Isolation Of Peripheral Blood Mononuclear Cells Research Articles

Investigation of conceptus stimulated gene expression in buffalo peripheral blood mononuclear cells as potential diagnostic markers of early pregnancy

Exploration of novel strategies for early pregnancy diagnosis is pivotal in enhancing the reproductive potential and monetary gains from dairy herds. In buffalo, the trophectoderm cells of the elongating conceptus secrete interferon-tau that stimulates the transcription of various genes in peripheral blood mononuclear cells (PBMC) during the peri-implantation period. We explored the differential expression of classical (ISG15) and novel (LGALS3BP and CD9) early pregnancy markers in PBMC of buffaloes during various stages of pregnancy. Natural heat was detected in buffaloes by assessing the vaginal fluid, and artificial insemination (AI) was done. Whole blood was collected from the jugular vein in EDTA-containing vacutainers for PBMC isolation before AI (0-day) and 20, 25 and 40 d post-AI. On day 40, transrectal ultrasonography examination was performed to confirm pregnancy. The inseminated non-pregnant animals served as control. Total RNA was extracted using the TRIzol method. The temporal abundance of ISG15, LGALS3BP and CD9 genes in PBMC was compared between pregnant and non-pregnant groups (n = 9 per group) using real time-qPCR. Results showed transcripts of ISG15 and LGALS3BP were more abundant at 20 d in the pregnant group compared to the 0 d and 20 d values of the non-pregnant group. However, due to variability in expression, threshold (Ct) cycle of RT-qPCR alone could not distinguish pregnant and non-pregnant animals. In conclusion, ISG15 and LGALS3BP transcripts abundance in PBMCs are potential candidate biomarkers for early prediction of buffalo pregnancy 20-days post-AI, but further work is required to allow the development of a reliable new methodology.

Implementing real-time immunometabolic assays and immune cell profiling to evaluate systemic immune response variations to Eimeria challenge in three novel layer genetic lines.

Evaluating differences in immune responses to Eimeria spp. between poultry genetic lines could be valuable for understanding favorable traits to address coccidiosis, a costly poultry disease. The objective was to compare peripheral blood mononuclear cell (PBMC) immunometabolism and composition during Eimeria challenge in three distinct and highly inbred genetic lines; Leghorn Ghs6, Leghorn Ghs13, and Fayoumi M5.1. At hatch, 180 chicks (60/ line) were placed in wire-floor cages (10 chicks/cage) and fed a commercial diet. Baseline PBMC were isolated on d21 (10 chicks/line) and 25 chicks/line were inoculated with 10X Merck CocciVac®-B52 (Kenilworth, NJ), creating 6 genetic line × Eimeria groups total. Chicks were euthanized on 1, 3, 7, and 10d post-inoculation (pi; 5 chicks/ line × Eimeria group) for PBMC isolation with body weight and feed intake recorded throughout. Immunometabolic assays to determine PBMC ATP production profiles and glycolytic activity were implemented along with flow cytometric immune cell profiling. Genetic line × Eimeria challenge, and line´challenge fixed effects were analyzed using the MIXED procedure (SAS 9.4; P ≤ 0.05). Before inoculation, M5.1 chicks had 14.4-25.4% greater average daily gain (ADG) with 19.0-63.6% increased monocyte/macrophage+, Bu-1+ B cell, and CD3+ T cell populations compared to both Ghs lines (P < 0.0001) but similar immunometabolic phenotype. The Eimeria main effect reduced ADG by 61.3% from 3-7dpi (P = 0.009) except in M5.1 chicks, where no ADG difference due to challenge was found. At 3dpi, Eimeria-challenged M5.1 chicks had 28.9 and 33.2% reduced PBMC CD3+ T cells and CD3+CD8α+ cytotoxic T cells than unchallenged chicks, suggesting early and preferential recruitment from systemic circulation to tissues local to Eimeria challenge (i.e., intestine; P ≤ 0.01). Both Ghs lines displayed 46.4-49.8% T cell reductions at 10dpi with 16.5-58.9% recruitment favoring underlying CD3+CD4+ helper T cells. Immunometabolic responses in Eimeria-challenged Ghs6 and Ghs13 chicks were characterized by a 24.0-31.8% greater proportion of ATP from glycolysis compared to unchallenged counterparts at 10dpi (P = 0.04). These results suggest that variable T cell subtype recruitment timelines in addition to altered systemic immunometabolic requirements may work synergistically to determine favorable immune responses to Eimeria challenge.

Determination of the autophagic flux in murine and human peripheral blood mononuclear cells.

The autophagy lysosomal system (ALS) is crucial for cellular homeostasis, contributing to maintain whole body health and alterations are associated with diseases like cancer or cardiovascular diseases. For determining the autophagic flux, inhibition of lysosomal degradation is mandatory, highly complicating autophagy measurement in vivo. To overcome this, herein blood cells were used as they are easy and routinely to isolate. Within this study we provide detailed protocols for determination of the autophagic flux in peripheral blood mononuclear cells (PBMCs) isolated from human and, to our knowledge the first time, also from murine whole blood, extensively discussing advantages and disadvantages of both methods. Isolation of PBMCs was performed using density gradient centrifugation. To minimize changes on the autophagic flux through experimental conditions, cells were directly treated with concanamycin A (ConA) for 2h at 37°C in their serum or for murine cells in serum filled up with NaCl. ConA treatment decreased lysosomal cathepsins activity and increased Sequestosome 1 (SQSTM1) protein and LC3A/B-II:LC3A/B-I ratio in murine PBMCs, while transcription factor EB was not altered yet. Aging further enhanced ConA-associated increase in SQSTM1 protein in murine PBMCs but not in cardiomyocytes, indicating tissue-specific differences in autophagic flux. In human PBMCs, ConA treatment also decreased lysosomal activity and increased LC3A/B-II protein levels, demonstrating successful autophagic flux detection in human subjects. In summary, both protocols are suitable to determine the autophagic flux in murine and human samples and may facilitate a better mechanistic understanding of altered autophagy in aging and disease models and to further develop novel treatment strategies.

Single Cell Transcriptome Analysis of Peripheral Blood Mononuclear Cells in Freshly Isolated versus Stored Blood Samples.

Peripheral blood mononuclear cells (PBMCs) are widely used as a model in the study of different human diseases. There is often a time delay from blood collection to PBMC isolation during the sampling process, which can result in an experimental bias, particularly when performing single cell RNA-seq (scRNAseq) studies. This study examined the impact of different time periods from blood draw to PBMC isolation on the subsequent transcriptome profiling of different cell types in PBMCs by scRNAseq using the 10X Chromium Single Cell Gene Expression assay. Examining the five major cell types constituting the PBMC cell population, i.e., CD4+ T cells, CD8+ T cells, NK cells, monocytes, and B cells, both common changes and cell-type-specific changes were observed in the single cell transcriptome profiling over time. In particular, the upregulation of genes regulated by NF-kB in response to TNF was observed in all five cell types. Significant changes in key genes involved in AP-1 signaling were also observed. RBC contamination was a major issue in stored blood, whereas RBC adherence had no direct impact on the cell transcriptome. Significant transcriptome changes were observed across different PBMC cell types as a factor of time from blood draw to PBMC isolation and as a consequence of blood storage. This should be kept in mind when interpreting experimental results.

Effect of withdrawing an immunomodulatory feed additive from lactating cow diets on peripheral blood mononuclear cell proliferation.

Supplementation of immunomodulatory feed additives, such as OmniGen AF (OG), helps to maintain immune competency; however, it is unknown if immune benefits persist in lactating cows after OG is removed from the diet. The aim of this trial was to evaluate the effect of withdrawing OG from the diet on peripheral blood mononuclear cells (PBMC) proliferation of midlactation dairy cows. Multiparous Holstein cows (N = 32), blocked by parity (2.7 ± 0.8) and days in milk (153 ± 39 d) were randomly assigned to one of the two dietary treatments within each block: diets were top dressed with either OG (56 g/d/cow) or placebo (CTL, 56 g/d/cow). Cows were housed in the same free-stall pen and individually fed once per day through Calan gates. All cows were fed the same diet containing OG for at least 1 yr before the onset of treatments. Cows were milked three time per day and milk yield was recorded at each milking. Milk samples were collected from three consecutive milkings weekly and composition analyzed. Body weight (BW) and condition score were measured weekly. Blood samples were collected at -1, 1, 3, 5, and 7 wk relative to the onset of treatments for the isolation of PBMC. The PBMC were cultured with concanavalin A (ConA) and lipopolysaccharides (LPS) for 72 h in vitro to determine proliferative responses. Prior to the experiment, cows in both treatments had similar disease incidence. During the experiment, cows did not show symptoms of disease. Withdrawing OG from the diet did not affect (P ≥ 0.20) milk yield or composition, intake, or BW. Compared with CTL, feeding OG maintained greater body condition score (2.83 vs. 2.92, P = 0.04). Regardless of time, relative to CTL, PBMC isolated from cows fed with OG had a greater proliferative rate when stimulated with LPS (stimulation index: 1.27 vs. 1.80, P = 0.05) and tended to have greater proliferation when stimulated with ConA (stimulation index: 5.24 vs. 7.80, P = 0.08). In conclusion, withdrawing OG from the diet of midlactation cows reduced proliferative response of PBMC suggesting that the immunomodulatory role of OG is lost as early as 1 wk after its withdrawal from the diet of lactating dairy cows.

A standardized method to study immune responses using porcine whole blood.

Peripheral blood mononuclear cells (PBMCs) are commonly used to assess in vitro immune responses. However, PBMC isolation is a time-consuming procedure, introduces technical variability, and requires a relatively large volume of blood. By contrast, whole blood assay (WBA) is faster, cheaper, maintains more physiological conditions, and requires less sample volume, laboratory training, and equipment. Herein, this study aimed to develop a porcine WBA for in vitro evaluation of immune responses. Heparinized whole blood (WB) was diluted (non-diluted, 1/2, 1/8, and 1/16) in RPMI-1640 media, followed by phorbol myristate acetate and ionomycin. After 24 h, cells were stained for interferon (IFN)-γ secreting T-cells followed by flow cytometry, and the supernatant was analyzed for tumor necrosis factor (TNF)-α. In addition, diluted WB was stimulated by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), reference strain KCTC3557 (RS), field isolate (FI), of heat-killed (HK) Streptococcus suis, and porcine reproductive and respiratory syndrome virus (PRRSV). The frequency of IFN-γ+CD3+ T-cells and concentration of TNF-α in the supernatant of WB increased with increasing dilution factor and were optimal at 1/8. WB TNF-α and interleukin (IL)-10 cytokine levels increased significantly following stimulation with LPS or poly I:C. Further, FI and RS induced IL-10 production in WB. Additionally, PRRSV strains increased the frequency of IFN-γ+CD4-CD8+ cells, and IFN-γ was non-significantly induced in the supernatant of re-stimulated samples. We propose that the WBA is a rapid, reliable, and simple method to evaluate immune responses and WB should be diluted to trigger immune cells.

Human Dendritic Cell Subset Isolation by Magnetic Bead Sorting: A Protocol to Efficiently Obtain Pure Populations.

Dendritic cells have been investigated for cell-based immunotherapy for various applications. The low abundance of dendritic cells in blood hampers their clinical application, resulting in the use of monocyte-derived dendritic cells as an alternative cell type. Limited knowledge is available regarding blood-circulating human dendritic cells, which can be divided into three subsets: type 2 conventional dendritic cells, type 1 conventional dendritic cells, and plasmacytoid dendritic cells. These subsets exhibit unique and desirable features for dendritic cell-based therapies. To enable efficient and reliable human research on dendritic cell subsets, we developed an efficient isolation protocol for the three human dendritic cell subsets, resulting in pure populations. The sequential steps include peripheral blood mononuclear cell isolation, magnetic-microbead lineage depletion (CD14, CD56, CD3, and CD19), and individual magnetic-microbead isolation of the three human dendritic cell subsets.

Isolation and cryopreservation of peripheral blood mononuclear cells.

The study of peripheral blood mononuclear cells (PBMCs) in immune-mediated diseases, such as celiac disease (CD), is important to uncover pathogenesis, find new biomarkers and discover and evaluate new treatments. Many studies have been published about the use and value of PBMCs in CD such as those including enzyme-linked immunospot (ELISPOT) assays, flow cytometry, peptide-MHC tetramers, genetic and proteomic analyses, and in vitro and proliferation assays. We present here and easy and efficient method for isolation of PBMCs using density gradient centrifugation. We also describe a simple way to freeze PBMCs in order to preserve their number and viability and a thawing procedure leading to high rates of viability of the cryopreserved cells to be used in subsequent applications.

SnoRNAs are Deregulated in Patients with Parkinson’s Disease

Purpose: Small nucleolar RNAs are ranging from 65 to 300 nucleotides in length that mediate post-transcriptional RNA modifications. They don’t have a 5′-Cap and a poly-A tail and are categorized as C/D box snoRNAs, H/ACA box snoRNAs, and small Cajal body-specific RNAs. snoRNAs have essential roles in important biological processes such as transcription, RNA splicing, cell cycle, and etc. In this study, we tried to reveal differential expressions of snoRNAs in PBMCs of patients with Parkinson’s Disease by microarray analysis.&#x0D; Materials and methods: Patients (n=3) who are considered to have a unilateral onset history and a good response to dopaminergic treatment in the first years were included in the study. 10 ml peripheral blood sample was taken for peripheral blood mononuclear cell isolation. Total RNA was extracted using GeneAll® Hybrid-R™ kit and microarray analysis was performed by using Affymetrix GeneChip Human ST 2.0 platform. Raw data were extracted using Affymetrix Command Console Software 1.1. KEGG pathway and GO terms analyses were performed and protein-protein interaction of host genes were determined by using STRING database.&#x0D; Results: Data from patients revealed that there were 28 snoRNAs were downregulated and 3 snoRNAs were upregulated. &#x0D; Conclusion: Here in this study, we evaluated the differential expressions of snoRNAs in patients with a definitive diagnosis of PD by microarray analysis and observed deregulated expressions of some snoRNAs. Differential expression of snoRNA may cause changes in the transcriptional activity of host genes and thus can serve as biomarkers for diseases.

Comparison of methods to isolate peripheral blood mononuclear cells from cattle blood

Peripheral blood mononuclear cells (PBMCs) are critical for assessment of host immune responses to infectious disease. The isolation of PBMCs from whole blood is a laborious process involving density gradients and multiple centrifugation steps. In the present study we compared a more traditional method of PBMC isolation used in our laboratory to two novel methods of cell isolation for efficiency, cell viability, and enumeration of cell subsets. Our laboratory method uses Histopaque-1077 density gradient in standard conical tubes and this was compared with isolation of cells using SepMate™ tubes, a novel conical tube containing an insert to separate the density gradient. Multiple experiments were performed to optimize the SepMate™ tubes for use with cattle blood. A final experiment was conducted to compare traditional methodology, the optimized SepMate™ method with a more novel method using cell preparation tubes (CPT-10 vacutainers containing density gradient). Results demonstrated that optimization of the SepMate™ tube methodology was necessary, including dilution of blood and addition of centrifugation steps to reduce platelet contamination. The CPT-10 tubes worked well but cell recovery was lower compared to other methods. Both of the newer methods were comparable to a modified version of our traditional laboratory method of PBMC isolation in terms of numbers of recovered viable cells and the frequency of immune cell subsets. Additionally, efficiency was improved, particularly with the SepMate™ tube method, resulting in reduced time in the laboratory as well as reduced usage of plasticware.

Vitamin D Presented In Vitro Immunomodulatory Property on T Lymphocyte-Related Cytokines in Systemic Lupus Erythematosus.

Inflammatory T lymphocyte cytokines contribute to tissue damage in SLE patients. Vitamin D (Vit D) has a well-established immunomodulatory action, but few studies have addressed the effect of 1,25 dihydroxyvitamin D3 (1,25 (OH)2D3) on peripheral blood mononuclear cells (PBMCs) in SLE patients. The aim of this study was to evaluate the immnunomodulatory effect of 1,25 (OH)2D3 on T lymphocyte-related cytokines. Blood from 27 female SLE patients was collected for PBMC isolation and anti-DNA, complement, and serum 25 (OH)D3 level measurements. PBMCs were stimulated with anti-CD3/anti-CD28 in the presence or absence of dexamethasone or various concentrations of 1,25 (OH)2D3 for 48h. We assessed IL-17A, IL-22, IL-21, IL-9, IFN-γ, IL-4, IL-10, IL-2, IL-6, and TNF by cytometric bead assay (CBA) and enzyme immune assay (ELISA) on culture supernatant. The mean age of patients was 36.2 (± 10.5years) and the median Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) was 4 (0-6). The addition of 1,25 (OH)2D3 in PBMC culture reduced IL-17 A, IL-22, IL-9, and IFN-γ levels at 100nM (p ≤ 0.0001). Furthermore, the addition of 1,25 (OH)2D3 at all concentrations increased IL-4 (p ≤ 0.0006), and 0.1 and 1nM increased IL-10 (p ≤ 0.0004) and 0.1nM increased IL-2 levels (p ≤ 0.0001). There was no difference regarding IL-21 and TNF levels. The addition of 1,25 (OH)2D3 in PBMC culture presented an inhibitory effect on proinflammatory cytokines and increased immunoregulatory cytokines in SLE patients, suggesting the beneficial effect of this vitamin.

CTIM-07. PHASE II RANDOMIZED, BLINDED, PLACEBO-CONTROLLED TRIAL TESTING PP65 CMV MRNA DENDRITIC CELL VACCINE AND TETANUS-DIPHTHERIA TOXOID FOR NEWLY DIAGNOSED GBM (ATTAC II, NCT02465268)

Abstract INTRODUCTION Immunotherapy is limited in efficacy for glioblastoma (GBM). The objective of this study was to evaluate the efficacy and immune effects of the pp65 mRNA dendritic cell (DC) vaccine with the use of lysosomal-associated membrane protein (LAMP) in two different formulations (full-length (fl) and short-length (sh). METHODS Patients who underwent resection of newly diagnosed GBM were enrolled and randomized to one of 3 arms: Arm 1) pp65-shLAMP mRNA DC with GM-CSF, Arm 2) pp65-flLAMP mRNA DC with GM-CSF, Arm 3) Unpulsed peripheral blood mononuclear cells (PBMC). After surgery, patients underwent leukapheresis for PBMC isolation and vaccines generation. Patients were treated with standard radiation and temozolomide followed by dose intensified adjuvant temozolomide at 75-100 mg/m2 x 21 days (aged less than 70) or standard dose at 150-200 mg/m2 x 5 days (patients 70 and older). Immunization started days 22-24 of cycle #1 with vaccines 1-3 delivered at 2 week intervals. All patients received tetanus-toxoid booster prior to vaccine 1. Patients continued to receive monthly vaccines for up to 10 vaccines with tetanus-diphtheria toxoid skin prep prior to vaccines 3, 6 and 9 (arms 1 and 2). Safety, efficacy and immune labs were evaluated. RESULTS Overall DC vaccines were well-tolerated with 2 SAEs possibly attributed to DC vaccination that met criteria as DLTs. Of recorded grade 2 or lower AEs, 2 were possibly attributable to DC vaccination. One patient was removed from the study due to failure to produce DC vaccines that met QA/QC release criteria. CONCLUSION In a large randomized and blinded patient cohort, delivery of CMV LAMP pp65 mRNA DC vaccines is feasible and safe in a multi-institutional setting. The primary endpoint of survival will be analyzed at the conclusion of the study.

Cellular Stress Assay in Peripheral Blood Mononuclear Cells: Factors Influencing Its Results.

Cellular stress is central to the understanding of pathological mechanisms and the development of new therapeutic strategies and serves as a biomarker for disease progression in neurodegeneration, diabetes, cancer, cardiovascular and other chronic diseases. The common cellular stress assay (CSA) based on Seahorse technology in peripheral blood mononuclear cells (PBMCs) shows inconsistent results, which prevents its use as a biomarker for the progression of chronic diseases. Therefore, the aim of this study was to investigate potential factors that affect the CSA in PBMCs. We measured the CSA parameters in PBMCs from study participants and compared the results according to the potential factors, namely, the PBMC isolation method, age, seasonal variation and the gender of the study participants. PBMCs were isolated by OptiPrep® and RobosepTM-S methods. PBMCs isolated with the OptiPrep method showed much higher extracellular acidification and higher respiration compared to Robosep-isolated cells. Moreover, OptiPrep-isolated cells showed a higher number of outliers for the proton production rate (PPR) and a high respiratory quotient, indicating impurities with other cells, such as platelets, and technical inconsistencies. PBMCs from older individuals showed higher maximal respiration, spare capacity and extracellular acidification than younger participants. Additionally, in winter, maximal respiration and spare capacity decreased. From spring until early autumn, spare capacity and maximal respiration continuously increased. Elderly males also showed higher basal respiration, spare capacity and extracellular acidification than females. In conclusion, the findings of this study clearly demonstrate that the results of CSA parameters measured in PBMCs are influenced by the PBMC isolation method, age, seasonal variation and gender. Therefore, we recommend that researchers and physicians properly interpret the results of CSA parameters in PBMCs by considering these factors. It is important to use separate CSA evaluation standards based on the isolation method, age, gender and season-dependent factors. To assess the cellular stress situation in PBMCs, both extracellular acidification and mitochondrial respiration should be taken into account. Further study of additional factors, such as mitochondrial mass, should be conducted to improve the measurement of CSA parameters for the assessment of the real mitochondrial fitness.

External Quality Assurance programs for processing methods provide evidence on impact of preanalytical variables

An annual External Quality Assurance (EQA) program has been provided to processing laboratories over the last ten years, allowing them to assess the performance of their processing methods, such as nucleic acid extractions or peripheral blood mononuclear cell (PBMC) isolation and cryopreservation. The objective of this study was to perform a global analysis on almost 1000 EQA scheme/participant data in order to assess (i) the impact of critical preanalytical factors on quantitative or qualitative attributes of different types of specimens and (ii) laboratory performance pattern over time. Statistical analysis was performed within each EQA scheme based on categorized preanalytical data provided by the participants and on centralized measurements of relevant quality attributes of the produced specimens (z-scores): DNA, cell-free (cf)DNA or RNA extraction from blood, DNA or RNA extraction from formalin fixed tissue, DNA or RNA extraction from frozen tissue, DNA extraction from saliva or stool, viable PBMC isolation and cryopreservation. The most critical preanalytical factors in nucleic acid extraction schemes were the nucleic acid extraction method and kit, the elution buffer, the enzymes used during extraction, the input material quantity and the storage temperature. Several indications of laboratory performance improvement over time could be seen. The conclusions are that EQA for processing methods provides unique evidence-based insights into the impact of preanalytical factors and the comparative performance of different processing methods and kits, while supporting laboratories in validating their processing methods.

Immunopharmacological Properties of VitD3: 1, 25VitD3 Modulates Regulatory T Cells and Th17 Cells and the Cytokine Balance in PBMCs from Women with Unexplained Recurrent Spontaneous Abortion (URSA).

VitD3 may contribute to a successful pregnancy through modulation of immune responses. Therefore, VitD3 deficiency may have a role in the immunopathogenesis of unexplained recurrent spontaneous abortion (URSA). However, the mechanisms of immunomodulatory actions of VitD3 in decreasing the risk of recurrent spontaneous abortion have not been understood well. The purpose of this research was to investigate the influence of 1,25VitD3 on regulatory T cells /Th17 axis, the gene expressions and concentrations of related cytokines including, TGF-β, IL-10, IL-6, IL-23, and IL-17A in peripheral blood mononuclear cells (PBMCs) of healthy women as a control group and women with URSA. Isolation of PBMCs was performed from peripheral blood of the subjects of the studied groups (20 women with URSA as a case group, and 20 control women). The effects of 1,25VitD3 (50 nM, for 24 hours) on the studied parameters were evaluated and were compared to the positive and negative controls in vitro. Flow cytometry analysis was used to determine the percentages of regulatory T cells and Th17 cells. For gene expression measurement and cytokines assay, Realtime PCR and ELISA were carried out. The proportion of regulatory T cells was markedly lower, while the proportion of Th17 cells in women with URSA was considerably higher than in the control group (P=0.01, P=0.01). The ratio of the frequency of Tregs to the baseline (1,25VitD3/Untreated) increased, while the ratio of the frequency of Th17 cells to the baseline decreased in women with URSA relative to the controls (P= 0.01, P=0.04). 1,25VitD3 increased IL-10 expressions at both the protein and mRNA levels in PBMCs in women with URSA relative to the control group (P=0.0001, P=0.04). TGF-β levels in the cultured supernatants decreased significantly in the case group in the presence of 1,25Vit- D3 relative to the controls (P=0.03). 1,25VitD3 treatment also significantly decreased gene expressions of IL-6, IL-17A, and IL-23 in PBMCs of women with URSA (P=0.01, P=0.001, P=0.0005), as well as the levels of those cytokines in cell culture supernatants (P=0.03, P=0.02, P=0.01, respectively) in women with URSA relative to the controls. According to the findings of this research, modulation of immune responses by 1,25VitD3 is accomplished by strengthening Tregs function and inhibiting inflammatory responses of Th17 cells, which may have a positive impact on pregnancy outcome. Thus, as an immunomodulating agent, VitD3 may be effective in reducing the risk of URSA.

The peripheral blood mononuclear cells preparation and the hematology of Varanus salvator.

The objectives of this study were to evaluate the proper anticoagulants coated in blood-collecting tube for the peripheral blood mononuclear cells (PBMCs) isolation and to evaluate the proper culture temperature for the Varanus salvator's PBMCs, in addition, the hematological characteristics also reported. The heparin treated blood (n = 10) and EDTA treated blood (n = 10) from Varanus salvator were obtained for PBMCs evaluation. The PBMCs obtained from the heparin treated blood was significantly higher than that of EDTA treated blood during the culture period (P < 0.05) indicated heparin would be more appropriated anticoagulant for blood collection. The PBMCs cultured under 37°C and 27°C were not significantly difference on first three days but 37°C showed significantly higher effect in the following days (P < 0.05) indicated both temperatures can be used which 37°C should be an optimal for PBMCs preparation. The peripheral blood cells of Varanus salvator (n = 49) were analyzed for hematological profiles and characteristics which the number of erythrocytes 1.19 ± 0.04 x 1012/L (1.17-1.35 x 1012/L) and WBC 2.41 ± 0.13 x 109/L (2.29-2.81 x 109/L) with absolute differential count of heterophils 0.92 ± 0.02 x 109/L (0.87-0.95 x 109/L), lymphocytes 1.17 ± 0.01 x 109/L (1.15-1.23 x 109/L), azurophils 0.40 ± 0.01 x 109/L (0.37-0.42 x 109/L), basophils 0.000 ± 0.001 x 109/L (0.004-0.011 x 109/L) and monocytes 0.027 ± 0.002 x 109/L (0.028-0.039 x 109/L). These results would play an important role on the cell immunological studies of the Varanus salvator in the future.

PB2000: LESSONS LEARNED FROM CERBA RESEARCH CHIMERIC ANTIGEN RECEPTOR T-CELL (CAR T) MULTIPLE MYELOMA RUNNING TRIALS

Background: The pipeline has 359 liquid tumor trials that are CAR T driven where Cerba Research (CR) understands that there is a demand to provide innovative specialty testing, such as flow cytometry, sequencing and downstream biomarker analysis. Herein, CR examined 38 of our active multiple myeloma (MM) trials since 2016 with a focus on CAR T therapy. We identified 15 of those trials where CR participated as a central laboratory. Aims: The aim of this evaluation is to identify gaps within current CAR T MM trial endpoints and to improve for the future of our liquid tumor trials. Methods: We analyzed internal trial matrix database and examined other documents such as laboratory specification documents, trial design, protocol, and schedule of activities. We used descriptive statistics to illustrate our specialty testing involvement and to identify gaps in our offerings. Results: CR currently has 38 running MM trials, in which 15 (39%) have a CAR T therapy. Sixteen (42%) of our MM trials are phase I/first-in-human (FIH), whereas 14 (37%) and 7 (18%) are phase II and III, respectively. Within our MM CAR T trials, we perform flow cytometry (FCM) in 87% of cases. Our involvement in FCM is exploratory in nature, and the design of the panels is performed with continued scientific discussions with sponsors. Some of the flow cytometry panels performed in our CAR T trials are: - Enumeration of CAR T cells in whole blood and bone marrow aspirates, including PK evaluation - Immunophenotyping of CAR T cells (memory, regulatory, activation status, exhaustion status) - Minimal residual disease (MRD) CR is often involved across full CAR T programs starting at FIH up to registration trials. We have robust upstream capabilities where we perform peripheral blood mononuclear cell (PBMC) isolation (60%), bone marrow mononuclear cell (BMMC) isolation (53%), DNA extraction (47%), cell sorting (53%), serum and urine protein electrophoresis/immunofixation (47%), and histopathology (decalcification, embedding, sectioning) (53%). We are also involved in routine testing such as hematology (27%), biochemistry (33%) and coagulation (27%). However, except for FCM (87%), immunohistochemistry (IHC 47%) and FISH (47%), we identified a gap in requests for sequencing within MM CAR T trials (0%.) Next generation sequencing (NGS), which was mostly for MRD assessment, was performed by referral labs. We also identified up to 11 referral labs participating in MM trials which significantly adds to a trial operation complexity. Summary/Conclusion: CR currently has strong FCM fit-for-purpose design and execution capabilities as demonstrated by our participation in CAR T programs as a central laboratory. Using FCM in CAR T trials is important as it can aid in determining effectiveness of therapy, any potential side effects, and general health of the immune system by performing longitudinal monitoring of the immune system. We also frequently isolate PBMCs/BMMCs, extract DNA and perform histopathology on a global scale for clinical trial purposes. Herein, we determined that we have good downstream efforts with FCM, IHC and FISH and plan on growing NGS offerings by creating a new MRD NGS oncopanel and compare it with FCM for validation and concordance in multiple myeloma.

Новая стратегия поиска гормональной терапии пролиферативных процессов эндометрия у пациенток в постменопаузе

The limited efficacy of hormone therapy for endometrial proliferative process (EPP) in postmenopausal patients and its side effects on the immune system functionalities have not been studied in detail. Here we assess the feasibility of hormone therapy for EPP in postmenopausal patients through evaluation of estradiol and progesterone receptor gene expression in endometrial tissue and peripheral blood mononuclear cells (PBMC). The study enrolled 92 postmenopausal patients with EPP, including 37 pts with glandular-fibrous polyps, 7 pts with non-atypical endometrial hyperplasia (EH), 8 pts with atypical endometrial hyperplasia (AEH), 31 pts with moderately differentiated adenocarcinoma and 9 pts with highly differentiated adenocarcinoma. The PBMC isolates and endometrial samples were tested for ER⍺, ERβ, mER, PRA, PRB, mPR and PGRmC1 expression by reverse real time polymerase chain reaction (RT–PCR). Differential changes in PBMC receptor profiles upon in vitro exposure to progesterone or mifepristone were determined for patients with endometrial polyps and healthy women. The results indicate elevated expression of ERα, ERβ, PRA, PRB, mPR and PGRmC1 by endometrial tissues in EH and elevated expression of mER, ER⍺ and PRA by PBMC in AEH, apparently reflecting suppressed functionalities of monocytes, macrophages, Т-cells and natural killer cells. Unaltered expression of the studied genes by PBMC in endometrial adenocarcinoma may reflect the incrementing tumor autonomy. In vitro, mifepristone inhibited ER⍺, ERβ, mPR, PGRmC1, PRA and PRB expression in PBMC isolated from patients with endometrial polyps. We suppose that such effects can mitigate the negative influence of sex steroid hormones on immunocompetent cells.

Investigation of the Toll-like receptors expression profile in peripheral blood mononuclear cells in COVID-19 patients in Iran

Introduction: Coronavirus disease 2019 (COVID-19) spreads all around the world and leads to several new infection cases and mortality. A better understanding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis could lead to more efficient therapeutic approaches. Objectives: The current study aimed to investigate the Toll-like receptors expression profile in peripheral blood mononuclear cells in COVID-19 patients in Iran. Patients and Methods: In this cross-sectional study, we evaluated 32 COVID-19 patients. At the admission time based on the disease severity patients were divided into two groups of severe and mild COVID-19. A group of 16 normal people was evaluated as the healthy control. Blood samples were collected before any treatment from patients at the admission time. Peripheral blood mononuclear cell isolation preformed on blood samples. RNA is extracted from human peripheral blood mononuclear cells and use for the cDNA synthesis. The expression level of TLR-3, 4, and 7 were evaluated by using semi-quantitative real-time polymerase chain reaction (PCR). Results: The patient’s mean age was 57.12±3.08 years and 15 (47%) were male. The background disorders represent two patients in the severe group with respiratory disorders, four patients with cardiovascular disease, and seven patients with diabetes. The TLRs expression levels represent a higher expression of TLR-4 in COVID-19 in comparison with controls for TLR-4 (P=0.007), TLR-3 and 7 also represent up-regulation however there were no statistically significant differences in the relative fold changes (RFCs) between groups for all other evaluated TLRs (P&gt;0.05). Conclusion: The study highlights the importance of the TLR-4 in COVID-19 patients in expression level. Further studies for a clear conclusion about TLRs expression levels are recommended.

The effect of hyperglycaemia on the macrophages in the cell culture.

The availability of glucose in tissue has a role in macrophages polarisation into an inflammatory phenotype. The overnutrition condition such as hyperglycaemia induces macrophage infiltration especially the inflammatory macrophages. The aim of this study was to analyse the effect of hyperglycaemia condition on cytokines production by human monocytes-derived macrophages. Monocyte cells obtained from peripheral blood mononuclear cells isolation from donors were incubated for 6 days in 37°C, 5% CO2. On day 4, the stimulating factors such as lipopolysaccharide (LPS) and interferon gamma were added to activate monocytes into macrophages. Then, on day 6, two doses of glucose; either normal or high doses along with low or high dose of LPS were given for 24 h, followed by collecting the culture media and cells then stored at -80°C until assayed. There was a significant difference in tumour necrosis factor alpha (TNF-a) levels among groups, with highest level found in group with high-dose glucose plus high-dose LPS. However, the concentration of interleukin-6 (IL-6) among groups was not significantly different. Macrophages treated with high-dose glucose plus high-dose LPS significantly increased production of TNF-a, but not of IL-6.