Increased endocytosis rate and enhanced lysosomal pathway of silica-coated superparamagnetic nanoparticles into M-HeLa cells compared with cultured primary motor neurons.

The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.

Unraveling viral drug targets: a deep learning-based approach for the identification of potential binding sites.

The coronavirus disease 2019 (COVID-19) pandemic has spurred a wide range of approaches to control and combat the disease. However, selecting an effective antiviral drug target remains a time-consuming challenge. Computational methods offer a promising solution by efficiently reducing the number of candidates. In this study, we propose a structure- and deep learning-based approach that identifies vulnerable regions in viral proteins corresponding to drug binding sites. Our approach takes into account the protein dynamics, accessibility and mutability of the binding site and the putative mechanism of action of the drug. We applied this technique to validate drug targeting toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein S. Our findings reveal a conformation- and oligomer-specific glycan-free binding site proximal to the receptor binding domain. This site comprises topologically important amino acid residues. Molecular dynamics simulations of Spike in complex with candidate drug molecules bound to the potential binding sites indicate an equilibrium shifted toward the inactive conformation compared with drug-free simulations. Small molecules targeting this binding site have the potential to prevent the closed-to-open conformational transition of Spike, thereby allosterically inhibiting its interaction with human angiotensin-converting enzyme 2 receptor. Using a pseudotyped virus-based assay with a SARS-CoV-2 neutralizing antibody, we identified a set of hit compounds that exhibited inhibition at micromolar concentrations.

Accuracy and User Acceptability of 24-hour Ambulatory Blood Pressure Monitoring by a Prototype Cuffless Multi-Sensor Device Compared to a Conventional Oscillometric Device.

OBJECTIVE: 24-hour ambulatory blood pressure monitoring (24ABPM) is state of the art in out-of-office blood pressure (BP) monitoring. Due to discomfort and technical limitations related to cuff-based 24ABPM devices, methods for non-invasive and continuous estimation of BP without the need for a cuff have gained interest. The main aims of the present study were to compare accuracy of a pulse arrival time (PAT) based BP-model and user acceptability of a prototype cuffless multi-sensor device (cuffless device), developed by Aidee Health AS, with a conventional cuff-based oscillometric device (ReferenceBP) during 24ABPM. METHODS: Ninety-five normotensive and hypertensive adults underwent simultaneous 24ABPM with the cuffless device on the chest and a conventional cuff-based oscillometric device on the non-dominant arm. PAT was calculated using the electrocardiogram (ECG) and photoplethysmography (PPG) sensors incorporated in the chest-worn device. The cuffless device recorded continuously, while ReferenceBP measurements were taken every 20 minutes during daytime and every 30 minutes during nighttime. Two-minute PAT-based BP predictions corresponding to the ReferenceBP measurements were compared with ReferenceBP measurements using paired t-tests, bias, and limits of agreement. RESULTS: Mean (SD) of ReferenceBP compared to PAT-based daytime and nighttime systolic BP (SBP) were 129.7 (13.8) mmHg versus 133.6 (20.9) mmHg and 113.1 (16.5) mmHg versus 131.9 (23.4) mmHg. Ninety-five % limits of agreements were [-26.7, 34.6 mmHg] and [-20.9, 58.4 mmHg] for daytime and nighttime SBP respectively. The cuffless device was reported to be significantly more comfortable and less disturbing than the ReferenceBP device during 24ABPM. CONCLUSIONS: In the present study, we demonstrated that a general PAT-based BP model had unsatisfactory agreement with ambulatory BP during 24ABPM, especially during nighttime. If sufficient accuracy can be achieved, cuffless BP devices have promising potential for clinical assessment of BP due to the opportunities provided by continuous BP measurements during real-life conditions and high user acceptability.

What is the context?Hypertension is a major risk factor for cardiovascular and cerebrovascular end-organ damage, morbidity, and mortality world-wide.Accurate measurement of blood pressure is essential for the diagnosis and management of hypertension.What is new?Cuffless blood pressure devices that allow measurement of blood pressure without a pressure cuff is a promising and novel method of blood pressure estimation.The objective of this study is to assess whether pulse arrival time alone can be used to estimate blood pressure accurately during 24-hour ambulatory blood pressure monitoring, using a prototype cuffless device placed on the chest.Our analysis shows that a general model based on pulse arrival time overestimated ambulatory blood pressure, especially during nighttime.User acceptability was higher with the cuffless device compared to a conventional cuff-based oscillometric device during 24-hour ambulatory blood pressure monitoring.What is the impact?This study provides further evidence that accurate blood pressure estimations cannot be achieved by using pulse arrival time alone as a surrogate for blood pressure measurements.

Targeted depletion of TRBV9+ T cells as immunotherapy in a patient with ankylosing spondylitis.

Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8+ TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9+ T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.

Control of the antitumour activity and specificity of CAR T cells via organic adapters covalently tethering the CAR to tumour cells.

On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.

Cropland Mapping Using Sentinel-1 Data in the Southern Part of the Russian Far East.

Crop identification is one of the most important tasks in digital farming. The use of remote sensing data makes it possible to clarify the boundaries of fields and identify fallow land. This study considered the possibility of using the seasonal variation in the Dual-polarization Radar Vegetation Index (DpRVI), which was calculated based on data acquired by the Sentinel-1B satellite between May and October 2021, as the main characteristic. Radar images of the Khabarovskiy District of the Khabarovsk Territory, as well as those of the Arkharinskiy, Ivanovskiy, and Oktyabrskiy districts in the Amur Region (Russian Far East), were obtained and processed. The identifiable classes were soybean and oat crops, as well as fallow land. Classification was carried out using the Support Vector Machines, Quadratic Discriminant Analysis (QDA), and Random Forest (RF) algorithms. The training (848 ha) and test (364 ha) samples were located in Khabarovskiy District. The best overall accuracy on the test set (82.0%) was achieved using RF. Classification accuracy at the field level was 79%. When using the QDA classifier on cropland in the Amur Region (2324 ha), the overall classification accuracy was 83.1% (F1 was 0.86 for soybean, 0.84 for fallow, and 0.79 for oat). Application of the Radar Vegetation Index (RVI) and VV/VH ratio enabled an overall classification accuracy in the Amur region of 74.9% and 74.6%, respectively. Thus, using DpRVI allowed us to achieve greater performance compared to other SAR data, and it can be used to identify crops in the south of the Far East and serve as the basis for the automatic classification of cropland.

Insulinoma in childhood: a retrospective review of 22 patients from one referral centre.

Background: Insulinomas are very rare in childhood with sparse knowledge on the clinical aspects and the presence of Multiple Endocrine Neoplasia type 1 (MEN1). Methods: We conducted a retrospective review of patients diagnosed with insulinoma between 1995 and 2021, presenting to one referral centre in Russia. Clinical, biochemical, genetic, imaging and histological data were collected. In addition, follow-up and family data were obtained. Results: A total of twenty-two children aged 5 to 16 years were identified. The median (range) gap between the first hypoglycaemia symptoms and diagnosis was 10 (1-46) months. Twelve children (55%) were misdiagnosed to have epilepsy and were treated with anticonvulsants before hypoglycemia was revealed. Contrast enhanced MRI and/or CT were accurate to localize the lesion in 82% (n=18). Five patients (23%) had multiple pancreatic lesions. All children underwent surgical treatment. The median (range) diameter of removed tumors was 1.5 (0.3-6) cm. Histopathological studies confirmed the presence of insulinoma in all cases. Immunohistochemical studies revealed G2 differentiation grade in 10 out of 17 cases. Two patients were diagnosed with metastatic insulinoma. One of them had metastases at the time of insulinoma diagnosis, while the other was diagnosed with liver metastases eight years after the surgery. Eight children (36%) were found to carry MEN1 mutations, inherited n=5, de novo n=1, no data, n=2. Children with MEN1 had significantly higher number of pancreatic tumors compared to sporadic cases. All of them developed additional MEN1 symptoms during the following 2-13 years. In the five patients with inherited MEN1, seven family members had hitherto undiscovered MEN1 manifestations. Conclusions: In this large cohort of children with rare pediatric insulinomas, MEN1 syndrome and G2 tumors were frequent, as well as hitherto undiscovered MEN1 manifestations in family members. Our data emphasize the need of genetic testing in all children with insulinoma and their relatives, even in the absence of any other features, as well as the importance of a prolonged follow-up observation.

Accuracy of non-invasive cuffless blood pressure in the intensive care unit: Promises and challenges.

Objective: Continuous non-invasive cuffless blood pressure (BP) monitoring may reduce adverse outcomes in hospitalized patients if accuracy is approved. We aimed to investigate accuracy of two different BP prediction models in critically ill intensive care unit (ICU) patients, using a prototype cuffless BP device based on electrocardiogram and photoplethysmography signals. We compared a pulse arrival time (PAT)-based BP model (generalized PAT-based model) derived from a general population cohort to more complex and individualized models (complex individualized models) utilizing other features of the BP sensor signals. Methods: Patients admitted to an ICU with indication of invasive BP monitoring were included. The first half of each patient's data was used to train a subject-specific machine learning model (complex individualized models). The second half was used to estimate BP and test accuracy of both the generalized PAT-based model and the complex individualized models. A total of 7,327 measurements of 15 s epochs were included in pairwise comparisons across 25 patients. Results: The generalized PAT-based model achieved a mean absolute error (SD of errors) of 7.6 (7.2) mmHg, 3.3 (3.1) mmHg and 4.6 (4.4) mmHg for systolic BP, diastolic BP and mean arterial pressure (MAP) respectively. Corresponding results for the complex individualized model were 6.5 (6.7) mmHg, 3.1 (3.0) mmHg and 4.0 (4.0) mmHg. Percentage of absolute errors within 10 mmHg for the generalized model were 77.6, 96.2, and 89.6% for systolic BP, diastolic BP and MAP, respectively. Corresponding results for the individualized model were 83.8, 96.2, and 94.2%. Accuracy was significantly improved when comparing the complex individualized models to the generalized PAT-based model in systolic BP and MAP, but not diastolic BP. Conclusion: A generalized PAT-based model, developed from a different population was not able to accurately track BP changes in critically ill ICU patients. Individually fitted models utilizing other cuffless BP sensor signals significantly improved accuracy, indicating that cuffless BP can be measured non-invasively, but the challenge toward generalizable models remains for future research to resolve.

Use of Nickel Oxide Catalysts (Bunsenites) for In-Situ Hydrothermal Upgrading Process of Heavy Oil.

In this study, Nickel oxide-based catalysts (NixOx) were synthesized and used for the in-situ upgrading process of heavy crude oil (viscosity 2157 mPa·s, and API gravity of 14.1° at 25 °C) in aquathermolysis conditions for viscosity reduction and heavy oil recovery. All characterizations of the obtained nanoparticles catalysts (NixOx) were performed through Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), X-Ray and Diffraction (XRD), and ASAP 2400 analyzer from Micromeritics (USA), methods. Experiments of catalytic and non-catalytic upgrading processes were carried out in a discontinuous reactor at a temperature of 300 °C and 72 bars for 24 h and 2% of catalyst ratio to the total weight of heavy crude oil. XRD analysis revealed that the use of nanoparticles of NiO significantly participated in the upgrading processes (by desulfurization) where different activated form catalysts were observed, such as α-NiS, ß-NiS, Ni3S4, Ni9S8, and NiO. The results of viscosity analysis, elemental analysis, and 13C NMR analysis revealed that the viscosity of heavy crude oil decreased from 2157 to 800 mPa·s, heteroatoms removal from heavy oil ranged from S-4.28% to 3.32% and N-0.40% to 0.37%, and total content of fractions (ΣC8-C25) increased from 59.56% to a maximum of 72.21%, with catalyst-3 thank to isomerization of normal and cyclo-alkanes and dealkylation of lateral chains of aromatics structures, respectively. Moreover, the obtained nanoparticles showed good selectivity, promoting in-situ hydrogenation-dehydrogenation reactions, and hydrogen redistribution over carbons (H/C) is improved, ranging from 1.48 to a maximum of 1.77 in sample catalyst-3. On the other hand, the use of nanoparticle catalysts have also impacted the hydrogen production, where the H2/CO provided from the water gas shift reaction has increased. Nickel oxide catalysts have the potential for in-situ hydrothermal upgrading of heavy crude oil because of their great potential to catalyze the aquathermolysis reactions in the presence of steam.

Specific nanoarchitecture of silica nanoparticles codoped with the oppositely charged Mn2+ and Ru2+ complexes for dual paramagnetic-luminescent contrasting effects.

The silica nanoparticles (SNs) co-doped with paramagnetic ([Mn(HL)]n-,) and luminescent ([Ru(dipy)3]2+) complexes are represented. The specific distribution of [Mn(HL)]n- within the SNs allows to achieve about ten-fold enhancing in magnetic relaxivities in comparison with those of [Mn(HL)]n- in solutions. The leaching of [Mn(HL)]n- from the shell can be minimized through the co-doping of [Ru(dipy)3]2+ into the core of the SNs. The co-doped SNs exhibit colloid stability in aqueous solutions, including those modeling a blood serum. The surface of the co-doped SNs was also decorated by amino- and carboxy-groups. The cytotoxicity, hemoagglutination and hemolytic activities of the co-doped SNs are on the levels convenient for "in vivo" studies, although the amino-decorated SNs cause more noticeable agglutination and suppression of cell viability. The co-doped SNs being intravenously injected into mice allows to reveal their biodistribution in both ex vivo and in vivo conditions through confocal microscopy and magnetic resonance imaging correspondingly.

Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Microbial Communities of Seawater and Coastal Soil of Russian Arctic Region and Their Potential for Bioremediation from Hydrocarbon Pollutants.

The development of Arctic regions leads to pollution of marine and coastal environments with oil and petroleum products. The purpose of this work was to determine the diversity of microbial communities in seawater, as well as in littoral and coastal soil, and the potential ability of their members to degrade hydrocarbons degradation and to isolate oil-degrading bacteria. Using high-throughput sequencing of the V4 region of the 16S rRNA gene, the dominance of bacteria in polar communities was shown, the proportion of archaea did not exceed 2% (of the total number of sequences in the libraries). Archaea inhabiting the seawater belonged to the genera Nitrosopumilus and Nitrosoarchaeum and to the Nitrososphaeraceae family. In the polluted samples, members of the Gammaproteobacteria, Alphaproteobacteria, and Actinomycetes classes predominated; bacteria of the classes Bacteroidia, Clostridia, Acidimicrobiia, Planctomycetia, and Deltaproteobacteria were less represented. Using the iVikodak program and KEGG database, the potential functional characteristics of the studied prokaryotic communities were predicted. Bacteria were potentially involved in nitrogen and sulfur cycles, in degradation of benzoate, terephthalate, fatty acids, and alkanes. A total of 19 strains of bacteria of the genera Pseudomonas, Aeromonas, Oceanisphaera, Shewanella, Paeniglutamicibacter, and Rhodococcus were isolated from the studied samples. Among them were psychrotolerant and psychrophilic bacteria growing in seawater and utilizing crude oil, diesel fuel, and motor oils. The data obtained suggest that the studied microbial communities could participate in the removal of hydrocarbons from arctic seawater and coastal soils and suggested the possibility of the application of the isolates for the bioaugmentation of oil-contaminated polar environments.

An armed oncolytic virus enhances the efficacy of tumor-infiltrating lymphocyte therapy by converting tumors to artificial antigen-presenting cells in situ.

The full potential of tumor-infiltrating lymphocyte (TIL) therapy has been hampered by the inadequate activation and low persistence of TILs, as well as inefficient neoantigen presentation by tumors. We transformed tumor cells into artificial antigen-presenting cells (aAPCs) by infecting them with a herpes simplex virus 1 (HSV-1)-based oncolytic virus encoding OX40L and IL12 (OV-OX40L/IL12) to provide local signals for optimum T cell activation. The infected tumor cells displayed increased expression of antigen-presenting cell-related markers and induced enhanced T cell activation and killing in coculture with TILs. Combining OV-OX40L/IL12 and TIL therapy induced complete tumor regression in patient-derived xenograft and syngeneic mouse tumor models and elicited an antitumor immunological memory. In addition, the combination therapy produced aAPC properties in tumor cells, activated T cells, and reprogrammed macrophages to a more M1-like phenotype in the tumor microenvironment. This combination strategy unleashes the full potential of TIL therapy and warrants further evaluation in clinical studies.

Blood pressure altering method affects correlation with pulse arrival time.

OBJECTIVE: Pulse arrival time (PAT) is a potential main feature in cuff-less blood pressure (BP) monitoring. However, the precise relationship between BP parameters and PAT under varying conditions lacks a complete understanding. We hypothesize that simple test protocols fail to demonstrate the complex relationship between PAT and both SBP and DBP. Therefore, this study aimed to investigate the correlation between PAT and BP during two exercise modalities with differing BP responses using an unobtrusive wearable device. METHODS: Seventy-five subjects, of which 43.7% had a prior diagnosis of hypertension, participated in an isometric and dynamic exercise test also including seated periods of rest prior to, in between and after. PAT was measured using a prototype wearable chest belt with a one-channel electrocardiogram and a photo-plethysmography sensor. Reference BP was measured auscultatory. RESULTS: Mean individual correlation between PAT and SBP was -0.82 ± 0.14 in the full protocol, -0.79 ± 0.27 during isometric exercise and -0.77 ± 0.19 during dynamic exercise. Corresponding correlation between PAT and DBP was 0.25 ± 0.35, -0.74 ± 0.23 and 0.39 ± 0.41. CONCLUSION: The results confirm PAT as a potential main feature to track changes in SBP. The relationship between DBP and PAT varied between exercise modalities, with the sign of the correlation changing from negative to positive between type of exercise modality. Thus, we hypothesize that simple test protocols fail to demonstrate the complex relationship between PAT and BP with emphasis on DBP.

Two-Step Targeted Drug Delivery via Proteinaceous Barnase-Barstar Interface and Doxorubicin-Loaded Nano-PLGA Outperforms One-Step Strategy for Targeted Delivery to HER2-Overexpressing Cells.

Nanoparticle-based chemotherapy is considered to be an effective approach to cancer diagnostics and therapy in modern biomedicine. However, efficient tumor targeting remains a great challenge due to the lack of specificity, selectivity, and high dosage of chemotherapeutic drugs required. A two-step targeted drug delivery strategy (DDS), involving cancer cell pre-targeting, first with a first nontoxic module and subsequent targeting with a second complementary toxic module, is a solution for decreasing doses for administration and lowering systemic toxicity. To prove two-step DDS efficiency, we performed a direct comparison of one-step and two-step DDS based on chemotherapy loaded PLGA nanoparticles and barnase*barstar interface. Namely, we developed and thoroughly characterized the two-step targeting strategy of HER2-overexpressing cancer cells. The first targeting block consists of anti-HER2 scaffold polypeptide DARPin9_29 fused with barstar. Barstar exhibits an extremely effective binding to ribonuclease barnase with Kaff = 1014 M-1, thus making the barnase*barstar protein pair one of the strongest known protein*protein complexes. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second targeting block. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and a chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers: IC50 of doxorubicin delivered via two-step DDS was more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. The obtained results demonstrate the significant efficiency of two-step DDS over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new generation cancer treatment strategies.

Multiple site place-of-care manufactured anti-CD19 CAR-T cells induce high remission rates in B-cell malignancy patients.

Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen are effective in treating adults and children with B-cell malignancies. Place-of-care manufacturing may improve performance and accessibility by obviating the need to cryopreserve and transport cells to centralized facilities. Here we develop an anti-CD19 CAR (CAR19) comprised of the 4-1BB co-stimulatory and TNFRSF19 transmembrane domains, showing anti-tumor efficacy in an in vivo xenograft lymphoma model. CAR19 T cells are manufactured under current good manufacturing practices (cGMP) at two disparate clinical sites, Moscow (Russia) and Cleveland (USA). The CAR19 T-cells is used to treat patients with relapsed/refractory pediatric B-cell Acute Lymphocytic Leukemia (ALL; n = 31) or adult B-cell Lymphoma (NHL; n = 23) in two independently conducted phase I clinical trials with safety as the primary outcome (NCT03467256 and NCT03434769, respectively). Probability of measurable residual disease-negative remission was also a primary outcome in the ALL study. Secondary outcomes include complete remission (CR) rates, overall survival and median duration of response. CR rates are 89% (ALL) and 73% (NHL). After a median follow-up of 17 months, one-year survival rate of ALL complete responders is 79.2% (95%CI 64.5‒97.2%) and median duration of response is 10.2 months. For NHL complete responders one-year survival is 92.9%, and median duration of response has not been reached. Place-of-care manufacturing produces consistent CAR-T cell products at multiple sites that are effective for the treatment of patients with B-cell malignancies.

Antigen-Specific Stimulation and Expansion of CAR-T Cells Using Membrane Vesicles as Target Cell Surrogates.

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy.

Impact of illumination spectrum and eye pigmentation on image quality from a fundus camera using transscleral illumination.

SIGNIFICANCE: The use of the transscleral illumination approach has the potential to simplify the optical design of fundus cameras. In particular, this approach could allow the use of smaller and cheaper cameras that are easier to use by non-specialists, thereby facilitating a wider spread of eye disease screening programs. AIM: Our aim was to investigate the suitability of transscleral illumination in a fundus camera system. In particular, we explored the impact of the illumination spectrum and the eye pigmentation on the quality of the image. These factors have never been systematically investigated before in the literature on transscleral illumination. APPROACH: A fundus camera was constructed using transscleral illumination. We studied the influence of eye pigmentation and choice of illumination spectra on the image quality for a group of 10 individuals with varied skin pigmentation, ranging from pale white (North-European) to darkest brown (African). The influence of the light source spectrum on the image quality was assessed using wavelength filters. RESULTS: There was a difference of a factor of 100 in the signal level of retinal images between individuals with low and high skin pigmentation. The image contrast was highest using illumination wavelengths of 500 to 600 nm. The illumination level can be adjusted to obtain high-quality images for highly pigmented eyes while keeping the system eye-safe. CONCLUSIONS: We have demonstrated that a fundus camera with transscleral illumination can provide high-quality images. However, the variations observed in scleral and retinal pigmentation in a practical setting require a system that must be able to adapt illumination and/or exposure to the individual patient.