Immunotherapy for Pediatric Cancer

AbstractImprovements in adult cancer survivorship can be achieved from behavioral changes and adopting screening programs. Yet, these approaches cannot be readily applied to lower the morbidity and mortality from childhood cancers. Rather, pediatric oncologists must rely on procedures and therapies to treat, rather than prevent malignancies. The systematic application of chemotherapy, radiation therapy, and surgery has led to remarkable advances in survival but these improvements have come at a cost. Children routinely receive chemotherapy agents that were designed decades ago, and these drugs have predictable side effects that result in the loss of potential for long-term survivors. The advent of targeted applications of immune-based therapies offers children with cancer a new class of oncolytic therapies that may be used to treat disease refractory to conventional approaches and lessen the toxicity of current treatment regimens without compromising remission. This review explores how 3 components of the immune system—T cells, natural killer (NK) cells, and antibodies—can be used for therapy of pediatric malignancies

Quantitative High-Throughput Single-Cell Cytotoxicity Assay for T cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5. Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8. Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells

New Eaxactly Solvable Hamiltonians: Shape Invariance and Self-Similarity

We discuss in some detail the self-similar potentials of Shabat and Spiridonov which are reflectionless and have an infinite number of bound states. We demonstrate that these self-similar potentials are in fact shape invariant potentials within the formalism of supersymmetric quantum mechanics. In particular, using a scaling ansatz for the change of parameters, we obtain a large class of new, reflectionless, shape invariant potentials of which the Shabat-Spiridonov ones are a special case. These new potentials can be viewed as q-deformations of the single soliton solution corresponding to the Rosen-Morse potential. Explicit expressions for the energy eigenvalues, eigenfunctions and transmission coefficients for these potentials are obtained. We show that these potentials can also be obtained numerically. Included as an intriguing case is a shape invariant double well potential whose supersymmetric partner potential is only a single well. Our class of exactly solvable Hamiltonians is further enlarged by examining two new directions: (i) changes of parameters which are different from the previously studied cases of translation and scaling; (ii) extending the usual concept of shape invariance in one step to a multi-step situation. These extensions can be viewed as q-deformations of the harmonic oscillator or multi-soliton solutions corresponding to the Rosen-Morse potential.Comment: 26 pages, plain tex, request figures by e-mai

Ultra-high resolution X-ray structure of orthorhombic bovine pancreatic Ribonuclease A at 100K

The crystal structure of orthorhombic Bovine Pancreatic Ribonuclease A has been determined to 0.85 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16000 keV (λ = 0.77 Å). This is the first ultra-high-resolution structure of a native form of Ribonuclease A to be reported. Refinement carried out with anisotropic displacement parameters, stereochemical restraints, inclusion of H atoms in calculated positions, five SO2−4 moieties, eleven ethanol molecules and 293 water molecules, converged with final R values of R1(Free) = 0.129 (4279 reflections) and R1 = 0.112 (85,346 reflections). The refined structure was deposited in the Protein Data Bank as structure 7p4r. Conserved waters, using four high resolution structures, have been investigated. Cluster analysis identified clusters of water molecules that are associated with the active site of Bovine Ribonuclease A. Particular attention has been paid to making detailed comparisons between the present structure and other high quality Bovine Pancreatic Ribonuclease A X-ray crystal structures with special reference to the deposited classic monoclinic structure 3RN3 Howlin et al. (Acta Crystallogr A 45:851–861, 1989). Detailed studies of various aspects of hydrogen bonding and conformation have been carried out with particular reference to active site residues Lys-1, Lys-7, Gln-11, His-12, Lys-41, Asn-44, Thr-45, Lys-66, His-119 and Ser-123. For the two histidine residues in the active site the initial electron density map gives a clear confirmation that the position of His-12 is very similar in the orthorhombic structure to that in 3RN3. In 3RN3 His-119 exhibited poor electron density which was modelled and refined as two distinct sites, A (65%) and B (35%) but with respect to His-119 in the present ultra-high resolution orthorhombic structure there is clear electron density which was modelled and refined as a single conformation distinct from either conformation A or B in 3RN3. Other points of interest include Serine-32 which is disordered at the end of the sidechain in the present orthorhombic form but has been modelled as a single form in 3RN3. Lysine-66: there is density indicating a possible conformation for this residue. However, the density is relatively weak, and the conformation is unclear. Three types of amino acid representation in the ultra-high resolution electron density are examined: (i) sharp with very clearly resolved features, for example Lys-37; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry, for example Tyr-76; (iii) poor density and difficult or impossible to model, an example is Lys-31 for which density is missing except for Cβ. The side chains of Gln-11, His-12, Lys-41, Thr-45 and His-119 are generally recognised as being closely involved in the enzyme activity. It has also been suggested that Lys-7, Asp-44, Lys-66, Phe-120, Asp-121 and Ser-123 may also have possible roles in this mechanism. A molecular dynamics study on both structures has investigated the conformations of His-119 which was modelled as two conformations in 3RN3 but is observed to have a single clearly defined conformation in the present orthorhombic structure. MD has also been used to investigate Lys-31, Lys-41 and Ser32. The form of the Ribonuclease A enzyme used in both the present study and in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989) includes a sulphate anion which occupies approximately the same location as the PO2−4 phosphate group in protein nucleotide complexes (Borkakoti et al. in J Mol Biol 169:743–755, 1983). The present structure contains 5 SO2−4 groups SO41151–SO41155 two of which, SO41152 and SO41153 are disordered, SO41152 being in the active site, and 11 EtOH molecules, EOH A 201–EOH A 211 all of which have good geometry. H atoms were built into the EtOH molecules geometrically. Illustrations of these features in the present structure are included here. The sulphates are presumably present in the material purchased for use in the present study. 293 water molecules are included in the present structure compared to 134 in 3RN3 (Howlin et al. in Acta Crystallogr A 45:851–861, 1989)

Rapid assembly and rejuvenation of a large silicic magmatic system : insights from mineral diffusive profiles in the Kidnappers and Rocky Hill deposits, New Zealand.

The timescales over which magmas in large silicic systems are reactivated, assembled and stored remains a fundamental question in volcanology. To address this question, we study timescales from Fe–Mg interdiffusion in orthopyroxenes and Ti diffusion in quartz from the caldera-forming 1200 km3 Kidnappers and 200 km3 Rocky Hill eruptions from the Mangakino volcanic centre (Taupo Volcanic Zone, New Zealand). The two eruptions came from the same source area, have indistinguishable 40Ar/39Ar ages (∼1.0 Ma) and zircon U–Pb age spectra, but their respective deposits are separated by a short period of erosion. Compositions of pumice, glass and mineral species in the collective eruption deposits define multiple melt dominant bodies but indicate that these shared a common magmatic mush zone. Diffusion timescales from both eruptions are used to build on chemical and textural crystal signatures and interpret both the crystal growth histories and the timing of magma accumulation. Fe–Mg interdiffusion profiles in orthopyroxenes imply that the three melt-dominant bodies, established through extraction of melt and crystals from the common source, were generated within 600 years and with peak accumulation rates within 100 years of each eruption. In addition, a less-evolved melt interacted with the Kidnappers magma, beginning ∼30 years prior to and peaking within 3 years of the eruption. This interaction did not directly trigger the eruption, but may have primed the magmatic system. Orthopyroxene crystals with the same zoning patterns from the Kidnappers and Rocky Hill pumices yield consistently different diffusion timescales, suggesting a time break between the eruptions of ∼20 years (from core–rim zones) to ∼10 years (outer rim zones). Diffusion of Ti in quartz reveals similarly short timescales and magmatic residence times of <30 years, suggesting quartz is only recording the last period of crystallization within the final eruptible melt. Accumulation of the eruptible magma for these two, closely successive eruptions was accomplished over centuries to decades, in contrast to the gestation time of the magmatic system of ∼200 kyr, as indicated by zircon age patterns. The magmatic system was able to recover after the Kidnappers eruption in only ∼10–20 years to accumulate enough eruptible melt and crystals for a second ∼200 km3 eruption. Our data support concepts of large silicic systems being stored as long-lived crystal mushes, with eruptible melts generated over extraordinarily short timescales prior to eruption

Quasirandom permutations are characterized by 4-point densities

For permutations π and τ of lengths |π|≤|τ| , let t(π,τ) be the probability that the restriction of τ to a random |π| -point set is (order) isomorphic to π . We show that every sequence {τj} of permutations such that |τj|→∞ and t(π,τj)→1/4! for every 4-point permutation π is quasirandom (that is, t(π,τj)→1/|π|! for every π ). This answers a question posed by Graham

Strong Spherical Asymptotics for Rotor-Router Aggregation and the Divisible Sandpile

The rotor-router model is a deterministic analogue of random walk. It can be used to define a deterministic growth model analogous to internal DLA. We prove that the asymptotic shape of this model is a Euclidean ball, in a sense which is stronger than our earlier work. For the shape consisting of $n=\omega_d r^d$ sites, where $\omega_d$ is the volume of the unit ball in $\R^d$, we show that the inradius of the set of occupied sites is at least $r-O(\log r)$, while the outradius is at most $r+O(r^\alpha)$ for any $\alpha > 1-1/d$. For a related model, the divisible sandpile, we show that the domain of occupied sites is a Euclidean ball with error in the radius a constant independent of the total mass. For the classical abelian sandpile model in two dimensions, with $n=\pi r^2$ particles, we show that the inradius is at least $r/\sqrt{3}$, and the outradius is at most $(r+o(r))/\sqrt{2}$. This improves on bounds of Le Borgne and Rossin. Similar bounds apply in higher dimensions.Comment: [v3] Added Theorem 4.1, which generalizes Theorem 1.4 for the abelian sandpile. [v4] Added references and improved exposition in sections 2 and 4. [v5] Final version, to appear in Potential Analysi

Bound state solutions of the Dirac-Rosen-Morse potential with spin and pseudospin symmetry

The energy spectra and the corresponding two- component spinor wavefunctions of the Dirac equation for the Rosen-Morse potential with spin and pseudospin symmetry are obtained. The $s-$wave ($\kappa = 0$ state) solutions for this problem are obtained by using the basic concept of the supersymmetric quantum mechanics approach and function analysis (standard approach) in the calculations. Under the spin symmetry and pseudospin symmetry, the energy equation and the corresponding two-component spinor wavefunctions for this potential and other special types of this potential are obtained. Extension of this result to $\kappa \neq 0$ state is suggested.Comment: 18 page

Individual motile CD4+ T cells can participate in efficient multikilling through conjugation to multiple tumor cells

T cells genetically modified to express a CD19-specific chimeric antigen receptor (CAR) for the investigational treatment of B-cell malignancies comprise a heterogeneous population, and their ability to persist and participate in serial killing of tumor cells is a predictor of therapeutic success. We implemented Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to provide direct evidence that CD4+CAR+ T cells (CAR4 cells) can engage in multikilling via simultaneous conjugation to multiple tumor cells. Comparisons of the CAR4 cells and CD8+CAR+ T cells (CAR8 cells) demonstrate that, although CAR4 cells can participate in killing and multikilling, they do so at slower rates, likely due to the lower granzyme B content. Significantly, in both sets of T cells, a minor subpopulation of individual T cells identified by their high motility demonstrated efficient killing of single tumor cells. A comparison of the multikiller and single-killer CAR+ T cells revealed that the propensity and kinetics of T-cell apoptosis were modulated by the number of functional conjugations. T cells underwent rapid apoptosis, and at higher frequencies, when conjugated to single tumor cells in isolation, and this effect was more pronounced on CAR8 cells. Our results suggest that the ability of CAR+ T cells to participate in multikilling should be evaluated in the context of their ability to resist activation-induced cell death. We anticipate that TIMING may be used to rapidly determine the potency of T-cell populations and may facilitate the design and manufacture of next-generation CAR+ T cells with improved efficacy. Cancer Immunol Res; 3(5); 473–82. ©2015 AACR