified, and the locations of each documented relative to the tibial plafond. Changes in the filling of these vessels along the medial aspect of the distal tibia were documented in a separate group of specimens ( = 12), which had undergone two different plating techniques. RESULTS: The proximal metaphysis of the tibia was found to have a rich extraosseous blood supply provided primarily from vessels from the popliteal artery, the anterior tibial artery (ATA) laterally, and the posterior tibial artery (PTA) medially. In comparison, the tibial diaphysis was found to have relatively few extraosseous vessels and a considerably hypovascular region, posteriorly. Branches of the ATA were found to supply the posterior aspect of the diaphysis with these branches passing through the interosseous membrane. The diaphysis also received a variable contribution from the PTA. The lateral aspect of the diaphysis was supplied by branches of the ATA. An anastomotic network of arteries from the ATA and PTA formed the rich extraosseous blood supply of the medial distal aspect of the tibia. Open plating of the medial aspect of the distal tibia caused a statistically significant ( < 0.05) greater disruption of the extraosseous blood supply of the metaphyseal region than did percutaneously applied plates. In each specimen, open plating prevented filling of each periosteal vessel in the region as opposed to percutaneous plates, which permitted filling of the extraosseous vessels up to the edge of the plate. CONCLUSIONS: The proximal and distal metaphyseal areas of the tibia have a rich extraosseous blood supply provided primarily by branches of the ATA and the PTA. Open plating of the medial aspect of the distal tibia caused a greater disruption of this extraosseous blood supply than did percutaneously applied plates. Disruption of these extraosseous vessels following fracture and subsequent operative stabilization may slow healing and increase the risk of delayed union and nonunion. These findings support current efforts to develop less invasive methods and implants for operative stabilization of distal tibia fractures.

Conducting Polyaniline Nanowire and Its Applications in Chemiresistive Sensing.

One dimensional polyaniline nanowire is an electrically conducting polymer that can be used as an active layer for sensors whose conductivity change can be used to detect chemical or biological species. In this review, the basic properties of polyaniline nanowires including chemical structures, redox chemistry, and method of synthesis are discussed. A comprehensive literature survey on chemiresistive/conductometric sensors based on polyaniline nanowires is presented and recent developments in polyaniline nanowire-based sensors are summarized. Finally, the current limitations and the future prospect of polyaniline nanowires are discussed.

An on-chip chemiresistive polyaniline nanowire-based pH sensor with self-calibration capability.

A pH sensor based on chemiresistive polyaniline nanowires, which makes use of the pH responsive conductivity of the polymer, requires an electrochemical system during measurements in order to maintain its oxidation state. In this work, we have developed an integrated electrochemical device where a reference, a counter, and a pair of working electrodes are all fabricated on a single chip. The polyaniline nanowires are grown between this pair of working electrodes that are separated by a microscale gap. This device can easily be connected to the external circuit for potential control and current measurements. Taking advantage of the fact that polyaniline becomes insulating when it is fully oxidized or fully reduced, the pH sensor can be electrically reset by controlling the electrochemical potential of the polymer. This paper presents the pH sensing results and the performance of the resetting function of the developed integrated device.

Reduction of high-grade isthmic and dysplastic spondylolisthesis in 5 adolescents.

Treatment of high-grade isthmic and dysplastic spondylolisthesis in children and adolescents remains a challenge. Surgical treatment of spondylolisthesis has been recommended in adolescents with pain refractory to nonoperative modalities, slippage progression, or > 50% slippage on presentation. Controversy exists as to the optimal surgical approach for high-grade spondylolisthesis. In this report, we describe 5 cases of high-grade isthmic and dysplastic spondylolisthesis in adolescents and review the literature on surgical treatment for this entity. Operative records, charts, x-rays, and Scoliosis Research Society outcome questionnaires (SRS-22) were retrospectively evaluated for 5 consecutive patients diagnosed with and treated for high-grade spondylolisthesis. Each patient received treatment consisting of decompression, reduction, and circumferential fusion with transpedicular and segmental fixation from a posterior approach. Two patients had transient L5 nerve root deficit, which resolved within 3 months. Reduction benefits include a decrease in shear stresses (and resulting decreased rates of postoperative pseudarthrosis and slip progression), restoration of sagittal alignment and lumbosacral spine balance, and improvement in clinical deformity.