Human Blood Research Articles

P1302 OCTN1 variants shape innate immunity and predict individual response to vedolizumab in ulcerative colitis patients: from preclinical models to AI

Abstract Background Limited evidence is available about predictors of response to specific medications for ulcerative colitis. Variants of OCTN1, an organic cation transporter, could modulate the inflammatory response and potentially predict individual response to immune-modulatory therapy. Methods We first assessed the ability of OCTN1 to differentially modulate IL1β secretion under different stimuli in a human leukemia monocytic cell line (THP-1) and in human peripheral blood mononuclear cells. We therefore prospectively enrolled a cohort of UC patients starting either anti-TNFα or vedolizumab, determined their OCTN1 genotype and evaluated their response to therapy after six months, as well as steroid free therapy persistence after 2 years. Finally, we performed an integrated machine learning based analysis including serum cytokines and gut microbiota analysis. Results OCTN1-deficient THP1 cells showed reduced IL-1β secretion in response to peptidoglycan (PGN) and Fusobacterium nucleatum (Fn), whilst primary monocytes bearing OCTN1 503F variant displayed an increased production of IL-1β in response to PGN and live bacteria. We therefore enrolled 90 patients, of which 50 starting anti-TNFα and 40 vedolizumab. At the multivariate analysis, a significant reduction of clinical response after six months was observed in patients with 503F variant treated with vedolizumab. The same trend was observed for steroid free therapy persistence after two years. The machine learning analysis revealed that changes in bacterial composition, notably the relative abundance of Gemellaceae, followed by Lachnospiracea, were significant predictors of lack of two-year steroid-free therapy persistence. Conclusion Mutated OCNT1 genotype (503F) could favor an increased IL1β secretion from human leukocytes, thus suggesting a worse disease course and lower response to vedolizumab as compared to 503L. ovel machine learning data analysis provides further insights into such topic.

Antioxidant and Anti-Inflammatory Effects of Vanillic Acid in Human Plasma, Human Neutrophils, and Non-Cellular Models In Vitro

Vanillic acid (VA) is a dietary benzoic acid derivative, flavoring agent, and food stabilizer. In this study, the antioxidant and anti-inflammatory potential of VA was explored in vitro and ex vivo in human immune cells and non-cellular models. In neutrophils, VA significantly downregulated the fMLP-induced oxidative burst and the generation of reactive oxygen species (ROS); it also suppressed the release of pro-inflammatory cytokines (TNF-α, IL-8) and the tissue-remodeling enzyme elastase-2 (ELA-2) in cells stimulated with LPS and fMLP+cytochalasin B. Additionally, VA showed good biocompatibility with human neutrophils and peripheral blood mononuclear cells (PBMCs) across the tested concentrations of 1–50 µg/mL. Furthermore, VA at 1–5 μg/mL enhanced the non-enzymatic antioxidant capacity of human plasma (NEAC) and prevented oxidative and nitrative damage to plasma proteins by protecting tyrosine moieties and thiols from peroxynitrite. VA also inhibited lipid peroxidation and the formation of thiobarbituric acid-reactive substances (at 50 μg/mL) and protein-bound carbonyls (at 5–50 μg/mL) in peroxynitrite-treated plasma. In non-cellular tests, VA acted as a hypochlorous acid and hydrogen peroxide scavenger and inhibited non-enzymatic protein glycation, outperforming the references Trolox and aminoguanidine. Along with existing data from animal models and studies on polyphenol intake, these results might support the synergic role of VA in dietary protection against chronic diseases related to oxidative stress and inflammation.

P0025 The effect of very small superparamagnetic nanoparticles on human blood cells as a diagnostic tool in Inflammatory Bowel Disease

Abstract Background The field of application of organic or inorganic nanoparticles (1-100 nm size) is extensive and involves their use in medicine. This includes using nanoparticles in human beings or animals (life-stock) which holds advantages but also bears risks. Therefore, nanoparticles used in diagnostics and therapy should be inert not affecting tissues or immune cells. Additionally, nanoparticles should not aggregate to form clusters exceeding the size of 100 nm or accumulate in sensitive structures like bone marrow or brain for long-term deposition. We developed in-house very small superparamagnetic iron oxide nanoparticles (VSOP) with a size of 7 nm that were successfully used in preclinical magnetic resonance imaging (MRI) for the detection of intestinal inflammation and sclerosis. This study elucidates the effect of nanoparticles on human blood cells, including monocytes as well as monocyte-derived macrophages, in vitro as a first step toward clinical application in inflammatory bowel disease (IBD). Methods Whole blood of healthy donors and patients suffering from IBD as well as monocytes and monocyte-derived macrophages were treated with VSOP in vitro and analysed for changes in their transcriptome, phenotype and function. For transcriptome analysis, RNA sequencing was performed. Cell abundance, activation and proliferation were measured by mass cytometry. Migratory behavior of monocytes was analyzed by live microscopy. Cell metabolism was detected by Seahorse assay. Results RNA sequencing of monocytes showed that the most significantly regulated gene is encoding for the transferrin receptor (Tcfr). As a response to VSOP treatment and VSOP uptake thereby increasing the levels of intracellular iron, Tcfr is probably downregulated to restrict further iron uptake. While RNA sequencing of whole blood showed only significant differences in non-coding genes (C3orf86P, ENSG00000278996, ENSG00000259002), CyTOF analyses revealed that VSOP-treated monocytes are neither activated nor showing increased proliferation. Also, the migratory behavior is not influenced by VSOP uptake. Additionally, VSOP uptake resulted in an enhanced oxygen consumption rate and extracellular acidification rate. In comparison to the uptake of Latex beads, these increased rates are rather attributed to phagocytosis. Conclusion Analyses of our data with PBMC, monocytes and monocyte-derived macrophages suggest, that treatment with VSOP has no major affect on phenotype and function of immune cells. Therefore, VSOP are a promising tool for the early detection of IBD through MRI.

Microenvironment-confined kinetic elucidation and implementation of a DNA nano-phage with a shielded internal computing layer

Multiple receptor analysis-based DNA molecular computation has been developed to mitigate the off-target effect caused by nonspecific expression of cell membrane receptors. However, it is quite difficult to involve nanobodies into molecular computation with programmed recognition order because of the “always-on” response mode and the inconvenient molecular programming. Here we propose a spatial segregation-based molecular computing strategy with a shielded internal computing layer termed DNA nano-phage (DNP) to program nanobody into DNA molecular computation and build a series of kinetic models to elucidate the mechanism of microenvironment-confinement. We explain the contradiction between fast molecular diffusion and effective DNA computation using a “diffusion trap” theory and comprehensively overcome the kinetic bottleneck of DNP by determining the rate-limiting step. We predict and verify that identifying trace amount of target cells in complex cell mixtures is an intrinsic merit of microenvironment-confined DNA computation. Finally, we show that DNP can efficiently work in complex human blood samples by shielding the interference of erythrocytes and enhance phagocytosis of macrophages toward target cells by blocking CD47-SIRPα pathway.

Distribution of ABO and Rhesus Blood Groups among Blood Donor Attending Transfusion Medicine Department of Chattagram Maa-O-Shishu Hospital Medical College

Introduction: The ABO blood group system was the first human blood group system to be discovered by Karl Landsteiner in 1900. Next to ABO, Rhesus system the second most significant blood group system. Blood group is applied to the genetically determined antigen which can be detected on the red cell surface of an individual by specific antibody. The aim of this study to examine the distribution patterns of ABO and Rh-D blood group among the blood donor attending at Transfusion Medicine Department in CMOSH. It is helpful to increase social awareness among the population and maintain safe blood transfusion to patient. Materials and methods: It was a cross sectional study conducted in the Department of Transfusion Medicine,Chattagram Maa O Shishu Hospital Medical College, from January to December 2022. After proper ethical consideration a total of 7,289 blood donors were included in this study. ABO and Rh blood groupings were carried out in our blood bank by standard tile techniques. Results: Among 7,289 blood donors male blood donors were 6,936(95.15%), female blood donors were 353(4.84%).2,775 (38.07%) blood donor were identified as having blood group B while 2,185 (29.97%) were blood group O, 1702( 23.35%) and 627(8.6%)were blood group A and AB respectively. Rh-D positive were 6,960(95.48%) and Rh-D negative were 329(4.51%). Conclusion: The knowledge of ABO and Rhesus D blood group is important in blood transfusion service.Study of blood grouping not only generates a simple database but also create a great social awareness about self-blood grouping and safe blood transfusion among the population of a country. Chatt Maa Shi Hosp Med Coll J; Vol.23 (1); January 2024; Page 52-55

Zinc Doped Akermanite: A Promising Biomaterial for Orthopedic Application with Enhanced Bioactivity, Mechanical Strength, and Bacterial Study.

Incorporating zinc into biocompatible materials has been identified as a potential strategy for promoting bone regeneration and osteogenic activity during hard tissue regeneration. This work aimed to investigate the impact of zinc doping on the structure of akermanite, which was synthesized using the sol-gel combustion method, with the goal of improving the biological response. Powder XRD and FT-IR analysis confirmed the phase purity and the respective functional groups associated with Zn-doped akermanite. Further XPS analysis confirmed the presence of zinc with the respective binding energies in the akermanite matrix. According to the results obtained from the analysis, the apatite-forming ability of Zn-doped akermanite demonstrated enhanced apatite deposition on the surface of the pellet after 9 days of immersion in the SBF medium. The measured mechanical parameters, including compressive strength (140-189 MPa) and Young's modulus (2505-3599 MPa), fall within the range of human cortical bone. Antimicrobial results showed an improved inhibition rate of the doped ceramics compared to pure akermanite with an inhibition percentage of 87% even at lower concentrations. The hemocompatibility of the materials showed hemolysis of human blood cells within the acceptable range without exhibiting toxicity. Cytotoxicity results demonstrate the biocompatibility of the materials with the MG-63 cell line. Based on the results, akermanite doped with zinc at optimal concentrations was found to be compatible and nontoxic promoting it as a potential alternative for bone regeneration in orthopedic applications.

An Evaluation of the Safety of Intravenous Injections of the Natural Extracellular Hemoglobin M101 in Dogs and Monkeys.

Hemoglobin-based oxygen carriers have been developed to compensate the needs of blood for transfusions. Most of them were based on intracellular hemoglobin extracted from bovine or human blood, but unfortunately, this type of hemoglobin did not pass through the last steps of clinical trials. In this context, HEMARINA discovered a natural extracellular hemoglobin, possessing several advantages avoiding intracellular hemoglobin-related side effects. Many preclinical studies assessed the safety of M101 used in intravenous (IV) injection in rodents. To explore the safety of IV injections of M101 in large mammals, six dogs received each a single injection of liquid M101 according to a dose escalation with a 48 h follow-up. Then, two monkeys received multiple IV injections of the same dose of M101 every hour for seven hours. This study showed that single and multiple IV injections in dogs and monkeys did not cause clinical or histological lesions, nor did they induce immunological reactions. This makes M101 the best candidate to date for human use in emergency situations requiring blood and, in several diseases, causing hypoxia problems.

Label-free Detection and Characterization of Metabolism in Fresh and Cryopreserved Macrophages.

Cryopreservation is a widely used technique to preserve biological samples for extended periods of time at low temperatures. Even though it is known to have significant effects on cell viability, its effect on their metabolism remains unexplored. Studying how cryopreservation influences the metabolism of cells is important to guarantee the reliability of samples transported between sites for analysis. Optical metabolic imaging allows for the study of cellular metabolism in a label-free manner by using the autofluorescence properties of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), two metabolic coenzymes. The goal of this research is to study the metabolic changes in macrophages after cryopreservation and compare these results with freshly isolated macrophage samples to evaluate if the metabolic data is retained after cryopreservation. The metabolism of macrophages was analyzed with fluorescence lifetime imaging microscopy (FLIM) using a multiphoton microscope. Monocytes were isolated from human whole blood and separated into two groups. In Group 1, freshly isolated monocytes were differentiated into macrophages using macrophage-colony stimulating factor (M-CSF) over 8 days. In Group 2, isolated monocytes were cryopreserved after separation from whole blood and then thawed and stimulated with M-CSF for 8 days. Single-cell analysis showed there are significant changes in FLIM parameters between the groups suggesting that the metabolic data of macrophages is altered after a cryopreservation and cell thawing cycle. Our research bridges the current gap by studying the metabolic changes of cells after cryopreservation using a non-invasive and label-free imaging technique.

Combating inflammation and oxidative stress: Exploring the cellular effects of Lonicera caerulea var. kamtschatica extract.

Lonicera caerulea var. kamtschatica (LCK), known as blue honeysuckle or haskap berry, is rich in bioactive compounds such as polyphenols, flavonoids, and anthocyanins, which are linked to various health benefits, including anti-inflammatory and antioxidant properties. The research specifically investigates the effects of an LCK extract that has been standardized to contain a minimum of 15% anthocyanins on inflammation and oxidative stress at the cellular level. In vitro studies using A549 human lung epithelial cells and peripheral blood mononuclear cells (PBMCs) demonstrated the extract's anti-inflammatory and antioxidant properties. LCK extract significantly inhibited the nuclear translocation of NF-κB p65 and reduced the production of IL-8 in A549 cells. It also downregulated the expression of pro-inflammatory genes (RELA and PTGS2) while upregulating antioxidant genes (CAT, HMOX1, and SOD2). In PBMCs, LCK extract decreased the phosphorylation of NF-κB p65 and reduced levels of pro-inflammatory cytokines IL-1β and IL-6 following LPS stimulation. Additionally, the extract inhibited reactive oxygen species (ROS) formation and nitric oxide (NO) production, demonstrating its potential to modulate oxidative stress. Furthermore, in vitro assays indicated that LCK extract could hinder the binding of SARS-CoV-2 spike protein to the hACE2 receptor, suggesting antiviral potential. These findings suggest that LCK extract exerts significant anti-inflammatory and antioxidant effects, indicating its potential as a functional food ingredient or dietary supplement to combat inflammation and oxidative stress.

Comprehensive Qualitative Drug Screening in Emergency Toxicology Using an Automated LC-MSn System:Simultaneous Quantification of Relevant Drugs and Metabolites in Blood Plasma.

Rapid and comprehensive qualitative and quantitative analytical procedures are crucial in 24/7 emergency toxicology (ET) to support diagnosis and treatment of acute intoxications and to monitor their progression and efficacy of detoxification strategies. This study aimed to develop the simultaneous qualitative and quantitative analysis of 62 drugs, as well as seven active metabolites in human blood plasma using an automated liquid chromatography (LC)-linear ion trap mass spectrometry (MS) screening system. Sample preparation was conducted by liquid-liquid extraction, and plasma concentrations were determined using an electronically stored 5-point calibration. Validation was performed according to international guidelines and recommendations for ET including selectivity, carry-over, accuracy, precision, and matrix effects. Finally, applicability was evaluated using case samples and proficiency tests. The method demonstrated selectivity for all analytes, with no significant carry-over or matrix effects. Accuracy and precision recommended for ET could be fulfilled for all tested analytes, except for 10 analytes. Patient plasma samples were analyzed and compared with results obtained by reference LC-tandem MS or gas chromatography-MS methods. Furthermore, the applicability of the method could be demonstrated. It provides a fast, robust, and reliable blood plasma screening for 69 analytes and an additional quantification of 59 analytes relevant in ET. The use of an electronically stored 5-point calibration and a simplified "push and print solution" allows for straightforward assessment of blood plasma levels of the analytes.

Inhibition of platelet activation process upon tris (2-chloroethyl) phosphate exposure: Role of PFKP-mediated glycolysis and the pentose phosphate pathway.

Tris (2-chloroethyl) phosphate (TCEP), recognized as an emerging pollutant, has been frequently detected in human blood. Maintenance of blood homeostasis is indispensable for regulating various physiological states and overall health, yet hematological toxicology of TCEP has not been extensively investigated. Platelets, a vital component of blood, are fundamental in the processes of hemostasis and thrombosis through their activation; thus, this study was designed to elucidate the effects and underlying mechanisms of TCEP on platelet activation. Utilizing an in vivo model, we conducted a proteomic analysis of platelets and found that TCEP exposure inhibited platelet activation. An ex vivo platelet evaluation system was employed to further dissect the processes of platelet activation, revealing that TCEP predominantly suppressed platelet aggregation, degranulation and clot retraction. These processes were highly dependent on energy metabolism, and TCEP was found to decrease ATP levels, primarily by impairing glycolysis and pentose phosphate pathways. Subsequent investigation into the molecular mechanisms revealed that TCEP decreased the activity of phosphofructokinase platelet (PFKP) by enhancing O-linked N-acetylglucosamine (O-GlcNAc) transferase interaction with PFKP. This study is the first to uncover the disruptive effects of TCEP on platelet activation process, providing valuable insights into the assessment of hematologic health risks associated with TCEP-like emerging pollutants exposure.

The Influence of Nanoparticles of Graphene Oxide‑PEG on Cytokine Profile of Monocytes from Human Blood In Vitro

The Influence of Nanoparticles of Graphene Oxide‑PEG on Cytokine Profile of Monocytes from Human Blood In Vitro

High-resolution, noninvasive single-cell lineage tracing in mice and humans based on DNA methylation epimutations.

In vivo lineage tracing holds great potential to reveal fundamental principles of tissue development and homeostasis. However, current lineage tracing in humans relies on extremely rare somatic mutations, which has limited temporal resolution and lineage accuracy. Here, we developed a generic lineage-tracing tool based on frequent epimutations on DNA methylation, enabled by our computational method MethylTree. Using single-cell genome-wide DNA methylation datasets with known lineage and phenotypic labels, MethylTree reconstructed lineage histories at nearly 100% accuracy across different cell types, developmental stages, and species. We demonstrated the epimutation-based single-cell multi-omic lineage tracing in mouse and human blood, where MethylTree recapitulated the differentiation hierarchy in hematopoiesis. Applying MethylTree to human embryos, we revealed early fate commitment at the four-cell stage. In native mouse blood, we identified ~250 clones of hematopoietic stem cells. MethylTree opens the door for high-resolution, noninvasive and multi-omic lineage tracing in humans and beyond.

A Simple, Low-Cost, and Efficient Protocol for Rapid Isolation of Pathogenic Bacteria from Human Blood.

Bacteremia is a serious clinical condition in which pathogenic bacteria enter the bloodstream, putting patients at risk of septic shock and necessitating antibiotic treatment. Choosing the most effective antibiotic is crucial not only for resolving the infection but also for minimizing side effects, such as dysbiosis in the healthy microbiome and reducing the selection pressure for antibiotic resistance. This requires prompt identification of the pathogen and antibiotic susceptibility testing, yet these processes are inherently slow in standard clinical microbiology labs due to reliance on growth-based assays. Although alternative methods exist, they are rarely adopted in clinical settings because they involve complex protocols and high costs for training and infrastructure. Here, we present an optimized, simple protocol for rapidly and efficiently isolating bacterial pathogens from blood without altering typical laboratory workflows. Our method is cost-effective and compatible with commonly available laboratory instruments, offering the advantage of isolating bacterial cells directly, which bypasses the delays associated with traditional blood culture methods and enables faster diagnostic results. The protocol achieved over 70% efficiency within 30 minutes, remained effective at low bacterial concentrations (1-10 bacteria/0.3 mL blood), and preserved bacterial viability with no notable change in growth lag times. We validated the protocol on several clinically relevant bacterial strains, including Escherichia coli, Klebsiella pneumoniae , and Staphylococcus aureus . These findings highlight our protocol's potential utility in clinical and research settings, facilitating timely cultures and minimizing diagnostic delays.

LC-MS-Based Global Metabolic Profiles of Alternative Blood Specimens Collected by Microsampling.

Blood microsampling (BμS) has recently emerged as an interesting approach in the analysis of endogenous metabolites but also in metabolomics applications. Their non-invasive way of use and the simplified logistics that they offer renders these technologies highly attractive in large-scale studies, especially the novel quantitative microsampling approaches such as VAMs or qDBS. Objectives: Herein, we investigate the potential of BµS devices compared to the conventional plasma samples used in global untargeted mass spectrometry-based metabolomics of blood. Methods: Two novel quantitative devices, namely, Mitra, Capitainer, and the widely used Whatman cards, were selected for comparison with plasma. Venous blood was collected from 10 healthy, overnight-fasted individuals and loaded on the devices; plasma was also collected from the same venous blood. An extraction solvent optimization study was first performed on the three devices before the main study, which compared the global metabolic profiles of the four extracts (three BµS devices and plasma). Analysis was conducted using reverse phase LC-TOF MS in positive mode. Results: BµS devices, especially Mitra and Capitainer, provided equal or even superior information on the metabolic profiling of human blood based on the number and intensity of features and the precision and stability of some annotated metabolites compared to plasma. Despite their rich metabolic profiles, BµS did not capture metabolites associated with biological differentiation of sexes. Conclusions: Overall, our results suggest that a more in-depth investigation of the acquired information is needed for each specific application, as a metabolite-dependent trend was obvious.

Acute Severe Hypoxia Decreases Mitochondrial Chain Complex II Respiration in Human Peripheral Blood Mononuclear Cells.

Peripheral blood mononuclear cells' (PBMCs) mitochondrial respiration is impaired and likely involved in myocardial injury and heart failure pathophysiology, but its response to acute and severe hypoxia, often associated with such diseases, is largely unknown in humans. We therefore determined the effects of acute hypoxia on PBMC mitochondrial respiration and ROS production in healthy volunteers exposed to controlled oxygen reduction, achieving an inspired oxygen fraction of 10.5%. We also investigated potential relationships with gene expression of key biomarkers of hypoxia, succinate and inflammation, as hypoxia and inflammation share common mechanisms involved in cardiovascular disease. Unlike global mitochondrial respiration, hypoxemia with a spO2 ≤ 80% significantly reduced PBMC complex II respiration (from 6.5 ± 1.2 to 3.1 ± 0.5 pmol/s/106 cell, p = 0.04). Complex II activity correlated positively with spO2 (r = 0.63, p = 0.02) and inversely correlated with the succinate receptor SUCNR1 (r = -0.68), the alpha-subunit of the hypoxia-inducible factor (HIF-1α, r = -0.61), the chemokine ligand-9 (r = -0.68) and interferon-stimulated gene 15 (r = -0.75). In conclusion, severe hypoxia specifically impairs complex II respiration in association with succinate, inflammation and HIF-1α pathway interactions in human PBMCs. These results support further studies investigating whether modulation of complex II activity might modify the inflammatory and metabolic alterations observed in heart failure.

Human PBMC-based humanized mice exhibit myositis features and serve as a drug evaluation model

Idiopathic inflammatory myopathies (IIMs) are a group of autoimmune disorders characterized by immune cell infiltration of muscle tissue accompanied by inflammation. Treatment of IIMs is challenging, with few effective therapeutic options due to the lack of appropriate models that successfully recapitulate the features of IIMs observed in humans. In the present study, we demonstrate that immunodeficient mice transplanted with human peripheral blood mononuclear cells (hPBMCs) exhibit the key pathologic features of myositis observed in humans and develop graft-versus-host disease. The hPBMC mice exhibit elevated serum levels of creatine kinase and aspartate transaminase, markers of myositis, and increased expression levels of myositis-related genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and serum amyloid A1, in muscle tissues. Histopathologic and flow cytometric analyses reveal the infiltration of CD8+ T cells in the muscle tissue of hPBMC mice, similar to that observed in patients with myositis, particularly in those with polymyositis. Transplantation of CD8+ T cell-depleted hPBMC leads to a significant reduction in polymyositis-like symptoms, in agreement with previous studies demonstrating CD8+ T cells as the main pathologic drivers of polymyositis. Furthermore, the transcriptome analysis of muscle tissue from hPBMC mice reveal extensive upregulation of characteristic genes of polymyositis, providing further support that hPBMC mice accurately reflect the pathophysiology of myositis in humans. Finally, administration of prednisolone or tacrolimus, which are commonly used for IIM treatment, leads to significant alleviation of myositis findings. Therefore, we propose that hPBMC mice should be considered as a model that accurately reflects the pathophysiology of myositis in human patients.

A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs.

Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns-particularly those specific to this class of drugs-is crucial. Here, we propose an in vitro approach to screen for potential adverse off-target effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NA-drugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticle-based delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.

Apolipoprotein А-I as a transport form of cytostatics in Ehrlich ascitic carcinoma cells

One of the pressing issues of modern pharmacology remains the development of drugs with targeted antitumor action, or the creation of dosage forms of selective action of already known cytostatics in order to increase the effectiveness of chemotherapy and reduce the overall toxic effect on the body. In this work, it is proposed to use apolipoprotein A-I as a transport form of the antitumor drugs actinomycin D, doxorubicin, vinblastine and melphalan. Material and methods. Apolipoprotein A-I was isolated from the high-density lipoprotein fraction of human blood plasma. Apolipoprotein A-I was labeled with 1 % fluoresceinisothiocyanate (FITC). For the experiments, we used male C57Bl mice weighing 20–25 g. Ehrlich ascites carcinoma cells were obtained from peritoneal exudate 9–10 days after transplantation. Fluorescent analysis was carried out on an AxioImager Z1 “Zeiss” microscope using an AxioCamMRc digital camera and AxioVision V.4.5 software. The absorption spectra of antitumor drugs in the optical region of electromagnetic radiation were studied using an Evolution 300 spectrophotometer (Thermo Fisher Scientific, USA). Results. The paper presents the results indicating the ability of FITC-labeled apolipoprotein A-I to enter tumor cells. Using the method of column chromatography and spectrofluorimetry, the formation of apolipoprotein A-Icytostatic complex (actinomycin D, doxorubicin, melphalan, vinblastine) was shown. Analysis of the content of drugs in tumor cell lysates showed that absorption (internalization) was more pronounced during incubation of cells with cytostatics in complex forms with apolipoprotein A-I compared with the absorption of cytostatics by cells without a carrier. Conclusions. It has been established that apolipoprotein A-I can be a direct carrier of cytostatics into the cells of Ehrlich ascites carcinoma.

In vitro functional validation of anti-CD19 chimeric antigen receptor T cells expressing lysine-specific demethylase 1 short hairpin RNA for the treatment of diffuse large B cell lymphoma.

Chimeric antigen receptor T (CAR-T) cell therapy is more effective in relapsed or refractory diffuse large B cell lymphoma (DLBCL) than other therapies, but a high proportion of patients relapse after CAR-T cell therapy owing to antigen escape, limited persistence of CAR-T cells, and immunosuppression in the tumor microenvironment. CAR-T cell exhaustion is a major cause of relapse. Epigenetic modifications can regulate T cell activation, maturation and depletion; they can be applied to reduce T cell depletion, improve infiltration, and promote memory phenotype formation to reduce relapse after CAR-T cell therapy. We propose to develop and validate in vitro the function of novel CAR-T cells for the treatment of DLBCL, which simultaneously express an anti-CD19 CAR with lysine-specific demethylase 1 (LSD1) short hairpin (sh)RNA to prevent depletion and prolong the survival of CAR-T cells. We designed an shRNA sequence targeting LSD1 mRNA, and created a vector with the following elements: the U6 promoter driving expression of the LSD1 shRNA sequence, the EF1a promoter driving a second-generation anti-CD19 CAR sequence encoding an anti-CD19 single-chain variable fragment (FMC63), the CD8 hinge and transmembrane structural domains, the CD28 co-stimulatory structural domain, and the CD3ζ-activating structural domain. The MFG-LSD1 shRNA anti-CD19 CAR plasmid was first constructed, then packaged in retroviral vectors and transduced into human primary peripheral blood mononuclear cell-derived T cells to generate the corresponding CAR-T cells. We examined by flow cytometry the efficiency of two CAR-T cells in killing U-2932 cells (a human DLBCL line) upon co-culture with RNAU6 anti-CD19 CAR-T cells or LSD1 shRNA anti-CD19 CAR-T cells. We analyzed Ki-67 staining of the CAR-T cells by flow cytometry on days 0, 5, and 10, and counted the cells to assess expansion. We also used flow cytometry to detect the central memory T cell (TCM) proportion. We detected the expression of the CAR in the CAR-T cells by flow cytometry, and observed transduction rates of 31.5% for RNAU6 anti-CD19 CAR-T cells and 60.7% for LSD1 shRNA anti-CD19 CAR-T cells. The killing efficiency of LSD1 shRNA anti-CD19 CAR-T cells was significantly higher than that of RNAU6 anti-CD19 CAR-T cells at the low effector target ratio. We further found that LSD1 shRNA anti-CD19 CAR-T cells secreted more IFN-γ and granzyme B than RNAU6 anti-CD19 CAR-T cells. CAR-T cells proliferated after U-2932 cell stimulation and were able to sustain proliferation. After stimulation via U-2932 cell co-culture, both RNAU6 anti-CD19 CAR-T and LSD1 shRNA anti-CD19 CAR-T populations had increased proportions of cells with the TCM phenotype, with a higher percentage among LSD1 shRNA anti-CD19 CAR-T cells. We developed a novel, feasible CD19-LSD1 shRNA CAR-T cell strategy for the treatment of DLBCL. Our in vitro assay results showed that LSD1 shRNA anti-CD19 CAR-T cells more effectively killed target cells than RNAU6 anti-CD19 CAR-T cells, and developed a higher proportion of TCM phenotype cells. LSD1 shRNA anti-CD19 CAR-T cells may represent a potential treatment for DLBCL.