Changes In Cell Phenotype Research Articles

Obesity and myeloma: Clinical and mechanistic contributions to disease progression.

Obesity and obesogenic behaviors are positively associated with both monoclonal gammopathy of unknown significance (MGUS) and multiple myeloma (MM). As the only known modifiable risk factor, this association has emerged as a new potential target for MM prevention, but little is known about the mechanistic relationship of body weight with MM progression. Here we summarize epidemiological correlations between weight, body composition, and the various stages of myeloma disease progression and treatments, as well as the current understanding of the molecular contributions of obesity-induced changes in myeloma cell phenotype and signaling. Finally, we outline groundwork for the future characterization of the relationship between body weight patterns, the bone marrow microenvironment, and MM pathogenesis in animal models, which have the potential to impact our understanding of disease pathogenesis and inform MM prevention messages.

Hydroquinone, an Environmental Pollutant, Affects Cartilage Homeostasis through the Activation of the Aryl Hydrocarbon Receptor Pathway.

Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.

YTHDF2 Regulates Cell Growth and Cycle by Facilitating KDM1A mRNA Stability

Breast cancer is the leading cause of cancer death in women. The physiological functions of N6-methyladenosine methylation in cancer have been the focus of studies in recent years. Herein, four data sets (GSE70947, GSE45827, GSE42586, and The Cancer Genome Atlas Breast Cancer) were analyzed to confirm the differentially expressed N6-methyladenosine genes. YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2) was found to be highly expressed in breast cancer tissues and cells. Invitro, YTHDF2 affects cell proliferation, cell cycle, and invasive ability. Tumorigenesis in xenograft nude mice confirmed that YTHDF2 interference reduced the tumor formation ability of cancer cells. Pearson correlation analysis demonstrated a positive correlation between YTHDF2 and lysine-specific histone demethylase 1A (KDM1A) expression. An online tool, Sequence-based RNA Adenosine Methylation Site Predictor (SRAMP), predicted eight methylation sites in the KDM1A mRNA sequence. The expression of KDM1A was dramatically increased in breast cancer tissues and cells. Down-regulation of YTHDF2 reduced KDM1A expression and the methylation level of KDM1A mRNA. YTHDF2 interference promoted the degradation of KDM1A mRNA, which suggested an interaction between YTHDF2 and KDM1A. KDM1A interference altered cell proliferation, cell cycle, and invasive ability, whereas YTHDF2 overexpression rescued KDM1A interference-induced cell phenotypic changes. In conclusion, YTHDF2 promotes breast cancer cell growth and cell cycle progression by facilitating KDM1A mRNA stability. This study provides new therapeutic targets for breast cancer treatment in the future.

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Cell phenotype changes and oxidative stress response in Vibrio spp. induced into viable but non-culturable (VBNC) state

PurposeAquatic bacteria of the genus Vibrio include animal and human pathogens. The occurrence of Vibrio-related diseases has been associated with the current climate change-driven increase of sea surface temperature. Vibrio spp. can enter into the viable but non-culturable (VBNC) state, as a consequence of starvation in seawater at low temperatures. In such physiological state, Vibrio cells are no longer culturable on standard media agar plates but can resuscitate if incubated at 30 °C prior to plating, retaining virulence. Since limited information is available on regards to this topic, in this work, we characterized the phenotypic changes of four Vibrio spp. strains (one laboratory strain and three environmental isolates) in cold seawater microcosms, investigating the relationship between resuscitation and a hydrogen peroxide-induced oxidative stress.MethodsCell phenotypic changes and the effect of hydrogen peroxide and/or catalase addition to the medium were studied on VBNC and resuscitated cells by flow cytometry in microcosm experiments, paralleled by culturability experiments by plating.ResultsThe cells of all the Vibrio strains changed their phenotype upon the induction of the VBNC state resulting in cell dwarfing and decrease in DNA quantity, losing the ability to grow on solid media. These features were partially or totally reverted when the cells were treated for resuscitation. Hydrogen peroxide at concentrations as low as 0.007 mM prevented resuscitation and a prolonged exposure to hydrogen peroxide at concentrations far under those inhibiting the growth of log-phase cells permanently damaged VBNC cells, which could not be resuscitated. However, the potential of culturability of VBNC cells could be preserved, at least for a part of the population, by plating the cells in the presence of catalase. The study also showed that during the resuscitation process, the cells gradually increased their resistance to hydrogen peroxide.ConclusionsThe timing and mode of induction of the VBNC state, as well as cell resuscitation and response to hydrogen peroxide, differed among Vibrio strains, indicating that induction and resuscitation from dormancy could vary in the context of species belonging to a single genus.

Trimethylamine-N-Oxide Promotes High-Glucose-Induced Dysfunction and NLRP3 Inflammasome Activation in Retinal Microvascular Endothelial Cells.

Along with blood glucose levels, diabetic retinopathy (DR) development also involves endogenous risk factors, such as trimethylamine-N-oxide (TMAO), a product of intestinal flora metabolic disorder, which exacerbates diabetic microvascular complications. However, the effect of TMAO on retinal cells under high-glucose conditions remains unclear. Therefore, this study examined the effects of TMAO on high-glucose-induced retinal dysfunction in the context of NLRP3 inflammasome activation, which is involved in DR. TMAO was assessed in the serum and aqueous humor of patients using ELISA. Human retinal microvascular endothelial cells (HRMECs) were treated for 72 h as follows: NG (normal glucose, D-glucose 5.5 mM), NG + TMAO (5 μM), HG (high glucose, D-glucose 30 mM), and HG + TMAO (5 μM). The CCK8 assay was then used to assess cell proliferation; wound healing, cell migration, and tube formation assays were used to verify changes in cell phenotype. ZO-1 expression was determined using immunofluorescence and western blotting. Reactive oxygen species (ROS) formation was assessed using DCFH-DA. NLRP3 inflammasome complex activation was determined using a western blot. The serum and aqueous humor from patients with PDR contained higher levels of TMAO compared to patients with nontype 2 diabetes (Control), non-DR (NDR), and non-PDR (NPDR). TMAO showed significant acceleration of high-glucose-induced cell proliferation, wound healing, cell migration, and tube formation. ZO-1 expression decreased remarkably with the combined action of TMAO and a high glucose compared to either treatment alone. TMAO also promoted high-glucose-activated NLRP3 inflammasome complex. The combination of TMAO and high-glucose results in increased levels of ROS and NLRP3 inflammasome complex activation in HRMECs, leading to exacerbated retinal dysfunction and barrier failure. Thus, TMAO can accelerate PDR occurrence and development, thus indicating the need for early fundus monitoring in diabetic patients with intestinal flora disorders.

Гиперпластические процессы эндометрия у пациенток с хронической болезнью почек: выбор тактики ведения

This article provides a literature review demonstrating modern paradigms concerning etiology, pathogenesis and management of endometrial hyperplasia processes in female patients with chronic kidney disease (CKD). Endometrial hyperplasia (EH) is a pathological process with various phenotypic cell changes and a characteristic predominance of glands over the stromal and epithelial components in the endometrium. Untimely diagnosis and treatment of EH can cause the pathology progression and the development of endometrium malignant transformation. Endometrial cancer (EC) refers to one of the most common malignant tumor forms of the female reproductive system, and EH, as a rule, is its precursor. In the gynecological morbidity structure, EH is 10 to 55%, and its incidence is steadily increasing. EH occurs as a result of excessive estrogen stimulation, when it is not compensated by the progesterone action. In addition, patients with CKD develop "immunosuppressive states" due to the use of various types of renal replacement therapy (hemodialysis, peritoneal dialysis, taking immunosuppressive drugs in patients with kidney transplant). Every year there are more and more such female patients. Over the past decade, there has been no reference in the Russian literature concerning a systematic approach to the diagnosis and treatment of EH in female patients with CKD. Considering that these were female patients of the reproductive period to a greater extent, who wanted to perform the childbearing function in the future, therefore, this problem is very relevant for the world medical community. KEYWORDS: endometrial hyperplasia, abnormal uterine bleeding, chronic kidney disease, hemodialysis, kidney transplant, peritoneal dialysis, immunosuppression. FOR CITATION: Dobrokhotova Yu.E., Lysenko M.A., Grabovsky V.M., Shevchenko N.A., Zapolskaya N.P. Endometrial hyperplasia process in female patients with chronic kidney disease: the choice of treatment tactics. Russian Journal of Woman and Child Health. 2023;6(4):380–384 (in Russ.). DOI: 10.32364/2618-8430-2023-6-4-9.

Macrophage regulation of Wnt pathway in canine and murine mammary cancer cells

Interactions between various cell types in a tumor microenvironment (TME) are important for cancer progression and metastasis. Tumor-associated macrophages (TAMs) play a key role in this intratumoral dialogue regulating Wnt signaling pathway in cancer cells. Co-culture of canine and murine tumor cells with TAMs lead to inhibition of the canonical Wnt pathway and activation of the non-canonical Wnt pathway in tumor cells resulting in change in tumor cell phenotype and subsequent higher potential to invasion and metastasis. These molecular changes in cancer cells that are induced by TAMs resemble the quercetin activity. This natural product is a plant flavonoid from the group of polyphenols. Its biological function is inhibition of the canonical Wnt pathway. As quercetin supplementation is broadly discussed in cancer patients, findings of this study show that this compound should be used with caution, as it may enhance cancer spread.

Senescent mesenchymal stem/stromal cells in pre-metastatic bone marrow of untreated advanced breast cancer patients.

Breast cancer is the predominant form of carcinoma among women worldwide, with 70% of advanced patients developing bone metastases, with a high mortality rate. In this sense, the bone marrow (BM) mesenchymal stem/stromal cells (MSCs) are critical for BM/bone homeostasis, and failures in their functionality, transform the BM into a pre-metastatic niche (PMN). We previously found that BM-MSCs from advanced breast cancer patients (BCPs, infiltrative ductal carcinoma, stage III-B) have an abnormal profile. This work aims to study some of the metabolic and molecular mechanisms underlying MSCs shift from a normal to an abnormal profile in this group of patients. A comparative analysis was undertaken, which included self-renewal capacity, morphology, proliferation capacity, cell cycle, reactive oxygen species (ROS) levels, and senescence-associated β‑galactosidase (SA‑β‑gal) staining of BM-derived MSCs isolated from 14 BCPs and 9 healthy volunteers (HVs). Additionally, the expression and activity of the telomerase subunit TERT, as well as telomere length, were measured. Expression levels of pluripotency, osteogenic, and osteoclastogenic genes (OCT-4, SOX-2, M-CAM, RUNX-2, BMP-2, CCL-2, M-CSF, and IL-6) were also determined. The results showed that MSCs from BCPs had reduced ,self-renewal and proliferation capacity. These cells also exhibited inhibited cell cycle progression and phenotypic changes, such as an enlarged and flattened appearance. Additionally, there was an increase in ROS and senescence levels and a decrease in the functional capacity of TERT to preserve telomere length. We also found an increase in pro-inflammatory/pro-osteoclastogenic gene expression and a decrease in pluripotency gene expression. We conclude that these changes could be responsible for the abnormal functional profile that MSCs show in this group of patients.

Variations in dynamic tumor-associated antigen-specific T cell responses correlate with HCC recurrence after thermal ablation.

Ablative therapy is a recommended treatment for hepatocellular carcinoma (HCC) not only for its effective eradication of tumors, but also for its induction of host immunity. However, the high 5-year recurrence rate after ablation underlines the poor understanding of the antitumor immunity response. Here, we investigated the effects of thermal ablation on antitumor immunity. We analyzed the dynamics of tumor-associated antigen (TAA)-specific immune responses and changes in peripheral blood mononuclear cell phenotype in patients with HCC before and after tumor ablation. We used the IFN-γ ELISPOT assay and immunophenotyping by flow cytometry to evaluate the effects of ablation on host immunity. The correlation between the T cell response and disease outcome was explored to uncover the efficacy of the immune response in inhibiting HCC recurrence. Different TAA-specific T cell responses were identified among patients before and after ablation. One week after ablation, there was an improved immune state, with a switch from the dominance of an AFP-specific T cell response to that of a SMNMS-specific T cell response, which was correlated with better survival. Furthermore, an improvement in immune status was accompanied by a lower level of PD1+ and Tim3+ T cells in CD8+ T cells. Although this functional state was not durable, there was a higher degree of AFP-specific T cell responses at 4-weeks post-ablation. Furthermore, T cells presented a more exhausted phenotype at 4-weeks post-ablation than at the 1-week timepoint. Ablation elicits a transient antitumor immune response in patients with HCC by changing the profile of the T cell response and the expression of immune checkpoint molecules, which correlated with longer recurrence-free survival of patients with HCC.

Degradable intracellular self-functionalized nanoprobes for in vitro diagnosis and monitoring of cancer

Despite various attempts to accelerate nanoparticle clearance from the cell, considerable amounts of nanomaterials remain in the cell, impairing cellular activities and hence precluding reliable invitro diagnosis. Retention of nanoparticles in cellular organelles disturbs cellular equilibrium and induces long-term consequences such as change in cell phenotype, genotype, cell mobility, toxicity, and even cell death. As a result, it is necessary to produce nanoparticles that degrade promptly and are then safely expelled from the cell while remaining biocompatible and fully functional. Here, we present a metabolizable organic carbon probe functionalized for Raman and fluorescence that can be used to track and monitor metabolizable organic carbon probe breakdown in lysosomes while simultaneously performing molecular level probing for invitro cancer diagnosis. The probes were stable in medium and other fluids and degraded in cancer cells within a week. The probes demonstrated an excellent Raman signal, which allowed surface-enhanced Raman spectroscopy-based monitoring of the degradation. Additionally, the probes showed the property of fluorescence which can be attributed to the inherent fluorescence of the probe on quantum scale. Experimental evidence of self-metabolization of the probe was further demonstrated. We tested the ability of the probes for cancer diagnosis with two cancer cell lines (breast and lung) and experimentally demonstrated the ability to discriminate between breast cancer and lung cancer without any ambiguity. These findings establish a novel type of functionalized degradable nanostructure for intracellular sensing and imaging cancer cells in cellular nanomedicine.

PDX MODEL OF BC IN THE STUDY OF PLASTICITY OF TUMOR CELLS IN THE PROCESS OF METASTASIZING

Mouse models using primary patient’s tumors (patient-derived xenograft, PDX) are an effective tool for studying the mechanisms of metastasizing. Tumor plasticity, i.e. the phenomenon of cell phenotype change in response to dynamic microenvironmental conditions is one of the key processes basic to metastasizing.

Age-dependent NK cell dysfunctions in severe COVID-19 patients.

Natural Killer (NK) cells are key innate effectors of antiviral immune response, and their activity changes in ageing and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we investigated the age-related changes of NK cell phenotype and function during SARS-CoV-2 infection, by comparing adult and elderly patients both requiring mechanical ventilation. Adult patients had a reduced number of total NK cells, while elderly showed a peculiar skewing of NK cell subsets towards the CD56lowCD16high and CD56neg phenotypes, expressing activation markers and check-point inhibitory receptors. Although NK cell degranulation ability is significantly compromised in both cohorts, IFN-γ production is impaired only in adult patients in a TGF-β-dependent manner. This inhibitory effect was associated with a shorter hospitalization time of adult patients suggesting a role for TGF-β in preventing an excessive NK cell activation and systemic inflammation. Our data highlight an age-dependent role of NK cells in shaping SARS-CoV-2 infection toward a pathophysiological evolution.

Adaptation of a multiple myeloma minimal residual disease multicolor flow cytometry assay for real-world practice.

Achieving minimal residual disease (MRD) negativity following treatment for multiple myeloma (MM) is associated with improved progression free and overall survival. In the UK, MRD assessments in MM are not incorporated into routine clinical use outside trials. Widely used in other haematological malignancies, there is a role for widening the availability of myeloma MRD assays to laboratories outside larger treating centers. We set up and assessed concordance of a multicolor flow cytometry (MCF) assay for MM MRD in collaboration with a reference center including validity following delayed processing of samples using an optimized fixation step. We then conducted a real-world snapshot of MRD results in a cohort of newly diagnosed transplant-eligible patients treated with UK standard induction therapies at the time of analysis. 43 MCF MRD samples run in parallel with a reference center showed high correlation and minimal bias. 24 samples were split and processed in duplicate both fixed and fresh, with strong correlation, minimal bias, and no change in plasma cell phenotype by flow markers confirming a 6-day delay in processing did not affect assay performance. A real-world snapshot found 17% (10/58) of patients were MRD-negative post-bortezomib-based triplet induction therapy. We successfully adopted a reference MCF MM MRD method which was stable for up to 6 days following sample collection potentially allowing broader access of this assay to smaller laboratories which would facilitate further investigation of the prognostic value and clinical utility of MRD assessments outside the trial setting in real-world practice.

Influence of PRRSV-1 vaccination and infection on mononuclear immune cells at the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8β T cells with CD8αposCD27dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46pos NK cells, CD8αhigh γδ T cells, as well as CD8αposCD27pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163highCD169pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection.

Targeting Immune Senescence in Atherosclerosis.

Atherosclerosis is one of the main underlying causes of cardiovascular diseases (CVD). It is associated with chronic inflammation and intimal thickening as well as the involvement of multiple cell types including immune cells. The engagement of innate or adaptive immune response has either athero-protective or atherogenic properties in exacerbating or alleviating atherosclerosis. In atherosclerosis, the mechanism of action of immune cells, particularly monocytes, macrophages, dendritic cells, and B- and T-lymphocytes have been discussed. Immuno-senescence is associated with aging, viral infections, genetic predispositions, and hyperlipidemia, which contribute to atherosclerosis. Immune senescent cells secrete SASP that delays or accelerates atherosclerosis plaque growth and associated pathologies such as aneurysms and coronary artery disease. Senescent cells undergo cell cycle arrest, morphological changes, and phenotypic changes in terms of their abundances and secretome profile including cytokines, chemokines, matrix metalloproteases (MMPs) and Toll-like receptors (TLRs) expressions. The senescence markers are used in therapeutics and currently, senolytics represent one of the emerging treatments where specific targets and clearance of senescent cells are being considered as therapy targets for the prevention or treatment of atherosclerosis.

Targeting the Crosstalk of Immune Response and Vascular Smooth Muscle Cells Phenotype Switch for Arteriovenous Fistula Maturation.

Plaque formation, thrombosis, and embolism are the underlying causes of acute cardiovascular events such as myocardial infarction and stroke while early thrombosis and stenosis are common pathologies for the maturation failure of arteriovenous fistula (AVF). Chronic inflammation is a common underlying pathogenesis mediated by innate and adaptive immune response involving infiltration of immune cells and secretion of pro- and anti-inflammatory cytokines. Impaired immune cell infiltration and change in vascular smooth muscle cell (VSMC) phenotype play a crucial role in the underlying pathophysiology. However, the change in the phenotype of VSMCs in a microenvironment of immune cell infiltration and increased secretion of cytokines have not been investigated. Since change in VSMC phenotype regulates vessel remodeling after intimal injury, in this study, we investigated the effect of macrophages and pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α, on the change in VSMC phenotype under in vitro conditions. We also investigated the expression of the markers of VSMC phenotypes in arteries with atherosclerotic plaques and VSMCs isolated from control arteries. We found that the inhibition of cytokine downstream signaling may mitigate the effect of cytokines on the change in VSMCs phenotype. The results of this study support that regulating or targeting immune cell infiltration and function might be a therapeutic strategy to mitigate the effects of chronic inflammation to attenuate plaque formation, early thrombosis, and stenosis, and thus enhance AVF maturation.

Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.

EFFECT ON MYOBLAST DIFFERENTIATION BY EXTREMELY LOW-FREQUENCY PULSED ELECTROMAGNETIC FIELDS

In the myoblasts differentiation process of C2C12 cells, it is imperative to research the expression and synthesis of the myogenic regulatory factors in extremely low-frequency pulsed electromagnetic fields (ELF-PEMFs) exposure. In the present study, using a cell cultivation method of C2C12 cells, the subcultured cells were divided into control and ELF-PEMF treated: A (0[Formula: see text]mT), B (0.5[Formula: see text]mT), C (1.0[Formula: see text]mT) and D (1.5[Formula: see text]mT) groups for five consecutive days training. Furthermore, cells were analyzed using immunochemical staining to detect the cell phenotypic changes, and RT-PCR and Western blot were used to test the expression levels of the myogenic regulatory factor D (MyoD) and the myogenic regulatory factor 5 (Myf5) genes and proteins synthesis in the process of myblasts differentiation. The results indicated that compared with the control group, different intensities of extremely low-frequency pulse electromagnetic fields could induce myoblast nuclear fusion, amplify MyoD and Mfy5 expression, and particularly the 1.5[Formula: see text]mT group at the 5th day exhibited the highest expression of these genes. Our study can pave a way for the development of a non-invasive and effective clinical prevention and treatment strategy in serious disorders like skeletal muscle atrophy.

Mechanobiological responses of astrocytes in optic nerve head due to biaxial stretch

BackgroundElevated intraocular pressure (IOP) is the main risk factor for glaucoma, which might cause the activation of astrocytes in optic nerve head. To determine the effect of mechanical stretch on the astrocytes, we investigated the changes in cell phenotype, proteins of interest and signaling pathways under biaxial stretch.MethodThe cultured astrocytes in rat optic nerve head were stretched biaxially by 10 and 17% for 24 h, respectively. Then, we detected the morphology, proliferation and apoptosis of the stretched cells, and performed proteomics analysis. Protein expression was analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. Proteins of interest and signaling pathways were screened using Gene Ontology enrichment analysis and pathway enrichment analysis, and the results were verified by western blot and the gene-chip data from Gene Expression Omnibus (GEO) database.ResultThe results showed that rearrangement of the actin cytoskeleton in response to stimulation by mechanical stress and proliferation rate of astrocytes decreased under 10 and 17% stretch condition, while there was no significant difference on the apoptosis rate of astrocytes in both groups. In the iTRAQ quantitative experiment, there were 141 differential proteins in the 10% stretch group and 140 differential proteins in the 17% stretch group. These proteins include low-density lipoprotein receptor-related protein (LRP6), caspase recruitment domain family, member 10 (CARD10), thrombospondin 1 (THBS1) and tetraspanin (CD81). The western blot results of LRP6, THBS1 and CD81 were consistent with that of iTRAQ experiment. ANTXR2 and CARD10 were both differentially expressed in the mass spectrometry results and GEO database. We also screened out the signaling pathways associated with astrocyte activation, including Wnt/β–catenin pathway, NF-κB signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Jak-STAT signaling pathway, ECM-receptor interaction, and transforming growth factor-β (TGF-β) signaling pathway.ConclusionMechanical stimulation can induce changes in cell phenotype, some proteins and signaling pathways, which might be associated with astrocyte activation. These proteins and signaling pathways may help us have a better understanding on the activation of astrocytes and the role astrocyte activation played in glaucomatous optic neuropathy.