Flow Cytometry Method Research Articles

Comprehensive analysis and identification of subtypes and hub genes of high immune response in lung adenocarcinoma

BackgroundThe advent of immunotherapy targeting immune checkpoints has conferred significant clinical advantages to patients with lung adenocarcinoma (LUAD); However, only a limited subset of patients exhibit responsiveness to this treatment. Consequently, there is an imperative need to stratify LUAD patients based on their response to immunotherapy and enhance the therapeutic efficacy of these treatments.MethodsThe differentially co-expressed genes associated with CD8 + T cells were identified through weighted gene co-expression network analysis (WGCNA) and the Search Tool for the Retrieval of Interacting Genes (STRING) database. These gene signatures facilitated consensus clustering for TCGA-LUAD and GEO cohorts, categorizing them into distinct immune subtypes (C1, C2, C3, and C4). The Tumor Immune Dysfunction and Exclusion (TIDE) model and Immunophenoscore (IPS) analysis were employed to assess the immunotherapy response of these subtypes. Additionally, the impact of inhibitors targeting five hub genes on the interaction between CD8 + T cells and LUAD cells was evaluated using CCK8 and EDU assays. To ascertain the effects of these inhibitors on immune checkpoint genes and the cytotoxicity mediated by CD8 + T cells, flow cytometry, qPCR, and ELISA methods were utilized.ResultsAmong the identified immune subtypes, subtypes C1 and C3 were characterized by an abundance of immune components and enhanced immunogenicity. Notably, both C1 and C3 exhibited higher T cell dysfunction scores and elevated expression of immune checkpoint genes. Multi-cohort analysis of Lung Adenocarcinoma (LUAD) suggested that these subtypes might elicit superior responses to immunotherapy and chemotherapy. In vitro experiments involved co-culturing LUAD cells with CD8 + T cells and implementing the inhibition of five pivotal genes to assess their function. The inhibition of these genes mitigated the immunosuppression on CD8 + T cells, reduced the levels of PD1 and PD-L1, and promoted the secretion of IFN-γ and IL-2.ConclusionsCollectively, this study delineated LUAD into four distinct subtypes and identified five hub genes correlated with CD8 + T cell activity. It lays the groundwork for refining personalized therapy and immunotherapy strategies for patients with LUAD.

Скрининг и характеристика антиоксидантных свойств психрофильных микроводорослей и цианобактерий Балтийского моря

At present, the issue of obtaining a complex of biologically active substances with antioxidant activity from psychrophilic Psychrophilic microalgae and cyanobacteria are a prospective source of biologically active antioxidant substances. New antioxidant complexes could expand the scope of their industrial application. The research objective was to identify psychrophilic microalgae and cyanobacteria from the Baltic Sea in order to study their antioxidant properties. The research featured psychrophilic microalgae and cyanobacteria obtained from the Curonian Lagoon and the Baltic Bay in the Baltic Sea, Kaliningrad Region, Russia, in March – May 2022. The authors used the Pasteur method and the flow cytometry method to isolate pure cultures of psychrophilic microalgae and cyanobacteria. The method of spectrophotometry made it possible to study the antioxidant activity by analyzing radical scavenging, reducing ability, and chelating. The psychrophilic microalga Skeletonema pseudocostatum demonstrated the highest antioxidant activity in all three methods: 17.62 μmol/g trolox equivalents according to the ABTS method, 58.16 μmol/g trolox equivalents according to the DPPH method, and 3.91 μmol/g trolox equivalents according to the FRAP method. The psychrophilic microalga Thalassiosira pseudonana exhibited the following values of antioxidant activity: 12.08, 12.42, and 3.13 μmol/g trolox equivalents, respectively. The antioxidant activity of the psychrophilic microalgae Fragilariopsis kerguelensis was 13.53, 11.84, and 1.09 μmol/g trolox equivalents, respectively. The antioxidant activity of the psychrophilic cyanobacterium Aphanizomenon gracile was 15.73, 19.89, and 2.47 μmol/g trolox equivalents, respectively. The antioxidant activity of the psychrophilic cyanobacterium Anabaena cylindrica was 12.62, 13.16, and 2.16 μmol/g trolox equivalents, respectively. The samples of psychrophilic microalgae and cyanobacteria obtained from natural environment in the Russian Baltic Sea demonstrated good antioxidant properties, which makes them a potential raw material for new pharmaceutical substances.

THE EXPERIENCE IN OBTAINING AND IMMUNOPHENOTYPING OF HEMATOPOIETIC STEM CELLS BY FLOW CYTOMETRY

The article presents the experience of obtaining hematopoietic stem cells (HSCs) and their immunophenotyping using the method of flow cytometry at the State Institution "Institute of Blood Pathology and Transfusion Medicine of the National Academy of Medical Sciences of Ukraine", Lviv. Lviv, Ukraine. Hematopoietic stem cells are key elements in the hematopoietic system and are the basis for bone marrow transplantation, which is used to treat various oncohematologic diseases, including leukemia, lymphoma, and others. The results of the analysis of HSCs in different patients with multiple myeloma showed variability in the concentration of HSCs in peripheral blood and in leukoconcentrate at different stages of apheresis. A high concentration of HSCs after stimulation with colony-stimulating factors was noted, as well as a case with no response to stimulation with colony-stimulating factors and the effectiveness of their cryofreezing. The results of immunophenotyping in the presence of HSCs populations at different stages of the differentiation and the experience of determining cell viability after cryofreezing and long-term storage are also presented.

Identification and validation of Cystatin A as a novel promising therapeutic target for gastric cancer.

The effect of pharmacological treatment of gastric cancer (GC) is limited, thus, it holds significant scientific importance to thoroughly investigate the molecular mechanisms underlying GC development and identify novel molecules capable of substantially extending patients' survival. This study utilized bioinformatics techniques to identify 11 genes associated with recurrence-free survival (RFS) in GC patients and investigated the potential biological functions of these genes through single-cell transcriptomic analysis. Subsequently, a single gene Cystatin A (CSTA) was selected for further analysis to explore its impact on signaling pathways and treatment. Differentially expressed genes (DEGs) were identified and overlapped in the analysis of RFS to identify potential prognostic genes for GC patients, based on data from the Cancer Genome Atlas-stomach adenocarcinoma (TCGA-STAD) and GSE54129. Subsequently, a prognostic model based on RFS in GC patients was established. Single-cell sequencing data were employed to explore the potential functions of these model genes. CSTA, one of the RFS-related genes, was further investigated using immunohistochemistry (IHC), Cell Counting Kit 8 (CCK-8), transwell, scratch, colony formation assays, flow cytometry, and Western blotting methods. Through bioinformatics analysis, we identified 23 RFS-related genes in GC. Using the least absolute shrinkage and selection operator (LASSO)-Cox method, an RFS prognostic model was developed which pinpointed 11 GC prognosis-related (GPR) genes as significant factors influencing RFS in GC patients. The single-cell analysis revealed their potential role in affecting differentiation and maturation of pre-fibroblasts thereby impacting RFS in GC patients. CSTA exhibited low expression levels in GC tissues. Overexpression of CSTA promoted apoptosis in GC cells through the caspase-dependent apoptotic pathway and enhanced their response to cisplatin via this same pathway. The 11 GPR genes are primarily enriched within a specific type of stromal cell exhibiting heightened communication, metabolism, and differentiation levels. The gene signature of these stromal cells has implications for patient prognosis. Additionally, CSTA, a gene related to prognosis, has been shown to influence apoptosis levels in GC cells.

A Label-Free Optical Flow Cytometry Based-Method for Rapid Assay of Disinfectants' Bactericidal Activity.

Selecting the appropriate disinfectant to control and prevent healthcare-associated infections (HAIs) is a challenging task for environmental health experts due to the large number of available disinfectant products. This study aimed to develop a label-free flow cytometry (FCM) method for the rapid evaluation of bactericidal activity and to compare its efficacy with that of standard qualitative/quantitative suspension tests. The bactericidal efficiency of eight commercial disinfectants containing quaternary ammonium compounds (QACs) was evaluated against four strains recommended by EN 13727 (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae) and four multidrug-resistant pathogens. The proposed FCM protocol measures changes in scattered light and counts following disinfectant exposure, neutralization, and culture steps. Unlike other available FCM-based methods, this approach does not rely on autofluorescence measurements, impedance cytometry, or fluorescent dyes. The FCM scattered light signals revealed both decreased count rates and morphological changes after treatment with minimum inhibitory concentrations (MICs) and higher concentrations for all tested bacteria. The results from the FCM measurements showed excellent correlation with those from standard assays, providing a rapid tool for monitoring the susceptibility profile of clinical, multidrug-resistant pathogens to chemical disinfectants, which could support infection prevention and control procedures for healthcare environments. This label-free FCM protocol offers a novel and rapid tool for environmental health experts, aiding in the optimization of disinfectant selection for the prevention and control of HAIs.

Comparison of plate counting with flow cytometry, using four different fluorescent dye techniques, for the enumeration of Bacillus cereus in milk

This study aimed to compare the performance of flow cytometry methods with plate counting for the enumeration of bacteria, using Bacillus cereus as a model organism. It was found that the cFDA-propidium iodide, CellROX™ Green-propidium iodide, and DiOC2 dye techniques had similar accuracy to plate counting, while the SYTO 24-propidium iodide dye technique was not as accurate. The four dye techniques had comparable precision to plate counting, with the CellROX™ Green-propidium iodide dye having the greatest precision. The consistency of the position and shape of the cell clusters on the flow cytometry plots, and the extent of separation of the cell from background clusters, was greatest with the DiOC2 and CellROX™ Green-propidium iodide dyes. Furthermore, the DiOC2 and CellROX™ Green-propidium iodide dyes performed well, even when a sample was measured containing reconstituted whole milk powder at a 10−1 dilution, without the use of sample preparation to specifically remove the milk constituents prior to measurement. Given gating of only one cell cluster was required to be managed with the DiOC2 dye, to determine the viable number of cells, it was found that the DiOC2 dye had the greatest ease-of-use. Overall, results indicated that the DiOC2 dye is an ideal candidate for the enumeration of viable bacteria in dairy samples on a high-throughput, routine basis.

Remodeling of anti-tumor immunity with antibodies targeting a p53 mutant

Backgroundp53, the most frequently mutated gene in cancer, lacks effective targeted drugs.MethodsWe developed monoclonal antibodies (mAbs) that target a p53 hotspot mutation E285K without cross-reactivity with wild-type p53. They were delivered using lipid nanoparticles (LNPs) that encapsulate DNA plasmids. Western blot, BLI, flow cytometry, single-cell sequencing (scRNA-seq), and other methods were employed to assess the function of mAbs in vitro and in vivo.ResultsThese LNP-pE285K-mAbs in the IgG1 format exhibited a robust anti-tumor effect, facilitating the infiltration of immune cells, including CD8+ T, B, and NK cells. scRNA-seq revealed that IgG1 reduces immune inhibitory signaling, increases MHC signaling from B cells to CD8+ T cells, and enriches anti-tumor T cell and B cell receptor profiles. The E285K-mAbs were also produced in the dimeric IgA (dIgA) format, whose anti-tumor activity depended on the polymeric immunoglobulin receptor (PIGR), a membrane Ig receptor, whereas that of IgG1 relied on TRIM21, an intracellular IgG receptor.ConclusionsTargeting specific mutant epitopes using DNA-encoded and LNP-delivered mAbs represents a potential precision medicine strategy against p53 mutants in TRIM21- or PIGR-positive cancers.

STUDY OF THE HUMAN BLOOD STATE AFTER THE EFFECT OF CORONAVIRUS INFECTION COVID-19

The publication reveals the relevance of the research, which is due to the expediency of obtaining the maximum spectrum of measured values based on the flow cytometry method for further analysis of the impact of the COVID-19 coronavirus infection on the human body. The paper describes the directions of application of the flow cytometry method based on hematological markers in scientific research: the causes of immune system disorders were identified, the processes of thrombus formation and fibrinolysis were analyzed, and the causes of the pathogenesis of arterial hypertension were studied. The results of the specified method implementation meant for studying the state of blood after exposure to the human body by the coronavirus infection COVID-19 are presented in the following areas: the occurrence of alveolar inflammation, the appearance of immunological changes, the effect on lymphocyte cells, the appearance of chronic fatigue syndrome based on secondary immunodeficiency after the coronavirus disease. The advantages and disadvantages of using the flow cytometry method for determining hematological parameters after exposure to the human body by the coronavirus infection are indicated, and the feasibility of using the VS-6000 MINDRAY hematological analyzer is substantiated. Reference values have been defined, on the basis of which the automatically measured hematological indicators are compared, the most important in the analysis during the effect of the Covid-19 coronavirus infection on the body of an adult: the absolute content of leukocytes, lymphocytes, monocytes, granulocytes, etc. The importance of identifying biomarkers of inflammatory reactions for patients with various forms and courses of the disease, which is associated with morphological and functional changes in blood cells, starting from the early stages of exposure to the body of the coronavirus infection, has been indicated. It is substantiated that the analysis of the obtained hematological indicators for patients with the COVID-19 coronavirus infection involves considering many factors, based on which the final conclusion regarding changes in the blood state should be formulated. The advantages of using the flow cytometry method in studying the effect of coronavirus infection on the human body are shown.

Broadening anticancer spectrum by preprocessing and treatment of T- lymphocytes expressed FcγRI and monoclonal antibodies for refractory cancers.

Chimeric antigen receptor T (CAR-T) cell therapies have achieved remarkable success in the treatment of hematological tumors. However, given the distinct features of solid tumors, particularly heterogeneity, metabolic aggressiveness, and fewer immune cells in tumor microenvironment (TME), the practical utility of CAR-T cells for solid tumors remains as a challenging issue. Meanwhile, although anti-PD-1 monoclonal antibody (mAb) has shown clinical efficacy, most mAbs also show limited clinical benefits for solid tumors due mainly to the issues associated with the lack of immune cells in TME. Thus, the infiltration of targeted immunological active cells into TME could generate synergistic efficacy for mAbs. We present a combinational strategy for solid tumor treatment, which combines armored-T cells to express Fc-gamma receptor I (FcγRI) fragment on the surfaces for targeting various tumors with therapeutically useful mAbs. Choosing CD20 and HER-2 as the targets, we characterized the in vitro and in vivo efficacy and latent mechanism of the combination drug by using flow cytometry, ELISA and other methods. The combination and preprocessing of armored T-cells with corresponding antibody of Rituximab and Pertuzumab exerted profound anti-tumor effects, which is demonstrated to be mediated by synergistically produced antibody-dependent cellular cytotoxicity (ADCC) effects. Meanwhile, mAb was able to carry armored-T cell by preprocessing for the infiltration to TME in cell derived xenograft (CDX) model. This combination strategy showed a significant increase of safety profiles from the reduction of antibody doses. More importantly, the present strategy could be a versatile tool for a broad spectrum of cancer treatment, with a simple pairing of engineered T cells and a conventional antibody.

Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability

The ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines.

The combination therapy using tyrosine kinase receptors inhibitors and repurposed drugs to target patient-derived glioblastoma stem cells

The lesson from many studies investigating the efficacy of targeted therapy in glioblastoma (GBM) showed that a future perspective should be focused on combining multiple target treatments. Our research aimed to assess the efficacy of drug combinations against glioblastoma stem cells (GSCs). Patient-derived cells U3042, U3009, and U3039 were obtained from the Human Glioblastoma Cell Culture resource. Additionally, the study was conducted on a GBM commercial U251 cell line. Gene expression analysis related to receptor tyrosine kinases (RTKs), stem cell markers and genes associated with significant molecular targets was performed, and selected proteins encoded by these genes were assessed using the immunofluorescence and flow cytometry methods. The cytotoxicity studies were preceded by analyzing the expression of specific proteins that serve as targets for selected drugs. The cytotoxicity study using the MTS assay was conducted to evaluate the effects of selected drugs/candidates in monotherapy and combinations. The most cytotoxic compounds for U3042 cells were Disulfiram combined with Copper gluconate (DSF/Cu), Dacomitinib, and Foretinib with IC50 values of 52.37 nM, 4.38 µM, and 4.54 µM after 24 h incubation, respectively. Interactions were assessed using SynergyFinder Plus software. The analysis enabled the identification of the most effective drug combinations against patient-derived GSCs. Our findings indicate that the most promising drug combinations are Dacomitinib and Foretinib, Dacomitinib and DSF/Cu, and Foretinib and AZD3759. Since most tested combinations have not been previously examined against glioblastoma stem-like cells, these results can shed new light on designing the therapeutic approach to target the GSC population.

Disinfection of sink drains to reduce a source of three opportunistic pathogens, during Serratia marcescens clusters in a neonatal intensive care unit.

Evaluate the effects of five disinfection methods on bacterial concentrations in hospital sink drains, focusing on three opportunistic pathogens (OPs): Serratia marcescens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. Over two years, three sampling campaigns were conducted in a neonatal intensive care unit (NICU). Samples from 19 sink drains were taken at three time points: before, during, and after disinfection. Bacterial concentration was measured using culture-based and flow cytometry methods. High-throughput short sequence typing was performed to identify the three OPs and assess S. marcescens persistence after disinfection at the genotypic level. This study was conducted in a pediatric hospitals NICU in Montréal, Canada, which is divided in an intensive and intermediate care side, with individual rooms equipped with a sink. Five treatments were compared: self-disinfecting drains, chlorine disinfection, boiling water disinfection, hot tap water flushing, and steam disinfection. This study highlights significant differences in the effectiveness of disinfection methods. Chlorine treatment proved ineffective in reducing bacterial concentration, including the three OPs. In contrast, all other drain interventions resulted in an immediate reduction in culturable bacteria (4-8 log) and intact cells (2-3 log). Thermal methods, particularly boiling water and steam treatments, exhibited superior effectiveness in reducing bacterial loads, including OPs. However, in drains with well-established bacterial biofilms, clonal strains of S. marcescens recolonized the drains after heat treatments. Our study supports thermal disinfection (>80°C) for pathogen reduction in drains but highlights the need for additional trials and the implementation of specific measures to limit biofilm formation.

Exploring heterogeneous expression of beta-actin (ACTB) in bladder cancer by producing a monoclonal antibody 6D6

BackgroundTo predict outcomes and identify potential therapeutic targets for cancers, it is critical to find novel specific biomarkers. The objective of this study was to search for and explore novel bladder cancer-associated protein biomarkers.MethodsA library of monoclonal antibodies (mAbs) against the JAM-ICR cell line was first generated, and clones with high affinity were selected. Hybridomas were screened using bladder cancer (BLCA) cell lines and normal cells. The target of the selected mAb was then characterized through immunoaffinity purification, western blotting, and mass spectrometry analysis. Expression of the target antigen was assessed by flow cytometry and IHC methods. Several databases were also used to evaluate the target antigen in BLCA and other types of cancers.ResultsBased on screenings, a 6D6 clone was selected that recognized an isoform of beta-actin (ACTB). Our data showed that ACTB expression on different cell lines was heterogeneous and varied significantly from low to high intensity. 6D6 bound strongly to epithelial cells while showing weak to no reactivity to stromal, endothelial, and smooth muscle cells. There was no association between ACTB intensity and related prognostic factors in BLCA. In silico evaluations revealed a significant correlation between ACTB and overexpressed genes and biomarkers in BLCA. Additionally, the differential expression of ACTB in tumor and healthy tissue as well as its correlation with survival time in a number of cancers were shown.ConclusionsThe heterogeneous expression of ACTB may suggest the potential value of this marker in the diagnosis or prognosis of cancer.

Combined oral low-dose cyclophosphamide endocrine therapy may improve clinical response among patients with metastatic breast cancer via Tregs in TLSs

Despite limited research on refractory and/or endocrine therapy failure in elderly metastatic breast cancer (MBC) patients, a prior study showed that low-dose oral cyclophosphamide (CY) can improve the overall survival rate of MBC patients, possibly through the immunoregulation of regulatory T cells (Tregs). We preliminarily investigated the combination of endocrine therapy (ET) with oral low-dose CY as salvage therapy in elderly patients via peripheral blood regulatory T-cell analyses. In addition, we evaluated the associations of tumor tertiary lymphoid structures (TLSs) with therapeutic outcomes. HR+/HER2− advanced breast cancer patients who received low-dose CY combined with ET or ET only from April 2015 to August 2021 were enrolled in this retrospective study. The primary outcome was the clinical control rate (CCR), and the secondary outcome was progression-free survival (PFS). Circulating T lymphocyte subpopulations represented by Tregs were monitored during treatment by flow cytometry methods. TLSs wereconfirmed by hematoxylin–eosin staining of pretreatment specimens, and CD3, CD4, and Foxp3 were detected using Opal multicolor immunofluorescence. A total of 85 patients who received CY + ET and 50 patients who received ET only were enrolled, the percentage of patients who received CCR was 73% (62/85) vs. 70% (45/50), and the objective response rate (ORR) was 28% (24/85) vs. 24% (12/50). No deaths occurred during the study period. The mean PFS time was 13 vs. 11 months (P = 0.03). In the CY + ET group, decreases in CD4+/CD25+/Foxp3+ T cells (P < 0.001) were favorable for both clinical control and prolonged PFS (P < 0.001). Compared with patients without TLSs, those with TLSs were more likely to have better clinical control and PFS (mean time = 6 months), and a greater number of Treg cells during TLS pretreatment correlated with longer PFS (P = 0.043). Oral low-dose CY combined with standard ET exerts immunological effects by decreasing Treg levels to achieve improved clinical responses. Moreover, patients with TLSs might benefit more from such therapy than those without TLSs, and a high Treg cell count in TLSs before treatment predicts better therapeutic efficacy.

Assessing the impact of green and roasted coffee extracts on colorectal cancer cells in a 3D cell culture model

BackgroundColorectal cancer (CRC) is one of the most common and deadly malignant neoplasms worldwide. It is associated with multiple lifestyle risk factors such as poor diet, physical inactivity, alcohol and red meat consumption, smoking, and obesity. Consequently, studies have been conducted on the protective activity of bioactive compounds present in some foods against CRC, making it necessary to understand how diet can affect human health. In this sense, coffee, the second most consumed beverage in the world, is a major source of phytochemicals associated with a possible antitumor effect on CRC cells. PurposeThis study aimed to analyze the chemopreventive potential of green and roasted coffee extracts on HT-29 CRC cells cultured in 2D and 3D models. MethodsFlow cytometry was employed to quantify propidium iodide and DiOC6 uptake, ROS production, phosphatidylserine exposure, and cell cycle progression. In addition, cells and spheroids were analyzed by light microscopy to identify morphological changes. ResultsThe results showed that, under the employed conditions, treatment with both extracts had a concentration-dependent cytotoxic effect. Decreased cell viability was associated with mitochondrial membrane hyperpolarization, prooxidant effect, increased phosphatidylserine exposure, and changes in the G1 phase of the cell cycle. ConclusionFor the evaluated cell markers, 3D cultures required higher doses to achieve significant cytotoxic effects when compared to monolayer cultures. Since 3D cultures favor the formation of complex cell–cell interactions, this model offers a closer look at in vivo conditions for evaluating the toxicity and preclinical efficacy of potential chemopreventive agents for CRC. The experimental evidence from this study supports the 3R principles of replacement, reduction, and refinement for toxicological research in the pharmaceutical industry.

IL-33-dependent NF-κB activation inhibits apoptosis and drives chemoresistance in acute myeloid leukemia

BackgroundDespite recent advances in therapeutic regimens, the prognosis of acute myeloid leukemia (AML) remains poor. Following our previous finding that interleukin-33 (IL-33) promotes cell survival along with activated NF-κB in AML, we further investigated the role of NF-κB during leukemia development. MethodsFlow cytometry was performed to value the apoptosis and proliferation. qRT-PCR and western blot were performed to detect the expression of IL-6, active caspase 3, BIRC2, Bcl-2, and Bax, as well as activated NF-κB p65 and AKT. Finally, xenograft mouse models and AML patient samples were used to verify the findings observed in AML cell lines. ResultsIL-33-mediated NF-κB activation in AML cell lines contributes to a reduction in apoptosis, an increase in proliferation rate as well as a decrease in drug sensitivity, which were reversed by NF-κB inhibitor, Bay-117085. Moreover, IL-33 decreased the expression of active caspase-3 while increasing the levels of BIRC2, Bcl-2, and Bax, and these effects were blocked by Bay-117085. Additionally, NF-κB activation induced by IL-33 increases the production of IL-6 and autocrine activation of AKT. Co-culture of bone marrow stroma with AML cells resulted in increased IL-33 expression by leukemia cells, along with decreased apoptosis level and reduced drug sensitivity. Finally, we confirmed the in vivo pro-tumor effect mediated by IL-33/ NF-κB axis using a xenograft model of AML. ConclusionOur data indicate that IL-33/IL1RL1-dependent signaling contributes to AML cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pAKT, supporting IL-33/NF-κB/pAKT as a potential target for AML therapy.

Correlation between VCAM-1 Level and Absolute Monocyte Count in Coronary Artery Disease

To prove the correlation between VCAM-1 level and absolute monocyte count among Coronary Artery Disease (CAD) patients. The study was an observational analytic study with a cross-sectional approach. The research subjects were 74 CAD patients who had been proven by coronary angiography and were treated in the Cardiology Department of Central General Hospital by Dr. Kariadi Semarang. This study was conducted from March to July 2021. VCAM-1 levels were examined with the ELISA method, using BioTek ELX800 Microplate reader (USA), and absolute monocyte count was analyzed with flow cytometry method, using Hematology Analyzer SYSMEX XN-1000 (Japan). Statistical analysis was done by Pearson's correlation coefficient test, in which p&lt;0.05 was considered significant. The mean VCAM-1 level was (361.78±128.01) ng/mL. The mean absolute monocyte count was (0.48±0.17) ×103/µL. The result of the Pearson correlation test showed a moderate positive correlation between VCAM-1 levels and absolute monocyte count in CAD patients (p=0.00; r=0.46). There was a moderate positive correlation between VCAM-1 level and absolute monocyte count in CAD patients. A positive correlation exists between VCAM-1 level and absolute monocyte count among CAD patients.

Time course of fluorescent-labelled oligonucleotide accumulation in cells with the use of folate receptor-targeted cationic liposomes

INTRODUCTION. The use of cationic liposomes is a promising approach to the delivery of therapeutic nucleic acids to target cells because liposomes can protect nucleic acids from degradation by extracellular nucleases. However, to ensure selective delivery to the site of action, this approach needs modification, including liposome surface functionalisation with targeting ligands.AIM. This study aimed to compare the time courses of the accumulation of a fluorescent-labelled oligonucleotide (FITC-ODN), which simulated a nucleic acid-based medicinal product, in cells with the use of folate receptor-targeted (F) and conventional (L) cationic liposomes.MATERIALS AND METHODS. F- and L-liposomes were prepared using the polycationic amphiphile 2X3, the zwitterionic helper lipid DOPE, and the folate lipoconjugate F12. Physicochemical characterisation of the liposomes was performed using dynamic light scattering and transmission electron microscopy. Liposome–FITC-ODN complexes were formed at various nitrogen to phosphate (N/P) charge ratios. Flow cytometry, fluorescence microscopy, and confocal microscopy methods were used to study the accumulation of liposome–FITC-ODN complexes in human cervical carcinoma (KB-3-1) and human embryonic kidney (HEK 293) cells.RESULTS. The prepared F- and L-liposomes were spherical particles with a diameter of 75–100 nm. The authors selected the optimal N/P ratio of 2/1 to obtain complexes of F- and L-liposomes with the FITC-ODN. This N/P ratio yielded homogeneous liposome–FITC-ODN complexes having a polydispersity index below 0.200 and a size of 112.4–125.1 nm. F-liposomes were 25% more efficient than L-liposomes in FITC-ODN delivery to KB-3-1 cells at 90, 120, and 240 minutes after transfection. In the first few minutes of cell transfection, fluorescence and confocal microscopy data on the distribution of liposome–FITC-ODN complexes showed that cationic liposome fluorescence signals colocalised with FITC-ODN signals. Later, FITC-ODN accumulation in the cytoplasm was observed.CONCLUSIONS. Cationic liposomes demonstrated efficient FITC-ODN delivery into the cytoplasm of cancer cells. F-liposomes enhanced the percentage of transfected cells and improved FITC-ODN delivery compared with L-liposomes. The results obtained can be used in the further development of targeted medicinal products based on therapeutic nucleic acids and liposomes.

Importance of CD71+ Erythrocyte Cell Levels in Prognosis in Patients With β-Thalassemia.

CD71+ erythroid cells (CECs) are immature red blood cells (proerythroblasts, erythroblasts, and reticulocytes). CECs play an important role in the development of sepsis and cancer by causing immunosuppression. We examined the CEC levels in the peripheral blood of beta thalassemia (βThal) patients and investigated the relationship between CECs and the clinical status of the patients, especially splenectomy. Sixty-eight patients with βThal (46 splenectomized and 22 nonsplenectomized) and 15 healthy controls were included in this study. The hemogram parameters, ferritin, and CECs (flow cytometry method) were measured. It was observed that the CEC level in the patient group was significantly higher than the control group (p < 0.05). CEC levels were found to be significantly higher in patients with splenectomy than in patients with nonsplenectomy (p < 0.05). CEC levels were higher in patients with nontransfusion-dependent βT (NTD-βThal) than in patients with transfusion-dependent βT (TD-βThal) (p < 0.05). CEC levels were found to be significantly higher in patients with splenectomy than in patients with nonsplenectomy in both TD-βThal and NTD-βThal groups (p < 0.05). There was a moderate-negative correlation was detected between CECs and Hb levels (r = -0.467; p < 0.05). High CEC levels in βThal patients develop as a result of ineffective erythropoiesis. We think that keeping CEC levels under control is important for prognosis, especially in patients with splenectomy.

Rapid and accurate quantification of viable Bifidobacterium cells in milk powder with a propidium monoazide-antibiotic fluorescence in situ hybridization-flow cytometry method

Due to its beneficial effects on human health, Bifidobacterium is commonly added to milk powder. Accurate quantification of viable Bifidobacterium is essential for assessing the therapeutic efficacy of milk powder. In this study, we introduced a novel propidium monoazide (PMA)-antibiotic fluorescence in situ hybridization (AFISH)-flow cytometry (FC) method to rapidly and accurately quantify viable Bifidobacterium cells in milk powder. Briefly, Bifidobacterium cells were treated with chloramphenicol (CM) to increase their rRNA content, followed by staining with RNA-binding oligonucleotide probes, based on the AFISH technique. Then, the DNA-binding dye PMA was used to differentiate between viable and nonviable cells. The PMA-AFISH-FC method, including sample pretreatment, CM treatment, dual staining, and FC analysis, required approximately 2 h and was found to be better than the current methods. This is the first study to implement FC combined with PMA and an oligonucleotide probe for detecting Bifidobacterium.