Ficoll Gradient Research Articles

TNF-alpha gene promoter's hypomethylation mediates a pro-inflammatory phenotype in peripheral blood monocytes from apical periodontitis individuals.

Epigenetic regulation of the key inflammatory genes plays a crucial role in controlling monocyte/macrophage-mediated local and systemic responses to bacterial challenges. However, it has not been addressed in apical periodontitis (AP). We aimed to explore the methylation pattern of the TNF-α gene promoter and its association with the inflammatory phenotype of peripheral blood monocytes from individuals with AP and controls. A cross-sectional study was conducted, including otherwise healthy individuals with AP (n = 25) and controls (n = 29). Monocytes were isolated from the volunteer's blood samples using a Ficoll gradient followed by negative immunoselection. RNA and DNA were extracted. The DNA methylation profiles of the TNF-α gene promoter region were analyzed using bisulfite sequencing PCR. The mRNA expression levels of DNA methyltransferases 3a (DNMT3a) and Ten Eleven Translocation enzymes 1(TET1) were assessed by qPCR. A fraction of primary monocytes was also cultured for 24 h, and the supernatant was collected to measure cytokine levels through a Luminex assay. Generalized structural equation models (GSEM) evaluated the association between AP, DNA methylation, and TNF-α protein expression controlled for potential covariates. Models included the effect of the methylation of TNF-α gene promoter as a mediator of the association between AP and TNF-α protein expression levels. Monocytes from AP individuals exhibited a heightened secretion of TNF-α and IL-1β and hypomethylation of the TNF gene promoter (p < .05). AP diagnosis was associated with the TNF-α gene promoter´s hypomethylated profile and enhanced pro-inflammatory cytokine levels, while lower methylation of the gene promoter region and -163 CpG single site mediated TNF-α overexpression (p < .05). DNA hypomethylation at the TNF-α gene mediates a proinflammatory phenotype in monocytes from AP patients, supporting a role in the systemic response.

Features of proinflammatory activation of macrophages in patients with rheumatoid arthritis and systemic lupus erythematosus

Autoimmune rheumatic diseases (ARDs) are chronic pathological conditions that arise from an abnormal immune response and are accompanied by systemic inflammation. The most common ARDs include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). The exact pathogenesis of ARDs remains unclear, but the complex influence of genetic, immunological and external environmental factors leads to the occurrence and further progression of ARDs. It has been shown that the cause of chronic inflammation may be proinflammatory activation of macrophages, in which an increase in the secretion of cytokines is observed. The aim of this study was to evaluate the inflammatory response of macrophages in patients with RA, SLE and SSc. Materials and methods. The study included 143 participants: 47 patients with RA, 45 patients with SLE, 34 patients with SSc, and 17 people without ARDs and other chronic diseases. Isolation of a primary culture of monocytes was carried out by centrifugation in a ficoll gradient using magnetic separation from the whole blood of study participants. Lipopolysaccharide (LPS) was added to stimulate cells along the proinflammatory pathway. Cell cultivation was carried out for 24 hours. Determination of basal and LPS-stimulated secretion of IL-8 by macrophages was carried out in the culture fluid using an enzyme-linked immunosorbent assay (ELISA). Proinflammatory activation of macrophages was calculated as the ratio of LPS-stimulated and basal IL-8 secretion. Research results. Basal secretion of IL-8 by macrophages was statistically significantly higher in the groups of patients with RA and SSc compared with the SLE and control groups. LPS-stimulated secretion of IL-8 by macrophages in the SSc group had statistically higher values compared to the RA and SLE groups. Proinflammatory activation of macrophages was reduced in the group of patients with RA compared to patients with SLE and the control group, and was also statistically significantly lower in patients with SSc compared to the control group.

Abstract B023: Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection

Abstract Pancreatic cancer is associated with late-stage diagnosis and poor patient prognosis. The disease is characterised by chronic dysregulated immunity and inflammation. Moreover, chronic pancreatitis is linked with an increased risk of pancreatic cancer. Tissue remodelling during disease progression involves a complex network of cells and a crosstalk between cancer cells, stromal components and immune cells, but it remains poorly explored. Thus, to improve the outcome of patients, there is a need for better understanding of biological changes associated with the initiation and progression of pancreatic cancer at the molecular and spatial levels. Using samples obtained from our Accelerated Diagnosis of neuroEndocrine and Pancreatic TumourS (ADEPTS) biobank, we have evaluated blood samples for the discovery of non- invasive biomarkers to discriminate patients with pancreatic cancer from symptomatic patients without cancer. In this pilot study, the transcriptome of peripheral blood mononuclear cells (PBMCs), isolated using a ficoll gradient was explored to identify PDAC-associated mRNA signatures. Moreover, the Olink® Explore Inflammation and Oncology panels were performed in a cohort of 40 patients with early stage (I-II) PDAC, 40 chronic pancreatitis samples and 40 control patients with benign symptomatic upper GI diseases. To validate and enrich these results, a panel of inflammatory and immune markers including immunoglobulins, cytokines and chemokines was also run using multiplex techniques. Symptoms observed in our cohort were also analysed together with clinical (e.g diabetic status, biochemistry profile) and demographic data (age, sex, ethnicity, post code etc). We have identified mRNA transcriptome signatures and differentially expressed proteins associated with inflammatory and immunological processes in PDAC compared to benign symptomatic samples. Our results offer potential diagnostic utility for early-stage disease. Additionally, our unique cohort of patient samples comprising of those with confirmed PDAC and those presenting to clinic with benign disease and similar non-specific symptoms, allows for the development of non-invasive diagnostic tests with better translational applicability. Future work will involve the validation of these findings in a larger patient cohort and the use of NanoString GeoMx® Digital Spatial Profiler to perform whole transcriptome and proteome profiling in human chronic pancreatic and pancreatic cancer tissue samples. Citation Format: Kyra Fraser, Maria Rosado, Stephen P Pereira, Pilar Acedo. Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B023.

Phase I Study of Intravitreal Injection of Autologous CD34+ Stem Cells from Bone Marrow in Eyes with Vision Loss from Retinitis Pigmentosa

PurposeTo evaluate the feasibility and safety of intravitreal injection of autologous CD34+ stem cells from bone marrow (BMSCs) in eyes with vision loss from retinitis pigmentosa (RP). DesignPhase I prospective open-labeled single center study. ParticipantsSeven eyes (7 patients) with RP with best corrected visual acuity (BCVA) of 20/100 to 20/400 or visual field constriction to within 10 degrees. MethodsA comprehensive exam with ETDRS BCVA, macular optical coherence tomography, perimetry, fluorescein angiography was performed at baseline, 1 to 3 months, and 6 months after study treatment. Bone marrow aspiration, isolation of CD34+ BMSCs under Good Manufacturing Practice (GMP) conditions, and intravitreal cell injection were performed on the same day. The CD34+ cells were isolated from bone marrow using a Ficoll gradient and the Miltenyi CliniMACs system. Isolated CD34+ cells were released for clinical use if viability, sterility and purity met the release criteria accepted by the Food and Drug Administration for this clinical study. Main Outcome MeasuresNumber of CD34+ cells isolated for injection and adverse events associated with study treatment during follow-up. Secondary outcome measures are changes in BCVA and perimetry. ResultsAll isolated CD34+ cells passed the release criteria. A mean of 3.26 + 0.66 million viable CD34+ cells (range 1.6 to 7.05 million) were injected intravitreally per eye. No adverse event was noted during the study follow-up except for one participant who was noted with transient cells in the anterior chamber with mild elevation in intraocular pressure at 18 hours after study injection which normalized by 24 hours. BCVA remained within 2 lines of baseline or improved in all participants at 6 months follow-up. Perimetry was stable or improved in all eyes during study follow-up except one eye with transient improvement at 1 month and worsening of both eyes at 6 months. ConclusionIntravitreal injection of autologous CD34+ BMSCs is feasible and appears to be well tolerated in eyes with vision loss from RP. A larger randomized prospective study would be needed to evaluate further the safety and potential efficacy of this cell therapy for vision loss associated with RP.

#1591 Analysis of microvesicles and their relationship with oxidative stress as biomarkers of cardiovascular damage in chronic kidney disease

Abstract Background and Aims Chronic kidney disease (CKD) is associated with a higher incidence of cardiovascular diseases. These diseases are preceded by endothelial dysfunction and alterations in coagulation systems. One of the challenges of research in this field is the search for diagnostic and predictive biomarkers of cardiovascular pathology. Our hypothesis is that endothelial dysfunction and cardiovascular risk could be evidenced by the increased release of endothelial microvesicles (EMVs) and platelet microvesicles (PMVs), as well as a greater expression of tissue factor (TF). We also propose a relationship between the release of microvesicles and the oxidative stress observed in these patients. The aim of the study was to analyze the levels of MVEs and MVPs, the expression of FT and its relationship with pro-oxidant and antioxidant parameters as possible early biomarkers of cardiovascular and thrombotic risk in different stages and treatments of CKD. Method It is a cross-sectional study consisted of 116 patients (40 with advanced chronic disease (CKD), 40 undergoing hemodialysis (HD) and 36 undergoing peritoneal dialysis (PD)), as well as 17 healthy subjects (CT). Microvesicle phenotyping was performed by direct immunofluorescence and flow cytometry in plasma isolated by centrifugation from peripheral blood. The analysis of oxidative stress parameters was carried out by spectrophotometry, after isolation of plasma and mononuclear and polymorphonuclear leukocytes by means of a ficoll gradient. Results Patients on PD have higher plasma PMVs levels (p = 0.0005 vs ACKD and p = 0,0002 vs HD), while those on HD and PD have higher levels of EMVs compared to ACKD (p = 0,0001 and p = 0,0125, respectively). FT expression in PMVs is lower in HD (p &amp;lt; 0.0001 vs ACKD) and FT expression in EMVs is lower in HD and PD compared to ACKD (p = 0,0004 and p= 0.0033, respectively) (Fig. 1). Patients with CKD have a higher activity of pro-oxidant enzymes (XO, GPx), as well as high levels of lipid peroxidation by-products (TBARS-MDA) and lower activity of antioxidant enzymes (GSH, CAT, SOD) at the plasma and cellular level. A positive relationship has been observed between pro-oxidant parameters and the release of MVP and MVE, as well as the expression of FT (Table 1). Patients with cardiovascular events during the years of follow-up have higher levels of FT expressed in microvesicles, and long-term survival decreases in patients with elevated FT levels. Conclusion Patients with CKD have higher plasma levels of PMVs and EMVs and higher FT expression, related to a state of oxidative stress. These parameters could be considered as biomarkers for predicting, diagnosing, and prognostic cardiovascular events in CKD.

DOP45 Blood-derived neutrophils give rise to differentially activated populations in the mucosa of active Inflammatory Bowel Disease patients

Abstract Background Infiltration of neutrophils is the main histological feature in active inflammatory bowel disease (IBD). Recent data suggests that tissue- and tumour-infiltrating neutrophils can adopt remarkably different phenotypes with potentially different functions. However, the heterogeneity of this population in IBD remains unknown. Methods Single-cell RNA sequencing (scRNA-seq) data was generated from colonic biopsies of 92 individuals including 6 healthy controls (HC), 36 inactive IBD, 19 active Crohn’s disease (CD) and 31 active ulcerative colitis (UC) patients. Neutrophils (6038 cells) found in the inflamed colon of CD and UC patients were analysed using a curated pipeline in Seurat (v4.0.2)1. Posterior analysis included the FindAllMarkers function (differentially expressed genes), Jaccard index similarity and the AddModuleScore function (comparisons with literature-described neutrophils), Monocle3 algorithm (developmental trajectory) and clusterProfiler (v3.0.4; enrichment and pathway analysis). For in vitro stimulation, neutrophils were isolated from the blood of HC donors (n=4) using a double Ficoll gradient 1. Cells and supernatants were analysed for qPCR and ELISA determinations. Results We identified 6 different clusters/states of colonic neutrophils (Figure 1A). In agreement with our previous results1 we found a population of S100A12/S100A8/S100A9 neutrophils (N0), IFN-response neutrophils (N3) and CCL3/CCL4 neutrophils (N2), in addition to N1.1 and N1.2 neutrophils. N3 neutrophils were enriched in the inflamed colon of CD patients compared to UC (17.2% vs 5.8%, p-value= 0.0021; Figure 1C). Comparative analysis with published datasets revealed that N0, N1.2 and N3 resemble populations of neutrophils found in blood2,3. In contrast, N1.1 and N2-like clusters showed some similarity to umbilical cord blood3 and tumour-infiltrating neutrophils4. Trajectory analysis, originating from N0 (peripheral-like) neutrophils, showed two potential branches of differentiation, with N1.2 leading towards N3 (IFN-response) and N1.1 differentiating into N2 (CCL3) neutrophils (Figure 1B). Finally, pathway analysis showed that N2 neutrophils presented enriched gene expression of the TNF signalling pathway, and together with the N1.1 state, an enrichment of the IL1β signalling pathway as well. In vitro experiments revealed that TNF, IL1β and LPS can all induce expression of CCL3 and CCL4, top differentially expressed genes of N2 neutrophils. Conclusion We show that active IBD is associated with a highly heterogeneous population of infiltrating neutrophils. Some of them resemble neutrophils in peripheral blood, while others most likely differentiate in response to inflammatory cues present in the colon of active IBD patients.

Immune Cell Alterations and PI3K-PKB Pathway Suppression in Patients with Allergic Rhinitis Undergoing Sublingual Immunotherapy.

Our prior clinical study assessed the efficacy and safety of sublingual immunotherapy (SLIT) with standardized Dermatophagoides farina drops on patients with allergic rhinitis (AR) while analyzing the characteristics of adverse reactions. This study was conducted to evaluate the immune cell composition alterations in AR patients before and after SLIT, and to comprehensively investigate the role and changes of antigen-specific immune cells associated with treatment efficacy. A total of 68 AR patients who completed 12months of SLIT were included in the study. Before the trial's initiation and after 1year of SLIT, 10ml of venous blood was collected. Peripheral blood mononuclear cells were isolated using the Ficoll gradient method. The mRNA transcriptome was analyzed using an Affymetrix microarray. The proportions of 22 immune cell types were calculated via the CIBERSORTx platform. Correlations between each immune cell type and SLIT were analyzed. PI3K-PKB pathway dysregulation were analyzed using quantitative PCR and Western blot. Flow cytometry was utilized to assess the percentages of Th1 and Th2 cells. Mono-sensitized AR patients exhibited marked increases in plasma cells, activated memory T cells, regulatory T cells, and activated dendritic cells, while experiencing decreased neutrophils and resting dendritic cells. In poly-sensitized AR patients, the most notable change was an increase in regulatory T cells, coupled with decreased T follicular helper cells, resting dendritic cells, and activated mast cells. These findings indicated that SLIT reshaped immune cell profiles in AR patients, and, notably, the specific changes differed between mono-sensitized and poly-sensitized individuals. Furthermore, SLIT appeared to shift the immune response towards a Th2 decrease profile in both groups. Importantly, suppression of the PI3K-PKB pathway was evidenced as inhibition of PKB phosphorylation and the decrease of glycogen synthase kinase 3 β (GSKβ) and mammalian target of rapamycin (mTOR) expression after SLIT. Our study has demonstrated that SLIT treatment led to distinct changes in immune cell profiles between mono-sensitized and poly-sensitized AR patients. Furthermore, SLIT appeared to reduce a Th2 immune response, highlighting its efficacy in AR treatment. Importantly, the study revealed the suppression of the PI3K-PKB pathway, shedding light on the immunological mechanisms underlying SLIT's effectiveness.

Abnormal Regulation of Mitochondrial Sphingolipids during Aging and Alzheimer's Disease.

During pathogenesis of Alzheimer's disease (AD), mitochondria suffer alterations that lead to low energy production and reactive oxygen species formation. However, the mechanism of impaired mitochondria homeostasis in AD is not fully understood. We hypothesized that abnormal sphingolipid metabolism in mitochondria could be one of the contributing factors to mitochondrial dysfunction. Synaptic and non-synaptic mitochondria were isolated from 5xFAD and wild type (WT) mice at 3 and 7 months using Ficoll gradient ultracentrifugation, and their function was analyzed using Seahorse assay. Additionally, mitochondria were analyzed using mass spectrometry for proteomics and sphingolipidomics analyses. Sphingolipid levels were also determined in synaptic and non-synaptic mitochondria isolated from AD patients and healthy controls. We found that synaptic mitochondria isolated from 3-months old 5xFAD mice manifest diminished oxygen consumption as compared to WT. Consistently, proteomics analysis showed that proteins related to respiratory electron transport and oxidative phosphorylation were altered in 5xFAD mice. When quantifying the main sphingolipids in mitochondria, we found that Cer 18:0, Cer 22:0, and Cer 24:1 were increased already at 3 months in 5xFAD mice. No increase in ceramides was detected in mitochondria isolated from AD patients. However, increased levels of sphingosine were found in both 5xFAD mice and AD patients when compared to respective controls. We report that the regulation of sphingolipids in mitochondria is abnormal at 3 months of age in 5xFAD mice, as indicated by the accumulation of long-chain ceramides, which increases with age. Sphingosine levels are increased in both the mitochondria of 5xFAD mice and AD patients. Our data suggest that the sphingolipid composition is dysregulated in mitochondria early during AD pathogenesis.

Extracellular DNA level as an indicator of the activity of inflammatory reactions in patients with rheumatoid arthritis and asthma

BACKGROUND: We performed a clinical and laboratory examination of patients with rheumatoid arthritis and bronchial asthma. Nosologies were selected to examine extracellular DNA changes in the blood during immunopathological processes based on generally accepted views on the role of helper balance in disease pathogenesis. The degree of disease activity in rheumatoid arthritis primarily depends on the severity of inflammatory changes in the joints; therefore, it is closely associated with a shift in the balance of helpers toward the Th1. According to today’s concepts, the pathogenesis of asthma is determined by the intensive production of specific antibodies, which are controlled by activated Th2 lymphocytes. AIM: To study extracellular DNA changes in the blood of patients during immunopathological processes depending on the Th1/Th2 balance ratio and compare these data with the parameters of standard laboratory tests and indicators of neutrophil activities and their ability to netosis. METHODS: Extracellular DNA concentration was determined using the Quant-ITTM PicoGreen fluorescent reagent for double-stranded DNA. Neutrophils isolated in a double-density gradient of ficoll–urografin were cultured with or without the addition of phorbol myristate acetate followed by Sytox Green. RESULTS: The studied immunopathological conditions had significantly decreased extracellular DNA levels in the plasma of patients with asthma, which sharply contrasted with its increase in patients with arthritis. Laboratory parameters confirmed the presence of local inflammation in patients with asthma without obvious symptoms of a systemic inflammatory response, while in patients with arthritis, inflammatory changes in the joints were accompanied by an increased C-reactive protein level, indicating a systemic reaction of the body in response to inflammatory stimuli. CONCLUSIONS: Thus, the state of the Th1/Th2 balance is assumed as one of the significant regulators that determined the extracellular DNA concentration in the blood. According to the proposed hypothesis, the shift in this ratio toward the predominance of Th1 cells should promote the development of inflammatory processes in the body and increase the amount of extracellular DNA, while a shift in the balance of helpers toward the dominance of Th2 lymphocytes should suppress these processes and consequently decrease the extracellular DNA in the blood.

Increased Proliferative Drive of Lymphocytes in Sickle Cell Disease

Introduction Patients with sickle cell disease (SCD) have higher levels of inflammation which plays a prominent role in disease pathogenesis. It is well known that individuals with SCD have a higher erythropoietic drive given the ~20 day life span of their red blood cells. We asked whether this increased proliferative drive also occurred in white blood cells (WBC). To characterize WBC turnover we focused on the longest-lived CD4+ and CD8+ T cells. Methods Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient from red cell waste products from consented individuals with sickle cell disease and from leukapheresis products of healthy donors. In ten healthy donors and ten donors with SCD, CD4 and CD8 T cells were purified using EasySep Cell Isolation, labeled with eFluor 670 proliferation dye, and cultured in RPMI with 30% FBS for seven days. Samples were collected daily and stained with naïve and memory T cell markers. CountBright beads were added to obtain absolute cell counts and eFluor 780 live/dead dye was used to capture daily snapshots of cell death. Flow cytometric analysis was performed to track absolute cell numbers, cell division and indirectly monitor cell death. B cells, NK, macrophages, and dendritic cells were similarly tracked. To further probe T cell turnover, T cell receptor (TCR) sequencing was performed on twelve separate aliquots from purified CD4+ T cells at two different timepoints in healthy donors and individuals with SCD. Turnover of clonal sequences was analyzed using the Morisita index. Results CD4+ and CD8+ lymphocytes from individuals with SCD experienced more death in culture by day 7 compared to healthy donor controls. In addition, T lymphocytes from donors with SCD were observed to undergo 15-fold more proliferation compared to healthy donors (p=0.0002, unpaired t-test). Immunophenotyping performed on the CD4 and CD8 T lymphocytes showed a propensity for memory cells to undergo proliferation. We also found individuals with SCD had higher turnover in TCR sequences in two timepoints compared to healthy donors (p=0.05, Chi-square). Notably, we also saw increased proliferation of B cells, NK cells, macrophages, and dendritic cells in individuals with SCD. Conclusion Our data suggest that T lymphocytes and other PBMCs undergo faster turnover in SCD than in healthy donors, suggesting a stronger hematopoietic drive. Our findings may have important implications for gene therapy since an increased proliferative drive may contribute to an increased risk of developing leukemia in SCD.

Recombinant Human Thrombopoietin Reduces the Risk of Acute Graft-Versus-Host-Disease and Its Mechanisms

Objective: Acute graft-versus-host-disease (aGVHD) is an important complication that is observed after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aimed to evaluate the effects of recombinant human thrombopoietin (TPO; rhTPO) on aGVHD using clinical data, mice models, and peripheral blood mononuclear cells (PBMCs). Methods: From 2016 to 2018, 162 cases of allo-HSCT in our hospital were recruited in this study. The correlation between TPO use and aGVHD was analyzed. After a GVHD model was constructed in Balb-C mice, HE staining was performed to compare the pathophysiological changes of the liver, intestine, lung, kidney, and other organs of the mice. The levels of lymphocyte subsets and cytokines in the mice spleens in the different groups were detected. PBMCs were isolated from the peripheral blood of healthy persons and patients with allo-HSCT via Ficoll gradient centrifugation and treated with different concentrations of rhTPO. The proliferation of PBMCs was detected via the CCK8 method, and the differentiation of PBMCs into lymphocytes was analyzed via flow cytometry. Results: 1) The occurrence of aGVHD was observed in 10 patients (47.6%) in the control group and 27 (23.1%) in the treatment group. The difference between the two groups was statistically significant (χ2=5.465, P=0.019). 2) The results of the aGVHD mice proved that rhTPO decreased the damage to the mouse liver, intestine, lung, and kidney tissues. In addition, splenic cell subpopulations CD4+cells, CD8+cells, T cells, and NK cells increased in the GVHD group but decreased due to rhTPO (P&amp;lt;0.05). After rhTPO treament, T cell subpopulations Th2 and Th17 cells decreased in the GVHD mice (P&amp;lt;0.05). Cytokines CD8+IL-17A+ and CD8+IL-4+IFNγ showed no difference between the four groups (P&amp;gt;0.05). Compared to GVHD mice, CD8+IL-4+ decreased in TPO-treated GVHD mice, and the effect of TPO at a high dose was more significant (P&amp;lt;0.05). 3) The results of the PMBCs suggested that a low dose of rhTPO could promote the proliferation of PMBCs, while a high dose could inhibit the proliferation. rhTPO promoted the differentiation of PBMCs into the CD3-PC5 subset. Conclusions: Altogether, this study indicates that rhTPO reduces the risk of aGVHD and is a protective factor against the occurrence of aGVHD.

Comparison of Myeloid-Derived Suppressor Cells (MDSC) in children with RSV vs. COVID-19

Abstract Introduction Lower respiratory tract infections (LRTI) caused by respiratory syncytial virus (RSV) result in over 100,000 deaths of infants globally. Though adults account for most COVID-19 hospitalizations, children also develop severe disease due to SARS-CoV-2. Children with severe illness due to LRTI demonstrate evidence of impaired adaptive immune function. MDSC are an immature, heterogenous cell population that expands significantly during inflammatory conditions. MDSC suppress T cell proliferation and function resulting in immunosuppression. MDSC subsets (granulocytic (G) MDSC and the monocytic (M-)MDSC) suppress T cells via distinct mechanisms. In a previous study, we observed a significant increase in G-MDSC in children with septic shock. We hypothesize that children with severe LRTI with RSV or COVID-19 will have expansion of MDSC that will be associated with differences in T cell subset composition and worse clinical outcomes. Methods Prospective, observational study in children admitted to the pediatric intensive care unit (PICU) with symptomatic RSV or COVID-19, defined by a positive polymerase chain reaction test. Children with multi-system inflammatory syndrome or positive for both RSV and COVID-19 were excluded. Pre-pandemic healthy control samples were obtained from children requiring IV placement for outpatient MRI. Peripheral blood mononuclear cells were isolated by ficoll gradient and stained for surface markers to identify MDSC (CD45+, HLA-DR-, CD33+ and CD11b+), including G- (CD15+, CD14-) and M-MDSC (CD14+, CD15-), and T cell populations. Clinical and demographic data were collected from the electronic medical record and stored in a secure database. Results We enrolled 30 children, 20 with RSV (50% male, median age 2 [1.2-5] months), 10 with COVID-19 (30 % male, median age 25 [ 3.8 – 215] months), and 30 healthy controls (64% male, median age 73.2 [57.6, 98.4] months. Children with severe LRTI due to RSV or COVID-19 have significantly increased percentage MDSC of PBMC compared to healthy controls (13% [5.6,22] vs 9.9% [5.2, 23] vs 0.95 [0.34, 2.8] respectively). Compared to children with COVID-19, children with RSV had higher percentage M-MDSC of total MDSC (90% [87,97] vs 45% (8.4, 71], p&amp;lt; 0.0001) and lower G-MDSC (1.1% [0.44, 3.0] vs 38% [17,90], p&amp;lt;0.0001). Children with RSV had a larger percentage of T regulatory cells of total CD4+ T cells (8.0% [6.1, 9.0] vs 4.3% [3.3, 5.8], p=0.01) and were more likely require non-invasive or invasive ventilatory respiratory support than those with COVID-19 (75% vs 30%, p=0.04). Conclusions Total MDSC are significantly expanded in children with severe LRTI due to RSV and COVID-19 compared to healthy controls, but with significantly different subset distributions. Children with RSV demonstrate a predominance of M-MDSC, while children with COVID-19 demonstrate significantly higher G-MDSC. Children with RSV were also more likely to have expanded Treg cell population and require higher level respiratory support. These findings suggest that MDSC composition and suppressive mechanisms may be partially driven by viral pathogen. Larger studies are needed to confirm these findings and to investigate the role of MDSC-mediated immune suppression in children with severe LRTI to identify this cell population as a potential therapeutic target.

Mitochondrial bioenergetics and cytometric characterization of a synaptosomal preparation from mouse brain cortex

Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described.Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools.Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively.Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition.As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.

From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid.

Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro. Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination.

Abstract 2995: Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma

Abstract Background: Immunotherapy is the standard of care for many solid tumor malignancies. However, due to a paucity of known tumor antigens in non-small cell lung cancer (NSCLC) and osteosarcoma (OS), identification of novel immunogenic antigens is needed. Our group has experience with neutrophil serine proteases (NSP) derived from the azurophilic granules of polymorphonuclear leukocytes (PMN), specifically, cathepsin (CG), proteinase 3 (P3), and neutrophil elastase (NE). We have identified two HLA-A2 (HLA-A*0201)-restricted nonameric peptides: CG1 (FLLPTGAEA), derived from CG, and PR1 (VLQELNVTV), derived from NE and P3. We have previously shown that CG1 and PR1 are effective targets in hematological malignancies and that PR1 can be a target for some solid tumor malignancies. Here, we extend our findings to demonstrate that CG1 and PR1 can be immunotherapeutic targets for NSCLC and OS. Methods: Healthy donor PMN were isolated from buffy coats using double Ficoll gradient centrifugation. To evaluate uptake, malignant cells were pulsed with PMN lysates, harvested, permeabilized, and stained with anti-CG, anti-NE or anti-P3. To evaluate cross-presentation, cells were cultured with PMN lysates, harvested and stained with CG1 TCR-like antibody, or PR1 TCR-like antibody (8F4). Flow cytometry was performed using the Fortessa X-20 Cell Analyzer. CG1- or PR1-specific cytotoxic T-lymphocyte (CTL) were expanded from healthy donor peripheral blood mononuclear cells. Standard cytotoxicity assays were used to evaluate the lysis of target cells co-cultured with CG1- or PR1-specific CTL. Results: We have previously demonstrated that NE and P3 are taken up and cross-presented by NSCLC and become targets for PR1-CTL, thus in this study, we investigate if NSCLC cells take up CG, and whether OS cells take up NE and P3, resulting in enhanced cytotoxicity by peptide-specific CTLs. We first employed The Cancer Cell Line Encyclopedia to show the absence of NSP’s in NSCLC and OS cell lines. RT-PCR confirmed lack of endogenous CG mRNA expression in NSCLC and OS, and lack of NE and P3 in OS cell lines. Next, we analyzed antigen cross-presentation and showed that NSCLC cells take up and cross-present CG, leading a 13.3-fold increase in CG1-CTL killing of NSCLC cells pulsed with CG versus non-pulsed cells. In addition, OS cells take up and cross-present NE and P3, with a 13.1-fold increase in PR1-CTL killing of OS cells that were pulsed with NE and P3 vs. non-pulsed cells. Conclusion: Our data define two novel HLA-A2 restricted peptides that can be targeted in NSCLC and OS. Specifically, CG1 and PR1 are presented on the surfaces of NSCLC and OS, increasing their susceptibility to killing by CG1 and PR1 specific CTL. Our work represents a novel link between innate and adaptive immune responses in solid tumors and identifies potential antigen targets for CTL, peptide vaccines, and TCR-like antibodies in the setting of NSCLC and OS. Citation Format: Amber Gibson, Pariya Sukhumalchandra, Celine Kerros, Na Qiao, Anne Philips, Mao Zhang, Sami Alkoutami, Scott Semelsberger, Yoshimi Lu, Lauren Byers, Ze Tian, Chunhua Shi, Jun Yan, Dongxing Zha, Alejandro Marinos, Anne Sergeeva, Hong He, Lisa St. John, Jeffrey J. Molldrem, Gheath Alatrash. Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2995.

Blood Inflammatory-like and Lung Resident-like Eosinophils Affect Migration of Airway Smooth Muscle Cells and Their ECM-Related Proliferation in Asthma

Airway remodeling is a hallmark feature of asthma, and one of its key structural changes is increased airway smooth muscle (ASM) mass and disturbed extracellular matrix (ECM) homeostasis. Eosinophil functions in asthma are broadly defined; however, we lack knowledge about eosinophil subtypes' interaction with lung structural cells and their effect on the airway's local microenvironment. Therefore, we investigated the effect of blood inflammatory-like eosinophils (iEOS-like) and lung resident-like eosinophils (rEOS-like) on ASM cells via impact on their migration and ECM-related proliferation in asthma. A total of 17 non-severe steroid-free allergic asthma (AA), 15 severe eosinophilic asthma (SEA) patients, and 12 healthy control subjects (HS) were involved in this study. Peripheral blood eosinophils were enriched using Ficoll gradient centrifugation and magnetic separation, subtyped by using magnetic separation against CD62L. ASM cell proliferation was assessed by AlamarBlue assay, migration by wound healing assay, and gene expression by qRT-PCR analysis. We found that blood iEOS-like and rEOS-like cells from AA and SEA patients' upregulated genes expression of contractile apparatus proteins, COL1A1, FN, TGF-β1 in ASM cells (p < 0.05), and SEA eosinophil subtypes demonstrated the highest effect on sm-MHC, SM22, and COL1A1 gene expression. Moreover, AA and SEA patients' blood eosinophil subtypes promoted migration of ASM cells and their ECM-related proliferation, compared with HS (p < 0.05) with the higher effect of rEOS-like cells. In conclusion, blood eosinophil subtypes may contribute to airway remodeling by upregulating contractile apparatus and ECM component production in ASM cells, further promoting their migration and ECM-related proliferation, with a stronger effect of rEOS-like cells and in SEA.

The density of bone marrow mononuclear cells and CD34+ cells in patients with three neurologic conditions

BackgroundThis study aimed to identify the density of mononuclear cells (MNCs) and CD34+ cells in the bone marrow of patients with three neurologic conditions.MethodsThe study included 88 patients with three neurologic conditions: 40 with cerebral palsy (CP) due to oxygen deprivation (OD), 23 with CP related to neonatal icterus (NI), and 25 with neurological sequelae after traumatic brain injury. Bone marrow aspiration was conducted from the patients’ bilateral anterior iliac crest under general anesthesia in an operating theater. MNCs were isolated by Ficoll gradient centrifugation and then infused intrathecally.ResultsThere was a significant difference in the average MNC per ml and percentage of CD34+ cells by the type of disease, age group, and infusion time (p value < 0.05). The multivariable regression model showed the percentage of CD34+ association with the outcome (gross motor function 88 items- GMFM-88) in patients with CP.ConclusionsThe density of MNCs was 5.22 million cells per mL and 5.03% CD34+ cells in patients with three neurologic conditions. The highest density of MNCs in each ml of bone marrow was found in patients with CP due to OD, whereas the percentage of CD34+ cells was the highest among patients with CP related to NI.

Effects of shear stress on the properties of colonyforming endothelial cells in comparison with coronary artery endothelial cells

Highlights. It is assumed that pre-colonization by endothelial cells of the inner surface of tissue-engineered vessels of small diameter can serve as an effective way to prevent thrombosis. The question of choosing the optimal source of endothelial cells for use in tissue engineering remains debatable. The paper considers the features of the culture of colony-forming endothelial cells obtained from the peripheral blood of patients with coronary heart disease, in comparison with mature endothelial cells from the coronary artery.Aim. To study the effect of laminar flow on the morphological and functional characteristics of mature endothelial cells and peripheral blood-derived endothelial colony-forming cells.Methods. Coronary artery endothelial cells were purchased from the Cell Applications, Inc. Colony-forming endothelial cells were obtained from the peripheral blood of patients with coronary artery disease who underwent percutaneous coronary intervention. The cells were isolated using a Ficoll gradient and cultured in EGM-2MV culture medium containing 5% fetal bovine serum. The cells of the experimental group were cultured in µ-Luer plates in a perfusion system with a shear stress of 3 dyn/cm2 . The cultivation time was 2 days. The cells of the control group were cultured under static conditions. At the end of the cultivation we performed immunofluorescent staining for CD31, KDR/CD309, CD144, vWF, type IV collagen, F-actin.Results. Colony-forming endothelial cells and coronary artery endothelial cells retained high density and viability both under static and laminar flow conditions. Shear stress stimulated a change in the phenotype of colony-forming endothelial cells towards a mature endothelial cells, in particular, a significant increased the expression of KDR/CD309 and CD31. The action of laminar flow reduced the synthesis of von Willebrand factor, stimulated the synthesis of type IV collagen. Shear stress promoted the development of structural rearrangements in cells in response to transduction, which manifested in a change in F-actin fibrils orientation on the flow direction.Conclusion. Colony-forming endothelial cells showed a characteristic response to the action of shear stress, consisting in a change in morphology, phenotype, and secretory activity of cells, comparable to that of coronary artery endothelial cells.

Predictive value of TRPV2 expression from peripheral blood mononuclear cells on the early recurrence of atrial fibrillation after radiofrequency catheter ablation

BackgroundRecent study has shown that the transient receptor potential vanilloid 2 (TRPV2) channel was exclusively upregulated in patients with atrial fibrillation (AF), and that this overexpression might be detrimental for occurrence and maintenance of AF. We aimed to characterize the expression levels of TRPV2 mRNA in peripheral blood mononuclear cells (PBMCs) with/without early recurrence of atrial fibrillation (ERAF) after radiofrequency catheter ablation (RFCA), and to find a reliable predictor for ERAF.Methods65 patients of AF, who underwent RFCA successfully, then divided into two groups according to ERAF during following 3 months. PBMCs were isolated from whole blood by Ficoll gradient centrifugation before and after RFCA. Gene set enrichment analysis was performed to evaluate TRPV channels expression levels and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping was used for pathway enrichment analysis.ResultsThere was no significant difference in the TRPV2 mRNA expression level between the two groups before RFCA, while without ERAF group of TRPV2 expression was markedly reduced compared to ERAF group after RFCA. Moreover, the number of TRPV2 expression was confirmed as an independent predictor for the first time through receiver operating characteristic and Kaplan–Meier survival curve analysis. It should be pointed out that the above results were only used to predict ERAF, and have no predictive significance for late recurrence of atrial fibrillation according to the current data. Additionally, ERAF was inversely correlated with P wave dispersion. KEGG mapping further clustered 41 pathways, revealing that ‘‘cyclic guanosine monophosphate-protein kinase G signaling pathway’’ was significantly enriched.ConclusionsWe firstly assume that downregulated expression of peripheral TRPV2 appear in patients without ERAF after RFCA. TRPV2 may thus represent a novel predictor of early phase after successful radiofrequency ablation.

Subpopulation composition of peripheral blood lymphocytes in COVID-19

The pathogenesis of severe coronavirus infection COVID-19 is associated with activation of immune system, cytokine storm, impaired blood clotting, microvascular thrombosis, organ ischemia and multiple organ dysfunction syndrome. The role of various lymphocyte subpopulations in COVID-19 is still debated. The aim of our study was to analyze the subpopulational profile of peripheral blood lymphocytes in COVID-19 patients as compared with healthy donors.The study included 20 COVID-19 patients (11 males and 9 females,) and 26 healthy donors. Average age of the patients was 52 and 56 years, respectively. Clinical examinations were performed by standard laboratory methods. Peripheral blood lymphocytes were isolated in the Ficoll gradient. The cells were stained with antibodies to specific antigens of main lymphocyte populations, endothelial cells, and apoptotic cell markers. The analysis was performed by flow cytometry. The results showed that all patients had elevated C-reactive protein (14- to 35-fold), ferritin (1.2- to 13-fold), D-dimers (1.2- to 90-fold). 55% of men had a decrease in the absolute number of lymphocytes, in women this index was at the low normal limit. Cytometric analysis showed that, among peripheral blood lymphocytes, the proportion of functional cells expressing the CD45 marker ranged from 2 to 12% in 70% of patients, as compared with 80-99% among the donors. The proportion of CD45+ lymphocytes significantly correlated with the level of hemoglobin, but not with the levels of inflammatory biochemical markers. Among the functional lymphocytes of patients, there was a decrease in the proportion of CD3+, CD4+, CD8+T cells, increased proportion of natural killer CD56+ and the apoptotic (AnnexinV+) cell contents, but the proportion of CD19 and HLA-DR+B cells was not changed. Analysis of the lymphocyte (LC) subpopulations that did not express CD45 marker showed that this fraction contained different lymphocyte subsets with reduced expression of CD4, CD8, CD19, CD56 etc. in the blood of patients and donors. Higher percentage of endothelial cells expressing CD62P marker made the difference between patients and donors.Laboratory determination of lymphocyte subsets in blood samples of COVID-19 patients does not reflect the real severity pattern of the disease, thus requiring studies of the CD45-expressing functional cell populations.