Ficoll-Paque Density Gradient Centrifugation Research Articles

Contribution of peripheral blood mononuclear cells isolated by advanced filtration system to myogenesis of human bone marrow mesenchymal stem cells co-cultured with myoblasts

BackgroundContribution of peripheral blood mononuclear cells (PBMCs) in myogenesis is still under debate, even though blood filtration systems are commonly used in clinical practice for successfully management of critic limb ischemia. ObjectivesA commercial blood filter used for autologous human PBMC transplantation procedures is characterized and used to collect PBMCs, that are then added to well-established 2D in vitro myogenic models assembled with a co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and skeletal myoblasts (hSkMs) whit the aim of investigating their potential contribution to stem cell myogenic commitment. MethodsA commercial blood filter was physically and chemically studied to understand its morphological characteristics and composition. PBMCs were concentrated using this system, further isolated by Ficoll-Paque density gradient centrifugation, and then added in an upper transwell chamber to a 2D co-culture of hBM-MSCs and hSkMs. Myogenic commitment was investigated by RT-PCR, immunofluorescence, and flow cytometry immunophenotyping. Cytokine levels were monitored by ELISA assay in culture media. ResultsThe blood filtration system was disassembled and appeared to be formed by twelve membranes of poly-butylene terephthalate fibers (diameters, 0.9–4.0 μm) with pore size distribution of 1–20 μm. Filter functional characterization was achieved by characterizing collected cells by flow cytometry. Subsequently, collected PBMCs fraction was added to an in-vitro model of hBM-MSC myogenic commitment. In the presence of PBMCs, stem cells significantly upregulated myogenic genes, such as Desmin and MYH2, as confirmed by qRT-PCR and expressed related proteins by immunofluorescence (IF) assay, while downregulated pro-inflammatory cytokines (IL12A at day 14) along the 21 days of culture. NoveltyOur work highlights chemical-physical properties of commercial blood filter and suggests that blood filtrated fraction of PBMC might modulate cytokine expression in response to muscle injury and promote myogenic events, supporting their clinical use in autologous transplantation.

Single-cell transcriptomics reveals peripheral immune responses in non-segmental vitiligo.

Vitiligo is a common autoimmune depigmented dermatology due to destruction of melanocytes. Much evidence suggests that vitiligo is associated with systemic immune activation. Previous studies have focused on immune cell infiltration in and around lesion areas, but few studies have investigated the cell types and function of circulating immune cells in peripheral blood. Here, single cell RNA-sequencing (scRNA-seq) was used to investigate the mechanisms of peripheral immune responses in vitiligo patients. Peripheral blood was collected from five patients with progressive non-segmental vitiligo and three healthy controls. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Paque density gradient centrifugation, and scRNA-seq was performed on isolated cell populations to obtain single cell transcriptomes and characterize important genes and intracellular signaling pathways. The key findings were validated with qPCR and flow cytometry assays. We identified 10 major cell types by scRNA-seq. Among these cell types, neutrophils were specifically observed in our scRNA-seq data from PBMCs. Peripheral blood effector CD8+ T cells from vitiligo patients did not show significant differences at the transcriptome level compared with healthy controls, whereas regulatory T cells showed pro-inflammatory TH1-like properties. Innate immune cells, including natural killer cells and dendritic cells, showed increased antigen processing and presentation as well as upregulated interferon responses. B cells, monocytes, and neutrophils all showed activation. B cells, especially memory B cells, had upregulated expression of genes related to humoral immunity. Monocytes showed production of proinflammatory cytokines and chemokines. Neutrophils showed strong chemokine ligand-receptor (L-R) pair (CXCR8-CXCR2) autocrine signaling pathway. This study revealed the genetic profile and signaling pathway characteristics of peripheral blood immune cells in vitiligo patients, providing new insights into its pathogenesis, which may facilitate identification of potential therapeutic targets.

The effect of influenza virus on the metabolism of peripheral blood mononuclear cells with a metabolomics approach.

Respiratory viruses have led to many deaths and hospitalizations per year in the world. The influenza virus is one of the most important respiratory viruses. Recently, metabolic studies in viral infections have been widely studied by scientists. Metabolomics states the metabolites present in a living organism under certain conditions. In this study, peripheral blood mononuclear cells were spinoculated using a virus produced by the Madin-Darby canine kidney cell culture system, and cells were harvested following spinoculation by the influenza virus. Isolation of peripheral blood mononuclear cells was performed by Ficoll-Paque density gradient centrifugation. Metabolites were extracted using organic and water approaches. Metabolic profiling was performed by a nontargeted technique using liquid chromatography with tandem mass spectrometry. Multivariate analysis methods were used to determine the main variables. the metabolic pathways involved were determined using databases. Results of the present study showed changes in biosynthesis pathways such as lipids, polyamines, catecholamines, and vitamins. Findings also showed that it is possible to explain the process of inflammation caused by the influenza virus by studying the metabolism of immune cells.

Analysis of the Cellular Immune Responses to Vaccines.

Flow cytometry, enzyme-linked immunospot (ELISpot), and cellular cytotoxicity assays are powerful tools for studying the cellular immune response toward intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity toward a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific toward cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium (51Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included, and a newly developed and simple assay to measure TCR avidity.

Phenotypic Changes of PD-1 and GITR in T Cells Are Associated With Hepatitis B Surface Antigen Seroclearance.

Regulatory T cells (Tregs) possess hepatitis B virus (HBV)-specific immunoregulatory effects in chronic HBV infection. The role of Tregs in spontaneous seroclearance of hepatitis B surface antigen (HBsAg) remains to be determined. We recruited treatment-naive chronic HBV patients achieving spontaneous HBsAg seroclearance (experimental group) and matched HBsAg-positive controls. Peripheral blood mononuclear cells were isolated using the Ficoll-Paque density gradient centrifugation method. The frequency of Tregs and inhibitory phenotypes and immunoregulatory cytokines of Tregs were detected by flow cytometry. Twenty-seven patients with HBsAg seroclearance (mean age: 52.40±6.00 y, 55.6% male) and 27 matched controls were recruited. Median HBsAg and HBV DNA levels in the control group were 2.80 (1.24 to 3.43) and 3.16 (1.68 to 3.85) log IU/mL, respectively. Mean frequencies of Tregs and expressions of FoxP3+ Tregs were comparable in both groups (both P>0.05). The mean expression of programmed death 1 (PD-1) and glucocorticoid-induced TNFR family-related gene (GITR) in total CD4+ T cells were significantly downregulated in the experimental group when compared with the control group (10.62% vs. 13.85%, P=0.003; 16.20% vs. 27.02%, P=0.002, respectively). When compared with the control group, PD-1+CD4+ Tregs expression in the experimental group was significantly downregulated (13.85% vs. 10.62%, P=0.003). A similar phenomenon was noted for GITR+CD8+ Tregs (20.16% vs. 14.08%, P=0.049). Intracellular cytokine productions showed no significant differences (all P>0.05). The reduced expression of PD-1 and GITR might attenuate the immunosuppressive capability of Tregs. Decreased expression on CD4+ T cells might represent an enhanced antiviral function, playing a role in initiating the "functional cure" of chronic HBV infection.

Differential expression of apoptosis related genes in the peripheral blood mononuclear cells of acute lymphoblastic/lymphocytic leukemia (ALL) patients

Apoptosis is the cell’s intrinsic death program which plays a crucial role in the regulation of many normal physiological processes in the body’s tissues. In leukemia patients, the extent of cancer cell susceptibility to apoptosis is correlated with clinical responses to chemotherapy and disease prognosis. The aim of this study was evaluation of the expression of apoptosis related genes in the peripheral blood mononuclear cells (PBMCs) of acute lymphoblastic/lymphocytic leukemia (ALL) patients. Peripheral blood samples were obtained from 20 patients with ALL and 20 healthy individuals. PBMCs were isolated using Ficoll-Paque density gradient centrifugation method. After RNA extraction and cDNA synthesis, gene expression levels of apoptosis related genes including caspase-3, 8, 9, BAX, and BAK genes were measured by real-time RT-PCR technique. Gene expression analysis showed that the expression levels of the initiator caspases-8 and -9 are increased in the PBMCs of adult ALL patients when compared with that in PBMCs of healthy individuals. Increased gene expression levels of the proapoptotic protein BAK was also detected in PBMCs of ALL patients. In contrast, decreased expression levels of the proapoptotic BAX and the executioner caspase-3 were observed in the PBMCs of ALL patients. These results suggest that the expression of genes involved in both extrinsic and intrinsic signaling pathways of apoptosis are induced in PBMCs of ALL patients while the gene expression of other proapoptotic molecule, BAK, and the executioner caspase-3 diminished in PBMCs cells of ALL patients. This findings indicate that resistance to apoptosis may be one of the hallmarks of ALL cells.

EVALUATION OF NATURAL KILLER CELLS USING DIFFERENT TECHNIQUES

Natural killer (NK) cells mediate spontaneous cytotoxicity toward abnormal and tumor cells and rapidly secrete numerous cytokines and chemokine`s which promote subsequent adaptive immune responses. The presented study aimed to compare between different methods for NK evaluation and determination. Ten samples of healthy individuals were collected from Al-Zahraa University hospital and evaluated for phenotypic identification for NK cells. Isolation procedure of NK was performed in Clinical Pathology Department, Hematology Unit, Flow-cytometry lab, Al-Azhar University. Protocols started with isolation of peripheral blood mononuclear cells (PBMCs) from fresh human whole blood depending on Ficoll-Paque density gradient centrifugation. Other protocol depended on flow cytometry sorting technique. All experiments were carried out in accordance with the approved guidelines. Sorting procedure using FACS Calibur machine yielded 64.97 % purity before cells were cultured, while mixed Ficoll separation of whole blood showed purity 22.74%. Through our study, it is clear to us that the percentage of purity of NK cells using the flow-cytometry sorting technique is much higher than the percentage of purity of NK cells using the separation method dependent on Ficoll-Paque density technique, Therefore, through our study, we see that it is better to rely on the flow-cytometry sorting technique using the flow cytometry device and CD56 +CD16 and CD3 antibodies in order to obtain the best purity of the NK cells (CD56+ CD16 positive /CD3 negative population) for use in medical purposes and laboratory research.

Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73.

CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Human Microglia Respond to Malaria-Induced Extracellular Vesicles.

Microglia are the chief immune cells of the brain and have been reported to be activated in severe malaria. Their activation may drive towards neuroinflammation in cerebral malaria. Malaria-infected red blood cell derived-extracellular vesicles (MiREVs) are produced during the blood stage of malaria infection. They mediate intercellular communication and immune regulation, among other functions. During cerebral malaria, the breakdown of the blood–brain barrier can promote the migration of substances such as MiREVs from the periphery into the brain, targeting cells such as microglia. Microglia and extracellular vesicle interactions in different pathological conditions have been reported to induce neuroinflammation. Unlike in astrocytes, microglia–extracellular vesicle interaction has not yet been described in malaria infection. Therefore, in this study, we aimed to investigate the uptake of MiREVs by human microglia cells and their cytokine response. Human blood monocyte-derived microglia (MoMi) were generated from buffy coats of anonymous healthy donors using Ficoll-Paque density gradient centrifugation. The MiREVs were isolated from the Plasmodium falciparum cultures. They were purified by ultracentrifugation and labeled with PKH67 green fluorescent dye. The internalization of MiREVs by MoMi was observed after 4 h of co-incubation on coverslips placed in a 24-well plate at 37 °C using confocal microscopy. Cytokine-gene expression was investigated using rt-qPCR, following the stimulation of the MoMi cells with supernatants from the parasite cultures at 2, 4, and 24 h, respectively. MiREVs were internalized by the microglia and accumulated in the perinuclear region. MiREVs-treated cells increased gene expression of the inflammatory cytokine TNFα and reduced gene expression of the immune suppressive IL-10. Overall, the results indicate that MiREVs may act on microglia, which would contribute to enhanced inflammation in cerebral malaria.

Reduced Antigen Presentation May Contribute to Immunomodulatory Drug Resistance in Multiple Myeloma

Introduction: Maintenance therapy with the immunomodulatory drug (IMiD) lenalidomide improves progression-free survival (PFS) and overall survival among patients with multiple myeloma (MM). It has been suggested that IMiDs enhance immune-mediated anti-tumor responses through increased activation and proliferation of T cells and natural killer (NK) cells as well as inhibition of regulatory T cells. Despite the effectiveness of this therapy, nearly all patients relapse for reasons that remain uncharacterized. We sought to elucidate mechanisms of resistance to lenalidomide maintenance through investigation of mutations in cell-free DNA (cfDNA) and identification of changes in the innate and adaptive immune system by single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs). While MM is a cancer of plasma cells that primarily reside in the bone marrow, we focused on the peripheral blood which offers a comprehensive view of multiple sites of disease including extramedullary locations commonly found at the time of relapse. Methods: Twenty-four patients with MM and no minimal residual disease by 10-color flow cytometry and IGHVnext-generation sequencing (Invivoscribe) after induction therapy with or without an autologous stem cell transplant were selected for analysis. All patients were enrolled in NCT02538198 at Memorial Sloan Kettering Cancer Center. Peripheral blood was collected serially including timepoints prior to lenalidomide maintenance and after disease progression. Plasma and PBMCs were isolated by Ficoll-Paque density gradient centrifugation. cfDNA was extracted from plasma and targeted next-generation sequencing using a 70-gene panel of recurrently mutated genes in myeloma was performed using the QIAseq Targeted DNA kit (QIAGEN). DNA sequencing was also performed using the same gene panel on granulocytes to exclude germline variants. On PBMC samples, 5' gene expression and T and B cell receptor single-cell sequencing were performed using the Chromium Single Cell V(D)J kit (10X Genomics). Analysis of single-cell sequencing was performed on the top 100 principal components and visualized by Uniform Manifold Approximation and Projection (UMAP). A regression-based classifier was trained to automatically assign clustered cells into 26 immune cell phenotypes using lists of genes reported to be uniquely expressed by each cell type. Results: cfDNA sequencing was performed on 12 patients characterized as durable responders (PFS > 57 months) and 12 early progressors (median PFS 25 months). Prior to maintenance therapy, no significant difference was observed in the number of cfDNA mutations between these groups. The number of non-synonymous somatic mutations in cfDNA tended to increase over time and was significantly higher among patients taking lenalidomide for more than 2 years compared to patients at the time of relapsed disease (mean number of mutations was 7 in responders versus 3 in progressors).ZNF292was among the genes most significantly mutated in responders compared to progressors. ZNF292encodes a zinc finger protein involved in RNA binding and has previously been identified as a potential source of tumor-associated antigens in patients with monoclonal gammopathy of undetermined significance. To identify changes in immune cell phenotype and function that associate with disease progression, we performed single-cell RNA sequencing on PBMCs from the 12 progressors before maintenance and at the time of relapse. The median number of cells sequenced per PBMC sample was 3,132. Over 90% of cells could automatically be classified into a specific immune cell type by their gene expression. We observed the frequency of NK cells and plasmablasts were significantly increased at the time progression on lenalidomide while monocytes, dendritic cells, and basophils were decreased. Conclusion: Mechanisms of resistance among patients receiving treatment with IMiDs is not well known. Using targeted sequencing of cfDNA, we identify patients responding to maintenance tended to have a higher mutational load in cfDNA including genes previously reported to encode tumor-associated antigens. Additionally, we show that a decline in antigen-presenting dendritic cells is more common at the time of disease progression. Taken together, these results suggest reduced neoantigen production and a decline in antigen presentation may contribute to IMiD resistance. Disclosures Landgren: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Green:Celgene: Consultancy; Cellectar Biosciences: Research Funding; Seattle Genetics: Research Funding; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; GSK: Consultancy.

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy

R. Jimenez-Saiz, Y. Ellenbogen and K. Bruton et al. developed a method to identify bona fide IgE+ MBCs in humans, demonstrated their extreme rarity in circulation and cautioned against the clinical utility of their assessment.

Isolation of Tumor-Infiltrating Lymphocytes by Ficoll-Paque Density Gradient Centrifugation.

With the renewed enthusiasm in immuno-oncology, characterization of the tumor immune microenvironment constitutes an essential and unique aspect to the assessment of therapeutics. The isolation of tumor-infiltrating lymphocytes (TILs) is a desirable approach toward the understanding of antitumor immune response. This chapter provides an effective protocol to mechanically dissociate tumor tissue and generate single-cell suspension from excised tumors. TILs are then isolated by Ficoll-Paque density gradient centrifugation. This protocol is applicable to both human and experimental tumors in immunocompetent murine models.

Combined Administration of Recombinant TGF-β1 and DMSO Decreases the in Vitro Inflammatory Properties of Neutrophils from Sickle Cell Anemia Individuals

Sickle cell anemia (SCA) is a chronic inflammatory disease, in which activated neutrophils play a role in initiating vaso-occlusion. Two populations of circulating neutrophils have been described, denominated as low-(LDN) and high-(HDN) density neutrophils. Circulating numbers of LDN (a less inflammatory subset) are normally minimal, but this population augments under inflammatory stress, such as that seen in cancer. Transforming growth factor beta-1 (TGF-β1) is a cytokine with anti-inflammatory properties that is elevated in SCA. In conditions such as Chron's disease, TGF-β1 has protective effects, mediated by its immuno-suppressive functions. In macrophages, it is thought to trigger the polarization from pro- (M1) to anti-inflammatory phenotypes (M2), which hypothetically occur in neutrophils too (N1 and N2). Moreover, dimethyl sulfoxide (DMSO) reportedly increases TGF-β receptors expression on epithelial cells. We aimed to characterize the subsets of circulating neutrophils in SCA patients and investigate the effects of TGF-β1 and DMSO on these cells. Neutrophils from healthy (CON) and SCA individuals, in steady state and without blood transfusion for 90 days, were isolated from peripheral blood by Ficoll-Paque density gradient centrifugation. HDN and LDN were obtained from the granulocyte and mononuclear layers, respectively, and stained with CD66b for neutrophil identification by flow cytometry. As no significant effect of hydroxyurea (HU) therapy on the data obtained was observed, patients' data were grouped together irrespective of HU use. Percentages of LDN were calculated based on the total of gated CD66b+ cells. SCA patients had higher levels of LDN than CON (3.2±0.9%, N=7 vs 1.3±0.3%, N=13; p=0.02). We next investigated the presence of CD66b+/CD206- and CD66b+/CD206+ cells, to infer the presence of N1 and N2 phenotypes, respectively. N2 were more frequent in the LDN than in the HDN subset (CON: 68.1±3.3% vs 52.0±4.4%, N=9, p=0.01; SCA: 77.6±8.9% vs 44.1±5.0%, N=3, p=0.03). To determine whether TGF-β1 and DMSO could shift HDN to a LDN profile, cells were treated (2h) with TGF-β1 (50pM) and/or DMSO (1.5%). Treatments with DMSO alone or combined with TGF-β1 increased the percentage of CD206+ cells in CON (45.7±2.1% vs 61.9±7.6 and 53.6±2.6% respectively, N=6, p=0.04), as well as CD206+ expression on each cell (mean fluorescence intensity, MFI) (137.5±16.9 MFI vs 293.6±71.2 MFI and 210.1±23.9 MFI, respectively, p=0.03). In SCA, only the combined TGF-β1/DMSO treatment increased the MFI of CD206 in HDN (115.7±10.2 vs 255.8±29.7 MFI, N=4, p=0.03). We next investigated whether TGF-β1/DMSO could reduce the adhesion of HDN to fibronectin ligand (FN, 20μg/mL) using static adhesion assays (30 min, 37ºC). HDN from CON and SCA were treated with TGF-β1 and/or DMSO (90min) and stimulated with TNF-α (200ng/mL, 30min). Although TGF-β1 alone did not reduce the adhesion of HDN to FN (p>0.05), the addition of DMSO decreased TNF-α-induced adhesion in CON (16.5±1.8% to 11.3±1.5%, p=0.03, N=10) and SCA HDN (38.9±23.9% to 13.9±1.5%, p=0.04, N=3). Subsequently, HDN were stimulated (4h) with LPS (100ng/mL) and INF-γ (20ng/mL), to induce N2 polarization, in the presence/absence of TGF-β1 and DMSO. The combined treatment again reduced adhesion in both groups (CON: 11.1±1.5% to 4.2±1.2%, p<0.01, N=4; SCA: 47.4±12.3% vs 21.9±4.5%, p=0.04, N=4). To assess whether TGF-β1 and DMSO could affect the production of proinflammatory cytokines by HDN after stimulation with LPS/INF-γ, TNF-α and IL-1β levels in cell supernatants were measured by ELISA. TGF-β1 and DMSO, in combination, decreased both TNF-α and IL-1β release from CON (TNF-α: 39.7±8.7pg/mL to 8.7±0.9pg/mL, p<0.01, N=6; IL-1β: 66.9±10.5pg/mL to 16.5±5.1pg/mL, p=0.02, N=4) and SCA HDN (TNF-α: 174.0±55.5pg/mL to 21.8±6.6pg/mL, p=0.01, N=8; IL-1β: 103.6±25.6pg/mL to 43.1±15.5pg/mL, p=0.01, N=8). Our results demonstrate for the first time the presence of elevated numbers of LDN in SCA patients, indicating an increased basal response to inflammatory stress. However, this shift in the anti-inflammatory subset does not appear enough to control inflammatory responses in the disease, and the use of agents capable of inducing this polarization may be a promising approach. Moreover, the anti-inflammatory effects of TGF-β1 on HDN seem to be enhanced by DMSO and suggest this combination as an effective modulator of the inflammatory profile of neutrophils. DisclosuresNo relevant conflicts of interest to declare.

Utility of Systematic Isolation of immune cell subsets from HIV-infected individuals for miRNA profiling

IntroductionPeripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune cell subsets by immunomagnetic separation and the RNA integrity after isolated cells from samples of HIV-infected patients. MethodsPBMC from Healthy Controls (HC, n=15), Elite Controllers (EC, n=15), Viremic Controllers (VC, n=15), Viremic Progressors (VP, n=15) and HIV-infected patients on therapy (ART, n=15) were isolated by Ficoll-Paque density gradient centrifugation. Subsets were separated with monoclonal antibodies (CD56, CD14, CD4, and CD8) conjugated to microbeads. We evaluated the yield and purity of each subset isolated from PBMCs under resting and activated conditions; LPS, anti-CD3/CD28 and anti-CD16 were used to activate monocytes, PBMC, T cells and NK cells, respectively. The quality of extracted RNA was tested by 2100 Bioanalyzer. ResultsIn resting conditions, the average yield of CD14+ (monocytes) was decreased (p=0.021) in HIV+ patients compared with healthy controls. CD56+ (Natural Killer-NKs; p=0.03) and CD8+ (Cytotoxic T lymphocytes-CTL p=0.001) cells were increased in HIV+ patients after 72h of activation. The purity assay detected significant differences in CD14+ (p≤0.001) and CD8+ (p=0.034) subpopulations when comparing PBMC isolated either from healthy controls or HIV+ patients. The number of activated cells in HIV+ presented differences in CD8 subset (p=0.003). Finally, similar quantities of high quality RNA were extracted from immune cells subsets obtained by our method. Specifically, we show that Bioanalyzer electrophenograms reveal optimal RIN values in HIV positive and negative patients in resting condition (EC:8;HC:6.5;VC:8.80;VP:8;HAART:7.5) and activated condition (EC:9;HC:6.7;VC:8.2;VP:7.2;HAART:8.6). ConclusionThis method allowed us to obtain a sufficient quantity of different isolated immune cell subsets from HIV–infected individuals at different disease stages. Moreover, the assessed qualities of nucleic acids allow us to perform subsequent molecular studies, such as microRNA profiling.

117. Development of Novel Cancer Therapy Combination of Ad-SOCS Gene Therapy and LAK Cell Immunotherapy for the Treatment of Prostate Cancer

Background Suppressor of cytokine signaling 3 (SOCS3) is a promising molecule for cancer gene therapy. SOCS3 suppresses tumor growth through inhibition of mutiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), and focal adhesion kinase (FAK). The constitutive activation of JAK/STAT3 pathway is known to be involved in oncogenesis of various types of cancers including prostate cancer. We investigated the anti-tumor activity of Ad-SOCS, which is the recombinant adenovirus vectors carrying the SOCS3 gene, against prostate cancer cell line in vitro. Furthermore, to improve the antitumor effect against prostate cancer, we combined LAK (Lymphokine activated killer) cell immunotherapy with Ad-SOCS gene therapy. Materials and Methods LNCaP, human prostate cancer cell line was used in this study. The cytotoxicity of Ad-SOCS against LNCaP was measured by XTT assay. LAK cells were generated from PBMCs isolated from fresh blood by Ficoll-paque (GE Healthcare, Munich, Germany) density gradient centrifugation. PBMC were incubated in medium with interleukin (IL)-2 and anti CD3 antibody (OKT3) to generate LAK cells. The cytotoxicity of LAK cells against LNCaP cells infected with Ad-SOCS was investigated by the in vitro cytotoxicity assay. Expression level of SOCS3, STAT3 and ULBPs were measured by real-time PCR. Results Ad-SOCS showed the significantly higher cell growth inhibitory effect compared to Ad-LacZ and PBS controls in LNCaP cells. In real-time PCR, Ad-SOCS significantly increased the level of SOCS3 mRNA expression. Sequentially, the expression of STAT3 mRNA was decreased by Ad-SOCS infection. Upregulation of SOCS3 mRNA and downregulation of STAT3 mRNA were not observed in both Ad-LacZ and PBS infected LNCaP cells. In cytotoxicity assay, LAK cells exhibited the significantly higher cytotoxicity against LNCaP cells infected with Ad-SOCS at all effector: target cell ratios, 1:1,5:1, 10:1, compared with that of non-infected or Ad-LacZ infected cells. The mRNA expression levels of ULBP 1-5, which are NKG2D ligands to stimulate LAK cell activities, were measured and the mRNA expressions of ULBP 1, 3 and 4, in LNCaP infected with Ad-SOCS were significantly increased compared to the treatments. Conclusion In the present study, we demonstrated that Ad-SOCS could inhibit the cell growth of LNCaP cells and increased the sensitivity of LAK cell in vitro. These findings suggested that the Ad-SOCS and LAK cell combination therapy warranted the further development of novel therapeutic approach for advanced prostate cancer.

Analysis of the Cellular Immune Responses to Vaccines.

Flow cytometry, enzyme-linked immunospot (ELISpot) and cellular cytotoxicity assays are powerful tools for studying the cellular immune response towards intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity towards a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific towards cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium ((51)Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included.

Redox-active biflavonoids from Garcinia brasiliensis as inhibitors of neutrophil oxidative burst and human erythrocyte membrane damage

Ethnopharmacological relevanceGarcinia brasiliensis, a plant native to the Brazilian Amazon Rainforest, is used in traditional medicine to treat inflammation of the urinary tract, peptic ulcers, arthritis and other conditions. Aim of the studyThe purposes of this study were to analyze the chemical constituents of G. brasiliensis branches and leaves and to evaluate the potential of isolated compounds to act as inhibitors of both the oxidative burst of stimulated neutrophils and oxidative damage in human erythrocyte membranes to verify the antioxidant and anti-inflammatory effects of this plant. Materials and methodsNeutrophils were isolated from the blood of healthy donors by Ficoll–Paque density gradient centrifugation. Superoxide anion and total reactive oxygen species (ROS) produced by stimulated neutrophils were measured by WST-1 reduction and luminol-enhanced chemiluminescence assays, respectively. Radical-induced lipoperoxidation and hemolysis were performed using erythrocytes from the blood of healthy donors. Compounds were isolated from G. brasiliensis branches and leaves by HPLC microfractionation, and structure elucidation of the isolated compounds was performed based on NMR and HR-MS analyses. ResultsThe biflavonoids procyanidin, fukugetin, amentoflavone and podocarpusflavone isolated from G. brasiliensis showed potent inhibitory effects on the oxidative burst of human neutrophils, inhibiting ROS production by 50% at 1μmolL−1. These biflavonoids also proved to be potent inhibitors of hemolysis (with 88±7% inhibition at 50µmolL−1 for procyanidin) and lipid peroxidation in human erythrocytes, with a malondialdehyde level (a biomarker of oxidative stress) of 8.5±0.3nmol/mg Hb at 50µmolL−1 for procyanidin. ConclusionsThese findings indicate that the biflavonoids extracted from G. brasiliensis branches and leaves modulate oxidative stress via inhibition of NADPH oxidase and ROS production by stimulated human neutrophils. Furthermore, the biflavonoids exhibited potent inhibition of oxidant hemolysis and lipid peroxidation induced by AAPH in human erythrocytes. Therefore, these studies suggest the use of G. brasiliensis extract as an antioxidant and anti-inflammatory agent.

Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

BackgroundHigh quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.ResultsThe mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.ConclusionsOur findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells’ handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0113-0) contains supplementary material, which is available to authorized users.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34+CD38+/CD34+CD38−). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34+CD38− hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.