Flow Cytometric Analysis Research Articles

Impact of breastfeeding and formula feeding on immune cell populations and blood cell parameters: an observational study.

Breastfeeding is associated with improved health outcomes in infancy and throughout adulthood as breast milk encompasses diverse immune-active factors that affect the ontogeny of the immune system in breastfed (BF) infants. Nevertheless, the impact of infant feeding on the immune system is poorly understood, and a comprehensive understanding of immune system development in human infants is lacking. In this observational study, we addressed the effects of different infant feeding approaches on cell populations and parameters in the peripheral blood of infants to gain insight into the innate and adaptive arms of the immune system. Using flowcytometric analysis, we performed complete blood counts and immunoprofiling of peripheral blood collected from BF and formula-fed (FF) infants at different ages. Our results showed that the blood of BF infants had a higher frequency of leukocytes and erythrocytes in early infancy. The hemoglobin concentration was enhanced in BF infants. However, the platelet count was comparable regardless of feeding regimen. We observed immunophenotypic differences between the two populations of infants, mirrored by improved frequency of innate and adaptive immune cells in BF infants.

ERRα is dispensable for hematopoietic stem cell function in the bone marrow

Hematopoietic stem cells (HSCs) have the ability to self-renew, differentiate into various blood cell types, and reside in the bone marrow (BM) niche. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that has a role in mitochondrial biogenesis and metabolic regulation. Previous research has shown that ERRα contributes to the development of acute myeloid leukemia (AML) by acting as a key regulator of mitochondrial processes, though its role in HSC regulation remains mostly unknown. Flow cytometric analysis determined the population of the hematopoietic stem and progenitor cells (HSPC) using Errα knockout (KO) and conditional KO (cKO) mice. Furthermore, we investigated reconstitution with BM transplantation assay. Here, the ablation of Errα in the BM demonstrated that the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the HSPC populations were similar to those of controls. In addition, the ablation of Errα did not perturb HSC function under the stress of transplantation. Collectively, ERRα is not necessary for the control of HSPC populations, as well as for the maintenance of HSC characteristics and functions within the BM.

Essential oil from Ocimum carnosum induces ROS mediated mitochondrial dysfunction and intrinsic apoptosis in HL-60 cells.

The present study demonstrates that essential oil from Ocimum carnosum (EOC), possesses potent cytotoxic properties against human promyelocytic leukemia HL-60 cells. The results demonstrated a concentration- and time-dependent reduction in cell viability, with an IC50 value of 0.029μl/ml after 24h. Further mechanistic studies revealed that EOC induces apoptosis, a regulated form of cell death in HL-60 cells. This was evidenced by morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, and nuclear condensation. Additionally, flow cytometric analysis demonstrated a significant increase in the sub-G0 cell population, indicative of DNA fragmentation. The mitochondrial pathway of apoptosis appears to be involved in EOC-induced cell death. A loss of mitochondrial membrane potential and the subsequent release of cytochrome c into the cytosol were observed. Pronounced quantity of cytosolic cytochrome c was associated with Bcl-2 depletion. Moreover, cytochrome c, in conjunction with other apoptotic factors, activates caspases, a family of cysteine proteases that execute cell death. These findings collectively indicate that EOC possesses promising anti-cancer properties through the induction of apoptosis via a mitochondrial-dependent pathway. However, further studies are required to elucidate the precise molecular mechanisms underlying EOC's cytotoxic effects and to evaluate its therapeutic potential in vivo.

Therapeutic JAK1 Inhibition Reverses Lupus Nephritis in a Mouse Model and Demonstrates Transcriptional Changes Consistent With Human Disease.

Janus kinase family members are essential for signaling by multiple cytokines, including many implicated in systemic lupus erythematosus (SLE) pathogenesis. To test whether inhibition of JAK1 signaling can be efficacious in SLE, we used a JAK1-selective inhibitor (ABT-317) and evaluated its ability to ameliorate disease in murine SLE. Efficacy of ABT-317 was evaluated using NZB/W-F1 mice treated prophylactically and therapeutically. Primary endpoints were proteinuria, survival, and saliva production. Other endpoints included histological analysis of kidneys and salivary glands, flow cytometric analysis of splenic cell populations, and gene expression analysis by RNA sequencing in the kidneys, salivary glands, and blood. Publicly available human kidney gene transcription data were used to assess the translatability of the mouse findings. ABT-317 was efficacious when dosed prophylactically and prevented disease for up to two months after treatment cessation. When dosed therapeutically, ABT-317 quickly reversed severe proteinuria and restored saliva production, as well as diminished kidney and salivary gland inflammation. ABT-317-induced changes in glomerular morphology coincided with normalization of a human nephrotic gene signature, suggesting translatability to human lupus nephritis (LN). JAK1 inhibition prevented and reversed kidney and salivary gland manifestations of murine lupus with long-lasting effects after treatment cessation. These data, along with the presence of JAK1 and nephrotic gene signatures in human LN glomeruli, suggest that a JAK1-selective inhibitor may be an effective therapeutic in the treatment of human SLE and LN.

NLRP3 Inflammasome Upregulates PD-L1 in Ovarian Cancer and Contributes to an Immunosuppressive Microenvironment.

The NLRP3 inflammasome has been implicated in the initiation of inflammation and tumorigenesis; however, its role in epithelial ovarian cancer (EOC) remains unclear. This study employed high-throughput sequencing data, ELISA, clone formation assay, Western blot, and flow cytometric analysis to investigate the specific role of the NLRP3 inflammasome in EOC. NLRP3 was highly expressed in human EOC tissues and correlated with an unfavorable prognosis. Activation of the NLRP3 inflammasome by LPS and ATP promoted EOC cell proliferation and increased IL-1 and PD-L1 levels. MCC950, a NLRP3 inflammasome blocker, reduced IL-1 and PD-L1 levels and diminished tumor-immune suppressive cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and PD-1+ CD4+ T cells, in a murine model of ovarian cancer. This intervention also suppressed tumor growth. Our investigation revealed the pro-tumorigenic role of the NLRP3 inflammasome and its regulation of PD-L1 expression in EOC. Blockade of the NLRP3 inflammasome led to reduced PD-L1 expression, fewer immunosuppressive cells, and suppressed tumor growth. These findings suggest that targeting the NLRP3 inflammasome-PD-L1 axis could be a novel treatment approach for ovarian cancer.

Comparative Analysis of Eugenol-Loaded Ag-Co and Unloaded Ag-Co Bimetallic Nanoparticles Against Escherichia coli.

Recent developments in nanotechnology have revealed the significance of bimetallic nanoparticles for various applications. This study reveals the facile green synthesis of Ag-Co bimetallic nanoparticles using eugenol. A comparative analysis of Eugenol-loaded Ag-Co and unloaded Ag-Co bimetallic nanoparticle (BNPs) was done to investigate their antibacterial and antioxidant activity using flow cytometry.The biosynthesized Eugenol-loaded and unloaded BNPs were evaluated for antibacterial activity againstEscherichia coli. The antioxidant activity was analyzed by using a DPPH scavenging activity assay and flow cytometry. UV-Vis spectroscopic analysis of synthesized eugenol-loaded and unloaded BNPs showed absorbance at 257nm and 240nm, respectively. FTIR analysis showed the peak range in the 500-4000cm-1 corresponds to different functional groups. Zeta potential confirms the formation of stable and uniformly synthesized BNPs. EDX and SEM analysis confirm the elemental compositions and the size of the BNPs, respectively. Flow cytometric analysis revealed the live-dead parameters ofEscherichia coli, when exposed to different concentrations of Eugenol-loaded Ag-Co (Eu@Ag-Co) and Unloaded Ag-Co BNPs. In addition, comparative MIC and MBC values of eugenol-loaded and unloaded BNPs were obtained forEscherichia coli. The Antioxidant activity revealed the comparative higher significant DPPH scavenging activity of Eu@Ag-Co.Moreover, the flow cytometric analysis confirmed the higher antibacterial efficacy of Eu@Ag-Co over unloaded Ag-Co BNPs by showing ahigher percentage of dead cells.The study determined the enhanced antibacterial and antioxidant activity of nanoparticles by Eugenol loading and advocated it to be a better therapeutic approach. This study encourages the use of biosynthesized loaded-phytochemicals BNPs over unloaded BNPs.

Dynamic Changes in the Intraepithelial Lymphocyte Numbers Following Salmonella Typhimurium Infection in Broiler Chickens.

At day 21 of age, Ross-308 broilers were orally gavaged with 7.5 × 106 CFU/mL S. Typhimurium (n = 30), and another 30 birds were kept as the control. The body weight of birds was recorded on days 0, 2, 7, and 14 days post-infection (dpi) to calculate body weight gains (BWGs). At each time point, seven birds per group were euthanized for sample collection to acquire IELs and lymphocytes from the ileum and spleen for flow cytometric analysis. A reduction in BWGs of the infected groups compared to the control group was observed only at 2 dpi. Additionally, there were no changes in the expression of IFN-γ, IL-1β, and TNF-α in the ileum at 2 and 7 dpi. The number of IELs increased significantly following Salmonella infection in the ileum at 2 and 7 dpi without any changes in spleen lymphocytes. The increase in the total number of IELs was derived from the elevated numbers of conventional CD8αβ+TCRαβ+ and natural IEL populations (CD4-CD8-TCRαβ+, CD8αα+TCRαβ+, TCRγδ+, non-T cells (TCRneg, and iCD8α cells)). The increase in regulatory IELs and the stable expression of proinflammatory cytokine genes during the first week of infection suggests the potential role of IELs in modulating intestinal inflammation.

Fasciola hepatica Soluble Antigen (FhAg)-Induced NETs Under Hypoxic Conditions Exert Cytotoxic Effects on Hepatic Cells In Vitro.

Fasciola hepatica is a parasitic trematode that causes fasciolosis in sheep, provoking a decrease in their reproductive capacity, weight gain, meat and milk production, and wool quality. In the pathogenesis of F. hepatica, the penetration and migration of parasitic stages through the liver provoke intense inflammatory immune responses and tissue damage. The aim of this study was to investigate the cytotoxic effects of Fascila hepatica-induced ovine NETs in exposed hepatocytes in vitro, and to analyze whether F. hepatica antigens (FhAg) trigger the release of ovine NETs under hypoxic conditions as well as the roles of matrix metalloproteinase-9 (MMP-9) and CD11b in this cellular process in vitro. Here, isolated ovine PMNs were co-cultured with FhAg under hypoxia (5% O2) and NETs were visualized via immunofluorescence analyses, confirming their classical characteristics. The quantification of NETs in response to FhAg in hypoxic conditions significantly enhanced the formation of anchored and cell-free NETs (p < 0.01), and NADPH oxidase (NOX) inhibitor diphenylene iodonium (DPI) significantly reduced their production (p < 0.05). Furthermore, the cytotoxic effect of NETs on hepatic cells was determined by using a live/dead-staining with Sytox Orange, thereby demonstrating that FhAg-induced NETs are cytotoxic for hepatic cells (p = 0.001). We additionally analyzed PMN supernatants to determine the enzymatic activity of MMP-9, observing that FhAg exposure enhances MMP-9 release in ovine PMNs (p < 0.05) but not in bovine PMNs. Interestingly, by using flow cytometric analysis, we determined that the exposure of PMNs to FhAg does not increase the CD11b surface expression of ovine PMNs. This could be an effect of the activation of other surface receptors or transcription factors involved in F. hepatica-induced NETosis. Consequently, we hypothesize that F. hepatica-induced NETs play a role in the pathogenesis of fasciolosis, contributing to liver tissue damage if released in an uncontrolled manner.

Taprenepag restores maternal–fetal interface homeostasis for the treatment of neurodevelopmental disorders

Background and purposeNeurodevelopmental disorders (NDDs) are characterized by abnormalities in brain development and neurobehaviors, including autism. The maternal–fetal interface (MFI) is a highly specialized tissue through which maternal factors affect fetal brain development. However, limited research exists on restoring and maintaining MFI homeostasis and its potential impact on NDDs. This study explores the role of placental indoleamine 2,3-dioxygenase (IDO-1) in MFI homeostasis and fetal brain development.Experimental ApproachThe maternal-fetal barrier was disrupted by sodium valproate (VPA) in pregnant mice, whose offspring show typical autism-like behaviors. Ultrastructural analysis and flow cytometric analysis were conducted to observe the morphological and immune system changes. Behavioral tests and immunofluorescence staining was used to investigate the ability and mechanism of taprenepag to alleviate the abnormal behaviors of VPA-exposed offspring and normalize the development of serotonergic neurons.Key resultsIn VPA-exposed pregnant mice, the downregulation of IDO-1 led to maternal immune overactivation and disruption of maternal-fetal barrier, resulting in excessive 5-HT synthesis in the placenta. This process disrupted the development of the serotonergic neuronal system in the offspring, resulting in impaired development of serotonergic neurons, thalamocortical axons, and NDDs in the progeny. However, a single injection of taprenepag at E13.5 ultimately upregulated placental IDO-1 through amplifying the positive feedback loop COX-2/PGE2/PTGER-2/IDO-1 and abolished these alterations.ConclusionTaprenepag improved autism-like behaviors in the offspring of VPA-exposed mice by addressing placental IDO-1 downregulation. This study highlights the potential of targeting IDO-1 to mitigate MFI disruption and NDD development.

5-methyl-N'-(thiophen-2-ylmethylene)-1H-pyrazole-3-carbohydrazide as potent anticancer agent: Synthesis, spectroscopic characterization, anticancer activity and DFT studies

A new pyrazole-hydrazide hydrazone derivative, named 5-methyl-N'-(thiophen-2-ylmethylene)-1H-pyrazole-3-carbohydrazide (TMPC) has been synthesized and characterized by FT-IR, UV–Vis, 1H NMR, 13C NMR, and HRMS-ESI. Its cytotoxic activity was tested on various cancer and healthy cell lines using the XTT method. In addition, the advanced anticancer mechanism was investigated by flow cytometry analysis. Hybrid B3LYP/6–311++G** calculations reveal two stable C1 and C2 conformers of TMPC with energy differences of 28.96 kJ/mol in the gas phase and 8.50 kJ/mol in ethanol solution. Comparisons among the experimental NMR, UV and infrared spectra with the predicted ones support the presence of both forms in solution. The presence of dimer C2 justify the intensities and numerous bands observed in the IR spectrum in the 2000–10 cm−1 region. Probably, the repulsion between the N7 and O1 atoms in C1 explain its lower stability and the higher solvation energy (-108.20 kJ/mol) observed for this form in ethanol than C2 (-88.01 kJ/mol). NBO calculations suggest that both forms are stable in the two media while the AIM analyses evidence the higher stability of C2 than C1 due to the N9-H22···N7 interaction observed only for this conformer in the gas phase. Gap values suggest high reactivities of both forms, as compared with similar pyrazole-carbohydrazide species. Full vibrational assignments are reported for both forms of TMPC together with the main scaled force constants. The XTT results showed that TMPC exhibited a selective cytotoxic effect on the tested cancer cell lines in a dose- and time-dependent manner, and also displayed the most potent cytotoxic activity against breast cancer MCF-7 cells with an IC50 value of 22.79 µM after 48 h Flow cytometric analyses showed that TMPC induced apoptosis and suppressed PI3K/Akt signaling in MCF-7 cells.

Molecular Mechanisms of Cell Death in Leishmania donovani Induced by Selected Steroidal Alkaloids.

We have earlier reported novel anti-leishmanial molecules, veratramine and hupehenine, targeting dephospho-coenzyme A kinase of the parasite. In our current investigation, we assessed the efficacy of these two steroidal alkaloids, veratramine and hupehenine, in combating the parasite. Contrary to expectations, our study did not detect the typical signs of apoptosis such as mitochondrial membrane potential loss and phosphatidylserine externalization. Instead, we observed a notable increase in acidic organelle formation, suggesting a pro-survival response in promastigotes. Through diverse flow cytometric analyses and imaging methods, we conclude that the parasitic death induced by these natural compounds does not follow the apoptosis pathway but likely involves autophagy. This discovery marks the first instance of autophagy-mediated cell death in Leishmania donovani triggered by veratramine and hupehenine.

Heterogeneous Surface CD79b Expression in Aggressive B-Cell Lymphomas Assessed by Flow Cytometry on Lymph Node Biopsies.

Background: CD79b is a B-cell-specific antigen that is crucial to the B-cell receptor and is considered a key target for treatment in aggressive B-cell lymphomas. Methods: While immunohistochemical studies have shown widespread expression of CD79b in mature B-cell-derived lymphomas, flow cytometry allows for precise measurement and differentiation between surface and intracellular localization. Results: In our comparative analysis, we discovered that CD79b expression percentages and mean fluorescence intensity (MFI) were lower in a group of 127 cases of aggressive B-cell lymphomas compared to a control group of benign reactive hyperplasia. We also observed significant variability in the surface expression of CD79b among lymphoma cases, with 18% showing predominantly intracellular positivity. There was a strong correlation between the surface expression of CD79b and clonal light chains. Notably, primary mediastinal B-cell lymphomas exhibited significantly lower surface CD79b expression compared to other lymphoma subtypes (median 0.8% IQR 0-48.5 vs. 80% IQR 24-97, p = 0.0005). Furthermore, patients over 60 years old and those with a higher Revised International Prognostic Index (R-IPI) had significantly higher CD79b expression, both of which are associated with a significant benefit from adding an anti-CD79b drug conjugate to first-line chemotherapy in diffuse large B-cell lymphomas. Conclusions: In conclusion, the quantitative flow cytometric analysis of CD79b surface expression in aggressive B-cell lymphomas provides clinically relevant information, highlighting its potential usefulness in guiding therapeutic decisions.

Human Blood Non-Classical/Classical Monocyte Cells Are Heterogeneously Presented in Severe COVID-19 and Correlate with Disease Activity

Introduction: COVID-19 is highly heterogeneous, ranging from cases with mild disease with an almost asymptomatic carrier to severe cases, in which the disease evolves rapidly. A better understanding of monocyte response during SARS-CoV-2 infection would highlight potential biomarkers and establish other possible approaches for severe cases. Methods: The study group consisted of 32 COVID-19 patients and 18 health controls from June 2023 to March 2024. The COVID-19 patients were further classified as mild and severe illnesses based on World Health Organization (WHO) criteria. For flow cytometric analysis, 50 µL of peripheral blood and 1 µL of specific monoclonal antibodies were added to each cytometric tube for surface marker detection. Results: Here, the promising finding was that the blood non-classical/classical monocyte (NC/CL) subset was skewed toward NChighCLlow and NClowCLhigh clusters among the severe COVID-19 patients. The NChighCLlow cluster in severe COVID-19 displayed a distinct clinical phenotype, implying a higher 7-day disease progression rate (p = 0.019) and a worse 28-day survival (p = 0.026). Moreover, the secretion of IL-1β and IFN-γ was primarily attributed to CL subset in monocytes, while IL-6 was secreted mainly by NC subset. Conclusion: As supported, regarding cytokine profile in context of SARS-CoV-2 infection, it was identified that circulating NC cells are proinflammatory cells most related to regulatory cells, while CL subset displayed an effective capacity to virus. These findings have implications toward optimizing evaluation in severe COVID-19, and developing strategies that target altered balance of NC/CL cell subsets.

Immune cells and the trajectories of depression, anxiety, and cognitive function among people with amyotrophic lateral sclerosis

BackgroundAmyotrophic lateral sclerosis (ALS) represents a complex syndrome characterized by motor, psychiatric, and cognitive symptoms, where associations between cellular immune features and non-motor manifestations remain unknown. MethodsIn this cohort study, we enrolled 250 incident people with ALS (pwALS) assessed with the Hospital Anxiety and Depression Scale, and 226 pwALS with the Montreal Cognitive Assessment, including 218 overlapping pwALS. All individuals were diagnosed between January 2015 and January 2023 in Stockholm, Sweden. We applied joint latent class models to delineate distinct trajectories of anxiety, depression, and cognition, incorporating survival outcomes. A majority of the pwALS had data on leukocyte counts and flow cytometric analyses using a comprehensive T cell panel. We then used immune cell subtypes measured at diagnosis to predict trajectories of these outcomes following ALS diagnosis. ResultsWe identified two distinct trajectories for anxiety, depression, and cognitive function following ALS diagnosis. PwALS with longer survival displayed more stable trajectories, while those with shorter survival showed decreasing anxiety symptom, increasing depressive symptom, and declining cognitive function. Higher count of leukocytes at the time of ALS diagnosis tended to associate with anxiety and depression trajectories related to shorter survival. Among T cell subpopulations, several CD8+ T cell subsets were associated with a stable trajectory of depressive symptom, and, in turn, better survival. ConclusionALS-associated psychiatric and cognitive trajectories vary significantly between pwALS with different prognosis. Certain T cell subsets measured at diagnosis might be indicative of depression trajectories post-diagnosis.

Exploring the anti-inflammatory and antiapoptotic properties of phloroglucinol on pancreatic cells in diabetic models: In silico and in vivo study.

Pancreatic cell damage in diabetes mellitus is closely linked to inflammation and apoptosis. This study aimed to investigate the protective effects of phloroglucinol on pancreatic cells in a streptozotocin-induced diabetic model by assessing its anti- inflammatory and anti-apoptotic mechanisms. Phloroglucinol ligand and the structures of Bax, Bcl-2, and caspase-3 proteins were sourced from the PubChem database. Molecular docking was performed using Autodock Tools and docking results were analyzed with PyRx software. In addition, during the in vivo study, the BALB/c mice were grouped into four categories: healthy control, untreated streptozotocin-induced diabetic, and streptozotocin-induced diabetic treated with two doses of oral phloroglucinol at 100 mg/kg and 200 mg/kg body weight. After 28 days, pancreatic tissues were collected for flow cytometric analysis of NF-κB, IL-6, TNF-α, and apoptotic markers (Bax, Bcl-2, and caspase-3). The docking simulations revealed specific binding interactions: phloroglucinol interacted with Bcl-2 via amino acid residues of ALA90 and TYR139, with Bax via ALA42, LEU45, ALA46, LEU47, PRO130, and ILE133, and with caspase-3 through ARG64, SER120, GLN161, CYS163, and ARG207. The binding affinities for Bax, Bcl-2, and caspase-3 were -5.0, -4.7, and -4.9 kcal/mol, respectively. In vivo, results showed that streptozotocin significantly elevated inflammatory cytokines NF-κB, TNF-α, and IL-6, along with apoptotic markers in pancreatic cells (p<0.05) compared to healthy controls. Phloroglucinol administration at 200 mg/kg significantly reduced TNF-α, NF-κB and IL- 6 levels. Phloroglucinol also prevented streptozotocin-induced pancreatic cell damage through anti-apoptotic effects by downregulating Bax and caspase-3 and upregulating Bcl-2. These findings suggest that phloroglucinol may offer protective benefits in diabetic conditions by modulating apoptotic and inflammatory pathways.

EZH2 promotes B-cell autoimmunity in primary Sjogren's syndrome via METTL3-mediated m6A modification

ObjectiveEnhancer of zeste homologue 2 (EZH2) plays an important role in promoting B-cell activation and differentiation. This study aimed to elucidate the role of EZH2 in the B-cell autoimmune response in primary Sjögren's syndrome (pSS) and to explore the therapeutic potential of inhibiting EZH2 in pSS. MethodsSingle-cell RNA sequencing analysis of B cells in peripheral blood from pSS patients was conducted to identify abnormal expression of EZH2 and METTL3 in B-cell subsets. The levels of EZH2 were further validated across multiple B-cell subsets and the salivary glands (SGs) of pSS patients, as well as three different mouse models of Sjögren's syndrome (SS). Correlation analyses were performed to explore the relationship between the expression of EZH2 and clinical features of pSS patients. Following EZH2 inhibition, SS-like signs and antibody production were assessed in an experimental Sjögren syndrome (ESS) mouse model. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data post-EZH2 inhibition were bioinformatically analyzed to identify the EZH2 targets in pSS. ChIP-qPCR was performed to validate the binding of H3K27me3 to the CDKN1A promoter. Flow cytometric apoptosis analysis and Carboxy Fluorescein Succinimidyl Ester (CFSE) assay were used to assess the impact of an EZH2 inhibitor on B-cell apoptosis and proliferation. Additionally, METTL3 expression and its correlation with disease activity were analyzed in pSS patients. EZH2 expression was examined after METTL3 knockdown. METTL3-RNA immunoprecipitation (RIP) and actinomycin D assays were conducted to confirm the direct binding of METTL3 to EZH2 mRNA and its impact on mRNA stability. M6A-RIP-qPCR was performed to validate the presence of m6A modifications on EZH2 mRNA. ResultsEZH2 was found upregulated in multiple B-cell subsets from the peripheral blood and SGs of pSS patients, as well as in three different animal models of SS. The expression of EZH2 in B cells was positively correlated with the ESSDAI score, which is a measure of disease activity. With treatment of EZH2 inhibitor, SS-like signs alleviated and autoantibody production reduced in ESS mice. Similarly, in pSS patients, METTL3 expression was increased in the SGs and peripheral blood CD19+ B cells, also showing a positively correlated with the ESSDAI score. With knockdown of METTL3, the expression of EZH2 reduced. Mechanistically, EZH2 inhibited B-cell apoptosis and promoted B-cell proliferation by catalyzing H3K27me3 modification at the CDKN1A locus. Furthermore, METTL3 bound to EZH2 mRNA and increased m6A modification on EZH2 mRNA, enhancing its stability and promoting EZH2 expression. ConclusionsThe upregulation of EZH2 mediated by METTL3 is implicated in the B-cell autoimmune response in pSS. Inhibition of EZH2 presents a promising therapeutic strategy for pSS treatment.

Mobilization of endothelial progenitor cells after implantation of CD34 antibody-covered sirolimus-eluting COMBO® stent: assessment with EPC colony-forming assay.

The COMBO® stent is a unique stent on which the CD34 antibody is mounted to capture CD34 + endothelial progenitor cells (EPCs) and from which sirolimus is eluted to suppress neointimal hyperplasia. The COMBO® stent aims to induce early re-endothelialization and vascular healing and to prevent restenosis. In the clinical setting, however, the effects of the COMBO® stent have not been validated in terms of EPC biology. In this study, we assessed the kinetics of circulating EPCs, not only quantitatively by flow cytometric analysis but also qualitatively by an EPC colony-forming assay, in 25 patients undergoing COMBO® stent implantation. Among all patients, flow cytometric analysis indicated that the number of circulating CD34 + /KDR + EPCs did not change after COMBO® stent implantation compared with baseline (before stent implantation). The EPC colony-forming assay demonstrated that the number of small-type EPC colonies increased on day 2 (3 [2, 9] to 6 [4, 9]/dish, P = 0.026) and that of large-type EPC colonies more prominently increased on day 2 (1 [0, 4] to 5 [1, 10]/dish, P < 0.001) and day 7 (to 2 [1, 7], P = 0.006) after COMBO® stent implantation. Based on the results of optical coherence tomography at 3months after stent implantation, the patients were divided into two groups: well (uncovered strut ratio < 10%, n = 14) and poor (uncovered strut ratio ≥ 10%, n = 10) stent coverage. Compared with baseline values, the number of large-type EPC colonies increased on day 2 (2.9 ± 0.8 to 7.3 ± 2.0, P = 0.026) and tended to increase on day 7 (6.8 ± 2.0/dish, P = 0.062) after COMBO® stent implantation in the well coverage group, while it did not change in the poor coverage group. Thus, the COMBO® stent might induce mature EPCs in the circulation, which might be associated with subsequent healing processes in vessel sites with stent-induced injury.

Multiparameter Flow Cytometric Analysis of the Conventional and Monocyte-Derived DC Compartment in the Murine Spleen.

Dendritic cells (DCs) are present in almost all tissues, where they act as sentinels involved in innate recognition and the initiation of adaptive immune responses. The DC family consists of several cell lineages that are heterogenous in their development, phenotype, and function. Within these DC lineages, further subdivisions exist, resulting in smaller, less characterized subpopulations, each with its unique immunomodulatory capabilities. Given the interest in utilizing DC for experimental studies and for vaccination purposes, it becomes increasingly crucial to thoroughly classify and characterize these diverse DC subpopulations. This understanding is vital for comprehending their relative contribution to the initiation, regulation, and propagation of immune responses. To facilitate such investigation, we here provide an easy and ready-to-use multicolor flow cytometry staining panel for the analysis of conventional DC, plasmacytoid DC, and monocyte-derived DC populations isolated from mouse spleens. This adaptable panel can be easily customized for the analysis of other tissue-specific DC populations, providing a valuable tool for DC research.

Establishment of a novel clonal GFP-expressing transgenic ginbuna crucian carp

The clonal triploid ginbuna crucian carp Carassius auratus langsdorfii, a naturally occurring gynogenetic fish, is suitable for cell transplantation studies to reveal the roles of stem cells and immune cells. To ensure long-term traceability of donor cells within recipient fish, we have established a transgenic ginbuna line that expresses green fluorescent protein (GFP). The Xenopus laevis ef1a promoter was introduced for regulating GFP expression. Tol2 transposon-based transgenesis to ginbuna embryos resulted in producing a putative founder fish (F0) in a mosaic fluorescent fashion; the frequency of germline transmission was 14.9%. All embryos of GFP-positive offspring (F1)-derived F2 generation expressed GFP widely across the body. The result of Southern blot analysis showed that the transgene was present on a single DNA fragment of equivalent size among F1 and F2 individuals tested, indicating that the transgene was stably transmitted without translocation. Analysis of the fluorescence intensity of organs obtained from F1 and F2 juveniles using fluorescence microscope showed that eyes, brain, skeletal muscle, heart and gonad exhibited a strong GFP fluorescence while gill, spleen and intestine gave a weak signal; no fluorescence was observed in erythrocytes. Flow cytometric analyses of peripheral leukocytes from F1 and F2 adult fish revealed all cell populations expressed GFP. Scale grafts from the transgenic fish to the wild-type fish exhibited persistent engraftment. Together, our transgenic line can be a powerful tool for studying cellular dynamics by cell transplantation and provide a solid basis for further immunological research advances in teleost.

Panel-reactive antibodies in kidney transplantation for patients with end-stage renal failure

BACKGROUND: Kidney transplantation is a recognized method of treating patients with end–stage kidney disease. However, the risk of graft loss is not excluded. In Azerbaijan, kidney transplantation is mainly performed from living donors; nevertheless, there is little data on the panel-reactive antibodies (PRA) and its use in the transplantation program. AIM: To evaluate the prevalence of positive panel-reactive antibodies (PRA) and short-term outcomes of living donor kidney transplantation. MATERIALS AND METHODS: Within 6 months after kidney transplantation, 113 recipients were followed up. All patients underwent their first transplantation. Complement-dependent cytotoxicity cross-matching, HLA typing, and flow cytometry cross-matching were carried out. Flow cytometric analysis was applied to detect PRA. The positive response percentage is presented by the percentage of events shifted to the right of the cutoff point. Data were analyzed using SPSS 16.0 statistical software. RESULTS: Among the examined patients, men predominated. In PRA-positive patients (n = 20), the dialysis duration was on average 22.3% longer than in PRA-negative patients (n = 93). 35.0% of PRA-positive patients had class I PRA, 30.0% of patients had class II PRA, and 35.0% had class I + II PRA. In 40.0% of cases, positive PRA had high sensitization 50% (predominantly class II), and the remaining 60.0% had PRA from 10% to 50%. Sensitizing factors were more common in the PRA-positive group (p 0.05). After 6 months of folow-up, the mean creatinine level was 183.4 ± 68.84 nmol/l in the PRA-positive group and 198.15 ± 108.23 nmol/l in the PRA-negative group (p = 0.91). The overall emptying rate was 4.4% (n = 5), with all 5 cases being in the PRA-negative group. No deaths were observed during the follow-up period. CONCLUSIONS: PRA positive patients usually have some sensitizing event such as pregnancy or blood transfusion. The transfusion itself was not a significant factor of sensitization in this study. Patients with high PRA levels require more thorough examination.