Proof Of Principle Studies Research Articles

Differentiation of SH-SY5Y neuroblastoma cells using retinoic acid and BDNF: a model for neuronal and synaptic differentiation in neurodegeneration.

There has been much interest in the use of cell culture models of neurones, to avoid the animal welfare and cost issues of using primary and human-induced pluripotent stem cell (hiPSC)-derived neurones respectively. The human neuroblastoma cell line, SH-SY5Y, is extensively used in laboratories as they can be readily expanded, are of low cost and can be differentiated into neuronal-like cells. However, much debate remains as to their phenotype once differentiated, and their ability to recapitulate the physiology of bona fide neurones. Here, we characterise a differentiation protocol using retinoic acid and BDNF, which results in extensive neurite outgrowth/branching within 10days, and expression of key neuronal and synaptic markers. We propose that these differentiated SH-SY5Y cells may be a useful substitute for primary or hiPSC-derived neurones for cell biology studies, in order to reduce costs and animal usage. We further propose that this characterised differentiation timecourse could be used as an in vitro model for neuronal differentiation, for proof-of principle studies on neurogenesis, e.g. relating to neurodegenerative diseases. Finally, we demonstrate profound changes in Tau phosphorylation during differentiation of these cells, suggesting that they should not be used for neurodegeneration studies in their undifferentiated state.

A general large-scale synthesis approach for crystalline porous materials

Crystalline porous materials such as covalent organic frameworks (COFs), metal-organic frameworks (MOFs) and porous organic cages (POCs) have been widely applied in various fields with outstanding performances. However, the lack of general and effective methodology for large-scale production limits their further industrial applications. In this work, we developed a general approach comprising high pressure homogenization (HPH), which can realize large-scale synthesis of crystalline porous materials including COFs, MOFs, and POCs under benign conditions. This universal strategy, as illustrated in the proof of principle studies, has prepared 4 COFs, 4 MOFs, and 2 POCs. It can circumvent some drawbacks of existing approaches including low yield, high energy consumption, low efficiency, weak mass/thermal transfer, tedious procedures, poor reproducibility, and high cost. On the basis of this approach, an industrial homogenizer can produce 0.96 ~ 580.48 ton of high-performance COFs, MOFs, and POCs per day, which is unachievable via other methods.

Fitting a deep generative hadronization model

Hadronization is a critical step in the simulation of high-energy particle and nuclear physics experiments. As there is no first principles understanding of this process, physically-inspired hadronization models have a large number of parameters that are fit to data. Deep generative models are a natural replacement for classical techniques, since they are more flexible and may be able to improve the overall precision. Proof of principle studies have shown how to use neural networks to emulate specific hadronization when trained using the inputs and outputs of classical methods. However, these approaches will not work with data, where we do not have a matching between observed hadrons and partons. In this paper, we develop a protocol for fitting a deep generative hadronization model in a realistic setting, where we only have access to a set of hadrons in data. Our approach uses a variation of a Generative Adversarial Network with a permutation invariant discriminator. We find that this setup is able to match the hadronization model in Herwig with multiple sets of parameters. This work represents a significant step forward in a longer term program to develop, train, and integrate machine learning-based hadronization models into parton shower Monte Carlo programs.

Quantifying Cell Heterogeneity and Subpopulations Using Single Cell Metabolomics.

Mass spectrometry (MS) has become an indispensable tool for metabolomics studies. However, due to the lack of applicable experimental platforms, suitable algorithm, software, and quantitative analyses of cell heterogeneity and subpopulations, investigating global metabolomics profiling at the single cell level remains challenging. We combined the Single-probe single cell MS (SCMS) experimental technique with a bioinformatics software package, SinCHet-MS (Single Cell Heterogeneity for Mass Spectrometry), to characterize changes of tumor heterogeneity, quantify cell subpopulations, and prioritize the metabolite biomarkers of each subpopulation. As proof of principle studies, two melanoma cancer cell lines, the primary (WM115; with a lower drug resistance) and the metastatic (WM266-4; with a higher drug resistance), were used as models. Our results indicate that after the treatment of the anticancer drug vemurafenib, a new subpopulation emerged in WM115 cells, while the proportion of the existing subpopulations was changed in the WM266-4 cells. In addition, metabolites for each subpopulation can be prioritized. Combining the SCMS experimental technique with a bioinformatics tool, our label-free approach can be applied to quantitatively study cell heterogeneity, prioritize markers for further investigation, and improve the understanding of cell metabolism in human diseases and response to therapy.

Robotised screening and characterisation for accelerated discovery of novel Lithium-ion battery electrolytes: Building a platform and proof of principle studies

A fast transition towards the use of clean and green energy sources requires accelerated discovery of new energy storage systems and devices. In this concept automation and robotics can play a key role. Here we present the development of a robotized platform, Poseidon, for the screening and discovery of new water-based electrolyte candidate systems for lithium-ion batteries (LIBs) systems. We have successfully demonstrated the Poseidon screening and characterisation capabilities for electrolytic discovery, which includes a range of steps such as electrolyte formulation, Raman spectroscopic characterization, coin-cell mounting/disassembling and electrochemical battery evaluation via an accelerated screening cycling procedure. A comparison with analogous manual laboratory experiments shows that relevant accuracy for robotized screening purposes has been established. Furthermore, the presented accelerated charge/discharge cycling procedure is shown to be adequate for screening purposes of the test system.

Progress in the Realization of µ-Brush W for Plasma-Facing Components

During the service life of plasma-facing components, they are exposed to cyclic stationary and transient thermal loads. The former causes thermal fatigue and potentially detachment between the plasma-facing material tungsten and the structural Cu-based materials (divertor) and steel (first wall). The latter causes surface roughening, cracking, or even melting, which could drastically increase the erosion rate. Employing thin flexible W wires (Ww) with a diameter of a few hundred µm can reduce mechanical stresses, and we demonstrated their crack resilience against transient loads within first proof of principle studies. Here, status and future paths towards the large-scale production of such Ww assemblies, including techniques for realizing feasible joints with Cu, steel, or W, are presented. Using wire-based laser metal deposition, we were able to create a homogeneous and shallow infiltration of about 200 µm of the Ww assembly with steel. A high-heat-flux test on such a µ-brush (10 × 10 × 5 mm3 Ww on a ~0.5 mm thick steel layer) using 5 MW/m2 for 2000 cycles was performed without loss of any wire. Microstructural examination after and infrared analysis during the test showed no significant signs of degradation of the joint.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Abstract P4-04-10: Pharmacological targeting of cholinergic receptors as a novel breast cancer immunotherapy

Abstract There is a general consensus that the devastating effect of malignant breast cancers (MBCs) is due in part to the ability of breast tumors to exploit host immune functions by subverting normal mechanisms of immunosurveillance. For example, while immunotherapies based on immune checkpoint inhibition in lymphocytes have been associated with better prognosis in other cancers, the efficacy in breast cancer has been limited. We focus here on activating biochemical pathways in antigen-presenting dendritic cells (DCs) that are otherwise immunosuppressive to stimulate the activity of tumor-infiltrating lymphocytes through a novel mechanism. To this end, we recently discovered using an immune-competent mouse model of MBC that breast cancer cells exploit adaptive immune cells such as DCs that express CHRNA7, the α7 nicotinic acetylcholine receptor. Based on the importance of the CHRNA7 signaling pathway in the control of normal inflammation in macrophages in general, we show here that CHRNA7 stimulation in DCs leads to an activation of lymphocytes associated with a reduction in MBC. Specifically, mice lacking the CHRNA7 gene have (a) decreased survival, (b) increased tumor growth kinetics, (c) immunophenotyping revealed a reduction in the “cancer-permissive” phenotype in CHRNA7 KO mice, and (d), a commensurate reduction in T cell proliferation. Based on these mechanistic proof of principle studies, we demonstrate that in contrast to the loss of CHRNA7 in KO, pharmacological activation of CHRNA7 with AR-R17779 has the opposite effect by stimulating DCs to activate the adaptive immune response which leads to tumor progression and increased survival. These findings demonstrate the preclinical potential of AR-R17779 as a clinically relevant approach to stimulate the adaptive immune system through a novel pathway that has not been examined to date, and with a small molecule drug that is stable, specific and well-tolerated. Citation Format: Brian P. Eliceiri, Jin Qian, Sarah Blair. Pharmacological targeting of cholinergic receptors as a novel breast cancer immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-10.

Modification of Single Cells Within Mouse Mammary Gland Derived Acini via Viral Transduction.

The growth of organoid cultures from primary donor tissue is able to recapitulate the original tissue morphology, heterogeneity, and characteristics. Close study of these cultures grants a deeper understanding of the chain of events occurring during disease progression and healthy tissue development. While patient derived organoids are particularly suited to assay for novel treatment options, organoids obtained from model organisms are perfectly suited to establish in-depth analysis technology, including longitudinal imaging approaches, as well as proof of principle studies that rely on a steady source of primary tissue. All these approaches profit from advancements in technology to manipulate cells within an organoid.Here we present an optimized protocol to generate, culture, and transduce 3D acini obtained from mouse primary mammary epithelial cells via viral vectors. Applying this method, a few cells within the preserved organoid can be marked, changed, and tracked within an unaltered neighboring environment of non-transduced cells to better understand processes like, for instance, tumor initiation.

In situ electromagnet with active cooling for real-time magneto-optic Kerr effect spectroscopy.

We present a compact in situ electromagnet with an active cooling system for use in ultrahigh vacuum environments. The active cooling enhances the thermal stability and increases the electric current that can be applied through the coil, promoting the generation of homogeneous magnetic fields, required for applications in real-time deposition experiments. The electromagnet has been integrated into a reflectance difference magneto-optic Kerr effect (RD-MOKE) spectroscopy system that allows the synchronous measurement of the optical anisotropy and the magneto-optic response in polar MOKE geometry. Proof of principle studies have been performed in real time during the deposition of ultra-thin Ni films on Cu(110)-(2 × 1)O surfaces, corroborating the extremely sharp spin reorientation transition above a critical coverage of 9 monolayers and demonstrating the potential of the applied setup for real-time and in situ investigations of magnetic thin films and interfaces.

Scale-up of cluster beam deposition to the gram scale with the matrix assembly cluster source for heterogeneous catalysis (propylene combustion)

Cluster beam deposition is a solvent-free method to prepare films of nanoparticles, one obvious application being heterogeneous catalysis. To address the problem of low cluster deposition rates, a novel cluster beam source, the “Matrix Assembly Cluster Source” was invented recently. Following the proof of principle studies, here, we demonstrate a further scale-up by 2 orders of magnitude, equivalent to reaching a production of ∼10 mg of clusters (Au100) per hour. This allows the preparation of cluster-decorated powder catalysts at the gram scale, comfortably sufficient for practical catalysis studies of novel materials at the research level, as demonstrated here by the catalytic combustion of propylene.

Barrett's Esophagus and Esophageal Carcinoma: Can Biomarkers Guide Clinical Practice?

Despite gastrointestinal societal recommendations for endoscopic screening and surveillance of Barrett's esophagus, the rates of esophageal adenocarcinoma continue to rise. Furthermore, this current practice is costly to patients and the medical system without clear evidence of reduction in cancer mortality. The use of biomarkers to guide screening, surveillance, and treatment strategies might alleviate some of these issues. Incredible advances in biomarker identification, biomarker assays, and minimally-invasive modalities to acquire biomarkers have shown promising results. We will highlight recently published, key studies demonstrating where we are with using biomarkers for screening and surveillance in clinical practice, and what is on the horizon regarding novel non-invasive and minimally invasive methods to acquire biomarkers. Proof-of principle studies using in silico models demonstrate that biomarker-guided screening, surveillance, and therapeutic intervention strategies can be cost-effective and can reduce cancer deaths in patients with Barrett's esophagus.

The Therapeutic Potential of Non-invasive Neurostimulation for Motor Skill Learning in Children with Neurodevelopmental Disorders

Non-invasive neurostimulation is becoming increasingly popular in rehabilitation, as it may enhance the brain’s natural learning processes, potentially increasing the effectiveness of movement interventions. The current paper provides a review of studies that have investigated the application of transcranial direct current stimulation (tDCS) in pediatric populations with neurodevelopmental disorders (NDD) complicated by motor impairment. Proof of principle studies indicate that tDCS can positively influence motor learning in children. The application of tDCS across pediatric populations with NDD is in its early stages. We emphasize the need for caution and further research investigating the therapeutic potential of tDCS in children with NDD and motor impairment. Indications of efficacy will require carefully designed trials before conclusions of effectiveness can be made. Optimal tDCS protocols may differ across both disorders and individuals. Protocol parameters which produce clinically significant improvements will become clearer with further high-quality evidence.

Alternative Strategy for a Quadrivalent Live Attenuated Influenza Virus Vaccine.

Influenza virus infections continue to pose a major public health threat worldwide associated with seasonal epidemics and sporadic pandemics. Vaccination is considered the first line of defense against influenza. Live attenuated influenza virus vaccines (LAIVs) may provide superior responses compared to inactivated vaccines because the former can better elicit a combination of humoral and cellular responses by mimicking a natural infection. Unfortunately, during the 2013-2014, 2014-2015, and 2015-2016 seasons, concerns emerged about the effectiveness of the only LAIV approved in the United States that prevented the Advisory Committee on Immunization Practices (ACIP) from recommending its use. Such drawbacks open up the opportunity for alternative LAIV strategies that could overcome such concerns. Previously, we developed a combined strategy of temperature-sensitive mutations in the PB2 and PB1 segments and an epitope tag in the C terminus of PB1 that effectively attenuates influenza A viruses of avian and mammalian origin. More recently, we adopted a similar strategy for influenza B viruses. The resulting attenuated (att) influenza A and B viruses were safe, immunogenic, and protective against lethal influenza virus challenge in a variety of animal models. In this report, we provide evidence of the potential use of our att strategy in a quadrivalent LAIV (QIV) formulation carrying H3N2 and H1N1 influenza A virus subtype viruses and two antigenic lineages of influenza B viruses. In naive DBA/2J mice, two doses of the QIV elicited hemagglutination inhibition (HI) responses with HI titers of ≥40 and effectively protected against lethal challenge with prototypical pandemic H1N1 influenza A and influenza B virus strains.IMPORTANCE Seasonal influenza viruses infect 1 billion people worldwide and are associated with ∼500,000 deaths annually. In addition, the never-ending emergence of zoonotic influenza viruses associated with lethal human infections and of pandemic concern calls for the development of better vaccines and/or vaccination strategies against influenza virus. Regardless of the strategy, novel influenza virus vaccines must aim at providing protection against both seasonal influenza A and B viruses. In this study, we tested an alternative quadrivalent live attenuated influenza virus vaccine (QIV) formulation whose individual components have been previously shown to provide protection. We demonstrate in proof-of principle studies in mice that the QIV provides effective protection against lethal challenge with either influenza A or B virus.

Genomic insights into Cushing syndrome

In the setting of Cushing syndrome, genomic analyses can be performed either in tumors responsible for endogenous Cushing, or in patients exposed to glucocorticoid excess. Genomics of tumors identified several new genes – including ZNRF3 in adrenocortical carcinomas, PRKACA in cortisol-producing adrenal adenomas, ARMC5 in primary macronodular adrenal hyperplasia and USP8 in pituitary corticotroph adenomas. These genes shed new lights on the mechanisms responsible for these tumors. Integrated genomic studies of adrenal carcinomas identified distinct molecular classes, with remarkably different prognostic outcome. Beyond the mechanistic novelties, a new generation of prognostic markers emerges, with potentially important impact on patients care. For the future, genomic efforts should be pursued, focusing on poorly characterized tumors responsible for Cushing syndrome – including endocrine tumors secreting ACTH. In addition, epigenomics is emerging as an outstanding set of tools for characterizing tumors, unraveling unprecedented aspects of tumorigenesis. Applying these tools to endocrine tumors responsible for Cushing syndrome may also lead to important discoveries. Genomics of patients exposed to glucocorticoid excess is an emerging research field. Proof of principle studies have been performed, identifying molecular markers of glucocorticoid excess in blood. Research efforts should now concentrate on markers of mild glucocorticoid excesses – endogenous or exogenous –, owing to their high prevalence in general population. In addition, markers of individual susceptibility to each type of glucocorticoid complication are needed. It remains to be determined whether genomics can identify such markers.

Abstract 2760: Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies

Abstract Background: During the past years, much progress has been made in detection and analysis of circulating tumor cells (CTCs) in several tumor entities, including prostate cancer, breast cancer or lung cancer. However, little is known about circulating tumor cells in patients suffering from clear cell renal cell carcinomas. The majority of technologies detecting CTCs is based on expression of epithelial markers on the surface of these cells, e.g. EpCAM. Additionally, biophysical approaches have been invented to detect CTCs based on size, invasive capacity or density. In order to be able to detect CTCs in patient samples, in vitro establishment of the most accurate isolation procedure followed by precise detection techniques has to be performed. Aim of our studies was to build a stable in vitro fundament of isolation and subsequent detection of CTCs in ccRCC patients. Methods: We made use of 4 different technologies, all of which have been approved for detection of CTCs in distinctive tumor entities. We used EpCAM based positive enrichment of CTCs, Ficoll densitiy centrifugation followed by CD45-positive cell depletion, rosette formation followed by CD45 positive cell depletion as well as size and deformability based enrichment technologies by using the Parsortix system. Furthermore, by using 4 phenotypically distinct ccRCC cell lines, we tried to detect markers unique for tumor cells in demarcation to blood cells. Results: By performing spiking experiments of renal cancer cells, we found the highest recovery rates by using the size based Parsortix system. Interestingly, the most established technique of EpCAM based isolation failed in three out of four cell lines to recover more than 40%. Expression of well-established markers for ccRCC, like carboanhydrase (CA)-9, could be detected in renal cancer cell lines. However, expression was also found in blood samples of healthy donors. Another marker used for immunohistochemical diagnosis of ccRCC, PAX8, showed weak to absent expression in established renal cancer cell lines. The highest specificity to detect renal cancer cells in blood samples was found when analyzing KRT8 or KRT 19 expression. Conclusion: Our results demonstrate that firstly, using the EpCAM based CTC enrichment, which is the basis of the CellSearch system, which up to now is the only methodology approved by the FDA, the majority of renal cancer cells will presumably not be detected in blood samples. This seems largely due to low or absent expression of EpCAM on renal cancer cells. The usage of the size based Parsortix system showed the highest recovery rates and should therefore be analyzed in more detail on samples of ccRCC patients. Secondly, an exclusive marker for defining a renal CTC is still missing. Some well-established ccRCC markers, like CA-9, failed to specifically detect renal cancer cells in blood samples, as they were either present also in healthy blood samples or absent in renal cancer cell lines. Citation Format: Yvonne Maertens, Verena Humberg, Julie Steinestel, Martin Boegemann, Andres J. Schrader, Christof Bernemann. Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2017-2760

Mechanisms of haem toxicity in haemolysis and protection by the haem‐binding protein, haemopexin

Haem (iron‐protoporphyrin IX) is vital to aerobic life. However, haemolysis generates massive amounts of free haem that can be maintained for several days. Haem is toxic because the chemical reactions of haem that occur in the presence of oxygen can destroy most biological molecules and haem activates cells of the immune system to drive inflammation. Haemopexin captures haem and disarms its chemical activity, thus protecting cells and molecules in biological fluids from haem toxicity. A surprisingly large number of disease states characterized by haemolysis or complicated by haemolysis (e.g. sickle cell disease, sepsis, haemolytic anaemias, viral haemorrhagic fevers) lack targeted therapies. Novel treatments from proof of principle studies in mice models of sickle cell disease and sepsis include replenishment therapies with haemopexin infusions or hepatic gene therapy to express haemopexin. Here, we characterize the key protective mechanisms that haemopexin instigates, explain how methaemoglobin and methemalbumin produce haem toxicity and evaluate the clinical importance of the haemopexin system in protecting against extracellular haem‐activated pathology in intravascular haemolysis. The main findings are that endocytosis of haem–haemopexin co‐regulates the safe trafficking of three redox‐active metals through subcellular compartments and, compared with the response to ‘free’ haem, uniquely activates several signalling pathways and transcription factors that induce the production of antioxidant enzymes and molecules known to maintain the intracellular redox state. Mounting evidence from patient longitudinal studies supports that impairments of haem and haemoglobin clearance are often unrecognized. Clinical data suggest that haemopexin is likely the best plasma biomarker for assessing a haem load as, unlike haptoglobin, it responds to heme and is not an acute‐phase reactant. Knowledge of the biology of haemopexin has yielded a potential novel therapeutic and also provided an impetus and means for improving diagnosis and longitudinal monitoring of patients with haemolysis.

Effects of Donor Vitamin A Deficiency and Pharmacologic Modulation of Donor T Cell Retinoic Acid Pathway on the Severity of Experimental Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). A combination of genetic and nongenetic factors dictates the incidence and severity of GVHD. Recent studies have identified the potential role of the retinoic acid (RA)/retinoic acid receptor (RAR) pathway in the pathogenesis of GVHD. RA is the active metabolite of vitamin A. Thus, a clinically relevant question is whether HSCT donor and/or recipient vitamin A status affects the development of GVHD. It has been previously reported that recipient vitamin A deficiency is associated with reduced intestinal GVHD and prolonged overall survival after experimental allogeneic HSCT. However, it is still unknown whether donor vitamin A status influences GVHD development. In the current study, we report that chronic vitamin A deficiency changes the composition of T cell compartment of donor mice with a reduction in the percentage of CD4+ T cells. We showed that although vitamin A deficiency does not affect donor T cell alloreactivity on a per cell basis, a decreased proportion of donor CD4+ T cells in marrow graft inoculums leads to reduced incidence and severity of GVHD. Furthermore, our proof of principle studies using a pan-RAR antagonist demonstrated that transient inhibition of donor T cell RAR signaling can reduce T cell alloreactivity and their ability to cause lethal GVHD. Our studies provide preclinical evidence that donor vitamin A deficiency may be a nongenetic factor that can modulate the severity of GVHD and pharmacologic interfering RA/RAR pathway in donor T cells might be a valuable approach for mitigating GVHD after allogeneic HSCT.

Local measurement of thermal conductivity and diffusivity.

Simultaneous measurement of local thermal diffusivity and conductivity is demonstrated on a range of ceramic samples. This was accomplished by measuring the temperature field spatial profile of samples excited by an amplitude modulated continuous wave laser beam. A thin gold film is applied to the samples to ensure strong optical absorption and to establish a second boundary condition that introduces an expression containing the substrate thermal conductivity. The diffusivity and conductivity are obtained by comparing the measured phase profile of the temperature field to a continuum based model. A sensitivity analysis is used to identify the optimal film thickness for extracting the both substrate conductivity and diffusivity. Proof of principle studies were conducted on a range of samples having thermal properties that are representatives of current and advanced accident tolerant nuclear fuels. It is shown that by including the Kapitza resistance as an additional fitting parameter, the measured conductivity and diffusivity of all the samples considered agreed closely with the literature values. A distinguishing feature of this technique is that it does not require a priori knowledge of the optical spot size which greatly increases measurement reliability and reproducibility.

Feasibility and safety of cCD20 RNA CAR-bearing T cell therapy for the treatment of canine B cell malignancies

CD19 chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating human hematological malignancies. However, the full potential of CAR technology has not been reached in solid malignancies and limitations exist in utilizing genetically identical murine models to interrogate the safety and efficacy of anti-tumor immune therapies. Canines are an outbred population that naturally develop spontaneous disease. Here we have established a treatment model for CAR T cell therapy in outbred dogs with spontaneous B cell lymphoma. We developed a protocol to isolate, activate, and express CAR in canine T cells. We characterized a methodology utilizing anti-cCD3 antibody with a K562 antigen-presenting cell (APC) line that expresses human CD32 and canine CD86 to activate canine lymphocytes. This method yields robust (50-120x) expansion of cCD4+ or cCD8+ T cells from normal or lymphoma-diseased dogs. Canine T cells were then engineered utilizing mRNA electroporation to express a CAR targeting the B cell antigen CD20. An anti-canine CD20 (cCD20) CAR was selected for proof of principle studies to establish feasibility of efficient CAR expression and redirected effector function in canine T cells from a healthy and lymphoma-diseased dogs. The cCD20-Z CAR construct was efficiently, but transiently, expressed in canine T cells after mRNA electroporation. cCD20 CAR T cells exhibited specific lysis and IFN-γ secretion in response to cCD20+ target cells. These data indicate successful establishment of a method to isolate, activate, and express a fully functional CAR in canine T cells for use in adoptive immunotherapy. These results rationalized testing of this CAR approach in canines with advanced CD20+ malignancies. In an IACUC approved protocol, we administered three doses of autologous cCD20-Z RNA CAR+ T cells to a companion canine with advanced B cell lymphoma. Treatment was well-tolerated and no evidence of serious adverse events was observed. The anti-tumor effect, however, was transient, suggesting a need for product optimization. Future study will establish a lenti- or retroviral-based method to modify canine T cells to promote T cell persistence and durable anti-tumor effects in vivo. In conclusion, our study establishes the methodologies and feasibility of anti-cCD20 CAR therapy in canines with spontaneous B cell lymphoma that may represent a future clinical option for such companion pets. This work also lays the foundation for study of a large, outbred animal model that better resembles spontaneous, human malignancies to allow for improved assessment for potential toxicity risks prior to translation into human studies.