Ficoll Density Gradient Centrifugation Research Articles

Population pharmacokinetics of tacrolimus whole blood and peripheral blood mononuclear cell concentrations in stable kidney-transplanted patients.

Therapeutic drug monitoring of tacrolimus based on whole blood drug concentrations is routinely performed. The concentration of tacrolimus in peripheral blood mononuclear cells (PMBCs) is likely to better reflect drug exposure at the treatment target site. We aimed to describe the relationship between tacrolimus whole blood and PBMC concentrations, and the influence of patient characteristics on this relationship by developing a population pharmacokinetic model. We prospectively enrolled 63 stable adult kidney-transplanted patients and collected dense (12-h, n = 18) or sparse (4-h, n = 45) pharmacokinetic profiles of tacrolimus. PBMCs were isolated from whole blood (Ficoll density gradient centrifugation), and drug concentrations in whole blood and PBMCs were analysed using liquid chromatography-mass spectrometry. Patient genotype (CYP3A4/5, ABCB1, NR1I2) was assessed with PCR. Population pharmacokinetic modelling and statistical evaluation was performed using NONMEM. Tacrolimus whole blood concentrations were well described using a two-compartment pharmacokinetic model with a lag-time and first-order absorption and elimination. Tacrolimus PBMC concentrations were best estimated from whole blood concentrations with the use of a scaling factor, the ratio of whole blood to PBMC concentrations (RC:PBMC), which was the extent of tacrolimus distribution into PBMC. CYP3A5*1 non-expressors and NR1I2-25 385T allele expressors demonstrated higher RC:PBMC ratios of 42.4% and 60.7%, respectively. Tacrolimus PBMC concentration could not be accurately predicted from whole blood concentrations and covariates because of significant residual unexplained variability in the distribution of tacrolimus into PBMCs and may need to be measured directly if required for future studies.

Effect of TLK2 Expression Regulated by MiR-21 on Proliferation and Apoptosis of Acute Myeloid Leukemia Cells

To investigate the effect of TLK2 expression regulated by miR-21 on proliferation and apoptosis of acute myeloid leukemia cells. Seventy patients with AML admitted to our hospital from January 2019 to July 2022 were selected, while 30 patients with iron deficiency anemia were selected as the control group. Bone marrow mononuclear cells (BMMNCs) of the patients were obtained using Ficoll density gradient centrifugation. RT-qPCR was used to determine the expression levels of miR-21 and TLK2 mRNA in BMMNCs. Mimics-miR-21, mimics-NC, inhibitor-miR-21, inhibitor-NC and NC were transfected into HL-60 cells using liposome-mediated transfection technology. CCK-8 method was used to determine the activity of transfected HL-60 cells after treatment with cytarabine. The apoptosis rate of HL-60 transfected cells was determined by TUNEL method. The expression of TLK2 mRNA in HL-60 cells transfected with inhibitor-miR-21 was determined by RT-qPCR. The relative expression levels of miR-21 and TLK2 mRNA in BMMNCs of AML patients were significantly higher than those of controls (both P < 0.05). After HL-60 cells were treated with cytarabine, both the cell activity of inhibitor-miR-21 group and mimics-miR-21 group decreased significantly with the increase of cytarabine concentration (both P < 0.05). However, at each concentration point of cytarabine, the cell activity of inhibitor-miR-21 group was lower than that of control group (P < 0.05), while mimics-miR-21 group was higher than control group (P < 0.05). After HL-60 cells were treated with cytarabine, the apoptosis rate of inhibitor-miR-21 group was significantly increased (P < 0.05), while that of mimics-miR-21 group was significantly decreased (P < 0.05). After HL-60 cells were treated with inhibitor-miR-21, the relative expression of TLK2 mRNA decreased significantly (P < 0.05). miR-21 is highly expressed in AML patients, which may promote the apoptosis of AML cells by inhibiting the expression of TLK2.

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation.

Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200g, 300g and 400g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. The 300g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. 300g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Label-Free CD34+ Cell Identification Using Deep Learning and Lens-Free Shadow Imaging Technology.

Accurate and efficient classification and quantification of CD34+ cells are essential for the diagnosis and monitoring of leukemia. Current methods, such as flow cytometry, are complex, time-consuming, and require specialized expertise and equipment. This study proposes a novel approach for the label-free identification of CD34+ cells using a deep learning model and lens-free shadow imaging technology (LSIT). LSIT is a portable and user-friendly technique that eliminates the need for cell staining, enhances accessibility to nonexperts, and reduces the risk of sample degradation. The study involved three phases: sample preparation, dataset generation, and data analysis. Bone marrow and peripheral blood samples were collected from leukemia patients, and mononuclear cells were isolated using Ficoll density gradient centrifugation. The samples were then injected into a cell chip and analyzed using a proprietary LSIT-based device (Cellytics). A robust dataset was generated, and a custom AlexNet deep learning model was meticulously trained to distinguish CD34+ from non-CD34+ cells using the dataset. The model achieved a high accuracy in identifying CD34+ cells from 1929 bone marrow cell images, with training and validation accuracies of 97.3% and 96.2%, respectively. The customized AlexNet model outperformed the Vgg16 and ResNet50 models. It also demonstrated a strong correlation with the standard fluorescence-activated cell sorting (FACS) technique for quantifying CD34+ cells across 13 patient samples, yielding a coefficient of determination of 0.81. Bland-Altman analysis confirmed the model's reliability, with a mean bias of -2.29 and 95% limits of agreement between 18.49 and -23.07. This deep-learning-powered LSIT offers a groundbreaking approach to detecting CD34+ cells without the need for cell staining, facilitating rapid CD34+ cell classification, even by individuals without prior expertise.

Cold Mechanical Isolation of Placental Macrophages as a Method to Limit Procedure-Induced Activation of Macrophages.

Isolation of placental macrophages using enzymatic digestion at warm temperatures is widely used for invitro studies. However, studies in brain and kidney tissue show that this method activates immune cells, immediate early genes, and heat shock proteins. Isolating placental macrophages while preserving their tissue-specific characteristics as much as possible is pivotal to reliably studying their functions. We therefore developed a mechanical dissociation protocol at low temperatures and compared this to enzymatic digestion at high temperatures. Decidual and villous macrophages were isolated from term human placentas. A cell suspension was generated by mechanical dissociation using a gentleMACS. For warm enzymatic digestion, Accutase was added, followed by incubation at 37°C. Macrophages were isolated after Ficoll density gradient centrifugation. Cell types were analyzed with flow cytometry (CD45, CD14, CD80, CD86, CD163, and CD206) and their activation status with real-time PCR (FOS, JUN, HSP27, HSP70, IL1β, TNFα, IL10, and TGFβ) after cell sorting. A higher proportion of leukocytes and macrophages was obtained from the villi with cold mechanical dissociation (p < 0.05). Compared to warm enzymatic digestion, cold mechanical dissociation resulted in a higher expression of CD163 in villous and decidual macrophages (p < 0.05). Warm enzymatic digestion showed higher levels of TNFα, IL1β, and IL10 in decidual and villous macrophages, and HSP70 in villous macrophages. Our data show that mechanical dissociation of placental tissue at low temperatures is associated with less activation of placental macrophages. This suggests that cold mechanical dissociation is a preferred method, resulting in macrophages that more closely resemble their in-tissue state.

Peripheral pre-existing cancer-antigen specific T-cell immunity as predictive biomarker for immunotherapy in NSCLC patients.

e20504 Background: Pre-existing Tumor Antigen specific T-cells describe the endogenous adaptive immunity before any treatment that may represent a valuable novel predictive biomarker for ICI treatment. We investigate the potential value of pre-existing cancer-antigen specific CD8+ T-cells as a circulating predictive biomarker. Additionally, we analyze the major differences of known immune-cell phenotypes between Pre-existing positive (PreI+) and Pre-existing negative immunity (PreI-) NSCLC patients. Here, we present the preliminary results of this study. Methods: Blood was collected from 24 patients with NSCLC, stage III PD-L1+, after completion of radical chemoradiation, before initiation of maintenance durvalumab. PBMCs were isolated with Ficoll density gradient centrifugation from patients and 10 healthy donors (HD). PreI was calculated by detecting endogenous IFNg expressing cells with FACS after in-vivo co-cultures of PBMCs with mixes of hTERT, MAGEA1, NY-ESO-1 και Survivin cancer-associated antigens. Immunophenotyping was performed by multi-color flow cytometry using antibodies against CD3, CD4, CD8, CD45RA, CD45RO, CCR7, PD-1, PD-L1 for T-cells. Results: From 24 patients receiving Durvalumab, 10/24 (41.6%) had peptide specific T-cells (PreI + patients), 60% (6/10) had detectable peptide specific T cells for all (4/4) antigens tested. Survival analysis revealed better PFS in PreI+ than PreI – patients (Log-rank = 0.06, median 333.5 and 185 days ) and better OS (p = 0.032, median not reached and 254 days). The levels of CD3CD4PD-1 were higher in patients compared to HD (p = 0.019) but there was not a difference between PreI+ and PreI- patients (p = 0.25). PreI+ patients had significantly higher numbers of CD3CD8PD-1 cells compared to PreI- (p = 0.024) but not CD3CD8PD-L1. Prei+ patients had high numbers of Teff (CD3+CD8+CD45RA+CD45RO-CCR7) compared to both HD (p = 0.0039) and PreI- (p = 0.032). Conclusions: Pre-existing tumor antigen specific immunity before initiation of ICI in NSCLC patients could serve as a good predictive factor of response. The study is ongoing.

Combinatorial analysis of CD4+Tregs and CD8+Teff to predict response to ICI in patients with ES-SCLC.

e20633 Background: Recently, the antiPD-L1 agent, durvalumab, was approved in first line treatment in combination with chemo. However, there is not a clear benefit from this regimen for the majority of patients. In this study, we explored the predictive role of regulatory T-cells in response of patients with ES-SCLC treated with the aforementioned regimen. Moreover, we evaluated the potential role of inflammatory hematological indices such as Neutrophil to Lymphocyte ratio (NLR) and CRP levels to predict responders. Methods: Blood was collected before initiation of immunotherapy from 24 patients with ES-SCLC. PBMCs were isolated with Ficoll density gradient centrifugation from patients and 1O healthy donors (HD). Immunophenotyping was performed by multi-color flow cytometry using antibodies against CD3,CD8,CD45RA,CD45RO,CCR7,PD-1, for CD8 T-cells and CD3,CD4,FoxP3,CD25,CD127,CTLA-4,CD39 for Tregs. Results: High percentages of CD39+ (p = 0.0061) and CTLA-4+ (p = 0.012) Tregs were detected in circulation of ES-SCLC compared to healthy individuals. Additionally, higher percentages of CD8+ Teff were detected in SCLC patients (p = 0.027). Patients with high numbers of Teff had significant higher PFS (p = 0.021, median 183 and 97 days) and better OS (p = 0.075, median 318 and 202 days) in response to immunotherapy compared to those with low numbers. Spearman analysis revealed a significant negative correlation between Tregs and Teff (Spearman r2 = -0.39,p = 0.043) that was accompanied with significant higher PFS (p = 0.041, median 207 and 130 days ) and OS (p = 0.029, median 318 and 202 days ) in patients with high levels of Teff and low Tregs comparing to the rest. Further analysis on inflammatory clinical signatures demonstrated that patients with better clinical response to immunotherapy have significantly lower NLR (p = 0.034) while no significant difference was revealed for CRP (p = 0.401) levels before therapy initiation. Conclusions: Combinatorial analysis of Tregs and Teff cells may be used as a predictive tool for response to immunotherapy in ES-SCLC patients.

Abstract P5-05-07: Assessment of immune checkpoint expression on the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC)

Abstract Background: Growing body of evidence highlights the role of the peripheral anti-tumor immune response in patients with solid tumors. Peripheral blood mononuclear cells (PBMCs) comprise all the key circulating immune cell subsets, and their analysis may inform on the peripheral anti-tumor immune response status in real-time. Programmed death-ligand 1 (PD-L1), toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) hold a key role in the cancer-associated inflammation and tumor immune evasion. We herein aimed to investigate the distribution of these molecules on PBMCs and their prognostic value among patients with breast cancer (BC). Methods: Peripheral blood (PB) was obtained from patients with early (n=99) and metastatic (n=99) BC, prior to the initiation of adjuvant and first-line treatment, respectively. PBMCs were isolated through ficoll density gradient centrifugation and PBMC cytospins were immunofluorescently stained using PD-L1, TLR4 and phosphorylated STAT3 (pSTAT3) antibodies. MDA.MB.231 BC cells served as controls to define the positivity of PBMCs for the respective markers via fluorescence microscopy. Results: PD-L1, TLR4 and pSTAT3 expression was identified on PBMCs of 27.8%, 27.1%, and 83.9% of all patients, respectively. The mean (± standard error of mean, SEM) percentage of positive PBMCs per patient was 13.8% ±1.8%, 10.5% ±1.6% and 37.1% ±1.9, respectively. A positive correlation was shown between the PD-L1pos, TLR4pos and pSTAT3pos PBMC proportions (PD-L1*TLR4; p=0.000, PD-L1*pSTAT3; p=0.001, TLR4*pSTAT3; p=0.002, Spearman’s rho analysis). Patients with metastatic disease displayed increased TLR4pos PBMC percentages as compared to early disease (mean: 15.8% versus 5.2%; p=0.008, Mann Whitney U test). In the early BC setting, the detection of TLR4pos PBMCs was associated with reduced disease-free survival (DFS; median: not reached; p=0.020), while PD-L1pos PBMCs were correlated to shorter overall survival (OS; median: not reached; p=0.009). Early BC patients with PD-L1pos/TLR4pos PBMCs showed significantly reduced survival times (median DFS: not reached; p=0.016; median OS: not reached; p=0.004, Kaplan Meier analysis). Conclusions: PD-L1, TLR4 and pSTAT3 molecules are frequently expressed on PBMCs of patients with BC and provide significant prognostic information for early-stage BC patients. The role of the immune-phenotyping of PBMCs as a source for biomarker discovery merits further investigation in BC. Citation Format: Maria A. Papadaki, Alexia Monastirioti, Christina Α Apostolopoulou, Sofia Agelaki, Dimitrios Mavroudis. Assessment of immune checkpoint expression on the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-07.

Bioenergetic changes and mitochondrial dysfunction in mania versus euthymia in bipolar disorder type I

IntroductionCurrent evidence has hypothesized the involvement of mitochondrial dysfunction during the acute episodes of BD compared to symptomatic remission. So far, no studies have compared mitochondrial and bioenergetic functions both in-vivo (respiratory parameters) and ex-vivo (cellular respiration) in different phases of the disease in the same individuals.ObjectivesThis multidisciplinary pilot study aims at assessing bioenergetic and mitochondrial intra-individual differences between manic and euthymic states.MethodsFour patients with a manic episode admitted to our acute psychiatric ward were recruited. Bioenergetic parameters were measured at admission (T0) and after symptomatic remission (T1).At admission (T0) and before discharge (T1), HAMD and YMRS total scores were obtained. For the assessment of cellular respiration, polymorphonuclear cells were obtained by a Ficoll density gradient centrifugation procedure. To determine oxygen consumption (at T0 and T1), a million of living peripheral blood mononuclear cells (PBMC) were used. High-resolution respirometry was performed at 37°C by polarographic oxygen sensors in a two-chamber Oxygraph-2k system.Specific oxygen uptakes (Routine: basal oxygen consumption; Proton Leak: oxygen consumption not coupled to ATP synthesis; and ETC: maximal capacity of the electron transport chain) rates were obtained using mitochondrial chain inhibitors and uncouplers. Oxygen consumption was normalized for protein concentration. Results are expressed as picomoles of oxygen per millilitre (pmol O2/s*μg prot).Also, a constant work rate exercise test was performed on a cycle ergometer and basal and effort respiratory variables were measured.Statistical analysis was performed with the SPSS v. 25.0 and GraphPad. Results were expressed as means and SD. Nonparametric tests (Mann–Whitney, Pearson) were used to determine differences (significant at p value &lt;0.05).ResultsOne patient was a man and three patients were women, with a mean age of 28 years old. HAMD initial and final mean scores were 11.0 and 7.0, and mean YMRS scores were 21.5 and 7.0 respectively.Results from mitochondrial oxygen consumption revealed that mean basal oxygen consumption tended to be higher in T1 (0.98±0.45) than in T0, and maximal respiratory capacity was significantly increased in T1 (2.26±0.33; p=0.028) compared to T0.Mean lactate levels and pH levels were similar in T0 and T1. Scales scores were not correlated to different pH or lactate changes after the effort task. Higher initial oxygen consumption was significantly correlated to higher maximal capacity (p&lt;0.05) in T0 and T1.Image:ConclusionsOur preliminary results suggest that mania could imply lower oxygen consumption capacity, which should be confirmed in future studies. A bigger study is planned to determine changes in bioenergetic patterns and capacity for aerobic response in manic and depressive episodes.Disclosure of InterestNone Declared

Mechanism of human peripheral blood-derived dendritic cells in the regulation of autophagy in carcinoma cells.

Dendritic cells (DC) are involved in immune recognition, response and immunomodulation mechanisms related to the onset of cancer. To explore DCs mechanism in the inhibition of autophagy in hepatoma cells. Human peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and induced into DCs, which were co-cultured with HepG2 cells by Transwell migration assay. HepG2 cell activity was determined using the CCK8 assay. LC3 autophagy index expression was measured with Western blot analysis, and the expression and secretion of cytokines, with qRT-PCR and ELISA. In the co-culture system, DCs were able to reduce HepG2 cells viability; IL-2, IL-12, IL-10 and IFN-γ expression in DCs was also significantly inhibited, although IL-2 and IFN-γ were still expressed 0.6 and 0.53 more than in the control group. DCs can regulate autophagy in hepatocellular carcinoma cells. The mechanism may be related to the synthesis and release of cytokines such as IL-2, IL-12 and IFN-γ by DCs.

Expression of scavenger receptors CD163, CD204, and CD206 on macrophages in patients with pulmonary tuberculosis

The aim of the study was to evaluate the expression of scavenger receptors (CD163, CD204, CD206) on macrophages in patients with pulmonary tuberculosis, depending on the clinical form of the disease and sensitivity of the pathogen to anti-tuberculosis drugs.Materials and methods. 64 patients with pulmonary tuberculosis (TB) were examined: 26 patients with disseminated pulmonary tuberculosis (DTB) and 38 patients with infiltrative pulmonary tuberculosis (ITB). Of these, 42 patients secreted Mycobacterium tuberculosis (MBT) sensitive to basic antituberculosis drugs (ATBD), and 22 patients secreted MBT resistant to first-line anti-TB drugs. Material for the study was venous blood. To isolate monocytes from the whole blood in order to transform them into macrophages, Ficoll density gradient centrifugation with a density of 1.077 g / cm3 was used followed by immunomagnetic separation of CD14+ cells. Monocytes were cultured in the X-VIVO 10 medium with gentamicin and phenol red with the addition of macrophage colony-stimulating factor (M-CSF) (5 ng / ml) at a concentration of 1×106 cells / ml with stimulators: interleukin (IL)-4 (10 ng / ml) and interferon (IFN) γ (100 ng / ml). Immunophenotyping of macrophages was performed using monoclonal antibodies to CD163, CD204, and CD206 on the Beckman Coulter CytoFLEX LX Flow Cytometer. The analysis of the obtained data was carried out using the CytExpert 2.0 software. The results were analyzed using statistical methods.Results. Switching the phenotype of macrophages from the M1-like proinflammatory phenotype to M2-like antiinflammatory one contributes to the chronic course of pulmonary TB, dissemination, and persistence of infection. In the present study, we analyzed the features of the expression of CD163, CD204, and CD206 scavenger receptors on macrophages in patients with pulmonary TB. An increase in the number of macrophages carrying markers of the M2 subpopulation (CD163, CD204, and CD206) on their surface was noted, regardless of the clinical form of pulmonary TB and drug resistance of M. tuberculosis.Conclusion. Studying the mechanisms underlying M1 or M2 activation of macrophages is necessary for a deeper understanding of the immunopathogenesis of TB and the role of innate immunity cells in protecting the body from mycobacteria. The analysis of the expression of scavenger receptors CD163, CD204, and CD206 on macrophages allowed to conclude that, in pulmonary TB, especially in patients with drug resistant M. tuberculosis and infiltrative TB, regulatory mechanisms that suppress the activation of innate immunity are implemented together with polarization of macrophage differentiation towards the M2 phenotype. It may be the cause of immune deficiency induced by the pathogen.

IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata

IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata

An approach for vacuole isolation and vacuolar protein identification by proteomics in apple

ABSTRACT Apple, one of the most common fruits consumed worldwide, has a high economic value. Vacuoles are membrane-bound organelles in plant cells that perform many important functions. An increasing number of studies have reported a variety of proteins in a vacuole, with crucial functions in the transport, storage and metabolism of energy and substances. Here, we successfully extracted the vacuoles and vacuolar proteins from the ‘Orin’ apple fruit calli. We collected intact vacuoles with a density of 100/mm2 by mild osmotic lysis and Ficoll density gradient centrifugation. The result of SDS-PAGE showed that lots of vacuolar proteins were isolated and enriched from vacuole samples, and western blotting showed that the purity of the vacuolar protein was high. Furthermore, we showed GO enrichment and KEGG enrichment of isolated vacuolar proteins and selected 41 vacuolar proteins with known functions and 10 vacuolar proteins with unknown functions for functional annotation from the mass spectrometry analysis. This approach for vacuole isolation and vacuolar protein identification will provide a theoretical basis for further study of the function of vacuoles and vacuolar proteins.

COMPARISON OF DIFFERENT METHODOLOGIES FOR EXPANSION OF NATURAL KILLER CELLS FROM PERIPHERAL BLOOD AND UMBILICAL CORD BLOOD FOR IMMUNOTHERAPY APPLICATIONS

In Immunotherapy, several clinical studies with Natural Killer (NK) cells have shown promising results in the treatment of onco-hematological diseases due to their high cytotoxicity against tumor cells. There are several protocols for the expansion of these cells, but there is no consensus of a gold standard protocol that provides large numbers of functional NK cells required for conducting clinical studies. Thus, we propose the development of a protocol for the expansion of these cells with the subsequent intention of applying them as cell therapy product in the Hematology Service of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP). Our aim was to compare different methodologies and establish a standardized protocol for the isolation and expansion of NK cells from peripheral blood (PB) and umbilical cord blood (UCB). We also characterized the expanded cells according to their functionality and cytotoxicity. The experiment was carried out at the department of Hematology and Cell Therapy of the HC-FMUSP, in the Laboratory of Medical Investigation (LIM-31), fresh PB samples were obtained from Fundação Pró-Sangue leukapheresis healthy donors and the cryopreserved UCB units were obtained from Hospital Sírio-Libanês Umbilical Cord Blood bank. Mononuclear cells were isolated by Ficoll density gradient centrifugation. The isolation and expansion of UCB-derived and PB-derived NK cells was performed with the selection of CD56+ CD3- cells by immunomagnetic separation. Cells were expanded ex vivo for 14 days in RPMI-1640 medium supplemented with different combinations of cytokines, such as interleukin IL-2 alone, IL-2 and IL-21, IL-15 and IL-18 and IL-12, co-cultured or not with CSTX002 feeder cells in a 1:2 ratio (NK:aAPC). The number of NK cells increased significantly when they were co-cultured with aAPC in comparison to conditions without feeder cells. Pre-activation conditions with IL-12, IL-15 and IL-18 for “cytokine-induced memory-like” (ML NK) cells in both PB-NK and UCB-NK expansion yielded a significantly greater fold expansion (12-fold and 8-fold) when compared to expansion with IL-2 alone or IL-2 and IL-21. Moreover, the addition of IL-21 did not have a significant impact in the expansion of CD56+ CD3- cells when compared to conditions that used IL-2 alone. Furthermore, aAPC-mediated expansion yielded a predominantly CD56bright CD16bright mature phenotype in both PB-NK and UCB-NK. To evaluate the functionality NK cells, in vitro assays against tumor target cells was performed and UCB-NK conditions with IL-2 alone or IL-2 and IL-21 showed enhanced degranulation capacity and lytic granules (Granzyme B and Perforin) and cytokine production (IFN-y and TNF-a) than PB-NK cells. ML NK cells from PB produced higher amounts of cytokines and lytic granules when compared to ML NK cells from UCB. This may be related to a lower expression of NKp46, NKG2D and CD69 activation markers in ML NK UCB, and possibly because the UCB units were cryopreserved. The results suggest that ML NK cells show good ex vivo expansion, but do not show proper functionality against tumor cells. It is possible that with the addition of IL-2, ML NK cells could improve the production of cytokines and lytic granules, as seen in the IL-2 alone and IL-2 and IL-21 conditions.

Determination of reference intervals for neutrophil granular enzymes is affected by cell isolation techniques

ObjectivesNeutrophils and their granular enzymes such as neutrophil elastase (NE) and myeloperoxidase (MPO) play important roles in inflammatory diseases, and might be utilized as biomarkers for disease severity and progression. The aim of this study was to determine reference intervals for NE and MPO activity in healthy volunteers comparing two methods of neutrophil isolation. MethodsNeutrophils were isolated using ficoll density gradient centrifugation or immunomagnetic negative selection in two separate volunteers' cohorts. Subsequently, cells were lysed and incubated with chromogens for NE and MPO activity measurements, then measured with a microplate reader at 415 or 450 nm respectively. ResultsThe enzymatic activity of NE and MPO depended on the neutrophil isolation technique. Both enzymatic activities were significantly higher (P < 0.001) after isolating neutrophils with ficoll density gradient centrifugation than using the immunomagnetic negative selection. ConclusionsWe demonstrated that neutrophil isolation is an important factor that influences the outcome of enzymatic activity measurements. Techniques based on immunomagnetic negative selection are favorable, specifically for investigations related to NE and MPO activity. When using NE and MPO activity measurements in clinical practice, care must be taken to interpret the data depending on the applied cell isolation technique.

Platelets correlate with false negative T-SPOT.TB results by inhibiting interferon-γ production in T cells via degranulation.

BackgroundT-SPOT.TB (T-SPOT) is widely used for the detection of Mycobacterium tuberculosis infection by detecting interferon-gamma (IFN-γ) release in T lymphocytes. This assay is performed on peripheral blood mononuclear cells (PBMCs) separated by Ficoll density gradient centrifugation, which often contain some residual platelets. Here, we investigated the impact of platelets on T-SPOT assay and related mechanisms.MethodsThe correlation between platelet count, platelet-to-lymphocyte ratio (PLR), and the IFN-γ secreting T cells (ISCs) in positive control wells of T-SPOT assay were retrospectively analyzed. T-SPOT assay was performed with un-treated PBMCs, platelets-removed PBMCs, and platelets-enriched PBMCs to confirm the impact of platelets on T-SPOT assay. The activation of platelets and their impact on IFN-γ production in T cells were detected by flow cytometry (FCM). Platelets and T cells were cultured in a mixed culture system and co-culture system respectively, followed by detection of the frequencies of IFN-γ-producing T cells and the levels of intracellular IFN-γ in T cells by FCM. Moreover, the effect of platelet releasate on the T-SPOT assay was evaluated.ResultsThe ISCs in positive control wells of the T-SPOT assay showed a significant decrease with the increase in platelet count. The PLR of the peripheral blood were negatively correlated with the ISCs in positive control wells of the T-SPOT assay. Removal or enrichment of platelets significantly increased or decreased the ISCs and the positive rate of T-SPOT. Inhibition of platelet activation significantly increased the ISCs of T-SPOT. The frequencies of IFN-γ-producing T cells in PBMCs and the levels of intracellular IFN-γ were significantly reduced by the addition of platelets, both in the mixed culture system and the co-culture system. Platelet releasate upon thrombin activation significantly decreased the ISCs of T-SPOT.ConclusionsPlatelets correlate with negative T-SPOT results by inhibiting IFN-γ production in T cells via degranulation.

Study of the anti-inflammatory and immunotropic activity of the secretome from multipotent mesenchymal stromal cells induced by erythropoietin, valproic acid or dexamethasone &lt;i&gt;in vitro&lt;/i&gt;

The aim of this study was to evaluate the effect of treatment with valproic acid, erythropoietin, and dexamethasone on the anti-inflammatory and immunosuppressive activity of the secretome of adipose-derived multipotent mesenchymal stromal cells (MMSCs) in an in vitro experiment.Materials and methods. MMSCs were isolated from the fat of 6 healthy donors. The cells were grown in the culture up to passage 4. Then they were treated with valproic acid, erythropoietin or dexamethasone for 3 hours, washed from preparations, and incubated in a serum-free medium for 48 hours. Some of the cells were not treated with preparations. Supernatants from the cell cultures were concentrated by ultrafiltration, and protein standardization was performed using a nanophotometer. Then the supernatants were sterilized and added to mononuclear cells from peripheral blood of 8 healthy donors. The mononuclear cells were isolated by Ficoll density gradient centrifugation according to the standard protocol. Concentrations of TNFα, IL-2, IL-4, IL-6, IL-10, and IFNγ cytokines in 24-hour cultures and IL-9, IL-10, IL-17A, and IL-21 cytokines in 48-hour cultures were determined using multiplex analysis.Results. The production of IL-2, IL-6, TNFα, and IL-10 was reduced by the secretome of MMSCs treated with valproic acid. The production of IL-2, IL-6, and TNFα decreased during incubation of the mononuclear cells with the secretome of MMSCs treated with erythropoietin. The secretome of dexamethasone-treated MMSCs suppressed the production of IFNγ, IL-1β, IL-1ra, IL-2, IL-6, IL-9, IL-10, and IL-17A. No statistically significant differences were revealed in the production of IL-4, IL-5, IL-9, and IL-21.Conclusion. Among the studied inducers, dexamethasone enhanced the anti-inflammatory and immunosuppressive activity of MMSCs the most, which was manifested through the effect of their supernatants on peripheral blood mononuclear cells.

Crocin restores the balance of Th1/Th2 immune cell response in ConA-treated human lymphocytes.

Following antigen stimulation, naive CD4+ T cells differentiate into different T helper (Th) subsets characterized by lineage-specific transcriptional factors and cytokines. The balance between cytokines from Th1 and Th2 cells is disrupted in autoimmune disorders, asthma, and allergic reactions. Crocin, the major carotenoid of saffron, has anti-inflammatory properties. We investigated crocin modifying effects on the human lymphocytes proliferation and Th1/Th2 balance as a possible mechanism of its anti-inflammatory effects. The human peripheral blood mononuclear cells were isolated using Ficoll density gradient centrifugation. MTT was used to evaluate the effect of 72-h treatment with different concentrations of crocin with or without ConA on lymphocytes proliferation. INF-γ/IL-4 cytokine secretion and T-bet/GATA-3 transcription factor expression ratios (as indicators of Th1/Th2 response status) were measured in non-stimulated and ConA-stimulated cells in the presence or absence of crocin byELISA and RT-qPCR methods, respectively. The results showed crocin at a concentration of 50μM and higher was toxic for human lymphocytes, and at a non-toxic concentration of 25µM, it did not affect cell proliferation. The ratio of T-bet/GATA-3 and INF-γ/IL-4 was higher in the culture supernatant of ConA-stimulated cells compared to non-stimulated ones. Crocin-treated cells showed slightly lower T-bet/GATA-3 and INF-γ/IL-4 ratios compared to untreated cells. Crocin (25μM) was also able to restore the increased ratio of Th1/Th2 immune response induced by ConA. Crocin can alleviate inflammatory-stimulant effects of ConA on human lymphocytes by decreasing T-bet/GATA-3 and INF-γ/IL-4 ratios, which are indicative of restoring the balance of Th1/Th2 responses.

Differentiation of Intracellular p16INK4a Expression in the Circulating Human Mononuclear Isolated Cells after ADMA and H2O2 Exposure

p16INK4a (CDKN2A) represent as primary cell-cycle regulation, which arranges the moment of continued or interrupt cell proliferation. Cells expressing p16INK4a accumulate in aging tissue and age-related accumulation DNA damage, yet their physiologic compensated effects in human peripheral blood mononuclear cells (PBMNCs) after different stress inducer are poorly understood. Blood samples were obtained from healthy and non-sedentary lifestyle volunteers. Human mononuclear cells (MNCs) were purified from peripheral blood with Ficoll-density gradient centrifugation subsequently seeded into a medium culture. 80% confluence cells were divided into untreated cells and four treated cells with Asymmetric dimethylarginine (ADMA) and H2O2 in different doses for 24 h. Quantification p16INK4a positive cells were analyzed by FACS. The difference of p16INK4a positive cells after ADMA treated cells, H2O2 treated cells and untreated cells were also analyzed with a statistical test. We found that ADMA and H2O2 treatment in human MNCs induce elevation of p16INK4a (p=0.001), continuous p16INK4a expression incline to increase attendant extension dose of cellular stress inducer (p=0.000). Expression of p16INK4a has been proven higher in ADMA treated cells (1.43% ± 0.21) than control cells (0.34% ± 0.125) after 24h, the number of p16INK4a positive cells tended to increase when the ADMA exposure dose is added at 500µM (2.76% ± 1.39) compare with H2O2 treated cells (1.22% ± 0.33). These findings showed that p16INK4a positive cells are a part of the cellular stress response that results in temporary adaptation to some stressors, and may promote inhibition of inappropriate cell division.

Revisiting miR-155 in intervertebral disc degeneration: blood cell signature and local cell-free profiles

We unraveled the expression profiles of coding-noncoding RNAs in intervertebral disc degeneration (IDD). However, it remains elusive regarding miR-155 expression in peripheral blood mononuclear cells (PBMCs) and local extracellular space in IDD. The study aimed for investigating the miR-155 expression of PBMCs, extracellular miRNAs (ex-miRNAs) of human nucleus pulposus (NP) tissues, and morphological changes of cell death in the IDD process. Here, we harvested peripheral blood and NP samples from scoliosis and IDD patients as control and degenerative groups, respectively. Then standard Ficoll density-gradient centrifugation was used to isolate PBMCs. The two subpopulations of PBMCs were divided based on the cellular difference in adherence, i.e., monocytes/ macrophages and lymphocytes. Quantitative real-time PCR was used to evaluate miR-155 relative expression in PBMCs. ex-miRNAs were screened by comparison between GSE19943, GSE63492, and extracellular RNA (exRNA) atlas. The morphological changes of cell death subtypes in IDD were observed in transmission electron microscopy (TEM). Results indicate that the lower expression of miR-155 in PBMCs from IDD patients ascribed to alterations in monocytes/macrophages. Moreover, we identified 594 shared hsa-miRNAs as the intracellular miRNA expression profile and 258 miRNAs as the ex-miRNA expression profile in human NP tissue. miR-155 was amongst intracellular miRNAs in NP. TEM observation presented multiple endocytic secretory vesicles within human NP cells, implicating exosome transporting machinery. Typical necroptosis and pyroptosis were noted in human NP. Collectively, this study reveals miR-155 expression in PBMCs from IDD patients. Moreover, we propose ex-miRNA expression profile and necroptosis/pyroptosis in human NP tissue, shedding novel light on the etiology of IDD.