Membrane Molecules Research Articles

Analysis of Phase Heterogeneity in Lipid Membranes Using Single-Molecule Tracking in Live Cells.

In live cells, the plasma membrane is composed of lipid domains separated by hundreds of nanometers in dynamic equilibrium. Lipid phase separation regulates the trafficking and spatiotemporal organization of membrane molecules that promote signal transduction. However, visualizing domains with adequate spatiotemporal accuracy remains challenging because of their subdiffraction limit size and highly dynamic properties. Here, we present a single lipid-molecular motion analysis pipeline (lipid-MAP) for analyzing the phase heterogeneity of lipid membranes by detecting the instantaneous velocity change of a single lipid molecule using the excellent optical properties of nanoparticles, high spatial localization accuracy of single-molecule localization microscopy, and separation capability of the diffusion state of the hidden Markov model algorithm. Using lipid-MAP, individual lipid molecules were found to be in dynamic equilibrium between two statistically distinguishable phases, leading to the formation of small (∼170 nm), viscous (2.5× more viscous than surrounding areas), and transient domains in live cells. Moreover, our findings provide an understanding of how membrane compositional changes, i.e., cholesterol and phospholipids, affect domain formation. This imaging method can contribute to an improved understanding of spatiotemporal-controlled membrane dynamics at the molecular level.

Identification of Hepatocellular Carcinoma-Specific Targets for Imaging and Therapy

Despite advances in the diagnosis and treatment of hepatocellular carcinoma (HCC), there are no tumor-selective agents that are clinically approved in the US for this disease. Here, we aimed to identify and validate molecules that are overexpressed on HCC plasma membrane compared to normal tissues, which could be facilitate the design, engineering, and testing of tumor-selective imaging and therapeutic agents. We analyzed next-generation sequencing (NGS) public datasets (TCGA and TIGER-LC) and NCI single cell RNA-sequencing datasets to identify overexpressed plasma membrane molecules and aimed to validate these targets using immunohistochemical staining (IHC) of patient tissue microarrays (TMAs), and flow cytometry using liver cancer cell lines (Huh7, HepG2, and Hep3B). NGS data identified GPC3, EGFR, MET, MUC13, and ROBO1 molecules overexpressed in HCC relative to non-tumor tissues. In HepG2 cell line, EGFR (p<0.05) and MET (p<0.01) demonstrated statistically significant increased median fluorescence intensity (MFI) relative to controls in flow cytometry. In the Hep3B cell line, MET, GPC3, and EGFR demonstrated an increased MFI relative to the control (p<0.01). No statistically significant difference was observed in Huh7 cell lines. IHC staining of TMAs for GPC3, MET, MUC14, and ROBO1 showed statistically significantly higher staining relative to the normal tumor tissue (p<0.001). We identified and validated plasma membrane molecules overexpressed in HCC compared to non-tumor tissue. Because GPC3, a well-known HCC-specific marker that is expressed in 75% of HCC, was identified using our approach, we are confident that that additional molecules may also represent promising HCC-selective targets. This work could facilitate the design, engineering, and testing of novel precision oncology imaging and therapeutic agents for HCC.

The Tetraspanin Tspan8 Associates with Endothelin Converting Enzyme ECE1 and Regulates Its Activity.

Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.

Molecular simulation study on permeation behavior of small molecules in graphyne/polypyrrole mixed matrix membrane

AbstractThe research on the permeation behavior of small molecules in mixed matrix membranes (MMMs) is an important part of the research of MMMs. Graphyne, a new two‐dimensional carbon material, was used as the filler, and polypyrrole was used as polymer matrix to construct a MMM. In order to clarify the permeation behavior of common small molecules in MMM at room temperature and constant pressure, the adsorption and diffusion properties of H2O, CO2, and O2 in graphyne/polypyrrole MMM were investigated by combining the Giant Canonical Monte Carlo (GCMC) and molecular dynamics (MD). The results show that: (1) the order of adsorption capacity of the three molecules is H2O &gt; CO2 &gt; O2; (2) the diffusion ability of the three molecules is influenced by the doping content of graphyne. Oxygen diffusion and permeation in MMMs are optimal when the graphyne filling level is 15%. The filling of graphyne affects the permeation of gases. It will shed light on application areas such as coating membranes for electronics in special environments, gas separation membranes, and gas sensor membranes.Highlights A new two‐dimensional carbon nano filler—graphyne was used. A simulation method using a combination of GCMC and MD. The diffusion and permeation behaviors are analyzed from multiple perspectives. The permeation behavior conforms to the solution‐diffusion theory. The graphyne‐filled MMM is conducive to oxygen molecular diffusion.

The alcohol biomarker phosphatidylethanol (PEth) – test performance and experiences from routine analysis and external quality assessment

Phosphatidylethanol (PEth) are membrane molecules formed from phosphatidylcholine and ethanol through transphosphatidylation catalyzed by phospholipase D. Measurement of the main PEth form 16:0/18:1 is used as a specific and sensitive alcohol biomarker, since its formation requires ethanol, it accumulates in the blood upon repeated ethanol exposure, and it is only slowly eliminated during abstinence. PEth formation correlates with alcohol intake at the population level, albeit with considerable inter-individual variation as for the half-life during withdrawal. Over the past decade, the use of PEth has increased significantly and the applications have broadened. In Sweden, routine decision limits and the interpretation of test results for PEth were harmonized in 2013, using < 0.05 µmol/L (∼35 µg/L) as the recommended lower reporting limit and values > 0.30 µmol/L (∼210 µg/L) to indicate regular high alcohol intake. Routine test results show a large variation with about half being < 0.05 µmol/L and some even exceeding 10 µmol/L. In 2013, an external quality assessment (EQA) scheme for PEth 16:0/18:1 measurement in whole blood was also started (Equalis, Uppsala, Sweden), presently involving 56 laboratories from 13 countries. The agreement of PEth results between the laboratories has gradually improved to a CV < 15%. The current clinical and scientific information suggests that PEth values below the lower reporting limit (typically ∼0.03–0.05 µmol/L, or ∼20–35 µg/L) indicates sobriety or only low or occasional alcohol consumption, while regular high alcohol intake at levels corresponding to harmful drinking is required in most cases to reach PEth values > 0.30 µmol/L.

Martini-3 Coarse-Grained Models for the Bacterial Lipopolysaccharide Outer Membrane of Escherichia coli.

The outer lipopolysaccharide (LPS) membrane of Gram-negative bacteria forms the main barrier for transport of antimicrobial molecules into the bacterial cell. In this study we develop coarse-grained models for the outer membrane of Escherichia coli in the Martini-3 framework. The coarse-grained model force field was parametrized and validated using all-atom simulations of symmetric membranes of lipid A and rough LPS as well as a complete asymmetric membrane of LPS with the O-antigen. The bonded parameters were obtained using an iterative refinement procedure with target bonded distributions obtained from all-atom simulations. The membrane thickness, area of the LPS, and density distributions for the different regions as well as the water and ion densities in Martini-3 simulations show excellent agreement with the all-atom data. Additionally the solvent accessible surface area for individual molecules in water was found to be in good agreement. The binding of calcium ions with phosphate and carboxylate moieties of LPS is accurately captured in the Martini-3 model, indicative of the integrity of the highly negatively charged LPS molecules in the outer membranes of Gram-negative bacteria. The melting transition of the coarse-grained lipid A membrane model was found to occur between 300 and 310 K, and the model captured variations in area per LPS, order parameter, and membrane thickness across the melting transition. Our study reveals that the proposed Martini-3 models for LPS are able to capture the physicochemical balance of the complex sugar architecture of the outer membrane of Escherichia coli. The coarse-grained models developed in this study would be useful for determining membrane protein interactions and permeation of potential antimicrobials through bacterial membranes at mesoscopic spatial and temporal scales.

Localization of the ibuprofen molecule in model lipid membranes revealed by spin-label-enhanced NMR relaxation

Non-steroidal anti-inflammatory drugs (NSAIDs) have antipyretic, anti-inflammatory and analgesic effects, and can be used in the treatment of various diseases. These drugs have also a number of side effects, which may be related to their interaction with lipid membranes. In this study, we use the spin-labeled NSAID ibuprofen (ibuprofen-SL) as a relaxation enhancer to study its interaction with model lipid membranes employing liquid-state 1H NMR at 500 MHz. The high magnetic moment of unpaired electron in the spin label made it possible to reduce the concentration of the studied drug in the membrane to tenths of a mole percent. As model membranes, unilamellar POPC liposomes and bicelles consisting of a 2:1 mixture of DHPC:DMPC or DHPC:POPC lipids were used. An increase in the rate of proton spin-lattice relaxation, T1−1, selectively detected for protons at different positions in the lipid molecule, showed that ibuprofen-SL is localized in the hydrophobic part of the lipid bilayer. As the concentration of ibuprofen-SL increases to 0.5 mol%, the distribution of positions of ibuprofen-SL across the bilayer becomes wider. In the presence of 20 mol% of cholesterol, ibuprofen-SL is displaced from the core of the membrane to a region closer to the head group of the bilayer. This displacement was also confirmed by the NMR NOESY experiment conducted with unlabeled ibuprofen. For bilayers containing unsaturated POPC lipids, the distribution of ibuprofen positions across the bilayer becomes narrower compared to the presence of saturated DMPC lipids.

Evaluation of Pt particles redeposition effect on gas transport in Nafion membrane

During the operation of proton exchange membrane fuel cell, Pt particles in the catalytic layer inevitably dissolve into the membrane, which affects the performance of fuel cell. In this work, molecular dynamics is employed to investigate the effect of the redeposition of Pt particles on gas diffusion and solvation characteristics in the hydration membrane. Gas diffusion, hydrophilic cluster and its connectivity are analyzed. The interaction between Pt particles, Nafion membrane and water molecules is discussed. The influence of Pt particles on the thermal conduction of membrane is also evaluated. The results demonstrate that the presence of Pt particles affects the solvation effect of membrane, leading to a weak water cluster connectivity of membrane, which in turn leads to a poor gas diffusion performance. In addition, the thermal conductivity gradually deteriorates during the redeposition of Pt particles.

Function and clinical application of exosome-how to improve tumor immunotherapy?

In recent years, immunotherapy has been increasingly used in clinical practice to treat tumors. However, immunotherapy's efficacy varies between tumor types and patient populations, and long-term drug resistance often occurs during treatment. Therefore, it is essential to explore the molecular mechanisms of immunotherapy to improve its efficacy. In this review, we focus on the significance of tumor-derived exosomes in the clinical treatment of tumors and how modifying these exosomes may enhance immune effectiveness. Specifically, we discuss exosome components, such as RNA, lipids, and proteins, and the role of membrane molecules on exosome surfaces. Additionally, we highlight the importance of engineered exosomes for tumor immunotherapy. Our goal is to propose new strategies to improve the efficacy of tumor immunotherapy.

Selectivity and permeability of gas separation in SILMs: Effect of collapsed structure

In this work, the influence of the intramembrane structure of SILMs on gas separation was demonstrated by molecular dynamics simulations. The five morphological characteristics of separation membranes bring different separation effects. For the selection process, interfacial characteristics have a significant effect on the selectivity by the analysis of density and energy barrier. The difference in the blocking effect on CO2 is the key to the separation. For the permeation process, the analysis of free volume and interaction energy revealed that the residence time and penetration of gas molecules in membranes are sensitive to distribution characteristics of ionic liquids. Lastly, the interaction energy and pressure difference were analyzed to reveal the influence of the membrane structure on the persistence of separation and flow interruption. This work can provide theoretical support for the high efficiency control of membrane separation and the development of two-dimensional membrane materials.

Distinctive aspects of the immune status of the Kola Saami and Russians living in the Far North.

The level of immunological reactivity of the indigenous inhabitants of the Far North may depend on the extreme-discomfort factors of the climatic environment and genetic traits of the population. Therefore, the aim of this study was to identify the distinctive elements of the immune status of the Kola Saami and Russians living in the Far North (Murmansk Region). A complex immunological study of peripheral venous blood composition was performed and involved 67 Saami and 81 Russian inhabitants of the Far North (Murmansk Region) who were aged 21-55. Blood was collected from the cubital vein on an empty stomach in the morning. For the peripheral venous blood, we obtained a hemogram and phagocytic activity of neutrophils, determined the content of lymphocytes with CD4, CD8, CD16, and CD71 phenotypes by indirect immunoperoxidase reaction and flow cytometry using an Epics XL apparatus (Beckman Coulter, United States), obtained concentrations of IL-6, IFN-γ, IL-10, IgA, M, G, E, sCD54, and sCD62L, transferrin, and sCD71 by enzyme immunoassay using the "Evolis" enzyme immunoassay analyzer from Bio-RAD (Germany), and obtained circulating immune complexes by precipitation using 3.5%, 4.0%, and 7.5% PEG-6000. The sIgA content was determined in morning saliva, urine, and gastrointestinal tract secretions. The results were analyzed using descriptive statistics, independent sample t-criterion, and Pearson correlation coefficient. A decrease in the total number of leukocyte cells in the Saami was associated with a high incidence of leukopenia, lymphopenia, and neutropenia. Deficiency in active phagocytes and serum IgA and sIgA concentrations was revealed as well as increased contents of CD4+ and CD8+ receptor lymphocytes, membrane and free adhesion molecules, and transferrin. Leukocytosis, lymphocytosis, neutrophilia, monocytosis, and eosinophilia with increased contents of natural killer cells, IL-6, and IL-10 were recorded more frequently in the Russians. High levels of IgE and IFN-γ, which have the most pronounced cytotoxicity and are indicative of strained immune mechanisms, were characteristic of the Saami and Russians. The distinctive aspects of the immune status of the Kola Saami is related to the deficit in the active phagocyte and serum IgA and sIgA concentrations as well as the increase in the contents of cytotoxic lymphocytes, intercellular adhesion molecules, transferrin, and IFN-γ proinflammatory cytokines. The Russian inhabitants of the Far North are characterized by an adaptive response manifested by an increase in antibody-dependent cytotoxicity, involving IL-6 and IL-10.

ATP and ssDNA aptamer-mediated peroxidase-like activity of rGO@PDA@CeO2 nanozyme: Exosomal proteins profiling and detection at physiological pH for colorimetric sensor

Exosomes are extracellular vesicles that inherit molecular markers from their parent cells for the transfer and delivery of membrane and cytosolic molecules between cells. Exosome detection and protein profiling provide noninvasive access to disease diagnosis and treatment. The nanozyme designed in this paper contains polydopamine-functionalization of graphene oxide (rGO@PDA) units that can adsorb ssDNA aptamers and CeO2 metal oxide nanosheet units with peroxidase activity. As auxiliary regulator, adenosine triphosphate (ATP) enhances peroxidase activity of rGO@PDA@CeO2 under a wide range of pH (4–7.4), which breaks through the limitation that traditional nanozymes can only exhibit peroxidase activity under acidic conditions. The ssDNA aptamers, which are recognition probes of exosome, can be adsorbed on the surface of rGO@PDA by aromatic stacking, competing with ATP molecules. Thus, the ssDNA aptamer acts as the switch for colorimetric sensor to flexibly regulate the peroxidase activity of the rGO@PDA@CeO2 under physiological conditions, and realizes sensitive detection of exosomes. This assay achieves a detection linear range of 0.46 × 107–108 particle/mL and a limit down to 3.81 × 105 particles/mL. In addition, this aptasensor sensitively profiles six exosomal protein across five cell types and clinical samples, suggesting their potential in clinic diagnosis.

The ligand interactions of B cell Siglecs are involved in the prevention of autoimmunity to sialylated self-antigens and in the quality control of signaling-competent B cells.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of membrane molecules that recognize sialic acid. Most of them are inhibitory receptors that inhibit immune-cell activation by recognizing sialic acid as a self-motif. Human B cells express CD22 (also known as Siglec-2), Siglec-5, Siglec-6 and Siglec-10 whereas mouse B cells express CD22 and Siglec-G (ortholog of human Siglec-10). Siglecs recognize both sialylated molecules expressed on the same cell (cis-ligands) and those expressed by other cells (trans-ligands). In Guillain-Barré syndrome (GBS), antibody production to gangliosides (which are sialic acid-containing glycolipids) expressed by neurons plays a pathogenic role. A Siglec-10 variant deficient in recognition of gangliosides is genetically associated with GBS, suggesting that Siglec-10 induces self-tolerance to gangliosides by recognizing gangliosides as trans-ligands. Recognition of the BCR as a cis-ligand by Siglec-G and CD22 suppresses BCR signaling in B-1 cells and conventional B cells, respectively. This signal suppression prevents excess expansion of B-1 cells and is involved in the quality control of signaling-competent B cells by setting a threshold for tonic signaling during B cell development. CD22 recognizes other cis-ligands including CD22 and β7 integrin. Interaction of CD22 with other CD22 molecules induces CD22 clustering that suppresses CD22-mediated signal inhibition upon BCR ligation, and interaction with β7 integrin maintains its function in the gut-homing of B cells. Taken together, interactions of B cell Siglecs with multiple trans- and cis-ligands play important roles in B cell homeostasis and immune responses.

A review of the mechanisms of blood-brain barrier disruption during COVID-19 infection.

Coronaviruses are prevalent in mammals and birds, including humans and bats, and they often spread through airborne droplets. In humans, these droplets then interact with angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), which are the main receptors for the SARS-CoV-2 virus. It can infect several organs, including the brain. The blood-brain barrier (BBB) is designed to maintain the homeostatic neural microenvironment of the brain, which is necessary for healthy neuronal activity, function, and stability. It prevents viruses from entering the brain parenchyma and does not easily allow chemicals to pass into the brain while assisting numerous compounds in exiting the brain. The purpose of this review was to examine how COVID-19 influences the BBB along with the mechanisms that indicate the BBB's deterioration. In addition, the cellular mechanism through which SARS-CoV-2 causes BBB destruction by binding to ACE2 was evaluated and addressed. The mechanisms of the immunological reaction that occurs during COVID-19 infection that may contribute to the breakdown of the BBB were also reviewed. It was discovered that the integrity of the tight junction (TJs), basement membrane, and adhesion molecules was damaged during COVID-19 infection, which led to the breakdown of the BBB. Therefore, understanding how the BBB is disrupted by COVID-19 infection will provide an indication of how the SARS-CoV-2 virus is able to reach the central nervous system (CNS). The findings of this research may help in the identification of treatment options for COVID-19 that can control and manage the infection.

Markers of Th1 polarized Th17 cells (literature review)

T helpers (Th) producing IL-17 (Th17) have high plasticity and under the influence of external conditions are able to redifferentiate into cells with a different phenotype, primarily in Th1-lymphocytes, forming a population that combines the characteristics of both Th17 and Th1 and has a high pro-inflammatory potential, as well as a unique ability to overcome histohematic barriers. These cells are currently assigned a key role in the pathogenesis of many inflammatory diseases, including autoimmune ones: they account for up to half of the lymphocytes present in infiltrates of inflamed tissues. The paper discusses the reasons for the increased plasticity of Th17 cells in comparison with the main T helper populations (Th1 and Th2) and considers in detail the mechanisms of formation of IFNγ producing Th17, taking into account not only the redifferentiation of mature Th17, but also possible alternative pathways, in particular, Th1 cell redifferentiation or naive CD4+T lymphocytes direct differentiation into cells with an intermediate Th1/Th17 phenotype. The main inducers of differentiation of IFNγ producing Th17 cells and the reversibility of this process are also discussed. Particular attention is paid to the methods for identifying Th1 polarized Th17 cells: this population is heterogeneous, and its size significantly depends on the type of markers used to characterize these cells – Th1/Th17-associated transcription factors, key cytokines, as well as chemokine receptors and other membrane molecules. As a result, the data in the works on this problem are poorly comparable with each other. The unification of approaches to identifying a population of Th1 like Th17 cells will solve this problem and make it possible to use an assessment of the size and activity of such a population as diagnostic or prognostic markers.

Features of innate immunity in children with hypertrophy of the palatine tonsils

Hypertrophy of pharyngeal lymphoid tissue is among the most common problems in pediatric otorhinolaryngology, in particular, hypertrophy of the palatine tonsils. This disorder is characterized by increased size of a single or both palatine tonsils combined with various clinical symptoms. The principles of treatment in children with this pathology remain debatable, since the long-term effects of bilateral tonsillotomy are still not fully understood. The aim of the study was to assess the expression levels of genes encoding the innate immunity molecules (TLR4, HBD1, HBD2, IL1β) in the mucous membranes of palatine tonsils in children with palatine tonsillitis before and after treatment.We conducted a study of 78 patients divided into three independent groups. The 1st group (comparison) included 20 somatically healthy children. The 2nd group included 28 children with grade 2 palatine tonsil hypertrophy who underwent local treatment. The 3rd group included 30 children with grade 3 hypertrophy of the palatine tonsils, who, by clinical indications, underwent bilateral tonsillotomy. Determination and evaluation of innate immunity indices was carried out both before starting the treatment, and one month later, using the real-time PCR method.In children with hypertrophy of palatine tonsils, the initial values of TLR4 gene expression and antimicrobial peptides differed from those of healthy children. A decreased expression of HBD1 and HBD2 genes, which provide immediate protection against pathogens, was revealed. The values of TLR4 gene expression differed in groups of children with varying degrees of palatine tonsillar hypertrophy. In patients with bilateral tonsillotomy, an increased expression of TLR4 gene and a decreased expression of antimicrobial peptide genes (HBD1, HBD2) were revealed, which may indicate a readiness for development of tonsillar inflammation in response to pathogens. One month after surgical treatment, the indices of innate immunity were comparable with those of healthy children thus confirming the validity of surgical treatment. In the 2nd group of patients, the TLR4 gene expression one month after conservative treatment remained reduced, the expression of β-defensin HBD1 gene increased and exceeded the indicators of the group of healthy children, the expression level of the IL-1β gene was reduced.The revealed imbalance between the TLR4, HBD1 and HBD2 expression levels confirms an important role of innate immunity mechanisms in pathogenesis of palatine tonsillitis. The assessment of innate immunity indices may be used as an additional criterion in administration of therapy for hypertrophy of palatine tonsils and evaluation of its efficiency.

Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology.

The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal resolution have been limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified. Using theoretical frameworks developed for the analysis of single-molecule trajectories in the plasma membrane (PM), this camera successfully detected fast hop diffusion of membrane molecules in the PM, previously detectable only in the apical PM using less preferable 40-nm gold probes, thus helping to elucidate the principles governing the PM organization and molecular dynamics. Furthermore, as described in the companion paper, this camera allows simultaneous data acquisitions for PALM/dSTORM at as fast as 1 kHz, with 29/19 nm localization precisions in the 640 × 640 pixel view-field.

Effect of dexamethasone therapy on factors of adhesiveness and coagulation in acute lower limb ischemia

Leukocyte-platelet adhesion during hypoxia, tissue damage, activation of inflammation and coagulation is associated with the expression of ICAM-1 membrane molecules and integrins by blood and tissue cells. At the same time, platelet adhesion receptors determine their adhesion to the endothelium and recruited lymphocytes. The role of platelets in the pathogenesis of ischemic cardiovascular diseases also consists in their ability to modulate both hemostasis and inflammatory reactions, which is accompanied by the secretion of inflammatory mediators and factors that promote the recruitment of leukocytes to tissue damage sites. Purpose of the study: to study the effect of the synthetic glucocorticoid dexamethasone on the expression of adhesion receptors CD18+ and CD54+ on leukocytes, the content of platelets and fibrinogen in the blood of patients with ALLI, the relationship of these indicators with the severity and outcome of the disease.To study the effect of anti-inflammatory therapy, a group of 32 patients treated with dexamethasone was formed; the comparison group was represented by 71 patients with basic therapy, the control group consisted of 15 volunteers. After revascularization, all patients received antiplatelet and anticoagulant therapy. Dexamethasone infusions were carried out in a course of 4 to 6 days after reconstructive surgery. In all patients, the content of C-reactive protein in the blood, the content of platelets and fibrinogen were determined. The number of lymphocytes expressing adhesion molecules ICAM-1 (CD54+) and integrins (CD18+) was counted using the immunocytochemical method. Studies were performed before surgery and on days 1, 3, 7, and 10 after surgery.With exacerbation of ischemia and damage to the endothelium, the accumulation of cytolysis products, the expression of adhesion molecules increases both on endotheliocytes and on inflammatory effector cells – leukocytes and platelets. Adhesion molecules conduct an activation signal inside the cell, which promotes adhesion of leukocytes and platelets to the endothelium, lymphocytic-platelet adhesion, the formation of a parietal thrombus, and possible occlusion of damaged vessels. Increased expression of adhesion molecules is associated with the activation of metabolism, inflammation, coagulation and oxidative stress, stimulates all hematopoietic lineages, including platelets. The level of involvement of cellular reactions in the pathogenesis of the disease affects the effectiveness and duration of treatment, the risk of recurrent thrombosis and death. Anti-inflammatory therapy with dexamethasone contributed to earlier remission, a decrease in the proportion of infectious complications, such as wound suppuration from 10% to 6%, the number of necessary amputations from 32% to 16%, the frequency of deaths from 31% to 6%, and a reduction in hospital stay from 13 days to 10.Inflammation, adhesiveness of effector cells and thrombosis are important factors in the pathogenesis of acute lower limb ischemia. Therapy with dexamethasone helps to reduce the level of systemic inflammatory response, the number of necessary amputations, the number of complications and adverse outcomes in the treatment of ALLI, and reduce the length of stay in the hospital.

Identification of Th1-polarized Th17 cells: solving the problem

Helper T cells producing IL-17 (Th17) have high plasticity: restimulation of lymphocytes in an inflammatory environment can induce their transformation into cells with another phenotype, and a shift towards Th1 is the most common. The result of this transformation is the appearance of cells expressing along with the classical markers of Th17 cells key Th1-associated molecules. In its most general form, this population is represented by CD4+CD161+CCR6+CXCR3+IL-17+IFNγ+Т cells, and in the current literature it is most often referred to as Th17.1. Some Th17.1 cells can completely lose the production of IL-17, while maintaining the expression of other Th17-associated molecules; these are the so-called ex-Th17 cells (CD4+CD161+CCR6+CXCR3+IL-17- IFNγ+Т cells). Consequently, the population of Th1-polarized Th17 includes Th17.1, ex-Th17 cells and a number of additional transitional forms. It has unique functional properties – an increased pro-inflammatory potential and the ability to overcome histohematic barriers. It is these cells that are currently assigned a key role in the pathogenesis of many autoimmune diseases, and the process of Th17 redifferentiation into Th1 is considered as a promising therapeutic target. However, the development of this direction is complicated by the weak comparability of data on the size of such a population. The analysis of methods for determining Th1-polarized Th17 in vivo and in vitro, carried out in this work, made it possible to resolve these contradictions and develop optimal approaches to identifying this population. In most studies, especially clinical ones, it is identified by co-expression of key cytokines (IL-17/IFNγ) or chemokine receptors (CCR6/CXCR3), rarely by their combination. In this approach, co-expression of CCR6/ CXCR3 marks the total population of Th1-like Th17, including both Th17.1 and ex-Th17, while co-expression of IL-17/IFNγ cytokines identifies only Th17.1 cells, and the subpopulation of ex-Th17 is misclassified as classic Th1 in this case. Such “underestimation” of the ex-Th17 subpopulation significantly marks down the results, since it is ex-Th17 that accounts for the bulk of Th1-like Th17. And only a simultaneous assessment of the co-expression of cytokines and Th17-associated membrane molecules allows identification Th17.1 and exTh17 cells separately, which is important to consider when interpreting data on the problem and when planning clinical trials.

&lt;i&gt;PD-L1&lt;/i&gt; and &lt;i&gt;PD-L2&lt;/i&gt; gene expression in human glioblastoma cells resistant to chemo- and radiotherapy

Membrane molecules PD-L1 and PD-L2, ligands of T lymphocytes PD1 receptor, perform immunoregulatory functions. Their binding to the receptor leads to inhibition of proliferation, reduction of cytokine production, cytotoxic response, and apoptosis of T lymphocytes. The cells of many tumors, regardless of their histogenesis, express PD-L1 molecules, thus limiting the development of an anti-tumor immune response. Glioblastomas are highly malignant recurrent tumors of the central nervous system. The main sources of glioblastoma recurrence are resistant tumor cells initially present in gliomas with heterogeneous cellular composition, as well as resistant cells that are formed during therapy. Increasing the dose of cytostatic drugs or radiation during relapse therapy is not effective in glioblastomas. It has been shown for a number of tumors, including ovarian cancer and non-small cell lung cancer, that drugs preventing PD-L1/PD1 interaction are effective in the treatment of neoplasms resistant to chemo- and radiotherapy. Immunotherapy using drugs that inhibit the binding of PD-L molecules to their receptor is considered as a way to overcome the resistance of glioblastomas to therapy. The aim of this work was to assess the level of PD-L1 and PD-L2 gene expression in resistant glioblastoma cells lines A172, R1, T2 and T98G, which resumed proliferation after exposure to the maximum for each line, sublethal doses of cytostatic drugs (fotemustine and temozolomide), fractionated or single gamma irradiation. A172 line belongs to glioblastomas that are highly sensitive to these influences, T98G is a highly resistant cell line, while R1 and T2 lines occupy an intermediate position. In intact glioblastoma A172, R1, and T2 cells the level of PD-L1 and PD-L2 gene expression was equally high, while in T98G cells it was significantly lower. Exposure of A172 and R1 glioblastoma lines to cytostatic drugs or irradiation did not significantly change the level of PD-L1 and PD-L2 genes expression typical for intact cells. In T2 glioblastoma cells, and especially in T98G cells, a significant increase in expression of these genes was found, most pronounced for PD-L2 gene. This increase in expression may indicate an enhanced malignancy of resistant T2 and T98G cells. High expression of the genes responsible for the production of PD-L1 and PD-L2, which limit the cytotoxic response against tumor cells, is a prerequisite for the use of drugs targeted against PD-L1 and PD-L2 for the elimination of resistant cells in glioblastoma.