Using high resolution X-ray computed tomography to create an image based model of a lymph node.

Lymph nodes are an important part of the immune system. They filter the lymphatic fluid as it is transported from the tissues before being returned to the blood stream. The fluid flow through the nodes influences the behaviour of the immune cells that gather within the nodes and the structure of the node itself. Measuring the fluid flow in lymph nodes experimentally is challenging due to their small size and fragility. In this paper, we present high resolution X-ray computed tomography images of a murine lymph node. The impact of the resulting visualized structures on fluid transport are investigated using an image based model. The high contrast between different structures within the lymph node provided by phase contrast X-ray computed tomography reconstruction results in images that, when related to the permeability of the lymph node tissue, suggest an increased fluid velocity through the interstitial channels in the lymph node tissue. Fluid taking a direct path from the afferent to the efferent lymphatic vessel, through the centre of the node, moved faster than the fluid that flowed around the periphery of the lymph node. This is a possible mechanism for particles being moved into the cortex.

Antithymidylate resistance enables transgene selection and cell survival for T cells in the presence of 5-fluorouracil and antifolates.

Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers.

Laparoscopic surgery improves pregnancy outcomes in women with suspected endometriosis with or without pathological confirmation.

PURPOSE OF THE INVESTIGATION: To verify whether histologic confirmation of endometriosis impacts fertility outcomes. MATERIALS AND METHODS: Women with unexplained infertility (UI) underwent laparoscopic excision or ablation with CO2 laser or electrocautery of all suspected endometriotic lesions, followed by clinical treatment between January 2007 and December 2013; pregnancy (> 12 weeks) within 12 months of monitored cycles was the main outcome measured. RESULTS: Women with histological confirmation (n = 74) did not differ from those not confirmed (n = 29) with age, body mass index, gravidity, parity, ovulation induction protocol, and past duration of infertility. Pregnancy outcome was similar in both groups (39/74 vs. 15/29-p = 0.9--Chi-square) and there was no statistical difference in time to conceive/deliver (p = 0.7) between groups. CONCLUSIONS: There is no difference in fertility outcomes in women with UI, whether or not suspected endometriosis is confirmed pathologically.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Adoptive cellular immunotherapy for childhood malignancies.

Clinical trials have established that T cells have the ability to prevent and treat pathogens and tumors. This is perhaps best exemplified by engraftment of allogeneic T cells in the context of hematopoietic stem-cell transplantation (HSCT), which for over the last 50 years remains one of the best and most robust examples of cell-based therapies for the treatment of hematologic malignancies. Yet, the approach to infuse T cells for treatment of cancer, in general, and pediatric tumors, in particular, generally remains on the sidelines of cancer therapy. This review outlines the current state-of-the-art and provides a rationale for undertaking adoptive immunotherapy trials with emphasis on childhood malignancies.

The effect of root exudates on rhizosphere water dynamics.

Most water and nutrients essential for plant growth travel across a thin zone of soil at the interface between roots and soil, termed the rhizosphere. Chemicals exuded by plant roots can alter the fluid properties, such as viscosity, of the water phase, potentially with impacts on plant productivity and stress tolerance. In this paper, we study the effects of plant exudates on the macroscale properties of water movement in soil. Our starting point is a microscale description of two fluid flow and exudate diffusion in a periodic geometry composed from a regular repetition of a unit cell. Using multiscale homogenization theory, we derive a coupled set of equations that describe the movement of air and water, and the diffusion of plant exudates on the macroscale. These equations are parametrized by a set of cell problems that capture the flow behaviour. The mathematical steps are validated by comparing the resulting homogenized equations to the original pore scale equations, and we show that the difference between the two models is ≲7% for eight cells. The resulting equations provide a computationally efficient method to study plant-soil interactions. This will increase our ability to predict how contrasting root exudation patterns may influence crop uptake of water and nutrients.

Fluid flow in porous media using image-based modelling to parametrize Richards' equation.

The parameters in Richards' equation are usually calculated from experimentally measured values of the soil-water characteristic curve and saturated hydraulic conductivity. The complex pore structures that often occur in porous media complicate such parametrization due to hysteresis between wetting and drying and the effects of tortuosity. Rather than estimate the parameters in Richards' equation from these indirect measurements, image-based modelling is used to investigate the relationship between the pore structure and the parameters. A three-dimensional, X-ray computed tomography image stack of a soil sample with voxel resolution of 6 µm has been used to create a computational mesh. The Cahn-Hilliard-Stokes equations for two-fluid flow, in this case water and air, were applied to this mesh and solved using the finite-element method in COMSOL Multiphysics. The upscaled parameters in Richards' equation are then obtained via homogenization. The effect on the soil-water retention curve due to three different contact angles, 0°, 20° and 60°, was also investigated. The results show that the pore structure affects the properties of the flow on the large scale, and different contact angles can change the parameters for Richards' equation.

Variable domain-identical antibodies exhibit IgG subclass-related differences in affinity and kinetic constants as determined by surface plasmon resonance.

We have analysed the binding of variable domain-identical mouse monoclonal antibodies (mAb) of the IgG3, IgG1 and IgG2b subclasses, as well as F(ab')2 fragments derived from the IgG3 and IgG1 mAb, to a multivalent glycoprotein target. Using a biosensor device (BIAcore, Pharmacia Biosensor) that measures the mass of the antibody (or other receptor molecule) deposited on a sensor chip displaying the relevant epitopes, we found that the IgG3 mAb binds more effectively than the other antibody species at a high but not a low epitope density. The greater functional affinity associated with the IgG3 mAb, at high epitope density, was correlated with both slower dissociation rate constants and faster association rate constants in comparison with the IgG1 and IgG2b mAb and the F(ab')2 fragments derived from the IgG3 and IgG1 mAb. Evidence for slower dissociation kinetics for the IgG3 mAb versus the IgG1 and IgG2b mAb was also obtained by ELISA and flow cytometry. These results demonstrate that: (1) differences in heavy chain constant (CH) domains can significantly influence apparent functional affinity for multivalent antigen, as determined without the use of covalently modified primary or secondary antibodies; (2) differences in CH domains can alter both association and dissociation rate constants for interactions between IgG antibodies and multivalent antigen; and (3) these effects of CH domains depend on epitope density. The effect of constant region differences on the apparent association rate constants suggests new approaches for achieving better binding or functional effectiveness through antibody engineering.

Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor.

T cells can be reprogrammed to redirect specificity to tumor-associated antigens (TAAs) through the enforced expression of chimeric antigen receptors (CARs). The prototypical CAR is a single-chain molecule that docks with TAA expressed on the cell surface and, in contrast to the T-cell receptor complex, recognizes target cells independent of human leukocyte antigen. The bioprocessing to generate CAR(+) T cells has been reduced to clinical practice based on two common steps that are accomplished in compliance with current good manufacturing practice. These are (1) gene transfer to stably integrate the CAR using viral and nonviral approaches and (2) activating the T cells for proliferation by crosslinking CD3 or antigen-driven numeric expansion using activating and propagating cells (AaPCs). Here, we outline our approach to nonviral gene transfer using the Sleeping Beauty system and the selective propagation of CD19-specific CAR(+) T cells on AaPCs.

Combining descriptive and experimental analyses of young children with behavior problems in preschool settings.

This investigation shows the merits of preceding experimental analyses with descriptive analyses of functional variables with preschool children who engage in problematic behavior. A two-phase descriptive analysis was conducted in daycare settings with three children. In Phase 1, the authors assessed the relation between child behavior and structural events. During Phase 2, the same behavior was replotted by functional variables. The results showed that when the descriptive data were plotted via functional variables, specific hypotheses could be generated regarding the variables controlling appropriate behavior. This procedure permitted the subsequent use of very brief experimental analyses to further identify functional relations. The use of descriptive analyses of functional rather than structural variables may be an important component in the assessment of problem behavior in community settings.

Use of a concurrent operants paradigm to evaluate positive reinforcers during treatment of food refusal.

The authors evaluated the responsiveness of 4 preschool-aged children to positive reinforcers within a concurrent operants paradigm during mealtimes. The children were presented with two identical, concurrently available sets of food. Each set differed in quantity and quality of positive reinforcement paired with acceptance of each bite of food or in the number of bites of food required to obtain positive reinforcement. Experiment 1 evaluated 1 child's responsiveness to positive reinforcement while permitting escape from bite offers. Experiment 2 evaluated 2 children's responsiveness to positive reinforcement when escape extinction occurred. Results from these experiments suggested that the children were responsive to positive reinforcers and chose more often the bites paired with the greater quantity and/or quality of reinforcement. Experiment 3 evaluated 1 child's responsiveness to positive reinforcement both without and with escape extinction. Results suggested that positive reinforcement affected choice behavior and that escape extinction affected amount of food consumed.

Increasing compliance with medical procedures: application of the high-probability request procedure to a toddler.

The effects of high-probability (high-p) requests on compliance with low-probability (low-p) responses have received increased attention from applied investigators. This study examined the effects of a high-p procedure on a toddler's compliance with medical procedures. Compliance to low-p requests occurred more frequently following compliance to high-p requests, suggesting that this procedure may be useful across different topographies of compliance.

The use of behavioral assessment to prescribe and evaluate treatments for severely handicapped children.

Behavioral assessment procedures were used to prescribe and evaluate treatments of maladaptive behavior for 2 children with severe multiple handicaps. In Experiment 1, the results of an assessment of reinforcer preference were used in conjunction with a functional analysis of the conditions maintaining self-injurious behavior to prescribe a treatment for a child with severe disabilities. The treatment procedure involved the use of a pressure-sensitive microswitch to activate reinforcing stimuli during two solitary conditions, during which self-injurious behavior had occurred at high rates. The results were evaluated with a multiple baseline across settings design and indicated that self-injury decreased with concomitant increases in microswitch activation. Results were maintained at 6 weeks, 8 weeks, and 6 months. In Experiment 2, the results of behavioral assessments of reinforcer preference and self-injurious behavior were combined to develop a treatment for a second severely handicapped child, who exhibited high rates of self-injury in demand situations. This treatment was evaluated with a multiple baseline across tasks design and resulted in the elimination of self-injury for up to 15 months.

Using parents as therapists to evaluate appropriate behavior of their children: application to a tertiary diagnostic clinic.

We conducted a preliminary analysis of maintaining variables for children with conduct disorders in an outpatient clinic. Eight children of normal intelligence between the ages of 4 and 9 years were evaluated during 90-min sessions. The children's parents conducted the assessments by varying task demands (easy and difficult) and parental attention (attention and no attention) within a multielement design. The assessment focused on appropriate child behavior and was conducted to formulate hypotheses regarding maintaining contingencies. Results demonstrated that the children's appropriate behavior varied across assessment conditions and, for 7 of the 8 children, occurred at a higher rate during one condition than during other conditions. In addition, treatment integrity data demonstrated that parents were able to implement the procedures as intended. The recommended treatments were rated as being both effective and acceptable to parents for up to 6 months following the evaluation. Our results extend previous studies of functional analytic procedures conducted by trained experimenters with severely handicapped children in more controlled settings.

Brief hierarchical assessment of potential treatment components with children in an outpatient clinic.

Seven patients conducted assessments in an outpatient clinic using a prescribed hierarchy of antecedent and consequence treatment components for their children's problem behavior. Brief assessment of potential treatment components was conducted to identify variables that controlled the children's appropriate behavior. Experimental control via a brief reversal was achieved for 6 of the 7 children, (1 child continued to behave appropriately following initial improvement in behavior). For these 6 children, improved behavior occurred with changes in treatment components. Our results extend previous studies of direct assessment procedures conducted in outpatient clinic settings.

Brief experimental analysis of stimulus prompts for accurate responding on academic tasks in an outpatient clinic.

Brief multielement designs were used to examine the effects of specific instructional strategies on accuracy of academic performance during outpatient evaluations of 4 children with learning disorders. Instructional strategies that improved accuracy on academic tasks were identified for all participants. These results suggest that the application of experimental analysis methodologies to instructional variables may facilitate the identification of stimulus prompts that are associated with enhanced academic performance.

Use of descriptive and experimental analyses to identify the functional properties of aberrant behavior in school settings.

We conducted descriptive and experimental analyses of aberrant behavior in school settings with 2 children with autism, using teachers as assessors. Experimental functional analyses carried out by the investigators were followed by training teachers to conduct a descriptive analysis and a classroom experimental analysis. A comparison of the assessment procedures showed that each procedure identified negative reinforcement as a maintaining variable for aberrant behavior. The teacher implemented an intervention based on the assessment with mixed results. We then replicated the initial results by having the first teacher train a second teacher to carry out the two assessment procedures. The results of these analyses were also in agreement, again identifying negative reinforcement as a variable maintaining aberrant behavior. An intervention based on negative reinforcement was then successfully implemented. These results suggest the applicability and utility of functional analyses carried out in school settings.

Analysis of the effects of task preferences, task demands, and adult attention on child behavior in outpatient and classroom settings.

Two studies were conducted with children who displayed behavior problems to evaluate the effects of task preference, task demands, and adult attention on child behavior. In Study 1, we conducted brief functional analyses in an outpatient clinic to identify variables that facilitated appropriate behavior. For 8 of 10 children, distinct patterns of performance occurred; 3 children displayed improved behavior with changes in task demands, 1 child displayed improved behavior with a preferred task, and 4 children displayed improved behavior with changes in adult attention. In most cases, the children's parents carried out the assessments with adequate procedural integrity. In Study 2, we applied similar assessment methods to a classroom setting over an extended period of time. We identified independent variables controlling appropriate, on-task, and academic behavior for 2 children on two tasks, with slightly different treatment procedures across tasks for both children. In addition, the results of brief functional analyses for both children corresponded to the extended classroom assessments.

The impact of functional analysis methodology on outpatient clinic services.

The impact of the article by Iwata, Dorsey, Slifer, Bauman, and Richman (1982) on research in severe behavior disorders has been impressive. Equally impressive, however, but not as fully recognized, has been the impact of this methodology on the routine professional activities of those who employ functional analysis methods in their daily work. As one example of this impact, we describe the evolution of assessment procedures based on "brief functional analysis" methodology in our outpatient clinics. Less apparent have been the collateral effects that occur from using these procedures. Interactions with clients and colleagues have changed in ways that result in increased positive reinforcement. In this article, we briefly discuss the positive impact functional analysis has had on one specific work behavior--outpatient clinic assessment--and describe some of the generalized effects we have experienced in related aspects of our daily professional activities.