Mononuclear Cell Fraction Research Articles

Extracellular traps in peripheral blood mononuclear cell fraction in childhood-onset systemic lupus erythematosus.

Although the role of low-density granulocytes (LDGs), neutrophils in the peripheral blood mononuclear cell (PBMC) fraction, and neutrophil extracellular traps (NETs) in assessing lupus disease severity is acknowledged, data specific to childhood-onset lupus remains scarce. This study analyzed 46 patients with childhood-onset systemic lupus erythematosus (82.6% females, mean age 14.5 ± 0.3 years), including 26 cases with normal complement levels and 20 with low complement levels, along with 20 healthy adult volunteers. Key parameters that distinguished healthy volunteers from lupus patients and differentiated between lupus patients with low and normal complement were serum interferon (IFN)-α, serum citrullinated histone 3 (CitH3), and extracellular traps (ETs) in LDGs. However, NETs (assessed by nuclear staining morphology), LDG abundance, and other parameters (such as endotoxemia, cytokines, and double-stranded (ds) DNA) did not show such differentiation. When lipopolysaccharide (LPS) was administered to LDGs in the PBMC fraction, it induced ETs in both low and normal complement groups, indicating the inducible nature of ETs. In adult healthy volunteers, activation by recombinant IFN-α or dsDNA in isolated neutrophils induced LDGs and NETs (identified using immunofluorescent staining for CitH3, myeloperoxidase, and neutrophil elastase) at 45min and 3h post-stimulation, respectively. Additionally, approximately half of the LDGs underwent late apoptosis at 3h post-stimulation, as determined by flow cytometry analysis. Activation by IFN-α or dsDNA in LDGs also led to a more pronounced expression of CD66b, an adhesion molecule, compared to regular-density neutrophils, suggesting higher activity in LDGs. In conclusion, IFN-α and/or dsDNA in serum may transform regular-density neutrophils into LDGs before progressing to NETosis and apoptosis, potentially exacerbating lupus severity through cell death-induced self-antigens. Therefore, LDGs and ETs in LDGs could provide deeper insights into the pathophysiology of childhood-onset lupus.

Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in β-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

TIM-3 expression on monocyte-derived dendritic cells

The T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), an inhibitory checkpoint receptor, has been identified as a crucial regulator of cellular immune responses. TIM-3 has been discovered as a receptor involved in the negative regulation of T cells. Recent studies have demonstrated that TIM-3 is expressed on innate immune cells, including dendritic cells (DCs), even at a higher level than T cells. In the tumor microenvironment, the majority of DCs have a monocytic origin. Models for studying such DCs in vitro are DC cultures generated from monocytes in the presence of growth factors. The present study aimed to investigate the expression of TIM-3 in IFNα-induced monocyte-derived DCs (IFN-DCs) and the impact of DC activation on TIM-3 expression. DCs were obtained by culturing the adherent fraction of mononuclear cells from healthy donors for 4 days in the presence of GM-CSF and IFNα, followed by LPS addition for 24 hours. Human double-stranded DNA (dsDNA, 5 μg/mL) was added as an activation stimulus to intact IFN-DCs at the stage of maturation, along with LPS. Expression of the membrane TIM-3 molecule was determined by flow cytometry, and the level of expression of TIM-3 mRNA – by real-time RT-PCR with reverse transcription. Intact donor IFN-DCs expressed the membrane TIM-3 molecule at a high level (more than 70% of cells). The addition of LPS as a maturation stimulus almost halved the expression of TIM-3 (pW < 0.05) without affecting the expression of HAVCR2/TIM-3 mRNA. Exogenous dsDNA (along with LPS) increased the expression of HAVCR2/TIM-3 mRNA by more than three times (pW = 0.05) with a decrease in the number of TIM-3+DCs (pW = 0.003). Our findings indicate the presence of mechanisms that support expression of this inhibitory checkpoint receptor under conditions of DC activation. Further studies of the regulation of TIM-3 expression by monocyte-derived dendritic cells will expand the understanding of the biological significance of inhibitory receptors on DCs from the point of view of the immune response, as well as, in the future, increase the effectiveness of current approaches in cancer immunotherapy using IFN-DCs and inhibitors of checkpoint molecules.

Comparison the impact of mesenchymal stromal cells, their microvesicles and plasma enriched with soluble platelet factors on survival and apoptosis of rat spleen lymphocytes <i>in vitro</i>

BACKGROUND: The trophic function is one of the important but insufficiently studied features of mesenchymal stromal cells (MSCs), their microvesicles (MVs), and plasma enriched with soluble platelet factors (PRPs), which ensure the viability of target cells. AIM: To analyze the capability of MSCs, MVs, and PRP to affect the viability and spontaneous and activation-induced apoptosis of rat spleen lymphocytes during culturing in vitro. MATERIALS AND METHODS: MSCs were isolated from the mononuclear cell fraction of the femoral bone marrow of Wistar rats using the plastic adhesion method. MVs were obtained from the conditioned medium of MSCs by centrifugation at 14,500 g. PRP was prepared by freezing/thawing rat peripheral blood platelet concentrate. Rat spleen lymphocytes were isolated on a density gradient of 1.077 g/cm3. The viability and degree of lymphocyte apoptosis in vitro were assessed by flow cytometry for 7-AAD incorporation and binding to annexin V. RESULTS: The presence of MSCs at 10 and 20% concentrations caused an increase in the number of living intact and PMA-activated lymphocytes by the end of 3-day in vitro cultivation. Compared with controls, the amount of necrotic cells decreased 8.3–13.5 times, and the number of apoptotic cells decreased 2.3–4.0 times, mainly due to lymphocytes at the late stage of apoptosis. MVs of MSCs at the indicated concentrations did not significantly affect the viability of lymphocytes cultured in vitro but reduced the level of apoptosis of intact and PMA-activated lymphocytes by 3.6 (р=0.03) and 4.8–5.2 (р=0.048; р=0.03) times respectively. Studies have shown that 1.25% rat PRP has a growth-stimulatory activity against MSCs but not against lymphocytes cultured in vitro. In the culture of intact lymphocytes, PRPs did not significantly affect cell viability with a slight decrease (2.7–2.9 fold, p 0.05) in the number of necrotic cells. In the culture of PMA-activated lymphocytes, 1.25–2.50% PRP increased the number of living cells by 1.6–2.2 times (р=0.002; р=0.01) and the number of necrotic cells by two times (р=0.02). CONCLUSION: MSCs, MVs of MSCs, and PRP, in decreasing order, enhanced the viability of rat spleen lymphocytes and suppressed late apoptosis during in vitro cultivation. Lymphocytes activated by phorbol myristate acetate are more sensitive to their vital action compared with resting cells.

DETERMINATION OF IL-1β mRNA GENE EXPRESSION AND HMGB-1 mRNA GENE EXPRESSION IN WOMEN WITH GENITAL ENDOMETRIOSIS ASSOCIATED WITH INFERTILITY AT THE STAGE OF PREPARATION FOR ASSISTED REPRODUCTIVE TECHNOLOGIES

Despite the large number of studies on the identification and search for noninvasive early diagnostic markers of genital endometriosis, this problem remains relevant today. It is important to introduce early diagnosis of genital endometriosis for the purpose of timely prevention and treatment.Objective: to study the expression levels of IL-1β and HMGB-1 mRNA genes in women with genital endometriosis associated with infertility, and to evaluate the effect of our proposed preparation for assisted reproductive technologies on the level of IL-1β and HMGB-1 mRNA gene expression.Material and methods. To analyze gene expression IL-1β and HMGB-1 and determine relative normalized mRNA expression IL-1β and HMGB-1, the polymerase chain reaction method was used with real-time reverse transcription(RT-PCR. The object of molecular genetic studies using the RT-PCR method was the fraction of mononuclear cells isolated from the whole blood of patients with endometriosis. In this study, we performed a retrospective analysis of the medical records of 30 women with infertility undergoing an assisted reproductive technology program. The main group consisted of 20 women diagnosed with external genital endometriosis who underwent assisted reproductive technologies. Women in the main group, in addition to standard training for assisted reproductive technologies, received a probiotic containing Lactobacilli 1010 manufactured by Unic Biotech Ltd, India. They took one pill twice a day for one month as part of a general treatment before undergoing assisted reproductive technologies. We determined the expression level of IL-1β and HMGB-1 before and after this preparation stage. The control group consisted of 10 women who had tubal infertility due to a previous inflammatory disease, but according to the results of a comprehensive clinical and laboratory examination, they were compared to healthy women. These women, aged 21 to 42 years, with a mean age of 29,75 years, did not receive our proposed preparation with the inclusion of a probiotic. This study was conducted at the Bukovyna State Medical University and the clinic "Yuzko medical center".Results. In the main group, the IL-1β mRNA gene expression level before training was (26,7877±0,01), which was significantly higher than the level after training (0,1610±0,01*). Consequently, the use of the probiotic led to a dramatic reduction in elevated mesothelin levels, underscoring its efficacy and potential application in assisted reproductive technology training programs. Also, a high level of expression of mRNA genes can be noted HMGB-1in whole blood of women of the main group before preparation for assisted reproductive technologies (table 2). Expression level of mRNA genes HMGB-1in whole blood of women of the main group before preparation for assisted reproductive technologies was (11,9198±0,01). After our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic, the mRNA gene expression level HMGB-1in the whole blood of women of the main group was (0,2821±0,01*). Therefore, our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic is quite effective, since the expression levels of IL-1β mRNA genes and HMGB-1sharply decreased.Conclusions. Extremely high expression of mRNA genes IL-1β and HMGB-1 indicates a close connection between the pathogenesis of endometriosis and inflammation. The inclusion of probiotics in the complex scheme of preparation for assisted reproductive technologies leads to a noticeable improvement in the well-being of the patient and a significant decrease in the expression of IL-1β and HMGB-1 mRNA genes. Therefore, we recommend our proposed preparation for assisted reproductive technologies with the inclusion of a probiotic.

Collection efficiency of mononuclear cells in offline extracorporeal photopheresis: can processing time be shortened?

Extracorporeal photopheresis (ECP) is a well-established but lengthy and burdensome cell-based therapy for various diseases such as cutaneous T-cell lymphoma, graft-versus-host disease and organ rejection after transplantation. The number of mononuclear cells (MNCs) that needs to be collected to obtain a clinical response to ECP is still under debate. The purpose of this retrospective study was to determine the number of lymphocytes, monocytes and neutrophils in mononuclear cell products (MCP) by flow cytometry and the collection efficiency in the offline ECP setting. We collected data from 10 different patients undergoing 162 ECP procedures using the Spectra Optia device for MNC collection. White blood cell (WBC) count of MCP was determined using a hematology analyzer. MNCs were analyzed for CD45 and CD14 expression by flow cytometry to exactly determine the collected lymphocyte and monocyte fractions. Collected MCP showed high cell yields with 55.3×106/kg MNCs and 41.1×106/kg lymphocytes. MCP were characterized by high MNC (81.3%) and low neutrophils (18.7%) percentage. Mean collection efficiency for WBCs and for MNCs was 23.9% and 62.0%, respectively. The MNC fraction showed a moderate to high correlation between peripheral blood cell count of patients and MCP count. This study is one of a few reports showing the monocyte-to-lymphocyte relation in MCP for ECP determined by flow cytometry. In comparison to historical data from inline ECP, the offline ECP processing one total blood volume results in considerably higher cell yields. For this reason, and to reduce the burden on patients, we propose that the offline ECP processing time can be substantially reduced.

Detecting thyrotropin receptor mRNA from peripheral blood of patients with differentiated thyroid cancer rules out non-aggressive cases.

Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify ∼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method - droplet digital PCR (ddPCR). Less than 5mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR. Thyrotropin receptor mRNA was detectable in 4mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases. Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment.

Clinical Relevance of Differential RARα and PPARβ/δ Expression in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are clinically heterogeneous hematological malignancies with an increased risk of transformation to acute myeloid leukemia, emphasizing the importance of identifying new diagnostic and prognostic markers. This study sought to investigate the predictive ability of all-trans retinoic acid (ATRA)-dependent nuclear transcription factors RARα and PPARβ/δ gene expression in MDS patients. Peripheral blood specimens were collected from 49 MDS patients and 15 healthy volunteers. The specimens were further separated in Ficoll density gradient to obtain the mononuclear cells fractions. Gene expression analysis was carried out using quantitative real-time polymerase chain reaction (qRT-PCR) technique. In the mononuclear cell fractions of MDS patients, RARα expression was increased (p<0.05) and PPARβ/δ expression was decreased (p<0.01) compared to healthy volunteers. When RARα and PPARβ/δ expression was compared in groups of MDS patients with different risks of disease progression, no statistically significant difference was found for RARα expression, while PPARβ/δ expression was significantly lower in the high-risk group of patients compared to the low-risk group (p<0.05). The expression of RARα was significantly associated with overall survival (p<0.05). ROC analysis showed that the expression of PPARβ/δ, rather than RARα expression, could have potential diagnostic value for MDS patients (AUC=0.75, p=0.003 and AUC=0.65, p=0.081, respectively). RARα and PPARβ/δ genes are putative biomarkers that may be associated with the diagnosis and prognosis of MDS.

Generation of a bank of clinical-grade, HLA-homozygous iPSC lines with high coverage of the Spanish population

BackgroundInduced pluripotent stem cell (iPSC)-derived cell therapies are an interesting new area in the field of regenerative medicine. One of the approaches to decrease the costs of iPSC-derived therapies is the use of allogenic homozygous human leukocyte antigen (HLA)-matched donors to generate iPSC lines and to build a clinical-grade iPSC bank covering a high percentage of the Spanish population.MethodsThe Spanish Stem Cell Transplantation Registry was screened for cord blood units (CBUs) homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes. Seven donors were selected with haplotypes covering 21.37% of the haplotypes of the Spanish population. CD34-positive hematopoietic progenitors were isolated from the mononuclear cell fraction of frozen cord blood units from each donor by density gradient centrifugation and further by immune magnetic labeling and separation using purification columns. Purified CD34 + cells were reprogrammed to iPSCs by transduction with the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit.ResultsThe iPSCs generated from the 7 donors were expanded, characterized, banked and registered. Master cell banks (MCBs) and working cell banks (WCBs) from the iPSCs of each donor were produced under GMP conditions in qualified clean rooms.ConclusionsHere, we present the first clinical-grade, iPSC haplobank in Spain made from CD34 + cells from seven cord blood units homozygous for the most common HLA-A, HLA-B and HLA-DRB1 haplotypes within the Spanish population. We describe their generation by transduction with Sendai viral vectors and their GMP-compliant expansion and banking. These haplolines will constitute starting materials for advanced therapy medicinal product development (ATMP).

Molecular Expression Differences in Blood Mononuclear Cells in Patients with AL Amyloidosis

Background Diagnosis of AL amyloidosis requires tissue biopsy using different surrogate and target biopsy sites with variable sensitivity leading to delay in diagnosis and worse patient outcomes. Mononuclear cells (MNC's) circulate in the peripheral blood and possess receptors for components in the tissue depositions of AL. Receptor triggered signaling is expected to result in characteristic changes in molecular expression levels and may be detected with a sensitive cell-specific technology. In this way, blood MNC's and receptor-mediated signaling could be used as a sensitive indicator of the presence of the AL deposits. The CellPrint™ platform, with the ability to assign molecular expression to specific cell lineages but with 10-100-fold enhanced sensitivity, is ideal for identifying the patterns of molecular expression in MNC's that are characteristic of AL. Herein, we are proposing a novel approach to the development of a biomarker for AL. Methods Peripheral blood samples were collected from patients with AL (n = 27) and multiple myeloma (MM) (n = 41) at the Cleveland Clinic. The samples were processed within four hours of venipuncture by isolating the mononuclear cell fraction via discontinuous gradient separation over ficoll/hypaque. The mononuclear cells were suspended in medium containing dimethyl sulfoxide, viably frozen, and stored in liquid nitrogen. The laboratory personnel were blinded to the clinical data associated with the samples. The code was not broken until the expression level dataset was finalized. Once accrual of the samples was completed, the cells were thawed, stained with antibodies to a lineage marker, fixed, permeabilized, and stained for the expression of selected analytes with signal amplification by CellPrint™. The expression levels of these analytes were assessed in CD4+ T lymphocytes and monocytes. Results Both differences in cell-type specific levels of expression and intermolecular organization, as indicated by bivariate correlations, were observed between samples from MM patients and AL. These differences were most pronounced in CD4+ T cells, but they also occurred in monocytes. CD4+ T cells from patients with AL demonstrated decreased expression of BDNF, calmodulin, and phospho-TBK1 as compared to those from patients with MM (figure 1). BDNF and phospho-TBK1 were found to be highly correlated in the CD4+ T cells from the patients with AL (r=0.75). In CD4+ T cells of patients without AL (MM patients), Vav expression correlated with phospho-Erk, phospho-GSK3β, phospho-cJun and other molecules. However, in these cells from patients with AL, Vav did not correlate with any of these molecules (table 1). Similar results were found with phospho-cJun. Thus, the contribution of Vav and phospho-cJun to various signal transduction pathways is altered in patients with AL. These analytes are known to be essential for the activation of T cells and the overall effect of the presence of amyloid on CD4+ T cells may be suppressive. Both cJun and Vav are known to be involved in the balance between T cell activation and exhaustion (Cells 9:2470, 2020). Alterations in Vav activation have also been observed because of PD1 ligation on T cells (Science 355:1428, 2017). Our findings show that both activated cJun (phospho-cJun) and Vav were disconnected to the major signaling pathways in CD4+ T cells. Conclusion Our results show molecular expression level and bivariate correlational changes in both CD4+ T cells and monocytes in patients with AL; the changes were more pronounced in the T cells. The finding of a possible exhaustion-type phenotype in CD4+ T cells in AL patients but not in MM patients requires further investigation and may have therapeutic implications. It will also be important to look at MNC molecular phenotype in patients with MGUS which could possibly serve as a predictor of progression to AL.

New Pattern of Emerging Somatic Mutations in Optimal Responders Following Tyrosine Kinase Inhibitor Therapy in Chronic Myeloid Leukemia Patients Evidenced from Mutational Kinetic Analysis Based on Pairwise Comparison

Introduction Recent advances in genomics in chronic myeloid leukemia (CML) patients (pts) provide new insights in understanding somatic mutation (SM) and their prognostic implications following tyrosine kinase inhibitor (TKI) therapy. DNMT3A, TET2, or ASXL1 (DTA) mutations are most frequently detected following TKI therapy and have adverse prognosis. Five longitudinal patterns of changes in SM after TKI therapy were suggested with its clinical significance (Kim, Blood 2017): Pattern 1, persistent mutation burden despite optimal TKI response; Pattern 2, emerging mutation which correlates with TKI resistance/CML progression; Pattern 3, mutational clearance with mixed clinical outcomes; and Pattern 4 and 5 having some mutation in germline control as well as CML cells. Clonal evolution (CE) in Philadelphia negative (Ph -) clone is found in up to 10% of CML pts who respond to TKI therapy optimally but develop +8 or -7 changes in Ph - clone. This is diagnosed only by metaphase cytogenetic test on bone marrow samples. Alternatively, fluorescence in situ hybridization (FISH) test can be used. With recent advances in next-generation sequencing (NGS), we hypothesize that SM profiling can capture a clone carrying a SM in the Ph - population. We sequenced and analyzed mutation profiles in paired samples taken prior to TKI therapy and at longitudinal follow-up. Patients and methods The present study adopted single molecule-tagging and molecular inversion probe (smMIP)-based sequencing methods to analyze the DNA extracted from the mononuclear cell fraction of peripheral blood samples collected from CML pts at initial diagnosis and during follow-up. While conventional NGS has a relatively high intrinsic sequencing error rate and is limited in detecting mutations with a variant allele frequency (VAF) of less than 2%, smMIP-based sequencing overcomes this issue by tracking all sequence reads from a single strand and can detect mutations with a limit of detection up to 0.1%. The in-house CML-specific smMIP panel used in this study included 40 genes with 332 amplicon probes covering genes involved in epigenetic modifiers, activation signaling, myeloid transcription factor, spliceosome, tumor suppressor, cohesion, and miscellaneous functions. To evaluate kinetics of mutations, we calculated doubling time of mutations. Using the mutational VAFs from 2 time points, t1 and t2, the doubling time was calculated according to the following equation: = (-2 -1) log(2) /log(-2) - log(-1), where VAF1 and VAF2 is the VAF of the mutant at time points t1 and t2, respectively. Results We performed targeted deep sequencing on a total of 119 serial samples from 51 CML pts (median of 405.5 days following TKI therapy). 41 pts had an optimal response, 7 were non-responders, and 3 were non-evaluable for response. Pattern 2 was observed in 1 of 7 non-responders. In 12 pts pattern 3 (mutation clearance with mixed clinical outcomes) was observed. 22 pts carried 36 mutations. At baseline 9 pts had somatic mutations. Emergence of somatic mutations in optimal responders was observed in 13 out of total 51 (25%) pts, with DNMT3A (n=5), TET2 (n=4), ASXL1 (n=4), and EZH2 (n=2). While JAK2, SF3B1, U2AF1, PHF6, TP53, BRAF, and CBL were observed in one patient each (Figure 1). Particularly, those with emerging DTA mutation were all optimal responders to TKI therapy. This pattern of mutational change is novel and has never been described before. The median doubling time was 77 days (range 21-1909 days) in the group with emergence of somatic mutation in follow-up samples. The doubling time was 56.1, 93.7, 74.8 days in DNMT3A, ASXL1, and TET2 mutations, respectively. Based on kinetics of mutations (doubling time), to capture 10-fold increase (i.e. 1 log increase) of certain mutation, NGS profile can be monitored every 6-7 months. This pattern of emergence of mutations (mostly DTA mutation) in pts responding optimally to TKI therapy is new and does not fit into the five patterns mentioned in our previous paper (Kim, Blood 2017). Thus, we propose this sixth novel pattern called “Emerging mutational clones in CML pts responding optimally to TKI therapy”. Conclusion We suggest a new pattern of emerging mutations in optimal responders to TKI therapy, which could derive from expansion of Ph- clone carrying some clones with emerging mutations. Longitudinal NGS monitoring in CML patients is feasible with frequency every 6-7 months.

250. Single cell profiling of blood immune cell kinetics in early sepsis reveals progressive decrease in a monocyte substate with immunosuppressive activity

Abstract Background Dysregulation of the immune response to bacterial infection is an essential but poorly understood component of sepsis. Kinetic changes in immune cell quantity during sepsis can reveal key elements of immunopathogenesis, yet these data are scant. Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from sepsis patients in our laboratory has demonstrated a novel monocyte transcriptional state (monocyte substate 1, or MS1) enriched in sepsis. The gene expression profile of MS1 cells is similar to that of myeloid-derived immune suppressor cells (MDSCs), which are key immune regulatory cells that inhibit T cell activation, proliferation, and cytotoxic activity in tumor microenvironments. In this study, we profiled blood samples obtained at multiple time points from patients with sepsis and controls comprising patients with non-infectious critical illness (sterile inflammation) and healthy individuals, to evaluate the kinetics of MS1 and other immune cell transcriptional states. Methods PBMCs were obtained from patients with sepsis (n=37), sterile inflammation (n=18) and healthy controls (n=8) at hospital presentation (Day 0), and on Day 1 and Day 3 of their clinical course. We analyzed ∼1500 single cells per sample and identified immune cell states by subclustering within immune cell lineages. Substate abundances were compared between patient phenotypes and across time using Wilcoxon rank sum testing with Benjamini-Hochberg correction. Results The fractional abundance of the MS1 substate was higher in sepsis at all time points (Figure 1) and decreased in sepsis patients from Day 0 to Day 3 (padj=0.027). Naïve and memory CD4+T and CD8+T fractions negatively correlated with MS1 fraction at Day 0 for sepsis patients (significant Pearson’s correlations &amp;lt; -0.4).Figure 1.Monocyte substate 1 (MS1) fractional abundance progressively decreases in sepsis patients. MS1 fraction of total peripheral blood mononuclear cells for each subject on Day 0, 1 and 3 of sampling. Subjects are aggregated by sepsis, sterile inflammation, and healthy control phenotypes; controls are sampled once but plotted at all time points to facilitate comparison. Boxes show the median and inter-quartile range (IQR) for each patient cohort, with whiskers extending to 1.5x the IQR in either direction from the top or bottom quartile. Adjusted p values are shown for inter-phenotype comparisons (two-tailed Wilcoxon rank-sum test with Benjamini-Hochberg correction for testing of multiple states). Conclusion This study demonstrates progressively decreasing abundance of monocytic MDSCs in early sepsis. Negative correlations between MS1 and T cell fractions on presentation, but not later, suggest that these monocytes may facilitate systemic immunosuppressive activity very early in sepsis; the subsequent decrease in abundance may limit prolonged immunosuppression and facilitate an adaptive microbicidal response to infection. Disclosures All Authors: No reported disclosures

Distinct T Cell Receptor Gene Repertoires and T Cell Subset Distribution in Peripheral Blood and Bone Marrow of Patients with Multiple Myeloma

Mounting evidence suggests that the T cell repertoire in multiple myeloma (MM) is actively shaped by antigen selection. However, the molecular underpinnings of this observation are not fully understood, while relevant studies have been mostly based on immunophenotypic characterization of T cell subsets, with considerably less information regarding T cell receptor (TR) gene repertoire profiles. Moreover, most available information derives from the analysis of a single tissue compartment [i.e. peripheral blood (PB) or bone marrow (BM)], thus hindering addressing the potential impact of local T cell responses. Here we sought to overcome these limitations through paired analysis of PB and BM aspirates of 20 newly diagnosed patients with MM before the administration of any treatment, from whom we isolated the PB and BM mononuclear cell (PBMC/BMMC) fraction, as well as CD4 + and CD8 + T cells. TR beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4 + and CD8 + T cells and then subjected to paired-end next generation sequencing (NGS). Raw reads (n=14,369,410 | median 179,618/sample) were processed through a purpose-built bioinformatics pipeline. Productive TRBV-TRBD-TRBJ gene rearrangements were taken into consideration (n=9,212,507 | median 115,156/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 1,005,150 TRΒ distinct clonotypes were assessed (median=12,565/sample). Both CD4 + and CD8 + T cell populations displayed skewed TRBV repertoires (7 TRBV genes and 3 TRBJ genes accounting for about ~40% and ~60% of the repertoire) in both BM and PB. All patients displayed oligoclonal T cell expansions in both CD4 + and CD8 + T cells, albeit these were significantly (p&amp;lt;0.001) more pronounced in CD8 + T cells for both BM and PB samples. That said, oligoclonality was more pronounced in BM vs. PB for both CD4 and CD8 T cells, reaching statistical significance in the former: in particular, the median cumulative frequency of the most expanded clonotypes/sample was ~40% higher in CD4 + BM T cells vs. CD4 + PB T cells (p&amp;lt;0.001). Additionally, a significant shift was noticed in the major clonotype repertoire of CD4 + and CD8 + T cells in both PB and BM. In more detail, the 10 most expanded clonotypes/sample in PB had significantly lower frequency in BM, and, vice versa, the 10 most expanded clonotypes in BM were represented at diminished frequency or were absent in the PB in 12/20 patients. Flow cytometry analysis revealed distinct T cell subset composition in CD4 + or CD8 + T cells, also in different tissue compartments (BM vs. PB). Significant (p&amp;lt;0.001) differences concerned the over-representation of: (i) effector T cells (CD45RA +/CCR7 -, Temra) in CD8 + vs. CD4 + T cells in both BM and PB (median frequencies in BM and PB: 22.5% and 7% for CD8 + T cells; 6% and 1.6% for CD4 + T cells); (ii) CD8 + Temra cells in BM vs. PB (median frequencies of 22.5% vs. 7% in BM and PB, respectively; p&amp;lt;0.001); (iii) CD4 + vs. CD8 + central memory T cells (CD45RO +/CCR7 +, Tcm) in both PB and BM (median frequencies in PB and BM: 42.7% and 49.1% for CD4 + Tcm and 14% and 18.8% for CD8 + Tcm); (iv) CD8 + effector memory T cells (CD45RO +/CCR7 -, Tem) in BM vs. PB (median frequencies of 21.8% vs. 2.7% in BM and PB, respectively); (v) CD8 + naïve T cells (CD45RA +/CCR7 +, Tn) in PB vs. BM (median frequencies of 67.7% vs. 24.2% in PB and BM, respectively). In conclusion, oligoclonal expansions of CD4 + and, particularly, CD8 + T cells in MM that are more pronounced in the BM (at least for CD4 + cells) argue for selection by BM-biased antigens, a claim also supported by the significant differences in the relative frequency of dominant clonotypes in BM vs. PB. The distinctive T cell subset distribution in the BM, characterized by a significant increase of effector at the expense of memory T cells, likely reflects an antigen-driven albeit ineffective immune response.

Distinct roles of estrone and estradiol in endothelial colony-forming cells.

Our current understanding of the relationship between estrogen and human endothelial colony-forming cell (hECFC) function is based almost exclusively on studies investigating estradiol action at nuclear estrogen receptors. In the current study the hypothesis was tested that the less potent estrogen receptor agonist, estrone, affects hECFC proliferation, migration, secretion, and tube formation in a way that is unique from that of estradiol. The relationship between the estrogens, estradiol and estrone, is clinically important, particularly in postmenopausal women where estradiol levels wane and estrone becomes the predominant estrogen. Cultured hECFCs from peripheral blood mononuclear cell fractions were treated with concentrations of estradiol and estrone ranging from 1 nM to 1 μM separately and in combination. Following treatment, proliferation, migration, ability to attract other hECFCs (autocrine secretion), and ability to enhance endothelial cell tube formation (tubulogenesis) were tested. Functional assays revealed unique, concentration-dependent physiological effects of estrone and estradiol. Estradiol exposure resulted in increased hECFC proliferation, migration, secretion of chemoattractant, and enhancement of tube formation as expected. As with estradiol, hECFC secretion of chemoattractant increased significantly with each increase in estrone exposure. Estrone treatment produced a biphasic, concentration-dependent relationship with proliferation and tube formation and relatively no effect on hECFC migration at any concentration. The quantitative relationship between the effects of estrone and estradiol and each hECFC function was analyzed. The extent to which estrone was similar in effect to that of estradiol was dependent on both the concentrations of estradiol and estrone and the hECFC function measured. Interestingly, when the two estrogens were present, differing ratios resulted in unique functional responses. hECFCs that were treated with combinations of estrone and estradiol with high estrone to estradiol ratios showed decreased proliferative capacity. Conversely, hECFCs that were treated with combinations that were relatively high in estradiol, showed increased proliferative capacity. Cells that were treated with estrone and estradiol in equal concentrations showed an attenuated proliferative response that was decreased compared to the proliferation that either estrone or estradiol produced when they were present alone. This co-inhibitory relationship, which has not been previously reported, challenges the prevailing understanding of estrone as solely a weak agonist at estrogen receptors. This study provides evidence that estrone signaling is distinct from that of estradiol and that further investigation of estrone's mechanism of action and the biological effect may provide important insight into understanding the dysfunction and decreased number of hECFCs, and the resulting cardiovascular disease risk observed clinically in menopausal women and women undergoing hormone replacement therapy.

Abstract 25 Cord Blood Mononuclear Cells and Cord Blood Plasma Alter Human Cardiac Endothelial Cell Angiogenic Sprouting and Growth In Vitro

Abstract Introduction The mononuclear cell fraction of human placental cord blood (CBMNC) comprises immune cells, hematopoietic, mesenchymal and other stem or progenitor cells. CBMNCs are proposed to afford therapeutic benefit based on their paracrine release of multiple cytokines, growth factors and other proteins that stimulate signalling for wound healing, including attenuation of inflammation and fibrosis. Whether CBMNCs augment angiogenic sprouting and tube formation by endothelial cells in human ventricular myocardium is not well characterised. Objectives The aim of this study was to investigate the in vitro influence of CBMNCs on endothelial cell angiogenic-like growth. Methods A tube formation assay for human donor primary ventricular myocardium microvascular endothelial cells (HMVECs), (3 female donors, age 48-50 yrs, 3 replicates per treatment group) was established using Matrigel-coated 96-well plates (62,500-78,125 cells/cm2). Cells from passage 5-8 were used for all experiments.. Angiogenic processes, as characterized by endothelial sprouting followed by tube extension into honeycomb-like mesh structures, were imaged over 24 hours. HMVECs were treated with CBMNCs at a concentration of 1:10 (MNC:HMVEC) or cord blood plasma (1μg/μL) at 0h, 3h, or 6h after the HMVECs were seeded. The cells were visualized by phase contrast microscope and images (4x magnification) were analysed using the Angiogenesis Analyzer plugin in FIJI ImageJ. Size exclusion chromatography and multiplex ELISAs were performed to determine the presence of analytes in media collected from each well. Results CBMNC-treated HMVECs exhibited greater average total elongated branching length and more tube-like structured meshes, compared to untreated controls. MMP-1, MMP-2, MMP-9, MMP-10, TIMP-1, TIMP-2, angiopoietin-2, EGF, endoglin, endothelin-1, FGF-2, follistatin, HB-EGF, IL-8, PLGF, VEGF-A, and VEGF-C were detected in extracellular media during in vitro tube formation. With CBMNC treatment, only MMP-9 was increased by 475.94%, whereas angiopoietin-2, follistatin, and PLGF levels were decreased by 27.90%, 15.90%, and 26.18% respectively. In contrast, CB plasma-treated HMVECs did not form tube sprouts, instead an intense endothelial cell proliferation was observed which was associated with increased MMP-2, MMP-9, endothelin-1 and IL-8 levels with decreased angiopoietin-2, HB-EGF, PLGF, and endoglin levels. Discussion CBMNC treatment enhanced pro-angiogenic processes in HMVECs in vitro through increased branching and network formation, whereas CB plasma evoked cell proliferation.

Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility.

Standard cell therapy equipment, including the gold standard cell processor to purify human islets for clinical transplantation, is rarely refrigerated, potentially exposing cells to elevated temperatures during the centrifugation step. Custom cooling systems have a direct benefit on human islet viability and function. The current study was designed to test the effectiveness of a newly developed, readily available cooled cell processor system requiring minimal modifications and to evaluate its impact on human cell viability and the GMP cleanroom environment.The cooler system, a mechanically refrigerated heat exchanger set at -30 °C was used to deliver cooled medical grade dry air to the cell processor bowl through a hole drilled in the centrifuge cover. With the limited availability of pancreas donors in Qatar, system validation was done with continuous density gradient purification of pooled human bone marrow buffy coat. Sterility, turbulence, and particle count were measured in class C and class B clean room environments. No turbulence developed around the cooled cell processor, and no excess 0.5 µm and 5 µm airborne particulates were generated as per cleanroom GMP standards. At the beginning and end of the collection steps, the temperature rose respectively to 21.50 °C ± 0.34 °C and 21.93 °C ± 0.20 °C in the non-cooled cell processor and to only 10.9 °C ± 0.17 °C and 11.16 °C ± 0.35 °C in the cooled- cell processor (p <0.05). The cooled cell processor led to both improved recovery (98%) of the mononuclear cell fraction and viability (100% ± 2%) post-processing. The new cooling system effectively reduces the heat produced by the cell processor while having no particulate impact on the GMP clean room environment. The cooled cell processor described here is an inexpensive ($16,000 without including taxes, customs clearance, and transportation) and minimally invasive method to provide robust cooling. Currently, this technology in the GMP cell therapy facilityis being applied to human islet cell isolation and transplantation for the clinical program.

G-CSF rescue of FOLFIRINOX-induced neutropenia leads to systemic immune suppression in mice and humans

BackgroundRecombinant granulocyte colony-stimulating factor (G-CSF) is routinely administered for prophylaxis or treatment of chemotherapy-induced neutropenia. Chronic myelopoiesis and granulopoiesis in patients with cancer has been shown to induce immature monocytes...

Peripheral blood mononuclear cells contribute to myogenesis in a 3D bioengineered system of bone marrow mesenchymal stem cells and myoblasts.

In this work, a 3D environment obtained using fibrin scaffold and two cell populations, such as bone marrow-derived mesenchymal stem cells (BM-MSCs), and primary skeletal muscle cells (SkMs), was assembled. Peripheral blood mononuclear cells (PBMCs) fraction obtained after blood filtration with HemaTrate® filter was then added to the 3D culture system to explore their influence on myogenesis. The best cell ratio into a 3D fibrin hydrogel was 1:1 (BM-MSCs plus SkMs:PBMCs) when cultured in a perfusion bioreactor; indeed, excellent viability and myogenic event induction were observed. Myogenic genes were significantly overexpressed when cultured with PBMCs, such as MyoD1 of 118-fold at day 14 and Desmin 6-fold at day 21. Desmin and Myosin Heavy Chain were also detected at protein level by immunostaining along the culture. Moreover, the presence of PBMCs in 3D culture induced a significant downregulation of pro-inflammatory cytokine gene expression, such as IL6. This smart biomimetic environment can be an excellent tool for investigation of cellular crosstalk and PBMC influence on myogenic processes.

The immunomodulatory activity of the betulonic acid based compound.

The immunomodulatory activity of a betulonic acid-based compound with furocoumarin (BABCF; 2-azido, 9-N-methylpiperazinomethyl oreozelone) has been investigated. Male C57BL/6 mice (aged 3 months) treated with the cytostatic agent cyclophosphamide (CP) and intact individuals served as experimental models. The expression of genes was studied in bone marrow (IL-12, IL-10, IL-1β, TNF-α, TGF-β, M-CSF, GM-CSF) or in the suspension of peritoneal cells (IL-12, IL-10; as the injection site). The surface markers of T-lymphocytes (CD3, CD4, and CD8) in fractions of venous blood mononuclear cells (MNCs) were determined by means of flow cytometry using antibodies. Histological and morphometric studies were performed to assess the impact of CP and BABCF on the thymus. BABCF caused a pronounced (about 3-fold) increase in relative expression of the GM-KSF gene. BABCF caused a local increase in the expression of IL-12 in the peritoneal cavity cells and restored the relative content of T-lymphocytes in the blood of CP-treated mice treated affecting mainly CD3⁺CD4⁺ lymphocytes. This substance reduced the tissue density of the thymic cortex and thymic medulla in CP-treated mice. Thus, results of this study suggest that BABCF exhibits a stimulating effect on the cellular link of immunity and promotes maintenance of the number of T-lymphocytes in the blood due to their migration from the central organs of the immune system.

A NEW APPROACH TO ASSISTED REPRODUCTIVE TECHNOLOGIES PREPARATION IN WOMEN WITH ENDOMETRIOSIS ASSOCIATED INFERTILITY USING PROBIOTICS.

Aim: To examine the expression Nlrp3-inflammasome in the blood of women with endometriosis-associated infertility using assisted reproductive technologies with included probiotic and estimate a new approach with included probiotic for preparation to assisted reproductive technologies. Materials and methods: For the purpose of the research we examined 30 infertile women. Control group consisted of 10 women with tubal infertility due to an inflammatory process in the anamnesis, in whom, as a result of a complex clinical and laboratory examination, no other diseases were detected and in terms of their health they could be equated with practically healthy women (age from 21 to 42 years, the average age was 29.75 years) and who did not take probiotic. The main group consists from 20 women with external genital endometriosis were included in assisted reproductive technologies. Patients from main group took probiotic produced by Unic Biotech Ltd, India. 1 tablet twice a day with 10×109 Lactobacillus during 1 month in complex preparation (treatment) before assisted reproductive technologies. The indices of Nlrp3 - Inflammasome determined before and after preparation. The study was performer in Bukovinian State Medical University and Centre of Reproductive Medicine. To analyze the expression of the NLPP3-inflammasome gene and determine the relative normalized expression of NLPP3 mRNA, we used the polymerase chain reaction with reverse transcription in real time (RT-PCR). The object for molecular genetic studies by the RT-PCR method was the fraction of mononuclear cells isolated from the whole blood of patients with endometriosis. The value of p (authenticity difference) was determined by Student's table-Fischer. Differences between contrasting averages were considered significant at p <0.05. Results: Analized the results of our research stated that in main group expression of Nlrp-3 inflammasome was 24,43, which is significantly higher than after preparation (0,70 accordingly). In control group expression of Nlrp3-inflammasome was 0,54. Expression of Nlrp-3 inflammasome increased in patients before preparation more than 34 times compare with patients after preparation accordingly. Conclusions: consequently, after using probiotic, the increased level of Nlrp3-inflammasome sharply decreases, indicating the effectiveness and the possibility of use in the programme for assisted reproductive technologies preparation.