A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro- and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D µComputer tomography bioluminescence tomography (µCT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (± IL-2) or control BDCA-2 CAR T cell (± IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 IU/mouse. CAR T cell distribution was measured in 2D BLI and 3D µCT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D µCT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D µCT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D- and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status

A Second-Generation Device for Automated Training and Quantitative Behavior Analyses of Molecularly-Tractable Model Organisms

A deep understanding of cognitive processes requires functional, quantitative analyses of the steps leading from genetics and the development of nervous system structure to behavior. Molecularly-tractable model systems such as Xenopus laevis and planaria offer an unprecedented opportunity to dissect the mechanisms determining the complex structure of the brain and CNS. A standardized platform that facilitated quantitative analysis of behavior would make a significant impact on evolutionary ethology, neuropharmacology, and cognitive science. While some animal tracking systems exist, the available systems do not allow automated training (feedback to individual subjects in real time, which is necessary for operant conditioning assays). The lack of standardization in the field, and the numerous technical challenges that face the development of a versatile system with the necessary capabilities, comprise a significant barrier keeping molecular developmental biology labs from integrating behavior analysis endpoints into their pharmacological and genetic perturbations. Here we report the development of a second-generation system that is a highly flexible, powerful machine vision and environmental control platform. In order to enable multidisciplinary studies aimed at understanding the roles of genes in brain function and behavior, and aid other laboratories that do not have the facilities to undergo complex engineering development, we describe the device and the problems that it overcomes. We also present sample data using frog tadpoles and flatworms to illustrate its use. Having solved significant engineering challenges in its construction, the resulting design is a relatively inexpensive instrument of wide relevance for several fields, and will accelerate interdisciplinary discovery in pharmacology, neurobiology, regenerative medicine, and cognitive science

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

On the influence of the aliphatic linker on fabrication of highly ordered and orientated self-assembled monolayers of aromatic selenols on AU(111)

Self-assembled monolayers (SAMs) formed by adsorption of 1,2-dibenzyldiselenide (DPMSe) and 1,2-diphenyldiselenide (DBSe) on Au(111) substrates at room temperature have been characterized using scanning tunnelling microscopy, X-ray photoelectron spectroscopy, infrared reflection absorption spectroscopy, near-edge X-ray absorption fine structure spectroscopy, and low-energy electron diffraction. Upon adsorption, the Se-Se bonds in DPMSe and DBSe were cleaved on the gold surface to form phenylmethaneselenolate (PMSe) and benzeneselenolate (BSe) species, respectively. Although both PMSe and BSe molecular entities only differ in their structure (an additional methyl group in PMSe), the resulting monolayer films revealed noteworthy dissimilarities regarding their adlayer SAM structure and surface morphology. The molecular adlayer structure and orientation of PMSe and BSe species were found to vary significantly with the immersion time (IT). The resulting PMSe films were poorly organized, and the structure was described by a (4√3 × 2) rectangular unit cell for the SAMs prepared with 24 h of IT. Moreover, the PMSe-SAMs were found to be unstable upon exposure to air for a long time. Our results showed that exposure to air for 48 h results in the formation of small bright ad-islands, which have a height corresponding to that of a single atomic step on the Au(111). Contrary, BSe-SAMs exhibited densely packed and well-ordered monolayers, and two different structural phases were resolved at short and long ITs. The most densely packed structure was obtained for SAMs prepared with very short ITs (10 min). Upon increasing the IT, the SAMs exhibited structural changes to a lower density of molecular packing structure. The spectroscopic data also confirmed this structural transformation by suggesting an upright orientation for BSe-SAMs prepared after short ITs and strongly inclined adsorption geometry for SAMs prepared after long ITs. © 2014 American Chemical Society

Photoacoustic imaging of tumor targeting with riboflavin-functionalized theranostic nanocarriers

Nataliia Beztsinna,1,* Yoanna Tsvetkova,2,* Jithin Jose,3 Boutayna Rhourri-Frih,1 Wa&rsquo;el Al Rawashdeh,2 Twan Lammers,2 Fabian Kiessling,2 Isabelle Bestel1 1Institute of Chemistry &amp; Biology of Membranes &amp; Nanoobjects (CBMN), UMR 5248, University of Bordeaux, Pessac, France; 2Institute for Experimental Molecular Imaging, RWTH Aachen University Clinic, Aachen, Germany; 3Fujifilm VisualSonics BV, Amsterdam, the Netherlands *These authors contributed equally to this work Abstract: Photoacoustic imaging is an emerging method in the molecular imaging field, providing high spatiotemporal resolution and sufficient imaging depths for many clinical applications. Therefore, the aim of this study was to use photoacoustic imaging as a tool to evaluate a riboflavin (RF)-based targeted nanoplatform. RF is internalized by the cells through a specific pathway, and its derivatives were recently shown as promising tumor-targeting vectors for the drug delivery systems. Here, the RF amphiphile synthesized from a PEGylated phospholipid was successfully inserted into a long-circulating liposome formulation labeled with the clinically approved photoacoustic contrast agent &ndash; indocyanine green (ICG). The obtained liposomes had a diameter of 124 nm (polydispersity index =0.17) and had a negative zeta potential of &ndash;26 mV. Studies in biological phantoms indicated a stable and concentration-dependent photoacoustic signal (Vevo&reg; LAZR) of the ICG-containing RF-functionalized liposomes. In A431 cells, a&nbsp;high uptake of RF-functionalized liposomes was found and could be blocked competitively. First, studies in mice revealed ~3 times higher photoacoustic signal in subcutaneous A431 tumor xenografts (P&lt;0.05) after injection of RF-functionalized liposomes compared to control particles. In this context, the application of a spectral unmixing protocol confirmed the initial quantitative data and improved the localization of liposomes in the tumor. In conclusion, the synthesized RF amphiphile leads to efficient liposomal tumor targeting and can be favorably detected by photoacoustic imaging with a perspective of theranostic applications. Keywords: photoacoustic imaging, long-circulating liposomes, active targeting, riboflavin, indocyanine green&nbsp

Management of the Most Common Functional Gastrointestinal Disorders in Infancy: The Middle East Expert Consensus

The occurrence of functional gastrointestinal disorders (FGIDs) is a formidable challenge for infants, parents, and healthcare professionals. Although data from the Middle East are scarce, experts consider FGIDs a prevalent condition in everyday clinical practice. The new Rome IV criteria revisited the definitions from a clinical perspective to provide a practical and consistent diagnostic protocol for FGIDs. However, the treatment practices for functional disorders vary considerably among Middle Eastern countries, often resulting in mismanagement with unnecessary investigations and treatments. In addition, the role of various treatment modalities, including probiotics such as Lactobacillus reuteri DSM 17938, in FGIDs requires further discussion and evaluation. During a consensus meeting, a locally relevant approach for treating common FGIDs such as infant regurgitation, infant colic, and functional constipation was discussed and approved by regional experts. The participants suggested a simplified treatment plan and protocol for general pediatricians and other primary care physicians managing FGIDs. This easy-to-follow standardized protocol will help streamline the initial management of this complex disorder in the Middle East region and even globally