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Sample records for activated mononuclear cells

  1. A description of an HPLC assay of coproporphyrinogen III oxidase activity in mononuclear cells.

    PubMed

    Gross, U; Gerlach, R; Kühnel, A; Seifert, V; Doss, M O

    2003-01-01

    Coproporphyrinogen III oxidase is deficient in hereditary coproporphyria. An activity assay for this enzyme in mononuclear cells, besides the preparation of the substrate, are presented. The separation conditions for the product of the test protoporphyrin IX by gradient, reversed-phase high-performance liquid chromatography are given. The normal value from mononuclear cells of healthy volunteers was 138 +/- 21 pkat/g total soluble protein (mean +/- SD). The enzyme activity of a family with hereditary coproporphyria was measured. The gene carriers exhibit a specific coproporphyrinogen III oxidase activity of 61-90 pkat/g total soluble protein. PMID:14605502

  2. Mitogen-activated Tasmanian devil blood mononuclear cells kill devil facial tumour disease cells.

    PubMed

    Brown, Gabriella K; Tovar, Cesar; Cooray, Anne A; Kreiss, Alexandre; Darby, Jocelyn; Murphy, James M; Corcoran, Lynn M; Bettiol, Silvana S; Lyons, A Bruce; Woods, Gregory M

    2016-08-01

    Devil facial tumour disease (DFTD) is a transmissible cancer that has brought the host species, the Tasmanian devil, to the brink of extinction. The cancer cells avoid allogeneic immune recognition by downregulating cell surface major histocompatibility complex (MHC) I expression. This should prevent CD8(+) T cell, but not natural killer (NK) cell, cytotoxicity. The reason why NK cells, normally reactive to MHC-negative cells, are not activated to kill DFTD cells has not been determined. The immune response of wild devils to DFTD, if it occurs, is uncharacterised. To investigate this, we tested 12 wild devils with DFTD, and found suggestive evidence of low levels of antibodies against DFTD cells in one devil. Eight of these devils were also analysed for cytotoxicity, however, none showed evidence for cytotoxicity against cultured DFTD cells. To establish whether mimicking activation of antitumour responses could induce cytotoxic activity against DFTD, Tasmanian devil peripheral blood mononuclear cells (PBMCs) were treated with either the mitogen Concanavalin A, the Toll-like receptor agonist polyinosinic:polycytidylic acid or recombinant Tasmanian devil IL-2. All induced the PBMC cells to kill cultured DFTD cells, suggesting that activation does not occur after encounter with DFTD cells in vivo, but can be induced. The identification of agents that activate cytotoxicity against DFTD target cells is critical for developing strategies to protect against DFTD. Such agents could function as adjuvants to induce functional immune responses capable of targeting DFTD cells and tumours in vivo. PMID:27089941

  3. HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

    PubMed Central

    Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

    2015-01-01

    Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

  4. Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

    PubMed Central

    Shih, Tsung-Chieh; Hsieh, Sen-Yung; Hsieh, Yi-Yueh; Chen, Tse-Chin; Yeh, Chien-Yu; Lin, Chun-Jung; Lin, Deng-Yn; Chiu, Cheng-Tang

    2008-01-01

    AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn’s disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (τ = 0.2145, P = 0.0281) and histologic grade (τ = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis. PMID:18350607

  5. Repetitive cryotherapy attenuates the in vitro and in vivo mononuclear cell activation response.

    PubMed

    Lindsay, Angus; Othman, Mohd Izani; Prebble, Hannah; Davies, Sian; Gieseg, Steven P

    2016-07-01

    What is the central question of this study? Acute and repetitive cryotherapy are routinely used to accelerate postexercise recovery, although the effect on resident immune cells and repetitive exposure has largely been unexplored and neglected. What is the main finding and its importance? Using blood-derived mononuclear cells and semi-professional mixed martial artists, we show that acute and repetitive cryotherapy reduces the in vitro and in vivo T-cell and monocyte activation response whilst remaining independent of the physical performance of elite athletes. We investigated the effect of repetitive cryotherapy on the in vitro (cold exposure) and in vivo (cold water immersion) activation of blood-derived mononuclear cells following high-intensity exercise. Single and repeated cold exposure (5°C) of a mixed cell culture (T cells and monocytes) was investigated using in vitro tissue culture experimentation for total neopterin production (neopterin plus 7,8-dihydroneopterin). Fourteen elite mixed martial art fighters were also randomly assigned to either a cold water immersion (15 min at 10°C) or passive recovery protocol, which they completed three times per week during a 6 week training camp. Urine was collected and analysed for neopterin and total neopterin three times per week, and perceived soreness, fatigue, physical performance (broad jump, push-ups and pull-ups) and training performance were also assessed. Single and repetitive cold exposure significantly (P < 0.001) reduced total neopterin production from the mixed cell culture, whereas cold water immersion significantly (P < 0.05) attenuated urinary neopterin and total neopterin during the training camp without having any effect on physical performance parameters. Soreness and fatigue showed little variation between the groups, whereas training session performance was significantly (P < 0.05) elevated in the cold water immersion group. The data suggest that acute and repetitive cryotherapy

  6. Activation-induced apoptosis in peripheral blood mononuclear cells during hepatosplenic Schistosoma mansoni infections.

    PubMed

    Ghoneim, H M; Demian, S R; Heshmat, M G; Ismail, N S; El-Sayed, Laila H

    2008-01-01

    It is well established that programmed cell death (apoptosis) is an important regulator of host responses during infection with a variety of intra- and extra-cellular pathogens. The present work aimed at assessment of in vitro spontaneous and phytohemagglutinin (PHA)-induced apoptosis in mononuclear cells isolated from patients with hepatosplenic form of S. mansoni infections. Cell death data were correlated to the degree of lymphoproliferative responses to PHA as well as to the serum anti-schistosomal antibody titers. A markedly significant increase in PHA-induced apoptosis in lymphocytes isolated from S. mansoni-infected patients was seen when compared to the corresponding healthy controls. However, a slight difference was recorded between the two studied groups regarding the spontaneous apoptosis. This was accompanied with a significant impairment of in vitro PHA-induced lymphoproliferation of T cells from S. mansoni patients. Data of the present study supports the hypothesis that activation-induced cell death (AICD) is a potentially contributing factor in T helper (Th) cell regulation during chronic stages of schistosomiasis, which represents a critically determinant factor in the host-parasite interaction and might influence the destiny of parasitic infections either towards establishment of chronic infection or towards host death. PMID:20306689

  7. ALDOSTERONISM AND PERIPHERAL BLOOD MONONUCLEAR CELL ACTIVATION: A NEUROENDOCRINE-IMMUNE INTERFACE

    PubMed Central

    Ahokas, Robert A.; Warrington, Kenneth J.; Gerling, Ivan C.; Sun, Yao; Wodi, Linus A.; Herring, Paula A.; Lu, Li; Bhattacharya, Syamal K.; Postlethwaite, Arnold E.; Weber, Karl T.

    2010-01-01

    Summary Aldosteronism eventuates in a proinflammatory/fibrogenic vascular phenotype of the heart and systemic organs. It remains uncertain whether peripheral blood mononuclear cells (PBMC) are activated prior to tissue invasion by monocytes/macrophages and lymphocytes as is the case for responsible pathogenic mechanisms. Uninephrectomized rats, treated for 4 wks with dietary 1%NaCl and aldosterone (0.75 μg/h, ALDOST) ± spironolactone (Spi, 100 mg/kg/daily gavage), were compared to unoperated/-untreated and uninephrectomized/salt-treated controls. Before intramural coronary vascular lesions appeared at wk 4 ALDOST, we found: 1) a reduction of PBMC cytosolic free [Mg2+]i, together with intracellular Mg2+ and Ca2+ loading while plasma and cardiac tissue Mg2+ were no different from controls; 2) increased H2O2 production by monocytes and lymphocytes together with upregulated PBMC gene expression of oxidative stress-inducible tyrosine phosphatase and Mn2+-superoxide dismutase, and the presence of 3-nitrotyrosine in CD4+ and ED-1-positive inflammatory cells that had invaded intramural coronary arteries; 3) B cell activation, including transcription of immunoglobulins, ICAM-1, CC and CXC chemokines and their receptors; 4) expansion of B lymphocyte subset and MHC Class II-expressing lymphocytes; and 5) autoreactivity with gene expression for antibodies to acetylcholine receptors and a downregulation of RT-6.2, which is in keeping with cell activation and associated with autoimmunity. Spi co-treatment attenuated the rise in intracellular Ca2+, the appearance of oxi/nitrosative stress in PBMC and invading inflammatory cells, and alterations in PBMC transcriptome. Thus, aldosteronism is associated with an activation of circulating immune cells induced by iterations in PBMC divalent cations and transduced by oxi/nitrosative stress. ALDO receptor antagonism modulates this neuroendocrine-immune interface. PMID:14576195

  8. Transcription factor expression in lipopolysaccharide-activated peripheral-blood-derived mononuclear cells

    PubMed Central

    Roach, Jared C.; Smith, Kelly D.; Strobe, Katie L.; Nissen, Stephanie M.; Haudenschild, Christian D.; Zhou, Daixing; Vasicek, Thomas J.; Held, G. A.; Stolovitzky, Gustavo A.; Hood, Leroy E.; Aderem, Alan

    2007-01-01

    Transcription factors play a key role in integrating and modulating biological information. In this study, we comprehensively measured the changing abundances of mRNAs over a time course of activation of human peripheral-blood-derived mononuclear cells (“macrophages”) with lipopolysaccharide. Global and dynamic analysis of transcription factors in response to a physiological stimulus has yet to be achieved in a human system, and our efforts significantly advanced this goal. We used multiple global high-throughput technologies for measuring mRNA levels, including massively parallel signature sequencing and GeneChip microarrays. We identified 92 of 1,288 known human transcription factors as having significantly measurable changes during our 24-h time course. At least 42 of these changes were previously unidentified in this system. Our data demonstrate that some transcription factors operate in a functional range below 10 transcripts per cell, whereas others operate in a range three orders of magnitude greater. The highly reproducible response of many mRNAs indicates feedback control. A broad range of activation kinetics was observed; thus, combinatorial regulation by small subsets of transcription factors would permit almost any timing input to cis-regulatory elements controlling gene transcription. PMID:17913878

  9. A method for production and determination of histamine releasing activity from human peripheral blood mononuclear cells.

    PubMed

    Kampen, G T; Poulsen, L K; Reimert, C M; Skov, P S

    1997-12-29

    Histamine releasing factors, i.e. cytokines capable of inducing histamine release from basophils or mast cells, have been suggested to be involved in the pathogenesis of, for example, allergic late-phase reactions. Here we describe a controlled method for production and determination of histamine releasing activity (HRA) from human peripheral blood mononuclear cells (MNC). MNC were incubated with concanavalin A (Con A) for 2 h and cultured for another 40 h in fresh serum free medium. The culture supernatants were concentrated 19-25 fold by ultrafiltration (molecular weight cut-off: 3000 Da). The preparations of HRA induced dose- and Ca2+-dependent histamine release from leukocytes. Supernatants of parallel cultures of unstimulated MNC did not induce histamine release. The HRA was neither due to exogenous histamine releasing compounds (e.g. Con A) nor to residual histamine in the preparations of HRA. The kinetics of HRA induced histamine release (half-maximal release after > 40 min) were slower and more protracted than those of anti-IgE induced histamine release. However, based on a comparison between HRA induced histamine release from leukocytes and purified (97%) basophils, this did not appear to be due to an indirect effect on the basophils. Finally, neither the production of nor the response to HRA was dependent on the allergic status of the donor. PMID:9520301

  10. Lactobacillus isolates from healthy volunteers exert immunomodulatory effects on activated peripheral blood mononuclear cells

    PubMed Central

    Sun, Keyi; Xie, Chao; Xu, Donghua; Yang, Xiaofan; Tang, James; Ji, Xiaohui

    2013-01-01

    As probiotics in the gut, Lactobacilli are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host. This study was aimed to investigate the immuno-modulatory function of candiate lactobacilli on T cells. Lactobacilli were isolated from healthy human feces and the microbiological characteristics were identified by API 50 CHL and randomly amplified polymorphic DNA (RAPD) assays. Anti-CD3 antibody activated peripheral blood mononuclear cells (PBMCs) were treated by viable, heat-killed lactobacilli and genomic DNA of lactobacilli, and cytokine profiles were tested by ELISA. Isolated lactobacilli C44 and C48 were identified as L. acidophilus and L. paracacei, which have properties of acid and bile tolerance and inhibitor effects on pathogens. Viable and heat-killed C44 and C48 induced low levels of proinflammatory cytokines (TNF-α, IL-6 and IL-8) and high levels of IFN-γ and IL-12p70 in PBMCs. In anti-CD3 antibody activated PBMCs, viable and heat-killed C44 increased Th2 cytokine levels (IL-5, IL-6 and IL-10), and simultaneously enhanced Th1 responses by inducing IFN-γ and IL-12p70 production. Different from that of lactabacillus strains, their genomic DNA induced low levels of IL-12p70, IFN-γ and proinflammatory cytokines in PBMCs with or without anti-CD3 antibody activation. These results provided in vitro evidence that the genomic DNA of strains of C44 and C48, especially C44, induced weaker inflammation, and may be potentially applied for treating allergic diseases. PMID:23554802

  11. The Elevated Secreted Immunoglobulin D Enhanced the Activation of Peripheral Blood Mononuclear Cells in Rheumatoid Arthritis

    PubMed Central

    Chen, Hengshi; Zhang, Lingling; Chang, Yan; Yan, Shangxue; Dai, Xing; Ma, Yang; Huang, Qiong; Wei, Wei

    2016-01-01

    Immunoglobulin D (IgD) is a surface immunoglobulin that is expressed as either membrane IgD (mIgD) or secreted IgD (sIgD). Researchers have shown that sIgD is often elevated in patients with autoimmune diseases. The possible roles of sIgD on the function of peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) are still unclear. In this study, we compared the expression of sIgD, mIgD and IgD receptor (IgDR) in RA patients and healthy controls, and investigated the effect of sIgD on the function of PBMCs. We found that the levels of sIgD, mIgD and IgDR were significantly higher in RA patients compared with healthy controls. The concentrations of sIgD were positively correlated with soluble receptor activator of nuclear factor-κB ligand (sRANKL), rheumatoid factor (RF) and C-reactive protein (CRP) in RA patients. Strikingly, IgD could enhance the proliferation of PBMCs and induce IL-1α, IL-1β, TNF-α, IL-6 and IL-10 production from PBMCs. Moreover, the percentage of activated T cell subsets (CD4+CD69+, CD4+CD154+) and activated B cell subsets (CD19+CD23+, CD19+CD21+, CD19+IgD+ and CD19-CD138+) were increased by IgD. The percentage of unactivated T cell subset (CD4+CD62L+) and immature B cell subset (CD19+IgM+IgD-) were decreased by IgD in PBMCs. Furthermore, the expressions of IgDR on T and B cells were significantly increased by treatment with IgD. Our results demonstrate that IgD enhanced the activation of PBMCs, which may contribute to RA pathogenesis. Therefore, IgD could be a potential novel immunotherapeutic target for the management of RA. PMID:26814717

  12. Effects of in vivo hyperthermia on natural killer cell activity, in vitro proliferative responses and blood mononuclear cell subpopulations.

    PubMed Central

    Kappel, M; Stadeager, C; Tvede, N; Galbo, H; Pedersen, B K

    1991-01-01

    This work was designed to test the hypothesis that elevations in body temperature of humans induce immunostimulation. Eight healthy volunteers were immersed in a water bath (water temperature 39.5 degrees C) for 2 h, during which their rectal temperature rose to 39.5 degrees C. On a later day they served as their own controls, being immersed into thermoneutral water (34.5 degrees C) for 2 h. Blood samples were collected before immersion, at body temperatures of 38 degree C, 39 degree C and 39.5 degree C, and 2 h after water immersion. The interleukin-2 (IL-2) enhanced natural killer (NK) cell activity (lysis per fixed number of mononuclear cells), as well as the proportion and total number of NK cells (CD16+ cells), increased significantly during hyperthermia compared with control values. The lymphocyte proliferative responses did not differ significantly between hyperthermia and thermoneutral conditions. The proportion of pan-T (CD3+) cells was maximally depressed 2 h after water immersion. The decreased proportion of CD3+ cells was mainly due to a decreased percentage of CD4+ cells (not significant). The proportion of B cells (CD19+ cells) did not fluctuate significantly, while a marked and significant increase in monocyte proportion (CD14+ cells) was found 2 h after hyperthermia. Two hours after hot water immersion the lymphocyte concentration declined while the neutrophil and monocyte concentrations were augmented. Induced hyperthermia causes significantly increased serum cortisol, plasma norepinephrine and plasma epinephrine concentrations compared to controls. It is possible that the altered immune functions induced by elevated body temperature can be ascribed to altered composition and function of blood mononuclear cells induced by elevated levels of stress hormones. PMID:2015709

  13. Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells.

    PubMed

    Kametani, Yoshie; Ohshima, Shino; Kita, Yuki F; Shimada, Shin; Kamiguchi, Hiroshi; Shiina, Takashi; Inoko, Hidetoshi; Kulski, Jerzy K; Ando, Asako

    2012-08-15

    The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72 h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48 h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72 h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar

  14. Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia.

    PubMed

    Walther, Ashley; Mohanty, Sujit K; Donnelly, Bryan; Coots, Abigail; Lages, Celine S; Lobeck, Inna; Dupree, Phylicia; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

    2015-09-15

    Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers. PMID:26206856

  15. Changes in the activity of the GPx-1 anti-oxidant selenoenzyme in mononuclear cells following imatinib treatment

    PubMed Central

    Terry, Emily N.; Gann, Peter H.; Molokie, Robert; Deininger, Michael; Diamond, Alan M.

    2011-01-01

    Imatinib inhibits the ABL tyrosine kinase and is effective for the treatment of chronic myeloid leukemia (CML). ABL activates GPx-1, an enzyme associated with protection against oxidative DNA damage and disease. Enzyme activity was assessed in sample pairs consisting of mononuclear cells obtained from patients before and after imatinib therapy. Control sample sets obtained from patients not receiving imatinib showed little change in GPx activity over a several month interval. Five of 7 sample sets obtained from imatinib-receiving patients showed changes in GPx activity greater than 30%. One sample decreased 42% while 4 others increased 33% to 208%. Patients with the largest increase in activity were female and had the lowest baseline levels of GPx activity. Changes in GPx activity may influence the clinical outcome of patients being treated for CML. PMID:21292323

  16. Beer down-regulates activated peripheral blood mononuclear cells in vitro.

    PubMed

    Winkler, C; Wirleitner, B; Schroecksnadel, K; Schennach, H; Fuchs, D

    2006-03-01

    Moderate consumption of alcoholic beverages is suggested to reduce cardiovascular risk. Within this context, most attention is drawn to antioxidant ingredients of wine, but also beer was found to be beneficial. Potential effects of three different types of beer including alcohol-free beer were investigated using freshly isolated human peripheral blood mononuclear cells stimulated with the mitogen phytohaemagglutinin in vitro. Neopterin production and tryptophan degradation were monitored in culture supernatants to determine effects of test substances on immunobiochemical pathways induced by interferon-gamma. In a subgroup of experiments also production of interferon-gamma was measured. Compared to unstimulated cells, phytohaemagglutinin increased production of neopterin and also triggered the degradation of tryptophan (all p < 0.01). All types of beer (2-4% dilution) were found to counteract these stimulation-induced effects and significant reduction of neopterin formation and tryptophan degradation was observed (p < 0.01). Data demonstrate that beer reduces production of neopterin and degradation of tryptophan, both these biochemical pathways are induced during cell-mediated immune response. Data suggest that the immunosuppressive capacity of beer may relate to its anti-inflammatory nature. PMID:16428074

  17. Immunocapture and microplate-based activity and quantity measurement of pyruvate dehydrogenase in human peripheral blood mononuclear cells

    PubMed Central

    Liu, Xiaowen; Pervez, Hira; Andersen, Lars W; Uber, Amy; Montissol, Sophia; Patel, Parth; Donnino, Michael W

    2015-01-01

    Background Pyruvate dehydrogenase (PDH) activity is altered in many human disorders. Current methods require tissue samples and yield inconsistent results. We describe a modified method for measuring PDH activity from isolated human peripheral blood mononuclear cells (PBMCs). Results/Methodology We found that PDH activity and quantity can be successfully measured in human PBMCs. Freeze-thaw cycles cannot efficiently disrupt the mitochondrial membrane. Processing time of up to 20 h does not affect PDH activity with proteinase inhibitor addition and a detergent concentration of 3.3% showed maximum yield. Sample protein concentration is correlated to PDH activity and quantity in human PBMCs from healthy subjects. Conclusion Measuring PDH activity from PBMCs is a novel, easy and less invasive way to further understand the role of PDH in human disease. PMID:25826140

  18. Bos taurus papillomavirus activity in peripheral blood mononuclear cells: demonstrating a productive infection.

    PubMed

    Melo, T C; Araldi, R P; Pessoa, N S D; de-Sá-Júnior, P L; Carvalho, R F; Beçak, W; Stocco, R C

    2015-01-01

    Bovine papillomavirus (BPV) is an oncogenic virus with mucous and epithelial tropism. Possible productive virus infection in other tissues, such as blood, has been hypothesized. In order to investigate this possibility, three samples of skin papillomas and blood were collected from bovines with BPV infection and five samples of peripheral blood and one sample of normal tissue were collected from a calf without BPV infection. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and examined by reverse transcription-polymerase chain reaction, immunofluorescence, in situ hybridization, and electron microscopy. The tissue samples were examined for histopathological and immunohistochemical features. The skin papillomas showed the presence of DNA sequences of BPV-2, BPV-11, and a putative virus type. The blood samples showed DNA sequences of BPV-1, 2, and 4 simultaneously. Immunohistochemistry showed BPV L1 protein in both epithelium and stroma and BPV E2 protein in koilocytes. In situ hybridization confirmed the presence of BPV DNA in PBMCs and immunofluorescence showed nuclear labeling of E2 and L1 BPV proteins in PBMCs. The transcription analysis revealed transcripts of BPV-1 L1, BPV-2 L2, and BPV-4 E7 in blood and papilloma samples of BPV-infected cattle. The comet assay revealed high levels of host cell DNA damage upon BPV infection. Electron microscopy analysis of PBMCs identified the presence of particles in the cytoplasm that are consistent with papillomavirus in size and shape. The productive infection of PBMCs with BPV has been previously discussed and this study provides evidence indicating that PBMCs are a target of BPV. PMID:26681018

  19. Effects of active bufadienolide compounds on human cancer cells and CD4+CD25+Foxp3+ regulatory T cells in mitogen-activated human peripheral blood mononuclear cells.

    PubMed

    Yuan, Bo; He, Jing; Kisoh, Keishi; Hayashi, Hideki; Tanaka, Sachiko; Si, Nan; Zhao, Hai-Yu; Hirano, Toshihiko; Bian, Baolin; Takagi, Norio

    2016-09-01

    The growth inhibitory effects of bufadienolide compounds were investigated in two intractable cancer cells, a human glioblastoma cell line U-87 and a pancreatic cancer cell line SW1990. Among four bufadienolide compounds, a dose-dependent cytotoxicity was observed in these cancer cells after treatment with gamabufotalin and arenobufagin. The IC50 values of the two compounds were 3-5 times higher in normal peripheral blood mononuclear cells (PBMCs) than these values for both cancer cell lines. However, similar phenomena were not observed for two other bufadienolide compounds, telocinobufagin and bufalin. These results thus suggest that gamabufotalin and arenobufagin possess selective cytotoxic activity against tumor cells rather than normal cells. Moreover, a clear dose-dependent lactate dehydrogenase (LDH) release, a well-known hallmark of necrosis, was observed in both cancer cells treated with gamabufotalin, suggesting that gamabufotalin-mediated cell death is predominantly associated with a necrosis-like phenotype. Of most importance, treatment with as little as 8 ng/ml of gamabufotalin, even an almost non-toxic concentration to PBMCs, efficiently downregulated the percentages of CD4+CD25+Foxp3+ regulator T (Treg) cells in mitogen-activated PBMCs. Given that Treg cells play a critical role in tumor immunotolerance by suppressing antitumor immunity, these results suggest that gamabufotalin may serve as a promising candidate, as an adjuvant therapeutic agent by manipulating Treg cells to enhance the efficacy of conventional anticancer drugs and lessen their side-effects. These findings provide insights into the clinical application of gamabufotalin for cancer patients with glioblastoma/pancreatic cancer based on its cytocidal effect against tumor cells as well as its depletion of Treg cells. PMID:27431260

  20. Immunopharmacological activity of Echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells.

    PubMed

    Rininger, J A; Kickner, S; Chigurupati, P; McLean, A; Franck, Z

    2000-10-01

    We have investigated the immunostimulatory, anti-inflammatory, and antioxidant activities of various Echinacea raw materials and commercially available products on murine macrophages and human peripheral blood mononuclear cells (PBMCs). To emulate oral dosing, a simulated digestion protocol was employed as a means of sample preparation. Echinacea-induced macrophage activation was used as a measure of immunostimulatory activity determined via quantitative assays for macrophage-derived factors including tumor necrosis factor alpha, interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, and nitric oxide. Echinacea herb and root powders were found to stimulate murine macrophage cytokine secretion as well as to significantly enhance the viability and/or proliferation of human PBMCs in vitro. In contrast, Echinacea extracts chemically standardized to phenolic acid or echinacoside content and fresh pressed juice preparations were found to be inactive as immunostimulatory agents but did display, to varying degrees, anti-inflammatory and antioxidant properties. PMID:11037971

  1. Active hepatitis C virus infection in bone marrow and peripheral blood mononuclear cells from patients with mixed cryoglobulinaemia.

    PubMed Central

    Gabrielli, A; Manzin, A; Candela, M; Caniglia, M L; Paolucci, S; Danieli, M G; Clementi, M

    1994-01-01

    The presence of hepatitis C virus (HCV) genomic sequences was checked in plasma, liver, peripheral blood mononuclear cells (PBMC) and bone marrow cells from 11 patients with mixed cryoglobulinaemia positive for anti-HCV antibodies, and from 11 patients with chronic HCV hepatitis without serological evidence of cryoglobulinaemia. HCV RNA sequences were demonstrated by reverse transcription polymerase chain reaction in seven plasma samples, in six PBMC samples, and in seven bone marrow cell samples from the 11 cryoglobulinaemic subjects; otherwise, viral specific nucleic acids were detected in 10 plasma samples, in one PBMC sample, and in two bone marrow cell samples from the 11 patients with chronic hepatitis. The HCV replicative intermediate was evidenced in four of the six PBMC and in five of the seven bone marrow aspirate HCV RNA-positive samples. Analysis of subpopulations isolated from bone marrow and peripheral blood samples showed HCV RNA sequences in mononuclear cells belonging either the CD2+ subset or to the CD19+ subpopulation or to the adherent cells. Finally, we compared the nucleotide sequences of a large portion (-270 to -59) of the HCV 5'-untranslated region from five patients with mixed cryoglobulinaemia and from seven patients with chronic hepatitis without cryoglobulinaemia; the degree of heterogeneity, compared with the prototype HCV sequence, was similar in both groups. These findings from two groups of HCV-infected patients indicate that transient or permanent active HCV infection of bone marrow and PBMC is frequent in anti-HCV-positive patients with mixed cryoglobulinaemia, and suggest that extra-hepatic infection may play a major role in influencing the pathophysiology of this infection as well as the viral persistence. Images Fig. 1 PMID:8033425

  2. Infection of SCID mice with Mycobacterium leprae and control with antigen-activated "immune" human peripheral blood mononuclear cells.

    PubMed Central

    Converse, P J; Haines, V L; Wondimu, A; Craig, L E; Meyers, W M

    1995-01-01

    The SCID (severe combined immunodeficient) mouse lacks both B and T cells and tolerates injected mononuclear cells from humans, the principal hosts of Mycobacterium leprae. A SCID mouse model of leprosy could be useful to investigate potential vaccine strategies using human cells in a context in which the growth of the organism is monitored. Initial experiments determined that SCID mice are more susceptible than normal mice to infection and dissemination of M. leprae. Cells from humans, either BCG vaccinated or from countries where leprosy is endemic, were stimulated in vitro with a number of mycobacterial antigens--whole M. leprae, M. leprae cell walls, purified protein derivative of M. tuberculosis, and Mycobacterium bovis BCG--and tested for proliferation and production of interleukin-6, tumor necrosis factor alpha, and gamma interferon. Cell walls were the most efficient and consistent in inducing all of these activities. In vitro-activated human cells retain function better after injection into SCID mice than nonactivated cells. To test the ability of cells to affect the growth of M. leprae in the footpads of SCID mice, cells from a known responder to mycobacterial antigens and from a nonresponder were activated by M. leprae cell wall antigens. The cells were harvested and coinjected with fresh M. leprae into the right hind footpads of SCID mice. After 3 months, there was no growth of M. leprae in the footpads of mice coinjected with cells from the mycobacterial antigen responder, while growth was uninhibited in mice receiving cells from the nonresponder. Future experiments will determine requirements for antigen specificity in inhibiting M. leprae multiplication. PMID:7868226

  3. Infection of SCID mice with Mycobacterium leprae and control with antigen-activated "immune" human peripheral blood mononuclear cells.

    PubMed

    Converse, P J; Haines, V L; Wondimu, A; Craig, L E; Meyers, W M

    1995-03-01

    The SCID (severe combined immunodeficient) mouse lacks both B and T cells and tolerates injected mononuclear cells from humans, the principal hosts of Mycobacterium leprae. A SCID mouse model of leprosy could be useful to investigate potential vaccine strategies using human cells in a context in which the growth of the organism is monitored. Initial experiments determined that SCID mice are more susceptible than normal mice to infection and dissemination of M. leprae. Cells from humans, either BCG vaccinated or from countries where leprosy is endemic, were stimulated in vitro with a number of mycobacterial antigens--whole M. leprae, M. leprae cell walls, purified protein derivative of M. tuberculosis, and Mycobacterium bovis BCG--and tested for proliferation and production of interleukin-6, tumor necrosis factor alpha, and gamma interferon. Cell walls were the most efficient and consistent in inducing all of these activities. In vitro-activated human cells retain function better after injection into SCID mice than nonactivated cells. To test the ability of cells to affect the growth of M. leprae in the footpads of SCID mice, cells from a known responder to mycobacterial antigens and from a nonresponder were activated by M. leprae cell wall antigens. The cells were harvested and coinjected with fresh M. leprae into the right hind footpads of SCID mice. After 3 months, there was no growth of M. leprae in the footpads of mice coinjected with cells from the mycobacterial antigen responder, while growth was uninhibited in mice receiving cells from the nonresponder. Future experiments will determine requirements for antigen specificity in inhibiting M. leprae multiplication. PMID:7868226

  4. Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways

    PubMed Central

    Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

    2016-01-01

    The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and

  5. Lymphokine-activated killer cell function of peripheral blood mononuclear cells, spleen cells and regional lymph node cells in gastric cancer patients.

    PubMed Central

    Karimine, N; Arinaga, S; Inoue, H; Nanbara, S; Ueo, H; Akiyoshi, T

    1994-01-01

    Lymphokine-activated killer (LAK) cells generated by culture of peripheral blood mononuclear cells (PBMC), spleen cells (SPC) and regional lymph node cells (LNC) with IL-2 for 4 days were examined for their functional capabilities in 29 patients with gastric carcinoma. The cytotoxic activity of LAK cells induced from LNC was significantly lower than that from either PBMC or SPC, although there was no difference between PBMC or SPC. The induction of mRNA of interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) and the production of these cytokines in the non-adherent LAK cells from LNC were also significantly reduced compared with those from PBMC or SPC. Further, the LAK cells from LNC secreted significantly lower levels of these cytokines when stimulated with tumour target, Raji cells, although the production of these cytokines was markedly increased by stimulation with the targets in all three cell populations. Phenotypic analysis of each cell population revealed a decreased proportion of the cells mediating natural killer (NK) activity, including CD16+, CD56+, and CD57+ cells in LNC either before or after culture, although OKIa1+ and CD25+ cells were uniformly increased in all cell populations after culture. Changes in subpopulations of CD4+ and CD8+ cells in LNC were not apparently different from PBMC or SPC. These results indicated the differential reactivity of each lymphocyte population to IL-2 and the reduced LAK cell function of LNC compared with PBMC or SPC in patients with gastric carcinoma. Images Fig. 2 PMID:8004819

  6. Human immunoglobulin production in immunodeficient mice: enhancement by immunosuppression of host and in vitro activation of human mononuclear cells.

    PubMed Central

    Cavacini, L A; Kennel, M; Lally, E V; Posner, M R; Quinn, A

    1992-01-01

    The affect of host and donor related factors on successful engraftment of human cells into mice was examined to minimize the variability that has been observed in successful development of human-mouse chimera for the study of human disease and immune physiology and regulation. Human immunoglobulin production in severe combined immunodeficiency (SCID) mice engrafted with human peripheral blood mononuclear cells (PBMC) was augmented by immunosuppressing recipient mice and activating donor PBMC. Immunosuppression of recipient mice with 3 Gy of gamma-irradiation induced a 10-fold increase in human IgG in the sera of engrafted SCID mice. Variation in production of human IgG in recipient mice correlated with preinjection phenotype and activation status of injected PBMC. Mice injected with PBMC with a low CD4/CD8 ratio (less than 0.5) produced no detectable circulating human immunoglobulin. When the CD4/CD8 ratio was greater than 1.5, human IgG was detected in sera of PBMC-recipient SCID mice. Serum IgG increased 10-fold following in vitro activation of donor PBMC with anti-CD3, IL-2 and Staphylococcus aureus. Successful engraftment and serum IgG production was evidenced by an increase in the recovery of activated human IgG+ cells in the spleens of mice with maximal IgG production. Optimization of functional engraftment required modification of both the host (SCID mice) and the donor cells. PMID:1395094

  7. Role of the mononuclear phagocyte as an antigen-presenting cell for human gamma delta T cells activated by live Mycobacterium tuberculosis.

    PubMed Central

    Boom, W H; Chervenak, K A; Mincek, M A; Ellner, J J

    1992-01-01

    gamma delta T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of gamma delta T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood gamma delta T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, gamma delta T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting gamma delta T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for gamma delta T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded gamma delta T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of gamma delta T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood gamma delta T cells and for activated expanded gamma delta T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for gamma delta T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for gamma delta T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas gamma delta T cells appeared to recognize only constituents associated with the whole organism and were not

  8. Modulation of transglutaminase activity in mononuclear phagocytes and macrophage-like tumor cell lines by differentiation agents

    SciTech Connect

    Goldman, R.

    1987-01-01

    The effect of glucocorticosteroids, retinoids, 1,25-dihydroxyvitamin D/sub 3/ (1,25(OH)/sub 2/D/sub 3/) and the tumor promoter phorbol myristate acetate (TPA) on the expression of transglutaminase activity in in vitro differentiating bone marrow-derived mouse and rat mononuclear phagocytes (BMDMP) and mouse and human myeloid leukemia cell lines was assessed. Dexamethasone was found to induce an increase of about 100% in transglutaminase activity in mouse and rat BMDMP. The effect was time- and dose-dependent, and specific for steroids with glucocorticoid activity. Retinoic acid (RA) suppressed transglutaminase activity in mouse BMDMP and enhanced it in rat BMDMP. In murine and human myeloid leukemia cell lines, dexamethasone enhanced transglutaminase activity to a varying degree, RA suppressed it in P388D1 cells and enhanced it in the other cell lines. 1,25(OH)/sub 2/D/sub 3/ induced a rather small augmentation of enzyme expression, whereas TPA suppressed enzyme expression (70-100%). The species-specific differences previously observed by the authors for the effect of RA, dexamethasone and 1,25(OH)/sub 2/D/sub 3/ on the formation of BMDMP from mouse and rat bone marrow progenitor cells are now shown to extend also to effects on expression of transglutaminase activity. From a mechanistic point of view it is of interest that dexamethasone uniformly enhanced transglutaminase activity, whereas TPA suppressed it. The data suggest that modulation of transglutaminase activity by the four agents occurs via disparate mechanisms.

  9. Surfactant protein D induces immune quiescence and apoptosis of mitogen-activated peripheral blood mononuclear cells.

    PubMed

    Pandit, Hrishikesh; Thakur, Gargi; Koippallil Gopalakrishnan, Aghila Rani; Dodagatta-Marri, Eswari; Patil, Anushree; Kishore, Uday; Madan, Taruna

    2016-02-01

    Surfactant protein D (SP-D) is an integral molecule of the innate immunity secreted by epithelial cells lining the mucosal surfaces. The C-type lectin domain of SP-D performs pattern recognition functions while it binds to putative receptors on immune cells to modify cellular functions. Activation of immune cells and increased serum SP-D is observed in a range of patho-physiological conditions including infections. We speculated if SP-D can modulate systemic immune response via direct interaction with activated PBMCs. In this study, we examined interaction of a recombinant fragment of human SP-D (rhSP-D) on PHA-activated PBMCs. We report a significant downregulation of activation receptors such as TLR2, TLR4, CD11c and CD69 upon rhSP-D treatment. rhSP-D inhibited production of Th1 (TNF-α and IFN-γ) and Th17 (IL-17A) cytokines along with IL-6. Interestingly, levels of IL-2, Th2 (IL-4) and regulatory (IL-10 and TGF-β) cytokines remained unaltered. Analysis of co-stimulatory CD28 and co-inhibitory CTLA4 receptors along with their ligands CD80 and CD86 revealed a selective up-regulation of CTLA4 in the lymphocyte subset. rhSP-D induced apoptosis in the activated but not in non-activated lymphocytes. Blockade of CTLA4 inhibited rhSP-D mediated apoptosis of activated lymphocytes, confirming involvement of CTLA4. We conclude that SP-D restores immune homeostasis. It regulates expression of immunomodulatory receptors and cytokines, which is followed by induction of apoptosis in activated lymphocytes. These findings suggest a critical role of SP-D in immune surveillance against activated immune cells. PMID:26563748

  10. Activation of human peripheral blood mononuclear cells by nitric oxide-generating compounds

    SciTech Connect

    Lander, H.M.; Sehajpal, P.; Levine, D.M.; Novogrodsky, A. )

    1993-02-15

    Recent work in this laboratory has identified immune-stimulatory properties of the oxidant hemin. In this study, the authors examined whether the nitrogen-based oxidant nitric oxide (NO) had inductive effects on human lymphocytes. They found that the NO-generating compounds sodium nitroprusside and S-nitroso-N acetylpenicillamine rapidly enhanced the rate of glucose transport in resting human PBMC. In addition, NF-[kappa]B binding activity was induced by these agents as was the secretion of TNF-[alpha]. The data suggest that a cGMP-independent mechanism is involved as the cell permeant cGMP analogue, 8-Br-cGMP, had no effect in eliciting these inductive events. Activation of lymphocytes by these NO-generating compounds may be mediated through the protein tyrosine phosphorylation signal transduction pathway. It was found that membrane-associated protein tyrosine phosphatase activity was enhanced in PBMC treated with sodium nitroprusside or S-nitroso-N-acetylpenicillamine and that the src family protein tyrosine kinase p56[sup lck] was activated in these cells. Inasmuch as p56[sup lck] activity is negatively controlled by tyrosine phosphorylation, its activation may be related to the enhancement of protein tyrosine phosphatase activity. 8Br-cGMP had no effect on these enzymes. Taken together, these data suggest that NO may have immune-stimulatory properties and may signal through a hitherto undescribed cGMP-independent pathway. 30 refs., 9 figs., 2 tabs.

  11. Fetal Immune Activation to Malaria Antigens Enhances Susceptibility to In Vitro HIV Infection in Cord Blood Mononuclear Cells

    PubMed Central

    Steiner, Kevin; Myrie, Latoya; Malhotra, Indu; Mungai, Peter; Muchiri, Eric; Dent, Arlene; King, Christopher L.

    2010-01-01

    Mother-to-child-transmission (MTCT) of human immunodeficiency virus (HIV) remains a significant cause of new HIV infections in many countries. To examine whether fetal immune activation as a consequence of prenatal exposure to parasitic antigens increases the risk of MTCT, cord blood mononuclear cells (CBMCs) from Kenyan and North American newborns were examined for relative susceptibility to HIV infection in vitro. Kenyan CBMCs were 3-fold more likely to be infected with HIV than were North American CBMCs (P = .03). Kenyan CBMCs with recall responses to malaria antigens demonstrated enhanced susceptibility to HIV when compared with Kenyan CBMCs lacking recall responses to malaria (P = .03). CD4+ T cells from malaria-sensitized newborns expressed higher levels of CD25 and human leukocyte antigen DR ex vivo, which is consistent with increased immune activation. CD4+ T cells were the primary reservoir of infection at day 4 after virus exposure. Thus, prenatal exposure and in utero priming to malaria may increase the risk of MTCT. PMID:20687848

  12. Mitochondrial activity and oxidative stress markers in peripheral blood mononuclear cells of patients with bipolar disorder, schizophrenia, and healthy subjects.

    PubMed

    Gubert, Carolina; Stertz, Laura; Pfaffenseller, Bianca; Panizzutti, Bruna Schilling; Rezin, Gislaine Tezza; Massuda, Raffael; Streck, Emilio Luiz; Gama, Clarissa Severino; Kapczinski, Flávio; Kunz, Maurício

    2013-10-01

    Evidence suggests that mitochondrial dysfunction is involved in the pathophysiology of psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD). However, the exact mechanisms underlying this dysfunction are not well understood. Impaired activity of electron transport chain (ETC) complexes has been described in these disorders and may reflect changes in mitochondrial metabolism and oxidative stress markers. The objective of this study was to compare ETC complex activity and protein and lipid oxidation markers in 12 euthymic patients with BD type I, in 18 patients with stable chronic SZ, and in 30 matched healthy volunteers. Activity of complexes I, II, and III was determined by enzyme kinetics of mitochondria isolated from peripheral blood mononuclear cells (PBMCs). Protein oxidation was evaluated using the protein carbonyl content (PCC) method, and lipid peroxidation, the thiobarbituric acid reactive substances (TBARS) assay kit. A significant decrease in complex I activity was observed (p = 0.02), as well as an increase in plasma levels of TBARS (p = 0.00617) in patients with SZ when compared to matched controls. Conversely, no significant differences were found in complex I activity (p = 0.17) or in plasma TBARS levels (p = 0.26) in patients with BD vs. matched controls. Our results suggest that mitochondrial complex I dysfunction and oxidative stress play important roles in the pathophysiology of SZ and may be used in potential novel adjunctive therapy for SZ, focusing primarily on cognitive impairment and disorder progression. PMID:23870796

  13. Dimethyl fumarate activates the prostaglandin EP2 receptor and stimulates cAMP signaling in human peripheral blood mononuclear cells.

    PubMed

    Fiedler, Sarah E; Kerns, Amelia R; Tsang, Catherine; Tsang, Vivian; Bourdette, Dennis; Salinthone, Sonemany

    2016-06-17

    Dimethyl fumarate (DMF) was recently approved by the FDA for the treatment of relapsing remitting MS. The pathology of MS is a result of both immune dysregulation and oxidative stress induced damage, and DMF is believed to have therapeutic effects on both of these processes. However, the mechanisms of action of DMF are not fully understood. To determine if DMF is able to activate signaling cascades that affect immune dysregulation, we treated human peripheral blood mononuclear cells with DMF. We discovered that DMF stimulates cyclic adenosine monophosphate (cAMP) production after 1 min treatment in vitro. cAMP is a small molecule second messenger that has been shown to modulate immune response. Using pharmacological inhibitors, we determined that adenylyl cyclase mediates DMF induced cAMP production; DMF activated the prostaglandin EP2 receptor to produce cAMP. This response was not due to increased endogenous production of prostaglandin E2 (PGE2), but was enhanced by addition of exogenous PGE2. Furthermore, we determined that the bioactive metabolite of DMF, monomethyl fumarate (MMF), also stimulates cAMP production. These novel findings suggest that DMF may provide protection against MS by inhibiting immune cell function via the cAMP signaling pathway. PMID:27157139

  14. Fetal Cord Blood Mononuclear Cells Collected At Term from HIV-1 Infected Women Harbor Transcriptionally Active Integrated Proviral DNA

    PubMed Central

    ELLIS, Jane E.; HAIR, Greg A.; LINDSAY, Michael K.; ANSARI, Aftab A.; SUNDSTROM, J. Bruce

    2007-01-01

    Objective To determine levels of intrauterine infection and transcriptional activity in cord blood mononuclear cells collected at term from fetuses born to HIV-infected pregnant women on highly active anti-retroviral therapy (HAART). Methods RNA and DNA were isolated from maternal placental tissues and fetal cord blood specimens obtained at term from HIV-infected pregnant women on HAART. Levels of integrated HIV provirus and mRNA transcripts were determined by real-time PCR. Results Detectable levels of transcriptionally active integrated provirus were present in approximately 27% of cord blood samples (n=22) collected from fetuses, born to HIV-positive mothers on HAART. Levels of HIV-p24 antigen in cultures detected in randomly selected cord blood samples confirmed the presence of inducible infectious virus. Conclusions These findings suggest that some fetuses from HIV-infected mothers on HAART who may present as HIV-negative infants postpartum, can harbor circulating leukocytes that are productively infected by intrauterine transmission (IUT) of HIV. PMID:17904964

  15. Autologous bone marrow mononuclear cells therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury

    PubMed Central

    Bedi, Supinder S.; Walker, Peter A.; Shah, Shinil K.; Jimenez, Fernando; Thomas, Chelsea P.; Smith, Philippa; Hetz, Robert A.; Xue, Hasen; Pati, Shibani; Dash, Pramod K.; Cox, Charles S.

    2014-01-01

    Background Autologous bone marrow-derived mononuclear cells (AMNC) have shown therapeutic promise for central nervous system insults such as stroke and traumatic brain injury (TBI). We hypothesized that intravenous injection of AMNC provides neuroprotection which leads to cognitive improvement after TBI. Methods A controlled cortical impact (CCI) rodent traumatic brain injury (TBI) model was used to examine blood-brain barrier permeability (BBB), neuronal and glial apoptosis and cognitive behavior. Two groups of rats underwent CCI with (CCI-Autologous) or without AMNC treatment (CCI-Alone), consisting of 2 million AMNC/kilogram body weight harvested from the tibia and intravenously injected 72 hr after injury. CCI-Alone animals underwent sham harvests and received vehicle injections. Results 96 hr after injury, AMNC significantly reduced the BBB permeability in injured animals, and there was an increase in apoptosis of pro-inflammatory activated microglia in the ipsilateral hippocampus. At 4 weeks after injury, we examined changes in spatial memory after TBI due to AMNC treatment. There was a significant improvement in probe testing of CCI-Autologous group in comparison to CCI-Alone in the Morris Water Maze paradigm. Conclusions Our data demonstrate that the intravenous injection of AMNC after TBI leads to neuroprotection by preserving early BBB integrity and increasing activated microglial apoptosis. In addition, AMNC also improves cognitive function. PMID:23928737

  16. Ability of peripheral blood mononuclear cells to activate interferon response in vitro is predictive of virological response in HCV patients.

    PubMed

    Lalle, E; Calcaterra, S; Horejsh, D; Abbate, I; D'Offizi, G; Abdeddaim, A; Vlassi, C; Antonucci, G; Capobianchi, M R

    2008-01-01

    The most reliable predictor of treatment efficacy in hepatitis C is HCV viremia decay at week 12 [early virological response (EVR)]. We investigated whether the ability of peripheral blood mononuclear cells (PBMC) to mount an interferon (IFN) response in vitro could be predictive of EVR. Fifteen patients treated with PEG IFNalpha + RBV, with pre-therapy frozen PBMC, were retrospectively selected. After a 3 hr PBMC exposure to IFNalpha in vitro, up-regulation of mRNA for IFN-stimulated genes (ISG) was measured by membrane super-array. ISG mRNA levels in unstimulated PBMC were low, but beta2M and CASP1 were significantly higher in EVR vs non-EVR. ISG mRNA up-regulation by IFN was more pronounced in EVR vs non-EVR. For 7 genes (IP-10, IFIT1, IFIT2, IFIT3, TRAIL, KIAA1628 and OAS2) cut-off levels were established, by ROC analysis, able to correctly classify all EVR and non-EVR. Early virological response to PEG IFNalpha +RBV is correlated with the pre-therapy ability of PBMC to activate an IFN response in vitro. If validated in a wider cohort of patients, the ability of this set of ISG to discriminate between EVR and non-EVR may be useful for pre-therapy evaluation, particularly in patients with unfavourable combinations of conventional response predictors. PMID:18842168

  17. Lysis of small cell carcinoma of the lung tumor cell lines by gamma interferon-activated allogeneic peripheral blood mononuclear cells: abrogation of killing by pretreatment of tumor cells with gamma interferon.

    PubMed

    Ball, E D; Nichols, K E; Pettengill, O S; Sorenson, G D; Fanger, M W

    1986-01-01

    Interferon has been shown to enhance the ability of nonspecific cytotoxic mononuclear cells to lyse some, but not all, tumor cells. We have examined the effect of recombinant human gamma interferon (rIFN gamma) on the cell-mediated cytolysis of tumor target cells derived from continuously cultured lines of small cell carcinoma of the lung (SCCL). Cells from the SCCL lines DMS 44, 53, 79, 92, and 406 were labeled with 51Cr and incubated with normal and rIFN gamma-stimulated peripheral blood mononuclear cells for 18 h at 37 degrees C and tumor cell lysis estimated by measuring 51Cr release. Although cells from certain SCCL lines were good targets for cell mediated cytotoxicity, susceptibility to lysis was heterogeneous among the different SCCL lines. DMS 406 and 79 were, on average, maximally lysed, while DMS 44, 53, and 92 showed less susceptibility to lysis by either control or rIFN gamma-stimulated effector cells. In addition, although pretreatment with rIFN gamma increased the cytolytic capacity of normal peripheral blood mononuclear cells from several different donors, preincubation of the tumor cell lines with rIFN gamma resulted in inhibition of cytolysis mediated by both control and IFN-activated effector cells. These findings suggest that although rIFN gamma may enhance cell-mediated lysis of SCCL tumor cells, it may also decrease susceptibility to lysis. PMID:3015408

  18. Circulating leptin and NF-κB activation in peripheral blood mononuclear cells across the menstrual cycle.

    PubMed

    Faustmann, Gernot; Tiran, Beate; Maimari, Theopisti; Kieslinger, Petra; Obermayer-Pietsch, Barbara; Gruber, Hans-Jürgen; Roob, Johannes M; Winklhofer-Roob, Brigitte M

    2016-07-01

    Using the menstrual cycle as a model, this study focused on longitudinal changes and associations within a physiological network known to play a role in female fertility, including, as biologically active nodes, NF-κB, leptin and adiponectin, β-carotene, adipose tissue, and progesterone. In 28 women, leptin, adiponectin, β-carotene, and progesterone concentrations, NF-κB p65 and p50 activation in peripheral blood mononuclear cells (known to possess estrogen, progesterone and leptin receptors), total body fat (TBF) and subcutaneous adipose tissue (SAT) mass were determined at early (T1) and late follicular (T2) and mid (T3) and late (T4) luteal phase. Leptin and adiponectin concentrations were higher, while NF-κB p65 activation was lower at T3 compared with T1. NF-κB p65 activation was inversely related to leptin concentrations at T1, T3, and T4. β-Carotene was inversely related to leptin (T1,T2,T4) and SAT (T1,T3,T4). NF-κB p50 activation was inversely related to TBF (T4) and SAT (T3,T4), and leptin was positively related to TBF and SAT (T1-T4). Progesterone was inversely related to leptin (T2,T3), adiponectin (T3), TBF (T3,T4), and SAT (T2,T3,T4). By providing evidence of luteal phase-specific reduced NF-κB p65 activation in women under physiological conditions, this study bridges the gap between existing evidence of a Th1-Th2 immune response shift induced by reduced NF-κB p65 activation and a Th1-Th2 shift previously observed at luteal phase. For the first time, inverse regressions suggest inhibitory effects of leptin on NF-κB p65 activation at luteal phase, along with inhibitory effects of leptin as well as adiponectin on progesterone production in corpus luteum. © 2016 The Authors BioFactors published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology. 24(4):376-387, 2016. PMID:27093900

  19. Human umbilical cord blood mononuclear cells activate the survival protein Akt in cardiac myocytes and endothelial cells that limits apoptosis and necrosis during hypoxia.

    PubMed

    Henning, Robert J; Dennis, Steve; Sawmiller, Darrell; Hunter, Lorynn; Sanberg, Paul; Miller, Leslie

    2012-06-01

    We have previously reported that human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic, mesenchymal, and endothelial stem cells, can significantly reduce acute myocardial infarction size. To determine the mechanism whereby HUCBC increase myocyte and vascular endothelial cell survival, we treated cardiac myocytes and coronary artery endothelial cells in separate experiments with HUCBC plus culture media or culture media alone and subjected the cells to 24 h of hypoxia or normoxia. We then determined in myocytes and endothelial cells activation of the cell survival protein Akt by Western blots. We also determined in these cells apoptosis by annexin V staining and necrosis by propidium iodide staining. Thereafter, we inhibited with API, a specific and sensitive Akt inhibitor, Akt activation in myocytes and endothelial cells cultured with HUCBC during hypoxia and determined cell apoptosis and necrosis. In cells cultured without HUCBC, hypoxia only slightly activated Akt. Moreover, hypoxia increased myocyte apoptosis by ≥ 226% and necrosis by 58% in comparison with myocytes in normoxia. Hypoxic treatment of endothelial cells without HUCBC increased apoptosis by 94% and necrosis by 59%. In contrast, hypoxia did not significantly affect HUCBC. Moreover, in myocyte + HUCBC cultures in hypoxia, HUCBC induced a ≥ 135% increase in myocyte phospho-Akt. Akt activation decreased myocyte apoptosis by 76% and necrosis by 35%. In endothelial cells, HUCBC increased phospho-Akt by 116%. HUCBC also decreased endothelial cell apoptosis by 58% and necrosis by 42%. Inhibition of Akt with API in myocytes and endothelial cells cultured with HUCBC during hypoxia nearly totally prevented the HUCBC-induced decrease in apoptosis and necrosis. We conclude that HUCBC can significantly decrease hypoxia-induced myocyte and endothelial cell apoptosis and necrosis by activating Akt in these cells and in this manner HUCBC can limit myocardial ischemia and injury. PMID

  20. In vitro activation of peripheral mononuclear cells by zinc in HIV-infected patients and healthy controls.

    PubMed Central

    Harrer, T; Wolf, B; Näger, W; Schwarz, W; Bergner, D; Kalden, J R

    1992-01-01

    Zinc is a mitogen for peripheral blood mononuclear cells (PBMC). The optimal mitogenic concentration was found to be 0.05 mmol/l (327 micrograms/dl), four times higher than physiological serum levels. Maximal proliferation was observed after 6 days. Limited dilution technique revealed a frequency of zinc reactive cells of 1:3467 (median; range 1:1628-1:6235). Cord blood mononuclear cells from four of six healthy children could be stimulated to proliferate by zinc. A normal zinc-induced proliferative response could be demonstrated in all six HIV-infected patients in the Walter-Reed-stage I, in nine of 11 patients in Walter-Reed II and in only two of five patients in Walter-Reed III. In Walter-Reed IV to VI all eight patients showed a weak response to zinc (less than 50% of the healthy day control). Decreased zinc serum levels were found in 10 of 28 patients and in one of 16 controls. There was a significant correlation of a diminished zinc-induced proliferation with lower serum levels of zinc and a reduced proportion of CD4 helper cells in HIV-1-infected men. Because of a suppression of mitogenesis by high dose of zinc an excessive intake of zinc as used by some HIV-1-infected patients can presently not be recommended. The value of zinc-induced proliferation for monitoring HIV-infected patients has still to be established. PMID:1638772

  1. Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. Characterization of a soluble suppressor of B cell immunoglobulin production

    SciTech Connect

    Fleisher, T.A.; Greene, W.C.; Blaese, R.M.; Waldmann, T.A.

    1981-03-01

    Human peripheral blood mononuclear cells (PBMC) activated with the mitogenic lectin concanavalin A (Con A) elaborate a soluble immune suppressor supernatant (SISS) that contains at least 2 distinct suppressor factors. One of these, SISS-B, inhibits polyclonal B cell immunoglobulin production, whereas the other, SISS-T, suppresses T cell proliferation to both mitogens and antigens. The latter mediator is discussed in the companion paper. Characteristics of the human soluble suppressor of B cell immunoglobulin production (SISS-B) include: 1) inhibition by a noncytotoxic mechanism, 2) loss of activity in the presence of the monosaccharide L-rhamnose, 3) appearance within 8 to 16 hr after the addition of Con A, 4) elaboration by cells irradiated with 500 or 2000 rads, 5) production by highly purified T cells, 6) stability at pH 2.5 but instability at 56/sup o/C, and 7) m.w. of 60 to 80,000. These data indicate that after Con A activation, selected T cells not only become potent suppressor cells, but also generate a soluble saccharide-specific factor(s) that inhibits polyclonal immunoglobulin production by human B cells.

  2. Infection by HIV-1 blocked by binding of dextrin 2-sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T-cells.

    PubMed Central

    Shaunak, S; Gooderham, N J; Edwards, R J; Payvandi, N; Javan, C M; Baggett, N; MacDermot, J; Weber, J N; Davies, D S

    1994-01-01

    1. Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV-1. Using the T-cell lines, C8166 and HPB-ALL, and the laboratory adapted strains of HIV-1.MN, HIV-1.IIIb and HIV-1.RF, dextrin 2-sulphate (D2S) combined the best combination of high anti-HIV-1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230-3700 nM depending upon the primary viral isolate tested. 2. In saturation binding studies, [3H]-D2S bound to a cell surface protein on HPB-ALL cells in a specific and saturable manner with a Kd of 82 +/- 14 nM and a Bmax of 4.8 +/- 0.3 pmol/10(6) cells. It bound to other human T-cell lines in a similar manner. 3. There was very little binding of [3H]-D2S to freshly isolated PBMN cells (Bmax 0.18 +/- 0.03 pmol/10(6) cells) and these cells could not be infected by HIV-1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL-2 did not significantly change the Bmax of [3H]-D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 micrograms ml-1) for 72 h had a Bmax of [3H]-D2S binding of 7.2 +/- 0.1 pmol/10(6) cells and these cells could be infected by HIV-1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL-2 resulted in a fall in the Bmax to 2.0 +/- 0.1 pmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7812605

  3. CD14+ cells are required for IL-12 response in bovine blood mononuclear cells activated with Toll-like receptor (TLR) 7 and TLR8 ligands.

    PubMed

    Buza, Joram; Benjamin, Ponn; Zhu, Jianzhung; Wilson, Heather L; Lipford, Grayson; Krieg, Arthur M; Babiuk, Lorne A; Mutwiri, George K

    2008-12-15

    Single-stranded viral RNA (ssRNA) was recently identified as the natural ligand for TLR7 and TLR8. ssRNA sequences from viruses, as well as their synthetic analogues stimulate innate immune responses in immune cells from humans and mice, but their immunostimulatory activity has not been investigated in ruminants. In the present investigations, we tested whether synthetic RNA oligoribonucleotides (ORN) can activate immune cells from cattle. In vitro incubation of bovine peripheral blood mononuclear cells (PBMCs) with ORN-induced production of IL-12, IFN-gamma and TNF-alpha. No significant induction of IFN-alpha was observed. Depletion of CD14+ cells from PBMC abrogated the IL-12 response and consequently the IFN-gamma response, suggesting that CD14+ cells are required for PBMC immune activation with ORN. Consistent with these findings, the putative receptors for ORN (TLR7 and TLR8) were expressed at higher levels in the CD14+ fraction than the CD14- PBMC fraction. Pre-treatment of PBMC with bafilomycin (an inhibitor of phagosomal acidification) prior to stimulation with ORN abolished the cytokine responses, confirming that the receptor(s) which mediate the ORN-induced responses are intracellular. These results demonstrate for the first time that the TLR7/8 agonist ORN's have strong immune stimulatory effects in cattle, and suggest that further investigation on the potential of TLR7/8 ligands to activate innate and adaptive immune responses in domestic animals are warranted. PMID:18789542

  4. Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

    SciTech Connect

    Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

    2008-06-01

    The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 {mu}g/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 {mu}g/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1{beta} (83%) or TNF-{alpha} (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity.

  5. In vitro augmentation of the cytotoxic activity of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes by famotidine in cancer patients.

    PubMed

    Tsunoda, T; Tanimura, H; Yamaue, H; Iwahashi, M; Tani, M; Tamai, M; Arii, K; Noguchi, K

    1992-01-01

    We investigated the in vitro effects of famotidine on the cytotoxic activity of peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TILs). The cytotoxic activity of PBMC was augmented by famotidine at a concentration of 10 ng/ml, which is equivalent to the serum level achieved by the intravenous administration of a dose of 20 mg. This response to famotidine was seen only in cancer patients. Both the cytotoxic activity and DNA synthesis of activated TILs were increased by the combination of interleukin-2 and 1 microgram/ml of famotidine. Augmentation of cytotoxic activity by famotidine occurred independently of any decrease in the population of suppressor T-cells. Thus, famotidine may have the potential to be used in adoptive immunotherapy with TILs for cancer patients. PMID:1582736

  6. Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.

    PubMed

    Reddy, Manjula; Eirikis, Edward; Davis, Cuc; Davis, Hugh M; Prabhakar, Uma

    2004-10-01

    Activation of lymphocytes is a complex, yet finely regulated cascade of events that results in the expression of cytokine receptors, production and secretion of cytokines and expression of several cell surface molecules that eventually lead to divergent immune responses. Assessing the qualitative and quantitative nature of lymphocyte function following immunotherapy provides valuable information about the immune responses mediated by a therapeutic agent. To facilitate evaluation of the immunomodulatory activity of therapeutic agents, we have established a platform of in vitro immunoassays with normal human peripheral blood mononuclear cells (PBMCs) treated with several polyclonal activators that are known to exhibit different modes of action. We evaluated the kinetics of cell surface marker expression and cytokine release from PBMCs stimulated in parallel with various activating agents over a time course. These stimulating agents induced early (CD69 and CD71) and late (CD25 and HLA-DR) activation markers to varying antigen densities, indicated different cytokine profiles, and showed differential inhibition with dexamethasone (DEX), an inhibitor of early signaling events. Based on the association or correlation of the kinetics of activation marker expression and secreted cytokines, the results of our study indicate the appropriate time points for the simultaneous measurement of both these activation products. This study defines the kinetics for both measures of T cell activation and provides a comprehensive review with various polyclonal activators that can serve as a reference for monitoring lymphocyte function in clinical study samples. PMID:15541283

  7. Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells.

    PubMed

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Carneiro, Karine Fernandes; Souza, Maria Aparecida de; Magalhães, Denildo

    2012-01-01

    This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (p<0.05). Statistically significant differences (p<0.05) were observed between the membranes and the control in the experimental period, although there was a significant reduction in viability over time (p<0.01). At 4 and 12 h, the porcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (p<0.01), and this difference was maintained at 24 h (p<0.05). This in vitro study showed that the porcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time. PMID:22460313

  8. Anti-apoptotic activity of caffeic acid, ellagic acid and ferulic acid in normal human peripheral blood mononuclear cells: a Bcl-2 independent mechanism.

    PubMed

    Khanduja, Krishan Lal; Avti, Pramod Kumar; Kumar, Surender; Mittal, Nidhi; Sohi, Kiranjit Kaur; Pathak, Chander Mohan

    2006-02-01

    Polyphenols have been shown to induce apoptosis in a variety of tumor cells including leukemia both in vitro and in vivo. However, their action on normal human peripheral blood mononuclear cells (PBMCs) during oxidative stress remains to be explored. In this study, we have evaluated the anti-apoptotic and radical scavenging activities of dietary phenolics, namely caffeic acid (CA), ellagic acid (EA) and ferulic acid (FA). H2O2-induced apoptosis in normal human PBMCs was assayed by phosphotidylserine externalization, nucleosomal damage and DNA fragmentation. Incubation of PBMCs with 5 mM H2O2 led to increased Annexin-V binding to externalized phosphatidyl serine (PS), an event of pre-apoptotic stage of the cell. Peripheral blood mononuclear cells pretreated with phenolics could resist H2O2-induced apoptotic damage. Caffeic acid (60 and 120 microM) and EA (100 and 200 microM) caused no change in externalization of PS, whereas FA (100 and 200 microM) increased externalization of PS in PBMCs treated with H2O2. The effects of phenolics were abolished to a large extent by culturing the PBMCs for 24 h after washing the phenolics from the medium. Inhibitory activities of these phenolics on lipid peroxidation were in the order of EAactivities of EA, CA and FA were found to be 31.2+/-1.36, 50+/-1.86 and 73.0+/-1.58 microM respectively. Although, the phenolics significantly inhibited DNA damage and lipid peroxidation, they could not alter the Bcl-2 expression in PBMCs. In conclusion, the anti-apoptotic effect of EA, CA and FA in PBMCs seems to be through the Bcl-2 independent mechanism. PMID:16459021

  9. Activation of the cholinergic anti-inflammatory system in peripheral blood mononuclear cells from patients with borderline personality disorder.

    PubMed

    Díaz-Marsá, Marina; Macdowell, Karina S; Guemes, Itziar; Rubio, Víctor; Carrasco, José L; Leza, Juan C

    2012-12-01

    A case-control study including patients (n = 20) with Borderline Personality Disorder (BPD) and healthy controls (n = 33) was carried out. To avoid interferences of other clinical conditions on biological findings, patients were free of current major depressive episodes or substance dependence disorders, and had no life history of schizophrenia, bipolar or neuropsychiatric disorders. Patients were free of medication for at least two weeks at the time of the study. Studies carried out in peripheral mononuclear blood cells and plasma evidence a systemic inflammatory condition in unstable-impulsive BPD patients. Specifically, a significant increase in some intracellular components of two main pro-inflammatory pathways such as iNOS and COX-2, as well as an increase in the plasma levels of the inflammatory cytokine IL1β. Interestingly, patients have an increase in the protein expression of the anti-inflammatory subtype of nicotinic receptor α7nAChR. This finding may reflect a possible mechanism trying to maintain intracellular inflammation pathways under control. All together, these results describe an imbalanced, pro-inflammatory and oxidant phenotype in BPD patients independent of plasma cotinine levels. Although more scientific evidence is needed, the determination of multiple components of pro- and anti-inflammatory cellular pathways have interesting potential as biological markers for BPD and other generalized impulsive syndromes, specially data obtained with α7nAChR and its lack of correlation with plasma levels of nicotine metabolites. Their pharmacological modulation with receptor modulators can be a promising therapeutic target to take into account in mental health conditions associated with inflammatory or oxido/nitrosative consequences. Also, identifying at-risk individuals would be of importance for early detection and intervention in adolescent subjects before they present severe behavioural problems. PMID:23083519

  10. Costimulation with anti-cluster of differentiation 3 and anti-cluster of differentiation 28 reduces the activity of mucin 1-stimulated human mononuclear cells

    PubMed Central

    WRIGHT, STEPHEN E.; REWERS-FELKINS, KATHLEEN A.; QUINLIN, IMELDA; ZOHRA, FATEMA; AHMED, JEWEL

    2016-01-01

    Cytotoxic T-lymphocyte activation and extension of the cell life span is necessary in order to enable immunotherapy to perform effectively against cancer. In the present study, mucin 1 (MUC1)-stimulated human mononuclear cells (M1SHMCs) were costimulated with bead-attached monoclonal antibodies specific for cluster of differentiation (CD)3 and CD28 receptors. The study was undertaken to determine whether costimulation was capable of enhancing the killing of cancer cells in vitro and of protecting non-obese diabetic severe combined immunodeficient mice from tumor development. Lysis of MCF-7 tumor cells by M1SHMCs was reduced following costimulation with anti-CD3 and anti-CD28. Furthermore, costimulation with anti-CD3 and anti-CD28 eliminated the protective effects of M1SHMCs on MCF-7 breast cancer cell growth in the non-obese diabetic severe combined immunodeficient mice. The present study suggested that costimulation with anti-CD3 and anti-CD28 is not advisable following antigen activation of lymphocytes under the conditions used here. Using a lower anti-CD3/CD28 bead to T-cell ratio may prevent immune suppression, however, further studies are required to support this hypothesis. PMID:26870234

  11. Isolation of equine peripheral blood mononuclear cells using Percoll.

    PubMed

    May, S A; Hooke, R E; Lees, P

    1991-01-01

    The concentration of Percoll required for isolating equine peripheral blood mononuclear cells has been reinvestigated. A poor cell yield was obtained at the 60 per cent concentration already reported. It is recommended that workers specifically interested in high yields of mononuclear cells, for investigation of lymphocyte and monocyte functions, use a concentration of 65 per cent Percoll. However, workers wishing to isolate pure populations of equine neutrophils might consider a concentration of 70 per cent in the upper layer of Percoll used to retain the mononuclear cells. PMID:1646471

  12. Defective mitochondrial respiration, altered dNTP pools and reduced AP endonuclease 1 activity in peripheral blood mononuclear cells of Alzheimer's disease patients

    PubMed Central

    Maynard, Scott; Hejl, Anne-Mette; Dinh, Thuan-Son T.; Keijzers, Guido; Hansen, Åse M.; Desler, Claus; Moreno-Villanueva, Maria; Bürkle, Alexander; Rasmussen, Lene J.; Waldemar, Gunhild; Bohr, Vilhelm A.

    2015-01-01

    AIMS Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics and DNA damage, in peripheral blood mononuclear cells (PBMCs) of AD and control participants, for biomarker discovery. METHODS PBMCs were isolated from 53 patients with AD of mild to moderate degree and 30 age-matched healthy controls. Tests were performed on the PBMCs from as many of these participants as possible. We measured glycolysis and mitochondrial respiration fluxes using the Seahorse Bioscience flux analyzer, mitochondrial ROS production using flow cytometry, dNTP levels by way of a DNA polymerization assay, DNA strand breaks using the Fluorometric detection of Alkaline DNA Unwinding (FADU) assay, and APE1 incision activity (in cell lysates) on a DNA substrate containing an AP site (to estimate DNA repair efficiency). RESULTS In the PBMCs of AD patients, we found reduced basal mitochondrial oxygen consumption, reduced proton leak, higher dATP level, and lower AP endonuclease 1 activity, depending on adjustments for gender and/or age. CONCLUSIONS: This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD. PMID:26539816

  13. Immunomodulating activities of cultivated maitake medicinal mushroom Grifola frondosa (Dicks.: Fr.) S.F. Gray (higher Basidiomycetes) on peripheral blood mononuclear cells.

    PubMed

    Svagelj, Mirjan; Berovic, Marin; Gregori, Andrej; Wraber, Branka; Simcic, Sasa; Boh, Bojana

    2012-01-01

    Grifola frondosa is a culinary-medicinal mushroom that contains several physiologically active compounds, of which polysaccharides, specifically β-glucans, are known to possess immunomodulating properties. Its extracts are studied for application as adjuncts for chemotherapy, and experiments in animal models support the use of this mushroom for cancer treatment. The effect of extracts obtained from mushrooms cultivated on different substrates and their capacity of inducing the secretion of cytokines from human peripheral blood mononuclear cells were studied. The activity of extracts at concentrations 12.5, 100, and 200 μg/mL on induction of TNF-α, IFN-γ, and IL-12 was screened. Two extracts from substrates fortified with olive oil press cakes showed appreciable activity and induced the secretion of TNF-α, IL-12, and INF-γ. The extracts differed from the others in the amount of sugar, protein, and β-glucans, which can explain their higher activity. Present results show that different substrates and different source materials can reasonably modify the bioactivity of cultivated G. frondosa. PMID:23510175

  14. Menstrual cycle and reproductive aging alters immune reactivity, NGF expression, antioxidant enzyme activities, and intracellular signaling pathways in the peripheral blood mononuclear cells of healthy women.

    PubMed

    Priyanka, Hannah P; Sharma, Utsav; Gopinath, Srinivasan; Sharma, Varun; Hima, Lalgi; ThyagaRajan, Srinivasan

    2013-08-01

    Reproductive senescence in women is a process that begins with regular menstrual cycles and culminates in menopause followed by gradual development of diseases such as autoimmune diseases, osteoporosis, neurodegenerative diseases, and hormone-dependent cancers. The age-associated impairment in the functions of neuroendocrine system and immune system results in menopause which contributes to subsequent development of diseases and cancer. The aim of this study is to characterize the alterations in immune responses, compensatory factors such as nerve growth factor (NGF) and antioxidant enzyme activities, and the molecular mechanisms of actions in the peripheral blood mononuclear cells (PBMCs) of young (follicular and luteal phases), middle-aged, and old healthy women. Peripheral blood mononuclear cells were isolated from young women in follicular and luteal phases of the menstrual cycle (n=20; 22.6±2.9 yrs), middle-aged women (n=19; 47.1±3.8 yrs; perimenopausal) and old (n=16; 63.2±4.7 yrs; post-menopausal) women and analyzed for Concanavalin (Con A)-induced proliferation of lymphocytes and cytokine (IL-2 and IFN-γ) production, expression of NGF, p-NF-κB, p-ERK, p-CREB, and p-Akt, antioxidant enzymes [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), glutathione-S-transferase (GST)], extent of lipid peroxidation, and nitric oxide (NO) production. Serum gonadal hormones (17β-estradiol and progesterone) were also measured. A characteristic age- and menstrual cycle-related change was observed in the serum gonadal hormone secretion (estrogen and progesterone), T lymphocyte proliferation and IFN-γ production. Salient features include the age-related decline observed in target-derived growth factors (lymphocyte NGF expression), signaling molecules (p-ERK/ERK and p-CREB/CREB ratios) and compensatory factors such as the activities of plasma and PBMC antioxidant enzymes (SOD and catalase) and NO production. Further, an age-associated increase in p

  15. Calcium ionophore and phorbol ester activation of proliferation and. gamma. -IFN production by neonatal mononuclear cells (MNCs)

    SciTech Connect

    Bryson, Y.J.; Kuhls, T.L.; Pineda, E.

    1986-03-01

    Human neonatal MNCs have a dissociation between prolif. and ..gamma..-IFN prod. Although cord MNCs display normal-high prolif. following lectin stim., ..gamma..-IFN prod. is greatly diminished compared to adult MNCs. Increasing data support a 2-stimuli requirement for human T-cell activation as noted in the T-cell line Jurkat as well as in peripheral T-cells. They have compared prolif. and ..gamma..-IFN responses of cord and adult MNCs to the calcium ionophore A23187, phorbol myristate acetate (PMA), PHA and their combinations. Cord and adult MNCs had similar prolif. responses to A23187, PMA and PHA. PMA alone acted as a weak mitogen compared to PHA. Optimal A23187 alone caused very low amts of prolif. Either PMA or A23187 suppressed PHA-stim. prolif. while A23187 augmented PMA-induced prolif. A23187, PMA or PHA alone prod. ..gamma..-IFN in adult but not cord MNCs. The addition of PMA or A23187 augmented the PHA-induced ..gamma..-IFN prod. in both cord and adult MNCs (6..-->..80 IU vs 240..-->..480 IU resp). When combined, A23187 and PMA stim. optimal and comparable amts of ..gamma..-IFN in adult and cord MNCs (480 IU). From these findings they conclude that although the stimuli for ..gamma..-IFN and prolif. may be similar, there is an absolute requirement for 2 stimuli (PMA/A23187) for ..gamma..-IFN prod. by cord cells and optimal prod. in adult MNCs. The defect of ..gamma..-IFN prod. observed in PHA stim. neonatal MNCs can be corrected using a calcium ionophore and protein kinase C activator.

  16. Decreased Cystathionine-γ-lyase (CSE) Activity in Livers of Type 1 Diabetic Rats and Peripheral Blood Mononuclear Cells (PBMC) of Type 1 Diabetic Patients*

    PubMed Central

    Manna, Prasenjit; Gungor, Neslihan; McVie, Robert; Jain, Sushil K.

    2014-01-01

    The liver plays a major role in the formation of H2S, a novel signaling molecule. Diabetes is associated with lower blood levels of H2S. This study investigated the activities of cystathionine-γ-lyase (CSE, the enzyme that catalyzes H2S formation) in livers of type 1 diabetic (T1D) animals and in peripheral blood mononuclear cells (PBMC) isolated from T1D patients. T1D is associated with both hyperketonemia (acetoacetate and β-hydroxybutyrate) and hyperglycemia. This study also examined the role of hyperglycemia and hyperketonemia per se in decreased CSE activity using U937 monocytes and PBMC isolated from healthy subjects. Livers from streptozotocin-treated T1D rats demonstrated a significantly higher reactive oxygen species production, lower CSE protein expression and activity, and lower H2S formation compared with those of controls. Studies with T1D patients showed a decrease in CSE protein expression and activity in PBMC compared with those of age-matched normal subjects. Cell culture studies demonstrated that high glucose (25 mm) and/or acetoacetate (4 mm) increased reactive oxygen species, decreased CSE mRNA expression, protein expression, and enzymatic activity, and reduced H2S levels; however, β-hydroxybutyrate treatment had no effect. A similar effect, which was also observed in PBMC treated with high glucose alone or along with acetoacetate, was prevented by vitamin D supplementation. Studies with CSE siRNA provide evidence for a relationship between impaired CSE expression and reduced H2S levels. This study demonstrates for the first time that both hyperglycemia and hyperketonemia mediate a reduction in CSE expression and activity, which can contribute to the impaired H2S signaling associated with diabetes. PMID:24610811

  17. Abnormal activation of calpain and protein kinase Cα promotes a constitutive release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients.

    PubMed

    Averna, Monica; Bavestrello, Margherita; Cresta, Federico; Pedrazzi, Marco; De Tullio, Roberta; Minicucci, Laura; Sparatore, Bianca; Salamino, Franca; Pontremoli, Sandro; Melloni, Edon

    2016-08-15

    Matrix metalloproteinase 9 (MMP9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies. We here report that peripheral blood mononuclear cells (PBMCs), isolated from cystic fibrosis (CF) patients homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR), express constitutively and release at high rate MMP9 due to the alteration in their intracellular Ca(2+) homeostasis. This spontaneous and sustained MMP9 secretion may contribute to the accumulation of this protease in fluids of CF patients. Conversely, in PBMCs isolated from healthy donors, expression and secretion of MMP9 are undetectable but can be evoked, after 12 h of culture, by paracrine stimulation which also promotes an increase in [Ca(2+)]i. We also demonstrate that in both CF and control PBMCs the Ca(2+)-dependent MMP9 secretion is mediated by the concomitant activation of calpain and protein kinase Cα (PKCα), and that MMP9 expression involves extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. Our results are supported by the fact that either the inhibition of Ca(2+) entry or chelation of [Ca(2+)]i as well as the inhibition of single components of the signaling pathway or the restoration of CFTR activity all promote the reduction of MMP9 secretion. PMID:27349634

  18. Cell-mediated cytotoxicity of bovine mononuclear cells to IBRV-infected cells: dependence on Sephadex G-10 adherent cells.

    PubMed

    Campos, M; Rossi, C R

    1985-04-01

    Following intranasal inoculation of cattle with infectious bovine rhinotracheitis virus (IBRV) mononuclear cells that produced a genetically unrestricted cytotoxic response against IBRV-infected, but not against uninfected cells, were present in peripheral blood. Cytotoxicity was detected between 6 and 14 days after primary infection in a 20 h, but not in a 5 h, 51Cr-release assay. Cytotoxic activity was present in peripheral blood mononuclear cells from infected and subsequently hyperimmunized cattle for a considerably longer time. Neither natural cytotoxicity, antibody-dependent cell cytotoxicity, nor antibody produced during the assay was responsible for the cytotoxicity. However, cytotoxicity was dependent upon an adherent mononuclear cell that was partially removed by passage over nylon wool and completely removed by passage over Sephadex G-10. PMID:2408374

  19. Analysis of mononuclear cell functions in Holstein cattle with leukocyte adhesion deficiency.

    PubMed

    Nagahata, H; Nochi, H; Sanada, Y; Tamoto, K; Noda, H; Kociba, G J

    1994-08-01

    Lymphocyte functions in cattle affected with leukocyte adhesion deficiency (LAD, termed BLAD in cattle) were evaluated by lymphocyte markers, blastogenic response, and immunoglobulin concentrations; mononuclear phagocyte functions were assessed by chemotactic and luminol-dependent chemiluminescent (CL) responses to determine the effects of impaired expression of leukocyte CD18 on mononuclear cell functions. Deficient CD18 expression on lymphocytes and mononuclear phagocytes from cattle with BLAD was clearly detected by use of flow cytometric analysis. There were no significant differences in the population of peanut agglutinin (PNA)-positive and surface immunoglobulin-bearing blood lymphocytes from clinically normal cattle and cattle with BLAD, as determined by flow cytometric analysis. Lymphocytes from cattle with BLAD had strong mitogen-induced blastogenic responses, which were greater than those from controls. Adherence of mononuclear phagocytes from cattle with BLAD was markedly impaired, and their chemotactic responses had diminished values, compared with those of controls. Luminol-dependent CL of mononuclear phagocytes from affected cattle, stimulated by opsonized zymosan, had significantly (P < 0.01) decreased values, compared with those of controls. Concentrations of IgG were markedly increased in serum from cattle with BLAD, compared with those in controls. These results indicated that impaired expression of leukocyte CD18 has marked effects on adhering activity of mononuclear phagocytes, and significantly inhibits CL response of mononuclear phagocytes mediated by inactivated-complement 3b-dependent functions. High selective immunoglobulin concentrations indicated that lymphocytes of B-cell lineage may have normal function. PMID:7978649

  20. NF-κB Repressing Factor Inhibits Chemokine Synthesis by Peripheral Blood Mononuclear Cells and Alveolar Macrophages in Active Pulmonary Tuberculosis

    PubMed Central

    Huang, Kuo-Hsiung; Wang, Chun-Hua; Lee, Kang-Yun; Lin, Shu-Min

    2013-01-01

    NF-κB repressing factor (NRF) is a transcriptional silencer implicated in the basal silencing of specific NF-κB targeting genes, including iNOS, IFN-β and IL-8/CXCL8. IP-10/CXCL10 and IL-8/CXCL8 are involved in neutrophil and lymphocyte recruitment against M. tuberculosis (MTb) and disease progression of pulmonary tuberculosis (TB). Alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMC) were used to study the regulatory role of NRF in pulmonary TB. AM and PBMC were purified from 19 TB patients and 15 normal subjects. To study the underlying mechanism, PBMC were exposed to heated TB bacilli. The regulation role of NRF in IP-10/CXCL10 and IL-8/CXCL8 was determined by NRF knock-down or over-expression. NRF binding capabilities in promoter sites were measured by chromatin immunoprecipitation (ChIP) assay. The levels of IP-10/CXCL10, IL-8/CXCL8 and NRF were significantly higher in AM and PBMC in patients with active TB. NRF played an inhibitory role in IP-10/CXCL10 and IL-8/CXCL8 inductions. We delineate the role of NRF in pulmonary TB, which inhibits the expressions of IP-10/CXCL10 and IL-8/CXCL8 in AM and PBMC of patients with high bacterial load. NRF may serve as an endogenous repressor to prevent robust increase in IP-10/CXCL10 and IL-8/CXCL8 when TB bacterial load is high. PMID:24223729

  1. In vitro activation of cord blood mononuclear cells and cytokine production in a remote coastal population exposed to organochlorines and methyl mercury.

    PubMed

    Bilrha, Houda; Roy, Raynald; Moreau, Brigitte; Belles-Isles, Marthe; Dewailly, Eric; Ayotte, Pierre

    2003-12-01

    Remote coastal populations that rely on seafood for subsistence often receive unusually high doses of organochlorines and methyl mercury. Immunosuppression resulting from prenatal exposure to organochlorines has been reported in wildlife species and humans. In this study, we assessed lymphocyte activation and associated cytokine secretion in 47 newborns from a remote maritime population living on the Mid and Lower North Shore regions of the St. Lawrence River (Québec, Canada; subsistence fishing group) and 65 newborns from nearby urban settings (reference group). Cord blood samples were collected for organochlorine and mercury analyses and also to isolate cord blood mononuclear cells (CBMCs) for the in vitro assessment of cytokine production and expression of surface markers after mitogenic stimulation (CD4(+)CD45RO(+), CD8(+)CD45RO(+), CD3(+)CD25(+), and CD8(+)HLA-DR(+)). Blood mercury and plasma concentrations of polychlorinated biphenyls (PCBs), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p'-DDE), and hexachlorobenzene (HCB) were significantly higher in the subsistence fishing group than in the reference group (p < 0.001). No difference was observed between the two groups regarding subsets of lymphocytes showing markers of activation. In vitro secretion of cytokines by CBMCs after mitogenic stimulation was lower in the subsistence fishing group than in the reference group (p < 0.05). Moreover, we found an inverse correlation between tumor necrosis factor-alpha (TNF-alpha) secretion and plasma PCB, p,p'-DDE, and HCB concentrations (p < 0.05). Our data support a negative association between TNF-alpha secretion by CBMCs and prenatal organochlorine exposure. If the relationship between organochlorine and TNF-alpha secretion is causal, it would suggest a role for this important proinflammatory cytokine in mediating organochlorine-induced immunotoxicity in infants developmentally exposed to these compounds. PMID:14644672

  2. Plasmids enriched with CpG motifs activate human peripheral blood mononuclear cells in vitro and enhance th-1 immune responses to hepatitis B surface antigen in mice.

    PubMed

    Chen, Zhihui; Cao, Jie; Liao, Xiaoling; Ke, Jinshan; Zhu, Shiying; Zhao, Ping; Qi, Zhongtian

    2011-06-01

    T helper-1 (Th-1)-type immune responses play an important role in viral clearance during infection with hepatitis B virus (HBV). Unmethylated CpG motifs present in bacterial DNA can activate toll-like receptor 9 (TLR9) signals and act as potent adjuvants to induce Th-1-type immune responses. Here, a mini-plasmid with 812 base pairs in length was constructed and used as a vector to prepare a series of plasmids containing 3-21 copies of D-type CpG motifs. In vitro, these CpG-enriched plasmids strongly stimulated proliferation of human peripheral blood mononuclear cells (PBMCs) and enhanced secretion of interferon-γ (IFN-γ) and interleukin-12 (IL-12). The responses of the PBMCs from healthy individuals to the plasmids were stronger than those obtained from HBV-infected individuals. Contrary to the strong Th-2-biased response induced by surface antigen of hepatitis B virus (HBsAg) plus alum adjuvant, immunization of BALB/c mice with HBsAg plus these plasmids induced a strong Th-1-biased response. The plasmids increased the titers of HBsAg-specific total immunoglobulin G (IgG) and IgG(2a). HBsAg-specific IL-2 and IFN-γ production and cytotoxic activity were also enhanced in the presence of the plasmids. The strength of the immune responses positively correlated with the number of CpG motifs in the plasmids. These results indicate that the use of CpG-enriched plasmids as an adjuvant to recombinant HBsAg could provide a promising and cost-effective approach for the development of efficacious therapeutic vaccines against HBV infection. PMID:21668361

  3. Interleukin-4 receptors on human blood mononuclear cells

    SciTech Connect

    Zuber, C.E.; Galizzi, J.P.; Harada, N.; Durand, I.; Banchereau, J. )

    1990-09-01

    We have studied regulation of the expression of the interleukin-4 receptor (IL-4R) on human blood mononuclear cells (PBMC) using both 125I-IL-4 binding assay and flow cytometric analysis of biotinylated IL-4 (B-IL-4) binding. PBMC express approximately 300 high-affinity IL-4R per cell (Kd = 25-100 pM). Activation of PBMC for 60-80 hr by phytohemagglutinin (PHA) or concanavalin A (Con A) results in a 2- to 4.5-fold increase of IL-4R number without alteration of IL-4R affinity for IL-4. Binding of B-IL-4 showed that IL-4R expression is upregulated on virtually all PHA-stimulated PBMC, whereas it mostly concerns larger cells among Con A-activated PBMC. Reculture of PHA-blasts with 1 nM IL-4 further upregulates IL-4R expression to a level approximately 10-fold higher than observed on freshly isolated PBMC. Interestingly, IL-4 is able to reinduce high IL-4R levels on cells that have been deprived of IL-4 for 20 hr and IL-2 is almost as efficient. Finally, SDS-PAGE analysis of IL-4-binding molecules on unstimulated, PHA- and PHA/IL-4-activated PBMC revealed the same three peptides of MW 140-130, 80-75, and 70-65 kDa, as shown on human cell lines.

  4. [Enhancement of the phagocytic activity of cells of the mononuclear phagocytosing system by the peroral administration of lactobacilli].

    PubMed

    Zaĭtseva, L G; Gorskaia, E M; Lentsner, A A; Shustrova, N M

    1986-09-01

    The phagocytic function of the peritoneal exudate cells after oral and intraperitoneal administration of Lactobacillus casei subsp. casei, L. plantarum and L. fermentum was studied on mice C57Bl and inbred rats CDF/F344/Crl. The phagocytic function was estimated by the capacity for absorbing and excreting 14C-labeled typhoid vaccine. Intraperitoneal administration of a mixture of various Lactobacillus strains resulted in two-fold increase of the bacteria absorption and digestion when the Lactobacillus strains were administered 4 days before administration of the test antigen. On their simultaneous administration the phagocytic function of the peritoneal exudate cells lowered. Oral administration of the Lactobacillus strains for 6 and 18 days resulted, respectively, in 1.5- and 3-fold increase of absorption and digestion of the labeled typhoid vaccine. This route of Lactobacillus administration provided more rapid increase of the level of the antibodies to the typhoid antigen. Possible immunomodulating action of Lactobacillus administered orally is discussed. PMID:3777898

  5. Infiltration of mononuclear inflammatory cells into primary colorectal carcinomas: an immunohistological analysis.

    PubMed Central

    Håkansson, L.; Adell, G.; Boeryd, B.; Sjögren, F.; Sjödahl, R.

    1997-01-01

    Local immunoregulation mediated by mononuclear tumour-infiltrating cells is considered of importance for tumour progression of colorectal cancer, although the balance between immunosuppressor and cytotoxic activities is unclear. Colorectal cancers from 26 patients were investigated using a panel of monoclonal antibodies in order to identify subsets of mononuclear inflammatory cells and to study their pattern of distribution in relation to tumour stage and cytotoxic immune reactivity against the tumour. In all but five tumours, mononuclear cells, lymphocytes or monocytes were present in fairly large numbers, particularly in the stroma. The infiltration of CD4+ mononuclear cells predominated over the CD8+ subset. Infiltration near the tumour cells was found in four cancers only. Stromal infiltration of CD11c+ macrophages was found in all but eight tumours. Small regressive areas, in which the histological architecture of the tumours was broken down, were found in 17 tumours with intense or moderate infiltration by CD4+ lymphocytes or CD11c+ macrophages. Probably this destruction of tumour tissue was caused by cytotoxic activity of the tumour-infiltrating mononuclear cells. In Dukes' class A and B tumours, CD4+ lymphocytes predominated over CD4+ cells with macrophage morphology, but the latter were increasingly found in Dukes' class C and D disease. The occurrence of MHC II-positive macrophages and lymphocytes in different Dukes' classes was similar to that of CD4+ cells. In contrast to this, CD11c+ and CD11a+ cells were more frequent in Dukes' A and B class tumours compared with Dukes' C and D. Four out of nine tumours of the latter stages showed a poor inflammatory reaction. The interpretation of our results is that the subsets of tumour-infiltrating mononuclear cells change with advancing Dukes' class and that the local immune control is gradually broken down in progressive tumour growth, even if some cytotoxic activity is still present. Images Figure 1 Figure 2

  6. Diagnostic values for the viral load in peripheral blood mononuclear cells of patients with chronic active Epstein-Barr virus disease.

    PubMed

    Ito, Yoshinori; Suzuki, Michio; Kawada, Jun-Ichi; Kimura, Hiroshi

    2016-04-01

    Chronic active Epstein-Barr virus disease (CAEBV) is a distinct EBV-associated lymphoproliferative disease with a poor prognosis. Although the viral load in blood samples has been widely used for diagnosing CAEBV, well-defined viral load thresholds to guide clinicians are currently lacking. The aim of the present study was to determine standardized diagnostic values for EBV load in blood samples of CAEBV patients using the World Health Organization international standard for reporting. Levels of EBV DNA in 103 peripheral blood mononuclear cells (PBMCs) and 95 plasma/serum samples from 107 cases with CAEBV were quantified and expressed in international units. Receiver operating characteristic curves were analyzed to assess the most appropriate cut-off values for levels of EBV DNA to distinguish CAEBV from EBV-associated infectious mononucleosis (IM) and controls with past EBV infection. Levels of EBV DNA in PBMCs were significantly higher in the CAEBV group (median, 10(4.2) IU/μgDNA) compared to the IM (median, 10(2.1) IU/μgDNA) and control groups. An inconsistent qualitative result was seen in 13 of 86 CAEBV patients; in these, EBV-DNA was positive in PBMCs, but negative in plasma. Diagnostic cut-off values for viral load in PBMCs from CAEBV patients, as compared to those of healthy controls and IM patients, were 10(2.0) IU/μgDNA and 10(3.2) IU/μgDNA, respectively. For diagnostic purposes, the viral load of PBMCs was better than of plasma/serum. A diagnostic cut-off EBV load for CAEBV may be useful for the management of CAEBV patients. PMID:26712582

  7. Basic surface properties of mononuclear cells from Didelphis marsupialis.

    PubMed

    Nacife, V P; de Meirelles, M de N; Silva Filho, F C

    1998-01-01

    The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5 degrees and 40.8 degrees, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis. PMID:9921307

  8. Establishment of an interleukin-1β-induced inflammation-activated endothelial cell-smooth muscle cell-mononuclear cell co-culture model and evaluation of the anti-inflammatory effects of tanshinone IIA on atherosclerosis.

    PubMed

    Li, Yujie; Guo, Yan; Chen, Ying; Wang, Yajie; You, Yun; Yang, Qing; Weng, Xiaogang; Li, Qi; Zhu, Xiaoxin; Zhou, Bingbing; Liu, Xucen; Gong, Zaipeng; Zhang, Ruijie

    2015-08-01

    Increasing evidence supports the hypothesis that inflammatory reactions serves an important function in the formation, progression and plaque rupture of atherosclerosis. Interleukin (IL)-1 primarily induces inflammation and is closely associated with the inflammatory environment and the formation of atherosclerosis. The present study aimed to establish an in vitro model for the evaluation of drug efficacy in the intervention of atherosclerosis from the inflammatory perspective, and to observe the anti-inflammatory effects of tanshinone IIA and andrographolide on atherosclerosis. The IL-1β-induced inflammation-activated endothelial cell (EC)-smooth muscle cell (SMC)-mononuclear cell (MC) co-culture model was established, based on the changes in a series of atherosclerosis-associated inflammatory markers secreted by ECs and SMCs. The expression of connexin in ECs, adhesion of MCs and changes in inflammatory signalling molecules were selected as evaluation indices for the inflammatory microenvironment of atherosclerosis. The use of this model revealed that tanshinone IIA exhibited significant efficacy against atherosclerosis and its inflammatory reactions. Inflammatory reactions were regarded as the primary mechanism underlying atherosclerosis. The established model simulated a series of relevant changes in the arterial wall under the inflammatory cytokines with oxidized low-density lipoprotein during the atherosclerotic process. The present study presented a reliable method for the identification of drugs with potential anti-inflammatory activity in atherosclerosis, for investigating the mechanisms of action, considering the improvement of the inflammatory state and the increase in plaque stability observed. PMID:25936371

  9. Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells

    PubMed Central

    Yang, Kuo H.; Kendrick, Racheal; Reddy, Y. Sunila; Kashuba, Angela D. M.; Troiani, Luigi; Bridges, Arlene S.; Fiscus, Susan A.; Forrest, Alan; Cohen, Myron S.

    2015-01-01

    The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure. PMID:26239974

  10. Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

    PubMed

    Lu, Ming-Chi; Lai, Ning-Sheng; Yin, Wen-Yao; Yu, Hui-Chun; Huang, Hsien-Bin; Tung, Chien-Hsueh; Huang, Kuang-Yung; Yu, Chia-Li

    2013-04-01

    In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α . PMID:23188524

  11. High Mobility Group Box 1-Protein expression in canine haematopoietic cells and influence on canine peripheral blood mononuclear cell proliferative activity.

    PubMed

    Altmann, S; Lange, S; Pommerencke, J; Murua Escobar, H; Bullerdiek, J; Nolte, I; Freund, M; Junghanss, C

    2008-12-15

    High Mobility Group Box 1-Protein (HMGB1) is a nuclear chromosomal protein occurring ubiquitary in mammalian tissues. HMGB1 demonstrates cytokine function and induces inflammation when actively released by haematopoietic cells or passively released during cell necrosis. This study aimed at the determination of HMGB1 expression in different cell types and at the evaluation of the role of HMGB1 in PBMC proliferation. Therefore we investigated the HMGB1 mRNA expression level in different canine haematopoietic cell types and the influence of exogenous rhHMGB1 on canine PBMC proliferation. Differentiated haematopoietic blood cells showed lower relative HMGB1 expression levels compared to CD34+ haematopoietic stem cells. Relative HMGB1 expression seemed also to decrease during differentiation of CD34+ stem cells into dendritic cells. Furthermore, peripheral blood CD14+ monocytes and granulocytes showed a lower relative HMGB1 expression in comparison to CD3+ T-lymphocytes. When exogenous rhHMGB1 at low concentrations was added to single PBMC cultures an increase of proliferation was obvious. However, in higher concentrations HMGB1 lost its stimulative effect. In conclusion, HMGB1 is broadly expressed in canine haematopoietic cells with highest levels in haematopoietic stem cells. HMGB1 induced directly PBMC proliferation. PMID:18762340

  12. Mononuclear cell secretome protects from experimental autoimmune myocarditis

    PubMed Central

    Hoetzenecker, Konrad; Zimmermann, Matthias; Hoetzenecker, Wolfram; Schweiger, Thomas; Kollmann, Dagmar; Mildner, Michael; Hegedus, Balazs; Mitterbauer, Andreas; Hacker, Stefan; Birner, Peter; Gabriel, Christian; Gyöngyösi, Mariann; Blyszczuk, Przemyslaw; Eriksson, Urs; Ankersmit, Hendrik Jan

    2015-01-01

    Aims Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. Methods and results BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. Conclusion MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases. PMID:23321350

  13. Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus.

    PubMed

    Soares, Filipa A C; Pedersen, Roger A; Vallier, Ludovic

    2016-01-01

    This protocol describes the efficient isolation of peripheral blood mononuclear cells from circulating blood via density gradient centrifugation and subsequent generation of integration-free human induced pluripotent stem cells. Peripheral blood mononuclear cells are cultured for 9 days to allow expansion of the erythroblast population. The erythroblasts are then used to derive human induced pluripotent stem cells using Sendai viral vectors, each expressing one of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. PMID:25687300

  14. Anti-inflammatory activity of probiotic Bifidobacterium: Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells

    PubMed Central

    Imaoka, Akemi; Shima, Tatsuichiro; Kato, Kimitoshi; Mizuno, Shigeaki; Uehara, Toshiki; Matsumoto, Satoshi; Setoyama, Hiromi; Hara, Taeko; Umesaki, Yoshinori

    2008-01-01

    AIM: To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk (BFM) which is effective against active ulcerative colitis (UC) and exacerbations of UC, and to explore the immunoregulatory mechanisms. METHODS: Peripheral blood mononuclear cells (PBMNC) from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult (BbrY) or Bifidobacterium bifidum strain Yakult (BbiY) to estimate the amount of IL-10 or IL-8 secreted. RESULTS: Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium (CM) and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-α, whereas no such effect was observed with heat-killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IκB-ζ mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM. CONCLUSION: Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis. PMID:18442197

  15. c-Jun NH2-Terminal Kinase Activity in Subcutaneous Adipose Tissue but Not Nuclear Factor-κB Activity in Peripheral Blood Mononuclear Cells Is an Independent Determinant of Insulin Resistance in Healthy Individuals

    PubMed Central

    Sourris, Karly C.; Lyons, Jasmine G.; de Courten, Maximilian P.J.; Dougherty, Sonia L.; Henstridge, Darren C.; Cooper, Mark E.; Hage, Michelle; Dart, Anthony; Kingwell, Bronwyn A.; Forbes, Josephine M.; de Courten, Barbora

    2009-01-01

    OBJECTIVE Chronic low-grade activation of the immune system (CLAIS) predicts type 2 diabetes via a decrease in insulin sensitivity. Our study investigated potential relationships between nuclear factor-κB (NF-κB) and c-Jun NH2-terminal kinase (JNK) pathways—two pathways proposed as the link between CLAIS and insulin resistance. RESEARCH DESIGN AND METHODS Adiposity (dual-energy X-ray absorptiometry), waist-to-hip ratio (WHR), and insulin sensitivity (M, hyperinsulinemic-euglycemic clamp) were measured in 22 healthy nondiabetic volunteers (aged 29 ± 11 years, body fat 28 ± 11%). NF-κB activity (DNA-binding assay) and JNK1/2 activity (phosphorylated JNK) were assessed in biopsies of the vastus lateralis muscle and subcutaneous adipose tissue and in peripheral blood mononuclear cell (PBMC) lysates. RESULTS NF-κB activities in PBMCs and muscle were positively associated with WHR after adjustment for age, sex, and percent body fat (both P < 0.05). NF-κB activity in PBMCs was inversely associated with M after adjustment for age, sex, percent body fat, and WHR (P = 0.02) and explained 16% of the variance of M. There were no significant relationships between NF-κB activity and M in muscle or adipose tissue (both NS). Adipose-derived JNK1/2 activity was not associated with obesity (all P> 0.1), although it was inversely related to M (r = −0.54, P < 0.05) and explained 29% of its variance. When both NF-κB and JNK1/2 were examined statistically, only JNK1/2 activity in adipose tissue was a significant determinant of insulin resistance (P = 0.02). CONCLUSIONS JNK1/2 activity in adipose tissue but not NF-κB activity in PBMCs is an independent determinant of insulin resistance in healthy individuals. PMID:19258436

  16. Alternatively Activated Mononuclear Phagocytes from the Skin Site of Infection and the Impact of IL-4Rα Signalling on CD4+T Cell Survival in Draining Lymph Nodes after Repeated Exposure to Schistosoma mansoni Cercariae.

    PubMed

    Prendergast, Catriona T; Sanin, David E; Mountford, Adrian P

    2016-08-01

    In a murine model of repeated exposure of the skin to infective Schistosoma mansoni cercariae, events leading to the priming of CD4 cells in the skin draining lymph nodes were examined. The dermal exudate cell (DEC) population recovered from repeatedly (4x) exposed skin contained an influx of mononuclear phagocytes comprising three distinct populations according to their differential expression of F4/80 and MHC-II. As determined by gene expression analysis, all three DEC populations (F4/80-MHC-IIhigh, F4/80+MHC-IIhigh, F4/80+MHC-IIint) exhibited major up-regulation of genes associated with alternative activation. The gene encoding RELMα (hallmark of alternatively activated cells) was highly up-regulated in all three DEC populations. However, in 4x infected mice deficient in RELMα, there was no change in the extent of inflammation at the skin infection site compared to 4x infected wild-type cohorts, nor was there a difference in the abundance of different mononuclear phagocyte DEC populations. The absence of RELMα resulted in greater numbers of CD4+ cells in the skin draining lymph nodes (sdLN) of 4x infected mice, although they remained hypo-responsive. Using mice deficient for IL-4Rα, in which alternative activation is compromised, we show that after repeated schistosome infection, levels of regulatory IL-10 in the skin were reduced, accompanied by increased numbers of MHC-IIhigh cells and CD4+ T cells in the skin. There were also increased numbers of CD4+ T cells in the sdLN in the absence of IL-4Rα compared to cells from singly infected mice. Although their ability to proliferate was still compromised, increased cellularity of sdLN from 4x IL-4RαKO mice correlated with reduced expression of Fas/FasL, resulting in decreased apoptosis and cell death but increased numbers of viable CD4+ T cells. This study highlights a mechanism through which IL-4Rα may regulate the immune system through the induction of IL-10 and regulation of Fas/FasL mediated cell death

  17. Alternatively Activated Mononuclear Phagocytes from the Skin Site of Infection and the Impact of IL-4Rα Signalling on CD4+T Cell Survival in Draining Lymph Nodes after Repeated Exposure to Schistosoma mansoni Cercariae

    PubMed Central

    Prendergast, Catriona T.; Sanin, David E.; Mountford, Adrian P.

    2016-01-01

    In a murine model of repeated exposure of the skin to infective Schistosoma mansoni cercariae, events leading to the priming of CD4 cells in the skin draining lymph nodes were examined. The dermal exudate cell (DEC) population recovered from repeatedly (4x) exposed skin contained an influx of mononuclear phagocytes comprising three distinct populations according to their differential expression of F4/80 and MHC-II. As determined by gene expression analysis, all three DEC populations (F4/80-MHC-IIhigh, F4/80+MHC-IIhigh, F4/80+MHC-IIint) exhibited major up-regulation of genes associated with alternative activation. The gene encoding RELMα (hallmark of alternatively activated cells) was highly up-regulated in all three DEC populations. However, in 4x infected mice deficient in RELMα, there was no change in the extent of inflammation at the skin infection site compared to 4x infected wild-type cohorts, nor was there a difference in the abundance of different mononuclear phagocyte DEC populations. The absence of RELMα resulted in greater numbers of CD4+ cells in the skin draining lymph nodes (sdLN) of 4x infected mice, although they remained hypo-responsive. Using mice deficient for IL-4Rα, in which alternative activation is compromised, we show that after repeated schistosome infection, levels of regulatory IL-10 in the skin were reduced, accompanied by increased numbers of MHC-IIhigh cells and CD4+ T cells in the skin. There were also increased numbers of CD4+ T cells in the sdLN in the absence of IL-4Rα compared to cells from singly infected mice. Although their ability to proliferate was still compromised, increased cellularity of sdLN from 4x IL-4RαKO mice correlated with reduced expression of Fas/FasL, resulting in decreased apoptosis and cell death but increased numbers of viable CD4+ T cells. This study highlights a mechanism through which IL-4Rα may regulate the immune system through the induction of IL-10 and regulation of Fas/FasL mediated cell death

  18. Mononuclear cell complement receptor blockade in primary biliary cirrhosis.

    PubMed Central

    Al-Aghbar, M N; Neuberger, J; Williams, R; Eddleston, A L

    1985-01-01

    Peripheral blood monocyte and lymphocyte receptors for Fc and C3b fragments were examined in vitro in patients with primary biliary cirrhosis and other chronic liver diseases using sheep red blood cells coated with anti-SRBC IgG1 (to detect Fc receptors) and with anti-SRBC IgM and complement (to detect C3b receptors). The number of C3b receptors detected on 100 monocytes was significantly lower in patients with primary biliary cirrhosis (23.0 +/- 12.0, mean +/- 1 SD) compared with normal controls (57.4 +/- 16.9) and other chronic liver disease (HBsAg negative chronic active hepatitis 62.0 +/- 17.0, alcoholic cirrhosis 50.9 +/- 4.0), while the number of Fc receptors detected on 100 monocytes was not significantly different in all the groups (primary biliary cirrhosis 72.8 +/- 28.6, chronic active hepatitis 74.7 +/- 14.0, alcoholic cirrhosis 58.0 +/- 13.5 and normal controls 69.6 +/- 19.9). When mononuclear cells isolated from normal individuals were pre-incubated with serum from patients with primary biliary cirrhosis before testing their receptor function there was a significant reduction in the number of C3b receptors detected per 100 monocytes (27.6 +/- 10.8) compared with pre-incubation with normal serum (72.0 +/- 18.0). This reduction in C3b-receptor function was again observed when the serum used for pre-incubation was depleted of circulating immune complexes; but when complement was further depleted from these sera, the number of C3b-receptors detected after pre-incubation was similar to normal values (64.0 +/- 11.8). Lymphocyte receptors showed a similar pattern of results. This implies a specific C3b receptor blockade on monocytes and lymphocytes from patients with primary biliary cirrhosis which appears to be because of blocking by serum factor(s) including complement fragments. PMID:3155513

  19. Characterization by mass cytometry of different methods for the preparation of muscle mononuclear cells.

    PubMed

    Spada, Filomena; Fuoco, Claudia; Pirrò, Stefano; Paoluzi, Serena; Castagnoli, Luisa; Gargioli, Cesare; Cesareni, Gianni

    2016-09-25

    Biological processes that are mediated by cell-cell interactions in heterogeneous populations are best approached by methods that have single cell resolution. Most of these methods rely on the preparation, from solid tissues, of cell suspensions by enzymatic digestion, followed by analysis of single cell reactivity to an antibody panel that allows the discrimination of cell populations and characterization of their activation state. Thus for any specific biological problem, both efficient and at the same time mild, protocols for cell separation, together with tissue specific panels of antibodies, need to be developed and optimized. Here we characterize an antibody panel that permits the discrimination of mononuclear muscle cell populations by mass cytometry and use it to characterize the cell populations obtained by three different cell extraction procedures from muscle fibers. We show that our panel of antibodies, albeit limited and incomplete, is sufficient to discriminate most of the mononuclear muscle cell populations and that each cell extraction method yields heterogeneous cell populations with a different relative abundance of the distinct cell types. PMID:26773739

  20. Generation of avian cells resembling osteoclasts from mononuclear phagocytes

    NASA Technical Reports Server (NTRS)

    Alvarez, J. I.; Teitelbaum, S. L.; Blair, H. C.; Greenfield, E. M.; Athanasou, N. A.; Ross, F. P.

    1991-01-01

    Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.

  1. High frequency of mononuclear myeloid-derived suppressor cells is associated with exacerbation of inflammatory bowel disease.

    PubMed

    Xi, Qinhua; Li, Yueqin; Dai, Juan; Chen, Weichang

    2015-01-01

    Exacerbation and relapse of inflammatory bowel disease (IBD) is associated with reduced antibacterial immunity and increased immune regulatory activity, but the source of increased immune regulation during episodes of disease activity is unclear. Myeloid-derived suppressor cells (MDSCs) are a cell type with a well-recognized role in limiting immune reactions. MDSC function in IBD and its relation to disease activity, however, remains unexplored. Here we show that patients with either ulcerative colitis (UC) or Crohn's disease (CD) have high peripheral blood levels of mononuclear MDSCs. Especially exacerbation of disease is associated with higher levels of mononuclear MDSC counts compared with those in remission of disease. Interestingly, chronic experimental colitis in mice coincides with increased MDCS mobilization. Thus, our results suggest that mononuclear MDCS are endogenous antagonists of immune system functionality in mucosal inflammation and the depression of antibacterial immunity associated with exacerbation of disease might involve increased activity of the MDSC compartment. PMID:25775229

  2. Effects of mononuclear phagocyte system modulating agents on Fc and C3 receptors of adherent cells.

    PubMed Central

    Hitomi, M.; Shimizu, F.

    1985-01-01

    Agents which modulate the mononuclear phagocyte system (MPS) were examined for their effects on Fc and C3 receptors of adherent cells (A-cells) as judged by rosette formation. Dextran sulphate, carrageenan, and immune complexes, known as MPS suppressants, reduced the percentage of receptor-positive A-cells, while levamisole, known as a MPS-activator, increased the percentage in vitro. The changes in the percentage of Fc receptor were parallel to those of the C3 receptor in vitro. The effects of these agents were also examined in vivo. PMID:2408651

  3. Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells

    PubMed Central

    Alves Melo, Tauá; Almeida, Milena Evangelista; Passos Rezende, Rachel

    2016-01-01

    Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of Lactobacillus plantarum Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-α, IL1-β, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4+CD25+ T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells. L. plantarum Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases. PMID:27446958

  4. Secretome of Peripheral Blood Mononuclear Cells Enhances Wound Healing

    PubMed Central

    Haider, Thomas; Gschwandtner, Maria; Werba, Gregor; Barresi, Caterina; Zimmermann, Matthias; Golabi, Bahar; Tschachler, Erwin; Ankersmit, Hendrik Jan

    2013-01-01

    Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SECPBMC). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SECPBMC containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SECPBMC on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SECPBMC containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SECPBMC induced proliferation and tube-formation in a matrigel-assay. In addition, SECPBMC treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers. PMID:23533667

  5. Fish peripheral blood mononuclear cells preparation for future monitoring applications.

    PubMed

    Pierrard, Marie-Aline; Roland, Kathleen; Kestemont, Patrick; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

    2012-07-15

    Fish species possess many specific characteristics that support their use in ecotoxicology. Widely used in clinical research, peripheral blood mononuclear cells (PBMCs) can reasonably be exploited as relevant target cells in the assessment of environmental chemical toxicity. The current article focuses on the methods necessary to isolate, characterize, and culture fish PBMCs. These procedures were successfully applied on an endangered species, the European eel (Anguilla anguilla L.), and on an economically important and worldwide exported species, the Asian catfish (Pangasianodon hypophthalmus S.). Proteomic approaches can be useful to screen xenobiotic exposure at the protein expression level, giving the opportunity to develop early warning signals thanks to molecular signatures of toxicity. To date, a major limitation of proteomic analyses is that most protein expression profiles often reveal the same predominant and frequently differentially expressed families of proteins regardless of the experimental stressing conditions. The current study describes a methodology to get a postnuclear fraction of high quality isolated from fish PBMCs in order to perform subsequent subproteomic analyses. Applied on samples from eel, the subproteomic analysis (two-dimensional differential in-gel electrophoresis) allowed the identification by liquid chromatography-tandem mass spectrometry and searches in the full NCBInr (National Center for Biotechnology Information nonredundant) database of 66 proteins representing 36 different proteins validated through Peptide and Protein Prophet of Scaffold software. PMID:22497769

  6. Interactions between the Fusarium toxin deoxynivalenol and lipopolysaccharides on the in vivo protein synthesis of acute phase proteins, cytokines and metabolic activity of peripheral blood mononuclear cells in pigs.

    PubMed

    Kullik, K; Brosig, B; Kersten, S; Valenta, H; Diesing, A-K; Panther, P; Reinhardt, N; Kluess, J; Rothkötter, H-J; Breves, G; Dänicke, S

    2013-07-01

    The in vivo effects of the Fusarium toxin deoxynivalenol (DON) on albumin and fibrinogen synthesis in pigs and metabolic activity of porcine peripheral blood mononuclear cells (PBMCs) were studied alone or in combination with lipopolysaccharides (LPSs) in order to examine proposed synergistic effects of both substances. A total of 36 male castrated pigs (initial weight of 26 kg) were used. Uncontaminated (Control) and naturally DON-contaminated (chronic oral DON, 3.1mg/kg diet) wheat was fed for 37 days. On the day of protein synthesis measurement, pigs recruited from the Control group were treated once intravenously with (iv) DON (100 μg/kg live weight (LW)/h), iv LPS (7.5 μg/kgLW/h) or a combination of both substances, and six pigs from the chronic oral group were treated once with iv LPS. A treatment with DON alone exhibited no alterations of acute phase protein synthesis and metabolic activity of PBMC. There was no evidence that the chosen dosing regimen of DON had influences on the induced sub-acute stage of sepsis, as the LPS challenge, irrespective of DON co-exposure, mediated an acute phase reaction with a typical decrease of albumin synthesis, as well as changes in cytokine concentration and a loss of metabolic activity in PBMC. PMID:23500770

  7. Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

    PubMed

    Neri, Tommaso; Pergoli, Laura; Petrini, Silvia; Gravendonk, Lotte; Balia, Cristina; Scalise, Valentina; Amoruso, Angela; Pedrinelli, Roberto; Paggiaro, Pierluigi; Bollati, Valentina; Celi, Alessandro

    2016-04-01

    Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity. PMID:26876346

  8. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    PubMed

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF. PMID:21170647

  9. CD11b(+) Mononuclear Cells Mitigate Hyperoxia-Induced Lung Injury in Neonatal Mice.

    PubMed

    Eldredge, Laurie C; Treuting, Piper M; Manicone, Anne M; Ziegler, Steven F; Parks, William C; McGuire, John K

    2016-02-01

    Bronchopulmonary dysplasia (BPD) is a common consequence of life-saving interventions for infants born with immature lungs. Resident tissue myeloid cells regulate lung pathology, but their role in BPD is poorly understood. To determine the role of lung interstitial myeloid cells in neonatal responses to lung injury, we exposed newborn mice to hyperoxia, a neonatal mouse lung injury model with features of human BPD. In newborn mice raised in normoxia, we identified a CD45(+) F4/80(+) CD11b(+), Ly6G(lo-int) CD71(+) population of cells in lungs of neonatal mice present in significantly greater percentages than in adult mice. In response to hyperoxia, surface marker and gene expression in whole lung macrophages/monocytes was biased to an alternatively activated phenotype. Partial depletion of these CD11b(+) mononuclear cells using CD11b-diphtheria toxin (DT) receptor transgenic mice resulted in 60% mortality by 40 hours of hyperoxia exposure with more severe lung injury, perivascular edema, and alveolar hemorrhage compared with DT-treated CD11b-DT receptor-negative controls, which displayed no mortality. These results identify an antiinflammatory population of CD11b(+) mononuclear cells that are protective in hyperoxia-induced neonatal lung injury in mice, and suggest that enhancing their beneficial functions may be a treatment strategy in infants at risk for BPD. PMID:26192732

  10. Perinatal hepatitis B virus infection caused by antihepatitis Be positive maternal mononuclear cells.

    PubMed

    Shimizu, H; Mitsuda, T; Fujita, S; Yokota, S

    1991-06-01

    To investigate the infectivity of hepatitis B virus (HBV) from mothers to their newborn offspring, HBV-DNA in plasma and peripheral mononuclear cells from 28 antihepatitis Be positive, hepatitis B surface antigen positive carrier mothers was examined by a highly sensitive polymerase chain reaction/Southern hybridisation technique. HBV specific DNA was detected in three maternal mononuclear cell samples, but was absent in plasma. Two of four infants born to the three mothers with HBV-DNA positive mononuclear cells developed acute or fulminant hepatitis within three months after birth. Two infants were effectively prevented from infection with HBV by combined hepatitis B immunoglobulin/HBV vaccine administration. The 25 infants born to the HBV-DNA negative mothers were free of HBV infection within the next seven months to 3.5 years. These results suggest that latent infection with HBV in maternal mononuclear cells is responsible for perinatal HBV infection. PMID:2053794

  11. The proliferative effects of asbestos-exposed peripheral blood mononuclear cells on mesothelial cells

    PubMed Central

    MAKI, YUHO; NISHIMURA, YASUMITSU; TOYOOKA, SHINICHI; SOH, JUNICHI; TSUKUDA, KAZUNORI; SHIEN, KAZUHIKO; FURUKAWA, MASASHI; MURAOKA, TAKAYUKI; UENO, TSUYOSHI; TANAKA, NORIMITSU; YAMAMOTO, HIROMASA; ASANO, HIROAKI; MAEDA, MEGUMI; KUMAGAI-TAKEI, NAOKO; LEE, SUNI; MATSUZAKI, HIDENORI; OTSUKI, TAKEMI; MIYOSHI, SHINICHIRO

    2016-01-01

    Malignant mesothelioma (MM) is thought to arise from the direct effect of asbestos on mesothelial cells. However, MM takes a long time to develop following exposure to asbestos, which suggests that the effects of asbestos are complex. The present study examined the effects of asbestos exposure on the cell growth of MeT-5A human mesothelial cells via cytokines produced by immune cells. Peripheral blood mononuclear cells (PBMCs) were stimulated with antibodies against cluster of differentiation (CD)3 and CD28 upon exposure to the asbestos chrysotile A (CA) or crocidolite (CR); the growth of MeT-5A cells in media supplemented with PBMC culture supernatants was subsequently examined. MeT-5A cells exhibited an increase in proliferation when grown in supernatant from the 7-day PBMC culture exposed to CA or CR. Analysis of cytokine production demonstrated increased levels of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1α, IL-1β, IL-3, IL-5, IL-13 and IL-17A in supernatants. Individual administration of these cytokines, excluding G-CSF and GM-CSF, led to an increase in cell growth of MeT-5A, whereas this effect was not observed following the combined administration of these cytokines. The results indicate that cytokines secreted by immune cells upon exposure to asbestos cause an increase in the growth activity of mesothelial cells, suggesting that alterations in the production of cytokines by immune cells may contribute to tumorigenesis in individuals exposed to asbestos. PMID:27123108

  12. Human mononuclear cell function after 4 C storage during 1-G and microgravity conditions of spaceflight

    NASA Technical Reports Server (NTRS)

    Meehan, Richard; Taylor, Gerald; Lionetti, Fabian; Neale, Laurie; Curren, Tim

    1989-01-01

    To investigate the possibility of restoring immune competence of crewmembers during a prolonged spaceflight by infusions of autologous blood components, the effect of storage at 4 C aboard Space Shuttle Columbia (Mission 61-c) on the activity of human peripheral blood mononuclear cells (PBMNCs), stored as leukocyte concentrates in autologous plasa, was investigated. The results of preflight storage at 4 C demonstrated a progressive daily loss in mitogen-stimulated protein synthesis, and thymidine uptake, as well as a progressive reduction in the percentage of PBMNCs expressing cell-surface phenotype markers. The ability of PBMNCs stored at 4 C for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained for 7 d on ground.

  13. Effect of surgical menopause and estrogen replacement on cytokine release from human blood mononuclear cells.

    PubMed Central

    Pacifici, R; Brown, C; Puscheck, E; Friedrich, E; Slatopolsky, E; Maggio, D; McCracken, R; Avioli, L V

    1991-01-01

    To determine whether mononuclear cell secretory products contribute to the changes in bone turnover that characterize the development of postmenopausal osteoporosis, we evaluated the effects of oophorectomy and subsequent estrogen replacement on the spontaneous secretion of interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and on the phytohemagglutinin A-induced secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) from peripheral blood mononuclear cells. In 15 healthy premenopausal women who underwent oophorectomy, increases in GM-CSF activity were observed as early as 1 week after surgery, whereas elevations in IL-1 and TNF-alpha and in hydroxyproline/creatinine and calcium/creatinine ratios, two urinary indices of bone resorption, were detectable 2 weeks after the surgical procedure. Six of the oophorectomized women received no estrogen therapy after surgery and in these subjects hydroxyproline/creatinine and calcium/creatinine ratios plateaued 6 weeks postoperatively, and all three cytokines reached the highest levels 8 weeks after oophorectomy, when the study ended. In the remaining 9 women, who were started on estrogen replacement therapy 4 weeks after oophorectomy, decreases in the indices of bone resorption paralleled decreases in the secretion of the cytokines, with lower levels detected after 2 weeks of therapy. In the women who did not receive estrogen therapy, circulating osteocalcin, a marker of bone formation, increased beyond preoperative levels 8 weeks after oophorectomy, whereas in the estrogen-treated subjects osteocalcin remained unchanged in the entire study period. In 9 female controls who underwent simple hysterectomy, cytokine release and biochemical indices of bone turnover did not change after surgery. These data indicate that changes in estrogen status in vivo are associated with the secretion of mononuclear cell immune factors in vitro and suggest that alterations in the local production of bone

  14. The Transcriptome of Equine Peripheral Blood Mononuclear Cells

    PubMed Central

    Pacholewska, Alicja; Drögemüller, Michaela; Klukowska-Rötzler, Jolanta; Lanz, Simone; Hamza, Eman; Dermitzakis, Emmanouil T.; Marti, Eliane; Gerber, Vincent

    2015-01-01

    Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome. PMID:25790166

  15. Beryllium Alters Lipopolysaccharide-Mediated Intracellular Phosphorylation and Cytokine Release in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Silva, Shannon; Ganguly, Kumkum; Fresquez, Theresa M.; Gupta, Goutam; McCleskey, T. Mark; Chaudhary, Anu

    2013-01-01

    Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide - mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We find that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1β is enhanced. Additionally, not all lipopolysaccharide - mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1β secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling. PMID:19894180

  16. The effects of retinoic acid on immunoglobulin synthesis by human cord blood mononuclear cells.

    PubMed

    Israel, H; Odziemiec, C; Ballow, M

    1991-06-01

    Derivatives of vitamin A have attracted considerable attention as agents which have immune potentiating properties and possibly tumor-suppressive effects. Recent investigations have shown that retinoic acid (RA) can augment immunoglobulin production of B-cell hybridomas from patients with immune deficiency. In this study we examined the ability of RA to modify the mitogen-induced polyclonal immunoglobulin synthesis of cord blood mononuclear cells (CBMC). RA in concentrations ranging from 10(-5) to 10(-7) M augmented IgM synthesis of CBMC in response to formalinized Cowans I strain Staphylococcus aureus (SAC) up to 45.6-fold which was greater at suboptimal responses to SAC. There were no changes in IgG or IgA synthesis and minimal effects on SAC-induced proliferative responses. RA did not produce similar changes in IgM synthesis of SAC-stimulated adult peripheral blood mononuclear cells (PBMC), and RA had no effect on the immunoglobulin synthesis of Epstein-Barr virus (EBV)-stimulated CBMC or adult PBMC. Time course studies showed that peak enhancement occurred when RA was added between 4 and 24 hr after culture initiation and required prior activation by SAC for augmentation of IgM synthesis. Cell separation experiments showed that prior incubation (18 hr) of an enriched T-cell fraction with RA enhanced the IgM synthesis of a T-cell-depleted B-cell fraction. These experiments and the findings that RA-induced augmentation of IgM production in response to SAC, but not to EBV suggest that the immunoregulatory effects of RA may be mediated by either T cells or T-cell products. Further studies will be necessary to understand the mechanism by which RA augments IgM synthesis of CBMC. PMID:2029794

  17. Ex vivo effects of naphthoquinones on allergen-sensitized mononuclear cells in mice.

    PubMed

    Tanaka, M; Inoue, K; Shimada, A; Takano, H

    2016-09-01

    Naphthoquinone (NQ), one of the extractable chemical compounds of diesel exhaust particles, enhances allergic asthma traits in mice. However, it remains unknown whether: (1) several types of NQs have the same potential to facilitate allergies; and (2) NQs synergistically disrupt the functional phenotypes of immune cells. The aim of the present study was to investigate the effects of two types (1,2- and 1,4-) of NQs on sensitized mononuclear cells using an ex vivo assay. Male BALB/c mice were repeatedly and intraperitoneally administered ovalbumin (OVA: 20 µg) plus alum with or without two different doses of each NQ. After the final administration, splenocytes (mononuclear cells) were isolated from these mice and cultured in the presence of OVA. Helper T-related cytokines in the culture supernatants and downstream molecules were then evaluated. Protein levels of interferon-γ were higher in the supernatants from 1,2-NQ and 1,4-NQ at low dose + OVA-exposed mononuclear cells following the OVA stimulation than in those from OVA-exposed mononuclear cells. Interleukin (IL)-13 levels were higher in the supernatants from low dose NQs + OVA-exposed mononuclear cells. IL-17 levels were significantly higher in the supernatants from low dose 1,2-NQ + OVA-exposed mononuclear cells. The quantity of phosphorylated STAT6 in the nuclei of these cells was significantly greater in the low dose NQ + OVA groups than in the OVA group. These findings suggest NQs differently enhance allergen sensitization in the context of the Th response against mononuclear cells such as lymphocytes. PMID:26884456

  18. Proteome signatures of inflammatory activated primary human peripheral blood mononuclear cells☆

    PubMed Central

    Haudek-Prinz, Verena J.; Klepeisz, Philip; Slany, Astrid; Griss, Johannes; Meshcheryakova, Anastasia; Paulitschke, Verena; Mitulovic, Goran; Stöckl, Johannes; Gerner, Christopher

    2012-01-01

    Proteome profiling is the method of choice to identify marker proteins whose expression may be characteristic for certain diseases. The formation of such marker proteins results from disease-related pathophysiologic processes. In healthy individuals, peripheral blood mononuclear cells (PBMCs) circulate in a quiescent cell state monitoring potential immune-relevant events, but have the competence to respond quickly and efficiently in an inflammatory manner to any invasion of potential pathogens. Activation of these cells is most plausibly accompanied by characteristic proteome alterations. Therefore we investigated untreated and inflammatory activated primary human PBMCs by proteome profiling using a ‘top down’ 2D-PAGE approach in addition to a ‘bottom up’ LC–MS/MS-based shotgun approach. Furthermore, we purified primary human T-cells and monocytes and activated them separately. Comparative analysis allowed us to characterize a robust proteome signature including NAMPT and PAI2 which indicates the activation of PBMCs. The T-cell specific inflammation signature included IRF-4, GBP1and the previously uncharacterized translation product of GBP5; the corresponding monocyte signature included PDCD5, IL1RN and IL1B. The involvement of inflammatory activated PBMCs in certain diseases as well as the responsiveness of these cells to anti-inflammatory drugs may be evaluated by quantification of these marker proteins. This article is part of a Special Issue entitled: Integrated omics. PMID:22813876

  19. Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors.

    PubMed

    Su, Ruijun Jeanna; Neises, Amanda; Zhang, Xiao-Bing

    2016-01-01

    Peripheral blood is the easy-to-access, minimally invasive, and the most abundant cell source to use for cell reprogramming. The episomal vector is among the best approaches for generating integration-free induced pluripotent stem (iPS) cells due to its simplicity and affordability. Here we describe the detailed protocol for the efficient generation of integration-free iPS cells from peripheral blood mononuclear cells. With this optimized protocol, one can readily generate hundreds of iPS cell colonies from 1 ml of peripheral blood. PMID:25403468

  20. Dequalinium induces apoptosis in peripheral blood mononuclear cells isolated from human chronic lymphocytic leukemia.

    PubMed

    Pajuelo, Lucia; Calviño, Eva; Diez, Jose Carlos; Boyano-Adánez, Maria Del Carmen; Gil, Juana; Sancho, Pilar

    2011-12-01

    B-cell chronic lymphocytic leukemia (B-CLL) is an abnormal neoplasic proliferation of B cells, which accumulate mainly in the bone marrow and blood preventing both B cells development in the lymph nodes and the ability to fight against infection. The antitumor agents used in chemotherapy are aimed at inducing malignant cell death, thus limiting the growth and spreading of these cells. However, the lack of specificity for tumor cells exhibited by these agents causes undesirable side effects that have led to the investigation of new therapeutic strategies designed to specifically target malignant cells and thus trigger selective cell destruction. Dequalinium (DQA) is an antitumoral agent that selectively accumulates in the mitochondria and has been shown to display anticancer activity in cells from different malignancies. In the present study, the DQA-induced cytotoxicity in B-CLL cells was analyzed by measuring cell viability and cell death, either by necrosis or apoptosis. Our results support the importance of DQA as a selective and potential antileukemic drug with a higher cytotoxic effect on peripheral blood mononuclear cells from B-CLL patients than in those from healthy donors and encourage the performance of further studies in combination with other agents. PMID:20524037

  1. Evidence of mononuclear cell preactivation in the fasting state in polycystic ovary syndrome

    PubMed Central

    GONZÁLEZ, Frank; KIRWAN, John P.; ROTE, Neal S.; MINIUM, Judi

    2014-01-01

    OBJECTIVE We evaluated mononuclear cell (MNC) preactivation in women with Polycystic Ovary Syndrome (PCOS) by examining the effect of in vitro lipopolysaccharide (LPS) exposure on cytokine release in the fasting state. STUDY DESIGN Twenty women with PCOS (10 lean, 10 obese) and 20 weight-matched controls (10 lean, 10 obese) volunteered for study participation. Tumor necrosis factor-α (TNF α) and interleukin-6 (IL-6) release was measured from mononuclear cell isolated from fasting blood samples and cultured in the presence and absence of lipopolysaccharide. Plasma IL-6 was measured from the same fasting blood samples. Insulin sensitivity was derived from an oral glucose tolerance test using the Matsuda index and truncal fat was measured by dual energy x-ray absorptiometry. RESULTS The percent change from baseline in TNFα and IL-6 release from mononuclear cell following lipopolysaccharide exposure was increased (pitalic>0.04) in lean and obese women with PCOS and obese controls compared with lean controls. Plasma IL-6 was increased (pbold>0.02) in obese women with PCOS compared with lean women with PCOS, which in turn was increased (p<0.02) compared with lean controls. The mononuclear cell -derived TNFα and IL-6 responses from mononuclear cell were negatively correlated with insulin sensitivity (p<0.03) and positively correlated with testosterone (p<0.03) and androstenedione (p<0.006) for the combined groups. Plasma IL-6 was positively correlated with percent truncal fat (p<0.008). CONCLUSIONS In PCOS, increased cytokine release from mononuclear cell following lipopolysaccharide exposure in the fasting state reveals the presence of mononuclear cell preactivation. Importantly, this phenomenon is independent of obesity and may contribute to the development of insulin resistance and hyperandrogenism in PCOS. In contrast, the source of plasma IL-6 elevations in PCOS may be excess adiposity. PMID:24956549

  2. Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

    PubMed Central

    Lu, Tzu-Li; Chuang, Jing-Yuan; Yang, Jai-Sing; Chiu, Shau-Ting; Hsiao, Nai-Wan; Wu, Mei-Chen; Wu, Shih-Hsiung; Hsu, Ching-Hsiang

    2011-01-01

    Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB) showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator. PMID:21584270

  3. Two inhibitors of neutrophil chemotaxis are produced by hyperimmunoglobulin E recurrent infection syndrome mononuclear cells exposed to heat-killed staphylococci.

    PubMed Central

    Donabedian, H; Gallin, J I

    1983-01-01

    Mononuclear cells from normal volunteers and from patients with the hyperimmunoglobulin E recurrent infection syndrome (HIE) were cultured for 18 h with and without opsonized, heat-killed Staphylococcus aureus (OS). The supernatants from normal mononuclear cell cultures without OS revealed no inhibitory activity for neutrophil chemotaxis, whereas those from HIE patients revealed the previously reported 61,000-dalton factor. However, when normal cells were cultured with OS, they produced a proteinaceous, 56 degrees C-stable, 30,000- to 45,000-dalton factor which preferentially inhibited neutrophil versus monocyte chemotaxis. When HIE cells were exposed to OS, they produced the same 30,000- to 45,000-dalton factor as normal cells, as well as the 61,000-dalton factor that they produced spontaneously. Assay of 1,000-fold dilutions of supernatants from cultures of normal mononuclear cells with OS revealed a mean production of 7.8 +/- 5.4% inhibition of chemotaxis, whereas assay of 1,000-fold dilutions of supernatants from cultures of HIE mononuclear cells (spontaneously producing the 61,000-dalton factor) with OS revealed a 26.6 +/- 3.6% inhibition (P less than 0.02). The data indicate that in short-term culture both normal and HIE mononuclear cells produce an inhibitor of neutrophil chemotaxis when exposed to particulate heat-killed staphylococci but that HIE cells produce qualitatively and quantitatively more inhibitory activity. PMID:6343237

  4. Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: Decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells

    SciTech Connect

    Hermann, G.G.; Petersen, K.R.; Steven, K.; Zeuthen, J. )

    1990-11-01

    The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in {sup 51}Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56+ or CD57+ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56+ and CD57+ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients.

  5. Propofol attenuates LPS-induced tumor necrosis factor-α, interleukin-6 and nitric oxide expression in canine peripheral blood mononuclear cells possibly through down-regulation of nuclear factor (NF)-κB activation.

    PubMed

    Pei, Zengyang; Wang, Jinqiu

    2015-02-01

    Sepsis is a major cause of mortality in intensive care medicine. Propofol, an intravenous general anesthetic, has been suggested to have anti-inflammatory properties and able to prevent sepsis induced by Gram-positive and Gram-negative bacteria by down-regulating the gene expression of pro-inflammatory cytokines. However, propofol's anti-inflammatory effects upon canine peripheral blood mononuclear cells (PBMCs) have not yet been clarified. Here, we isolate canine PBMCs and investigate the effects of propofol on the gene expressions of both lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor (TNF)-α and upon the production of nitric oxide (NO). Through real-time quantitative PCR and the Griess reagent system, we found that non-cytotoxic levels of propofol significantly inhibited the release of NO and IL-6 and TNF-α gene expression in LPS-induced canine PBMCs. Western blotting revealed that LPS does significantly increase the expression of inducible NO synthase (iNOS) protein in canine PBMCs, while pretreatment with propofol significantly decreases the LPS-induced iNOS protein expression. Propofol, at concentration of 25 µM and 50 µM, also significantly inhibited the LPS-induced nuclear translocation of nuclear factor (NF)-κB p65 protein in canine PBMCs. This diminished TNF-α, IL-6 and iNOS expression, and NO production was in parallel to the respective decreased NF-κB p65 protein nuclear translocation in the LPS-activated canine PBMCs pretreated with 25 µM and 50 µM propofol. This suggests that non-cytotoxic levels of propofol pretreatment can down-regulate LPS-induced inflammatory responses in canine PBMCs, possibly by inhibiting the nuclear translocation of the NF-κB p65 protein. PMID:25312048

  6. Whole blood leukocyte vs. separated mononuclear cell blastogenesis in calves: time-dependent changes after shipping.

    PubMed Central

    Kelley, K W; Osborne, C A; Evermann, J F; Parish, S M; Hinrichs, D J

    1981-01-01

    The blastogenic response of peripheral blood mononuclear cells to mitogenic stimulation by concanavalin A was lower (P less than 0.01) after transporting 60 dairy calves 480 km than it was either one or two weeks later. The response was similar for phytohemagglutinin. There was a decrease (P less than 0.05) in the number of peripheral blood monocytes and neutrophils two weeks after shipping. The transportation of calves did not affect plasma IgG1 or IgM level. The mitogenic stimulation of peripheral blood leukocytes by both phytohemagglutinin and concanavalin A in whole blood cultures was more variable than with the culture of peripheral blood mononuclear cells. Technique variation, which was defined as the coefficient of variation among quadruplicate cultures, was greater than 20% for while blood assays and less than 10% for cultures of peripheral blood mononuclear cells. The variation among different calves tested at the same time and the variation within single calves tested at different times were also lower in peripheral blood mononuclear cell cultures than in whole blood mononuclear cell cultures than in whole blood assays. It is suggested that the variation among replicate cultures be reported in blastogenesis studies. PMID:7340911

  7. Differential MSC activation leads to distinct mononuclear leukocyte binding mechanisms

    NASA Astrophysics Data System (ADS)

    Kota, Daniel J.; Dicarlo, Bryan; Hetz, Robert A.; Smith, Philippa; Cox, Charles S.; Olson, Scott D.

    2014-04-01

    Advances in the field of Multipotent Mesenchymal Stromal cell (MSC) biology have demonstrated that MSCs can improve disease outcome when `activated' to exert immunomodulatory effects. However, the precise mechanisms modulating MSC-immune cells interactions remain largely elusive. In here, we activated MSC based on a recent polarization paradigm, in which MSCs can be polarized towards a pro- or anti-inflammatory phenotype depending on the Toll-like receptor stimulated, to dissect the mechanisms through which MSCs physically interact with and modulate leukocytes in this context. Our data show that MSCs activated through the Toll-like receptor (TLR) 4 pathway increased VCAM-1 and ICAM-1 dependent binding of leukocytes. On the other hand, TLR3 stimulation strongly increases leukocytes affinity to MSC comparatively, through the formation of cable-like hyaluronic acid structures. In addition, TLR4 activation elicited secretion of pro-inflammatory mediators by MSCs, whereas TLR3-activated MSCs displayed a milder pro-inflammatory phenotype, similar to inactivated MSCs. However, the differently activated MSCs maintained their ability to suppress leukocyte activation at similar levels in our in vitro model, and this immunomodulatory property was shown here to be partially mediated by prostaglandin. These results reinforce the concept that alternate activation profiles control MSC responses and may impact the therapeutic use of MSCs.

  8. Viable mononuclear cell stability study for implementation in a proficiency testing program: impact of shipment conditions.

    PubMed

    Kofanova, Olga A; Davis, Kristine; Glazer, Barbara; De Souza, Yvonne; Kessler, Joseph; Betsou, Fotini

    2014-06-01

    The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed, in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature, dry ice, and liquid nitrogen) and in different preservation media (serum with cryoprotectant, commercial cryopreservation solution, and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion, flow cytometry, Cell Analysis System cell counting (CASY)), and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However, we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions, satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. PMID:24955735

  9. Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells.

    PubMed

    Jeurink, Prescilla V; Noguera, Cristina Lull; Savelkoul, Huub F J; Wichers, Harry J

    2008-08-01

    Immunomodulation by fungal compounds can be determined by the capacity of the compounds to influence the cytokine production by human peripheral blood mononuclear cells (hPBMC). These activities include mitogenicity, stimulation and activation of immune effector cells. Eight mushroom strains (Agaricus blazei, Coprinus comatus, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Volvariella volvacea, Lentinus edodes, and Pleurotus ostreatus) were tested for the immunomodulating activity of the isolated protein fractions and polysaccharides fractions present in mycelia and culture liquid. The fungal proteins and polysaccharides have been investigated for their in vitro effect on the cytokine profile (IFN-gamma, IL-4, IL-10, IL-12 and TNF-alpha) of unstimulated or hPBMC stimulated with the polyclonal stimulations PMA/Ca-I, ConA or LPS. In addition to their influence on the cytokine profile, the hemagglutination activity of the fungal proteins on rabbit red blood cells was determined. Proteins from V. volvacea and G. lucidum showed immunomodulating activity without the presence of any mitogen, however, neither of them decreased the production of IL-4 and IFN-gamma in combination with a stimulus. All used stimuli resulted in an induction of IL-12 in the presence of the protein extracts, suggesting a direct effect on monocytes. This effect might lead to the indirect immunomodulation of T cell activation and cytokine production. In addition, both protein extracts showed more hemagglutination activity after trypsin treatment of the rabbit red blood cells, indicating the presence of carbohydrate-binding proteins, like lectins and FIPs. In conclusion, the protein extracts of V. volvacea and G. lucidum contain immunomodulating activity by acting directly on monocytes and thereby modulating T cell activation. Further purification of the fungal extracts is needed to clarify whether there are FIPs or lectins present that are responsible for this immunomodulating activity

  10. Influence of Delivery Method on Neuroprotection by Bone Marrow Mononuclear Cell Therapy following Ventral Root Reimplantation with Fibrin Sealant

    PubMed Central

    Barbizan, Roberta; Castro, Mateus V.; Barraviera, Benedito; Ferreira, Rui S.; Oliveira, Alexandre L. R.

    2014-01-01

    The present work compared the local injection of mononuclear cells to the spinal cord lateral funiculus with the alternative approach of local delivery with fibrin sealant after ventral root avulsion (VRA) and reimplantation. For that, female adult Lewis rats were divided into the following groups: avulsion only, reimplantation with fibrin sealant; root repair with fibrin sealant associated with mononuclear cells; and repair with fibrin sealant and injected mononuclear cells. Cell therapy resulted in greater survival of spinal motoneurons up to four weeks post-surgery, especially when mononuclear cells were added to the fibrin glue. Injection of mononuclear cells to the lateral funiculus yield similar results to the reimplantation alone. Additionally, mononuclear cells added to the fibrin glue increased neurotrophic factor gene transcript levels in the spinal cord ventral horn. Regarding the motor recovery, evaluated by the functional peroneal index, as well as the paw print pressure, cell treated rats performed equally well as compared to reimplanted only animals, and significantly better than the avulsion only subjects. The results herein demonstrate that mononuclear cells therapy is neuroprotective by increasing levels of brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF). Moreover, the use of fibrin sealant mononuclear cells delivery approach gave the best and more long lasting results. PMID:25157845

  11. Transcriptomic analysis of mononuclear phagocyte differentiation and activation.

    PubMed

    Hume, David A; Freeman, Tom C

    2014-11-01

    Monocytes and macrophages differentiate from progenitor cells under the influence of colony-stimulating factors. Genome-scale data have enabled the identification of the sets of genes that are associated with specific functions and the mechanisms by which thousands of genes are regulated in response to pathogen challenge. In large datasets, it is possible to identify large sets of genes that are coregulated with the transcription factors that regulate them. They include macrophage-specific genes, interferon-responsive genes, early inflammatory genes, and those associated with endocytosis. Such analyses can also extract macrophage-associated signatures from large cancer tissue datasets. However, cluster analysis provides no support for a signature that distinguishes macrophages from antigen-presenting dendritic cells, nor the classification of macrophage activation states as classical versus alternative, or M1 versus M2. Although there has been a focus on a small subset of lineage-enriched transcription factors, such as PU.1, more than half of the transcription factors in the genome can be expressed in macrophage lineage cells under some state of activation, and they interact in a complex network. The network architecture is conserved across species, but many of the target genes evolve rapidly and differ between mouse and human. The data and publication deluge related to macrophage biology require the development of new analytical tools and ways of presenting information in an accessible form. PMID:25319328

  12. Bisphenol A suppresses Th1-type immune response in human peripheral blood mononuclear cells in vitro.

    PubMed

    Gostner, Johanna M; Raggl, Emanuel; Becker, Kathrin; Überall, Florian; Schennach, Harald; Pease, James E; Fuchs, Dietmar

    2015-12-01

    Bisphenol A (BPA) is a widely used plasticizer, which came into focus because of its genotoxic and sensitizing potential. Besides its toxic properties, BPA is also well-known for its antioxidant chemical properties. This in vitro study investigated the interference of BPA with interferon-γ (IFN-γ)-induced tryptophan breakdown and neopterin production in human peripheral blood mononuclear cells (PBMC). The pro-inflammatory cytokine IFN-γ induces the conversion of the essential amino acid tryptophan into kynurenine via the enzyme indoleamine-2,3-dioxygenase (IDO-1). In parallel, GTP-cyclohydrolase produces neopterin, a marker of immune activation. A model system of phytohaemagglutinin (PHA)-stimulated PBMC was used to assess potential immunomodulatory properties of BPA. Treatment of cells with BPA [12.5-200μM] resulted in a significant and dose-dependent suppression of mitogen-induced tryptophan breakdown and neopterin formation along with a decrease of IFN-γ levels. Similar but less pronounced effects were observed in unstimulated cells. We postulate that the inhibitory effects of BPA on both T-cell activation and IDO-1 activity that we describe here may be critical for immune surveillance and is likely to influence T helper (Th) type 1/Th2 balance. Such immunosuppressive effects likely contribute to counteract inflammation. Further studies are required to address the in vivo relevance our in vitro findings. PMID:26475400

  13. Multiparameter analysis of stimulated human peripheral blood mononuclear cells: A comparison of mass and fluorescence cytometry.

    PubMed

    Nicholas, Katherine J; Greenplate, Allison R; Flaherty, David K; Matlock, Brittany K; Juan, Juan San; Smith, Rita M; Irish, Jonathan M; Kalams, Spyros A

    2016-03-01

    Mass and fluorescence cytometry are quantitative single cell flow cytometry approaches that are powerful tools for characterizing diverse tissues and cellular systems. Here mass cytometry was directly compared with fluorescence cytometry by studying phenotypes of healthy human peripheral blood mononuclear cells (PBMC) in the context of superantigen stimulation. One mass cytometry panel and five fluorescence cytometry panels were used to measure 20 well-established lymphocyte markers of memory and activation. Comparable frequencies of both common and rare cell subpopulations were observed with fluorescence and mass cytometry using biaxial gating. The unsupervised high-dimensional analysis tool viSNE was then used to analyze data sets generated from both mass and fluorescence cytometry. viSNE analysis effectively characterized PBMC using eight features per cell and identified similar frequencies of activated CD4+ T cells with both technologies. These results suggest combinations of unsupervised analysis programs and extended multiparameter cytometry will be indispensable tools for detecting perturbations in protein expression in both health and disease. PMID:26599989

  14. The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults.

    PubMed

    Zenarruzabeitia, Olatz; Vitallé, Joana; García-Obregón, Susana; Astigarraga, Itziar; Eguizabal, Cristina; Santos, Silvia; Simhadri, Venkateswara R; Borrego, Francisco

    2016-01-01

    Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates' qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses. PMID:27595670

  15. The expression and function of human CD300 receptors on blood circulating mononuclear cells are distinct in neonates and adults

    PubMed Central

    Zenarruzabeitia, Olatz; Vitallé, Joana; García-Obregón, Susana; Astigarraga, Itziar; Eguizabal, Cristina; Santos, Silvia; Simhadri, Venkateswara R.; Borrego, Francisco

    2016-01-01

    Neonates are more susceptible to infections than adults. This susceptibility is thought to reflect neonates’ qualitative and quantitative defects in the adaptive and innate immune responses. Differential expression of cell surface receptors may result in altered thresholds of neonatal immune cell activation. We determined whether the expression and function of the lipid-binding CD300 family of receptors are different on neonatal immune cells compared to adult immune cells. A multiparametric flow cytometry analysis was performed to determine the expression of CD300 receptors on adult peripheral blood mononuclear cells and neonatal cord blood mononuclear cells. The expression of the CD300a inhibitory receptor was significantly reduced on cells from the newborn adaptive immune system, and neonatal antigen presenting cells exhibited a different CD300 receptors expression pattern. We also found differential LPS-mediated regulation of CD300 receptors expression on adult monocytes compared to cord blood monocytes, and that CD300c and CD300e-mediated activation was quantitatively different in neonatal monocytes. This is the first complete study examining the expression of CD300 receptors on human neonatal immune cells compared with adult immune cells. Significant differences in the expression and function of CD300 receptors may help to explain the peculiarities and distinctness of the neonatal immune responses. PMID:27595670

  16. A novel subpopulation of peripheral blood mononuclear cells presents in major burn patients.

    PubMed

    Liu, Hongbin; Ding, Jie; Ma, Zengshuan; Zhu, Zhenshen; Shankowsky, Heather A; Tredget, Edward E

    2015-08-01

    Hypertrophic scars (HTS) are generally believed to result from proliferation and activation of resident connective tissue fibroblasts after burns. To demonstrate a potential role of blood-borne cells, the peripheral blood mononuclear cells (PBMCs) and the effect of PBMCs on dermal fibroblast behavior was investigated. Flow cytometry was used to analyze the surface and intracellular protein expression of PBMCs and fibroblasts. Transwell migration assay, enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction was performed to assess fibroblast functions. We identified a novel subpopulation of PBMCs in burn patients in vivo that appears at an early stage following major thermal injuries, which primarily express procollagen 1, leukocyte specific protein 1, CD204, toll-like receptor 4 and stromal cell-derived factor 1 (SDF-1) receptor CXCR4. In vitro, the conditioned media from burn patient PBMCs up-regulated the expression of fibrotic growth factors and extracellular matrix molecules, down-regulated antifibrotic factor decorin, enhanced cell chemotaxis and promoted cell differentiation into contractile myofibroblasts in dermal fibroblasts. After thermal injury, this novel subpopulation of PBMCs is systemically triggered and attracted to the wounds under SDF-1/CXCR4 signaling where they appear to modulate the functions of resident connective tissue cells and thus contribute to the development of HTS. PMID:25683215

  17. [Detection of stimulator-induced cytotoxicity of human mononuclear cells in short-term culture].

    PubMed

    von Baehr, R; Timm, M; Fröbe, I

    1983-01-01

    A new test modification for the detection of stimulant-induced cytotoxicity of human mononuclear blood cells is described. Papain-treated human erythrocytes were used as indicator cells. The effector cells were lymphocytes. The ratio of target cells to effector cells was 10/1. Haemoglobin as a marker of lysis of the erythrocytes, released in the supernatant, was measured quantitatively in form of its pseudoperoxidase-activity. PHA, ConA and tannic acid were ascertained and tested as stimulants of cytotoxicity. The reaction was inhibitable by anti-human-lymphocyte-globulin. The test conditions were optimized in regard to incubation time, -temperature, -vessels, culture medium and target cells. The technique is easy to manipulate, has only slight pretensions to the equipment of the laboratory and appears to be very effective. We recommend to apply this method of stimulant-induced cytotoxicity within the detection of the immune state, especially in the progress of immunopathological diseases and the analysis of efficiency of immunosuppressive therapy. PMID:6224413

  18. Mycoplasma genitalium promotes epithelial crossing and peripheral blood mononuclear cell infection by HIV-1

    PubMed Central

    Das, Kishore; De la Garza, Georgina; Siwak, Edward B.; Scofield, Virginia L.; Dhandayuthapani, Subramanian

    2014-01-01

    Summary Background Mycoplasma genitalium co-infection in HIV-infected individuals has been reported to increase the shedding of HIV in the urogenital region of females. To better understand this relationship, we investigated the influence of M. genitalium on the transmission and replication of HIV using an in vitro model. Methods The Transwell co-culture system was employed to assess the crossing of an endocervical cell barrier by HIV-1. Immunocytochemistry and confocal microscopy were used to assess the distribution of the nectin-1 molecule on M. genitalium-infected epithelial cells of the End1/E6E7 endocervical cell line, grown as monolayers in the insert wells. Peripheral blood mononuclear cells (PBMC) were cultured in the bottom wells to assess the effects of M. genitalium, passing through the semipermeable culturing membrane, on subsequent HIV infection of susceptible target cells. Results Infection of the endocervical cells with the adhesion-positive M. genitalium G37 strain (wild-type) significantly elevated the passage of HIV across the epithelial cell barrier relative to HIV transfer across endocervical cells infected with the adhesion-negative M. genitalium JB1 strain. Immunostaining of the M. genitalium-G37-infected epithelial cells disclosed capping and internalization of the junctional regulatory protein nectin-1, in association with reduced transepithelial resistance (TER) in the cell monolayer. When PBMC were cultured beneath insert wells containing M. genitalium-G37-infected epithelial cell monolayers, we observed significantly enhanced infectivity and replication of HIV added afterward to the cultures. Conclusions M. genitalium influences events on both sides of a cultured mucosal epithelial monolayer: (1) by infecting the epithelial cells and reducing the integrity of the barrier itself, and (2) by activating HIV target cells below it, thereby promoting HIV infection and progeny virus production. PMID:24661929

  19. Enhancement of antigen-induced leukocyte histamine release by a mononuclear cell--derived factor.

    PubMed

    Bamzai, A K; Kretschmer, R R

    1978-09-01

    Supernatant fluids of phytohemagglutinin-stimulated mononuclear cells from ragweed-sensitive patients significantly enhanced the release of histamine from antigen-triggered leukocytes of ragweed-sensitive as well as control individuals. Supernatants of mononuclear cells from control individuals did not reveal this enhancing effect, nor was it found with the use of supernatants of unstimulated mononuclear cells of ragweed-sensitive patients or culture media with PHA alone. Supernatant fluids of phytohemagglutinin-stimulated mononuclear cells of patients sensitive to trees and grass also revealed this enhancing effect. The factor(s) responsible for the enhancement of antigen-induced histamine is heat labile and has a molecular weight of less than 10,000 daltons. The mechanism and site of action of the enhancing factor could involve initiating and/or modulating steps of the leukocyte histamine release reaction. This factor, presumably a lymphokine or a monokine, may constitute a regulating link between cell-mediated immunity and histamine-mediated hypersensitivity reactions in allergic patients. PMID:79584

  20. Up-regulation of DNAM-1 and NKp30, associated with improvement of NK cells activation after long-term culture of mononuclear cells from patients with ovarian neoplasia.

    PubMed

    da Silva, Rodrigo Fernandes; Petta, Carlos Alberto; Derchain, Sophie Françoise; Alici, Evren; Guimarães, Fernando

    2014-08-01

    This study aimed at evaluating the functional activation and activating receptors expression on resting, short- and long-term NK and NK-like T cells from blood of ovarian neoplasia patients. Blood from patients with adnexal benign alterations (n = 10) and ovarian cancer (grade I-IV n = 14) were collected after signed consent. Effector cells activation was evaluated by the expression of the CD107a molecule. Short-term culture was conducted overnight with IL-2 and long-term culture for 21 days, by a method designed to expand CD56(+) lymphocytes. Short-term culture significantly increased NK cells activation compared to resting NK cells (p<0.05), however, the long-term procedure supported an even higher increase (p<0.001). Resting NK-like T cells showed poor activation, which was not altered by the culture procedures. The long-term culture effectively increased the expression of the activating receptors on NK and NK-like T cells, either by increasing the number of cells expressing a given receptor and/or by up-regulating their expression intensity. As a conclusion, the long-term culture system employed, resulted in a high number of functional NK cells. The culture system was particularly efficient on the up-regulation of NKp30 and DNAM-1 receptors on NK cells. PMID:24882570

  1. A Hitchhiker's Guide to Myeloid Cell Subsets: Practical Implementation of a Novel Mononuclear Phagocyte Classification System.

    PubMed

    Guilliams, Martin; van de Laar, Lianne

    2015-01-01

    The classification of mononuclear phagocytes as either dendritic cells or macrophages has been mainly based on morphology, the expression of surface markers, and assumed functional specialization. We have recently proposed a novel classification system of mononuclear phagocytes based on their ontogeny. Here, we discuss the practical application of such a classification system through a number of prototypical examples we have encountered while hitchhiking from one subset to another, across species and between steady-state and inflammatory settings. Finally, we discuss the advantages and drawbacks of such a classification system and propose a number of improvements to move from theoretical concepts to concrete guidelines. PMID:26322042

  2. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (...

  3. Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, res...

  4. Global DNA hypomethylation in peripheral blood mononuclear cells as a biomarker of cancer risk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. From an original sample-set of 753 male and...

  5. Transcriptome Characterization of Bovine Peripheral Blood Mononuclear Cells Stimulated by LPS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study aimed to identify transcription networks and signatures of bovine peripheral blood mononuclear cells in response to LPS stimulation. A total of 464 genes including at least 17 transcription factors were identified to be significantly induced by LPS using a high density bovine oligonucleo...

  6. Lentinula edodes (Shiitake) mushroom extract protects against hydrogen peroxide induced cytotoxicity in peripheral blood mononuclear cells.

    PubMed

    Kuppusamy, U R; Chong, Y L; Mahmood, A A; Indran, M; Abdullah, Noorlidah; Vikineswary, S

    2009-04-01

    Lentinula edodes (Berk) Pegler, commonly known as Shiitake mushroom has been used as medicinal food in Asian countries, especially in China and Japan and is believed to possess strong immunomodulatory property. In the present study, the methanolic extract of the fruit bodies of L. edodes was investigated for cytoprotective effect against H2O2-induced cytotoxicity in human peripheral blood mononuclear cells (PBMCs) by measuring the activities of xanthine oxidase (XO) and glutathione peroxidase (GPx) . H2O2 at a concentration of 5 microM caused 50% inhibition of PBMCs viability. The extract improved the PBMC viability and exerted a dose-dependent protection against H2O2-induced cytotoxicity. At 100 microg/ml of extract concentration, the cell viability increased by 60% compared with the PBMCs incubated with H2O2 alone. The extract also inhibited XO activity in PBMC, while showing moderate stimulatory effect on GPx. However, in the presence of H2O2 alone, both the enzyme activities were increased significantly. The GPx activity increased, possibly in response to the increased availability of H2O2 in the cell. When the cells were pretreated with the extract and washed (to remove the extract) prior to the addition of H2O2, the GPx and XO activities as well as the cell viability were comparable to those when incubated with the extract alone. Thus, it is suggested that one of the possible mechanisms via which L. edodes methanolic extract confers protection against H2O2-induced oxidative stress in PBMC is by inhibiting the superoxide-producing XO and increasing GPx activity which could rapidly inactivate H2O2. PMID:19517993

  7. Effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (zidovudine) in human mononuclear cells

    SciTech Connect

    Szebeni, J.; Patel, S.S.; Hung, K.; Wahl, L.M.; Weinstein, J.N. )

    1991-01-01

    The effects of thymidine and uridine on the phosphorylation of 3'-azido-3'-deoxythymidine (AZT) were studied in various human mononuclear cell preparations. Thymidine suppressed ({sup 3}H)AZT phosphorylation in the same concentration range (20 to 100 microM) in which it antagonizes the anti-human immunodeficiency virus activity of AZT. Uridine, in turn, had no influence on AZT phosphorylation, just as it has no effect on the anti-human immunodeficiency virus activity of AZT. These findings are consistent with a close relationship between the inhibition of AZT phosphorylation and the influence of physiological nucleosides on the antiviral activity of AZT.

  8. Developing mononuclear copper-active-oxygen complexes relevant to reactive intermediates of biological oxidation reactions.

    PubMed

    Itoh, Shinobu

    2015-07-21

    Active-oxygen species generated on a copper complex play vital roles in several biological and chemical oxidation reactions. Recent attention has been focused on the reactive intermediates generated at the mononuclear copper active sites of copper monooxygenases such as dopamine β-monooxygenase (DβM), tyramine β-monooxygenase (TβM), peptidylglycine-α-hydroxylating monooxygenase (PHM), and polysaccharide monooxygenases (PMO). In a simple model system, reaction of O2 and a reduced copper(I) complex affords a mononuclear copper(II)-superoxide complex or a copper(III)-peroxide complex, and subsequent H(•) or e(-)/H(+) transfer, which gives a copper(II)-hydroperoxide complex. A more reactive species such as a copper(II)-oxyl radical type species could be generated via O-O bond cleavage of the peroxide complex. However, little had been explored about the chemical properties and reactivity of the mononuclear copper-active-oxygen complexes due to the lack of appropriate model compounds. Thus, a great deal of effort has recently been made to develop efficient ligands that can stabilize such reactive active-oxygen complexes in synthetic modeling studies. In this Account, I describe our recent achievements of the development of a mononuclear copper(II)-(end-on)superoxide complex using a simple tridentate ligand consisting of an eight-membered cyclic diamine with a pyridylethyl donor group. The superoxide complex exhibits a similar structure (four-coordinate tetrahedral geometry) and reactivity (aliphatic hydroxylation) to those of a proposed reactive intermediate of copper monooxygenases. Systematic studies based on the crystal structures of copper(I) and copper(II) complexes of the related tridentate supporting ligands have indicated that the rigid eight-membered cyclic diamine framework is crucial for controlling the geometry and the redox potential, which are prerequisites for the generation of such a unique mononuclear copper(II)-(end-on)superoxide complex

  9. Mitochondrial Alterations in Peripheral Mononuclear Blood Cells from Alzheimer's Disease and Mild Cognitive Impairment Patients

    PubMed Central

    Delbarba, A.; Abate, G.; Prandelli, C.; Marziano, M.; Buizza, L.; Arce Varas, N.; Novelli, A.; Cuetos, F.; Martinez, C.; Lanni, C.; Memo, M.; Uberti, D.

    2016-01-01

    It is well recognized that mitochondrial dysfunction contributes to neurodegeneration occurring in Alzheimer's disease (AD). However, evidences of mitochondrial defects in AD peripheral cells are still inconclusive. Here, some mitochondrial-encoded and nuclear-encoded proteins, involved in maintaining the correct mitochondria machine, were investigated in terms of protein expression and enzymatic activity in peripheral blood mononuclear cells (PBMCs) isolated from AD and Mild Cognitive Impairment (MCI) patients and healthy subjects. In addition mitochondrial DNA copy number was measured by real time PCR. We found some differences and some similarities between AD and MCI patients when compared with healthy subjects. For example, cytochrome C and cytochrome B were decreased in AD, while MCI showed only a statistical reduction of cytochrome C. On the other hand, both AD and MCI blood cells exhibited highly nitrated MnSOD, index of a prooxidant environment inside the mitochondria. TFAM, a regulator of mitochondrial genome replication and transcription, was decreased in both AD and MCI patients' blood cells. Moreover also the mitochondrial DNA amount was reduced in PBMCs from both patient groups. In conclusion these data confirmed peripheral mitochondria impairment in AD and demonstrated that TFAM and mtDNA amount reduction could be two features of early events occurring in AD pathogenesis. PMID:26881032

  10. Effects of vitamin E on human platelet and mononuclear cell responses in vitro

    PubMed Central

    Williams, J C; Forster, L A; Tull, S P; Ferns, G A A

    1999-01-01

    Recent epidemiological studies have provided evidence supporting the potential benefits of antioxidants in coronary prevention. We have investigated the effects of vitamin E on platelets, monocytes and endothelial cells in vitro. Pre-incubation of platelets with vitamin E inhibited subsequent thrombin- (P < 0.05, n = 5), collagen- (P < 0.0001, n = 5) and ADP-(P < 0.05, n = 4) induced platelet aggregation measured using a microtitre plate method, or conventional aggregometry. The adhesion of thrombin-activated platelets to collagen was also inhibited by vitamin E (P < 0.05, n = 8), but not by vitamin C (P > 0.05, n = 8); nor was the adhesion of unstimulated platelets significantly affected (P > 0.05, n = 8). Pre-incubation of monocytes with vitamin E inhibited their subsequent adhesion to plastic (P < 0.05, n = 9), and was also associated with an 18% reduction in adhesion to EA.hy 926 endothelial cells (n = 8), although this failed to reach statistical significance. Pre-incubation of the endothelial cells with vitamin E also significantly reduced subsequent mononuclear cell adhesion by 56% (P < 0.05, n = 3). PMID:10583632

  11. Tucaresol down-modulation of MUC1-stimulated human mononuclear cells.

    PubMed

    Wright, Stephen E; Rewers-Felkins, Kathleen A; Chowdhury, Nazrul I; Ahmed, Jewel; Srivastava, Sanjay K; Lockwood-Cooke, Pamela R

    2014-01-01

    Interaction between antigen presenting cells and T lymphocytes, through receptors and co-stimulatory molecules present on the surface of these cells, is one of the key means to modulate the adaptive immune system. Tucaresol, a Schiff-base-forming chemical, can be used as a substitute for the physiological donor of carbonyl groups of antigen presenting cells, which can interact with the amine groups of T lymphocytes to modulate the adaptive immune system. This study was done to determine whether tucaresol can enhance killing of cancer cells in vitro as well as protect non-obese diabetic severe combined immunodeficient mice from tumor development by mucin 1 stimulated human mononuclear cells through the adaptive immune system. The expected hypothesis was not supported. Percent specific lysis of MCF-7 tumor cells by mucin 1 stimulated human mononuclear cells was reduced by tucaresol. Furthermore, tucaresol abolished the protective effect of mucin 1 stimulated human mononuclear cells against MCF-7 breast cancer cell growth in non-obese diabetic severe combined immunodeficient mice. This study implies that tucaresol may be of use as an immunosuppressive agent. PMID:24303799

  12. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

    PubMed

    Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro; Seko, Yuko

    2016-01-01

    Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration. PMID:27170256

  13. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

    PubMed Central

    Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro

    2016-01-01

    ABSTRACT Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration. PMID:27170256

  14. Analysis of cytotoxic effects of silver nanoclusters on human peripheral blood mononuclear cells 'in vitro'.

    PubMed

    Orta-García, Sandra Teresa; Plascencia-Villa, Germán; Ochoa-Martínez, Angeles Catalina; Ruiz-Vera, Tania; Pérez-Vázquez, Francisco Javier; Velázquez-Salazar, J Jesús; Yacamán, Miguel José; Navarro-Contreras, Hugo Ricardo; Pérez-Maldonado, Iván N

    2015-10-01

    The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most used nanomaterials in consumer products. Therefore, an understanding of the interactions (unwanted toxicity) between nanoparticles and human cells is of significant interest. The aim of this study was to assess the in vitro cytotoxicity effects of silver nanoclusters (AgNC, < 2 nm diameter) on peripheral blood mononuclear cells (PBMC). Using flow cytometry and comet assay methods, we demonstrate that exposure of PBMC to AgNC induced intracellular reactive oxygen species (ROS) generation, DNA damage and apoptosis at 3, 6 and 12 h, with a dose-dependent response (0.1, 1, 3, 5 and 30 µg ml(-1)). Advanced electron microscopy imaging of complete and ultrathin-sections of PBMC confirmed the cytotoxic effects and cell damage caused by AgNC. The present study showed that AgNC produced without coating agents induced significant cytotoxic effects on PBMC owing to their high aspect ratio and active surface area, even at much lower concentrations (<1 µg ml(-1)) than those applied in previous studies, resembling what would occur under real exposure conditions to nanosilver-functionalized consumer products. PMID:26281020

  15. Improved Quality of Life in A Case of Cerebral Palsy after Bone Marrow Mononuclear Cell Transplantation.

    PubMed

    Sharma, Alok; Sane, Hemangi; Kulkarni, Pooja; D'sa, Myola; Gokulchandran, Nandini; Badhe, Prerna

    2015-01-01

    Cerebral palsy (CP) is a non progressive, demyelinating disorder that affects a child's development and posture and may be associated with sensation, cognition, communication and perception abnormalities. In CP, cerebral white matter is injured resulting in the loss of oligodendrocytes. This causes damage to the myelin and disruption of nerve conduction. Cell therapy is being explored as an alternate therapeutic strategy as there is no treatment currently available for CP. To study the benefits of this treatment we have administered autologous bone marrow mononuclear cells (BMMNCs) to a 12-year-old CP case. He was clinically re-evaluated after six months and found to demonstrate positive clinical and functional outcomes. His trunk strength, upper limb control, hand functions, walking stability, balance, posture and coordination improved. His ability to perform activities of daily living improved. On repeating the Functional Independence Measure (FIM), the score increased from 90 to 113. A repeat positron emission tomography-computed tomography (PET-CT) scan of the brain six months after intervention showed progression of the mean standard deviation values towards normalization which correlated to the functional changes. At one year, all clinical improvements have remained. This indicated that cell transplantation may improve quality of life and have a potential for treatment of CP. PMID:26199918

  16. Improved Quality of Life in A Case of Cerebral Palsy after Bone Marrow Mononuclear Cell Transplantation

    PubMed Central

    Sharma, Alok; Sane, Hemangi; Kulkarni, Pooja; D’sa, Myola; Gokulchandran, Nandini; Badhe, Prerna

    2015-01-01

    Cerebral palsy (CP) is a non progressive, demyelinating disorder that affects a child’s development and posture and may be associated with sensation, cognition, communication and perception abnormalities. In CP, cerebral white matter is injured resulting in the loss of oligodendrocytes. This causes damage to the myelin and disruption of nerve conduction. Cell therapy is being explored as an alternate therapeutic strategy as there is no treatment currently available for CP. To study the benefits of this treatment we have administered autologous bone marrow mononuclear cells (BMMNCs) to a 12-year-old CP case. He was clinically re-evaluated after six months and found to demonstrate positive clinical and functional outcomes. His trunk strength, upper limb control, hand functions, walking stability, balance, posture and coordination improved. His ability to perform activities of daily living improved. On repeating the Functional Independence Measure (FIM), the score increased from 90 to 113. A repeat positron emission tomography-computed tomography (PET-CT) scan of the brain six months after intervention showed progression of the mean standard deviation values towards normalization which correlated to the functional changes. At one year, all clinical improvements have remained. This indicated that cell transplantation may improve quality of life and have a potential for treatment of CP. PMID:26199918

  17. Secretomes of apoptotic mononuclear cells ameliorate neurological damage in rats with focal ischemia

    PubMed Central

    Altmann, Patrick; Mildner, Michael; Haider, Thomas; Traxler, Denise; Beer, Lucian; Ristl, Robin; Golabi, Bahar; Gabriel, Christian; Leutmezer, Fritz; Ankersmit, Hendrik Jan

    2014-01-01

    The pursuit of targeting multiple pathways in the ischemic cascade of cerebral stroke is a promising treatment option. We examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC), rMNC apo sec and hMNC apo sec, in experimental stroke. We performed middle cerebral artery occlusion on Wistar rats and administered apoptotic MNC-secretomes intraperitoneally in two experimental settings. Ischemic lesion volumes were determined 48 hours after cerebral ischemia. Neurological evaluations were performed after 6, 24 and 48 hours. Immunoblots were conducted to analyze neuroprotective signal-transduction in human primary glia cells and neurons. Neuronal sprouting assays were performed and neurotrophic factors in both hMNC apo sec and rat plasma were quantified using ELISA. Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNC apo sec resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNC apo sec and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF). Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke. PMID:25383184

  18. p-Cresol Affects Reactive Oxygen Species Generation, Cell Cycle Arrest, Cytotoxicity and Inflammation/Atherosclerosis-Related Modulators Production in Endothelial Cells and Mononuclear Cells

    PubMed Central

    Chan, Chiu-Po; Yeung, Sin-Yuet; Hsien, Hsiang-Chi; Lin, Bor-Ru; Yeh, Chien-Yang; Tseng, Wan-Yu; Tseng, Shui-Kuan; Jeng, Jiiang-Huei

    2014-01-01

    Aims Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells. Methods EA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2′,7′-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F2α (PGF2α), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA). Results Exposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF2α, and uPA production in EAHY cells. Conclusions p-Cresol may contribute to atherosclerosis and thrombosis in patients with

  19. Concise Review: Prospects of Bone Marrow Mononuclear Cells and Mesenchymal Stem Cells for Treating Status Epilepticus and Chronic Epilepsy.

    PubMed

    Agadi, Satish; Shetty, Ashok K

    2015-07-01

    Mononuclear cells (MNCs) and mesenchymal stem cells (MSCs) derived from the bone marrow and other sources have received significant attention as donor cells for treating various neurological disorders due to their robust neuroprotective and anti-inflammatory effects. Moreover, it is relatively easy to procure these cells from both autogenic and allogenic sources. Currently, there is considerable interest in examining the usefulness of these cells for conditions such as status epilepticus (SE) and chronic epilepsy. A prolonged seizure activity in SE triggers neurodegeneration in the limbic brain areas, which elicits epileptogenesis and evolves into a chronic epileptic state. Because of their potential for providing neuroprotection, diminishing inflammation and curbing epileptogenesis, early intervention with MNCs or MSCs appears attractive for treating SE as such effects may restrain the development of chronic epilepsy typified by spontaneous seizures and learning and memory impairments. Delayed administration of these cells after SE may also be useful for easing spontaneous seizures and cognitive dysfunction in chronic epilepsy. This concise review evaluates the current knowledge and outlook pertaining to MNC and MSC therapies for SE and chronic epilepsy. In the first section, the behavior of these cells in animal models of SE and their efficacy to restrain neurodegeneration, inflammation, and epileptogenesis are discussed. The competence of these cells for suppressing seizures and improving cognitive function in chronic epilepsy are conferred in the next section. The final segment ponders issues that need to be addressed to pave the way for clinical application of these cells for SE and chronic epilepsy. PMID:25851047

  20. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 C or 41 C...

  1. Inactivation of O6-alkylguanine-DNA alkyltransferase in human peripheral blood mononuclear cells by temozolomide.

    PubMed Central

    Lee, S. M.; Thatcher, N.; Crowther, D.; Margison, G. P.

    1994-01-01

    O6-alkylguanine-DNA-alkyltransferase (ATase) activity was measured in extracts of peripheral blood mononuclear cells (PMCs) taken from eight patients at various times during 5 days of oral treatment with temozolomide (150 mg m-2, days 1-5). Pretreatment ATase levels ranged from approximately 70 to 600 fmol per mg of protein. Depletion of PMC ATase was seen within 4 h of the first dose of temozolomide and had a median nadir of 52.9% and values ranging from 44.4% to 71.0% of pretreatment levels. There was a correlation between the extent of ATase depletion (pretreatment minus nadir level) and the pretreatment ATase level (r = 0.97). A progressive depletion of ATase was observed during the 5 days of continuous temozolomide therapy with median ATase activities of 66.3%, 52.5%, 39.5%, 30.5% and 28.9% of the pretreatment values at days 2, 3, 4, 5 and 6 respectively. This suggests that the schedule-dependent anti-tumour activity of temozolomide seen in experimental models and clinics may be related to a cumulative depletion of ATase. PMID:8123472

  2. Interleukin-6 and interleukin-8 production by mononuclear cells of chronic alcoholics during treatment.

    PubMed

    Martinez, F; Thomas, N M; Darban, H; Cox, T J; Wood, S; Watson, R R

    1993-12-01

    Chronic alcohol consumption has been associated with suppression of a number of immune parameters. This study was designed to investigate the relationship between chronic alcohol ingestion and cessation with respect to release of interleukin-6 (IL-6) and interleukin-8 (IL-8) using highly specific and sensitive ELISA assays, as well as a functional assay, natural killer cell cytotoxic activity. ELISAs were developed to determine the amount of IL-6 and IL-8 release by peripheral blood mononuclear cells (PBMCs). Two groups of subjects were recruited: young (18-22 years old), nonalcoholic users (controls) and long-term alcoholics (35-55 years old). Blood samples were collected at time 0 from all subjects and from alcoholics 28 days after treatment had begun and alcohol use had ceased. Then mitogen-stimulated release of cytokines by peripheral blood cells was determined. The abstaining controls, and the alcoholics, after 30 days of abstinence, tended to produce lower amounts of IL-6 and IL-8, although these differences were not statistically significant. Natural killer cell activity was not statistically different between the young groups, yet appeared to increase once alcohol use discontinued. Some of the cells from the controls (abstainers) were incubated with ethanol (EtOH). Its content in sealed wells was measured after the time of incubation of PBMCs. When EtOH was serially diluted in plates, some well-well diffusion was noted, but the maximum concentration of EtOH never fell below 0.3% from an initial concentration of 0.5%, and at no time was the EtOH concentration gradient completely lost, even after 66 hr of incubation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8116830

  3. Regulation of Mycobacterium-specific mononuclear cell responses by 25-hydroxyvitamin D3.

    PubMed

    Nelson, Corwin D; Nonnecke, Brian J; Reinhardt, Timothy A; Waters, W Ray; Beitz, Donald C; Lippolis, John D

    2011-01-01

    The active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)(2)D(3) from 25-hydroxyvitamin D(3) (25(OH)D(3)) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D(3) down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D(3) down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3) down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion. PMID:21738762

  4. Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells.

    PubMed

    Su, Ming-Wei; Yu, Sung-Liang; Lin, Wen-Chang; Tsai, Ching-Hui; Chen, Po-Hua; Lee, Yungling Leo

    2016-08-15

    Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. PMID:27321975

  5. Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients.

    PubMed Central

    Stoll-Becker, S; Repp, R; Glebe, D; Schaefer, S; Kreuder, J; Kann, M; Lampert, F; Gerlich, W H

    1997-01-01

    Hepatitis B virus (HBV) has been reported to exist in peripheral blood mononuclear cells (PBMC), but it is not clear whether it replicates there. A precondition for replication should be the formation of covalently closed viral DNA and transcription of all essential viral mRNAs. The mRNAs of HBV form a nested box with common 3' ends. In order to detect even low levels of potential replication, we developed a quantitative reverse transcription-PCR method for detection of a smaller HBV mRNA species in the presence of the larger ones. All three highly viremic patients tested so far had mRNAs for the large and the small surface proteins and the X protein of the virus within PBMC but not in the virus from their sera. Furthermore, we detected by PCR covalently closed viral DNA in their PBMC. These data suggest that HBV may be not only taken up but also replicated by mononuclear blood cells and that these cells may be an extrahepatic site of viral persistence. X mRNA was detected in the largest amount. Possibly, X protein interferes with functions of the mononuclear cells during the immune response against the virus. PMID:9188611

  6. Effects of endotoxin exposure on cationic amino acid transporter function in ovine peripheral blood mononuclear cells.

    PubMed

    Clark, Megan F; Reade, Michael C; Boyd, C A R; Young, J Duncan

    2003-03-01

    Rodent models of sepsis differ from clinical human disease in that humans make substantially less whole-body nitric oxide and have different cellular responses to endotoxin. Sheep, when exposed to endotoxin, behave in a manner more similar to humans. Many studies of rodent peripheral blood mononuclear cells (PBMCs) exposed to endotoxin demonstrate increased cationic amino acid transporter function (particularly through the y+ transporter) to supply arginine substrate to upregulated nitric oxide synthase. Whether this is true in sheep is not known. We have studied cationic amino acid transport in sheep PBMCs stimulated with endotoxin, using labelled lysine. PBMCs stimulated both in vitro and in vivo show an initial reduction in total and y+ lysine transport (after 1-2 h exposure to endotoxin): a previously undescribed effect of endotoxin. In in vitro activated cells, the reduction in y+ transport was prevented by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), and the phospholipase inhibitor 4-bromophenacyl bromide (4-BPAB), but not cyclohexamide or a number of other inhibitors of intracellular second-messenger pathways. In contrast after 14 h incubation, the expected increase in total and y+ lysine transport was seen. The increase in y+ transport could be prevented by cyclohexamide, dexamethasone, ibuprofen, the protein kinase C inhibitor sphingosine, NDGA and 4-BPAB. These results suggest that in response to endotoxin exposure there is an initial decrease in y+ activity mediated by a lipoxygenase product, followed by a substantial increase in y+ activity mediated by the products of either cyclo-oxygenase or lipoxygenase. Cyclo-oxygenase and/or lipoxygenase inhibition might be useful in reducing arginine transport, and hence nitric oxide production, in these cells. PMID:12621525

  7. M cells and granular mononuclear cells in Peyer's patch domes of mice depleted of their lymphocytes by total lymphoid irradiation.

    PubMed Central

    Ermak, T. H.; Steger, H. J.; Strober, S.; Owen, R. L.

    1989-01-01

    The cytoarchitecture of Peyer's patches that were depleted of their lymphocytes by total lymphoid irradiation (TLI) was examined with particular attention to the effects on M cells in the follicle epithelium and on mononuclear cells in follicle domes underlying the epithelium. Five-month-old, specific pathogen-free Balb/c mice were irradiated with 200-250 rad/day, five times a week to a total dose of 3400-4250, and their Peyer's patches were either fixed for electron microscopy or frozen for immunohistochemistry 1-4 days after completion of irradiation. Control mice were examined at the same time intervals. Follicle domes of TLI mice had approximately one fourth the epithelial surface area of domes of control mice. Within the epithelium, lymphoid cells were virtually depleted after TLI, and yet the epithelium contained M cells. In control mice, most M cells were accompanied by lymphoid cells in invaginations of the apical-lateral cell membrane. In TLI mice, most M cells did not have such apical-lateral invaginations and were columnar shaped. Other than lacking lymphocytes, these cells appeared to be mature M cells. Some M cells did have lymphoid cells or granular mononuclear cells below their basal membranes, adjacent to the basal lamina. Below the epithelium, the proportion of granular mononuclear cells was greatly increased following TLI. The retention of M cells and the increase in proportion of granular mononuclear cells in follicle domes are consistent with selective depletion of lymphocytes following TLI. Persistence of M cells without lymphocytic invaginations after TLI suggests that M cells can differentiate in the absence of, or at least in the presence of very few, lymphocytes, and that invagination by lymphocytes is not necessary to maintain mature M cell morphology. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:2923183

  8. Differential proteomics analysis of mononuclear cells in cerebrospinal fluid of Parkinson’s disease

    PubMed Central

    Xing, Lifei; Wang, Dongtao; Wang, Lihong; Lan, Wenjie; Pan, Suyue

    2015-01-01

    Parkinson’s disease (PD) is one common neurodegenerative disease featured with degeneration of dopaminergic neurons in substantia nigra. Multiple factors participate in the pathogenesis and progression of PD. In this study, we investigated the proteomics profiles of mononuclear cells in cerebrospinal fluids from both PD patients and normal people, in order to explore the correlation between disease factors and PD. Cerebrospinal fluid samples were collected from both PD and normal people and were separated for mononuclear cells in vitro. Proteins were then extracted and separated by 2-dimensional gel electrophoresis. Proteins with differential expressions were identified by comparison to standard proteome expression profile map, followed by software and database analysis. In PD patients, there were 8 proteins with consistent expression profile and 16 proteins with differential expressions. Those differential proteins identified include cytoskeleton proteins (actin, myosin), signal transduction proteins (adenosine cyclase binding protein 1, calcium binding protein, talin) and anti-oxidation factor (thioredoxin peroxide reductase). PD patients had differential protein expressional profiles in the mononuclear cells of cerebrospinal fluids compared to normal people, suggesting the potential involvement of cytoskeleton and signal transduction proteins in apoptosis of neuronal apoptosis and PD pathogenesis. PMID:26823915

  9. Interleukin-8 Transcripts in Mononuclear Cells Determine Impaired Graft Function after Kidney Transplantation

    PubMed Central

    Borst, Christoffer; Xia, Shengqiang; Bistrup, Claus; Tepel, Martin

    2015-01-01

    Objective Interleukin-8 (IL-8) has been associated with ischemia reperfusion injury after renal allograft transplantation. Impaired allograft function may cause major impact on patient morbidity and health care costs. We investigated whether transcript levels in mononuclear cells including IL-8 on the first postoperative day may be involved in immediate allograft dysfunction as defined by reduced relative change in plasma creatinine at the first postoperative day. Methods We performed a single center, prospective-cohort study of 113 patients receiving kidney transplants. Peripheral blood mononuclear cells were harvested within 24 hours after transplantation. Transcripts were measured using quantitative RT-PCR. Results Transcript levels of IL-8 and S100A8 were significantly lower in patients with relative change in plasma creatinine less than 10% at the first postoperative day. Receiver-operator characteristic curves showed that IL-8 predicted the relative change in plasma creatinine less than 10% (area under curve (AUC), 0.80; P = 0.0007). Multivariate analyses showed that lower IL-8 transcripts, longer time on dialysis, higher recipient body mass index and deceased donor type were associated with relative change in plasma creatinine at the first postoperative day less than 10%. Conclusion Reduced levels of IL-8 transcripts in peripheral mononuclear cells predict immediate graft dysfunction and delayed graft function. PMID:25689147

  10. [THE EXPRESSION OF TLR-4 GENE MONONUCLEAR CELLS PERIPHERAL BLOOO IN PATIENTS BY HIGH RISK OF PURULENT-INFLAMMATORY COMPLICATIONS AFTER SURGERY FOR ACUTE DISEASES OF THE ABDOMINAL ORGANS].

    PubMed

    Sheyko, V D; Sytnik, D A; Pryhidko, R A; Shkurupiy, O A; Shlykova, O A; Izmailova, O V

    2015-06-01

    The specified level of gene expression TLR-4 in peripheral blood mononuclear cells in 77 patients operated on acute diseases of the abdominal organs in the 1st and the 4th day after surgery was determined. Established dynamic changes of gene expression TLR-4. Adverse course early postoperative period in patients initially high and medium risk of purulent-septic complications was accompanied by activation of gene expression TLR-4 in peripheral blood mononuclear cells. PMID:26521462

  11. Comparative analysis of mononuclear cells isolated from mucosal lymphoid follicles of the human ileum and colon

    PubMed Central

    Junker, Y; Bode, H; Wahnschaffe, U; Kroesen, A; Loddenkemper, C; Duchmann, R; Zeitz, M; Ullrich, R

    2009-01-01

    Studies of human mucosal lymphoid follicles are rare and have been limited to children's Peyer's patches, which are visible at endoscopy. We investigated lymphoid follicles in ileum biopsies of 87 patients and surgical colon specimens from 66 cancer patients, and examined phenotype and function of isolated follicular immune cells. Two (0–10) and 12 (0–117) follicles per patient were found in ileum and colon samples respectively (P < 0·001). The number of lymphoid follicles mononuclear cells (LFMC) that could be isolated per patient was higher from colon compared with ileum specimens [725 000 (0–23 Mio) versus 100 000 (0–1·3 Mio), P < 0·001]. T cells were predominant in both LFMC and lamina propria mononuclear cells (LPMC), but B cells were more and plasma cells less frequent in LFMC. T cells from mucosal follicles were more frequently CD4-positive and CD62L-positive, but less frequently CD8-positive, CD103-positive and CD69-positive than lamina propria T cells. LFMC from ileum compared with colon showed no differences in mononuclear cell composition. Anti-CD3/CD28 stimulation induced similar proliferation of LFMC and LPMC from ileum and colon, as well as secretion of high levels of interferon-γ, tumour necrosis factor-α and interleukin (IL)-2, but lower levels of IL-4, IL-6 and IL-10. LFMC from colon secreted more IL-2 than those from ileum. Our study shows that mucosal lymphoid follicles can be identified clearly in adult human colon and yield viable immune cells sufficient for phenotypical and functional analysis. The cellular composition of LFMC from ileum and colon is similar, and both secrete predominantly T helper type 1 cytokines. PMID:19250280

  12. The effects of retinol on in vitro immunoglobulin synthesis by cord blood and adult peripheral blood mononuclear cells.

    PubMed

    Wang, W; Napoli, J L; Ballow, M

    1993-04-01

    In this study we examined the effects of retinol (ROH), a metabolic precursor of retinoic acid (RA), on Staphylococcus aureus Cowan I (SAC)-induced immunoglobulin synthesis of cord blood mononuclear cells (CBMC) and adult peripheral blood mononuclear cells (PBMC). ROH augmented SAC-induced IgM synthesis of CBMC by 5.9 +/- 1.5-fold (n = 7, mean +/- s.d.), and IgG synthesis of adult PBMC by 16.3 +/- 5.1-fold (n = 3) at optimal concentrations of 10(-6) M and 10(-11) M, respectively. No augmenting effects could be demonstrated for the other immunoglobulin isotypes. Time-course studies showed that the synthesis of IgM by CBMC was accelerated with detectable immunoglobulin in supernatant fluids starting on day 3. ROH augmented immunoglobulin synthesis of CBMC stimulated by Epstein-Barr virus (EBV), a T cell-independent polyclonal activator, and of EBV-transformed B cell clones (2.5 +/- 0.2 and 4.1 +/- 1.5-fold increase, respectively), which suggests that ROH can act directly on B cells to enhance immunoglobulin synthesis. In contrast, when ROH was preincubated with cord blood T cells, washed and added to the B cell-enriched fraction with SAC, no increase (0.9-1.8-fold) in IgM synthesis was obtained. Thus, the principal mechanism(s) by which ROH augments immunoglobulin synthesis is by acting on B cells. This is in contrast to the immunoglobulin-enhancing effects of RA which is mediated by T cells, or T cell products, e.g. cytokine. Our studies suggest that RA and ROH may have different pathways of immunoglobulin-enhancing effects, perhaps mediated by different retinoid binding proteins resulting in gene activation and immunoglobulin synthesis. PMID:8385583

  13. Alteration of the electrophoretic mobility of human peripheral blood mononuclear cells following treatment with dimethyl sulfoxide

    SciTech Connect

    Skrabut, E.M.; Catsimpoolas, N.; Kurtz, S.R.; Griffith, A.L.; Valeri, C.R.

    1983-12-01

    Studies have been conducted to determine the effects of DMSO and freezing on the electrophoretic distribution of peripheral blood mononuclear cells. Sodium (/sup 51/Cr)chromate was used to label the cells, and the distributions of cell number and cell-associated radioactivity were determined. Cells treated with DMSO had a narrower distribution of electrophoretic mobilities when compared with those not treated. DMSO-treated cells also demonstrated a more homogeneous distribution of radioactivity relative to the cell distribution than did the nontreated cells. The freezing of DMSO-treated cells did not result in any additional alteration of electrophoretic pattern compared to DMSO treatment alone. Analysis by linear categorization techniques indicated that the DMSO-treated and nontreated cells were completely distinguished by their electrophoretic behavior.

  14. A mononuclear Ni(II) complex with 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine: DNA- and BSA-binding and anticancer activity against human breast carcinoma cells

    NASA Astrophysics Data System (ADS)

    Anjomshoa, Marzieh; Hadadzadeh, Hassan; Fatemi, Seyed Jamilaldin; Torkzadeh-Mahani, Masoud

    2015-02-01

    DNA- and BSA-binding properties of a mononuclear Ni(II) complex, [Ni(dppt)2Cl2] (dppt = 5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine), have been investigated under physiological conditions. The interaction of the complex with the fish sperm DNA (FS-DNA) has been studied by UV-Vis absorption, thermal denaturation, viscosity measurement, competitive DNA-binding studies with ethidium bromide (EB) by fluorescence, and gel electrophoresis technique. The experimental results indicate that the complex interacts with DNA by intercalative binding mode. The competitive study with ethidium bromide (EB) shows that the complex competes for the DNA-binding sites with EB and displaces the DNA-bound EB molecule. The interactions of the dppt ligand and the complex with BSA have been studied by UV-Vis absorption and fluorescence spectroscopic techniques. The values of Kb for the BSA-dppt and the BSA-complex systems at room temperature were calculated to be 0.14 × 104 M-1 and 0.32 × 105 M-1, respectively, indicating that the complex has stronger tendency to bind with BSA than the dppt ligand. The quenching constants (Ksv), binding constants (Kbin), and number of binding sites (n) at different temperatures, as well as the binding distance (r) and thermodynamic parameters (ΔH°, ΔS° and ΔG°) have been calculated for the BSA-dppt and the BSA-complex systems. The cytotoxicities of the dppt ligand and the complex have been also tested against the human breast adenocarcinoma (MCF-7) cell line using the MTT assay. The results indicate that the dppt ligand and the complex display cytotoxicity against human breast cancer cell lines (MCF-7) with the IC50 values of 17.35 μM and 13.00 μM, respectively. It is remarkable that the complex can introduce as a potential anticancer drug.

  15. Reduced pCREB in Alzheimer's disease prefrontal cortex is reflected in peripheral blood mononuclear cells.

    PubMed

    Bartolotti, N; Bennett, D A; Lazarov, O

    2016-09-01

    Cyclic-AMP response element-binding protein (CREB) signaling has a critical role in the formation of memories. CREB signaling is dysfunctional in the brains of mouse models of Alzheimer's disease (AD), and evidence suggests that CREB signaling may be disrupted in human AD brains as well. Here, we show that both CREB and its activated form pCREB-Ser(133) (pCREB) are reduced in the prefrontal cortex of AD patients. Similarly, the transcription cofactors CREB-binding protein (CBP) and p300 are reduced in the prefrontal cortex of AD patients, indicating additional dysfunction of CREB signaling in AD. Importantly, we show that pCREB expression is reduced in peripheral blood mononuclear cells (PBMC) of AD subjects. In addition, pCREB levels in PBMC positively correlated with pCREB expression in the postmortem brain of persons with AD. These results suggest that pCREB expression in PBMC may be indicative of its expression in the brain, and thus offers the intriguing possibility of pCREB as a biomarker of cognitive function and disease progression in AD. PMID:27480489

  16. Reduced pCREB in Alzheimer's disease prefrontal cortex is reflected in peripheral blood mononuclear cells

    PubMed Central

    Bartolotti, N; Bennett, D A; Lazarov, O

    2016-01-01

    Cyclic-AMP response element-binding protein (CREB) signaling has a critical role in the formation of memories. CREB signaling is dysfunctional in the brains of mouse models of Alzheimer's disease (AD), and evidence suggests that CREB signaling may be disrupted in human AD brains as well. Here, we show that both CREB and its activated form pCREB-Ser133 (pCREB) are reduced in the prefrontal cortex of AD patients. Similarly, the transcription cofactors CREB-binding protein (CBP) and p300 are reduced in the prefrontal cortex of AD patients, indicating additional dysfunction of CREB signaling in AD. Importantly, we show that pCREB expression is reduced in peripheral blood mononuclear cells (PBMC) of AD subjects. In addition, pCREB levels in PBMC positively correlated with pCREB expression in the postmortem brain of persons with AD. These results suggest that pCREB expression in PBMC may be indicative of its expression in the brain, and thus offers the intriguing possibility of pCREB as a biomarker of cognitive function and disease progression in AD. PMID:27480489

  17. Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro.

    PubMed

    Henrich, Dirk; Verboket, René; Schaible, Alexander; Kontradowitz, Kerstin; Oppermann, Elsie; Brune, Jan C; Nau, Christoph; Meier, Simon; Bonig, Halvard; Marzi, Ingo; Seebach, Caroline

    2015-01-01

    Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on β-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and β-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo. PMID:25802865

  18. Conversion of adult human peripheral blood mononuclear cells into induced neural stem cell by using episomal vectors.

    PubMed

    Tang, Xihe; Wang, Shuyan; Bai, Yunfei; Wu, Jianyu; Fu, Linlin; Li, Mo; Xu, Qunyuan; Xu, Zhi-Qing David; Alex Zhang, Y; Chen, Zhiguo

    2016-03-01

    Human neural stem cells (NSCs) hold great promise for research and therapy in neural diseases. Many studies have shown direct induction of NSCs from human fibroblasts, which require an invasive skin biopsy and a prolonged period of expansion in cell culture prior to use. Peripheral blood (PB) is routinely used in medical diagnoses, and represents a noninvasive and easily accessible source of cells. Here we show direct derivation of NSCs from adult human PB mononuclear cells (PB-MNCs) by employing episomal vectors for transgene delivery. These induced NSCs (iNSCs) can expand more than 60 passages, can exhibit NSC morphology, gene expression, differentiation potential, and self-renewing capability and can give rise to multiple functional neural subtypes and glial cells in vitro. Furthermore, the iNSCs carry a specific regional identity and have electrophysiological activity upon differentiation. Our findings provide an easily accessible approach for generating human iNSCs which will facilitate disease modeling, drug screening, and possibly regenerative medicine. PMID:26826927

  19. The effects of calpain inhibition upon IL-2 and CD25 expression in human peripheral blood mononuclear cells.

    PubMed

    Schaecher, K E; Goust, J M; Banik, N L

    2001-10-01

    Calcium is an important contributor to T cell activation; it is also the major factor in the activation of the calcium-activated neutral proteinase, calpain. For this reason, we wanted to investigate if calpain has a role in T cell activation and what aspects of this activation calpain affects. As measured by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), calpain inhibition decreased interleukin-2 (IL-2) and CD25 mRNA expression in a dose-dependent manner, at early time points following the initial activation, and over extended periods of time in activated human peripheral blood mononuclear cells (PBMCs). Using an enzyme-linked immuno-sorbent assay (ELISA) specific for human IL-2, we found that calpain inhibition decreased IL-2 secretion in a dose-dependent manner, shortly after activation, and continuously over time. Inhibiting calpain caused a dose-dependent inhibition of CD25 cell surface expression and also inhibited expression shortly after activation and for at least 48 h. This study showed that calpain has an integral role in the synthesis of the two important T cell activation factors, IL-2 and CD25. PMID:11585637

  20. Modulation of TNF-α Secretion in Peripheral Blood Mononuclear Cells by Cocoa Flavanols and Procyanidins

    PubMed Central

    Mao, T. K.; van de Water, J.; Keen, C. L.; Schmitz, H. H.; Gershwin, M. E.

    2002-01-01

    Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines and eicosanoids. To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory (–1.5 and –15%, respectively), while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels (13, 19 and 15%, respectively). The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis. PMID:12885154

  1. Evaluating mononuclear cells as nanoparticle delivery vehicles for the treatment of breast tumors

    NASA Astrophysics Data System (ADS)

    Murton, Jaclyn K.; Hu, Chelin; Ahmed, Mona M.; Hathaway, Helen J.; Nysus, Monique; Anderson Daniels, Tamara; Norenberg, Jeffrey P.; Adolphi, Natalie L.

    2015-08-01

    In breast cancer, certain types of circulating immune cells respond to long-range chemical signals from tumors by leaving the blood stream to actively infiltrate tumor tissue. The aim of this study was to evaluate whether immune cells could be used to deliver therapeutic nanoparticles into breast tumors in mice. Mononuclear splenocytes (MS) were harvested from donor mice, labeled with Indium-111, injected intravenously into immune-competent recipient mice (3 tumor-bearing and 3 control), and imaged longitudinally by SPECT/CT. For comparison, the biodistribution of bonemarrow derived macrophages (BMDM) in one pair of mice was also imaged. Quantitative analysis of the SPECT images demonstrates that, after 24 hours, the concentration of MS detected in mammary tumors is more than 3-fold higher than the concentration detected in normal mammary glands. The ratio of MS concentration in mammary tissue to MS concentration in non-target tissues (muscle, lung, heart, liver, spleen, and kidney) was enhanced in tumor-bearing mice (compared to controls), with statistical significance achieved for mammary/muscle (p<0.01), mammary/lung (p<0.05), and mammary/kidney (p<0.05). By contrast, BMDM did not show a different affinity for tumors relative to normal mammary tissue. MS were incubated with 100 nm red fluorescent nanoparticles, and flow cytometry demonstrated that ~35% of the MS population exhibited strong phagocytic uptake of the nanoparticles. After intravenous injection into tumor-bearing mice, fluorescence microscopy images of tumor sections show qualitatively that nanoparticle-loaded MS retain the ability to infiltrate mammary tumors. Taken together, these results suggest that MS carriers are capable of actively targeting therapeutic nanoparticles to breast tumors.

  2. Alzheimer patients treated with an AchE inhibitor show higher IL-4 and lower IL-1 beta levels and expression in peripheral blood mononuclear cells.

    PubMed

    Gambi, Francesco; Reale, Marcella; Iarlori, Carla; Salone, Anatolia; Toma, Lucia; Paladini, Carlo; De Luca, Giovanna; Feliciani, Claudio; Salvatore, Mirella; Salerno, Rosa M; Theoharides, Theoharis C; Conti, Pio; Exton, Michael; Gambi, Domenico

    2004-06-01

    The study evaluates the expression and production of cytokines in peripheral blood mononuclear cells of patients with Alzheimer disease treated or not treated with acetylcholinesterase inhibitor, which enhances neuronal transmission. Cytokines associated with brain inflammation such as interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha have been implicated in the regulation of amyloid peptide protein synthesis. The anti-inflammatory cytokine, IL-4, may suppress the activity of IL-1beta. Patients were assessed for clinical and immunologic features at baseline and after 1 month of treatment with Donepezil, an acetylcholinesterase inhibitor. Peripheral blood mononuclear cells were cultured with and without phytohemagglutinin stimulation. IL-1beta and IL-4 levels were measured by enzyme-linked immunosorbent assay. Reverse transcriptase-polymerase chain reaction was used to determine the expression of cytokines in peripheral mononuclear cells. Compared with untreated patients and healthy control subjects, IL-1beta levels and expression decreased in Alzheimer disease patients treated with Donepezil (P < 0.001). In contrast, IL-4 levels and expression were significantly higher in Alzheimer patients treated with the acetylcholinesterase inhibitor. This increment was observed in both unstimulated and phytohemagglutinin-stimulated peripheral blood mononuclear cells. PMID:15118486

  3. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    SciTech Connect

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O.

    1995-05-01

    Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet {beta}-cells following islet filtration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in {beta}-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1{beta}, IL-2, IL-4, IL-10, and IFN-{gamma} in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (>13 wk). However, only IFN-{gamma} mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-{gamma} and IL-4 were not different in the four groups of mice. These results suggest that islet {beta}-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-{gamma} production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-{gamma} production in the islets. 56 refs., 4 figs., 3 tabs.

  4. Canine PHA-stimulated adherent cell enhance interferon-gamma production and proliferation of autologous peripheral blood mononuclear cells.

    PubMed

    Ide, Kaori; Momoi, Yasuyuki; Iwasaki, Toshiroh

    2005-03-01

    Dendritic cells are specialized antigen-presenting cells with immuno-modulating functions that are attractive for clinical applications for cancer immunotherapy. This study examined immunostimulatory functions of phytohemagglutinin (PHA)-stimulated adherent cells (PHA-Ad cells) from peripheral blood mononuclear cells (PBMCs) in dogs. PHA-Ad cells enhanced interferon-gamma from autologous PBMC in vitro. PHA-Ad cells also stimulated antigen-independent proliferation of peripheral blood lymphocytes. These results suggest that PHA-Ad cells from PBMC possess a stimulatory function to evoke anti-tumour immunity and that they demonstrate potential for therapeutic applications in dogs. PMID:19379211

  5. In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

    NASA Astrophysics Data System (ADS)

    Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

    2013-11-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin

  6. Nanoparticles with Therapeutic Properties Generate Various Response of Human Peripheral Blood Mononuclear Cells.

    PubMed

    Szwed, Marzena; Santos-Oliveira, Ralph

    2016-06-01

    In the present study we report the interactions of four types of different nanoparticles with normal peripheral blood mononuclear cells. To our research we chose four types of nanoparticles which possess therapeutic properties (Trastuzumab, ethylene-diamine-tetra-methylene-phosphonic for breast and bone cancers treatment, respectively) or can be used as the ingredients of sun-protected films (nanoemulsions with or without chitosan). By carrying out XTT survival assay we observed that both types of tested nanoemulsions suppressed the proliferation of normal lymphocytes. However, the survival of peripheral blood mononuclear cells after incubation neither with Trastuzumab nor with ethylene-diamine-tetra-methylene-phosphonic nanoparticles decreased below 80%. If the investigated nanoparticles were analyzed for their effectiveness to the induction of programmed cell death, we proved that only nanoemulsions with or without chitosan provoked an increase of the fraction of apoptotic cells. Moreover we noticed the characteristic, typical for apoptosis changes of cells morphology, which appeared in lymphocytes after all tested nanoparticles treatment. Interestingly, representative for necrosis swollen, enlarged cells were observed after nanoemulsions treatment. PMID:27427750

  7. Peripheral blood mononuclear cell infiltration and neuroinflammation in the HexB−/− mouse model of neurodegeneration

    PubMed Central

    Kyrkanides, Stephanos; Miller, Ann W.; Miller, Jen-nie H.; Tallents, Ross H.; Brouxhon, Sabine M.; Olschowka, Malory E.; O’Banion, M. Kerry; Olschowka, John A.

    2008-01-01

    Myeloid-derived immune cells, including microglia, macrophages and monocytes, have been previously implicated in neurodegeneration. We investigated the role of infiltrating peripheral blood mononuclear cells (PBMC) in neuroinflammation and neurodegeneration in the HexB−/− mouse model of Sandhoff disease. Ablation of the chemokine receptor CCR2 in the HexB−/− mouse resulted in significant inhibition of PBMC infiltration into the brain, decrease in TNFα and MHC-II mRNA abundance and retardation in clinical disease development. There was no change in the level of GM2 storage and pro-apoptotic activity or astrocyte activation in HexB−/−;Ccr2−/− double knockout mice, which eventually succumbed secondary to GM2 gangliosidosis. PMID:18657867

  8. Interferon-α curbs production of interleukin-22 by human peripheral blood mononuclear cells exposed to live Borrelia burgdorferi.

    PubMed

    Berner, Anika; Bachmann, Malte; Pfeilschifter, Josef; Kraiczy, Peter; Mühl, Heiko

    2015-10-01

    Cytokine networks initiated by means of innate immunity are regarded as a major determinant of host defence in response to acute infection by bacteria including Borrelia burgdorferi. Herein, we demonstrate that interferon (IFN)-α, either endogenously produced after exposure of cells to toll-like receptor-9-activating CpG oligonucleotides or provided as recombinant cytokine, weakens activation of the anti-bacterial interleukin (IL)-1/IL-22 axis in human peripheral blood mononuclear cells exposed to viable B. burgdorferi. As IFN-α has been related to pathological dissemination of the spirochaete, data suggest an immunoregulatory role of type I IFN in this context that is able to significantly modify cytokine profiles thereby possibly determining early course of B. burgdorferi infection. PMID:26152778

  9. Autologous red blood cells potentiate antibody synthesis by unfractionated human mononuclear cell cultures.

    PubMed

    Rugeles, M T; La Via, M; Goust, J M; Kilpatrick, J M; Hyman, B; Virella, G

    1987-08-01

    We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear cells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH, and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (1:50 ratio of PBMNC/RBC). The cultures were harvested on day 11; immunoglobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-TT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in the presence of autologous RBC. Similarly, anti-KLH responses were easier to induce with PBMNC from an immune donor; maximal response was observed after stimulation with PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-gamma). However, addition of IFN-gamma in different doses and at different times to PWM-stimulated PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody. PMID:3114872

  10. Non-Anticoagulant Fractions of Enoxaparin Suppress Inflammatory Cytokine Release from Peripheral Blood Mononuclear Cells of Allergic Asthmatic Individuals

    PubMed Central

    Horne, James; Zaidi, Syed Tabish R.; Sohal, Sukhwinder Singh; Peterson, Gregory M.; Korner, Heinrich; Patel, Rahul P.

    2015-01-01

    Background Enoxaparin, a low-molecular-weight heparin, is known to possess anti-inflammatory properties. However, its clinical exploitation as an anti-inflammatory agent is hampered by its anticoagulant effect and the associated risk of bleeding. Objective The aim of the current study was to examine the ability of non-anticoagulant fractions of enoxaparin to inhibit the release of key inflammatory cytokines in primed peripheral blood mononuclear cells derived from allergic mild asthmatics. Methods Peripheral blood mononuclear cells from allergic asthmatics were activated with phytohaemag glutinin (PHA), concanavalin-A (ConA) or phorbol 12-myristate 13-acetate (PMA) in the presence or absence of enoxaparin fractions before cytokine levels were quantified using specific cytokine bead arrays. Together with nuclear magnetic resonance analysis,time-dependent and target-specific effects of enoxaparin fractions were used to elucidate structural determinants for their anti-inflammatory effect and gain mechanistic insights into their anti-inflammatory activity. Results Two non-anticoagulant fractions of enoxaparin were identified that significantly inhibited T-cell activation. A disaccharide fraction of enoxaparin inhibited the release of IL-4, IL-5, IL-13 and TNF-α by more than 57% while a tetrasaccharide fraction was found to inhibit the release of tested cytokines by more than 68%. Our data suggest that the observed response is likely to be due to an interaction of 6-O-sulfated tetrasaccharide with cellular receptor(s). Conclusion and Clinical Relevance The two identified anti-inflammatory fractions lacked anticoagulant activity and are therefore not associated with risk of bleeding. The findings highlight the potential therapeutic use of enoxaparin-derived fractions, in particular tetrasaccharide, in patients with chronic inflammatory disorders. PMID:26046354

  11. Gene expression profiling of peripheral blood mononuclear cells in the setting of peripheral arterial disease

    PubMed Central

    2012-01-01

    Background Peripheral arterial disease (PAD) is a relatively common manifestation of systemic atherosclerosis that leads to progressive narrowing of the lumen of leg arteries. Circulating monocytes are in contact with the arterial wall and can serve as reporters of vascular pathology in the setting of PAD. We performed gene expression analysis of peripheral blood mononuclear cells (PBMC) in patients with PAD and controls without PAD to identify differentially regulated genes. Methods PAD was defined as an ankle brachial index (ABI) ≤0.9 (n = 19) while age and gender matched controls had an ABI > 1.0 (n = 18). Microarray analysis was performed using Affymetrix HG-U133 plus 2.0 gene chips and analyzed using GeneSpring GX 11.0. Gene expression data was normalized using Robust Multichip Analysis (RMA) normalization method, differential expression was defined as a fold change ≥1.5, followed by unpaired Mann-Whitney test (P < 0.05) and correction for multiple testing by Benjamini and Hochberg False Discovery Rate. Meta-analysis of differentially expressed genes was performed using an integrated bioinformatics pipeline with tools for enrichment analysis using Gene Ontology (GO) terms, pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), molecular event enrichment using Reactome annotations and network analysis using Ingenuity Pathway Analysis suite. Extensive biocuration was also performed to understand the functional context of genes. Results We identified 87 genes differentially expressed in the setting of PAD; 40 genes were upregulated and 47 genes were downregulated. We employed an integrated bioinformatics pipeline coupled with literature curation to characterize the functional coherence of differentially regulated genes. Conclusion Notably, upregulated genes mediate immune response, inflammation, apoptosis, stress response, phosphorylation, hemostasis, platelet activation and platelet aggregation. Downregulated genes included several genes from

  12. Gene expression of peripheral blood mononuclear cells is affected by cold exposure.

    PubMed

    Reynés, Bàrbara; García-Ruiz, Estefanía; Oliver, Paula; Palou, Andreu

    2015-10-15

    Because of the discovery of brown adipose tissue (BAT) in humans, there is increased interest in the study of induction of this thermogenic tissue as a basis to combat obesity and related complications. Cold exposure is one of the strongest stimuli able to activate BAT and to induce the appearance of brown-like (brite) adipocytes in white fat depots (browning process). We analyzed the potential of peripheral blood mononuclear cells (PBMCs) to reflect BAT and retroperitoneal white adipose tissue (rWAT) response to 1-wk cold acclimation (4°C) at different ages of rat development (1, 2, 4, and 6 mo). As expected, cold exposure increased fatty acid β-oxidation capacity in BAT and rWAT (increased Cpt1a expression), explaining increased circulating nonesterified free fatty acids and decreased adiposity. Cold exposure increased expression of the key thermogenic gene, Ucp1, in BAT and rWAT, but only in 1-mo-old animals. Additionally, other brown/brite markers were affected by cold during the whole developmental period studied in BAT. However, in rWAT, cold exposure increased studied markers mainly at early age. PBMCs did not express Ucp1, but expressed other brown/brite markers, which were cold regulated. Of particular interest, PBMCs reflected adipose tissue-increased Cpt1a mRNA expression in response to cold (in older animals) and browning induction occurring in rWAT of young animals (1 mo) characterized by increased Cidea expression and by the appearance of a high number of multilocular CIDE-A positive adipocytes. These results provide evidence pointing to PBMCs as an easily obtainable biological material to be considered to perform browning studies with minimum invasiveness. PMID:26246506

  13. Changes in transcriptional output of human peripheral blood mononuclear cells following resistance exercise.

    PubMed

    Carlson, Lara A; Tighe, S W; Kenefick, R W; Dragon, J; Westcott, N W; Leclair, R J

    2011-12-01

    Various types of exercise alter the population of circulating peripheral blood mononuclear cells (PBMCs) and change their transcriptional output. This work examines changes in PBMC populations and transcription in response to resistance exercise training (RET), and identify key transcriptional changes in PBMCs that may play a role in altering peripheral tissues in response to RET. Ten resistance-trained men (20-24 years), performed an acute bout of RET for ~30 min following a 12 h fast. Venous blood was sampled at rest, immediately following exercise, and at 2 h post-exercise and analyzed for total and differential leukocytes and global gene expression using Affymetrix Genechips. Results showed elevated leukocytes, monocytes, lymphocytes, and lactate values immediately post-exercise (P < 0.05) over baseline. At 2 h post-exercise, leukocytes, and granulocytes remained elevated (P < 0.05), whereas lymphocytes were lower than (P < 0.05) baseline values. Initial microarray results showed the greatest transcriptional changes in pathways related to immune response, inflammation, and cellular communication. The change in PBMC population (2 h time point) correlated with a dramatic decrease in the expression of CD160, and XCL1, markers of lymphocyte populations. At the 2 h recovery time point upregulation of matrix metalloproteinase 9, orosomucoid 1, dishevelled-associated activator of morphogenesis 2, and arginase 1 suggest an induction in muscle damage and repair during this time frame. These results demonstrate that an acute bout of RET disrupts cellular homeostasis, induces a transient redistribution of certain leukocytes, and results in transcriptional changes in PBMCs translating into systemic changes in response to RET. PMID:21437602

  14. Relationship between T cell subpopulations and the mitogen responsiveness and suppressor cell function of peripheral blood mononuclear cells in normal individuals.

    PubMed Central

    Victorino, R M; Hodgson, H J

    1980-01-01

    A simultaneous analysis was made of numbers and proportions of T cell subsets (T mu and T gamma cells), lymphocyte responsiveness to non-specific mitogens in vitro and 'short-lived suppressor cell activity' in peripheral blood mononuclear cells (PBMC) of normal individuals. No correlation was found between either T gamma or T mu cells and the 'short-lived suppressor cell activity', suggesting that suppression in this system is not a reflection of quantitative alteration in these subsets. However, a highly significant positive correlation was found between numbers of T mu cells and PBMC responses to the mitogens phytohaemagglutinin, concanavalin A and pokeweek mitogen. This may reflect either a helper effect of T mu cells on lymphocyte proliferation in response to mitogens or the presence of the majority of mitogen-responsive cells within this subpopulation. As in normal individuals lymphocyte responsiveness correlates with the number of circulating T mu cells, it is possible that a reduction in these cells in disease states may contribute to defects in cell-mediated immunity. PMID:6452237

  15. Omega-3 fatty acids, EPA and DHA induce apoptosis and enhance drug sensitivity in multiple myeloma cells but not in normal peripheral mononuclear cells.

    PubMed

    Abdi, J; Garssen, J; Faber, J; Redegeld, F A

    2014-12-01

    The n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to enhance the effect of chemotherapeutic drugs in clinical studies in cancer patients and to induce apoptotic tumor cell death in vitro. Until now, EPA and DHA have never been investigated in multiple myeloma (MM). Human myeloma cells (L363, OPM-1, OPM-2 and U266) and normal peripheral blood mononuclear cells were exposed to EPA and DHA, and effects on mitochondrial function and apoptosis, caspase-3 activation, gene expression and drug toxicity were measured. Exposure to EPA and DHA induced apoptosis and increased sensitivity to bortezomib in MM cells. Importantly, they did not affect viability of normal human peripheral mononuclear cells. Messenger RNA expression arrays showed that EPA and DHA modulated genes involved in multiple signaling pathways including nuclear factor (NF) κB, Notch, Hedgehog, oxidative stress and Wnt. EPA and DHA inhibited NFκB activity and induced apoptosis through mitochondrial perturbation and caspase-3 activation. Our study suggests that EPA and DHA induce selective cytotoxic effects in MM and increase sensitivity to bortezomib and calls for further exploration into a potential application of these n-3 polyunsaturated fatty acids in the therapy of MM. PMID:25277647

  16. Peripheral Blood Mononuclear Cell Proteome Changes in Patients with Myelodysplastic Syndrome

    PubMed Central

    Majek, Pavel; Cermak, Jaroslav; Dyr, Jan E.

    2015-01-01

    Our aim was to search for proteome changes in peripheral blood mononuclear cells (PBMCs) of MDS patients with refractory cytopenia with multilineage dysplasia. PBMCs were isolated from a total of 12 blood samples using a Histopaque-1077 solution. The proteins were fractioned, separated by 2D SDS-PAGE (pI 4–7), and double-stained. The proteomes were compared and statistically processed with Progenesis SameSpots; then proteins were identified by nano-LC-MS/MS. Protein functional association and expression profiles were analyzed using the EnrichNet application and Progenesis SameSpots hierarchical clustering software, respectively. By comparing the cytosolic, membrane, and nuclear fractions of the two groups, 178 significantly (P < 0.05, ANOVA) differing spots were found, corresponding to 139 unique proteins. Data mining of the Reactome and KEGG databases using EnrichNet highlighted the possible involvement of the identified protein alterations in apoptosis, proteasome protein degradation, heat shock protein action, and signal transduction. Western blot analysis revealed underexpression of vinculin and advanced fragmentation of fermitin-3 in MDS patients. To the best of our knowledge, this is the first time that proteome changes have been identified in the mononuclear cells of MDS patients. Vinculin and fermitin-3, the proteins involved in cell adhesion and integrin signaling, have been shown to be dysregulated in MDS. PMID:25969835

  17. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease

    PubMed Central

    Soman, Kizhake V.; Zago, Maria P.; Koo, Sue-Jie; Spratt, Heidi; Stafford, Susan; Blell, Zinzi N.; Gupta, Shivali; Nuñez Burgos, Julio; Barrientos, Natalia; Brasier, Allan R.

    2016-01-01

    Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30–40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting

  18. Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

    PubMed

    Garg, Nisha Jain; Soman, Kizhake V; Zago, Maria P; Koo, Sue-Jie; Spratt, Heidi; Stafford, Susan; Blell, Zinzi N; Gupta, Shivali; Nuñez Burgos, Julio; Barrientos, Natalia; Brasier, Allan R; Wiktorowicz, John E

    2016-02-01

    Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive

  19. Pancratistatin induces apoptosis in clinical leukemia samples with minimal effect on non-cancerous peripheral blood mononuclear cells

    PubMed Central

    2010-01-01

    Background Pancratistatin, a natural compound extracted from Hymenocallis littoralis, can selectively induce apoptosis in several cancer cell lines. In this ex vivo study, we evaluated the effect of pancratistatin on peripheral blood mononuclear cells obtained from 15 leukemia patients prior to clinical intervention of newly diagnosed patients, as well as others of different ages in relapse and at various disease progression states. Results Mononuclear cells from healthy volunteers and leukemia patients were exposed to 1 μM pancratistatin for up to 48 h. Irrespective of leukemia type, pancratistatin induced apoptosis in the leukemic samples, with minimal effects on non-cancerous peripheral blood mononuclear control cells. Conclusion Our results show that pancratistatin is an effective and selective anti-cancer agent with potential for advancement to clinical trials. PMID:20205924

  20. TGF-β receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation.

    PubMed

    Lee, Yong-Ung; de Dios Ruiz-Rosado, Juan; Mahler, Nathan; Best, Cameron A; Tara, Shuhei; Yi, Tai; Shoji, Toshihiro; Sugiura, Tadahisa; Lee, Avione Y; Robledo-Avila, Frank; Hibino, Narutoshi; Pober, Jordan S; Shinoka, Toshiharu; Partida-Sanchez, Santiago; Breuer, Christopher K

    2016-07-01

    Stenosis is a critical problem in the long-term efficacy of tissue-engineered vascular grafts (TEVGs). We previously showed that host monocyte infiltration and activation within the graft drives stenosis and that TGF-β receptor 1 (TGF-βR1) inhibition can prevent it, but the latter effect was attributed primarily to inhibition of mesenchymal cell expansion. In this study, we assessed the effects of TGF-βR1 inhibition on the host monocytes. Biodegradable TEVGs were implanted as inferior vena cava interposition conduits in 2 groups of C57BL/6 mice (n = 25/group): unseeded grafts and unseeded grafts with TGF-βR1 inhibitor systemic treatment for the first 2 wk. The TGF-βR1 inhibitor treatment effectively improved TEVG patency at 6 mo compared to the untreated control group (91.7 vs. 48%, P < 0.001), which is associated with a reduction in classic activation of mononuclear phagocytes. Consistent with these findings, the addition of rTGF-β to LPS/IFN-γ-stimulated monocytes enhanced secretion of inflammatory cytokines TNF-α, IL-12, and IL-6; this effect was blocked by TGF-βR1 inhibition (P < 0.0001). These findings suggest that the TGF-β signaling pathway contributes to TEVG stenosis by inducing classic activation of host monocytes. Furthermore, blocking monocyte activation by TGF-βR1 inhibition provides a viable strategy for preventing TEVG stenosis while maintaining neotissue formation.-Lee, Y.-U., de Dios Ruiz-Rosado, J., Mahler, N., Best, C. A., Tara, S., Yi, T., Shoji, T., Sugiura, T., Lee, A. Y., Robledo-Avila, F., Hibino, N., Pober, J. S., Shinoka, T., Partida-Sanchez, S., Breuer, C. K. TGF-β receptor 1 inhibition prevents stenosis of tissue-engineered vascular grafts by reducing host mononuclear phagocyte activation. PMID:27059717

  1. The role of Card9 overexpression in peripheral blood mononuclear cells from patients with aseptic acute pancreatitis.

    PubMed

    Yang, Zhi-wen; Weng, Cheng-zhao; Wang, Jing; Xu, Ping

    2016-03-01

    Activated mononuclear cells are an early event in the course of severe acute pancreatitis (SAP). To date, the molecular mechanism triggering peripheral blood mononuclear cells (PBMCs) is poorly understood. The aim of this paper was to determine the potential role of Card9 in SAP. We collected data from 72 subjects between January 2013 and June 2014. Subsequently, PBMCs were isolated on day 1, 3 and 5 of pancreatitis. Immunofluorescence staining, quantitative real-time PCR, Western blotting, immunoprecipitation and ELISA were used to determine the role of Card9 in SAP. Microbial culture showed that SAP patients at the early period did not develop any bacteria and fungi infection. Card9 expression in SAP patients was higher than that in mild acute pancreatitis and volunteer healthy controls, up to the peak on day 1. The monocyte-derived cytokines interleukin (IL)-17, IL-1β, IL-6 and tumour necrosis factor-α mediated by the induction of Card9 markedly increased in SAP patients compared with the control group. Furthermore, the inducible formation of Card9-Bcl10 complex was found in PBMCs, which may be involved in nuclear factor kappa B (NF-κB) and p38 activation in SAP. Receiver operating characteristic curve indicated that Card9 levels had a high sensitivity of 87.5% and specificity of 67.7%, showing the close correlation with SAP patients. Card9 overexpression was firstly found in aseptic SAP, which may be played an important role in NF-κB and p38 activation in PBMCs. It also provided the new insights into therapeutic interventions by targeting monocytes activation in SAP patients. PMID:26893103

  2. Interleukin-5 is the predominant cytokine produced by peripheral blood mononuclear cells in alveolar echinococcosis.

    PubMed Central

    Sturm, D; Menzel, J; Gottstein, B; Kern, P

    1995-01-01

    An involvement of cellular immunity in alveolar echinococcosis is strongly suggested by the intense granulomatous infiltrations observed around the hepatic parasite lesions. However, the basis of cellular immunoregulation in patient with alveolar echinococcosis is poorly understood. The present report shows a comparative analysis of lymphoid cell function in peripheral blood mononuclear cells (PBMC) of 16 patients with alveolar echinococcosis and of healthy individuals. Our in vitro restimulation studies with crude Echinococcus multilocularis antigen demonstrated that PBMC from patients with alveolar echinococcosis were responsive to challenge with parasitic antigen as measured by lymphoid cell proliferation. In this system, we also evaluated cytokine expression at the gene and protein levels after stimulation with E. multilocularis antigen. Analysis of cytokine mRNA expression revealed distinct patterns of cytokine expression in patients and normal donors. By using reverse transcriptase PCR, we could demonstrate that the TH1 cytokine transcripts interleukin-2 (IL-2) and gamma interferon (IFN-gamma) are present in PBMC from patients with alveolar echinococcosis. Moreover, it was found that stimulation with E. multilocularis antigen induced or enhanced the expression of the TH2 cytokine IL-3, IL-4, IL-10, and especially IL-5 mRNAs in PBMC from 13 of 16 patients with alveolar echinococcosis. Two patients who were examined after radical surgery, as well as another patient with a stable course of the disease under continuous chemotherapy, were not able to generate the same pattern of cytokine response and had no evidence of IL-5 mRNA synthesis. In contrast to the frequent expression of TH2 cytokine mRNAs observed in patients with alveolar echinococcosis, PBMC cultures from normal donors showed prominent IL-2 and IFN-gamma mRNA expression but weak IL-3, IL-4, and IL-10 mRNA expression. Most interestingly, IL-5 mRNA was substantially absent in PBMC from healthy

  3. Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure

    PubMed Central

    Ariemma, Fabiana; Cimmino, Ilaria; Bruzzese, Dario; Scerbo, Roberta; Picascia, Stefania; D’Esposito, Vittoria; Beguinot, Francesco; Formisano, Pietro

    2016-01-01

    Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly

  4. Human Peripheral Blood Mononuclear Cell Function and Dendritic Cell Differentiation Are Affected by Bisphenol-A Exposure.

    PubMed

    Camarca, Alessandra; Gianfrani, Carmen; Ariemma, Fabiana; Cimmino, Ilaria; Bruzzese, Dario; Scerbo, Roberta; Picascia, Stefania; D'Esposito, Vittoria; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental pollutants, including endocrine disruptor chemicals (EDCs), interfere on human health, leading to hormonal, immune and metabolic perturbations. Bisphenol-A (BPA), a main component of polycarbonate plastics, has been receiving increased attention due to its worldwide distribution with a large exposure. In humans, BPA, for its estrogenic activity, may have a role in autoimmunity, inflammatory and allergic diseases. To this aim, we assessed the effect of low BPA doses on functionality of human peripheral blood mononuclear cells (PBMCs), and on in vitro differentiation of dendritic cells from monocytes (mDCs). Fresh peripheral blood samples were obtained from 12 healthy adult volunteers. PBMCs were left unstimulated or were activated with the mitogen phytohemagglutinin (PHA) or the anti-CD3 and anti-CD28 antibodies and incubated in presence or absence of BPA at 0.1 and 1nM concentrations. The immune-modulatory effect of BPA was assessed by evaluating the cell proliferation and the levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-13 (IL-13) secreted by PBMCs. mDCs were differentiated with IL-4 and GC-CSF with or without BPA and the expression of differentiation/maturation markers (CD11c, CD1a, CD86, HLA-DR) was evaluated by flow cytometry; furthermore, a panel of 27 different cytokines, growth factors and chemokines were assayed in the mDC culture supernatants. PBMCs proliferation significantly increased upon BPA exposure compared to BPA untreated cells. In addition, a significant decrease in IL-10 secretion was observed in PBMCs incubated with BPA, either in unstimulated or mitogen-stimulated cells, and at both 0.1 and 1nM BPA concentrations. Similarly, IL-13 was reduced, mainly in cells activated by antiCD3/CD28. By contrast, no significant changes in IFN-γ and IL-4 production were found in any condition assayed. Finally, BPA at 1nM increased the density of dendritic cells expressing CD1a and concomitantly

  5. Evaluation of apoptosis induction in human peripheral blood mononuclear cells and synovial cells in patients with rheumatoid arthritis.

    PubMed

    Demian, Soheir R; Abo-Shousha, Seham A; Sultan, Hussein E; Zarka, Wael El

    2005-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory destructive disease involving the joint and characterized by T-lymphocyte accumulation within the synovial compartment. It is dominated by the presence of macrophages, plasma cells and synovial fibroblasts which are the main pathogenic factors leading to the destruction of bone and cartilage. The survival of these cells may be promoted by inadequate apoptosis leading to synovial hyperplasia. So, the aim of the present study was to evaluate the apoptosis levels before and after induction of apoptosis using anti-Fas mAb, both in peripheral blood (PB) and synovial fluid (SF) infiltrating mononuclear cells (MCs) of patients with RA. CD4+ T cell subsets and cell survival assays were also done to investigate correlations between these parameters. The study was conducted on 15 patients with RA, 10 individual volunteers as a control group and 10 patients with osteoarthritis (OA) as a control group for SF evaluations (have defective Fas expression on their cells). Results of this work revealed that in vitro induction of apoptosis by anti-Fas mAb resulted in increase of: percent (%) reduction of cell viability in PBMCs and SFMCs, % reduction of CD4+ T cell subsets and apoptotic cell % in all studied groups than before induction. The increase in the three parameters is only significant in SF of RA group compared to PB while it is non significant in OA group due to the defective Fas expression on OA cells. Our results also showed a significant positive correlation between CD4+ T cell and viability percentages before induction of apoptosis in SF of RA and between apoptosis levels and CD4+ T cell percentage after induction of apoptosis in the SF of RA group. In conclusion, activated T cells infiltrating SF of RA patients have functional Fas antigen which enable them to undergo in vitro apoptosis using anti-Fas mAb. The cytotoxicity of which is more specific to local lesion such as SF of RA patients suggesting that local

  6. Impact of fexofenadine, osthole and histamine on peripheral blood mononuclear cell proliferation and cytokine secretion.

    PubMed

    Karolina Kordulewska, Natalia; Kostyra, Elżbieta; Matysiewicz, Michał; Cieślińska, Anna; Jarmołowska, Beata

    2015-08-15

    This paper compares results of peripheral blood mononuclear cell (PBMC) incubation with fexofenadine (FXF) and osthole. FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. To our best knowledge, this is the first comparative study on FXF, osthole and histamine cytokine secretion and cytotoxicity in PBMC in vitro cultures using cell proliferation ELISA BrdU. The cultures were treated 12, 42, 48 and 72h with FXF and osthole at 150, 300 and 450ng/ml concentrations and histamine at 50, 100 and 200ng/ml. Our study results confirm that FXF, osthole and histamine exert no cytotoxic effect on PBMCs and that IL-6, IL-10 and TNF-α cytokine secretion following osthole cell stimulation was similar to that by FXF stimulation.This confirms our hypothesis that osthole is a natural histamine antagonist, and can therefore be beneficially applied in antihistamine treatment. PMID:26048308

  7. Integrated Bioinformatics Approach Reveals Crosstalk Between Tumor Stroma and Peripheral Blood Mononuclear Cells in Breast Cancer.

    PubMed

    He, Lang; Wang, Dan; Wei, Na; Guo, Zheng

    2016-01-01

    Breast cancer is now the leading cause of cancer death in women worldwide. Cancer progression is driven not only by cancer cell intrinsic alterations and interactions with tumor microenvironment, but also by systemic effects. Integration of multiple profiling data may provide insights into the underlying molecular mechanisms of complex systemic processes. We performed a bioinformatic analysis of two public available microarray datasets for breast tumor stroma and peripheral blood mononuclear cells, featuring integrated transcriptomics data, protein-protein interactions (PPIs) and protein subcellular localization, to identify genes and biological pathways that contribute to dialogue between tumor stroma and the peripheral circulation. Genes of the integrin family as well as CXCR4 proved to be hub nodes of the crosstalk network and may play an important role in response to stroma-derived chemoattractants. This study pointed to potential for development of therapeutic strategies that target systemic signals travelling through the circulation and interdict tumor cell recruitment. PMID:27039717

  8. The role of mononuclear cell tissue factor and inflammatory cytokines in patients with chronic thromboembolic pulmonary hypertension.

    PubMed

    Yang, Minxia; Deng, Chaosheng; Wu, Dawen; Zhong, Zhanghua; Lv, Xiaoting; Huang, Zhihua; Lian, Ningfang; Liu, Kaixiong; Zhang, Qiaoxian

    2016-07-01

    Thrombosis and inflammation are two major factors underlying chronic thromboembolic pulmonary hypertension (CTEPH). Tissue factor (TF), C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) may play critical roles in the process of CTEPH thrombosis and pulmonary vascular remodeling. Ten patients with a confirmed diagnosis of CTEPH, 20 patients with acute pulmonary thromboembolism and 15 patients with other types of pulmonary hypertension were enrolled in this study, along with 20 healthy subjects as the control group. The immunoturbidimetric method was used to determine the plasma content of CRP. The plasma levels of TNF-α, MCP-1, and TF antigen were measured by an enzyme-linked immunosorbent assay, and TF activity was measured by the chromogenic substrate method. Percoll density gradient centrifugation was used to separate peripheral blood mononuclear cells from plasma. The level of monocyte TF mRNA was examined by reverse transcriptase-polymerase chain reaction. The correlations between all indices described above were analyzed. In CTEPH patients, the expression of CRP, TNF-α, and MCP-1 was significantly higher than that in controls (P < 0.05). The levels of TF activity, TF antigen, and TF mRNA in monocyte cells were increased in CTEPH patients when compared with control subjects, but only the TF antigen and TF mRNA levels were significantly different (P < 0.05). In CTEPH patients, levels of CRP, MCP-1, and TNF-α significantly correlated with the level of TF antigen in plasma. TF gene expression was increased in patients with CTEPH, suggesting that blood-borne TF mainly comes from mononuclear cells. TF expression significantly correlated with levels of CRP, TNF-α and MCP-1. These factors may play an important role in the development of CTEPH via the inflammation-coagulation-thrombosis cycle. PMID:26667361

  9. THE CONTRIBUTION OF TYRO3 FAMILY RECEPTOR TYROSINE KINASES TO THE HETEROGENEITY OF APOPTOTIC CELL UPTAKE BY MONONUCLEAR PHAGOCYTES

    PubMed Central

    Curtis, Jeffrey L.; Todt, Jill C.; Hu, Bin; Osterholzer, John J.; Freeman, Christine M.

    2014-01-01

    Mononuclear phagocytes comprise a mobile, broadly dispersed and highly adaptable system that lies at the very epicenter of host defense against pathogens and the interplay of the innate and adaptive arms of immunity. Understanding the molecular mechanisms that control the response of mononuclear phagocytes to apoptotic cells and the anti-inflammatory consequences of that response is an important goal with implications for multiple areas of biomedical sciences. This review details current understanding of the heterogeneity of apoptotic cell uptake by different members of the mononuclear phagocyte family in humans and mice. It also recounts the unique role of the Tyro3 family of receptor tyrosine kinases, best characterized for Mertk, in the signal transduction leading both to apoptotic cell ingestion and the anti-inflammatory effects that result. PMID:19273223

  10. Bone marrow and bone marrow derived mononuclear stem cells therapy for the chronically ischemic myocardium

    SciTech Connect

    Waksman, Ron; Baffour, Richard

    2003-09-01

    Bone marrow stem cells have been shown to differentiate into various phenotypes including cardiomyocytes, vascular endothelial cells and smooth muscle. Bone marrow stem cells are mobilized and home in to areas of injured myocardium where they are involved in tissue repair. In addition, bone marrow secretes multiple growth factors, which are essential for angiogenesis and arteriogenesis. In some patients, these processes are not enough to avert clinical symptoms of ischemic disease. Therefore, in vivo administration of an adequate number of stem cells would be a significant therapeutic advance. Unfractionated bone marrow derived mononuclear stem cells, which contain both hematopoietic and nonhematopoietic cells may be more appropriate for cell therapy. Studies in animal models suggest that implantation of different types of stem cells improve angiogenesis and arteriogenesis, tissue perfusion as well as left ventricular function. Several unanswered questions remain. For example, the optimal delivery approach, dosage and timing of the administration of cell therapy as well as durability of improvements need to be studied. Early clinical studies have demonstrated safety and feasibility of various cell therapies in ischemic disease. Randomized, double blind and placebo-controlled clinical trials need to be completed to determine the effectiveness of stem cell.

  11. The effect of catechol on human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Bukowska, Bożena; Michałowicz, Jaromir; Marczak, Agnieszka

    2015-01-01

    Catechol also known as pyrocatechol or 1,2-dihydroxybenzene is formed endogenously in the organism from neurotransmitters including adrenaline, noradrenaline, and dopamine. It is also a metabolite of many drugs like DOPA, isoproterenol or aspirin and it is also formed in the environment during transformation of various xenobiotics. We evaluated in vitro the effect of catechol on the structure and function of human peripheral blood mononuclear cells (PBMCs). The cells were incubated with xenobiotic at concentration range from 2 to 500μg/mL for 1h. Human blood mononuclear cells were obtained from leucocyte-platelet buffy coat taken from healthy donors in the Blood Bank of Łódź, Poland. Using flow cytometry we have evaluated necrotic, apoptotic and morphological changes in PBMCs incubated with catechol. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, protein carbonylation and lipid peroxidation in the cells studied. The compound studied provoked necrotic (from 250μg/mL), apoptotic (from 100μg/mL), and morphological changes (from 250μg/mL) in the incubated cells. We have also noted that catechol decreased H2DCF oxidation at 2 and 10μg/mL but at higher concentrations of 250 and 500μg/mL it caused statistically significant increase in the oxidation of this probe. We also observed an increase in lipid peroxidation (from 250μg/mL) and protein carbonylation (from 50μg/mL) of PBMCs. It was observed that catechol only at high concentrations was capable of inducing changes in PBMCs. The obtained results clearly showed that catechol may induce change in PBMCs only in the caste of poisoning with this compound. PMID:25528409

  12. Maternal Gene Expression Profiling During Pregnancy And Preeclampsia In Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Rajakumar, Augustine; Tianjiao, Chu; Handley, Daniel E.; Bunce, Kimberly D.; Burke, Brian; Hubel, Carl A.; Jeyabalan, Arundhathi; Peters, David G.

    2010-01-01

    Preeclampsia is a major obstetrical complication affecting maternal and fetal health. While it is clear that there is a substantial placental contribution to preeclampsia pathogenesis, the maternal contribution is less well characterized. We therefore performed a genome wide transcriptome analysis to explore disease-associated changes in maternal gene expression patterns in peripheral blood mononuclear cells (PBMCs). Methods Preeclampsia was defined as gestational hypertension, proteinuria and hyperurecimia. Total RNA was isolated from PBMCs obtained from women with uncomplicated pregnancies (n=5) and women with preeclamptic pregnancies (n=5). Gene expression analysis was carried out using Agilent oligonucleotide microarrays. Biological pathway analysis was undertaken using Ingenuity Pathway Analysis software. Quantitative real time PCR (QRTPCR) was performed to validate the gene expression changes of selected genes in normotensive and preeclamptic patients (n=12 each). Results We identified a total of 368 genes that were differentially expressed in women with preeclampsia compared to normal controls with false discovery rate (FDR) controlled at 10%. In follow up experiments we further analyzed the expression levels of a number of genes that were identified as altered by the microarray data including survivin (BIRC5), caveolin (CAV1), GATA binding protein-1 (GATA1), signal tranducer and activator of transcription 1 (STAT1), E2F transcription factor-1 (E2F1), fibronectin-1 (FN1), interleukin-4 (IL-4), matrix metalloprotease-9 (MMP-9) and WAP four-disulfide domain protein (WFDC-1) by QRTPCR. Additionally we performed immunoblot analysis and zymography to verify some of these candidate genes at the protein level. Computational analysis of gene function identified an anti-proliferative and altered immune function cellular phenotype in severe preeclamptic samples. Conclusions We have characterized the genome-wide mRNA expression changes associated with preeclampsia

  13. Activation of RAGE induces elevated O2- generation by mononuclear phagocytes in diabetes.

    PubMed

    Ding, Yong; Kantarci, Alpdogan; Hasturk, Hatice; Trackman, Philip C; Malabanan, Alan; Van Dyke, Thomas E

    2007-02-01

    Oxidative stress has been found to play a role in the pathogenesis of diabetic complications. The aim of this study was to define the oxidative burst of diabetic monocytes to characterize the phenotype associated with poor diabetic control. Superoxide (O(2)(-)) is the first molecule generated during the respiratory burst of phagocytes by NADPH oxidase, and its generation by monocytes from 26 controls and 34 diabetic subjects was evaluated in this study. Under resting states or stimulation by PMA or opsonized zymosan, diabetic monocytes produce significantly more O(2)(-) than nondiabetic cells. The increased O(2)(-) generation was found to be correlated with glycemic control (glycated hemoglobin) of patients. To clarify the effects of hyperglycemia on O(2)(-) generation, normal human monocytes were treated with receptor for advanced glycation endproducts (RAGE) ligands (AGE protein and S100B) or high glucose media before stimulation. RAGE ligands and high glucose concentration increased O(2)(-) generation from human mononuclear phagocytes. RAGE ligands, specifically and potently, increased O(2)(-) generation from mononuclear phagocytes, and high-glucose effects were associated with correspondingly increased osmotic pressure. Differentiated THP-1 cells, from a human monocytic cell line, were used as a model of human monocytes to study the effects of S100B, the RAGE ligand. It was confirmed that RAGE is involved in the priming of O(2)(-) generation by S100B. This study demonstrates that RAGE ligands can contribute significantly to the hyper-responsive phenotype of diabetic monocytes, which might be reversible by blocking the RAGE or controlling the presence of RAGE ligands by controlling hyperglycemia. PMID:17095613

  14. Correlation of the content of hepatitis B core antigen in peripheral blood mononuclear cells with HBV virus load.

    PubMed

    Peng, Yanzhong; Xiong, Xiaojia; Tong, Xindeng; Li, Min; Yang, Xifei; Hu, Guoxin; Geng, Yijie; Li, Jianxin

    2016-06-01

    The dysfunction of peripheral blood mononuclear cell (PBMC) plays an important role in hepatitis B virus (HBV) chronic infection and tolerance. This study is aimed to explore the changes of expression and distribution of Hepatitis B virus core antigen (HBcAg) in the PBMCs of patients infected with chronic hepatitis B virus by using confocal laser scanning microscopy (CLSM). The levels of HBcAg in PBMCs were correlated with the HBV load in serum, and the change of HBcAg distribution in different PBMC organelles may represent various HBV infection states. HBcAg was mainly distributed in the nuclei of PBMC in the cases of immune tolerance and no inflammatory activity. Taken together, our data suggest that the measurement of HBcAg and its distribution in PBMCs using CLSM may serve as an alternative approach to monitor HBV load and the immune states of HBV infection with ease of using and improved sensitivity. PMID:26680298

  15. Limits of Peripheral Blood Mononuclear Cells for Gene Expression-Based Biomarkers in Juvenile Idiopathic Arthritis

    PubMed Central

    Wong, Laiping; Jiang, Kaiyu; Chen, Yanmin; Hennon, Teresa; Holmes, Lucy; Wallace, Carol A.; Jarvis, James N.

    2016-01-01

    Juvenile Idiopathic Arthritis (JIA) is one of the most common chronic disease conditions affecting children in the USA. As with many rheumatic diseases, there is growing interest in using genomic technologies to develop biomarkers for either diagnosis or to guide treatment (“personalized medicine”). Here, we explore the use of gene expression patterns in peripheral blood mononuclear cells (PBMC) as a first step approach to developing such biomarkers. Although PBMC carry many theoretical advantages for translational research, we have found that sample heterogeneity makes RNASeq on PBMC unsuitable as a first-step method for screening biomarker candidates in JIA. RNASeq studies of homogeneous cell populations are more likely to be useful and informative. PMID:27385437

  16. Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

    NASA Astrophysics Data System (ADS)

    Martinakova, Z.; Horilova, J.; Lajdova, I.; Marcek Chorvatova, A.

    2014-12-01

    Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

  17. Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism.

    PubMed

    Liu, Jing; Brzeszczynska, Joanna; Samuel, Kay; Black, Jim; Palakkan, Anwar; Anderson, Richard A; Gallagher, Ronald; Ross, James A

    2015-11-01

    The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function. PMID:26256888

  18. The mononuclear phagocyte system and lymphocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This book chapter outlines the cellular processes that are activated and interact during host immune responses including: (a) phagocytosis and antigen presentation by cells of the mononuclear phagocyte system (monocytes, macrophages, and dendritic cells), and (b) cell-mediated and humoral immunity ...

  19. Excess apoptosis of mononuclear cells contributes to the depressed cytomegalovirus-specific immunity in HIV-infected patients on HAART

    SciTech Connect

    Weinberg, Adriana . E-mail: Adriana.Weinberg@uchsc.edu; Jesser, Renee D.; Edelstein, Charles L.; Bill, Jerome R.; Wohl, David A.

    2004-12-05

    HIV-infected patients on highly active antiretroviral therapy (HAART) have persistently decreased cytomegalovirus (CMV)-specific proliferative responses [lymphocyte proliferation assay (LPA)] in spite of increases in CD4+ T cell counts. Here we demonstrate an association between apoptosis of unstimulated peripheral blood mononuclear cells (uPBMC) and decreased CMV-LPA. HAART recipients had more apoptosis of uPBMC than controls when measured by caspases 3, 8, and 9 activities and by annexin V binding. Patients with undetectable HIV replication maintained significantly higher apoptosis of CD4+ and CD14+ cells compared to controls. CMV-LPA decreased with higher apoptosis of uPBMC in patients only. This association was independent of CD4+ cell counts or HIV replication. Furthermore, rescuing PBMC from apoptosis with crmA, but not with TRAIL- or Fas-pathway blocking agents or with other caspase inhibitors, increased CMV-LPA in HAART recipients. This effect was not observed in uninfected controls, further indicating that the down regulatory effect of apoptosis on cell-mediated immunity (CMI) was specifically associated with the HIV-infected status.

  20. Human mononuclear cell function after 4 degrees C storage during 1-G and microgravity conditions of spaceflight

    NASA Technical Reports Server (NTRS)

    Meehan, R.; Taylor, G.; Lionetti, F.; Neale, L.; Curren, T.

    1989-01-01

    Future space missions of long duration may require that autologous leukocytes be stored in flight for infusion to restore normal immune competence in crewmembers. Peripheral blood mononuclear cells (PBMNCs), as leukocyte concentrates in autologous plasma and 2% dextrose, were stored in the microgravity conditions provided by the U.S. Space Shuttle Columbia (Mission 61-C). Activity of PBMNC after space flight was compared with that from a series of preflight ground control experiments, which demonstrated in culture a progressive daily loss in mitogen-stimulated protein synthesis at 24 h and thymidine uptake at 72 h after storage for 7 d at 4 degrees C. Post-storage viabilities were at least 90% as determined by trypan dye exclusion. A progressive reduction in the percentage of PBMNC expressing cell-surface phenotype markers, which was similar for monocytes, B cells, and T-cell subsets, also occurred after storage. The ability of PBMNC, stored for 8 d in Columbia's middeck, to become activated and proliferate in vitro was similar to that of cells that remained in identical flight lockers on the ground as 1-G controls, thus indicating that PBMNCs were not adversely affected by storage under microgravity conditions.

  1. Productive Infection of Human Peripheral Blood Mononuclear Cells by Feline Immunodeficiency Virus: Implications for Vector Development

    PubMed Central

    Johnston, James; Power, Christopher

    1999-01-01

    Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V1CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 101.3 to 102.1 50% tissue culture infective doses/106 cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy. PMID:9971834

  2. Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts.

    PubMed

    Ueno, Koji; Takeuchi, Yuriko; Samura, Makoto; Tanaka, Yuya; Nakamura, Tamami; Nishimoto, Arata; Murata, Tomoaki; Hosoyama, Tohru; Hamano, Kimikazu

    2016-01-01

    The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers. PMID:27329845

  3. Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs.

    PubMed

    Tani, Hiroyuki; Nabetani, Tomoyo; Sasai, Kazumi; Baba, Eiichiroh

    2005-04-01

    The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism. PMID:15876785

  4. Treatment of refractory cutaneous ulcers with mixed sheets consisting of peripheral blood mononuclear cells and fibroblasts

    PubMed Central

    Ueno, Koji; Takeuchi, Yuriko; Samura, Makoto; Tanaka, Yuya; Nakamura, Tamami; Nishimoto, Arata; Murata, Tomoaki; Hosoyama, Tohru; Hamano, Kimikazu

    2016-01-01

    The purpose of this study was to confirm the therapeutic effects of mixed sheets consisting of peripheral blood mononuclear cells (PBMNCs) and fibroblasts on cutaneous skin ulcers. Vascular endothelial growth factor (VEGF) secretion in mixed cell sheets was much higher than in PBMNCs and fibroblasts. Concerning the mechanism, transforming growth factor beta 1 and platelet-derived growth factor BB secreted from PBMNCs enhanced VEGF production in fibroblasts. In wounds created on the backs of diabetic mice, the therapeutic effect of mixed cell sheets was similar to that of daily treatment with trafermin, a recombinant human basic fibroblast growth factor. Although abnormal granulation tissue and inflammatory cell infiltration were observed in trafermin-treated wounds, the transplantation of mixed cell sheets resulted in the natural anatomy of subcutaneous tissues. The expression patterns of identical wound-healing factors in wounds were different between mixed sheet-transfected and trafermin-treated animals. Because mixed cell sheets transplanted into full-thickness skin defects were eliminated in hosts by day 21 in syngeneic transplantation models, allogeneic transplantation was performed using mice with different genetic backgrounds. The wound-healing rates were similar between the mixed cell sheet and trafermin groups. Our data indicated that mixed cell sheets represent a promising therapeutic material for cutaneous ulcers. PMID:27329845

  5. Human Mycobacterium tuberculosis-reactive CD4+ T-cell clones: heterogeneity in antigen recognition, cytokine production, and cytotoxicity for mononuclear phagocytes.

    PubMed Central

    Boom, W H; Wallis, R S; Chervenak, K A

    1991-01-01

    CD4+ T cells regulate the protective immune response which follows exposure to Mycobacterium tuberculosis by activating macrophages through the cytokines the CD4+ T cells secrete. In addition CD4+ T cells have been shown to be directly cytotoxic for antigen-pulsed mononuclear phagocytes (monocytes-macrophages). To explore the functional interaction between mycobacterial antigen-specific CD4+ T cells and mononuclear phagocytes further, CD4+ T-cell clones were derived from healthy purified protein derivative-positive individuals. Five T-cell clones were selected for detailed analysis. None responded to the purified recombinant or native mycobacterial antigens of 14, 19, 65, 71, and 30 (alpha-antigen/Ag6) kDa. However, the T-cell clones demonstrated heterogeneity in antigen recognition as measured by their Western blot (immunoblot) responses. Some T-cell clones made only interleukin 2, while others made only interleukin 4; all produced gamma interferon, although in differing amounts. Four of five T-cells clones were cytotoxic for purified protein derivative-pulsed monocytes at 1:1 and 10:1 effector-target cell ratios. When monocytes infected with live M. tuberculosis were used as targets, comparable levels of cytotoxicity were observed. The cytotoxicity was major histocompatibility complex class II restricted and inhibited by antibodies to ICAM-1 and LFA-1 and not by antibodies to tumor necrosis factor alpha, lymphotoxin, and gamma interferon. Cytotoxicity by CD4+ T cells for monocytes pulsed with mycobacterial antigens or infected with live M. tuberculosis is a common property of these cells and appears to be independent of the repertoire of lymphokines produced and not limited to recognition of defined mycobacterial heat shock proteins. Lysis of heavily infected mononuclear phagocytes may be one manner in which CD4+ T cells regulate host immune response to M. tuberculosis. PMID:1713198

  6. Comparative phenotypical analysis of B cells in fresh and cryopreserved mononuclear cells from blood and tissue of rhesus macaques.

    PubMed

    Klippert, Antonina; Neumann, Berit; Stahl-Hennig, Christiane

    2016-06-01

    Cryopreservation of peripheral blood mononuclear cells (PBMCs) is common for large clinical trials for which phenotypical characterization of lymphocytes is retrospectively performed in specialized core laboratories. It is therefore essential to assess the comparability between fresh and frozen samples. No side-by-side comparison of B and plasma cells of rhesus macaques (RM), which serve as useful models for several human diseases has been conducted until now. Hence, we performed an extensive comparative analysis between fresh and thawed mononuclear cells (MNCs) from blood and various tissues of healthy RM to analyze for the possible effects of cryopreservation on phenotype and functionality. Our data demonstrate that -80°C cryopreservation induces profound changes compared to fresh ex vivo-derived material. Percentages of B cells were stable in PBMCs, but were increased in all organs analyzed. The expression of CD27, a marker for differentiation between naïve and memory B cells, was massively reduced in PBMCs and MNC from organs with the most severe changes observed in cells from bone marrow (BM). Additionally, similar low percentages of CD27(+) memory B cells were detected in PBMCs and BM samples stored in liquid nitrogen. Therefore, cryopreservation is not suitable for the phenotypical and functional characterization of B cells. Further optimization of cryoconservation protocols monitoring the surface expression of CD27, which was identified as a marker for the quality of cryopreserved material of RM, will be essential. PMID:26970138

  7. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture.

    PubMed

    Majumdar, M; Ratho, R; Chawla, Y; Singh, M P

    2014-01-01

    The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs) were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA) and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001), even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET) technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures. PMID:24713904

  8. Interleukin-15 Constrains Mucosal T Helper 17 Cell Generation: Influence of Mononuclear Phagocytes

    PubMed Central

    Yu, Huifeng; Sui, Yongjun; Wang, Yichuan; Sato, Noriko; Frey, Blake; Xia, Zheng; Waldmann, Thomas A.; Berzofsky, Jay

    2015-01-01

    Interleukin (IL)-15 has multiple roles in innate and adaptive immunity, especially regarding CD8+ T cells and natural killer cells. However, the role of IL-15 in regulating differentiation of T helper cell subsets and mononuclear phagocytes (MPs) in different tissues in vivo is unknown. Here we report that IL-15 indirectly regulates Th17 but not other Th subsets in the intestinal lamina propria (LP), apparently through effects on MPs. Th17 cells in the LP were more prevalent in IL-15 KO mice than their wild-type counterparts, and less prevalent in IL-15 transgenic mice than their wild-type littermates, even co-caged. MPs from the LP of these mice were sufficient to mimic the in vivo finding in vitro by skewing of cocultured wild type OVA-specific CD4+ T cells. However, production of IL-15 or lack thereof by these MPs was not sufficient to explain the skewing, as addition or blockade of IL-15 in the cultures had no effect. Rather, a skewing of the relative proportion of CD11b+, CD103+ and double positive LP MP subsets in transgenic and KO could explain the differences in Th17 cells. Thus, IL-15 may influence MP subsets in the gut in a novel way that alters the frequency of LP Th17 cells. PMID:26600079

  9. Mononuclear phagocytes rapidly clear apoptotic epithelial cells in the proximal epididymis

    PubMed Central

    Smith, T. B.; Cortez-Retamozo, V.; Grigoryeva, L. S.; Hill, E.; Pittet, M. J.; Da Silva, N.

    2015-01-01

    SUMMARY We have shown previously that a network of mononuclear phagocytes (MPs) expressing macrophage and dendritic cell markers such as CD11c, F4/80 and CX3CR1, lines the base of the epididymal tubule. However, in the initial segment (IS) and only in that particular segment, epididymal MPs establish extremely close interactions with the epithelium by projecting slender dendrites between most epithelial cells. We undertook the present study to determine how epididymal phagocytes respond to the transient wave of apoptosis initiated by unilateral efferent duct ligation (EDL) in the epididymal epithelium. We show profound morphological and phenotypical changes restricted to the MPs populating the proximal epididymis following EDL. Within 48 h, a large subset of IS epithelial cells had entered an apoptotic state, visualized by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and CD11c+ and CX3CR1+ MPs readily engulfed TUNEL-positive cells and other debris. Despite the high levels of apoptosis and the rapid clearance of apoptotic cells occurring after EDL, the epithelium preserved its overall architecture and maintained tight junctions of the blood–epididymis barrier (BEB). The discovery of a functional population of MPs in the epididymal epithelium responsible for maintaining the integrity of the BEB raises further questions regarding the role of these cells in clearing defective epithelial cells in the steady-state epididymis, as well as pathogens and abnormal spermatozoa in the lumen. PMID:25082073

  10. Docosahexaenoic diet supplementation, exercise and temperature affect cytokine production by lipopolysaccharide-stimulated mononuclear cells.

    PubMed

    Capó, Xavier; Martorell, Miquel; Sureda, Antoni; Batle, Juan Miguel; Tur, Josep Antoni; Pons, Antoni

    2016-09-01

    Acute exercise induces changes in peripheral mononuclear cells' (PBMCs) capabilities to produce cytokines. The aim was to investigate the effect of docosahexaenoic acid (DHA) diet supplementation on cytokine production, by lipopolysaccharide (LPS)-stimulated PBMCs after exercise, and the in vitro influence of temperature. Fifteen male soccer players were randomly assigned to a placebo or an experimental group. The experimental group consumed an almond-based beverage enriched with DHA (1.16 g DHA/day) for 8 weeks, whereas the placebo group consumed a similar non-enriched beverage. Blood samples were taken before and after the nutritional intervention in basal conditions and 2 h after acute exercise. Nutritional intervention significantly increased the DHA content in erythrocytes only in experimental group (from 34 ± 3.6 to 43 ± 3.6 nmols DHA/10(9) erythrocytes). Exercise significantly increased Toll-like receptor 4 (TLR4) in PBMCs but only in the placebo group (203 %). Exercise also significantly increased IL6, IL8, VEGF, INFγ, TNFα, IL1α, IL1β, MCP1, and EGG production rates by LPS-stimulated PBMCs, and this response was attenuated by DHA supplementation. Temperature but not DHA also affected the pattern of cytokine production increasing IL6, IL8, IL1β, and MCP1 synthesis. The higher change was evidenced in IL1β increasing the production rate at 39.5 °C from 3.19 ± 0.77 to 22.4 ± 6.1 pg/h 10(6) PBMC in placebo and from 2.36 ± 0.11 to 10.6 ± 0.38 pg/h 10(6) PBMC in the supplemented group. The profile of affected cytokines differs between temperature and exercise, suggesting a different PBMC activation pathway. DHA diet supplementation only attenuated cytokine production after exercise and not that induced by temperature. PMID:27139422

  11. Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation

    PubMed Central

    Natan, Dity; Nagler, Arnon; Arav, Amir

    2009-01-01

    Background We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. Methodology/Principal Findings We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs) derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34+-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%–91%. The total number of CD34+-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4×104±4.7, 3.49×104±6 and 6.31×104±12.27 cells, respectively, and 31±25.15, 47±45.8 and 23.44±13.3 colonies, respectively). Conclusions This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells. PMID:19381290

  12. Inverse relationship of tumors and mononuclear cell leukemia infiltration in the lungs of F344 rats

    SciTech Connect

    Lundgren, D.L.; Griffith, W.C.; Hahn, F.F.

    1995-12-01

    In 1970 and F344 rat, along with the B6C3F{sub 1} mouse, were selected as the standard rodents for the National Cancer Institute Carcinogenic Bioassay program for studies of potentially carcinogenic chemicals. The F344 rat has also been used in a variety of other carcinogenesis studies, including numerous studies at ITRI. A major concern to be considered in evaluating carcinogenic bioassay studies using the F344 rat is the relatively high background incidence of mononuclear cell leukemia (MCL) (also referred to as large granular lymphocytic leukemia, Fischer rat leukemia, or monocytic leukemia). Incidences of MCL ranging from 10 to 72% in male F344 rats to 6 to 31% in female F344 rats have been reported. Gaining the understanding of the mechanisms involved in the negative correlations noted should enhance our understanding of the mechanisms involved in the development of lung cancer.

  13. Decreased mononuclear cell response to mitogens in artificially reared neonatal pigs.

    PubMed Central

    Blecha, F; Pollmann, D S; Kluber, E F

    1986-01-01

    The influence of two rearing methods for neonatal pigs on mononuclear cell responses to mitogens was studied. Littermate pigs were reared artificially or on the sow. Artificially reared pigs displayed a leukocytosis (P less than 0.05) characterized by an increase (P less than 0.05) in mature and immature neutrophils. Skin-test responses to intradermal injections of phytohemagglutinin were less (P less than 0.05) in artificially reared pigs than in sow-reared controls. Lymphocyte proliferative responses to mitogenic stimulation were approximately 50% lower (P less than 0.05) in artificially reared pigs than in sow-reared pigs. These data suggest that artificial rearing lowers in vivo and in vitro lymphocyte responses in neonatal pigs. PMID:3791077

  14. Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

    PubMed

    Adams, E M; Lubrano, T M; Gordon, J; Fields, J Z

    1992-09-01

    We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor. PMID:1392105

  15. [Research advances on DNA extraction methods from peripheral blood mononuclear cells].

    PubMed

    Wang, Xiao-Ying; Yu, Chen-Xi

    2014-10-01

    DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized. PMID:25338615

  16. Kinetic study of cytokines production by human peripheral blood mononuclear cells in response to Brucella DNA.

    PubMed

    Lashkarbolouki, Taghi; Ardestani, Sussan K; Kariminia, Amina; Ziaee, Abed-Ali; Torkabadi, Ebrahim; Ebrahimi, Mohammad

    2008-01-01

    In spite of reports on cytokines induction by the Brucella DNA in murine model, there is no comparison between pathogenic and appropriate vaccine strains in human. We investigated the cytokines profile of human peripheral blood mononuclear cells (PBMCs) induced by DNA extracted from pathogenic isolates of Brucella melitensis and B. abortus as well as Rev1 and S19; the appropriate vaccine strains. It was observed that despite differential induction of Interleukin(IL)-12 and IL-10 production, identical IL-12/IL-10 concentration ratio was obtained by all Brucella strains DNAs that was 2 after 24 h and 4 after 5 days of incubation. In addition, IL-2 and Interferon(IFN)-gamma production were profoundly increased compared to the medium at day 3 and 5 respectively but IFN-alpha was not induced. Therefore, Brucella strains DNAs are Th1 inducing component with similar pattern in human PBMCs. PMID:17008080

  17. A Hitchhiker’s Guide to Myeloid Cell Subsets: Practical Implementation of a Novel Mononuclear Phagocyte Classification System

    PubMed Central

    Guilliams, Martin; van de Laar, Lianne

    2015-01-01

    The classification of mononuclear phagocytes as either dendritic cells or macrophages has been mainly based on morphology, the expression of surface markers, and assumed functional specialization. We have recently proposed a novel classification system of mononuclear phagocytes based on their ontogeny. Here, we discuss the practical application of such a classification system through a number of prototypical examples we have encountered while hitchhiking from one subset to another, across species and between steady-state and inflammatory settings. Finally, we discuss the advantages and drawbacks of such a classification system and propose a number of improvements to move from theoretical concepts to concrete guidelines. PMID:26322042

  18. 15-deoxyspergualin treatment of graft rejection in man: effect on mononuclear cells.

    PubMed

    Borg, A J; Ohlman, S

    1992-09-01

    We studied the effects of 15-deoxyspergualin (DSG) on human mononuclear cells in blood when DSG was administered as anti-rejection treatment to kidney-transplant patients in combination with methylprednisolone (MP) in a safety study. The numbers of leucocytes, lymphocytes and monocytes, the percentages of T and B cells and the expression of the interleukin 2 receptor and the human leucocyte antigen DR locus (HLA-DR) were determined in blood from these patients and from patients treated with MP only. We found that the number of lymphocytes and monocytes decreased in all groups, as also did the CD4/CD8 ratio and the HLA-DR expression on monocytes. DSG counteracted the increase in the number of leucocytes observed in patients treated with MP only. Different effects of DSG and MP were also observed on B cells. While the percentage of CD20+ cells increased in the MP group, it remained unaltered in patients given low-dose DSG and/or was decreased in those given higher doses. Since available evidence suggests an effect of DSG on B-cell reactivity, this drug may become an important addition to the arsenal of immunosuppressive drugs in clinical transplantation. PMID:1418313

  19. Effect of arsenic, cadmium and lead on the induction of apoptosis of normal human mononuclear cells

    PubMed Central

    DE LA FUENTE, H; PORTALES-PÉREZ, D; BARANDA, L; DÍAZ-BARRIGA, F; SAAVEDRA-ALANÍS, V; LAYSECA, E; GONZÁLEZ-AMARO, R

    2002-01-01

    The aim of this work was to investigate the effect of cadmium, lead and arsenic on the apoptosis of human immune cells. Peripheral blood mononuclear cells (MNC) were incubated with increasing concentrations of these metals and then cellular apoptosis was determined by flow cytometry and by DNA electrophoresis. We found that arsenic induced a significant level of apoptosis at 15 μm after 48h of incubation. Cadmium had a similar effect, but at higher concentrations (65 μm). In addition, cadmium exerted a cytotoxic effect on MNC that seemed to be independent of the induction of apoptosis. In contrast, concentrations of lead as high as 500 μm were nontoxic and did not induce a significant degree of apoptosis. Additional experiments showed that arsenic at concentrations as low as 1·0 μm had a significant pro-apoptotic effect when cells were cultured in the presence of this pollutant for more than 72. Non-T cells were more susceptible than T lymphocytes to the effect of arsenic and cadmium. Interestingly, MNC from children chronically exposed to arsenic showed a high basal rate of apoptosis and a diminished in vitro sensibility to this metalloid. Our results indicate that both arsenic and cadmium are able to induce apoptosis of lymphoid cells, and suggest that this phenomenon may contribute to their immunotoxic effect in vivo. PMID:12100024

  20. Presepsin (sCD14-ST) secretion and kinetics by peripheral blood mononuclear cells and monocytic THP-1 cell line.

    PubMed

    Chenevier-Gobeaux, Camille; Bardet, Valérie; Poupet, Hélène; Poyart, Claire; Borderie, Didier; Claessens, Yann-Erick

    2016-02-01

    Presepsin could help for early diagnosis of systemic infection. Little is known regarding its kinetics. We studied presepsin concentration after challenge with bacterial agonist lipopolysaccharide (LPS) stimulation in peripheral mononuclear cells (PMNC) collected from 5 healthy volunteers and in a human cell line of monocytic cells (THP1). In PMNC, an exposure to LPS (100 ng/mL) induced an increase of median presepsin levels as early as hour 1 (+31%, p=0.007), concomitantly to IL-6 synthesis. In THP1 cells, presepsin was detected at 1 hour after LPS exposure, and peaked at 3 hours, in THP1 cells. In conclusion, we report here that presepsin, a surrogate marker of the host response to bacteria, increases early in PMNC and in a monocytic cell lineage. Our findings might confirm the potential usefulness of presepsin bedside as an early marker of infectious diseases. PMID:26743983

  1. Functional characteristics of histamine receptor-bearing mononuclear cells. I. Selective production of lymphocyte chemoattractant lymphokines with histamine used as a ligand.

    PubMed

    Center, D M; Cruikshank, W W; Berman, J S; Beer, D J

    1983-10-01

    Mitogens and antigens have been the traditional ligands for activating lymphocytes in vitro for the elaboration of lymphokines. Recently, histamine, by interaction with histamine-type 2 receptors on T lymphocytes, has been found to induce the production of one lymphokine, histamine-induced suppressor factor (HSF), that inhibits lymphocyte proliferation and lymphokine production in vitro. Because the biologic effects of HSF appear to be confined to alterations in lymphocyte function, we assessed the ability of soluble products of histamine-stimulated human blood mononuclear cells to affect another lymphocyte function, motility. Utilizing a modified Boyden chamber assay to assess lymphocyte migration, we identified chemoattractant activity for human blood and rat splenic T lymphocytes in histamine-induced mononuclear cell supernatants. No neutrophil or monocyte chemoattractant activity was present. Sephadex G-100 gel filtration of histamine-induced supernatants showed the lymphotactic activity eluted with a 56,000 m.w. This activity was cationic as determined by its elution pattern from a Sephadex QAE anion exchange matrix with a single pl of 9.0 to 9.4 determined by isoelectric focusing in sucrose. Its biologic activity is predominantly chemokinetic in nature, is stable to heating at 56 degrees C for 30 min, but is sensitive to the effects of trypsin and neuraminidase. These physicochemical and functional characteristics establish it as identical to a recently described concanavalin A-induced (Con A) lymphotactic lymphokine (LCF). Mononuclear cells that did not adhere to a histamine affinity matrix were unable to produce LCF when subsequently stimulated with histamine or Con A. Mononuclear cells incubated with histamine and diphenhydramine produced LCF; the addition of cimetidine eliminated LCF production. In fact, supernatants from cells incubated with histamine and cimetidine significantly inhibited lymphocyte migration, a phenomenon explainable by the two regions

  2. A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

    PubMed Central

    Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

    2016-01-01

    Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies. PMID:26813960

  3. Cytokine production by mononuclear cells following stimulation with a peptide-containing, endotoxin-free Escherichia coli extract.

    PubMed

    Thomsen, A; Loppnow, H

    1995-05-01

    The beneficial effects of the E. coli extract Colibiogen inj. N (Cb) observed in therapy of inflammatory bowel diseases, allergies, or gastrointestinal tumors are possibly mediated by the induction of cytokines in human leukocytes or vascular cells. Thus, the induction of the cytokines interleukin 1 (IL1), IL6 and tumor necrosis factor (TNF) in human mononuclear cells (MNC) and vascular cells was investigated in vitro. Various administration forms of the extract (including Cb-inj. N, Cb-oral, and Cb-infantibus N) induced the release of IL1 and IL6 from MNC. The compounds stimulated TNF production less potently, possibly due to a lower sensitivity of the TNF assay system, as compared to the IL1 and IL6 detection system. The MNC produced the cytokines with a kinetics similar to that observed with other stimuli. Monospecific antibodies abolished the respective cytokine activity in the biological assays. Addition of submaximal amounts of endotoxin potently enhanced the IL1- and IL6-inducing activity of the bacterial extract, indicating synergism of the extract and endotoxin. These results provide evidence that cytokines produced by MNC following administration of the tested bacterial extract may contribute to the regulation of the immune response during therapy of gastrointestinal tumors. At present the in vivo production of cytokines following treatment with the bacterial extract tested is under investigation in a phase III study. PMID:7612070

  4. [Autotransfusion of UV-irradiated blood and the morphofunctional status of mononuclear cells in neurodermitis patients].

    PubMed

    Finkel'shteĭn, B B; Zver'kova, F A

    1989-01-01

    Structural changes in the blood mononuclear glycocalyx has been revealed in children suffering from neurodermatitis. These shifts correlated with the cellular ability to respond to mitogenic stimulation. Treatment by UV-irradiated blood autotransfusions normalized the structure and function of mononuclears whereas routine therapy has been ineffective. PMID:2728605

  5. Bisphenol A and its analogs exhibit different apoptotic potential in peripheral blood mononuclear cells (in vitro study).

    PubMed

    Mokra, Katarzyna; Kocia, Magdalena; Michałowicz, Jaromir

    2015-10-01

    There are only a few studies that have assessed the effect of bisphenol A (BPA) on human blood cells and no study has been conducted to analyze the impact of BPA analogs on human leucocytes. In this study, we have investigated the effect of BPA and its analogs like bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF) on apoptosis induction in human peripheral blood mononuclear cells (PBMCs). In order to clarify the mechanism of bisphenols-induced programmed cell death, changes in various signaling molecules of this process have been assessed. We observed an increase in cytosolic calcium ions (Ca(2+)) level and reduction of transmembrane mitochondrial potential (ΔΨm) in PBMCs incubated with all compounds examined, and particularly BPA and BPAF. All compounds studied changed PBMCs membrane permeability, activated caspase-8, -9, -3 and induced PARP-1 cleavage and chromatin condensation, which confirmed that they were capable of inducing apoptosis both via intrinsic and extrinsic pathway. Moreover, we have found that modus operandi of bisphenols studied was different. We noticed that BPAF and BPS caused mainly necrotic and apoptotic changes, respectively, whereas BPA induced comparable apoptotic and necrotic effects in the incubated cells. PMID:26271707

  6. Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

    PubMed Central

    Baker, Gregory J.; Castro, Maria G.; Lowenstein, Pedro R.

    2016-01-01

    Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4–6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists’ discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the

  7. In vivo administration of the frog skin peptide frenatin 2.1S induces immunostimulatory phenotypes of mouse mononuclear cells.

    PubMed

    Pantic, Jelena M; Radosavljevic, Gordana D; Jovanovic, Ivan P; Arsenijevic, Nebojsa N; Conlon, J Michael; Lukic, Miodrag L

    2015-09-01

    Host-defense peptides secreted by epithelial cells exhibit cytotoxic and immunoregulatory effects in order to protect the organism against invading microorganisms. Antimicrobial peptides derived from frog skin display both immunostimulatory and immunosuppressive actions as demonstrated by in vitro cytokine production by macrophages. Frenatin 2.1S, first isolated from skin secretions of the frog, Sphaenorhynchus lacteus (Hylidae), enhances the in vitro production of pro-inflammatory IL-1β, TNF-α and IL-23 by mouse peritoneal cells. In order to test whether the immunostimulatory action of frenatin 2.1S may be reproduced in vivo, effects of intraperitoneal injections of this peptide on mononuclear cells in the peritoneum and spleen were determined 24h after administration. The data indicate that frenatin 2.1S enhances the activation state and homing capacity of Th1 type lymphocytes and NKT cells in the mouse peritoneal cavity, as evaluated by increased expression of early activation marker CD69 among T and NKT cells and chemokine receptor CXCR3 among T cells. Frenatin 2.1S significantly increases the percentage of (F4/80(+)CD11c(+)CD206(+)) pro-inflammatory M1 macrophages and enhances the expression of MHC class II molecules on F4/80(+)CD11c(+) macrophages in the mouse peritoneal cavity. Additionally, injection of frenatin 2.1S, in the presence or absence of lipopolysaccharide, increases the percentage of peritoneal B cells of the (CD19(+)CD11b(+)CD5(+)) B1a phenotype thus contributing to an inflammatory milieu. We suggest that the immunostimulatory effect of frenatin 2.1S may have therapeutic relevance in disease states, such as certain types of cancer, in which an enhanced inflammatory response may be beneficial. PMID:25861850

  8. Functional and Transcriptomic Recovery of Infarcted Mouse Myocardium Treated with Bone Marrow Mononuclear Cells

    PubMed Central

    Lachtermacher, Stephan; Esporcatte, Bruno L. B.; da Silva de Azevedo Fortes, Fábio; Rocha, Nazareth Novaes; Montalvão, Fabrício; Costa, Patricia C.; Belem, Luciano; Rabischoffisky, Arnaldo; Neto, Hugo C. C. Faria; Vasconcellos, Rita; Iacobas, Dumitru A.; Iacobas, Sanda; Spray, David C.; Thomas, Neil M.; Goldenberg, Regina C. S.; de Carvalho, Antonio C. Campos

    2011-01-01

    Although bone marrow-derived mononuclear cells (BMNC) have been extensively used in cell therapy for cardiac diseases, little mechanistic information is available to support reports of their efficacy. To address this shortcoming, we compared structural and functional recovery and associated global gene expression profiles in post-ischaemic myocardium treated with BMNC transplantation. BMNC suspensions were injected into cardiac scar tissue 10 days after experimental myocardial infarction. Six weeks later, mice undergoing BMNC therapy were found to have normalized antibody repertoire and improved cardiac performance measured by ECG, treadmill exercise time and echocardiography. After functional testing, gene expression profiles in cardiac tissue were evaluated using high-density oligonucleotide arrays. Expression of more than 18% of the 11981 quantified unigenes was significantly altered in the infarcted hearts. BMNC therapy restored expression of 2099 (96.2%) of the genes that were altered by infarction but led to altered expression of 286 other genes, considered to be a side effect of the treatment. Transcriptional therapeutic efficacy, a metric calculated using a formula that incorporates both recovery and side effect of treatment, was 73%. In conclusion, our results confirm a beneficial role for bone marrow-derived cell therapy and provide new information on molecular mechanisms operating after BMNC transplantation on post ischemic heart failure in mice. PMID:21671060

  9. Modifications of the endosomal compartment in peripheral blood mononuclear cells and fibroblasts from Alzheimer's disease patients.

    PubMed

    Corlier, F; Rivals, I; Lagarde, J; Hamelin, L; Corne, H; Dauphinot, L; Ando, K; Cossec, J-C; Fontaine, G; Dorothée, G; Malaplate-Armand, C; Olivier, J-L; Dubois, B; Bottlaender, M; Duyckaerts, C; Sarazin, M; Potier, M-C

    2015-01-01

    Identification of blood-based biomarkers of Alzheimer's disease (AD) remains a challenge. Neuropathological studies have identified enlarged endosomes in post-mortem brains as the earliest cellular change associated to AD. Here the presence of enlarged endosomes was investigated in peripheral blood mononuclear cells from 48 biologically defined AD patients (25 with mild cognitive impairment and 23 with dementia (AD-D)), and 23 age-matched healthy controls using immunocytochemistry and confocal microscopy. The volume and number of endosomes were not significantly different between AD and controls. However, the percentage of cells containing enlarged endosomes was significantly higher in the AD-D group as compared with controls. Furthermore, endosomal volumes significantly correlated to [C(11)]PiB cortical index measured by positron emission tomography in the AD group, independently of the APOE genotype, but not to the levels of amyloid-beta, tau and phosphorylated tau measured in the cerebrospinal fluid. Importantly, we confirmed the presence of enlarged endosomes in fibroblasts from six unrelated AD-D patients as compared with five cognitively normal controls. This study is the first, to our knowledge, to report morphological alterations of the endosomal compartment in peripheral cells from AD patients correlated to amyloid load that will now be evaluated as a possible biomarker. PMID:26151923

  10. Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine Peripheral Artery Disease

    PubMed Central

    van der Bogt, Koen E.A.; Hellingman, Alwine A.; Lijkwan, Maarten A.; Bos, Ernst-Jan; de Vries, Margreet R.; Fischbein, Michael P.; Quax, Paul H.; Robbins, Robert C.; Hamming, Jaap F.; Wu, Joseph C.

    2013-01-01

    Introduction Bone marrow mononuclear cell (MNC) therapy is a promising treatment for peripheral artery disease (PAD). This study aims to provide insight into cellular kinetics using molecular imaging following different transplantation methods. Methods and Results MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n=50) underwent femoral artery ligation and were randomized into 4 groups receiving: (1) single intramuscular (i.m.) injection of 2×106 MNC; (2) four weekly i.m. injections of 5×105 MNC; (3) 2×106 MNCs intravenously (i.v.); and (4) PBS. Cellular kinetics, measured by in vivo bioluminescence imaging (BLI), revealed near-complete donor cell death 4 weeks after i.m. transplantation. Following i.v. transplantation, BLI monitored cells homed in on the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularisation were observed as monitored by Laser-Doppler-Perfusion-Imaging and histology. Conclusion This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short lived and MNCs do not significantly stimulate perfusion in this model. PMID:22239892

  11. Cytotoxic and inflammatory responses of TiO2 nanoparticles on human peripheral blood mononuclear cells.

    PubMed

    Kongseng, Supunsa; Yoovathaworn, Krongtong; Wongprasert, Kanokpan; Chunhabundit, Rodjana; Sukwong, Patinya; Pissuwan, Dakrong

    2016-10-01

    Titanium dioxide nanoparticles (TiO2 -NPs) have been widely used in many applications. Owing to their nanoscale size, interactions between cells and NPs have been expansively investigated. With the health concerns raised regarding the adverse effects of these interactions, closer examination of whether TiO2 -NPs can induce toxicity towards human cells is greatly needed. Therefore, in this study, we investigated the cytotoxicity of TiO2 -NPs towards human blood cells (peripheral blood mononuclear cells [PBMCs]) in serum-free medium, for which there is little information regarding the cytotoxic effects of TiO2 -NPs. Our results provide evidence that PBMCs treated with TiO2 -NPs (at concentrations ≥25 μg ml(-1) ) for 24 h significantly reduced cell viability and significantly increased production of toxic mediators such as reactive oxygen species and inflammatory response cytokines such as interleukin-6 and tumor necrosis factor-α (P < 0.05). Cell apoptosis induction also occurred at these concentrations. Significant expressions of cyclooxygenase-2 and interleukin-1β were also observed in PBMCs treated with TiO2 -NPs at concentrations ≥125 μg ml(-1) . Our data presented here clearly indicate that the concentration of TiO2 -NPs (at size ~26.4 ± 1.2 nm) applied to human blood cells has a strong impact on cytotoxic induction. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27225715

  12. In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells

    PubMed Central

    Norkina, Oxana; Lum, Lawrence G.

    2013-01-01

    In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG2 (P < 0.001) and cytokines that promote IgG2 synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses. PMID:21713642

  13. Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris.

    PubMed

    Sweat, J M; Johnson, C M; Marikar, Y; Gibbs, E P

    2005-12-15

    An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin. PMID:16112745

  14. Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration

    PubMed Central

    Beer, Lucian; Zimmermann, Matthias; Mitterbauer, Andreas; Ellinger, Adolf; Gruber, Florian; Narzt, Marie-Sophie; Zellner, Maria; Gyöngyösi, Mariann; Madlener, Sibylle; Simader, Elisabeth; Gabriel, Christian; Mildner, Michael; Ankersmit, Hendrik Jan

    2015-01-01

    We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine. PMID:26567861

  15. A sensitive LC-MS/MS method for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells.

    PubMed

    Tu, Xiaowei; Lu, Youming; Zhong, Dafang; Zhang, Yifan; Chen, Xiaoyan

    2014-08-01

    Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography-tandem mass spectrometry method (LC-MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25-100 ng/10(7) cells with acceptable accuracy and precision using 50 μL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze-thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine. PMID:24529342

  16. Evaluation of proliferation and cytokines production by mitogen-stimulated bovine peripheral blood mononuclear cells

    PubMed Central

    Norian, Reza; Delirezh, Nowruz; Azadmehr, Abbas

    2015-01-01

    This in vitro study was conducted to evaluate lymphocyte blastogenic and cytokine production by bovine peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (Con A) mitogens, by using tetrazolium salt and ELISA tests, respectively. The results presented that Interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-17 and IFN-γ production in response to PWM mitogens was the highest and Con A the lowest amount and the median values of three mitogens were in the following order: PWM > PHA > Con A > cell control. In the case of IL-6, the production of this cytokine was the same amount for PWM and Con A and a lower amount for PHA stimulation. The results of this study not only showed a normal range for the production of these cytokines from PBMCs that were affected by mitogens, but it demonstrated that the bovine immune system at 2.5 to 3 months was post-natally matured enough to mount an effective immune response to mitogens as well as specific antigens. PMID:26973760

  17. Differential expression and interaction of host factors augment HIV-1 gene expression in neonatal mononuclear cells

    SciTech Connect

    Sundaravaradan, Vasudha; Mehta, Roshni; Harris, David T.; Zack, Jerome A.; Ahmad, Nafees

    2010-04-25

    We have previously shown a higher level of HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells (CBMC) compared with adult blood cells (PBMC), which could be due to differential expression of host factors. We performed the gene expression profile of CBMC and PBMC and found that 8013 genes were expressed at higher levels in CBMC than PBMC and 8028 genes in PBMC than CBMC, including 1181 and 1414 genes upregulated after HIV-1 infection in CBMC and PBMC, respectively. Several transcription factors (NF-kappaB, E2F, HAT-1, TFIIE, Cdk9, Cyclin T1), signal transducers (STAT3, STAT5A) and cytokines (IL-1beta, IL-6, IL-10) were upregulated in CBMC than PBMC, which are known to influence HIV-1 replication. In addition, a repressor of HIV-1 transcription, YY1, was down regulated in CBMC than PBMC and several matrix metalloproteinase (MMP-7, -12, -14) were significantly upregulated in HIV-1 infected CBMC than PBMC. Furthermore, we show that CBMC nuclear extracts interacted with a higher extent to HIV-1 LTR cis-acting sequences, including NF-kappaB, NFAT, AP1 and NF-IL6 compared with PBMC nuclear extracts and retroviral based short hairpin RNA (shRNA) for STAT3 and IL-6 down regulated their own and HIV-1 gene expression, signifying that these factors influenced differential HIV-1 gene expression in CBMC than PBMC.

  18. Proteomic analysis of chicken peripheral blood mononuclear cells after infection by Newcastle disease virus

    PubMed Central

    Deng, Xiaoyu; Cong, Yanlong; Yin, Renfu; Yang, Guilian; Ding, Chan; Yu, Shengqing; Liu, Xiufan; Wang, Chunfeng

    2014-01-01

    Characteristic clinical manifestations of Newcastle disease include leukopenia and immunosuppression. Peripheral blood mononuclear cells (PBMCs) are the main targets of Newcastle disease virus (NDV) infection. To survey changes in proteomic expression in chicken PBMCs following NDV infection, PBMC proteins from 30 chickens were separated using two-dimensional electrophoresis (2-DE) and subjected to mass spectrometry analysis. Quantitative intensity analysis showed that the expression of 78 proteins increased more than two-fold. Thirty-five proteins exhibited consistent changes in expression and 13 were identified as unique proteins by matrix assisted laser desorption ionization-time of flight mass spectrometer/mass spectrometer including three that were down-regulated and 10 that were up-regulated. These proteins were sorted into five groups based on function: macromolecular biosynthesis, cytoskeleton organization, metabolism, stress responses, and signal transduction. Furthermore, Western blot analysis confirmed the down-regulation of integrin-linked kinase expression and up-regulation of lamin A production. These data provide insight into the in vivo response of target cells to NDV infection at the molecular level. Additionally, results from this study have helped elucidate the molecular pathogenesis of NDV and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods. PMID:25234324

  19. Proteomic analysis of chicken peripheral blood mononuclear cells after infection by Newcastle disease virus.

    PubMed

    Deng, Xiaoyu; Cong, Yanlong; Yin, Renfu; Yang, Guilian; Ding, Chan; Yu, Shengqing; Liu, Xiufan; Wang, Chunfeng; Ding, Zhuang

    2014-12-01

    Characteristic clinical manifestations of Newcastle disease include leukopenia and immunosuppression. Peripheral blood mononuclear cells (PBMCs) are the main targets of Newcastle disease virus (NDV) infection. To survey changes in proteomic expression in chicken PBMCs following NDV infection, PBMC proteins from 30 chickens were separated using two- dimensional electrophoresis (2-DE) and subjected to mass spectrometry analysis. Quantitative intensity analysis showed that the expression of 78 proteins increased more than two-fold. Thirty-five proteins exhibited consistent changes in expression and 13 were identified as unique proteins by matrix assisted laser desorption ionization-time of flight mass spectrometer/mass spectrometer including three that were down-regulated and 10 that were up-regulated. These proteins were sorted into five groups based on function: macromolecular biosynthesis, cytoskeleton organization, metabolism, stress responses, and signal transduction. Furthermore, Western blot analysis confirmed the down-regulation of integrin-linked kinase expression and up-regulation of lamin A production. These data provide insight into the in vivo response of target cells to NDV infection at the molecular level. Additionally, results from this study have helped elucidate the molecular pathogenesis of NDV and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods. PMID:25234324

  20. Intermittent Hypoxia Alters Gene Expression in Peripheral Blood Mononuclear Cells of Healthy Volunteers

    PubMed Central

    Polotsky, Vsevolod Y.; Bevans-Fonti, Shannon; Grigoryev, Dmitry N.; Punjabi, Naresh M.

    2015-01-01

    Obstructive sleep apnea is associated with high cardiovascular morbidity and mortality. Intermittent hypoxia of obstructive sleep apnea is implicated in the development and progression of insulin resistance and atherosclerosis, which have been attributed to systemic inflammation. Intermittent hypoxia leads to pro-inflammatory gene up-regulation in cell culture, but the effects of intermittent hypoxia on gene expression in humans have not been elucidated. A cross-over study was performed exposing eight healthy men to intermittent hypoxia or control conditions for five hours with peripheral blood mononuclear cell isolation before and after exposures. Total RNA was isolated followed by gene microarrays and confirmatory real time reverse transcriptase PCR. Intermittent hypoxia led to greater than two fold up-regulation of the pro-inflammatory gene toll receptor 2 (TLR2), which was not increased in the control exposure. We hypothesize that up-regulation of TLR2 by intermittent hypoxia may lead to systemic inflammation, insulin resistance and atherosclerosis in patients with obstructive sleep apnea. PMID:26657991

  1. Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells.

    PubMed

    Matt, Katja; Burger, Katharina; Gebhard, Daniel; Bergemann, Jörg

    2016-03-01

    Caloric restrictive feeding prolongs the lifespan of a variety of model organisms like rodents and invertebrates. It has been shown that caloric restriction reduces age-related as well as overall-mortality, reduces oxidative stress and influences DNA repair ability positively. There are numerous studies underlining this, but fewer studies involving humans exist. To contribute to a better understanding of the correlation of calorie reduction and DNA repair in humans, we adapted the host cell reactivation assay to an application with human peripheral blood mononuclear cells. Furthermore, we used this reliable and reproducible assay to research the influence of a special kind of calorie reduction, namely F. X. Mayr therapy, on DNA repair capacity. We found a positive effect in all persons with low pre-existing DNA repair capacity. In individuals with normal pre-existing DNA repair capacity, no effect on DNA repair capacity was detectable. Decline of DNA repair, accumulation of oxidative DNA damages, mitochondrial dysfunction, telomere shortening as well as caloric intake are widely thought to contribute to aging. With regard to that, our results can be considered as a strong indication that calorie reduction may support DNA repair processes and thus contribute to a healthier aging. PMID:26879629

  2. Evaluation of proliferation and cytokines production by mitogen-stimulated bovine peripheral blood mononuclear cells.

    PubMed

    Norian, Reza; Delirezh, Nowruz; Azadmehr, Abbas

    2015-01-01

    This in vitro study was conducted to evaluate lymphocyte blastogenic and cytokine production by bovine peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM) and concanavalin A (Con A) mitogens, by using tetrazolium salt and ELISA tests, respectively. The results presented that Interleukin-2 (IL-2), IL-4, IL-5, IL-10, IL-17 and IFN-γ production in response to PWM mitogens was the highest and Con A the lowest amount and the median values of three mitogens were in the following order: PWM > PHA > Con A > cell control. In the case of IL-6, the production of this cytokine was the same amount for PWM and Con A and a lower amount for PHA stimulation. The results of this study not only showed a normal range for the production of these cytokines from PBMCs that were affected by mitogens, but it demonstrated that the bovine immune system at 2.5 to 3 months was post-natally matured enough to mount an effective immune response to mitogens as well as specific antigens. PMID:26973760

  3. Human cord blood mononuclear cell transplantation for the treatment of premature ovarian failure in nude mice

    PubMed Central

    Dang, Jianhong; Jin, Zhijun; Liu, Xiaojun; Hu, Dian; Wang, Zhifeng

    2015-01-01

    Objective: This study explored the potential of human cord blood mononuclear cell (HCMNC) transplantation as a treatment for premature ovarian failure (POF) in a nude mouse model. Methods: Female nude mice were randomly divided into three groups; a normal control group (n = 35), a POF group (POF plus vehicle, n = 35) and a POF plus cell transplantation group (HCMNCs were implanted into the ovaries, n = 35). HCMNCs were isolated by Ficoll density gradient centrifugation and labeled with BrdU. Four weeks after transplantation, the nude mice were sacrificed to determine serum levels of E2, FSH and LH as indicators of ovarian function, and the ovaries were examined both histologically and immunochemically. Results: The transplanted HCMNCs survived in the transplantation group and were detected by BrdU. In the transplantation group, serum levels of E2 significantly increased while serum levels of FSH and LH significantly decreased compared to the POF control group. Additionally, the transplantation group had a recovery in follicle number. Conclusion: HCMNCs can be successfully transplanted into the ovaries of nude mice and can improve ovarian function in POF. PMID:26064319

  4. In vitro gene expression profile of bovine peripheral blood mononuclear cells in early Mycobacterium bovis infection

    PubMed Central

    CHENG, YAFEN; CHOU, CHUNG-HSI; TSAI, HSIANG-JUNG

    2015-01-01

    The intracellular parasite Mycobacterium bovis (M. bovis) causes tuberculosis in cattle and humans. Understanding the interactions between M. bovis and host cells is essential in developing tools for the prevention, detection, and treatment of M. bovis infection. Gene expression profiles provide a large amount of information regarding the molecular mechanisms underlying these interactions. The present study analyzed changes in gene expression in bovine peripheral blood mononuclear cells (PBMCs) at 0, 4 and 24 h following exposure to M. bovis. Using bovine whole-genome microarrays, a total of 420 genes were identified that exhibited significant alterations in expression (≥2-fold). Significantly enriched genes were identified using the Kyoto Encyclopedia of Genes and Genomes database, of which the highest differentially expressed genes were associated with the immune system, signal transduction, endocytosis, cellular transport, inflammation, and apoptosis. Of the genes associated with the immune system, 84.85% displayed downregulation. These findings support the view that M. bovis inhibits signaling pathways of antimycobacterial host defense in bovine PBMCs. These in vitro data demonstrated that molecular alterations underlying the pathogenesis of tuberculosis begin early, during the initial 24 h following M. bovis infection. PMID:26668602

  5. Comparison of osteoclast precursors in peripheral blood mononuclear cells from rheumatoid arthritis and osteoporosis patients.

    PubMed

    Nose, Michinari; Yamazaki, Hidetoshi; Hagino, Hiroshi; Morio, Yasuo; Hayashi, Shin-Ichi; Teshima, Ryota

    2009-01-01

    Osteolytic disorders cause serious problems for quality of life with aging. Osteolysis is performed by osteoclasts of the hematopoietic lineage that share some characteristics with monocytes and macrophages. As osteoclast precursors (pOCs) are present in peripheral blood, their characterization in osteolytic diseases may help us to understand risk factors. Although essential factors for osteoclastogenesis have been reported, the effective induction from pOCs in human peripheral blood mononuclear cells (PBMCs) to mature osteoclasts in culture requires further improvement. The aim of this study was development of an efficient culture system for human osteoclastogenesis and providing a simple system for the enrichment of pOCs from PBMCs. We employed coculturing of human PBMCs with a mouse stromal cell line. Significant numbers of tartrate-resistant acid phosphatase-positive (TRAP(+)) multinucleated osteoclasts (MNCs), which could resorb dentine slices, were efficiently induced in this culture condition. pOCs were enriched in an anti-CD16 antibody column-passed anti-CD14 antibody-bound cell population isolated by magnetic cell sorting. We compared the percentage of the CD14(high) CD16(dull) cell population, which mainly contained pOCs in PBMCs, from age-matched patients with rheumatoid arthritis (RA) and osteoporosis (OP), but it was comparable. However, the mean number of TRAP(+) MNCs generated in cultures from PBMCs of RA was higher. In contrast, the frequency of pOCs in PBMCs from OP was relatively higher. These results suggest the characteristics of pOCs from RA and OP may be different, because single pOCs from OP gave rise to lower numbers of osteoclasts than those from RA. PMID:19082778

  6. Phagocytosis-induced 51Cr release from activated macrophages and blood mononuclears. Effect of colchicine and antioxidants

    SciTech Connect

    McGee, M.P.; Hale, A.H.

    1981-09-01

    The chromium-release test was adapted to the measurement of the cellular injury induced when activated macrophages phagocytose particulates. Macrophages obtained from rabbit lungs undergoing BCG-induced chronic inflammation released more chromium when incubated in the presence of phagocytosable particles than when incubated under resting conditions. Blood mononuclear cells, 40-60% monocytes, procured from the same BCG-injected animals, were less susceptible to phagocytosis-induced injury than the macrophages obtained from the lungs. The amount of chromium released by the activated macrophages was proportional to the number of particles present during incubation. In the presence of catalase, the amounts of chromium released by phagocytosing and resting macrophages were similar; in the presence of superoxide dismutase and cytochrome c, the amount of chromium released by phagocytosing macrophages was 13-35% less than the amount of chromium released by macrophages incubated without the antioxidants. In addition, colchicine, an inhibitor of degranulation also exerted partial inhibition of the chromium release. These results suggest that oxygen radicals and lysosomal contents contribute to the cellular injury that results from phagocytosis.

  7. Peripheral blood mononuclear cell-converted induced pluripotent stem cells (iPSCs) from an early onset Alzheimer's patient.

    PubMed

    Lee, Han-Kyu; Morin, Peter; Xia, Weiming

    2016-03-01

    Improvement in transduction efficiency makes it possible to convert blood cells into induced pluripotent stem cells (iPSC). In this study, we generated an iPSC line from peripheral blood mononuclear cells (PBMC) donated by a patient who exhibited memory deficit at age 59; outcome of positron emission tomography scan is consistent with a diagnosis of Alzheimer's disease. Integration-free CytoTune-iPS Sendai Reprogramming factors which include Sendai virus particles of the four Yamanaka factors Oct4, Sox2, Klf4, and c-Myc were introduced to PBMC to convert them to iPSCs without retention of virus. Three germ layer differentiation was induced to demonstrate the pluripotency of these iPSCs. PMID:27345971

  8. Low expression of Toll-like receptors in peripheral blood mononuclear cells of pediatric patients with acute lymphoblastic leukemia.

    PubMed

    Sánchez-Cuaxospa, María; Contreras-Ramos, Alejandra; Pérez-Figueroa, Erandi; Medina-Sansón, Aurora; Jiménez-Hernández, Elva; Torres-Nava, José R; Rojas-Castillo, Emilio; Maldonado-Bernal, Carmen

    2016-08-01

    Cancer is the second most common cause of death among children aged 1-14 years. Leukemia accounts for one-third of all childhood cancers, 78% of which is acute lymphoblastic leukemia (ALL). The development of cancer has been associated with malignant cells that express low levels of immunogenic molecules, which facilitates their escape from the antineoplastic immune response. It is thought that it may be possible to rescue the antineoplastic immune response through the activation of recognition receptors, such as Toll-like receptors (TLRs), which activate the innate immune system. TLRs are type I membrane glycoproteins expressed mainly in immune system cells such as monocytes, neutrophils, macrophages, dendritic cells, T, B and natural killer cells. The aim of the present study was to evaluate the expression of TLR1, TLR3, TLR4, TLR7 and TLR9 in peripheral blood mononuclear cells (PBMCs) in patients with ALL and prior to any treatment. PBMCs were obtained from 50 pediatric patients diagnosed with ALL and from 20 children attending the ophthalmology and orthopedics services. The mean fluorescence intensity was obtained by analysis of immunofluorescence. We found lower expression levels of TLR1, TLR3, TLR4, TLR7 and TLR9 in PBMCs from patients with ALL compared with those from control patients. We also observed that the PBMCs from patients with Pre-B and B ALL had lower TLR4 expression than controls and patients with Pro-B, Pre-B, B and T ALL had lower TLR7 expression than controls. The present study is the first to demonstrate reduced expression of TLRs in PBMCs from pediatric patients with ALL. This finding is of great relevance and may partly explain the reduction in the antineoplastic immune response in patients with ALL. PMID:27277333

  9. Overexpression of MMP-9 and Its Inhibitors in Blood Mononuclear Cells after Myocardial Infarction - Is It Associated with Depressive Symptomatology?

    PubMed Central

    Jönsson, Simon; Lundberg, Anna K.; Jonasson, Lena

    2014-01-01

    Background Matrix metalloproteinase (MMP)-9 may play a central role in the development and progression of atherosclerosis. Emerging evidence also indicates an association between MMP-9 and depressive symptomatology. Here, we investigated whether expression of MMP-9 and its inhibitors in blood mononuclear cells and plasma were related to depressive symptoms in patients with a recent myocardial infarction (MI). Methods and Results Blood sampling was performed between 6 and 18 months after MI in 57 patients. Forty-one clinically healthy subjects were included as controls. Gene expression of MMP-9 and its main tissue inhibitors TIMP-1 and -2 were analyzed in freshly isolated or cultured blood mononuclear cells. Corresponding protein levels were assessed in cell supernatants and plasma. In post-MI patients, mRNA levels of MMP-9 and TIMP-1 and -2 were significantly higher than in controls while protein levels in cell supernatants and plasma did not differ between groups. The Center for Epidemiological Studies - Depression (CES-D) scale was used to assess depressive symptomatology. Repeated assessments during the first 18 months after MI showed significantly higher CES-D scores in patients compared with controls. However, there were no relationships between depressive mood and any of the measurements of MMP-9 or TIMPs. Conclusion Our findings indicate that overexpression of MMP-9 and TIMPs in blood mononuclear cells and elevated depressive symptoms represent two unrelated phenomena after MI. PMID:25153995

  10. Can Melatonin Act as an Antioxidant in Hydrogen Peroxide-Induced Oxidative Stress Model in Human Peripheral Blood Mononuclear Cells?

    PubMed Central

    Emamgholipour, Solaleh; Hossein-Nezhad, Arash; Ansari, Mohammad

    2016-01-01

    Purpose. We aimed to investigate the possible effects of melatonin on gene expressions and activities of MnSOD and catalase under conditions of oxidative stress induced by hydrogen peroxide (H2O2) in peripheral blood mononuclear cells (PBMCs). Materials and Methods. PBMCs were isolated from healthy subjects and treated as follows: (1) control (only with 0.1% DMSO for 12 h); (2) melatonin (1 mM) for 12 h; (3) H2O2 (250 μM) for 2 h; (4) H2O2 (250 μM) for 2 h following 10 h pretreatment with melatonin (1 mM). The gene expression was evaluated by real-time PCR. MnSOD and catalase activities in PBMCs were determined by colorimetric assays. Results. Pretreatment of PBMCs with melatonin significantly augmented expression and activity of MnSOD which were diminished by H2O2. Melatonin treatment of PBMCs caused a significant upregulation of catalase by almost 2-fold in comparison with untreated cells. However, activity and expression of catalase increased by 1.5-fold in PBMCs under H2O2-induced oxidative stress compared with untreated cell. Moreover, pretreatment of PBMCs with melatonin resulted in a significant 1.8-fold increase in catalase expression compared to PBMCs treated only with H2O2. Conclusion. It seems that melatonin could prevent from undesirable impacts of H2O2-induced oxidative stress on MnSOD downregulation. Moreover, melatonin could promote inductive effect of H2O2 on catalase mRNA expression. PMID:26881079

  11. Synthesis, crystal structure, and biological activities of two chiral mononuclear Mn((III)) complexes.

    PubMed

    Wang, Bi-Wei; Jiang, Lin; Shu, Si-Sheng; Li, Bo-Wen; Dong, Zhang; Gu, Wen; Liu, Xin; Tian, Jin-Lei

    2015-02-01

    Two new chiral mononuclear Mn((III)) complexes, [MnL((R)) Cl (C2 H5 OH)]•C2 H5 OH () and [MnL((S)) (CH3 OH)2 ]Cl•CH3 OH (), {H2 L = (R,R)-or (S,S)-N,N'-bis-(2-hydroxy-1-naphthalidehydene)-cyclohexanediamine} were synthesized and characterized by various physicochemical techniques. Bond valence sum (BVS) calculations and the Jahn-Teller effect indicate that the Mn centers are in a +3 oxidation state. The statuses of the two complexes in the solution were confirmed as a pair of enantiomers by electrospray ionization, mass spectrometry (ESI-MS) spectrum. The binding ability of the complexes with calf thymus CT-DNA was investigated by spectroscopic and viscosity measurements. Both of the complexes could interact with CT-DNA via an intercalative mode with the order of (R-enantiomer) > (S-enantiomer). Under the physiological conditions, the two compounds exhibit efficient DNA cleavage activities without any external agent, which also follows the order of R-enantiomer > S-enantiomer. Interestingly, the concentration-dependent DNA cleavage experiments indicate an optimal concentration of 17.5 μM. In addition, the interaction of the compounds with bovine serum albumin (BSA) was also investigated, which indicated that the complexes could quench the intrinsic fluorescence of BSA by a static quenching mechanism. PMID:25403736

  12. Tumour growth inhibition in mice by glycosylated recombinant human lymphotoxin: analysis of tumour-regional mononuclear cells involved with its action.

    PubMed Central

    Funahashi, I.; Watanabe, H.; Abo, T.; Indo, K.; Miyaji, H.

    1993-01-01

    We compared the antitumour effects of glycosylated LT (gLT), nonglycosylated LT and TNF against a solid tumour in mice. We found that: (a) The systemic administration of gLT showed significant antitumour activity. These effects were, however, quite small in nude mice. Nonglycosylated LT and TNF attained the same degree of effectiveness as gLT, but at a 5-times higher dose. The serum half-life of gLT was 3-fold longer than that of nonglycosylated LT and 22-fold longer than that of TNF. (b) The effect of gLT was significantly blocked by pretreatment with anti-asialo GM1 antibody. Treatment with gLT produced a significant reduction in numbers of tumour-regional mononuclear cells, which in turn, produced increases intensive necrosis. (c) Mononuclear cells in the tumour tissues before gLT-injection were predominantly IL-2 receptor +/CD3- cells and CD3+ cells. Pretreatment with the anti-asialo GM1 antibody produced a drastic reduction of IL-2 receptor +/CD3- cells. These findings suggest that the efficient antitumour effect of gLT is due to a longer serum half-life than that of nonglycosylated LT or TNF in vivo, and its function is largely mediated by IL-2 receptor +/CD3- cells. Images Figure 6 Figure 7 PMID:8439496

  13. Time-Course Study of the Transcriptome of Peripheral Blood Mononuclear Cells (PBMCs) from Sheep Infected with Fasciola hepatica

    PubMed Central

    Scheerlinck, Jean-Pierre; Ansell, Brendan R. E.; Hall, Ross S.; Gasser, Robin B.; Jex, Aaron R.

    2016-01-01

    Fasciola hepatica is a parasitic trematode that infects a wide range of mammalian hosts, including livestock and humans, in temperate and tropical regions globally. This trematode causes the disease fascioliasis, which consists of an acute phase (≤ 12 weeks) during which juvenile parasites migrate through the host liver tissues, and a chronic phase (> 12 weeks) following the establishment of adult parasites in the liver bile ducts. Few studies have explored the progression of the host response over the course of Fasciola infection in the same animals. In this study, we characterized transcriptomic changes in peripheral blood mononuclear cells (PBMCs) collected from sheep at three time points over the first eight weeks of infection relative to uninfected controls. In total, 183 and 76 genes were found to be differentially transcribed at two and eight weeks post-infection respectively. Functional and pathway analysis of differentially transcribed genes revealed changes related to T-cell activation that may underpin a Th2-biased immune response against this parasite. This first insight into the dynamics of host responses during the early stages of infection improves the understanding of the pathogenesis of acute fascioliasis, informs vaccine development and presents a set of PBMC markers with diagnostic potential. PMID:27438474

  14. Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.

    PubMed

    Vatta, Adriano F; Dzimianski, Michael; Storey, Bob E; Camus, Melinda S; Moorhead, Andrew R; Kaplan, Ray M; Wolstenholme, Adrian J

    2014-11-15

    The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100 nM and adherence of neutrophils was increased in drug concentrations ≥10 nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40 h incubation. Neutrophil adherence was maximal after 16 h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the host's innate immune system. PMID:24594213

  15. Effects of spironolactone on human blood mononuclear cells: mineralocorticoid receptor independent effects on gene expression and late apoptosis induction

    PubMed Central

    Sønder, Søren Ulrik Salling; Mikkelsen, Marianne; Rieneck, Klaus; Hedegaard, Chris Juul; Bendtzen, Klaus

    2006-01-01

    Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. To elucidate the mechanism behind this, the four MR-binding steroids SPIR, canrenone, 7α-thiomethyl-spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide- and phytohemagglutinin-A-activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR-related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene-regulatory effect independent of MR. The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3-, CD14- and CD19-positive cells, but only after 18 h of exposure to SPIR. The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF-κB, CEBPβ and MYC. These observations provide new insight into the non-MR-mediated effects of SPIR. PMID:16520746

  16. Immunological effects of the anti-programmed death-1 antibody on human peripheral blood mononuclear cells.

    PubMed

    Akiyama, Yasuto; Nonomura, Chizu; Kondou, Ryota; Miyata, Haruo; Ashizawa, Tadashi; Maeda, Chie; Mitsuya, Koichi; Hayashi, Nakamasa; Nakasu, Yoko; Yamaguchi, Ken

    2016-09-01

    Immune checkpoint antibody-mediated blockade has gained attention as a new cancer immunotherapy strategy. Accumulating evidence suggests that this therapy imparts a survival benefit to metastatic melanoma and non-small cell lung cancer patients. A substantial amount of data on immune checkpoint antibodies has been collected from clinical trials; however, the direct effect of the antibodies on human peripheral blood mononuclear cells (PBMCs) has not been exclusively investigated. In this study, we developed an anti-programmed death-1 (PD-1) antibody (with biosimilarity to nivolumab) and examined the effects of the antibody on PBMCs derived from cancer patients. Specifically, we investigated the effects of the anti-PD-1 antibody on proliferation, cytokine production, cytotoxic T lymphocytes (CTL) and regulatory T cells. These investigations yielded several important results. First, the anti-PD-1 antibody had no obvious effect on resting PBMCs; however, high levels of the anti-PD-1 antibody partly stimulated PBMC proliferation when accompanied by an anti-CD3 antibody. Second, the anti-PD-1 antibody restored the growth inhibition of anti-CD3 Ab-stimulated PBMCs mediated by PD-L1. Third, the anti-PD-1 antibody exhibited a moderate inhibitory effect on the induction of myeloid-derived suppressor cells (MDSCs) by anti-CD3 antibody stimulation. Additionally, the presence of the anti-PD-1 antibody promoted antigen-specific CTL induction, which suggests that combining anti-PD-1 antibody and conventional immunotherapy treatments may have beneficial effects. These results indicate that specific cellular immunological mechanisms are partly responsible for the antitumor effect exhibited by the anti-PD-1 antibody against advanced cancers in clinical trials. PMID:27573705

  17. Reactions of peripheral blood mononuclear cells (PBMC) of camels with monoclonal antibodies against ruminant leukocytes.

    PubMed

    Ungar-Waron, H; Yagil, R; Brenner, J; Paz, R; Partosh, N; Van Creveld, C; Lubashevsky, E; Trainin, Z

    2003-03-01

    The particular immune system of the camel has been but little investigated. In this work circulating camel peripheral blood mononuclear cells (PBMC) were studied by flow cytometry. Monoclonal antibodies (mAbs) raised against ruminant leukocytes were used for the detection of cell surface antigens. Monoclonals to T-cell markers, CD4 (CACT138A) and CD8 (CACT80C), exhibited no reactivity towards camel PBMC in contrast to their reactivity to PBMC of other ruminant species and those of cattle in particular. A relatively high percentage (29.1+/-8.9%) of camel PBMC reacted with a non-immunoglobulin cell surface marker, B-B2, comparable to the reactivity of bovine PBMC. The B-B7 cell marker revealed 22.4+/-10.0% of reactive camel PBMC while the CD45 leukocyte common antigen was identified only on 19.4+/-3.1% of camel PBMC as compared to 74.7+/-4.9% for bovine PBMC. IgM (PIg45A) was detected on 9.1+/-1.4% of camel PBMC and on 46.6+/-19.5% of the bovine PBMC. Double fluorescent labeling with two B-cell markers and an anti-ruminant lambda light-chain mAb revealed 7-9% of cells bearing both B and lambda L-chain markers. Light chain reactivity was also assessed using an anti-goat F(ab')(2) antiserum. The values obtained, 14.3+/-5.8% for the camel and 47.8+/-2.7% for the cattle, are close to the values observed for surface IgM. These data suggest that camels, like other ruminants, possess L-chain bearing cells of the B-cell lineage. However, in the camel, Igs are different in that in addition to regular four chain Igs, about 65% of them possess two heavy chain Igs devoid of light chains. Because different sets of V(H) gene segments are used by four and two chain Igs, it is possible that there might be two lineages of B-cells each secreting a different form of antibodies. PMID:12493494

  18. Various Cell Populations Within the Mononuclear Fraction of Bone Marrow Contribute to the Beneficial Effects of Autologous Bone Marrow Cell Therapy in a Rodent Stroke Model.

    PubMed

    Yang, Bing; Parsha, Kaushik; Schaar, Krystal; Xi, XiaoPei; Aronowski, Jaroslaw; Savitz, Sean I

    2016-08-01

    Cell-based therapies including bone-marrow derived mononuclear cells (MNCs) are now widely being studied because of their pleotropic effects and promising results to improve recovery after stroke in animal models. Unlike other types of cell therapies, MNCs is a mixture of lymphoid, myeloid, erythroid, and stem cell populations. Which cell population(s) accounts for the beneficial effects of MNCs in stroke recovery is unclear. In this paper, we employed a mouse stroke model with middle cerebral artery occlusion (MCAo), and used positively and negatively sorted autologous MNCs by MACs to determine which fractions of the MNCs contribute to their beneficial effects. We evaluated the benefits of neurofunctional recovery produced by individual cell lineages within MNCs in a long-term observation study up to 28 days after stroke. Mortality and modulation of inflammation were also compared among different sub-populations. We further studied the impact of neurotoxicity posed by activated microglia in the presence of different cell lineages within MNCs. We concluded that myeloid cell lineage and stem cell/progenitors appeared to be important components within MNCs that contribute to improved outcomes after stroke. PMID:26997513

  19. Chlamydia pneumoniae enhances the Th2 profile of stimulated peripheral blood mononuclear cells from asthmatic patients.

    PubMed

    Smith-Norowitz, Tamar A; Chotikanatis, Kobkul; Erstein, David P; Perlman, Jason; Norowitz, Yitzchok M; Joks, Rauno; Durkin, Helen G; Hammerschlag, Margaret R; Kohlhoff, Stephan

    2016-05-01

    Chlamydia pneumoniae is a cause of respiratory infection in adults and children. There is evidence for an association between atypical bacterial respiratory pathogens and the pathogenesis of asthma. We compared T helper (Th) responses in C. pneumoniae - infected peripheral blood mononuclear cells (PBMC) in patients with or without asthma. PBMC (1×10(6)/mL) from asthmatic patients (N=11) and non-asthmatic controls (N=12) were infected or mock-infected for 1h +/- C. pneumoniae TW-183 at a multiplicity of infection (MOI)=1 and MOI=0.1, or cultured for 24h +/- Lactobacillus rhamnosus GG (LGG). Interleukin (IL)-4, IL-10, IL-12, Interferon (IFN)-gamma and total IgE levels were measured in supernatants (ELISA). C. pneumoniae infection led to an increase (>50%) of IgE levels in PBMC from asthmatics, compared with mock-infected on day 10; IgE wasn't detected in non-asthmatics. C. pneumoniae - infected PBMC from asthmatics increased levels of IL-4 and IFN-gamma after 24h, compared with PBMC alone; levels of IL-10 and IL-12 were low. When uninfected-PBMC from asthmatics were LGG-stimulated, after 24h, IL-4 was undetectable, but IL-10, IL-12, and IFN-gamma increased, compared with PBMC alone. Thus, C. pneumoniae infection has the ability to induce allergic responses in PBMC of asthmatics, as evidenced by production of Th2 responses and IgE. PMID:26924667

  20. Differential proteomic analysis of respiratory failure in peripheral blood mononuclear cells using iTRAQ technology

    PubMed Central

    SUN, GUOPING; CAO, CUIHUI; CHEN, WENBIAO; ZHANG, YANG; DAI, YONG

    2016-01-01

    Respiratory failure (RF) is a state in which the respiratory system fails by its gas exchange functions. Failure of the lung, which is caused by all types of lung diseases, leads to hypoxaemia with type I respiratory failure. Failure of the pump leads to hypercapnia or type II respiratory failure. Using isobaric tags for relative and absolute quantification (iTRAQ) technology to identify and quantify the total proteins in peripheral blood mononuclear cells (PBMCs) of RF patients and identify the differentially expressed proteome. The present study analyzed the total proteins in the PBMCs of RF patients and healthy controls using the eight-plex iTRAQ added with strong cation-exchange chromatography and liquid chromatography coupled with tandem mass spectrometry. The differentially expressed proteins were identified by MASCOT. A total of 4,795 differentially expressed proteins were identified, and 403 proteins were upregulated and 421 were downregulated. Among them, 4 proteins were significantly differentially expressed, which were upregulated KIAA1520 protein and γ fibrinogen type B (AA at 202) and downregulated chain A, crystal structure of recombinant human platelet factor 4 and myosin regulatory light polypeptide 9. iTRAQ technology is suitable for identifying and quantifying the proteome in the PBMCs of RF patients. The differentially expressed proteins of RF patients have been identified in the present study, and further research of the molecular mechanism of the differentially expressed proteins is required to clarify the pathogenesis and identify novel biomarkers of RF. PMID:27123249

  1. Autologous Bone Marrow Mononuclear Cell Therapy for Autism: An Open Label Proof of Concept Study

    PubMed Central

    Sharma, Alok; Gokulchandran, Nandini; Sane, Hemangi; Nagrajan, Anjana; Kulkarni, Pooja; Shetty, Akshata; Mishra, Priti; Kali, Mrudula; Biju, Hema; Badhe, Prerna

    2013-01-01

    Cellular therapy is an emerging therapeutic modality with a great potential for the treatment of autism. Recent findings show that the major underlying pathogenetic mechanisms of autism are hypoperfusion and immune alterations in the brain. So conceptually, cellular therapy which facilitates counteractive processes of improving perfusion by angiogenesis and balancing inflammation by immune regulation would exhibit beneficial clinical effects in patients with autism. This is an open label proof of concept study of autologous bone marrow mononuclear cells (BMMNCs) intrathecal transplantation in 32 patients with autism followed by multidisciplinary therapies. All patients were followed up for 26 months (mean 12.7). Outcome measures used were ISAA, CGI, and FIM/Wee-FIM scales. Positron Emission Tomography-Computed Tomography (PET-CT) scan recorded objective changes. Out of 32 patients, a total of 29 (91%) patients improved on total ISAA scores and 20 patients (62%) showed decreased severity on CGI-I. The difference between pre- and postscores was statistically significant (P < 0.001) on Wilcoxon matched-pairs signed rank test. On CGI-II 96% of patients showed global improvement. The efficacy was measured on CGI-III efficacy index. Few adverse events including seizures in three patients were controlled with medications. The encouraging results of this leading clinical study provide future directions for application of cellular therapy in autism. PMID:24062774

  2. Gene Expression Profiles from Peripheral Blood Mononuclear Cells Are Sensitive to Short Processing Delays

    PubMed Central

    Grom, Alexei A.; Griffin, Thomas A.; Colbert, Robert A.; Thompson, Susan D.

    2010-01-01

    In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMCs) was assessed by isolating and stabilizing PBMC-derived RNA from 3 individuals either immediately after phlebotomy or after a 4 h delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133 Plus 2.0 GeneChip®. Comparison of gene expression levels (≥2-fold expression change and P < 0.05) identified 307 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 h. These differentially expressed genes include many that are important to inflammatory, immunologic, and cancer pathways. Among others, CCR2, CCR5, TLR10, CD180, and IL-16 have decreased expression, whereas VEGF, IL8, SOCS2, SOCS3, CD69, and CD83 have increased expression after a 4 h processing delay. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis. PMID:21743826

  3. Gene Expression Profiles from Peripheral Blood Mononuclear Cells Are Sensitive to Short Processing Delays.

    PubMed

    Barnes, Michael G; Grom, Alexei A; Griffin, Thomas A; Colbert, Robert A; Thompson, Susan D

    2010-09-29

    In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMCs) was assessed by isolating and stabilizing PBMC-derived RNA from 3 individuals either immediately after phlebotomy or after a 4 h delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133 Plus 2.0 GeneChip(®). Comparison of gene expression levels (≥2-fold expression change and P < 0.05) identified 307 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 h. These differentially expressed genes include many that are important to inflammatory, immunologic, and cancer pathways. Among others, CCR2, CCR5, TLR10, CD180, and IL-16 have decreased expression, whereas VEGF, IL8, SOCS2, SOCS3, CD69, and CD83 have increased expression after a 4 h processing delay. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis. PMID:21743826

  4. Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

    PubMed Central

    Cassart, Dominique; Fett, Thomas; Sarlet, Michaël; Baise, Etienne; Coignoul, Freddy; Desmecht, Daniel

    2007-01-01

    Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction. PMID:17903245

  5. Mitochondrial respiration in peripheral blood mononuclear cells correlates with depressive subsymptoms and severity of major depression

    PubMed Central

    Karabatsiakis, A; Böck, C; Salinas-Manrique, J; Kolassa, S; Calzia, E; Dietrich, D E; Kolassa, I-T

    2014-01-01

    Mitochondrial dysfunction might have a central role in the pathophysiology of depression. Phenotypically, depression is characterized by lack of energy, concentration problems and fatigue. These symptoms might be partially explained by reduced availability of adenosine triphosphate (ATP) as a consequence of impaired mitochondrial functioning. This study investigated mitochondrial respiration in peripheral blood mononuclear cells (PBMCs), an established model to investigate the pathophysiology of depression. Mitochondrial respiration was assessed in intact PBMCs in 22 individuals with a diagnosis of major depression (MD) compared with 22 healthy age-matched controls using high-resolution respirometry. Individuals with MD showed significantly impaired mitochondrial functioning: routine and uncoupled respiration as well as spare respiratory capacity, coupling efficiency and ATP turnover-related respiration were significantly lower in the MD compared with the control group. Furthermore, mitochondrial respiration was significantly negatively correlated with the severity of depressive symptoms, in particular, with loss of energy, difficulties concentrating and fatigue. The results suggest that mitochondrial dysfunction contributes to the biomolecular pathophysiology of depressive symptoms. The decreased immune capability observed in MD leading to a higher risk of comorbidities could be attributable to impaired energy supply due to mitochondrial dysfunction. Thus mitochondrial respiration in PBMCs and its functional consequences might be an interesting target for new therapeutical approaches in the treatment of MD and immune-related comorbidities. PMID:26126180

  6. Influence of gender on ABCC2 expression in peripheral blood mononuclear cells.

    PubMed

    Sudchada, P; Chareanchim, W; Assawamakin, A; Thaipiya, P; Choochaimongkhol, W; Thiplui, N; Sukmangsa, P

    2015-01-01

    It is known that several factors, including gender, may influence the expression of multidrug resistance associated proteins 2 (MRP2/ABCC2) in the peripheral blood mononuclear cells (PBMCs). This study aims to compare ABCC2 gene expression in PBMCs of healthy males and females. PBMCs were extracted from 48 females and 44 males, and gene expression was measured using real-time quantitative polymerase chain reaction (real-time QPCR). Multiple housekeeping genes (Actin-β, β2-M, GAPDH) were utilized as endogenous controls. The stability of housekeeping genes was verified using the Excel-based Bestkeeper® program. Our results showed that expression level of ABCC2 in PBMCs was 1.2-1.4 fold higher in males compared to that in females, depending on the endogenous control(s) used. However, this difference was not statistically significant. When considering using a single endogenous control gene, GAPDH and Actin-βwere found to be more suitable than β2-M. Moreover, GAPDH + Actin-β, or the combination of all three housekeeping gene as endogenous control(s) showed greater stability than other endogenous control genes for normalization of ABCC2 expression in PBMCs. This study suggests that ABCC2 expression in PBMCs may be, in part, influenced by gender, and that at least two endogenous control genes should be utilized for gene expression normalization. PMID:26681017

  7. DNA Damage and Augmented Oxidative Stress in Bone Marrow Mononuclear Cells from Angiotensin-Dependent Hypertensive Mice

    PubMed Central

    Campagnaro, Bianca P.; Tonini, Clarissa L.; Nogueira, Breno V.; Casarini, Dulce E.; Vasquez, Elisardo C.; Meyrelles, Silvana S.

    2013-01-01

    It has been proposed that the nonhemodynamic effects of angiotensin II are important for the damage observed in the two-kidney, one-clip (2K1C) renovascular hypertension model. Much evidence confirms that angiotensin II is directly involved in NAD(P)H oxidase activation and consequent superoxide anion production, which can damage DNA. The current study was performed to examine the effects of angiotensin-II-dependent hypertension in bone marrow mononuclear cells (BM-MNC); dihydroethidium staining was used to assess reactive oxygen species (ROS) production, and the comet assay was used to assess DNA fragmentation in 2K1C hypertensive mice 14 days after renal artery clipping. In this study we demonstrated that 2K1C hypertensive mice have an elevated lymphocyte count, while undifferentiated BM-MNC counts were diminished. 2K1C mice also showed an augmented ROS production and marked BM-MNC DNA fragmentation. In conclusion, endogenous renin angiotensin system activation-induced arterial hypertension is characterized by excessive ROS production in BM-MNC, which might cause marked DNA damage. PMID:23476745

  8. Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells.

    PubMed

    Appavoo, Elamurugan; Hajam, Irshad Ahmed; Muneeswaran, Narayanan Selvaraj; Kondabattula, Ganesh; Bhanuprakash, Veerakyathappa; Kishore, Subodh

    2016-03-01

    High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations. PMID:26639899

  9. Autologous Bone Marrow Mononuclear Cell Transplantation Delays Progression of Carotid Atherosclerosis in Rabbits.

    PubMed

    Cui, Kefei; Ma, Xiao; Yu, Lie; Jiang, Chao; Fu, Chao; Fu, Xiaojie; Yu, Xiaofang; Huang, Yuanjing; Hou, Suyun; Si, Caifeng; Chen, Zhengguang; Yu, Jing; Wan, Jieru; Wang, Jian

    2016-09-01

    Bone marrow mononuclear cells (BMMNCs) can counteract oxidative stress and inhibit the inflammatory response in focal ischemic stroke models. However, the effect of BMMNC transplantation on carotid atherosclerosis needs to be determined. The carotid atherosclerotic plaque model was established in New Zealand White rabbits by balloon injury and 8 weeks of high-fat diet. Rabbits were randomized to receive an intravenous injection of autologous bromodeoxyuridine (BrdU)-labeled BMMNCs or an equal volume of phosphate-buffered saline. Plaques were evaluated for expression of proinflammatory and anti-inflammatory cytokines, anti-oxidant proteins, and markers of cell death. BMMNCs migrated into atherosclerotic plaque on the first day after cell transplantation. BMMNC-treated rabbits had smaller plaques and more collagen deposition than did the vehicle-treated controls on day 28 (p < 0.05). BMMNC treatment significantly increased endothelial nitric oxide synthase and the anti-oxidant enzymes glutathione peroxidase and superoxide dismutase in plaques compared to vehicle treatment on day 7. BMMNC-treated rabbits also had lower levels of cleaved caspase-3 expression; lower levels of proinflammatory cytokines interleukin-1β, tumor necrosis factor alpha, and matrix metalloproteinase 9; and higher levels of insulin-like growth factor-1 and its receptor (p < 0.05). Autologous BMMNC transplantation can suppress the process of atherosclerotic plaque formation and is associated with enhanced anti-oxidative effect, reduced levels of inflammatory cytokines and cleaved caspase-3, and increased expression of insulin-like growth factor-1 and its receptor. BMMNC transplantation represents a novel approach for the treatment of carotid atherosclerosis. PMID:26232064

  10. Comparative genomics analysis of mononuclear phagocyte subsets confirms homology between lymphoid tissue-resident and dermal XCR1(+) DCs in mouse and human and distinguishes them from Langerhans cells.

    PubMed

    Carpentier, Sabrina; Vu Manh, Thien-Phong; Chelbi, Rabie; Henri, Sandrine; Malissen, Bernard; Haniffa, Muzlifah; Ginhoux, Florent; Dalod, Marc

    2016-05-01

    Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1(+) DCs, and between mouse and human Langerhans cells. PMID:26966045

  11. Comparative genomics analysis of mononuclear phagocyte subsets confirms homology between lymphoid tissue-resident and dermal XCR1+ DCs in mouse and human and distinguishes them from Langerhans cells

    PubMed Central

    Carpentier, Sabrina; Vu Manh, Thien-Phong; Chelbi, Rabie; Henri, Sandrine; Malissen, Bernard; Haniffa, Muzlifah; Ginhoux, Florent; Dalod, Marc

    2016-01-01

    Dendritic cells (DC) are mononuclear phagocytes which exhibit a branching (dendritic) morphology and excel at naïve T cell activation. DC encompass several subsets initially identified by their expression of cell surface molecules and later shown to possess distinct functions. DC subset differentiation is orchestrated by transcription factors, growth factors and cytokines. Identifying DC subsets is challenging as very few cell surface molecules are uniquely expressed on any one of these cell populations. There is no standard consensus to identify mononuclear phagocyte subsets; varying antigens are employed depending on the tissue and animal species studied and between laboratories. This has led to confusion in how to accurately define and classify DCs across tissues and between species. Here we report a comparative genomics strategy that enables universal definition of DC and other mononuclear phagocyte subsets across species. We performed a meta-analysis of several public datasets of human and mouse mononuclear phagocyte subsets isolated from blood, spleen, skin or cutaneous lymph nodes, including by using a novel and user friendly software, BubbleGUM, which generates and integrates gene signatures for high throughput gene set enrichment analysis. This analysis demonstrates the equivalence between human and mouse skin XCR1+ DCs, and between mouse and human Langerhans cells. PMID:26966045

  12. Presence of human herpesvirus 6 variants A and B in saliva and peripheral blood mononuclear cells of healthy adults.

    PubMed Central

    Aberle, S W; Mandl, C W; Kunz, C; Popow-Kraupp, T

    1996-01-01

    Saliva and peripheral blood mononuclear cells (PBMCs) from 44 healthy young adults were tested for human herpesvirus 6 variants A and B (HHV-6A and -6B) DNA by a sensitive nested PCR. HHV-6B infection was ascertained in 98% of the subjects, and 95% were found to excrete variant B in their saliva. HHV-6A was found in the PBMCs of 16%, but was not detected in saliva samples. PMID:8940478

  13. Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated proliferation of human peripheral blood mononuclear cells.

    PubMed Central

    Bruzzone, Santina; De Flora, Antonio; Usai, Cesare; Graeff, Richard; Lee, Hon Cheung

    2003-01-01

    Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 microg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD+ levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2-3-fold increases of intracellular calcium ([Ca2+]i), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs. PMID:12852785

  14. Clearance and binding of radiolabeled glycoproteins by cells of the murine mononuclear phagocyte system

    SciTech Connect

    Imber, M.J.

    1982-01-01

    The clearance and binding of radiolabeled lactoferrin and fast ..cap alpha../sub 2/-macroglobulin were studied. Both glycoproteins cleared rapidly following intravenous injection in mice, and both bound specifically to discrete receptors on murine peritoneal macrophages. The simultaneous presence of excess, unlabeled ligands specific for receptors recognizing terminal fucose, mannose, N-acetylglucosamine or galactose residues did not inhibit the clearance or binding of either lactoferrin or fast-..cap alpha../sub 2/M. The clearance and binding of enzymatically defucosylated lactoferrin was indistinguishable from native lactoferrin, indicating that terminal ..cap alpha..(1-3)-linked fucose on lactoferrin is not necessary for receptor recognition. The clearance and binding of two fast -..cap alpha../sub 2/M forms, ..cap alpha../sub 2/M-trypsin and ..cap alpha../sub 2/M-MeNH/sub 2/ cross compete with each other. Saturation binding studies indicated that the total binding of mannosyl -BSA, fusocyl-BSA, and N-acetylglucosaminyl-BSA to macrophages activated by BCG was approximately 15% of the levels observed with inflammatory macrophages elicited by thioglycollate broth. Cross-competition binding studies demonstrated a common surface receptor mediated binding of all three neoglycoprotein ligands and was identical to the receptor on mononuclear phagocytes that binds mannosyl- and N-acetylglucosaminyl-terminated glycoproteins. These results suggest that difference between discrete states of macrophage function may be correlated with selective changes in levels of the surface receptor for mannose-containing glycoproteins.

  15. Lipopolysaccharide-induced early response genes in bovine peripheral blood mononuclear cells implicate GLG1/E-selectin as a key ligand–receptor interaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study uses a systems biology approach, integrating global gene expression information and knowledge of the regulatory events in cells to identify transcription networks controlling peripheral blood mononuclear cells’ (PBMCs) immune response to lipopolysaccharide (LPS) and to identify the molecu...

  16. Peripheral blood mononuclear cells and regulatory T cells in acute viral hepatitis.

    PubMed Central

    Barnaba, V; Tamburrini, E; Laghi, V; Cauda, R; Levrero, M; Ruocco, G; Ortona, L; Balsano, F

    1985-01-01

    During acute viral hepatitis, we observed a significant decrease in OKT4/OKT8 ratio with a significant increase in the OKT8 positive subset in acute type B and non-A-non-B hepatitis. This altered ratio persisted in type B for a long time until HBsAg antibody became detectable, while it soon returned to normal in type A and non-A-non-B hepatitis. In the majority of acute hepatitis the altered ratio is because of an increase and not to a decrease in the whole T cell population, as described in chronic HBV infection. The number of HNK-1 positive cells remained raised during the recovery phase of type B and non-A-non-B hepatitis, a finding consistent with the hypothesis that NK cells play a role in the host defence against B and non-A-non-B virus infections. Serum beta 2-microglobulin concentrations were increased only in acute hepatitis B and non-A-non-B where immunological mechanisms are suspected to be involved, and showed a good correlation with the population of activated OKIa positive cells. PMID:2862096

  17. Utilizing the Trispyrazolyl Borate Ligand for the Mimicking of O2-Activating Mononuclear Nonheme Iron Enzymes.

    PubMed

    Sallmann, Madleen; Limberg, Christian

    2015-10-20

    Mononuclear, O2-activating nonheme iron enzymes are a fascinating class of metalloproteines, capable of realizing the most different reactions, ranging from C-H activation, via O atom transfer to C-C bond cleavage, in the course of O2 activation. They can lead us the way to achieve similar reactions with comparable efficiency and selectivity in chemical laboratories, which would be highly desirable aiming at accessing value-added products or to achieve degradation of unwanted compounds. Hence, these enyzmes motivate attempts to construct artificial low-molecular weight analogues, mimicking structural or functional characteristics. Such models can, for instance, provide insights about which of the features inherent to an active site are essential and guarantee the enzyme function, and from this kind of information the minimal requirements for a biomimetic or bioinspired complex that may be applied in catalysis can be derived. On the other hand, they can contribute to an understanding of the enzyme functioning. In order to create such replicates, it is important to faithfully mimic the surroundings of the iron centers in their active sites. Most of them feature two histidine residues and one carboxylate donor, while a few exhibit a deceptively simple (His)3Fe active site. For the simulation of these, the trispyrazolyl borate ligand (Tp) particularly offers itself, as the facial arrangement of three pyrazole donors is reminiscent of the three histidine-derived imidazole donors. The focus of this Account will be on bioinorganic/biomimetic research from our laboratory utilizing Tp ligands to develop molecular models for (i) two representatives of the (His)3Fe-enzyme family, namely, the cysteine dioxygenase (CDO) and acetyl acetone dioxygenase (Dke1), (ii) a related but less well-explored variant of the CDO-the 2-aminoethanethiol dioxygenase-as well as (iii) the 2-His-1-carboxylate representative 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). The CDO catalyzes the

  18. Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells

    PubMed Central

    Kariuki, Silvia N.; Maranville, Joseph C.; Baxter, Shaneen S.; Jeong, Choongwon; Nakagome, Shigeki; Hrusch, Cara L.; Witonsky, David B.; Sperling, Anne I.; Di Rienzo, Anna

    2016-01-01

    The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax) induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10−8) and rs6451692 on chromosome 5 (p = 2.55 x 10−8), which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDR<0.10 for transcriptional response to 1,25D treatment, which include the transcriptional regulator ets variant 3-like (ETV3L) and EH-domain containing 4 (EHD4). In addition, we identified response eQTLs in vitamin D receptor binding sites near genes differentially expressed in response to 1,25D, such as FERM Domain Containing 6 (FRMD6), which plays a critical role in regulating both cell proliferation and apoptosis. Combining information from the GWAS of Imax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis. PMID:27454520

  19. Mapping Variation in Cellular and Transcriptional Response to 1,25-Dihydroxyvitamin D3 in Peripheral Blood Mononuclear Cells.

    PubMed

    Kariuki, Silvia N; Maranville, Joseph C; Baxter, Shaneen S; Jeong, Choongwon; Nakagome, Shigeki; Hrusch, Cara L; Witonsky, David B; Sperling, Anne I; Di Rienzo, Anna

    2016-01-01

    The active hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is an important modulator of the immune system, inhibiting cellular proliferation and regulating transcription of immune response genes. In order to characterize the genetic basis of variation in the immunomodulatory effects of 1,25D, we mapped quantitative traits of 1,25D response at both the cellular and the transcriptional level. We carried out a genome-wide association scan of percent inhibition of cell proliferation (Imax) induced by 1,25D treatment of peripheral blood mononuclear cells from 88 healthy African-American individuals. Two genome-wide significant variants were identified: rs1893662 in a gene desert on chromosome 18 (p = 2.32 x 10-8) and rs6451692 on chromosome 5 (p = 2.55 x 10-8), which may influence the anti-proliferative activity of 1,25D by regulating the expression of nearby genes such as the chemokine gene, CCL28, and the translation initiation gene, PAIP1. We also identified 8 expression quantitative trait loci at a FDR<0.10 for transcriptional response to 1,25D treatment, which include the transcriptional regulator ets variant 3-like (ETV3L) and EH-domain containing 4 (EHD4). In addition, we identified response eQTLs in vitamin D receptor binding sites near genes differentially expressed in response to 1,25D, such as FERM Domain Containing 6 (FRMD6), which plays a critical role in regulating both cell proliferation and apoptosis. Combining information from the GWAS of Imax and the response eQTL mapping enabled identification of putative Imax-associated candidate genes such as PAIP1 and the transcriptional repressor gene ZNF649. Overall, the variants identified in this study are strong candidates for immune traits and diseases linked to vitamin D, such as multiple sclerosis. PMID:27454520

  20. Analysis of Various Subsets of Circulating Mononuclear Cells in Asymptomatic Coronary Artery Disease

    PubMed Central

    Berezin, Alexander E.; Kremzer, Alexander A.

    2013-01-01

    The objective of this study was to evaluate the correlation between multiple cardiovascular risk factors (MCRFs) and circulating mononuclear cells (CMCs) in asymptomatic coronary artery disease patients. Design and Methods: 126 subjects (54 male), aged 48 to 62 years, with asymptomatic coronary artery disease (CAD) documented previously with angiography, and 25 healthy volunteers were enrolled in the study. The flow cytometric technique was used for predictably distinguishing cell subsets that depend on the expression of CD14, CD34, Tie-2, CD45, and CD309 (VEGFR2). Results: The analysis of the outcome obtained shows a trend of an increase in circulating CD45−CD34+ CMCs and a reduction in CMC population defined as CD14+CD309+ and CD14+CD309+Tie2+ in known asymptomatic CAD patients in comparison with healthy volunteers. Substantial correlations between CD45−CD34+ and conventional cardiovascular risk factors (hs-CRP, T2DM, serum uric acid and hypertension) were found in the patient cohort. The concentrations of CD14+CD309+ and CD14+CD309+Tie2+ CMCs had effect on such factors as T2DM (RR = 1.21; 95% CI = 1.10–1.40; p = 0.008), hs-CRP > 2.54 mg/L (RR = 1.29; 95% CI = 1.12–1.58; p = 0.006), Agatston score index (RR = 1.20; 95% CI = 1.15–1.27; p = 0.034), and occurrence of three and more cardiovascular risk factors (RR = 1.31; 95% CI = 1.12–1.49; p = 0.008). Conclusion: It is postulated that the reduction in circulating CD14+CD309+ and CD14+CD309+Tei2+ CMCs is related to a number of cardiovascular risk factors in asymptomatic patients with known CAD. PMID:26237060

  1. Increased apoptosis of peripheral blood mononuclear cells (PBMC) during general and epidural anaesthesia in dogs.

    PubMed

    Simeonova, Galina P; Slavov, Emil; Usunov, Roustislav; Halacheva, Krasimira; Dinev, Dinco N

    2008-12-01

    The objective of the study was to investigate the hypothesis that perioperative lymphocytopenia was due to apoptosis of these cells induced by either halothane or epidural anaesthesia in dogs. The relationship between apoptosis induction and plasma concentrations of the stress hormone cortisol and the cytokines TNF-alpha and IL-10 was examined as well. The study was performed on 22 healthy mongrel dogs, equal numbers from both genders, weighing 18.3 +/- 2.9 kg, and aged between 3-5 years. Dogs were divided in three groups. Eight of the animals were anaesthetized with halothane, another eight received epidural anaesthesia using lidocaine, and six served as controls. Venous blood samples were obtained immediately before (0 minute) anaesthesia, during deep anaesthesia (120 minute), and on the next day (24 hour) in order to determine the following parameters: the total lymphocyte counts, the percentage of apoptotic peripheral blood mononuclear cells (PBMC) by flow cytometry, plasma concentrations of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) by enzyme-linked immunosorbent assay (ELISA), and plasma cortisol levels by radioimmune assay. Both halothane and epidural anaesthesia in dogs induces apoptosis of PBMC with slight decrease in total lymphocyte counts. These immunomodulatory effects were transient and faded till the 24th hour. Concerning the mechanism of inducing lymphocyte apoptosis by general or epidural anaesthesia, it seemed that neither cortisol, nor the tested cytokines TNF-alpha and IL-10 were implicated in this process. Further investigations are necessary to confirm this assumption. PMID:18491210

  2. Cytokines profile and peripheral blood mononuclear cells morphology in Rett and autistic patients.

    PubMed

    Pecorelli, Alessandra; Cervellati, Franco; Belmonte, Giuseppe; Montagner, Giulia; Waldon, PhiAnh; Hayek, Joussef; Gambari, Roberto; Valacchi, Giuseppe

    2016-01-01

    A potential role for immune dysfunction in autism spectrum disorders (ASD) has been well established. However, immunological features of Rett syndrome (RTT), a genetic neurodevelopmental disorder closely related to autism, have not been well addressed yet. By using multiplex Luminex technology, a panel of 27 cytokines and chemokines was evaluated in serum from 10 RTT patients with confirmed diagnosis of MECP2 mutation (typical RTT), 12 children affected by classic autistic disorder and 8 control subjects. The cytokine/chemokine gene expression was assessed by real time PCR on mRNA of isolated peripheral blood mononuclear cells (PBMCs). Moreover, ultrastructural analysis of PBMCs was performed using transmission electron microscopy (TEM). Significantly higher serum levels of interleukin-8 (IL-8), IL-9, IL-13 were detected in RTT compared to control subjects, and IL-15 shows a trend toward the upregulation in RTT. In addition, IL-1β and VEGF were the only down-regulated cytokines in autistic patients with respect to RTT. No difference in cytokine/chemokine profile between autistic and control groups was detected. These data were also confirmed by ELISA real time PCR. At the ultrastructural level, the most severe morphological abnormalities were observed in mitochondria of both RTT and autistic PBMCs. In conclusion, our study shows a deregulated cytokine/chemokine profile together with morphologically altered immune cells in RTT. Such abnormalities were not quite as evident in autistic subjects. These findings indicate a possible role of immune dysfunction in RTT making the clinical features of this pathology related also to the immunology aspects, suggesting, therefore, novel possible therapeutic interventions for this disorder. PMID:26471937

  3. Characterisation of the Immunomodulatory Effects of Meningococcal Opa Proteins on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells

    PubMed Central

    Lewis, Susan; Payne, Isabelle; Saleem, Muhammad; Derrick, Jeremy P.; Pollard, Andrew J.

    2016-01-01

    Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins. PMID:27111850

  4. An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics.

    PubMed

    Reed, Daniel M; Paschalaki, Koralia E; Starke, Richard D; Mohamed, Nura A; Sharp, Giles; Fox, Bernard; Eastwood, David; Bristow, Adrian; Ball, Christina; Vessillier, Sandrine; Hansel, Trevor T; Thorpe, Susan J; Randi, Anna M; Stebbings, Richard; Mitchell, Jane A

    2015-06-01

    There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine. Current assays also do not fully delineate mild (such as Campath) and severe (such as TGN1412) cytokine storm-inducing drugs. Here, we report a novel bioassay where endothelial cells grown from stem cells in the peripheral blood (blood outgrowth endothelial cells) and PBMCs from the same donor can be used to create an autologous coculture bioassay that responds by releasing a plethora of cytokines to authentic TGN1412 but only modestly to Campath and not to control antibodies such as Herceptin, Avastin, and Arzerra. This assay performed better than the traditional mixed donor assay in terms of cytokine release to TGN1412 and, thus, we suggest provides significant advancement and a definitive system by which biologics can be tested and paves the way for personalized medicine. PMID:25746794

  5. A series of mononuclear Co(III) complexes using tridentate N,O-donor ligands: chemical properties and cytotoxicity activity.

    PubMed

    Souza, Elizabeth Teixeira; Maia, Paulo José Sousa; Azevedo, Erica Melo; Kaiser, Carlos Roland; Resende, Jackson Antônio Lamounier Camargos; Pinheiro, Carlos Basílio; Heinrich, Tassiele Andrea; da Silva, Roberto Santana; Scarpellini, Marciela

    2011-12-01

    Continuing our interest in tridentate ligands to develop new prototypes of cobalt-based metallodrugs for combating cancer, modifications in the backbone of HL1, [(2-hydroxybenzyl)(2-(pyridil-2-yl)ethyl]amine) were proposed in order to modulate the redox potential of new Co(III) complexes. Three ligands with electron withdrawing groups were synthesized: HL2: [(2-hydroxy-5-nitrobenzyl)(2-(pyridil-2-yl)ethyl]amine); HL3: [(2-hydroxybenzyl)(2-(pyridil-2-yl)ethyl]imine) and HL4: [(2-hydroxy-5-nitrobenzyl)(2-(pyridil-2-yl)ethyl]imine). They were used to obtain the respective mononuclear complexes 2, 3 and 4, which are discussed compared to the previous reported complex 1 (obtained from HL1). The new complexes were characterized and studied by several techniques including X-ray crystallography, elemental and conductimetric analysis, IR, UV-vis and (1)H NMR spectroscopies, and electrochemistry. The substitutions of the group in the para position of the phenol (HL1 and HL2) and the imine instead of the amine (HL3 and HL4), promote anodic shifts in the complexes reduction potentials. The influence of these substitutions in the biological activities of the Co(III) complexes against the murine melanoma cell line (B16F10) was also evaluated. Little effect was observed on cellular viability decrease for all free ligands, however the coordination to Co(III) enhances their activities in the following range: 1>4≈2>3. The data suggest that no straight correlation can be addressed between the reduction potential of the Co(III) center and the cell viability. PMID:22001498

  6. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells

    PubMed Central

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H. Lie; Smit, Jolanda M.; Rodenhuis-Zybert, Izabela A.

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  7. Effect of Vitamin D on Peripheral Blood Mononuclear Cells from Patients with Psoriasis Vulgaris and Psoriatic Arthritis

    PubMed Central

    Cubillos, Susana; Krieg, Nadine; Norgauer, Johannes

    2016-01-01

    Background Psoriasis, a chronic skin disease with or without joint inflammation, has increased circulating proinflammatory cytokine levels. Vitamin D is involved in calcium homeostasis, bone formation, osteoclastogenesis and osteoclast activity, as well as regulation of immune response. We aimed to study osteoclast differentiation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris and psoriatic arthritis, in response to 1,25(OH)2D3. Methods Serum levels of bone turnover markers were measured by ELISA in patients with psoriasis vulgaris and psoriatic arthritis, and healthy controls. PBMCs were isolated and cultured with or without RANKL/M-CSF and 1,25(OH)2D3. Osteoclast differentiation and cytokine secretion were assessed. Results Psoriatic arthritis patients had lower osteocalcin, as well as higher C-telopeptide of type I collagen and cathepsin K serum levels compared with psoriasis vulgaris patients and controls. RANKL/M-CSF-stimulated PBMCs from psoriatic arthritis patients produced higher proinflammatory cytokine levels and had a differential secretion profile in response to 1,25(OH)2D3, compared with psoriasis vulgaris and control PBMCs. Conclusions Our data confirmed altered bone turnover in psoriatic arthritis patients, and demonstrated increased osteoclastogenic potential and proinflammatory cytokine secretion capacity of these PBMCs compared with psoriasis vulgaris and controls. 1,25(OH)2D3 abrogated these effects. PMID:27050092

  8. Posttraumatic stress disorder is associated with an enhanced spontaneous production of pro-inflammatory cytokines by peripheral blood mononuclear cells

    PubMed Central

    2013-01-01

    Background Posttraumatic stress disorder (PTSD) is associated with an enhanced risk for cardiovascular and other inflammatory diseases. Chronic low-level inflammation has been suggested as a potential mechanism linking these conditions. Methods We investigated plasma cytokine levels as well as spontaneous and lipopolysaccharide (LPS)-stimulated cytokine production by peripheral blood mononuclear cells (PBMCs) in a group of 35 severely traumatized PTSD patients compared to 25 healthy controls. Results Spontaneous production of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α by isolated PBMCs was significantly higher in the PTSD compared to the control group and even correlated with PTSD symptom severity within the PTSD group. In contrast, circulating plasma levels of pro- and anti-inflammatory cytokines such as IL-6, IL-8, IL-10, TNF-α, or monocyte chemotactic protein (MCP)-1 were not significantly altered in PTSD patients compared to healthy controls. Conclusions Our findings indicate that PBMCs of PTSD patients are already pre-activated in vivo, providing further evidence for low-grade inflammation in PTSD. This might possibly represent one psychobiological pathway from PTSD to poor physical health. PMID:23360282

  9. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells.

    PubMed

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H Lie; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  10. Inhibitor treatment of peripheral mononuclear cells from Parkinson’s disease patients further validates LRRK2 dephosphorylation as a pharmacodynamic biomarker

    PubMed Central

    Perera, G.; Ranola, M.; Rowe, D. B.; Halliday, G. M.; Dzamko, N.

    2016-01-01

    Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with increased risk of Parkinson’s disease (PD). Thus, LRRK2 kinase inhibitors are in development as potential Parkinson’s disease therapeutics. A reduction in the constitutive levels of phosphorylation on leucine-rich repeat kinase 2 (LRRK2) is currently used to measure target engagement of LRRK2 kinase inhibitors in cell and animal models. We aimed to determine if reduced phosphorylation of LRRK2 following inhibitor treatment is also a valid measure of target engagement in peripheral mononuclear cells from Parkinson’s disease patients. Peripheral mononuclear cells from idiopathic Parkinson’s disease patients and controls were treated ex vivo with two structurally distinct inhibitors of LRRK2, at four different doses, and immunoblotting was used to assess the reduction in LRRK2 phosphorylation at Ser910, Ser935, Ser955 and Ser973. Both inhibitors showed no acute toxicity in primary cells and both inhibitors reduced the constitutive phosphorylation of LRRK2 at all measured residues equally in both control and Parkinson’s disease groups. Measuring the reduction in LRRK2 phosphorylation resulting from LRRK2 kinase inhibition, is thus a valid measure of acute peripheral target engagement in Parkinson’s disease patients. This is important if LRRK2 kinase inhibitors are to be used in a clinical setting. PMID:27503089

  11. Interleukin-12 suppresses immunoglobulin E production but enhances immunoglobulin G4 production by human peripheral blood mononuclear cells.

    PubMed Central

    de Boer, B A; Kruize, Y C; Rotmans, P J; Yazdanbakhsh, M

    1997-01-01

    The effect of interleukin-12 (IL-12) on human immunoglobulin G4 (IgG4) and IgE production was examined with cells derived from filarial patients and European controls. IL-12 inhibited IgE release but enhanced IgG4 production in cultures of peripheral blood mononuclear cells stimulated with anti-CD2 plus IL-2. When purified T- and B-cell cocultures were examined, IL-12 again markedly enhanced IgG4, whereas IgE production was no longer inhibited. PMID:9038328

  12. Radiofrequency currents exert cytotoxic effects in NB69 human neuroblastoma cells but not in peripheral blood mononuclear cells

    PubMed Central

    HERNÁNDEZ-BULE, MARÍA LUISA; ROLDÁN, ERNESTO; MATILLA, JOAQUÍN; TRILLO, MARÍA ÁNGELES; ÚBEDA, ALEJANDRO

    2012-01-01

    Recently, a number of electric and electrothermal therapies have been applied to the treatment of specific cancer types. However, the cellular and molecular mechanisms involved in the response to such therapies have not been well characterized yet. Capacitive-resistive electric transfer (CRET) therapy uses electric currents at frequencies within the 0.45–0.6 MHz range to induce hyperthermia in target tissues. Preliminary trials in cancer patients have shown consistent signs that CRET could slow down growth of tumor tissues in brain gliomas, without inducing detectable damage in the surrounding healthy tissue. Previous studies by our group have shown that subthermal treatment with 0.57-MHz electric currents can induce a cytostatic, not cytotoxic response in HepG2 human hepatocarcinoma cells; such effect being mediated by cell cycle alterations. In contrast, the study of the response of NB69 human neuroblastoma cells to the same electric treatment revealed consistent indications of cytotoxic effects. The present study extends the knowledge on the response of NB69 cells to the subthermal stimulus, comparing it to that of primary cultures of human peripheral blood mononuclear cells (PBMC) exposed to the same treatment. The results showed no sensitivity of PBMC to the 0.57 MHz subthermal currents and confirmed that the treatment exerts a cytotoxic action in NB69 cells. The data also revealed a previously undetected cytostatic response of the neuroblastoma cell line. CRET currents affected NB69 cell proliferation by significantly reducing the fraction of cells in the phase G2/M of the cell cycle at 12 h of exposure. These data provide new information on the mechanisms of response to CRET therapy, and are consistent with a cytotoxic and/or cytostatic action of the electric treatment, which would affect human cells of tumor origin but not normal cells with a low proliferation rate. PMID:22843038

  13. The Peripheral Blood Mononuclear Cell Count Is Associated With Bone Health in Elderly Men

    PubMed Central

    Lin, Xianfeng; Yu, Hejun; Zhao, Chenchen; Qian, Yu; Hong, Dun; Huang, Kangmao; Mo, Jian; Qin, An; Fang, Xiangqian; Fan, Shunwu

    2016-01-01

    Abstract The peripheral blood mononuclear cell (PBMC) count is a routinely used and meaningful index for infection and blood diseases. PBMCs may be closely related to osteoclasts and include osteoclast precursors; we examined the association between the PBMC count and bone health. This research included 2806 community men aged ≥50 years who underwent full health examinations from October 2007 through December 2011 in four medical centers. The PBMC count was significantly high among subjects with “at least osteopenia” compared with controls. In analysis of covariance adjusted for potential confounders, the bone mineral density (BMD) value and T-score had a significant decreasing trend across the quartiles of PBMC count. In univariate analysis, the PBMC count had a strong association with “at least osteopenia” (odds ratio [OR] = 2.520, 95% confidence interval [CI]: 1.397–4.547). After adjustment for confounding factors (multivariate analysis) from Model 1 to 4, PBMC count remained as an independent risk factor for “at least osteopenia” (OR = 2.481, 95% CI: 1.176–5.236). Moreover, after adjusting for all confounding variables, participants had a significantly high OR in the body mass index (BMI) <25 group (OR = 2.798, CI: 1.122–6.973; P = 0.027) and systolic blood pressure (SBP) <140 group (OR = 2.519, CI: 1.059–5.993; P = 0.037). In conclusion, the PBMC count is significantly associated with bone loss in elderly men and the exact mechanism requires further clarification. PMID:27082593

  14. Serum Uric Acid Predicts Declining of Circulating Proangiogenic Mononuclear Progenitor Cells in Chronic Heart Failure Patients

    PubMed Central

    Berezin, Alexander E.; Kremzer, Alexander A.; Samura, Tatyana A.; Berezina, Tatyana A.; Martovitskaya, Yulia V.

    2014-01-01

    Introduction: Serum uric acid (SUA) is considered a marker for natural progression of chronic heart failure (CHF) mediated cardiovascular remodelling. CHF associates with declining of circulating mononuclear progenitor cells (MPCs). The objective of this study was to evaluate the interrelationship between SUA concentrations and proangiogenic MPCs in ischemic CHF patients. Methods: The study population was structured retrospectively after determining the coronary artery disease (CAD) by contrast-enhanced spiral computed tomography angiography in 126 subjects with symptomatic ischemic mild-to-severe CHF and 128 CAD subjects without CHF. Baseline biomarkers were measured in all patients. Cox proportional multivariate hazard ratio was calculated for predictors of MPCs declining in both CHF and non-CHF patient population predictors of MPCs declining in CHF subjects were examined in stepwise logistic regression. C-statistics, integrated discrimination indices (IDI) and net-reclassification improvement were utilized for prediction performance analyses. Results: Cox proportional adjusted hazard ratio analyses for CD14+CD309+ and CD14+CD309+Tie2+ MPCs by SUA has shown that the higher quartiles (Q3 and Q4) of SUA compared to the lower quartiles (Q1 and Q2) are associated with increased risks of depletion of both CD14+CD309+ and CD14+CD309+Tie2+ MPCs. The addition of Q4 SUA to the ABC model improved the relative IDI by 13.8% for depletion of CD14+CD309+ MPCs and by 14.5% for depletion of CD14+CD309+Tie2+ MPCs. Conclusion: Circulating levels of proangiogenic MPCs are declined progressively depending on the levels of SUA in the HF subjects with CHF. We suggest that even mild elevations of SUA might be used to predict of relative depletion of proangiogenic MPCs among chronic HF patients. PMID:25320662

  15. Monocyte chemoattractant protein 1 and interleukin-8 production in mononuclear cells stimulated by oral microorganisms.

    PubMed Central

    Jiang, Y; Russell, T R; Graves, D T; Cheng, H; Nong, S H; Levitz, S M

    1996-01-01

    Chemokines are a family of low-molecular-weight proinflammatory cytokines that stimulate recruitment of leukocytes. The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) are relatively specific chemoattractants for neutrophils and monocytes, respectively. Chemokine expression contributes to the presence of different leukocyte populations observed in normal and pathologic states. In the present studies, peripheral blood mononuclear cells (PBMC) were stimulated by microbes (Candida albicans, Streptococcus mutans, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans) selected based upon their importance as oral pathogens. IL-8 and MCP-1 gene expression and protein release were determined by Northern blot (RNA blot) analysis and enzyme-linked immunosorbent assay. C. albicans, P. gingivalis, and A. actinomycetemcomitans induced high levels of production of both MCP-1 and IL-8. S. mutans was a strong inducer of MCP-1, but it did not stimulate significant production of IL-8. C. albicans, S. mutans, and A. actinomycetemcomitans were 500 to 5,000 times more potent than P. gingivalis in terms of MCP-1 production. In general, the microbe-to-PBMC ratios required for maximum gene expression of MCP-1 were lower than those for IL-8. However, for actual protein release of MCP-1 versus IL-8, differences in the effects of various microbe concentrations were observed only for A. actinomycetemcomitans. These results demonstrate that different oral pathogens induce specific dose-dependent patterns of chemokine gene expression and release. Such patterns may help explain the immunopathology of oral infections, particularly with regard to inflammatory leukocyte recruitment. PMID:8890191

  16. Synergistic Effects of Calcineurin Inhibitors and Steroids on Steroid Sensitivity of Peripheral Blood Mononuclear Cells.

    PubMed

    Takeuchi, Hironori; Iwamoto, Hitoshi; Nakamura, Yuki; Hirano, Toshihiko; Konno, Osamu; Kihara, Yu; Chiba, Naokazu; Yokoyama, Takayoshi; Takano, Kiminori; Toraishi, Tatsunori; Okuyama, Kiyoshi; Ikeda, Chie; Tanaka, Sachiko; Onda, Kenji; Soga, Akiko; Kikuchi, Yukiko; Kawaguchi, Takashi; Kawachi, Shigeyuki; Unezaki, Sakae; Shimazu, Motohide

    2015-02-01

    The steroid receptor (SR) complex contains FKBP51 and FKBP52, which bind to tacrolimus (TAC) and cyclophilin 40, which, in turn, bind to cyclosporine (CYA); these influence the intranuclear mobility of steroid-SR complexes. Pharmacodynamic interactions are thought to exist between steroids and calcineurin inhibitors (CNIs) on the SR complex. We examined the effect of CNIs on steroid sensitivity. Methylprednisolone (MPSL) sensitivity was estimated as the concentration inhibiting mitosis in 50% (IC50) of peripheral blood mononuclear cells and as the area under the MPSL concentration-proliferation suppressive rate curves (CPS-AUC) in 30 healthy subjects. MPSL sensitivity was compared between the additive group (AG) as the MPSL sensitivity that was a result of addition of the proliferation suppressive rate of CNIs to that of MPSL and the mixed culture group (MCG) as MPSL sensitivity of mixed culture with both MPSL and CNIs in identical patients. IC50 values of MPSL and cortisol sensitivity were examined before and 2 months after CNI administration in 23 renal transplant recipients. IC50 and CPS-AUC values of MPSL were lower in the MCG than in the AG with administration of TAC and CYA. The CPS-AUC ratio of MCG and AG was lower in the TAC group. IC50 values of MPSL and cortisol tended to be lower after administration of TAC and CYA, and a significant difference was observed in the IC50 of cortisol after TAC administration. Steroid sensitivity increased with both TAC and CYA. Furthermore, TAC had a greater effect on increasing sensitivity. Thus, concomitant administration of CNIs and steroids can increase steroid sensitivity. PMID:26858893

  17. Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris.

    PubMed

    Walsh, Catherine J; Stuckey, Joyce E; Cox, Heather; Smith, Brett; Funke, Christina; Stott, Jeff; Colle, Clarence; Gaspard, Joseph; Manire, Charles A

    2007-08-15

    Florida manatees (Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-gamma, or TNF-alpha, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), L-NIL (L-N(6)-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal. PMID:17614139

  18. Cultures and co-cultures of human blood mononuclear cells and endothelial cells for the biocompatibility assessment of surface modified AISI 316L austenitic stainless steel.

    PubMed

    Stio, Maria; Martinesi, Maria; Treves, Cristina; Borgioli, Francesca

    2016-12-01

    Samples of AISI 316L austenitic stainless steel were subjected either to grinding and polishing procedure, or to grinding and then low temperature glow-discharge nitriding treatment, or to grinding, nitriding and subsequently coating with collagen-I. Nitrided samples, even if only ground, show a higher corrosion resistance in PBS solution, in comparison with ground and polished AISI 316L. Biocompatibility was evaluated in vitro by incubating the samples with either peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC), tested separately or in co-culture. HUVEC-PBMC co-culture and co-incubation of HUVEC with PBMC culture medium, after the previous incubation of PBMC with metallic samples, allowed to determine whether the incubation of PBMC with the different samples might affect HUVEC behaviour. Many biological parameters were considered: cell proliferation, release of cytokines, matrix metalloproteinases (MMPs) and sICAM-1, gelatinolytic activity of MMPs, and ICAM-1 protein expression. Nitriding treatment, with or without collagen coating of the samples, is able to ameliorate some of the biological parameters taken into account. The obtained results point out that biocompatibility may be successfully tested in vitro, using cultures of normal human cells, as blood and endothelial cells, but more than one cell line should be used, separately or in co-culture, and different parameters should be determined, in particular those correlated with inflammatory phenomena. PMID:27612806

  19. Altered expression of intracellular Toll-like receptors in peripheral blood mononuclear cells from patients with alopecia areata

    PubMed Central

    Alzolibani, Abdullateef A.; Rasheed, Zafar; Saif, Ghada Bin; Al-Dhubaibi, Mohammed S.; Al Robaee, Ahmad A.

    2016-01-01

    Background Toll-like receptors (TLRs) are pattern-recognition-receptors that sense a variety of pathogens and initiation of innate and adaptive immune responses. This study was undertaken to investigate the expression of TLRs in peripheral blood-mononuclear cells (PBMCs) of AA patients and to determine whether TLR-mediated inflammatory signals are important for the perspective of AA management. Methods Gene expression of TLRs and T-helper (Th) type-1, Th-2, Th-17 and regulatory T-cell cytokines in PBMCs was quantified by TaqMan Assays. Production of these cytokines in serum samples was determined by sandwich ELISAs. Results All TLRs (TLRs 1–10) were expressed in PBMCs of AA patients. Importantly intracellular TLRs (TLRs 3, 7, 8 and 9) were significantly up-regulated in AA patients as compared with controls (p < 0.05). Interleukin (IL)-2, TNF-α, and IL-17A gene expression in patients' PBMCs and their secretion in patients' sera were significantly higher as compared with their respective controls (p < 0.05). Whereas, TGF-β gene expression in patients' PBMCs and TGF-β protein level in patients' sera were significantly lower as compared with their controls (p < 0.05). Conclusion This is the first report that shows the comprehensive expression profile of TLRs in AA patients. We conclude that up-regulated expression of intracellular TLRs in PBMCs of AA patients may play an active role in abnormal regulation of Th-1, Th-17 and regulatory T-cell cytokines in alopecia areata. General significance Targeting of TLRs and their associated inflammatory signaling will open new areas of research; this may lead to the development of novel therapeutic targets for the treatment of AA or other skin disorders. PMID:27114923

  20. Sperm Cells Induce Distinct Cytokine Response in Peripheral Mononuclear Cells from Infertile Women with Serum Anti-Sperm Antibodies

    PubMed Central

    Kverka, Miloslav; Ulcova-Gallova, Zdenka; Bartova, Jirina; Cibulka, Jan; Bibkova, Katarina; Micanova, Zdenka; Tlaskalova-Hogenova, Helena

    2012-01-01

    Background and Aims Anti-sperm antibodies in can markedly reduce the likelihood of natural conception. The etiology of this anti-sperm immunity in human females is unknown. We compared the cytokine response of peripheral blood mononuclear cells (PBMCs) from infertile patients with or without anti-sperm antibodies (ASA) and fertile women. Methodology/Principal Findings We cultivated the PBMCs together with sperm antigens (whole cells or cell lysate), and screened the supernatants for 40 cytokines by antibody array. When stimulated with whole sperm cells, the PBMCs from patients with ASA produce less IL-3, IL-11, IL-13, ICAM-1, GCSF and more IL-2, IL-4 and IL-12p70 as compared to healthy women. PBMCs from patients with ASA produce typically less IL-13, IL-7, IL-17 and MIG, and more MIP-1β and IL-8, as compared to PBMCs from patients without ASA. In response to sperm cell lysate, PBMCs from infertile women without ASA respond initially by increase in production of growth factors (GCSF, GM-CSF and PDGF-BB) followed by increase in chemokines (e.g. IL-8, MCP-1 and MIP-1β). Conclusions Cellular immune responses to sperm antigens, measured by production of cytokines, differ among infertile women with ASA, infertile women without ASA and healthy women. This difference could play an important role in the initial steps of the infertility pathogenesis. PMID:22952917

  1. Functional canine dendritic cells can be generated in vitro from peripheral blood mononuclear cells and contain a cytoplasmic ultrastructural marker.

    PubMed

    Ibisch, C; Pradal, G; Bach, J M; Lieubeau, B

    2005-03-01

    For physiological and practical reasons the dog is a large animal model used increasingly to study the pathogenesis of human diseases and new therapeutic approaches, in particular for immune disorders. However, some immunological resources are lacking in this model, especially concerning dendritic cells. The aim of our study was to develop an efficient method to generate dendritic cells (DC) in vitro from dog peripheral blood mononuclear cells (PBMC) and to characterize their functional, structural and ultrastructural properties. PBMC were cultured in vitro with IL-4 and GM-CSF. After 1 week of culture, a great proportion of non-adherent cells displayed typical cytoplasmic processes, as evidenced both by optical and electron microscopy. Cytometric analysis revealed the presence of 41.7+/-24.6% CD14+ cells expressing both CD11c and MHC class II molecules. Allogeneic mixed lymphocyte reactions confirmed the ability of these cultures to stimulate the proliferation of allogeneic lymphocytes as already reported as a characteristic of DC in other species. In addition, we describe for the first time the presence in canine DC of cytoplasmic periodic microstructures (PMS) that could represent ultrastructural markers of canine DC. In conclusion, our study provides an easy method to generate DC from PBMC in sufficient numbers for immunological in vitro investigations in dogs, a pre-clinical model for many human diseases. PMID:15847807

  2. 1,25-Dihydroxyvitamin D increases the gene expression of enzymes protecting from glucolipotoxicity in peripheral blood mononuclear cells and human primary endothelial cells.

    PubMed

    Kuricova, Katarina; Pleskacova, Anna; Pacal, Lukas; Kankova, Katerina

    2016-06-15

    Besides its classical function as an orchestrator of calcium and phosphorus homeostasis, vitamin D also affects insulin secretion and tissue efficiency. A number of studies have consistently reported the inverse relationship between vitamin D deficiency and type 2 diabetes. Activation of certain metabolic pathways and down-stream transcription factors may protect from glucolipotoxicity and their targeted activation -e.g. by vitamin D - might explain the detrimental role of vitamin D deficiency in diabetes. The aim of the study was to quantify gene and protein expression of selected enzymes involved in the protection from glucolipotoxicity, specifically glyoxalase 1 (GLO1), and other enzymes with antioxidant activity - hemoxygenase (HMOX), thiamin pyrophosphokinase (TPK1) and transketolase (TKT), under normo- and hyperglycemic conditions and upon addition of vitamin D in peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC). The results of our study indicate that the active form of vitamin D regulates gene expression of enzymes opposing the harmful effect of glucolipotoxicity whose activities appear to be suppressed by hyperglycemia. However, we were unable to confirm this effect on protein expression. While we cannot speculate on the effect of vitamin D on diabetes itself our results support its role in the protection against existing glucolipotoxicity therefore possibly translating into the prevention of development of diabetic complications. PMID:26952188

  3. Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects.

    PubMed

    Wallace, Fiona A; Miles, Elizabeth A; Calder, Philip C

    2003-05-01

    Studies on animal and human subjects have shown that greatly increasing the amount of linseed (also known as flaxseed) oil (rich in the n-3 polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALNA)) or fish oil (FO; rich in the long-chain n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) in the diet can decrease a number of markers of immune function. The immunological effects of more modest doses of n-3 PUFA in human subjects are unclear, dose-response relationships between n-3 PUFA supply and immune function have not been established and whether ALNA has the same effects as its long-chain derivatives is not known. Therefore, the objective of the present study was to determine the effect of enriching the diet with different doses of FO or with a modest dose of ALNA on a range of functional responses of human monocytes and lymphocytes. In a randomised, placebo-controlled, double-blind, parallel study, forty healthy males aged 18-39 years were randomised to receive placebo or 3.5 g ALNA/d or 0.44, 0.94 or 1.9 g (EPA+DHA)/d in capsules for 12 weeks. The EPA:DHA ratio in the FO used was 1.0:2.5. ALNA supplementation increased the proportion of EPA but not DHA in plasma phospholipids. FO supplementation decreased the proportions of linoleic acid and arachidonic acid and increased the proportions of EPA and DHA in plasma phospholipids. The interventions did not alter circulating mononuclear cell subsets or the production of tumour necrosis factor-alpha, interleukin (IL) 1beta, IL-2, IL-4, IL-10 or interferon-gamma by stimulated mononuclear cells. There was little effect of the interventions on lymphocyte proliferation. The two higher doses of FO resulted in a significant decrease in IL-6 production by stimulated mononuclear cells. It is concluded that, with the exception of IL-6 production, a modest increase in intake of either ALNA or EPA+DHA does not influence the functional activity of mononuclear cells. The threshold of EPA+DHA intake that

  4. Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

    PubMed

    Maslova, E V; Andreeva, E R; Andrianova, I V; Bobyleva, P I; Romanov, Yu A; Kabaeva, N V; Balashova, E E; Ryaskina, S S; Dugina, T N; Buravkova, L B

    2014-02-01

    We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine. PMID:24771453

  5. Telomete length in peripheral blood mononuclear cells is associated with folate status in men

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human chromosomes are capped by tandem repeats of DNA and associated proteins termed telomeres. The length of the telomeres is reduced with increasing cell divisions except when the enzyme telomerase is active as seen in stem cells and germ cells. Telomere dysfunction has been associated with deve...

  6. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    SciTech Connect

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  7. Coxsackievirus group B type 3 infection upregulates expression of monocyte chemoattractant protein 1 in cardiac myocytes, which leads to enhanced migration of mononuclear cells in viral myocarditis.

    PubMed

    Shen, Yan; Xu, Wei; Chu, Yi-Wei; Wang, Ying; Liu, Quan-Sheng; Xiong, Si-Dong

    2004-11-01

    Coxsackievirus group B type 3 (CVB3) is an important cause of viral myocarditis. The infiltration of mononuclear cells into the myocardial tissue is one of the key events in viral myocarditis. Immediately after CVB3 infects the heart, the expression of chemokine(s) by infected myocardial cells may be the first trigger for inflammatory infiltration and immune response. However, it is unknown whether CVB3 can induce the chemokine expression in cardiac myocytes. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemokine that stimulates the migration of mononuclear cells. The objective of the present study was to investigate the effect of CVB3 infection on MCP-1 expression in murine cardiac myocytes and the role of MCP-1 in migration of mononuclear cells in viral myocarditis. Our results showed that the expression of MCP-1 was significantly increased in cardiac myocytes after wild-type CVB3 infection in a time- and dose-dependent manner, which resulted in enhanced migration of mononuclear cells in mice with viral myocarditis. The migration of mononuclear cells was partially abolished by antibodies specific for MCP-1 in vivo and in vitro. Administration of anti-MCP-1 antibody prevented infiltration of mononuclear cells bearing the MCP-1 receptor CCR2 in mice with viral myocarditis. Infection by UV-irradiated CVB3 induced rapid and transient expression of MCP-1 in cardiac myocytes. In conclusion, our results indicate that CVB3 infection stimulates the expression of MCP-1 in myocardial cells, which subsequently leads to migration of mononuclear cells in viral myocarditis. PMID:15507642

  8. Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair

    PubMed Central

    Fernandes, Sarah; Chong, James J.H.; Paige, Sharon L.; Iwata, Mineo; Torok-Storb, Beverly; Keller, Gordon; Reinecke, Hans; Murry, Charles E.

    2015-01-01

    Summary Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts. We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBM-MNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart. PMID:26607951

  9. Regulation of human mononuclear phagocyte migration by cell surface-binding proteins for advanced glycation end products.

    PubMed Central

    Schmidt, A M; Yan, S D; Brett, J; Mora, R; Nowygrod, R; Stern, D

    1993-01-01

    Nonenzymatic glycation of proteins occurs at an accelerated rate in diabetes and can lead to the formation of advanced glycation end products of proteins (AGEs), which bind to mononuclear phagocytes (MPs) and induce chemotaxis. We have isolated two cell surface-associated binding proteins that mediate the interaction of AGEs with bovine endothelial cells. One of these proteins is a new member of the immunoglobulin superfamily of receptors (termed receptor for AGEs or RAGE); and the second is a lactoferrin-like polypeptide (LF-L). Using monospecific antibodies to these two AGE-binding proteins, we detected immunoreactive material on Western blots of detergent extracts from human MPs. Radioligand-binding studies demonstrated that antibody to the binding proteins blocked 125I-AGE-albumin binding and endocytosis by MPs. Chemotaxis of human MPs induced by soluble AGE-albumin was prevented in a dose-dependent manner by intact antibodies raised to the AGE-binding proteins, F(ab')2 fragments of these antibodies and by soluble RAGE. When MP migration in response to N-formyl-Met-Leu-Phe was studied in a chemotaxis chamber with AGE-albumin adsorbed to the upper surface of the chamber membrane, movement of MPs to the lower compartment was decreased because of interaction of the glycated proteins with RAGE and LF-L on the cell surface. The capacity of AGEs to attract and retain MPs was shown by implanting polytetrafluoroethylene (PTFE) mesh impregnated with AGE-albumin into rats: within 4 d a florid mononuclear cell infiltrate was evident in contrast to the lack of a significant cellular response to PTFE with adsorbed native albumin. These data indicate that RAGE and LF-L have a central role in the interaction of AGEs with human mononuclear cells and that AGEs can serve as a nidus to attract MPs in vivo. Images PMID:8387541

  10. PI3K-Akt/PKB signaling pathway in neutrophils and mononuclear cells exposed to N-nitrosodimethylamine.

    PubMed

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr; Iwaniuk, Agnieszka; Grubczak, Kamil

    2014-01-01

    Neutrophils (PMN) play diverse regulatory and effector functions in the immune system through the release of reactive nitrogen species, including nitric oxide (NO). The enzyme responsible for NO synthesis in PMN is inducible nitric oxide synthase (iNOS) that is regulated by various signaling pathways, e.g. PI3K-Akt/PKB, and transcription factors. N-Nitrosodimethylamine (NDMA), a xenobiotic widespread in the human environment, affects immune cells. The study objective here was to examine the role of the PI3K-Akt/PKB pathway in induction of NO synthesis (with involvement of iNOS) in human PMN, as well as in autologous mononuclear cells (PBMC), exposed to NDMA. Isolated cells were incubated for 2 h with a sub-lethal dose of NDMA and then the expression of several select proteins in the cell cytoplasmic and nuclear fractions were determined by Western blot analyses. The results indicated that NDMA enhanced expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasmic fraction of the PMN and PBMC. The nuclear fraction of these cells also had a higher NF-κB expression. Moreover, in PMN, NDMA caused an increased expression of phospho-Akt (T308), phospho-Akt (S473), and phospho-IKKαβ in the cytoplasm, and c-Jun and FosB in the nuclear fraction. Blocking of PI3K caused a decrease in expression of all these proteins in NDMA-exposed PMN. However, inhibition of PI3K led to a drop in expression of iNOS, phospho-PI3K, and phospho-IκBα in the cytoplasm, and in NF-κB in the nuclear fraction, of PBMC. The results of these studies indicated to us that NDMA activates the PI3K-Akt/PKB pathway in human PMN and that this, in turn, contributes to the activation of transcription factors NF-κB, c-Jun, and FosB involved in NO production (through modulation of iNOS expression). PMID:23971717

  11. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Shao, Jia; Loveren, Henk van; Peijnenburg, Ad

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  12. Antioxidant compounds in the seaweed Gelidiella acerosa protects human Peripheral Blood Mononuclear Cells against TCDD induced toxicity.

    PubMed

    Ilavarasi, K; Chermakani, P; Arif Nisha, S; Sheeja Malar, D; Pandima Devi, K

    2015-04-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxin formed as an unintentional by-product of incomplete combustion. Several therapeutic approaches have evolved to combat its toxicity since it elicits immunotoxicity, neurotoxicity, hepatotoxicity, carcinogenicity and lethality. Search for drugs from natural resources especially from seaweeds has become intense due to their enormous pharmacological potential. Hence, the present study aims at revealing the protective effect of methanolic extract of G. acerosa (MEGA) in Peripheral Blood Mononuclear Cells (PBMC) against TCDD induced toxicity, by assessing the antioxidant, anti-apoptotic and cytoprotective activities. The results of antioxidant assays suggests that MEGA reverted TCDD induced toxicity by causing an alteration in the levels of antioxidant enzymes (Catalase [CAT], Superoxide dismutase [SOD], Glutathione peroxidase [GPx], Glutathione-S-transferase [GST]) and Glutathione [GSH]. The results of lipid peroxidation assay and protein carbonyl content reveal that MEGA protects PBMC from TCDD induced macromolecular damage. MEGA was found to exhibit significant (p < 0.05) anti-apoptotic activity as verified by evaluation of mitochondrial membrane potential and AO-EtBr dual staining. In addition, PBMC co-treated with MEGA prevented TCDD induced oxidative DNA damage. Levels of phase-I detoxification enzymes determined by EROD assay and semi-quantitative RT-PCR showed that TCDD up-regulates the expression of CYP1A1 and upon co-treatment with MEGA, the expression got slightly decreased suggesting its protective role. Preliminary phytochemical analysis demonstrates that the extract is rich in cardiac glycosides and terpenoids. LC-MS analysis revealed the presence of antioxidants including caffeic acid, phytol and mannoheptulose in MEGA, which could be attributed for the observed protective effect against TCDD induced toxicity. PMID:24844840

  13. Influence of Genes Suppressing Interferon Effects in Peripheral Blood Mononuclear Cells during Triple Antiviral Therapy for Chronic Hepatitis C

    PubMed Central

    Iijima, Sayuki; Matsuura, Kentaro; Watanabe, Tsunamasa; Onomoto, Koji; Fujita, Takashi; Ito, Kyoko; Iio, Etsuko; Miyaki, Tomokatsu; Fujiwara, Kei; Shinkai, Noboru; Kusakabe, Atsunori; Endo, Mio; Nojiri, Shunsuke; Joh, Takashi; Tanaka, Yasuhito

    2015-01-01

    The levels of expression of interferon-stimulated genes (ISGs) in liver are associated with response to treatment with pegylated interferon (PEG-IFN) plus ribavirin (RBV). However, associations between the responses of ISGs to IFN-based therapy and treatment efficacy or interleukin-28B (IL28B) genotype have not yet been determined. Therefore, we investigated the early responses of ISGs and interferon-lambdas (IFN-λs) in peripheral blood mononuclear cells (PBMCs) during PEG-IFN/RBV plus NS3/4 protease inhibitor (PI) therapy. We prospectively enrolled 50 chronic hepatitis C patients with HCV genotype 1, and collected PBMCs at baseline, 8 and 24 h after the initial administration of PEG-IFN/RBV/PI. Levels of mRNAs for selected ISGs and IFN-λs were evaluated by real-time PCR. All 31 patients with a favorable IL28B genotype and 13 of 19 with an unfavorable genotype achieved sustained virological responses (SVR). Levels of mRNA for A20, SOCS1, and SOCS3, known to suppress antiviral activity by interfering with the IFN signaling pathway, as well as IRF1 were significantly higher at 8 h in patients with an unfavorable IL28B genotype than in those with a favorable one (P = 0.007, 0.026, 0.0004, 0.0006, respectively), especially in the non-SVR group. Particularly, the fold-change of IRF1 at 8 h relative to baseline was significantly higher in non-SVR than in SVR cases with an unfavorable IL28B genotype (P = 0.035). In conclusion, levels of several mRNAs of genes suppressing antiviral activity in PBMCs during PEG-IFN/RBV/PI differed according to IL28B genotypes, paralleling treatment efficacy. PMID:25706116

  14. Cyclosporine A inhibits transcription of cytokine genes and decreases the frequencies of IL-2 producing cells in feline mononuclear cells.

    PubMed

    Kuga, Kazufumi; Nishifuji, Koji; Iwasaki, Toshiroh

    2008-10-01

    Cyclosporine A (CsA) has been widely used for suppression of transplant rejection and controlling pruritus in allergic dermatitis in humans, dogs and cats. CsA is known to suppress the expression of inflammatory cytokines, including IL-2, IL-4, IFN-gamma and TNF-alpha in humans, dogs and experimental mice. However, little is known about the immunomodulating effect of CsA in cats. The aim of this study was to evaluate the effects of CsA on the expression of inflammatory cytokines in feline peripheral blood mononuclear cells (PBMC). Real-time PCR analyses with Concanavalin A (ConA)-stimulated PBMC obtained from 5 cats revealed that the expression of mRNAs for IL-2, IL-4, IFN- gamma and TNF-alpha was inhibited by CsA in a dose-dependent manner. Moreover, an enzyme-linked immunospot (ELISPOT) assay, which is capable of detecting IL-2 secreting cells as single spots, revealed that the frequency of IL-2 secreting cells in ConA-stimulated feline PBMC was significantly reduced in the presence of CsA. These results might provide an explanation for the mechanisms of action of CsA in the suppression of transplant rejection and the control of pruritus in cats. PMID:18981654

  15. Population Pharmacokinetics of Azithromycin in Whole Blood, Peripheral Blood Mononuclear Cells, and Polymorphonuclear Cells in Healthy Adults

    PubMed Central

    Sampson, M R; Dumitrescu, T P; Brouwer, K L R; Schmith, V D

    2014-01-01

    Azithromycin's extensive distribution to proinflammatory cells, including peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs), may be important to its antimicrobial and anti-inflammatory properties. The need to simultaneously predict azithromycin concentrations in whole blood (“blood”), PBMCs, and PMNs motivated this investigation. A single-dose study in 20 healthy adults was conducted, and nonlinear mixed effects modeling was used to simultaneously describe azithromycin concentrations in blood, PBMCs, and PMNs (simultaneous PK model). Data were well described by a four-compartment mamillary model. Apparent central clearance and volume of distribution estimates were 67.3 l/hour and 336 l (interindividual variability of 114 and 122%, respectively). Bootstrapping and visual predictive checks showed adequate model performance. Azithromycin concentrations in blood, PBMCs, and PMNs from external studies of healthy adults and cystic fibrosis patients were within the 5th and 95th percentiles of model simulations. This novel empirical model can be used to predict azithromycin concentrations in blood, PBMCs, and PMNs with different dosing regimens. PMID:24599342

  16. Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study

    PubMed Central

    Joehanes, Roby; Johnson, Andrew D.; Barb, Jennifer J.; Raghavachari, Nalini; Liu, Poching; Woodhouse, Kimberly A.; O'Donnell, Christopher J.; Munson, Peter J.

    2012-01-01

    Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL. PMID:22045913

  17. Damage Associated Molecular Pattern Molecule-Induced microRNAs (DAMPmiRs) in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Unlu, Sebnem; Tang, Siuwah; Wang, E. na; Martinez, Ivan; Tang, Daolin; Bianchi, Marco E.; Zeh, Herbert J.; Lotze, Michael T.

    2012-01-01

    Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10−4 and p<3.7×10−3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1+/+) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1−/− MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic “PAMPmiR”. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries

  18. Selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines.

    PubMed Central

    Greenway, A L; Overall, M L; Sattayasai, N; Rowley, M J; Hertzog, P J; McMullen, G L; Cheetham, B F; Marzuki, S

    1992-01-01

    The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology. Images Figure 1 Figure 2 Figure 3 PMID:1537595

  19. HIV Vasculopathy: Role of Mononuclear cell-associated Krüppel-like Factors 2 and 4

    PubMed Central

    Hale, Andrew T.; Longenecker, Chris T.; Jiang, Ying; Debanne, Sara M.; Labatto, Danielle E.; Storer, Norma; Hamik, Anne; McComsey, Grace A.

    2015-01-01

    Objective To determine the relationships between Krüppel-like Factors (KLF) 2 and 4, immune-activation, and sub-clinical vascular disease in HIV-infected patients on antiretroviral therapy. Design Double blind, randomized, placebo-controlled trial Methods We studied 74 HIV-infected adults on antiretroviral therapy enrolled in a randomized clinical trial of statin therapy. KLF2 and KLF4 gene expression was measured by quantitative polymerase chain reaction from peripheral blood mononuclear cells (PBMCs) at baseline and after 24 weeks of 10mg daily rosuvastatin or placebo. At the same time points, T-cell and monocyte activation were assessed by flow cytometry and vascular health was assessed by cardiac computed tomography and carotid ultrasound. Results KLF4 expression was negatively correlated with duration of antiretroviral therapy (r=−0.351, p=0.004) and positively correlated with measures of immune activation: pro-inflammatory monocytes [CD14+CD16+ (r=0.343, p=0.003)], patrolling monocytes [CD14dimCD16+ (r=0.276, p=0.017] and activated CD8+ T-lymphocytes [CD8+DR+CD38+ (r=0.264, p=0.023)]. KLF2 expression was negatively correlated with subclinical atherosclerosis: mean-mean common carotid artery intima media thickness (CIMT) (r= −0.231, p=0.048), mean-max CIMT (r= −0.271, p=0.020) and coronary artery calcium score (r=−0.254, p=0.029). There were no statistically significant changes in KLF2/4 expression in PBMCs after 24 weeks of rosuvastatin. Conclusions Expression of KLF4 in PBMCs positively correlates with cellular markers of immune activation, while KLF2 expression negatively correlates with markers of subclinical atherosclerosis in this HIV-infected population on antiretroviral therapy. Additional studies are needed to determine if targeted interventions might alter KLF2/4 expression to reduce inflammation and vascular risk in humans. PMID:26372274

  20. Interleukin-4 and interleukin-10 are chondroprotective and decrease mononuclear cell recruitment in human rheumatoid synovium in vivo.

    PubMed Central

    Jorgensen, C; Apparailly, F; Couret, I; Canovas, F; Jacquet, C; Sany, J

    1998-01-01

    We used the severe combined immunodeficient (SCID) mouse model to assess the effect of interleukin-4 (IL-4) or IL-10 injection on cartilage degradation and mononuclear cell (MNC) recruitment to human rheumatoid synovium in vivo. Human rheumatoid synovium and cartilage from five rheumatoid arthritis patients, obtained after joint replacement surgery, were engrafted subcutaneously to 6-8-week-old SCID CB17 mice. Synovial tissues were injected with recombinant human IL-4 (rhIL-4, 100 ng; rhIL-10, 100 ng), both cytokines, or tumour necrosis factor-alpha (TNF-alpha) (1000 U), or phosphate-buffered saline twice a week for 4 weeks. The graft was removed and immunochemical analysis was carried out to assess intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression. Moreover, cartilage degradation was assessed through the quantification of the erosion surface on a computerized image of the engrafted cartilage at high power view. MNC recruitment in the synovial tissue was determined by labelling blood MNC with indium-111 before their intraperitoneal injection. The activity obtained in the region of the graft were determined with a gamma camera 72 hr postinjection. The results are expressed as a percentage of initial injected activity. After 4 weeks we observed a decrease of cartilage area in controls (77 +/- 8%), inhibited after injection of IL-4, IL-10, or both cytokines (90 +/- 3%, 89.1 +/- 4%, 89.2 +/- 5% respectively), and 57 +/- 17% after TNF-alpha injection. The % MNC activity in the graft decreased to 77 +/- 81% (NS), 9 +/- 4% (P < 0.003) and 19 +/- 6% (P < 0.007) compared with untreated synovial tissue after treatment with IL-4, IL-10, or both cytokines, respectively. Moreover, IL-10 but not IL-4 decreased the expression of ICAM-1 but not VCAM-1 or E-selectin by synovial cells. These results suggest that IL-10 and IL-4 could have chondroprotective properties, and that IL-10 but not IL-4 inhibits MNC traffic

  1. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

    PubMed Central

    Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

    2015-01-01

    Background Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated. PMID:26396966

  2. SOCS1 and SOCS3 are expressed in mononuclear cells in human cytomegalovirus viremia after allogeneic hematopoietic stem cell transplantation

    PubMed Central

    Shin, Seung-Hwan; Lee, Ji Yoon; Lee, Tae Hyang; Park, So-Hye; Yahng, Seung-Ah; Yoon, Jae-Ho; Lee, Sung-Eun; Cho, Byung-Sik; Lee, Dong-Gun; Kim, Yoo-Jin; Lee, Seok; Min, Chang-Ki; Cho, Seok-Goo; Kim, Dong-Wook; Lee, Jong-Wook; Min, Woo-Sung; Park, Chong-Won

    2015-01-01

    Background The expression of the SOCS genes in cytomegalovirus (CMV) viremia after hematopoietic stem cell transplantation (HSCT) remains largely unexplored. Methods Using quantitative RT-PCR of mononuclear cells, we conducted pairwise comparison of SOCS1 and SOCS3 expression levels among a healthy donor group (N=55), a pre-HSCT group (N=17), and the recipient subgroup (N=107), which were divided according to the occurrence of CMV viremia and acute graft-versus-host disease (aGVHD). Results Compared to that in the healthy donor group, SOCS1 expression was higher in the CMV+ subgroup, especially in the CMV+GVHD- group, but decreased in the other subgroups. When compared to the expression in the pre-HSCT group, SOCS1 expression was significantly higher in the CMV+ subgroup, especially in the CMV+GVHD+ subgroup. Meanwhile, compared to that in the healthy donor group, SOCS3 expression was significantly lower in all other groups. The CMV-GVHD- subgroup showed significantly lower SOCS3 expression compared to the CMV+ subgroup, the CMV+GVHD+ subgroup, and the CMV+GVHD- subgroup. Conclusion We report differential expression of SOCS genes according to CMV viremia with acute GVHD occurrence after HSCT, suggesting that regulation of SOCS expression is associated with CMV viremia. PMID:25830129

  3. Clonal analysis of multipotent stromal cells derived from CD271+ bone marrow mononuclear cells: functional heterogeneity and different mechanisms of allosuppression

    PubMed Central

    Kuçi, Zyrafete; Seiberth, Julia; Latifi-Pupovci, Hatixhe; Wehner, Sibylle; Stein, Stefan; Grez, Manuel; Bönig, Halvard; Köhl, Ulrike; Klingebiel, Thomas; Bader, Peter; Kuçi, Selim

    2013-01-01

    Previous reports demonstrated a relationship between proliferation potential and trilineage differentiation in mesenchymal stromal cell-derived clones generated using plastic adherence (PA-MSCs). However, there are no reports presenting a clonal analysis of the proliferative potential, differentiation potential and allosuppressive effects of human mesenchymal stromal cell subsets. In this study, we performed a clonal analysis of mesenchymal stromal cells generated from human CD271+ bone marrow mononuclear cells (CD271-MSCs). After transfection with the gene encoding green fluorescent protein, the cells were single-cell sorted and cultured for 2–4 weeks. A population doubling analysis demonstrated that 25% of CD271-MSC clones are fast-proliferating clones compared to only 10% of PA-MSC clones. Evaluation of the allosuppressive potential demonstrated that 81.8% of CD271-MSC clones were highly allosuppressive compared to only 58% of PA-MSC clones. However, no consistent correlation was observed between allosuppression and proliferative potential. Prostaglandin E2 levels were positively correlated with the allosuppressive activity of individual clones, suggesting that this molecule may be a useful predictive biomarker for the allosuppressive potential of mesenchymal stromal cells. In contrast, inhibitory studies of indoleamine 2,3 dioxygenase indicated that none of the clones used this enzyme to mediate their allosuppressive effect. Differentiation studies revealed the presence of tripotent, bipotent and unipotent CD271-MSC and PA-MSC clones which suppressed the allogeneic reaction to differing extents in vitro. In conclusion, our findings demonstrate differences between CD271-MSCs and PA-MSCs and indicate that neither proliferation potential nor differentiation potential represents a consistent predictive parameter for the immunomodulatory effects of either type of mesenchymal stromal cells. PMID:23975178

  4. Tumor necrosis factor-alpha (TNF-alpha) concentrations from whole blood cultures correlate with isolated peripheral blood mononuclear cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many cellular immune assays are impractical because they require labor-intensive isolation of cells from their natural environment. The objectives of this study were to determine the relationship between cell culture supernatant TNF-alpha from isolated peripheral blood mononuclear cells (PBMC) and w...

  5. Inhibition of endotoxin-induced interleukin-6 production by synthetic lipid A partial structures in human peripheral blood mononuclear cells.

    PubMed Central

    Wang, M H; Flad, H D; Feist, W; Brade, H; Kusumoto, S; Rietschel, E T; Ulmer, A J

    1991-01-01

    The effect of two synthetic lipid A partial structures, compound 406 (or LA-14-PP, identical in structure to the lipid A precursor, known as Ia or IVa) and compound 401 (lipid X), on the in vitro modulation of endotoxin (lipopolysaccharide)-induced interleukin-6 production by human blood mononuclear cells was investigated. Lipopolysaccharide of Salmonella abortus equi and synthetic Escherichia coli-type lipid A (compound 506, or LA-15-PP) had potent interleukin-6-inducing capacities. The maximum release of interleukin-6 was found after stimulation with 1 to 10 ng of lipopolysaccharide or 10 to 100 ng of synthetic E. coli-type lipid A per ml. Both synthetic lipid A partial structures (compounds 406 and 401) failed to induce interleukin-6 release. However, they inhibited lipopolysaccharide- or lipid A-induced interleukin-6 production in a dose-dependent manner. Inhibition was found not only in mononuclear cells but also in purified monocytes and was not due to a shift in the kinetics of cytokine production. Suppression was manifested in the early stage of interleukin-6 production. Inhibition was also found in the presence of recombinant gamma interferon, indicating that compound 406 and recombinant gamma interferon act in different, independent pathways. Our data, therefore, indicate that the inhibition of interleukin-6 production by lipid A partial structures may help elucidate the mechanism of interaction of the lipid A component of lipopolysaccharide with immune cells in the inflammatory reaction during gram-negative infection. PMID:1937825

  6. Preliminary results on intersexual differences in gene expression of chemokine K203 in mononuclear cells of chicken.

    PubMed

    Kaňková, Zuzana; Zeman, Michal; Schwarz, Susanne; Kaspers, Bernd

    2016-03-01

    Sex steroid levels increase during sexual maturation and cause alterations in many physiological and morphological traits. Some of these changes may be connected with age-dependent and intersexual differences in the immune system. This topic is still insufficiently understood, especially in avian species, partially due to methodological limitations. In this study we measured the gene expression of proinflammatory cytokines (IL-1β, IL-6, IL-18) and chemokines [K60 (IL-8-like chicken chemokine - CXCLi1), CAF (IL-8-like chicken chemokine - CXCLi2), and K203] in mononuclear cells isolated from blood and spleen after in vitro stimulation with lipopolysaccharide (LPS). Samples were collected from chickens at two ages (from pullets before sexual maturity and from sexually mature egglaying hens). After LPS stimulation, a substantial increase was recorded in the gene expression of IL-6 and K203. All other measured genes were expressed at low levels in mononuclear cells irrespective of cell sources. We found a trend toward intersexual differences in K203 expression, but the expression of other cytokines and chemokines did not differ between the two sexes. The effect of stimulation was more pronounced in monocytes than in spleen macrophages, mainly in IL-6, IL-1β and K203 gene expression. Our findings represent a basis for further studies on the effects exerted by sexual hormones on the immune phenotype of birds. PMID:26919142

  7. The effects of acute oral glutamine supplementation on exercise-induced gastrointestinal permeability and heat shock protein expression in peripheral blood mononuclear cells.

    PubMed

    Zuhl, Micah; Dokladny, Karol; Mermier, Christine; Schneider, Suzanne; Salgado, Roy; Moseley, Pope

    2015-01-01

    Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N = 7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO2max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06 ± 0.01 vs. 0.02 ± 0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715 ± 0.046 pg/ml vs. 7.952 ± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64 ± 0.09 pg/ml vs. 1.87 ± 0.12 pg/ml). PBMC expression of IkBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29 ± 0.43 vs. 0.8892 ± 0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000 ± 0.000 and 1.65 ± 0.21 vs. 1.27 ± 0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells. PMID:25062931

  8. Increased expression of human T-cell immunoglobulin- and mucin-domain-containing molecule-4 in peripheral blood mononuclear cells from patients with system lupus erythematosus

    PubMed Central

    Zhao, Peiqing; Xu, Liyun; Wang, Piming; Liang, Xiaohong; Qi, Jianni; Liu, Peng; Guo, Chun; Zhang, Lining; Ma, Chunhong; Gao, Lifen

    2010-01-01

    Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease. Innate and adaptive immunity cooperatively contribute to the development of SLE. Antigen-presenting cells (APCs) have been suggested to link innate and adaptive immunity. T-cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4; also known as Timd4), expressed primarily on the surface of APCs, is a member of the TIM family, a recently described group of molecules that have received much attention as potential regulators of the immune system. In this study, we used quantitative real-time reverse transcription-polymerase chain reaction to examine the mRNA expression of Tim-4 in peripheral blood mononuclear cells (PBMCs) from SLE patients and further analyzed the correlation between the expression of Tim-4 and Tim-1 (a potential ligand for Tim-4) in PBMCs and serum tumor necrosis factor (TNF)-α levels. The results showed that Tim-4 mRNA expression in PBMCs was significantly higher in SLE patients than in healthy controls, especially those patients in the active phase of disease. Moreover, Tim-4 mRNA levels were closely correlated with Tim-1 mRNA levels in PBMCs and with serum TNF-α levels in SLE patients but not in the control group. Taken together, these results demonstrate that Tim-4 may be involved in the pathogenesis of SLE. PMID:20140011

  9. Transendocardial Mesenchymal Stem Cells and Mononuclear Bone Marrow Cells for Ischemic Cardiomyopathy: The TAC-HFT Randomized Trial

    PubMed Central

    Heldman, Alan W.; DiFede, Darcy L.; Fishman, Joel E; Zambrano, Juan P.; Trachtenberg, Barry H.; Karantalis, Vasileios; Mushtaq, Muzammil; Williams, Adam R.; Suncion, Viky Y.; McNiece, Ian K.; Ghersin, Eduard; Soto, Victor; Lopera, Gustavo; Miki, Roberto; Willens, Howard; Hendel, Robert; Mitrani, Raul; Pattany, Pradip; Feigenbaum, Gary; Oskouei, Behzad; Byrnes, John; Lowery, Maureen H.; Sierra, Julio; Pujol, Mariesty V; Delgado, Cindy; Gonzalez, Phillip J.; Rodriguez, Jose E.; Bagno, Luiza Lima; Rouy, Didier; Altman, Peter; Foo, Cheryl Wong Po; da Silva, Jose; Anderson, Erica; Schwarz, Richard; Mendizabal, Adam; Hare, Joshua M.

    2014-01-01

    Importance Whether culture expanded mesenchymal stem cells or whole bone marrow mononuclear cells are safe and effective in chronic ischemic cardiomyopathy (ICM) remains controversial. Objective To demonstrate the safety of transendocardial stem cell injection with autologous mesenchymal stem cells (MSCs) and whole bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy. Design, Setting and Patients A phase 1 and 2 randomized blinded placebo-controlled study involving 65 patients with ischemic cardiomyopathy and left ventricular (LV) ejection fraction less than50%(September 1, 2009-July 12, 2013). The study compared injection of MSCs (N=19) and placebo (N=11) or BMCs (N=19) with placebo (N=10) with 1-year of follow up. Interventions Injections into 10 LV sites with an infusion catheter. Main Outcomes and Measures Treatment-emergent 30 day serious adverse event rate defined as composite of death, myocardial infarction, stroke, hospitalization for worsening heart failure, perforation, tamponade or sustained ventricular arrhythmias. Results No patient had a treatment-emergent serious adverse events at day 30. The 1-year incidence of serious adverse events was 31.6% (95% CI, 12.6%-56.6%) for MSCs, 31.6% (95% CI, 12.6%-56.6%) for BMCs, and 38.1% (95% CI, 18.1%-61.6%) for placebo. Over 1-year the Minnesota Living with Heart Failure (MLHF) score improved with MSCs (repeated measures ANOVA P= .02) and BMCs (P= .005) but not placebo (P= .38), and 6-minute walk distance increased with MSCs only (repeated measures model P= .03). Infarct size as a percentage of LV Mass was reduced by MSCs (-18.9%; 95% CI, -30.4 to -7.4; within-group P= .004) but not by BMCs (-7.0%; 95% CI, -15.7%-1.7%; within-group P= .11) or placebo (-5.2; 95% CI, -16.8%-6.5%; within-group P=.36). Regional myocardial function as peak Eulerian circumferential strain at the site of injection improved with MSCs (-4.9; 95% CI, -13.3-3.5; within-group repeated measures P=.03) but not BMCs (-2

  10. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury

    PubMed Central

    Mitra, Srabani

    2015-01-01

    Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC) apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1) induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control) nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury. PMID:26710067

  11. Flavin fluorescence lifetime imaging of living peripheral blood mononuclear cells on micro and nano-structured surfaces

    NASA Astrophysics Data System (ADS)

    Teplicky, T.; Horilova, J.; Bruncko, J.; Gladine, C.; Lajdova, I.; Mateasik, A.; Chorvat, D.; Marcek Chorvatova, A.

    2015-03-01

    Fabricated micro- and nano-structured surfaces were evaluated for use with living cells. Metabolic state was tested by means of endogenous flavin fluorescence of living peripheral blood mononuclear cells (PBMC) positioned on a coverslip, non-covered, or covered with micro- or nano-structured surfaces (OrmoComp polymer structures produced by 2-photon photopolymerisation, or Zinc Oxide (ZnO) layer fabricated by pulsed laser deposition). Confocal microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) were employed to gather flavin fluorescence lifetime images of living PBMC on structured surfaces. Gathered data are the first step towards monitoring of the live cell interaction with different micro/nano-structured surfaces and thus evaluate their potential applicability in the biomedical field.

  12. Gene expression changes in mononuclear cells in patients with metabolic syndrome after acute intake of phenol-rich virgin olive oil

    PubMed Central

    2010-01-01

    Background Previous studies have shown that acute intake of high-phenol virgin olive oil reduces pro-inflammatory, pro-oxidant and pro-thrombotic markers compared with low phenols virgin olive oil, but it still remains unclear whether effects attributed to its phenolic fraction are exerted at transcriptional level in vivo. To achieve this goal, we aimed at identifying expression changes in genes which could be mediated by virgin olive oil phenol compounds in the human. Results Postprandial gene expression microarray analysis was performed on peripheral blood mononuclear cells during postprandial period. Two virgin olive oil-based breakfasts with high (398 ppm) and low (70 ppm) content of phenolic compounds were administered to 20 patients suffering from metabolic syndrome following a double-blinded, randomized, crossover design. To eliminate the potential effect that might exist in their usual dietary habits, all subjects followed a similar low-fat, carbohydrate rich diet during the study period. Microarray analysis identified 98 differentially expressed genes (79 underexpressed and 19 overexpressed) when comparing the intake of phenol-rich olive oil with low-phenol olive oil. Many of these genes seem linked to obesity, dyslipemia and type 2 diabetes mellitus. Among these, several genes seem involved in inflammatory processes mediated by transcription factor NF-κB, activator protein-1 transcription factor complex AP-1, cytokines, mitogen-activated protein kinases MAPKs or arachidonic acid pathways. Conclusion This study shows that intake of virgin olive oil based breakfast, which is rich in phenol compounds is able to repress in vivo expression of several pro-inflammatory genes, thereby switching activity of peripheral blood mononuclear cells to a less deleterious inflammatory profile. These results provide at least a partial molecular basis for reduced risk of cardiovascular disease observed in Mediterranean countries, where virgin olive oil represents a main

  13. Lactobacilli, bifidobacteria and E. coli nissle induce pro- and anti-inflammatory cytokines in peripheral blood mononuclear cells

    PubMed Central

    Helwig, Ulf; Lammers, Karen M; Rizzello, Fernando; Brigidi, Patricia; Rohleder, Verena; Caramelli, Elisabetta; Gionchetti, Paolo; Schrezenmeir, Juergen; Foelsch, Ulrich R; Schreiber, Stefan; Campieri, Massimo

    2006-01-01

    AIM: To investigate whether the stimulation of peripheral blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or species specific. METHODS: Three strains of bifidobacteria, 4 strains of lactobacilli, and E. coli nissle were sonicated and centrifuged in order to divide them into cell extract and cell debris. PBMNC were separated by density gradient and incubated for 36 h with either the cell debris or the cell extract of single strains of probiotic bacteria in doses from 102 to 108 CFU/mL. Cell supernatants were taken and interleukin (IL)-10, IL-1β, and tumor necosis factor (TNF)-α were determined by ELISA. RESULTS: Depending on the species super-family, the strains had different stimulation patterns. Except for both L. casei strains, the cell extract of bifidobacteria and lactobacilli had less stimulating capacity than cell debris, whereas the cell extract of E. coli nissle had similar stimulating properties to that of the cell debris of the strain and significantly more stimulating capacity than that of bifidobacteria and lactobacilli. The cell debris of bifidobacteria stimulated more cytokine release than the cell debris of lactobacilli. The cell debris of lactobacilli did not have a stimulating capacity when lower concentrations were used. Neither cell extraction nor cell debris had an inhibitory effect on the production of the tested cytokines by stimulated PBMNC. CONCLUSION: The incubation of probiotic strains, which have been used in clinical trials for inflammatory diseases, with immunocompetent cells leads to different species specific reactions. High IL-10 response to cell debris of bifidobacteria and E. coli nissle can be found. This corresponds to positive effects of bifidobacteria and E. coli nissle in clinical trials for inflammatory bowel disease compared to negative outcomes obtained with lactobacilli. PMID:17009396

  14. Proinflammatory effect of trivalent arsenical species in a co-culture of Caco-2 cells and peripheral blood mononuclear cells.

    PubMed

    Calatayud, Marta; Gimeno-Alcañiz, José V; Devesa, Vicenta; Vélez, Dinoraz

    2015-04-01

    Chronic exposure to inorganic arsenic (As) is associated with type 2 diabetes, cardiovascular diseases and cancer. Ingested inorganic As is transformed within the gastrointestinal tract and can give rise to more toxic species such as monomethylarsonous acid [MMA(III)] and dimethylarsinous acid [DMA(III)]. Thus, the intestinal epithelium comes into contact with toxic arsenical species, and the effects of such exposure upon epithelial function are not clear. The present study has evaluated the effect of 1 µM arsenite [As(III)], 0.1 µM MMA(III) and 1 µM DMA(III) upon the release of cytokines [interleukin-6 (IL6), IL8, tumor necrosis factor alpha (TNFα)], using a compartmentalized co-culture model with differentiated Caco-2 cells in the apical compartment and peripheral blood mononuclear cells in the basolateral compartment. In addition, the combined effect of arsenical species and lipopolysaccharide (LPS), both added into the apical compartment, has been analyzed. The results indicate that exposure to the arsenical forms induces a proinflammatory response. An increase in cytokine secretion into the basolateral compartment was observed, particularly as regards TNFα (up to 1,600 %). The cytokine levels on the apical side also increased, though to a lesser extent. As/LPS co-exposure significantly affected the proinflammatory response as compared to treatment with As alone. Treatment with DMA(III) and As/LPS co-exposure increased the permeability of the intestinal monolayer. In addition, As/LPS treatments enhanced As(III) and MMA(III) transport through the intestinal monolayer. PMID:24862236

  15. West Nile Virus Challenge Alters the Transcription Profiles of Innate Immune Genes in Rabbit Peripheral Blood Mononuclear Cells

    PubMed Central

    Uddin, Muhammad J.; Suen, Willy W.; Prow, Natalie A.; Hall, Roy A.; Bielefeldt-Ohmann, Helle

    2015-01-01

    The peripheral innate immune response to West Nile virus (WNV) is crucial for control of virus spread to the central nervous system. Therefore, transcriptomes encoding the innate immune response proteins against WNV were investigated in peripheral blood mononuclear cells (PBMCs) of New Zealand White rabbits, a recently established novel rabbit model for WNV pathogenesis studies. PBMCs were challenged with an Australian WNV strain, WNVNSW2011, in vitro, and mRNA expression of selected immune response genes were quantified at 2-, 6-, 12-, and 24-h post-infection (pi) using qRT-PCR. Compared to mock-inoculated PBMCs, WNV-stimulated PBMCs expressed high levels of interferon (IFN) alpha (IFNA), gamma (IFNG), IL6, IL12, IL22, CXCL10, and pentraxin 3 (PTX3) mRNA. Likewise, TLR1, 2, 3, 4, 6, and 10 mRNA became up-regulated with the highest expression seen for TLR3, 4, and 6. TLRs-signaling downstream genes (MyD88, STAT1, TRAF3, IRF7, and IRF9) subsequently became up-regulated. The high expression of IFNs, TLR3, TLR4, TRAF3, STAT1, IRF7, and IRF9 are in accordance with antiviral activities, while expression of TNFA, HO1, iNOS, caspase 3, and caspase 9 transcripts suggests the involvement of oxidative stress and apoptosis in WNV-stimulated rabbit PBMCs, respectively. The level of WNVNSW2011 RNA increased at 24-h pi in PBMCs challenged with virus in vitro compared to input virus. The expression dynamics of selected genes were validated in PBMCs from rabbits experimentally infected with WNV in vivo. Higher expression of IFNA, IFN beta (IFNB), IFNG, TNFA, IL6, IL22, PTX3, TLR3 and TLR4, IRF7, IRF9, STST1, TRAF3, caspase 3, and caspase 9 were seen in PBMCs from WNV-infected rabbits on day 3 post-intradermal virus inoculation compared to PBMCs from uninfected control rabbits. This study highlights the array of cytokines and TLRs involved in the host innate immune response to WNV in the rabbit leukocytes and suggests that these cells may be a useful in vitro model for WNV

  16. Quality control on mononuclear cells collected for extracorporeal photochemotherapy: comparison between two UV-A irradiation devices.

    PubMed

    Cervio, M; Scudeller, L; Viarengo, G; Del Fante, C; Perotti, C

    2015-11-01

    Recently, MacoPharma released a new UV-A cell irradiator device (Macogenic G2) for extracorporeal photopheresis (ECP), smaller and lighter than the Macogenic G1 but with no integrated cooling system. We compared the two devices at different working temperatures (G1 at standard irradiation temperature - 21°C - and G2 set by purpose at 34°C) in patients affected with chronic graft-versus-host disease and chronic lung allograft dysfunction treated by ECP. We demonstrate that both G1 and G2 devices are efficient in inducing the inhibition of lymphocytic proliferation and mononuclear cells apoptosis after 48 h even when G2 is set at higher-than-standard temperature. PMID:25975883

  17. Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares.

    PubMed

    Maeda, Yousuke; Ohtsuka, Hiromichi; Tomioka, Michiko; Tanabe, Taishi; Nambo, Yasuo; Uematsu, Haruki; Oikawa, Masa-aki

    2012-05-01

    Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was shown to have significantly inhibited E. coli induced proliferation and expression of IFN-γ in PBMCs. These results indicate that P(4) inhibits the immune response to E. coli in mares. PMID:22167103

  18. Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells

    PubMed Central

    Cohen, Jeffrey I.; Lekstrom, Kristen

    1999-01-01

    The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus. PMID:10438853

  19. Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

    NASA Technical Reports Server (NTRS)

    Lawless, DeSales

    2003-01-01

    We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

  20. Intraarterial transplantation of human umbilical cord blood mononuclear cells is more efficacious and safer compared with umbilical cord mesenchymal stromal cells in a rodent stroke model

    PubMed Central

    2014-01-01

    Introduction Stroke is the second leading cause of death worldwide, claims six lives every 60 seconds, and is a leading cause of adult disability across the globe. Tissue plasminogen activator, the only United States Food and Drug Administration (FDA)-approved drug currently available, has a narrow therapeutic time window of less than 5 hours. In the past decade, cells derived from the human umbilical cord (HUC) have emerged as a potential therapeutic alternative for stroke; however, the most effective HUC-derived cell population remains unknown. Methods We compared three cell populations derived from the human umbilical cord: cord blood mononuclear cells (cbMNCs); cord blood mesenchymal stromal cells (cbMSCs), a subpopulation of cbMNCs; and cord matrix MSCs (cmMSCs). We characterized these cells in vitro with flow cytometry and assessed the cells’ in vivo efficacy in a 2-hour transient middle cerebral artery occlusion (MCAo) rat model of stroke. cbMNCs, cbMSCs, and cmMSCs were each transplanted intraarterially at 24 hours after stroke. Results A reduction in neurologic deficit and infarct area was observed in all three cell groups; however, this reduction was significantly enhanced in the cbMNC group compared with the cmMSC group. At 2 weeks after stroke, human nuclei-positive cells were present in the ischemic hemispheres of immunocompetent stroke rats in all three cell groups. Significantly decreased expression of rat brain-derived neurotrophic factor mRNA was observed in the ischemic hemispheres of all three cell-treated and phosphate-buffered saline (PBS) group animals compared with sham animals, although the decrease was least in cbMNC-treated animals. Significantly decreased expression of rat interleukin (IL)-2 mRNA and IL-6 mRNA was seen only in the cbMSC group. Notably, more severe complications (death, eye inflammation) were observed in the cmMSC group compared with the cbMNC and cbMSC groups. Conclusions All three tested cell types promoted recovery

  1. Adequate Selection of a Therapeutic Site Enables Efficient Development of Collateral Vessels in Angiogenic Treatment With Bone Marrow Mononuclear Cells

    PubMed Central

    Nemoto, Masaru; Koyama, Hiroyuki; Nishiyama, Ayako; Shigematsu, Kunihiro; Miyata, Tetsuro; Watanabe, Toshiaki

    2015-01-01

    Background Induction of angiogenic mechanisms to promote development of collateral vessels is considered promising for the treatment of peripheral arterial diseases. Collateral vessels generally develop from preexisting arteriolar connections, bypassing the diseased artery. We speculated that induction of angiogenic mechanisms should be directed to such arteriolar connections to achieve efficient collateral development. The aim of this study was to verify this hypothesis using autologous transplantation of bone marrow mononuclear cells in the rabbit model of chronic limb ischemia. Methods and Results The left femoral artery was excised to induce limb ischemia in male rabbits. In this model, arteriolar connections in the left coccygeofemoral muscle tend to develop into collateral vessels, although this transformation is insufficient to alleviate the limb ischemia. In contrast, arteriolar connections in the closely located adductor muscle do not readily develop into collateral vessels. At 21 days after ischemia initiation, a sufficient number of automononuclear cells were selectively injected in the left coccygeofemoral muscle (coccygeo group) or left adductor muscle (adductor group). Evaluation of calf blood pressure ratios, blood flow in the left internal iliac artery, and angiographic scores at day 28 after injection revealed that collateral development and improvement of limb ischemia were significantly more efficient in the coccygeo group than in the adductor group. Morphometric analysis of the coccygeofemoral muscle at day 14 showed similar results. Conclusions Specific delivery of mononuclear cells to the coccygeofemoral but not the adductor muscle effectively improves collateral circulation in the rabbit model of limb ischemia and suggests that adequate site selection can facilitate therapeutic angiogenesis. PMID:26370447

  2. CD38 Ligation in Peripheral Blood Mononuclear Cells of Myeloma Patients Induces Release of Protumorigenic IL-6 and Impaired Secretion of IFNγ Cytokines and Proliferation

    PubMed Central

    Fedele, Giorgio; Di Girolamo, Marco; Recine, Umberto; Palazzo, Raffaella; Urbani, Francesca; Horenstein, Alberto L.; Malavasi, Fabio; Ausiello, Clara Maria

    2013-01-01

    CD38, a surface receptor that controls signals in immunocompetent cells, is densely expressed by cells of multiple myeloma (MM). The immune system of MM patients appears as functionally impaired, with qualitative and quantitative defects in T cell immune responses. This work answers the issue whether CD38 plays a role in the impairment of T lymphocyte response. To this aim, we analyzed the signals implemented by monoclonal antibodies (mAb) ligation in peripheral blood mononuclear cells (PBMC) obtained from MM patients and compared to benign monoclonal gammopathy of undetermined significance (MGUS). PBMC from MM both failed to proliferate and secrete IFNγ induced by CD38 ligation while it retained the ability to respond to TCR/CD3. The impaired CD38-dependent proliferative response likely reflects an arrest in the progression of cell cycle, as indicated by the reduced expression of PCNA. CD38 signaling showed an enhanced ability to induce IL-6 secretion. PBMC from MM patients displays a deregulated response possibly due to defects of CD38 activation pathways and CD38 may be functionally involved in the progression of this pathology via the secretion of high levels of IL-6 that protects neoplastic cells from apoptosis. PMID:24489445

  3. Comparison of immunomodulative effects of histamine-2 receptor antagonists in gastric cancer patients: focus on the lymphoblastogenesis and cytotoxicity of peripheral blood mononuclear cells.

    PubMed

    Hahm, K B; Lee, S I; Chung, J P; Kim, W H; Kim, J H; Park, I S

    1994-12-01

    A proposed mechanism of the immunomodulative effects of histamine-2 receptor antagonist (H2-RA) has been considered to be the inhibition of suppressor T-lymphocyte activity, an increase in interleukin-2 production of helper T-lymphocytes, and an enhancement of natural killer cell activity. Since there is a lack of comparative data about the immunomodulative effects of various H2-RAs, cimetidine, ranitidine and famotidine on peripheral blood mononuclear cells (PBMC), study of the comparison of the actions of H2-RA will be required. We compared the immunomodulative effect of each H2-RA on PBMC in patients with gastric cancer. DNA synthesis, cytotoxicity of PBMC against K562 cells and gastric cancer cell lines, and the levels of supernatant soluble interleukin-2 receptor (sIL-2R) were measured after the addition of each H2-RA, respectively. Increased suppressor cell activities were attenuated and restored to the levels of normal controls by the addition of cimetidine to H2-RA. Statistically significant lymphoblastogenesis and cytotoxicity against K562 cells were observed only in cimetidine-treated PBMC (P < 0.05). Such effects were not observed in ranitidine- or famotidine-treated PBMC. Neither cimetidine- nor ranitidine-activated activated PBMC showed any significant cytotoxicity against gastric cancer cells. Significantly increased levels of sIL-2R were found in supernatants obtained from culture flasks treated with cimetidine or ranitidine and phytohemagglutinin (P < 0.01). A significant correlation was found between the cytotoxicity of cimetidine- or ranitidine-treated PBMC and supernatant sIL-2R (P < 0.05). In conclusion, the most strongly modulative substance among H2-RAs was cimetidine and the least modulative drug was famotidine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7705971

  4. Bacillus Calmette-Guérin enhances production and secretion of type IV collagenases in peripheral blood mononuclear cells.

    PubMed

    Kageyama, Y; Kawakami, S; Fujii, Y; Kihara, K; Oshima, H

    1997-03-01

    Intravesical administration of bacillus Calmette-Guérin (BCG) is an effective and widely accepted treatment for superficial bladder cancer. Rapid progression of the disease after BCG therapy, however, has been reported in some cases refractory to the treatment. We examined whether BCG treatment and coexistence of peripheral blood mononuclear cells (PBMCs) alter the invasive potential of bladder cancer cells. Production and secretion of two type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP 9, by PBMCs from five healthy donors or bladder cancer cells (T24, JTC 30, and JTC 32) were evaluated by gelatin zymography, western blot analysis, and northern blot analysis. Invasion of bladder cancer cells was also examined using reconstituted basement membrane (Matrigel). BCG (5, 50, and 500 micrograms/ml) had no effect on secretion of MMP 2 and MMP 9 by bladder cancer cells, but increased the production and secretion of MMP 9 by PBMCs in a dose-dependent manner. The coexistence of PBMCs increased invasion of T24 cells and BCG further enhanced the invasion. Thus, BCG promotes invasion of bladder cancer cells under certain conditions. An increase in the secretion of MMP 9 by PBMCs may account in part for the effect. PMID:9140113

  5. Intravenous administration of bone marrow mononuclear cells alleviates hearing loss after transient cochlear ischemia through paracrine effects.

    PubMed

    Takagi, Taro; Yoshida, Tadashi; Okada, Masahiro; Hata, Ryuji; Hato, Naohito; Gyo, Kiyofumi; Hakuba, Nobuhiro

    2014-05-16

    Bone marrow mononuclear cells (BMMCs) are known to enhance recovery from ischemic insults by secreting angiogenic factors and inducing the expression of angiogenic factors from host tissues. Therefore, the transplantation of BMMCs is considered a potential approach to promoting the repair of ischemic damaged organs. Here, we investigated the influence of BMMCs on progressive hair cell degeneration after transient cochlear ischemia in gerbils. Transient cochlear ischemia was produced by extracranial occlusion of the bilateral vertebral arteries immediately before their entry into the transverse foramen of the cervical vertebra. An intravenous injection of BMMCs prevented ischemia-induced hair cell degeneration and ameliorated hearing impairment. A tracking study showed that BMMCs injected into the femoral vein were limited in the spiral artery of the cochlea, suggesting that, although transplanted BMMCs were retained within the spiral ganglion area of the cochlea, they were neither transdifferentiated into cochlear cells nor fused with the injured hair cells and supporting cells in the organ of Corti to restore their functions. We also showed that the protein level of neurotrophin-3 and glial cell line-derived neurotrophic factor in the organ of Corti was upregulated after treatment with BMMCs. These results suggested that BMMCs have therapeutic potential possibly through paracrine effects. Thus, we propose the use of BMMCs as a potential new therapeutic strategy for hearing loss. PMID:24840930

  6. Effects of pentoxifylline on TNF-alpha production by peripheral blood mononuclear cells in patients with nonalcoholic steatohepatitis.

    PubMed

    Duman, Deniz G; Ozdemir, Filiz; Birben, Esra; Keskin, Ozlem; Ekşioğlu-Demiralp, Emel; Celikel, Cigdem; Kalayci, Omer; Kalayci, Cem

    2007-10-01

    Pentoxifylline (POF) is a new candidate for the treatment of nonalcoholic steatohepatitis (NASH). Its effects on the cytokine production in patients with NASH are not completely understood. This study was designed to investigate the effect of POF on TNF-alpha production by peripheral blood mononuclear cells (PBMC) in patients with NASH. After preliminary experiments in healthy control subjects to determine the range of POF concentration to be used in NASH patients, PBMCs from patients with NASH (n = 13) were cultured in the presence of lipopolysaccharide (LPS, 100 ng/ml) and various concentrations of POF for 24 hr. Concentrations of TNF-alpha in culture supernatants were measured by ELISA and the transcriptional activity was determined by RT-PCR. As dictated by the results of our preliminary study in PBMC from healthy control subjects, we treated LPS stimulated PBMCs from NASH patients with 10, 100, and 500 microg/ml of POF. Stimulation of PBMCs from NASH patients with LPS resulted in a strong up-regulation of TNF-alpha production from median 355.9 (interquartile range, 206.7-463.5) pg/ml to 1,670 pg/ml (interquartile range, 1,121-2,414) pg/ml. In this LPS-stimulated culture system, POF caused a dose-dependent suppression of TNF-alpha levels (P < 0.001, ANOVA on ranks for repeated measures). TNF-alpha levels in culture supernatants decreased to 870.3 (range, 598.3-2,077) pg/ml with 10 microg/ml of POF treatment, and to levels similar to those obtained in baseline unstimulated cultures (133.4 (range, 95.8-1518.5) pg/ml) at 100 microg/ml. At 500 microg/ml, POF suppressed TNF-alpha production to levels significantly lower than that obtained in unstimulated (baseline) culture supernatants (76.3 (range, 33-94.5) pg/ml; P = 0.001). The mRNA expression was consistent with the effects on protein concentration. Demographic characteristics of the patients, laboratory results, such as AST, ALT, alkaline phosphatase, GGT, and triglyceride levels, and the liver histology did

  7. Genome wide expression profiling of human peripheral blood mononuclear cells stimulated with BAY 50-4798, a novel T cell selective interleukin-2 analog.

    PubMed

    Steppan, Sonja; Kupfer, Kenneth; Mayer, Anja; Evans, Mark; Yamasaki, Glenn; Greve, Jeffrey M; Eckart, Michael R; Cassell, Delanie J

    2007-01-01

    BAY 50-4798, a novel, engineered form of interleukin (IL)-2, is a selective agonist for the high-affinity IL-2 receptor and induces the proliferation of activated human T cells with potency similar to recombinant IL-2 (rIL-2), but has reduced proliferative activity on natural killer cells and is associated with a diminished secondary cytokine cascade. In the current study, the transcriptional profiles of human peripheral blood mononuclear cells (PBMCs) stimulated in vitro with BAY 50-4798 and rIL-2 were compared using Affymetrix microarray technology in combination with Ingenuity Pathway Analysis (IPA) to determine whether there are quantitative or qualitative differences in the molecular networks activated by these IL-2 analogs. A total of 299 genes were differentially expressed in response to the two IL-2 analogs, with an increase in the number of differences over time. Consistent with the fact that BAY 50-4798 interacts with fewer forms of the IL-2 receptor than rIL-2 to activate fewer cell types, 169 genes were expressed at lower levels in PBMCs cultured with BAY 50-4798 compared with IL-2. These genes were mainly categorized as cytokines and chemokines, and were used to build multiple molecular interaction networks, the most significant of which centered around a subunit of NF-kappaB, which is known to play a pivotal role in inflammation, and was associated with cell death. Of the genes induced in response to BAY 50-4798, only 25% were expressed at lower levels than those induced by rIL-2. Moreover, despite its more selective receptor targeting compared with rIL-2, BAY 50-4798 caused higher levels of expression of 130 genes, which predominantly fell into categories associated with metabolism and transcription. We interpret these results as consistent with the expected transcriptional profile of a mutein engineered and demonstrated to have diminished inflammatory effects yet fully retain selected features of IL-2 activity. In addition to demonstrating that the

  8. Early Antiretroviral Therapy in South African Children Reduces HIV-1-Infected Cells and Cell-Associated HIV-1 RNA in Blood Mononuclear Cells.

    PubMed

    van Zyl, Gert U; Bedison, Margaret A; van Rensburg, Anita Janse; Laughton, Barbara; Cotton, Mark F; Mellors, John W

    2015-07-01

    We measured cell-associated human immunodeficiency virus (HIV)-1 DNA (CAD) and RNA (CAR) and plasma HIV-1 RNA in blood samples from 20 children in the Children with HIV Early Antiretroviral (CHER) cohort after 7-8 years of suppressive combination antiretroviral therapy (cART). Children who initiated cART early (<2 months; n = 12) had lower HIV-1 CAD (median, 48 vs 216; P < .01) and CAR (median, 5 vs 436; P < .01) per million peripheral blood mononuclear cells than children who started later (≥ 2 months; n = 8). Plasma HIV-1 RNA levels were not significantly lower in early-treated children (0.5 vs 1.2 copies/mL; P = .16). Early treatment at <2 months of age reduces the number of HIV-infected cells and HIV CAR. PMID:25538273

  9. Quantitation of microbicidal activity of mononuclear phagocytes: an in vitro technique.

    PubMed

    Rege, N N; Dahanukar, S A

    1993-01-01

    An in vitro assay technique was set up to determine the phagocytic and microbicidal activity of a monocyte-macrophage cell line using Candida species as test organisms. The norms were determined for the activity of peritoneal macrophages of rats (24.69 +/- 2.6% phagocytosis and 35.4 +/- 5.22% ICK) and human (27.89 +/- 3.63% phagocytosis and 50.91 +/- 6.3% ICK). The assay technique was used to test the degree of activation of macrophages induced by metronidazole, Tinospora cordifolia and Asparaqus racemousus and to compare their effects with a standard immunomodulator muramyl-dipeptide. All the three test agents increased the phagocytic and killing capacity of macrophages in a dose dependent manner upto a certain dose, beyond which either these activities were found to have plateaued or decreased. The optimal doses for MDP, Metronidazole, Asparagus racemosus and Tinospora cordifolia were found to be 100 micrograms, 300 mg/kg, 200 mg/kg and 100 mg/kg respectively. Patients with cirrhosis were screened for defects in monocyte function. The depressed monocyte function (20.58 +/- 5% phago and 41.24 +/- 12.19% ICK; P < 0.05) was observed indicating a compromised host defense. The utility of this candidicidal assay in experimental and clinical studies is discussed. PMID:8295140

  10. Selenium supplementation restores the decreased albumin level of peripheral blood mononuclear cells in streptozotocin-induced diabetic mice.

    PubMed

    Ahn, Taeho; Bae, Chun-Sik; Yun, Chul-Ho

    2016-05-01

    Previously, it has been suggested that the phenotypic level of albumin in peripheral blood mononuclear cells (PBMC) decreased in streptozotocin (STZ)-induced diabetic rats. Concomitantly, the production of oxidative stresses was also elevated in the diabetic PBMC compared to that of normal control. These results suggest the close relationship between PBMC-albumin and its antioxidant roles. Here, we expanded the previous studies and investigated the effect of selenium supplementation as inorganic (sodium selenate) forms on the levels of albumin expression and oxidative stress in PBMC of STZ-induced diabetic mice. Selenium intake recovered the decreased albumin levels to those of normal mice and reduced the production of reactive oxygen species (ROS). These results support that selenium intake may alleviate the etiology and pathology of PBMC in type 1 diabetic mice by restoring the decrease in albumin contents and the production of ROS. PMID:26726102

  11. Selenium supplementation restores the decreased albumin level of peripheral blood mononuclear cells in streptozotocin-induced diabetic mice

    PubMed Central

    AHN, Taeho; BAE, Chun-Sik; YUN, Chul-Ho

    2016-01-01

    Previously, it has been suggested that the phenotypic level of albumin in peripheral blood mononuclear cells (PBMC) decreased in streptozotocin (STZ)-induced diabetic rats. Concomitantly, the production of oxidative stresses was also elevated in the diabetic PBMC compared to that of normal control. These results suggest the close relationship between PBMC-albumin and its antioxidant roles. Here, we expanded the previous studies and investigated the effect of selenium supplementation as inorganic (sodium selenate) forms on the levels of albumin expression and oxidative stress in PBMC of STZ-induced diabetic mice. Selenium intake recovered the decreased albumin levels to those of normal mice and reduced the production of reactive oxygen species (ROS). These results support that selenium intake may alleviate the etiology and pathology of PBMC in type 1 diabetic mice by restoring the decrease in albumin contents and the production of ROS. PMID:26726102

  12. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    NASA Technical Reports Server (NTRS)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  13. Assessment of the cytokine profile in peripheral blood mononuclear cells of naturally Sarcoptes scabiei var. canis infested dogs.

    PubMed

    Singh, Shanker K; Dimri, Umesh; Sharma, Bhaskar; Saxena, Meeta; Kumari, Priyambada

    2014-12-15

    The mechanism of cytokine secretion from T lymphocytes plays an important role in the immune response of dogs and parasitic skin infestations. Assessment of the cytokine profile of naturally S. scabiei var. canis infested dogs could augment understanding of the pathobiology of canine sarcoptic mange. Therefore, the present study examined the cytokines in peripheral blood mononuclear cells of dogs suffering from sarcoptic mange. Thirteen dogs naturally infected with sarcoptic mange participated in the study. The dogs were found positive for S. scabiei var. canis mites in skin scraping examinations and revealed at least three clinical inclusion criteria. Another five clinically healthy dogs were kept as healthy controls. Peripheral blood mononuclear cells were isolated from heparinized blood samples and used for extraction of mRNA. Further, cDNA was synthesized by using 1 mg of mRNA by reverse transcription using oligonucleotide primers. Relative levels of cytokine expression were compared with normalized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. The levels of interleukin-4, interleukin-5 and transforming growth factor beta (TGF-β) mRNA expression in dogs with sarcoptic mange were significantly higher (P ≤ 0.01), whereas the level of tumor necrosis factor alpha (TNF-α) was significantly lower (P ≤ 0.01) in comparison with the healthy dogs. No remarkable difference was seen for interleukin-2 mRNA expression between these animals. An overproduction IL-4 and IL-5 might be involved in immuno-pathogenesis of canine sarcoptic mange. S. scabiei var. canis mites possibly induce an overproduction of TGF-β and reduced expression of TNF-α and thus could be conferring the immune suppression of infested dogs. PMID:25468023

  14. Phosphorylation of Transcription Factor Specificity Protein 4 Is Increased in Peripheral Blood Mononuclear Cells of First-Episode Psychosis

    PubMed Central

    Fusté, Montserrat; Meléndez-Pérez, Iria; Villalta-Gil, Victoria; Haro, Josep Maria; Gill, Grace; Ramos, Belén

    2015-01-01

    Background Altered expression of transcription factor specificity protein 4 (SP4) has been found in the postmortem brain of patients with psychiatric disorders including schizophrenia and bipolar disorder. Reduced levels of SP4 protein have recently been reported in peripheral blood mononuclear cells in first-episode psychosis. Also, SP4 levels are modulated by lithium treatment in cultured neurons. Phosphorylation of SP4 at S770 is increased in the cerebellum of bipolar disorder subjects and upon inhibition of NMDA receptor signaling in cultured neurons. The aim of this study was to investigate whether SP4 S770 phosphorylation is increased in lymphocytes of first-episode psychosis patients and the effect of lithium treatment on this phosphorylation. Methods A cross-sectional study of S770 phosphorylation relative to total SP4 immunoreactivity using specific antibodies in peripheral blood mononuclear cells in first-episode psychosis patients (n = 14, treated with lithium or not) and matched healthy controls (n = 14) by immunoblot was designed. We also determined the effects of the prescribed drugs lithium, olanzapine or valproic acid on SP4 phosphorylation in rat primary cultured cerebellar granule neurons. Results We found that SP4 S770 phosphorylation was significantly increased in lymphocytes in first-episode psychosis compared to controls and decreased in patients treated with lithium compared to patients who did not receive lithium. Moreover, incubation with lithium but not olanzapine or valproic acid reduced SP4 phosphorylation in rat cultured cerebellar granule neurons. Conclusions The findings presented here indicate that SP4 S770 phosphorylation is increased in lymphocytes in first-episode psychosis which may be reduced by lithium treatment in patients. Moreover, our study shows lithium treatment prevents this phosphorylation in vitro in neurons. This pilot study suggests that S770 SP4 phosphorylation could be a peripheral biomarker of psychosis, and may

  15. Select human milk oligosaccharides directly modulate peripheral blood mononuclear cells isolated from 10-d-old pigs.

    PubMed

    Comstock, Sarah S; Wang, Mei; Hester, Shelly N; Li, Min; Donovan, Sharon M

    2014-03-14

    Infant formulas lack the complex mixture of oligosaccharides found in human milk. These human milk oligosaccharides (HMO) may be pivotal to the development of the neonatal immune system. Few comprehensive analyses of the effects of HMO on immune cells from neonates have been undertaken. Herein, the direct effects of HMO on immune cells were analysed ex vivo. Peripheral blood mononuclear cells (PBMC) isolated from 10-d-old sow-reared (SR) or colostrum-deprived formula-fed (FF) pigs were stimulated for 72 h with single HMO, mixtures of single HMO or a complex mixture of HMO isolated from human milk (iHMO). T-cell phenotype, cytokine production and proliferation were measured by flow cytometry, immunoassay and [³H]thymidine incorporation, respectively. Stimulation with HMO had direct effects on PBMC. For instance, cells stimulated with iHMO produced more IL-10 than unstimulated cells, and cells stimulated with fucosylated HMO tended to proliferate less than unstimulated cells. Additionally, co-stimulation with HMO mixtures or single HMO altered PBMC responses to phytohaemagglutinin (PHA) or lipopolysaccharide (LPS) stimulation. Compared with PBMC stimulated with PHA alone, cells co-stimulated with iHMO and PHA proliferated more and had fewer detectable CD4⁺CD8⁺ T cells. Compared with PBMC stimulated by LPS alone, cells co-stimulated with a mixture of sialylated HMO and LPS proliferated more and tended to have fewer detectable CD4⁺ T cells. Differences in the baseline responses of PBMC isolated from the SR or FF pigs were observed. In summary, HMO directly affected PBMC populations and functions. Additionally, ex vivo measurements of PBMC phenotype, cytokine production and proliferation were influenced by the neonate's diet. PMID:24131853

  16. Sickle red cells as danger signals on proinflammatory gene expression, leukotriene B4 and interleukin-1 beta production in peripheral blood mononuclear cell.

    PubMed

    Pitanga, Thassila N; Oliveira, Ricardo R; Zanette, Dalila L; Guarda, Caroline C; Santiago, Rayra P; Santana, Sanzio S; Nascimento, Valma M L; Lima, Jonilson B; Carvalho, Graziele Q; Maffili, Vitor V; Carvalho, Magda O S; Alcântara, Luiz C J; Borges, Valéria M; Goncalves, Marilda S

    2016-07-01

    This study tested the hypothesis that sickle red blood cell (SS-RBC) induce Toll-like receptors (TLR) and Nod-like receptor family, pyrin domain containing 3 (NLRP3)- inflammasome expression in peripheral blood mononuclear cells (PBMC). TLR and NLRP3 inflammasome could contribute to the maintenance of the inflammatory status in sickle cell anemia (SCA) patients, since SS-RBC act as danger signals activating these pathways. In this study, first, we evaluated TLR (2, 4, 5 and 9), NLRP3, Caspase-1, interleukin (IL)-1β and IL-18 expression in PBMC freshly isolated from SCA patients (SS-PBMC) in comparison with PBMC from healthy individuals (AA-PBMC). In the second moment, we investigated whether SS-RBC could interfere with the expression of these molecules in PBMC from healthy donor, in the absence or presence of hydroxyurea (HU) in vitro. TLRs and NLRP3 inflammasome expression were investigated by qPCR. IL-1β, Leukotriene-B4 (LTB4) and nitrite production were measured in PBMC (from healthy donor) culture supernatants. TLR2, TLR4, TLR5, NLRP3 and IL-1β were highly expressed in SS-PBMC when compared to AA-PBMC. Additionally, SS-RBC induced TLR9, NLRP3, Caspase-1, IL-1β and IL-18 expression and induced IL-1β, LTB4 and nitrite production in PBMC cultures. HU did not prevent TLR and NLRP3 inflammasome expression, but increased TLR2 and IL-18 expression and reduced nitrite production. In conclusion, our data suggest that TLR and inflammasome complexes may be key inducers of inflammation in SCA patients, probably through SS-RBC; also, HU does not prevent NLRP3 inflammasome- and TLR-dependent inflammation, indicating the need to develop new therapeutic strategies to SCA patients that act with different mechanisms of those observed for HU. PMID:27045344

  17. Synthesis, characterization, and in vitro anti-neoplastic activity of novel vic-dioximes bearing thiosemicarbazone side groups and their mononuclear complexes.

    PubMed

    Babahan, İlknur; Özmen, Ali; Orhan, Nil; Kazar, Didem; Değirmenci, Esin Hafize

    2014-04-01

    Two novel vicinal dioxime ligands containing thiosemicarbazone units, (2E)-2-[4-(diethylamino)benzylidene]-N-[(1Z,2E)-N-hydroxy-2-(hydroxyimino)ethanimidoyl]hydrazine carbothioamide (L(1)H2) and (2E)-2-[4-(dimethylamino)benzylidene]-N-[(1Z,2E)-N-hydroxy-2-(hydroxyimino)ethanimidoyl]hydrazinecarbothioamide (L(2)H2), were synthesized. Using the HL-60 human leukemia cell line, the in vitro anti-neoplastic activity of these thiosemicarbazone-oxime derivatives was evaluated. Mononuclear nickel(II), copper(II), and cobalt(II) complexes with a metal:ligand ratio of 1:2 for both the L(1)H2 and L(2)H2 ligands were also synthesized. To characterize these compounds, Fourier transform-infrared spectroscopy (FT-IR), mass spectrometry (MS), magnetic susceptibility measurements, (1)H and (13)C nuclear magnetic resonance (NMR), ultraviolet-visible (UV-Vis) absorption spectroscopy, heteronuclear multiple-bond correlation (HMQC), and elemental analysis were performed. For L(1)H2, L(2)H2, and each of their derivatives, antiproliferative effects against HL-60 cells were exhibited and the associated IpC50 values ranged from 5μM to 20μM. Furthermore, L(1)H2 and its derivatives inhibited the proliferation of HL-60 cells more effectively than L(2)H2, and 5μM [Cu(L(1)H)2] exhibited the strongest antiproliferative activity. PMID:24651042

  18. Mononuclear phagocyte proliferation, maturation and function.

    PubMed

    Territo, M C; Cline, M J

    1975-10-01

    The mononuclear phagocytic system is a continuum of cells beginning with the bone marrow monoblast and promonocyte, through the monocyte to the larger tissue macrophages and multinucleate giant cells. This system of cells is widely distributed throughout the body in the blood and bone marrow; the pleural, peritoneal, and alveolar spaces; the lymph nodes, spleen, liver, and other parenchymal organs. The activity and composition of the cell varies with the level of maturation, changes in cellular environment, and with various cellular activities. The monocyte-macrophage group of cells plays an active role in defense reactions against certain microorganisms, and in the removal of dying cells and cell debris. They are an integral part of both the inductive phase of the immune response, and of cell-mediated immune reactions. In addition, they probably play a role in the defence against spontaneously arising tumours, in the control of granulopoiesis, and possibly in erythropoiesis. PMID:53114

  19. Bisphenol A and its analogs induce morphological and biochemical alterations in human peripheral blood mononuclear cells (in vitro study).

    PubMed

    Michałowicz, Jaromir; Mokra, Katarzyna; Bąk, Agata

    2015-10-01

    Few studies have addressed the cellular effects of bisphenol S (BPS) and bisphenol AF (BPAF) on cells, and no study has been conducted to analyze the mechanism of action of bisphenols in blood cells. In this study, the effect of bisphenol A (BPA), bisphenol F (BPF), BPS and BPAF on human peripheral blood mononuclear cells (PBMCs) was analyzed. It was shown that BPA, BPF and BPAF in particular, decreased cell viability, which was associated with depletion of intracellular ATP level and alterations in PBMCs size and granulation. Bisphenols enhanced ROS (including OH˙) formation, which led to damage to lipids and proteins in PBMCs. The most significant alterations in ROS level were induced by BPF, and particularly BPAF. Moreover, it was shown that BPAF most strongly provoked lipid peroxidation, while BPA and BPS caused the greatest damage to proteins. It may be concluded that BPA and its analogs were capable of inducing oxidative stress and damage in PBMCs in the concentrations ranging from 0.06 to 0.5 μM (0.02-0.1 μg/ml), which may be present in human blood as a result of environmental exposure. Although, most of bisphenols studied decreased cell viability, size and ATP level at higher concentrations, BPAF exhibited its cytotoxic potential at low concentrations ranging from 0.3 to 3 μM (0.1-1.0 μg/ml) that may correspond to concentrations in humans following occupational exposure. PMID:26028149

  20. Quantification and characterization of ACTH-related peptides produced by human peripheral blood mononuclear cells

    SciTech Connect

    Pepper, G.M.; Maimon, J.; Schneider, B.S. )

    1991-04-01

    The authors have used a sensitive radioimmunoassay to quantify and characterize PBMC-associated immunoreactive ACTH (ACTH-IR). Mean ACTH content of freshly isolated human PBMCs was 3.8 {plus minus} 0.72 pg (SEM) per 10(6) cells. During 3 days of incubation ACTH-IR in conditioned media of control PBMCs increased significantly, p less than 0.02. Gel filtration chromatography revealed a minor peak of ACTH-IR coeluting with ACTH (1-39) and a major peak coeluting with ACTH (11-24). Treatment with 15 nM CRH did not alter the amount of ACTH-IR secreted or its gel pattern. Synthetic ACTH (11-24), was radioiodinated and was used for binding experiments that demonstrated specific high- and low-affinity binding sites for ACTH (11-24) on a human T cell line. These results add support for a role of ACTH and related peptides in immune regulatory systems and suggest that cell-specific post-translational processing of PBMC may generate an expanding number of biologically active moieties.

  1. Ceramide induces a loss in cytosolic peroxide levels in mononuclear cells.

    PubMed Central

    Phillips, Darren C; Griffiths, Helen R

    2003-01-01

    Ceramide (a sphingolipid) and reactive oxygen species are each partly responsible for intracellular signal transduction in response to a variety of agents. It has been reported that ceramide and reactive oxygen species are intimately linked and show reciprocal regulation [Liu, Andreieu-Abadie, Levade, Zhang, Obeid and Hannun (1998) J. Biol. Chem. 273, 11313-11320]. Utilizing synthetic, short-chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide formation or using stimulation of CD95 to induce ceramide formation, we found that the principal redox-altering property of ceramide is to lower the [peroxide](cyt) (cytosolic peroxide concentration). Apoptosis of Jurkat T-cells, primary resting and phytohaemagglutinin-activated human peripheral blood T-lymphocytes was preceded by a loss in [peroxide](cyt), as measured by the peroxide-sensitive probe 2',7'-dichlorofluorescein diacetate (also reflected in a lower rate of superoxide dismutase-inhibitable cytochrome c reduction), and this was not associated with a loss of membrane integrity. Where growth arrest of U937 monocytes was observed without a loss of membrane integrity, the decrease in [peroxide](cyt) was of a lower magnitude when compared with that preceding the onset of apoptosis in T-cells. Furthermore, decreasing the cytosolic peroxide level in U937 monocytes before the application of synthetic ceramide by pretreatment with either of the antioxidants N -acetyl cysteine or glutathione conferred apoptosis. However, N -acetyl cysteine or glutathione did not affect the kinetics or magnitude of ceramide-induced apoptosis of Jurkat T-cells. Therefore the primary redox effect of cellular ceramide accumulation is to lower the [peroxide](cyt) of both primary and immortalized cells, the magnitude of which dictates the cellular response. PMID:12877656

  2. Cryopreservation of blood mononuclear leukocytes and stem cells suspended in a large fluid volume. A preclinical model for a blood stem cell bank.

    PubMed

    Fliedner, T M; Körbling, M; Calvo, W; Bruch, C; Herbst, E

    1977-09-29

    It was the purpose of this study to establish and evaluate a freezing-and-thawing method for preservation of hemopoietic stem cells from the peripheral blood. Blood leukocytes collected by means of an IBM Blood-Cell-Separator were frozen in plastic bags using 10% DMSO and controlled cooling rates. Thawing was performed rapidly, and DMSO was diluted and removed prior to the in-vitro and in-vivo assays. The mean recovery of mononuclear cells collected from 82 leukaphereses was 86%. To assess the recovery of cryopreserved hemopoietic stem cells, the soft agar culture method adapted for the dog was used. There was no significant difference in the CFUc recovery per 1 X 10(6) mononuclear cells or in per leukapheresis after different cryopreservation times (1--6 and 7--27 months). To evaluate the hemopoietic repopulation capability of cryopreserved blood stem cells, leukapheresis-derived leukocytes were transfused into 1200 R whole body x-irradiated dogs. The hemopoietic repopulation pattern at day 10 after transfusion of comparable numbers of fresh or frozen leukocytes was not significantly different, as measured in bone marrow smears and sections and by granulocyte concentration in the peripheral blood. PMID:912104

  3. Autologous Bone Marrow Mononuclear Cell Therapy is Safe and Promotes Amputation Free Survival in Patients with Critical Limb Ischemia

    PubMed Central

    Murphy, Michael P.; Lawson, Jeffrey H.; Rapp, Brian M.; Dalsing, Michael C.; Klein, Janet; Wilson, Michael G.; Hutchins, Gary D.; March, Keith L.

    2011-01-01

    Objective The purpose of this phase I open label non-randomized trial was to assess the safety and efficacy of autologous bone marrow mononuclear cell (ABMNC) therapy in promoting amputation free survival (AFS) in patients with critical limb ischemia (CLI). Methods Between September 2005 and March 2009 twenty-nine patients (30 limbs), with a median age of 66 (range 23–84) (14 male,15 female) with CLI were enrolled . Twentyone limbs presented with rest pain (RP), six with RP and ulceration, and three with ulcer only. All patients were not candidates for surgical bypass due to absence of a patent artery below the knee and/or endovascular approaches to improving perfusion was not possible as determined by an independent vascular surgeon. Patients were treated with an average dose of 1.7 ± 0.7 × 109 ABMNC injected intramuscularly in the index limb distal to the anterior tibial tuberosity. The primary safety endpoint was accumulation of serious adverse events and the primary efficacy endpoint was AFS at one year. Secondary endpoints at 12 weeks post-treatment were changes in first toe pressure (FTP), toe-brachial index (TBI), ankle-brachial index (ABI), and transcutaneous oxygen measurements (TcPO2). Perfusion of the index limb was measured with PET-CT with intra-arterial infusion of H2O15. Rest pain (RP), using a 10-cm visual analog scale, quality of life using the VascuQuol questionnaire, and ulcer healing were assessed at each follow-up interval. Subpopulations of endothelial progenitor cells were quantified prior to ABMNC administration using immunocytochemistry and fluorescent activated cell sorting. Results There were two serious adverse events however there no procedure related deaths. Amputation-free survival at one-year was 86.3%. There was a significant increase in FTP (10.2+ 6.2 mmHg, P=.02) and TBI (0.10± 0.05, P=.02) and a trend in improvement in ABI (0.08±0.04, P=.73). Perfusion Index by PET-CT H2O15 increased by 19.3 ± 3.1 and RP decreased

  4. Characterization of Epstein-Barr virus-induced lymphoproliferation derived from human peripheral blood mononuclear cells transferred to severe combined immunodeficient mice.

    PubMed

    Okano, M; Taguchi, Y; Nakamine, H; Pirruccello, S J; Davis, J R; Beisel, K W; Kleveland, K L; Sanger, W G; Fordyce, R R; Purtilo, D T

    1990-09-01

    Mice with severe combined immunodeficiency (SCID) received 6 X 10(7) fresh human peripheral blood mononuclear cells (PBMC) intraperitoneally from Epstein-Barr virus (EBV)-seropositive and -seronegative donors. B95-8 EBV was inoculated intraperitoneally and intravenously to the mice 6 weeks after transfer of seronegative PBMC. Three of four mice transferred with PBMC from two EBV-seropositive donors and two of four mice from two EBV-seronegative donors inoculated with EBV developed fatal EBV-induced lymphoproliferative disease within 6 to 10 weeks. These tumors were oligoclonal or polyclonal by cytoplasmic immunoglobulin expression. Furthermore no consistent clonal chromosomal abnormalities were shown. Cell lines established from these tumors showed low cloning efficiency in soft agarose. In addition, latent membrane protein, B-lymphocyte activation antigen (CD23), and cell-adhesion molecules (ICAM-1, CD18) all were expressed in the EBV-positive infiltrating lymphoproliferative lesions in each mouse. These results suggest that lymphoid tumors are comparable to lymphoblastoid cell lines immortalized by EBV and are not malignant lymphomas such as Burkitt's lymphoma. This model may be useful for investigating mechanisms responsible for the growing numbers of lymphoproliferative diseases that are occurring in patients with inherited or acquired immunodeficiencies. PMID:1975985

  5. Requirement of dual stimulation by homologous recombinant IL-2 and recombinant IL-12 for the in vitro production of interferon gamma by canine peripheral blood mononuclear cells

    PubMed Central

    2014-01-01

    Background Very few studies have been carried out so far aiming at modulating cellular immune responses in dogs. In this study, we evaluated the ability of recombinant canine IL-2 (rcaIL-2) and IL-12, in the form of a single-chain fusion protein (rsccaIL-12), to stimulate peripheral blood mononuclear cells (PBMC) of healthy mongrel dogs. Results Recombinant canine IL-2 purified from Escherichia coli or present in the supernatant of COS-7 cells transfected with pcDNA3.1-caIL-2 (COS-7 caIL-2 supernatant) was able to induce proliferation of CTLL-2 cells, thus showing their functional activity. In addition, purified rcaIL-2 and COS-7 caIL-2 supernatant stimulated resting canine PBMC proliferation to a level higher than baseline level. Neither COS-7 sccaIL-12 supernatant nor COS-7 caIL-2 supernatant alone was able to induce significant production of interferon gamma by resting PBMC. However, COS-7 sccaIL-12 supernatant in combination with COS-7 caIL-2 supernatant induced production of IFN-γ by those cells. Conclusions The data shown herein suggest that the combination of canine recombinant IL-12 and IL-2 can be useful to promote cellular immune responses in dogs. PMID:25037233

  6. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

    PubMed Central

    Riedel, Michael; Jou, Chuanchau J.; Lai, Shuping; Lux, Robert L.; Moreno, Alonso P.; Spitzer, Kenneth W.; Christians, Elizabeth; Tristani-Firouzi, Martin; Benjamin, Ivor J.

    2014-01-01

    Summary Advances in induced pluripotent stem cell (iPSC) technology have set the stage for routine derivation of patient- and disease-specific human iPSC-cardiomyocyte (CM) models for preclinical drug screening and personalized medicine approaches. Peripheral blood mononuclear cells (PBMCs) are an advantageous source of somatic cells because they are easily obtained and readily amenable to transduction. Here, we report that the electrophysiological properties and pharmacological responses of PBMC-derived iPSC CM are generally similar to those of iPSC CM derived from other somatic cells, using patch-clamp, calcium transient, and multielectrode array (MEA) analyses. Distinct iPSC lines derived from a single patient display similar electrophysiological features and pharmacological responses. Finally, we demonstrate that human iPSC CMs undergo acute changes in calcium-handling properties and gene expression in response to rapid electrical stimulation, laying the foundation for an in-vitro-tachypacing model system for the study of human tachyarrhythmias. PMID:25068127

  7. Proteomic responses of peripheral blood mononuclear cells in the European eel (Anguilla anguilla) after perfluorooctane sulfonate exposure.

    PubMed

    Roland, Kathleen; Kestemont, Patrick; Hénuset, Laurence; Pierrard, Marie-Aline; Raes, Martine; Dieu, Marc; Silvestre, Frédéric

    2013-03-15

    Since the 1980s, the stocks of European eel have been declining in most of their geographical distribution area. Many factors can be attributed to this decline such as pollution by xenobiotics like perfluorooctane sulfonate (PFOS). This study aimed at evaluating the in vitro toxicity of eel peripheral blood mononuclear cells (PBMC) exposed to PFOS. Exposure time and two concentrations were chosen to avoid cell mortality (48 h exposure at 10 μg PFOS/L and 1mg PFOS/L). After in vitro contaminations, the post-nuclear fraction was isolated and a proteomic analysis using 2D-DIGE was performed to compare PBMC from the control group with cells exposed to the pollutant. On the 158 spots that were significantly affected by PFOS exposure, a total of 48 different proteins were identified using nano-LCESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. These proteins can be categorized into diverse functional classes, related to cytoskeleton, protein folding, cell signaling, proteolytic pathway and carbohydrate and energy metabolism, which provide clues on the cellular pathways mainly affected by PFOS. Some of the identified proteins are rarely found in other ecotoxicological proteomic studies and could constitute potential biomarkers of exposure to PFOS in fish. PMID:23261670

  8. Intravenous transplantation of bone marrow-derived mononuclear cells prevents memory impairment in transgenic mouse models of Alzheimer's disease.

    PubMed

    Kanamaru, Takuya; Kamimura, Naomi; Yokota, Takashi; Nishimaki, Kiyomi; Iuchi, Katsuya; Lee, Hyunjin; Takami, Shinya; Akashiba, Hiroki; Shitaka, Yoshitsugu; Ueda, Masayuki; Katsura, Ken-Ichiro; Kimura, Kazumi; Ohta, Shigeo

    2015-04-24

    Stem cell transplantation therapy is currently in clinical trials for the treatment of ischemic stroke, and several beneficial aspects have been reported. Similarly, in Alzheimer's disease (AD), stem cell therapy is expected to provide an efficient therapeutic approach. Indeed, the intracerebral transplantation of stem cells reduced amyloid-β (Aβ) deposition and rescued memory deficits in AD model mice. Here, we show that intravenous transplantation of bone marrow-derived mononuclear cells (BMMCs) improves cognitive function in two different AD mouse models, DAL and APP mice, and prevents neurodegeneration. GFP-positive BMMCs were isolated from tibiae and femurs of 4-week-old mice and then transplanted intravenously into DAL and APP mice. Transplantation of BMMCs suppressed neuronal loss and restored memory impairment of DAL mice to almost the same level as in wild-type mice. Transplantation of BMMCs to APP mice reduced Aβ deposition in the brain. APP mice treated with BMMCs performed significantly better on behavioral tests than vehicle-injected mice. Moreover, the effects were observed even with transplantation after the onset of cognitive impairment in DAL mice. Together, our results indicate that intravenous transplantation of BMMCs has preventive effects against the cognitive decline in AD model mice and suggest a potential therapeutic effect of BMMC transplantation therapy. PMID:25698614

  9. Glycoconjugates Effects: do Gender and Ethnicity Influence Exposure of Pathogen by Peripheral Mononuclear Cells ?

    NASA Astrophysics Data System (ADS)

    Lahiani, Mohamed; Tarasenko, Olga

    2010-04-01

    Members of the Bacillus cereus group demonstrate different pathological effects. B. cereus is a spore-forming, gram positive bacterium responsible for most foodborne illnesses. It was shown that susceptibility to infection and response to vaccines or treatments can be attributed to specific immunogenetic factors including gender and ethnicity. Glycoconjugate polymers (GCs) are potentially important in pharmaceutical and biomedical research. Our group has shown that GCs activate murine macrophages and promote killing of Bacillus cereus spores during phagocytosis. We hypothesized that the GCs effects are independent from gender and race. The goal of the present study was two-folds: A) determine whether GCs influence on human PMNC exposure of B. cereus spores and B) analyze whether gender and ethnicity influence of the effect of GCs. GCs were studied during exposure and post-exposure conditions. Phagocytosis was performed during exposure of PMNC to Bacillus spores. Post-exposure analysis involved cytotoxicity, cell viability and activation, and colonies forming unit. GC1 and GC3 enhance Bacillus spore killing. GC1 proved more effective than GC3 in spore killing while activating PMNC. Results demonstrate GCs effect were independent from ethnicity or gender. Findings of this research demonstrated that GC can be used as ligands to stimulate PMNC and kill B. cereus spores.

  10. Tryptamine and dimethyltryptamine inhibit indoleamine 2,3 dioxygenase and increase the tumor-reactive effect of peripheral blood mononuclear cells.

    PubMed

    Tourino, Melissa Cavalheiro; de Oliveira, Edson Mendes; Bellé, Luziane Potrich; Knebel, Franciele Hinterholz; Albuquerque, Renata Chaves; Dörr, Felipe Augusto; Okada, Sabrina Sayori; Migliorini, Silene; Soares, Irene Silva; Campa, Ana

    2013-07-01

    Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced tryptophan-degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N-dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non-competitive inhibitors, with Ki values of 156 and 506 μM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN-γ-induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co-culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor-reactive response by the PBMCs. PMID:23754498

  11. Immunoregulatory function of human intestinal mucosa lymphoid cells: evidence for enhanced suppressor cell activity in inflammatory bowel disease.

    PubMed Central

    Fiocchi, C; Youngman, K R; Farmer, R G

    1983-01-01

    Abnormalities in immune regulation at the gut level may be relevant to the pathogenesis of inflammatory bowel disease, but little is known about the immunoregulatory properties of intestinal mononuclear cells. Therefore, we wished to see if lymphoid cells derived from the lamina propria of surgically resected bowel specimens have any modulatory effect upon the immune response of peripheral blood mononuclear cells from patients with ulcerative colitis or Crohn's disease. When autologous peripheral blood and intestinal lamina propria lymphoid cells were mixed at different ratios and cultured in the presence of phytohaemagglutinin, we were able to show that intestinal mononuclear cells had the capacity to modify the mitogenic response of the cultured cells. These intestinal immunoregulatory cells, when obtained from mucosa affected by inflammatory bowel disease, express a significantly enhanced suppressor cell activity as compared with those from non-inflamed control mucosa. Such suppressor cell activity varies with cell concentration and requires cell proliferation, but it is independent of anatomical origin (small vs large bowel), type of inflammatory bowel disease (ulcerative colitis vs Crohn's disease) or immunosuppressive therapy. These findings point to an important functional difference between inflammatory bowel disease and control intestinal mucosa mononuclear cells. The enhanced suppressor activity of lamina propria mononuclear cells may be associated with impairment of cell-mediated immunity at the gut level. This may be related to the pathogenesis of inflammatory bowel disease by leading to defective intestinal immune regulatory events, which may not be detectable at the peripheral level. PMID:6223862

  12. An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells.

    PubMed

    Davies, Richard; Vogelsang, Petra; Jonsson, Roland; Appel, Silke

    2016-09-01

    Phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in the treatment and monitoring of disease and therapy. The ability to measure numerous phospho-protein targets simultaneously at a single cell level accurately and rapidly provides significant advantages over other methods. We here discuss important considerations required to successfully implement these methods. Three different blood collection tubes (lithium-heparin tubes, CPT with sodium citrate and CPT with sodium heparin) were evaluated, with PBMC isolated through lithium-heparin tubes/lymphoprep displaying reduced basal and increased stimulation induced phosphorylation compared to the other two methods. Further, we provide a protocol outlining an 8 color assay developed for the study of intracellular signaling in peripheral blood mononuclear cells. The assay allows for the quantitative measurement of the phospho-proteins ERK1/2, NF-κB p65, Stat1 (Y701), Stat1 (S727), Stat3 (Y705), Stat3 (S727), Stat4 (Y693), p38 and Stat5 (Y694), as well as the identification of T cells, B cells, natural killer cells and monocytes. The assay additionally incorporates fluorescent cell barcoding, reducing assay costs and increasing throughput while increasing data robustness. Inter-assay precision was assessed over a month long period for all experimental variables (phospho-protein measured, cell type and stimulant). Coefficient of variations (CVs) calculated from process triplicates of normalized median fluorescence intensity (MFI) of the phospho-proteins displayed median CVs under 10% when grouped according to cell type, stimulation agent and phospho-protein measured, while the CV for each triplicate did not exceed 20%. PMID:27369043

  13. Viability and Recovery of Peripheral Blood Mononuclear Cells Cryopreserved for up to 12 Years in a Multicenter Study

    PubMed Central

    Kleeberger, Cynthia A.; Lyles, Robert H.; Margolick, Joseph B.; Rinaldo, Charles R.; Phair, John P.; Giorgi, Janis V.

    1999-01-01

    The Multicenter AIDS Cohort Study (MACS), an ongoing prospective study of the natural history of human immunodeficiency virus (HIV), has stored biologic specimens, including peripheral blood mononuclear cells (PBMC), from 5,622 participants for up to 12 years. The purpose of the present analysis was to evaluate the quality of the PBMC in the MACS repository in order to test the validity and feasibility of nested retrospective studies and to guide the planning of future repositories. PBMC were collected from MACS participants at four centers at 6-month intervals from 1984 to 1995, cryopreserved, and transported to a central repository for storage. A total of 596 of these specimens were subsequently tested for viability and used to evaluate cell function, to conduct immunophenotype analysis, or to isolate HIV. Simple linear regression models were applied to evaluate trends in recovery and viability over time and by center. Results indicated that from a nominal 107 cells cryopreserved per vial at all four centers, the median number of viable cells recovered was at least 5 × 106 (50% of the number stored) and the median viability was at least 90%. Results suggested that cryopreserved cells can be stored for at least 12 years with no general tendency toward cell loss over time. Furthermore, there were no statistically significant changes in the percent cell viability according to the length of time frozen, regardless of HIV serostatus or the level of CD4+ lymphocytes. Storing 107 PBMC per vial yields sufficient viable cells for phenotypic and/or functional analysis. Results from the MACS provide the basis for the planning of future repositories for use by investigators with similar research goals. PMID:9874657

  14. Therapeutic efficacy of bone marrow-derived mononuclear cells in diabetic polyneuropathy is impaired with aging or diabetes

    PubMed Central

    Kondo, Masaki; Kamiya, Hideki; Himeno, Tatsuhito; Naruse, Keiko; Nakashima, Eitaro; Watarai, Atsuko; Shibata, Taiga; Tosaki, Takahiro; Kato, Jiro; Okawa, Tetsuji; Hamada, Yoji; Isobe, Ken-ichi; Oiso, Yutaka; Nakamura, Jiro

    2015-01-01

    Aims/Introduction Recent studies have shown that cell transplantation therapies, such as endothelial precursor cells, bone marrow-derived mononuclear cells (BM-MNCs) and mesenchymal stem cells, are effective on diabetic polyneuropathy through ameliorating impaired nerve blood flow in diabetic rats. Here, we investigated the effects of BM-MNCs transplantation in diabetic polyneuropathy using BM-MNCs derived from adult (16-week-old) diabetic (AD), adult non-diabetic (AN) or young (8-week-old) non-diabetic (YN) rats. Materials and Methods BM-MNCs of AD and AN were isolated after an 8-week diabetes duration. The BM-MNCs were characterized using flow cytometry analysis of cell surface markers and reverse transcription polymerase chain reaction of several cytokines. BM-MNCs or saline were injected into hind limb muscles. Four weeks later, the thermal plantar test, nerve conduction velocity, blood flow of the sciatic nerve and capillary-to-muscle fiber ratio were evaluated. Results The number of CD29+/CD90+ cells that host mesenchymal stem cells in BM-MNCs decreased in AD compared with AN or YN, and transcript expressions of basic fibroblast growth factor and nerve growth factor in BM-MNCs decreased in AD compared with AN or YN. Impaired thermal sensation, decreased blood flow of the sciatic nerve and delayed nerve conduction velocity in 8-week-diabetic rats were significantly ameliorated by BM-MNCs derived from YN, whereas BM-MNCs from AD or AN rats did not show any beneficial effect in these functional tests. Conclusions These results show that cytokine production abilities and the mesenchymal stem cell population of BM-MNCs would be modified by aging and metabolic changes in diabetes, and that these differences could explain the disparity of the therapeutic efficacy of BM-MNCs between young and adult or diabetic and non-diabetic patients in diabetic polyneuropathy. PMID:25802721

  15. S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

    PubMed Central

    Spratt, Heidi M.; Gupta, Shivali; Petersen, John R.; Kuyumcu-Martinez, Muge N.

    2016-01-01

    Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

  16. Bone marrow mononuclear cells enhance anti-inflammatory effects of pravastatin against isoproterenol-induced myocardial infarction in rats.

    PubMed

    El-Mahdy, Nageh; Salem, Mohamed L; El-Sayad, Magda; El-Desouky, Karima I; Zaghow, Nesma

    2016-05-01

    The current study investigated the combinatorial effect of pravastatin (PRAV) and bone marrow mononuclear cells (BM-MNC) on acute myocardial infarction (AMI) induced experimentally in rats. After induction of MI, rats were given oral PRAV (20 mg/kg/day) for 28 days or a bolus intravenous injection (via lateral vein) of a total of 14 × 10(6) autologous BM-MNC or a combination of both. Serum brain natriuretic peptide (BNP) and histologic changes in cardiac tissues were assessed. Cardiac contents of lipid peroxides, superoxide dismutase (SOD) and inflammatory biomarkers including tumor necrosis factor (TNF)-α and interleukin (IL)-1β as well as vascular endothelial growth factor (VEGF) and nitric oxide (NO) were also measured. Combined PRAV and BM-MNC treatment significantly suppressed serum BNP. Cardiac cell apoptosis and inflammatory cell infiltration in heart tissue decreased significantly in both the PRAV and the PRAV + BM-MNC groups. Cardiac lipid peroxides along with TNFα and IL-1β levels were significantly reduced in both the PRAV and PRAV + BM-MNC hosts with an increase in SOD levels. However, the combined treatment increased cardiac NO levels and did not modify cardiac VEGF levels. The current results indicated that administration of BM-MNC improved the therapeutic efficacy of PRAV treatment by improving the morphology of infarcted hearts as well as decreasing inflammation in a host, but did not do so by inducing therapeutic angiogenesis. PMID:26606075

  17. Autologous transplantation of CD34(+) bone marrow derived mononuclear cells in management of non-reconstructable critical lower limb ischemia.

    PubMed

    Ismail, Ahmed M; Abdou, Said M; Aty, Hassan Abdel; Kamhawy, Adel H; Elhinedy, Mohammed; Elwageh, Mohammed; Taha, Atef; Ezzat, Amal; Salem, Hoda A; Youssif, Said; Salem, Mohamed L

    2016-08-01

    Patients with a decrease in limb perfusion with a potential threat to limb viability manifested by ischemic rest pain, ischemic ulcers, and/or gangrene are considered to have critical limb ischemia (CLI). Because of this generally poor outcome, there is a strong need for attempting any procedure to save the affected limb. The aim of this work is to evaluate the possibility to use stem cell therapy as a treatment option for patients with chronic critical lower limb ischemia with no distal run off. This study includes 20 patients with chronic critical lower limb ischemia with no distal run off who are unsuitable for vascular or endovascular option. These patients underwent stem cell therapy (SCT) by autologous transplantation of bone marrow derived mononuclear cells. 55 % of patients treated with SCT showed improvement of the rest pain after the first month, 60 % continued improvement of the rest pain after 6 months, 75 % after 1 year and 80 % after 2 years and continued without any deterioration till the third year. Limb salvage rate after STC was 80 % after the first year till the end of the second and third years. SCT can result in angiogenesis in patients with no-option CLI, providing a foundation for the application of this therapy to leg ischemia. PMID:25511801

  18. Effects of aspirin-triggered resolvin D1 on peripheral blood mononuclear cells from patients with Chagas' heart disease.

    PubMed

    Ogata, Haline; Teixeira, Maxelle Martins; de Sousa, Rodrigo Cunha; da Silva, Marcos Vinícius; Correia, Dalmo; Rodrigues Junior, Virmondes; Levy, Bruce David; de Paula Rogério, Alexandre

    2016-04-15

    Chagas disease is caused by Trypanosoma cruzi (T. cruzi). In some patients with Chagas disease, symptoms progress to chronic chagasic cardiomyopathy. Endogenously, inflammation is resolved in the presence of lipid mediators such as aspirin-triggered RvD1 (AT-RvD1) which has anti-inflammatory and pro-resolution effects. Here, we demonstrated, for the first time, the effects of AT-RvD1 on T. cruzi antigen-stimulated peripheral blood mononuclear cells (PBMCs) from patients with Chagas heart disease. The levels of IFN-γ, TNF-α, IL-10, and IL-13 increased in PBMCs from cardiac-form Chagas patients in stage B1 (patients with fewer heart abnormalities) stimulated with T. cruzi antigen compared to those in non-stimulated PBMCs. AT-RvD1 reduced the IFN-γ concentrations in PBMCs from patients with Chagas disease stimulated with T. cruzi antigen compared to stimulated with T. cruzi antigen cells. AT-RvD1 treatment resulted in no observable changes in TNF-α, IL-10, and IL-13 levels. AT-RvD1 significantly decreased the percentage of necrotic cells and caused a significant reduction in the proliferation rate of T. cruzi antigen-stimulated PBMCs from patients with Chagas disease. These findings demonstrate that AT-RvD1 modulates the immune response in Chagas disease patients and might have potential to be used as an alternative approach for slowing the development of further heart damage. PMID:26927755

  19. HSP27 modulates survival signaling in endosulfan-exposed human peripheral blood mononuclear cells treated with curcumin.

    PubMed

    Ahmed, T; Banerjee, B D

    2016-07-01

    Endosulfan, a well-known organochlorine pesticide, induces apoptosis and depletion of reduced glutathione (GSH) in human peripheral blood mononuclear cells (PBMC). Thus, for the amelioration of its effect, antioxidant and antiapoptotic potential of curcumin was evaluated. For ascertaining the attenuating effect of curcumin, various biochemical indices of cell damage such as cytotoxicity, oxidative stress, apoptosis (phosphatidylserine externalization, DNA fragmentation, and cytochrome c) in human PBMC was evaluated following endosulfan exposure (0-100 µM). To assess the role of HSP27 on endosulfan-induced apoptosis, the expression of HSP27 was examined. Curcumin (25 µM) increased cell viability significantly. As evident from the restoration of GSH, antiapoptotic potential was directly proportional to their antioxidant nature of curcumin. The present study indicates that the beneficial effect of curcumin on endosulfan-induced cytotoxicity is related to the induced synthesis of HSP27, emphasizing its antioxidant and therapeutic potential as well as underscoring the mechanism of pesticide-induced toxicity at cellular level. Taken together, these findings suggest that curcumin protects against endosulfan-induced immunotoxicity in human PBMC by attenuating apoptosis. PMID:26242398

  20. Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

    PubMed

    Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

    2016-03-01

    Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus. PMID:26775814

  1. Cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leucocytes from patients with atopic dermatitis: correlation with respiratory atopy.

    PubMed

    Sawai, T; Ikai, K; Uehara, M

    1998-05-01

    We determined the cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) activity in peripheral blood mononuclear leucocytes from 100 patients with atopic dermatitis (AD) aged 13-57 years (mean +/- SD, 29.8 +/- 17.7 years). The correlation between cAMP-PDE activity and clinical parameters such as the severity of eczema and a personal or family predisposition to atopic respiratory diseases (ARD) (asthma or allergic rhinitis) was examined. Although the enzymic activity varied from normal to very high in the AD patients, cAMP-PDE activity was significantly (P < 0.005) elevated in AD patients (42.1 +/- 22.0 units) as compared with the normal controls (12.4 +/- 5.6) and clinical control subjects (13.4 +/- 9.5). In contrast, we found no correlation between cAMP-PDE activity and the severity of eczema when AD patients were classified into four categories (remission, mild, moderate and severe) according to the extent of their skin involvement. Furthermore, we found that systemic corticosteroid therapy in severe AD patients did not alter the cAMP-PDE activity. cAMP-PDE activity was significantly (P < 0.01) higher in those AD patients who had a personal history of ARD (47.2 +/- 11.2) than in AD patients with a family history of ARD (37.2 +/- 17.4) and those without a personal or family history ('pure' AD) (34.4 +/- 19.8). Nevertheless, the cAMP-PDE activity was significantly higher even in 'pure' AD patients than in the controls. These results suggest that an elevation of cAMP-PDE activity is closely related to a predisposition to respiratory atopy, and does not follow inflammation in AD patients. PMID:9666832

  2. Dynamics of mononuclear phagocyte system Fc receptor function in systemic lupus erythematosus. Relation to disease activity and circulating immune complexes.

    PubMed

    Kimberly, R P; Parris, T M; Inman, R D; McDougal, J S

    1983-02-01

    Seventeen pairs of longitudinal studies of mononuclear phagocyte system (MPS) Fc receptor function in 15 patients with systemic lupus were performed to explore the dynamic range of Fc receptor dysfunction in lupus and to establish the relationships between MPS function, clinical disease activity and circulating immune complexes (CIC). Fc receptor function was measured by the clearance of IgG sensitized autologous erythrocytes. At the time of first study the degree of MPS dysfunction was correlated with both clinical activity (P less than 0.05) and CIC (P less than 0.05). At follow-up patients with a change in clinical status show significantly larger changes in clearance function compared to clinically stable patients (206 min vs 7 min; P less than 0.001). MPS function changed concordantly with a change in clinical status in all cases (P = 0.002). Longitudinal assessments did not demonstrate concordance of changes in MPS function and CIC, measured by three different assays. The MPS Fc receptor defect in systemic lupus is dynamic and closely associated with disease activity. The lack of concordance of the defect with changes in CIC suggests that either CIC does not adequately reflect receptor site saturation or that other factors may also contribute to the magnitude of MPS dysfunction. PMID:6839542

  3. Increased Expression of IL-37 in Patients with Graves' Disease and Its Contribution to Suppression of Proinflammatory Cytokines Production in Peripheral Blood Mononuclear Cells

    PubMed Central

    Li, Yanqun; Wang, Zi; Yu, Ting; Chen, Bingni; Zhang, Jinshun; Huang, Kunzhao; Huang, Zhong

    2014-01-01

    Background Intreleukin-37 (IL-37), a member of IL-1 family, is primarily an anti-inflammatory cytokine, which reduces systemic and local inflammation. However, the expression and role of IL-37 in Graves' disease (GD) remains unknown. This study aims to measure the levels of serum and peripheral blood mononuclear cells (PBMCs) IL-37 in patients with Graves' disease and to examine its association with disease activity. Furthermore, we investigate the effect of IL-37 on proinflammatory cytokines involved in the pathogenesis of GD. Methods The expressions of IL-37, TNF-α, IL-6, and IL-17 mRNA in peripheral blood mononuclear cells (PBMCs) of 40 patients with Graves' disease were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR), and the levels of IL-37, TNF-α, IL-6, and IL-17 in serum were detected by enzyme-linked immunoassay (ELISA). The correlation of serum IL-37 levels with cytokines and disease activity in Graves' disease patients were investigated. The expressions of cytokines TNF-α, IL-6, and IL-17 in PBMCs under recombinant IL-37 stimulation were determined by RT-PCR and ELISA respectively. Results The levels of IL-37, TNF-α, IL-6, and IL-17 in PBMCs and serum were significantly increased in patients with GD compared with healthy controls (HC). Serum IL-37 were closely correlated with TNF-α, IL-6, IL-17, thyrotropin (TSH), free thyroxine (FT4),free triiodothyronine (FT3) and thyrotropin receptor antibody (TRAB). GD patients with active disease showed higher IL-37 mRNA and serum protein levels compared with those with inactive disease as well as HC. Moreover, IL-37 suppressed the production of IL-6, IL-17 and TNF-α in PBMCs of patients with GD. Conclusions Increased level of IL-37 in patients with GD are associated with TNF-α, IL-6, IL-17 and disease activity, and it plays a protective role against inflammatory effect in GD by inhibiting the production of proinflammatory cytokines. Thus, IL-37 may provide a novel research

  4. Anti-ovarian tumor response of donor peripheral blood mononuclear cells is due to infiltrating cytotoxic NK cells

    PubMed Central

    Pandey, Veethika; Oyer, Jeremiah L.; Igarashi, Robert Y.; Gitto, Sarah B.; Copik, Alicja J.; Altomare, Deborah A.

    2016-01-01

    Treatment of ovarian cancer, a leading cause of gynecological malignancy, has good initial efficacy with surgery and platinum/taxane-based chemotherapy, but poor long-term survival in patients. Inferior long-term prognosis is attributed to intraperitoneal spreading, relapse and ineffective alternate therapies. Adoptive cell therapy is promising for tumor remission, although logistical concerns impede widespread implementation. In this study, healthy PBMCs were used to examine the immune response in a mouse model with human ovarian cancer, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion in mice treated intraperitoneally with PBMC+Interleukin-2 (IL-2), as compared to no expansion in non-tumor-bearing mice given the same treatment. PBMC+IL-2 treated mice exhibiting NK cell expansion had complete tumor remission. To validate NK cell mediated anti-tumor response, the intratumoral presence of NK cells and their cytotoxicity was confirmed by immunohistochemistry and granzyme activity of NK cells recovered from the tumor. Collectively, this study highlights the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population, as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients. PMID:26802025

  5. Vasculogenic Conditioning of Peripheral Blood Mononuclear Cells Promotes Endothelial Progenitor Cell Expansion and Phenotype Transition of Anti‐Inflammatory Macrophage and T Lymphocyte to Cells With Regenerative Potential

    PubMed Central

    Masuda, Haruchika; Tanaka, Rica; Fujimura, Satoshi; Ishikawa, Masakazu; Akimaru, Hiroshi; Shizuno, Tomoko; Sato, Atsuko; Okada, Yoshinori; Iida, Yumi; Itoh, Jobu; Itoh, Yoshiko; Kamiguchi, Hiroshi; Kawamoto, Atsuhiko; Asahara, Takayuki

    2014-01-01

    Background Cell‐based therapies involving mononuclear cells (MNCs) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNCs has not been evaluated. We investigated the therapeutic potential of peripheral blood (PB) MNCs, operated by recently developed quality and quantity (QQ) culture of endothelial progenitor cells (EPCs). Methods and Results PBs were collected from healthy volunteers; peripheral blood mononuclear cells (PBMNCs) isolated from these PBs were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt‐3 ligand, vascular endothelial growth factor, and interleukin‐6. The resulting cells (QQMNCs) in EPC colony‐forming assay generated significantly more definitive EPC colonies than PBMNCs. In flow cytometry, macrophages and helper T lymphocytes of QQMNCs became phenotypically polarized into angiogenic, anti‐inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T‐cell expansion. Quantitative real‐time polymerase chain reaction (qRT‐PCR) assay revealed the predominant proangiogenic gene expressions in QQMNCs versus PBMNCs. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured “early EPC” Tx (eEPCTx), and granulocyte colony‐stimulating factor mobilized CD34+ cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx. Histological evaluations and qRT‐PCR assays in ischemic hindlimbs demonstrated that QQMNCTx, similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx. Conclusions QQ culture potentiates the ability of PBMNCs to promote regeneration of injured tissue

  6. In vitro response of human peripheral blood mononuclear cells to AISI 316L austenitic stainless steel subjected to nitriding and collagen coating treatments.

    PubMed

    Stio, Maria; Martinesi, Maria; Treves, Cristina; Borgioli, Francesca

    2015-02-01

    Surface modification treatments can be used to improve the biocompatibility of austenitic stainless steels. In the present research two different modifications of AISI 316L stainless steel were considered, low temperature nitriding and collagen-I coating, applied as single treatment or in conjunction. Low temperature nitriding produced modified surface layers consisting mainly of S phase, which enhanced corrosion resistance in PBS solution. Biocompatibility was assessed using human peripheral blood mononuclear cells (PBMC) in culture. Proliferation, lactate dehydrogenase (LDH) levels, release of cytokines (TNF-α, IL-1β, IL-12, IL-10), secretion of metalloproteinase (MMP)-9 and its inhibitor TIMP-1, and the gelatinolytic activity of MMP-9 were determined. While the 48-h incubation of PBMC with all the sample types did not negatively influence cell proliferation, LDH and MMP-9 levels, suggesting therefore a good biocompatibility, the release of the pro-inflammatory cytokines was always remarkable when compared to that of control cells. However, in the presence of the nitrided and collagen coated samples, the release of the pro-inflammatory cytokine IL-1β decreased, while that of the anti-inflammatory cytokine IL-10 increased, in comparison with the untreated AISI 316L samples. Our results suggest that some biological parameters were ameliorated by these surface treatments of AISI 316L. PMID:25655502

  7. Human umbilical cord blood mononuclear cells and chorionic plate-derived mesenchymal stem cells promote axon survival in a rat model of optic nerve crush injury

    PubMed Central

    CHUNG, SOKJOONG; RHO, SEUNGSOO; KIM, GIJIN; KIM, SO-RA; BAEK, KWANG-HYUN; KANG, MYUNGSEO; LEW, HELEN

    2016-01-01

    The use of mesenchymal stem cells (MSCs) in cell therapy in regenerative medicine has great potential, particularly in the treatment of nerve injury. Umbilical cord blood (UCB) reportedly contains stem cells, which have been widely used as a hematopoietic source and may have therapeutic potential for neurological impairment. Although ongoing research is dedicated to the management of traumatic optic nerve injury using various measures, novel therapeutic strategies based on the complex underlying mechanisms responsible for optic nerve injury, such as inflammation and/or ischemia, are required. In the present study, a rat model of optic nerve crush (ONC) injury was established in order to examine the effects of transplanting human chorionic plate-derived MSCs (CP-MSCs) isolated from the placenta, as well as human UCB mononuclear cells (CB-MNCs) on compressed rat optic nerves. Expression markers for inflammation, apoptosis, and optic nerve regeneration were analyzed, as well as the axon survival rate by direct counting. Increased axon survival rates were observed following the injection of CB-MNCs at at 1 week post-transplantation compared with the controls. The levels of growth-associated protein-43 (GAP-43) were increased after the injection of CB-MNCs or CP-MSCs compared with the controls, and the expression levels of hypoxia-inducible factor-1α (HIF-1α) were also significantly increased following the injection of CB-MNCs or CP-MSCs. ERM-like protein (ERMN) and SLIT-ROBO Rho GTPase activating protein 2 (SRGAP2) were found to be expressed in the optic nerves of the CP-MSC-injected rats with ONC injury. The findings of our study suggest that the administration of CB-MNCs or CP-MSCs may promote axon survival through systemic concomitant mechanisms involving GAP-43 and HIF-1α. Taken together, these findings provide further understanding of the mechanisms repsonsible for optic nerve injury and may aid in the development of novel cell-based therapeutic strategies with

  8. Therapeutic Benefit of Intravenous Administration of Human Umbilical Cord Blood- Mononuclear Cells Following Intracerebral Hemorrhage in Rat

    PubMed Central

    Seghatoleslam, Masoumeh; Jalali, Mehdi; Nikravesh, Mohammad Reza; Hosseini, Mahmoud; Hamidi Alamdari, Daryoush; Fazel, Alireza

    2012-01-01

    Objective(s) Human umbilical cord blood (HUCB) is now considered as a valuable source for stem cell–based therapies. Previous studies showed that intravascular injection of the HUCB significantly improves neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). In the present study, we hypothesize transplanted HUCB derived mononuclear cells (UC-MCs) can decrease injured volume and also ameliorate neurological function in ICH rats. Materials and Methods Experimental ICH was induced by intrastriatal administration of collagenase in rats. One day after surgery, the rats were divided into 3 groups to receive intravenously either BrdU positive human UC-MCs [(4×106 and 8×106 cells in 1 ml saline, n=10 respectively) as treated groups] or the same amount of saline [as lesion group (n=10)]. There was also one group (control) that received only vehicle solution of collagenase. The animals were evaluated for 14 days with behavioral tests. Transplanted UC-MCs were detected by immunohistochemistry. Histological data and scores of functional tests were analyzed using ANOVA. Cellular co-localization of BrdU+ cells in the histological slides was determined by software Image J. Results Intravenously transplanted UC-MCs migrated selectively to the hematomal area and reduce injured volume. The UC-MCs transplanted groups showed better performance on functional tests after 2 weeks compared with the lesion and control groups (P< 0.05). There was no difference in the functional recovery and injured volume improvement between the 2 treated groups. Conclusion Intravenously transplanted UC-MCs accelerate neurological function recovery of ICH rat and diminish the striatum lesion size. Thus these cells may provide a potential cell candidate for cell-based therapy in ICH. PMID:23492836

  9. The renal mononuclear phagocytic system.

    PubMed

    Nelson, Peter J; Rees, Andrew J; Griffin, Matthew D; Hughes, Jeremy; Kurts, Christian; Duffield, Jeremy

    2012-02-01

    The renal mononuclear phagocytic system, conventionally composed of macrophages (Mø) and dendritic cells (DCs), plays a central role in health and disease of the kidney. Overlapping definitions of renal DCs and Mø, stemming from historically separate research tracks and the lack of experimental tools to specifically study the roles of these cells in vivo, have generated confusion and controversy, however, regarding their immunologic function in the kidney. This brief review provides an appraisal of the current state of knowledge of the renal mononuclear phagocytic system interpreted from the perspective of immunologic function. Physical characteristics, ontogeny, and known functions of the main subsets of renal mononuclear phagocytes as they relate to homeostasis, surveillance against injury and infection, and immune-mediated inflammatory injury and repair within the kidney are described. Gaps and inconsistencies in current knowledge are used to create a roadmap of key questions to be answered in future research. PMID:22135312

  10. Type I interferon subtypes produced by human peripheral mononuclear cells from one normal donor stimulated by viral and non-viral inducing factors.

    PubMed

    Palmer, Pierre; Tovey, Michael G; Raschilas, Franck; Brassart, Lilia; Meritet, Jean-François; Porcher, Raphaël; Lebon, Pierre

    2007-06-01

    Through the activation of Toll-like receptors (TLRs) or cytosolic RNA helicases, a large number of pathogenic or synthetic components can induce the transcription of genes coding for type I interferons (IFNs). This family of related cytokines includes notably, a single IFN-beta protein and 13 different IFN-alpha subtypes, whose biological activities are probably not the same. The aim of this study was to characterize the type I IFN subtypes produced in vitro by human peripheral blood mononuclear cells (PBMCs) in response to specific inducers. Thus, PBMCs obtained from a single donor, were exposed to various agents including Sendai virus, Herpes simplex virus-1 (HSV-1), poliovirus-IgG complexes and serum from a patient with systemic lupus erythematosus (SLE). Six hours later, mRNA was extracted and amplified by RT-PCR using primers which recognize IFN-B mRNA and the different IFN-A mRNA subtypes. IFN-A subtypes were identified by cloning and sequencing the amplification product. Antiviral activity was assayed in supernatant at 18 hours. Human PBMCs were found to express constitutively type I IFNs mRNA. Antiviral activity and expression of IFN-A and IFN-B mRNA increased with each inducing agent. Although almost all the IFN-A subtypes were detected, their relative abundance appeared to be dependent upon the inducing agent. Incubation of PBMCs with a neutralizing monoclonal antibody directed against the type I IFN receptor (IFNAR) did not affect the level of antiviral activity in the supernatant of induced PBMCs. Our results suggest that the level of IFN-alpha expressed by PBMCs cells is independent of IFNAR feedback signalling and that the nature of the inducing agent modifies the pattern of IFN-A subtypes preferentially expressed by these cells. PMID:17594944

  11. Intrauterine insemination of cultured peripheral blood mononuclear cells prior to embryo transfer improves clinical outcome for patients with repeated implantation failures.

    PubMed

    Madkour, Aicha; Bouamoud, Nouzha; Louanjli, Noureddine; Kaarouch, Ismail; Copin, Henri; Benkhalifa, Moncef; Sefrioui, Omar

    2016-02-01

    Implantation failure is a major limiting factor in assisted reproduction improvement. Dysfunction of embryo-maternal immuno-tolerance pathways may be responsible for repeated implantation failures. This fact is supported by immunotropic theory stipulating that maternal immune cells, essentially uterine CD56+ natural killer cells, are determinants of implantation success. In order to test this hypothesis, we applied endometrium immuno-modulation prior to fresh embryo transfer for patients with repeated implantation failures. Peripheral blood mononuclear cells were isolated from repeated implantation failure patients undergoing assisted reproductive technology cycles. On the day of ovulation induction, cells were isolated and then cultured for 3 days and transferred into the endometrium cavity prior to fresh embryo transfer. This immunotherapy was performed on 27 patients with repeated implantation failures and compared with another 27 patients who served as controls. Implantation and clinical pregnancy were increased significantly in the peripheral blood mononuclear cell test versus control (21.54, 44.44 vs. 8.62, 14.81%). This finding suggests a clear role for endometrium immuno-modulation and the inflammation process in implantation success. Our study showed the feasibility of intrauterine administration of autologous peripheral blood mononuclear cells as an effective therapy to improve clinical outcomes for patients with repeated implantation failures and who are undergoing in vitro fertilization cycles. PMID:25613318

  12. Interferon (IFN) production by peripheral blood mononuclear (PBM) cells of an elderly population

    SciTech Connect

    Nelson, B.J.; Murasko, D.M.

    1986-03-05

    Previous investigations in the laboratory have reported decreased mitogen responses of PBM's from elderly individuals compared to responses of young adults to PHA and ConA. Current studies have investigated the role of IFN in this decreased T cell responsiveness of the elderly. Supernatants of PBM's from 80 elderly (mean age 85) and 50 young individuals (mean age 28) were assayed for antiviral activity, after incubation with optimum and supraoptimum concentrations of mitogen for 24-120 hrs. IFN levels were maximum for both elderly and young populations at 72 hrs coinciding with time of maximum proliferation as determined by uptake of /sup 3/H thymidine. IFN levels declined with longer incubation periods. All IFN produced was IFN-gamma as determined by sensitivity to pH 2 and by neutralizations with monoclonal antibody specific for human IFN-gamma and polyclonal antiserum specific for IFN-alpha. The elderly population's mean IFN titers for both PHA and ConA were about 39% of the mean titers of the young (p less than or equal to 0.02). Both elderly and young groups displayed significant positive correlation between the amount of IFN produced and the level of proliferation in response to the mitogens (p less than or equal to 0.036). Therefore, the above data suggests the decreased levels of IFN produced by elderly PBM's may be one of the factors responsible for the observed decreased proliferative response to mitogens.

  13. Mononuclear and dinuclear peroxotungsten complexes with co-ordinated dipeptides as potent inhibitors of alkaline phosphatase activity.

    PubMed

    Hazarika, Pankaj; Kalita, Diganta; Islam, Nashreen S

    2008-08-01

    New molecular peroxotungstate(VI) complexes with dipeptides as ancillary ligands of the type, [WO(O(2))(2)(dipeptide)(H(2)O)].3H(2)O, dipeptide = glycyl-glycine or glycyl-leucine, have been synthesized and characterized by elemental analysis, spectral and physico-chemical methods including thermal analysis. The complexes contain side-on bound peroxo groups and a peptide zwitterion bonded to the metal centre unidentately through an O(carboxylate) atom. Investigations on certain biologically important key properties of these compounds and a set of dimeric compounds in analogous co-ligand environment, Na(2)[W(2)O(3)(O(2))(4)(dipeptide)(2)].3H(2)O, dipeptide = glycyl-glycine and glycyl-leucine, reported previously by us revealed interesting features of the compounds. Each of the compounds despite having a 7 co-ordinated metal centre exerts a strong inhibitory effect on alkaline phosphatase activity with a potency higher than that of the free dipeptide, tungstate or peroxotungstate. The compounds exhibit remarkable stability in solutions of acidic as well as physiological pH and are weaker as substrate to the enzyme catalase, compared to H(2)O(2). The mononuclear and dinuclear peroxotungsten compounds are efficient oxidants of reduced glutathione (GSH), a reaction in which only one of the peroxo groups of a diperoxotungsten moiety of the complexes was found to be active. PMID:18665997

  14. Human eosinophils modulate peripheral blood mononuclear cell response to Schistosoma mansoni adult worm antigen in vitro.

    PubMed

    Tweyongyere, R; Namanya, H; Naniima, P; Cose, S; Tukahebwa, E M; Elliott, A M; Dunne, D W; Wilson, S

    2016-08-01

    High numbers of eosinophils are observed in parasitic infections and allergic diseases, where they are proposed to be terminally differentiated effector cells that play beneficial role in host defence, or cause harmful inflammatory response. Eosinophils have been associated with killing of schistosomulae in vitro, but there is growing evidence that eosinophils can play additional immuno-regulatory role. Here, we report results of a study that examines peripheral blood mononuclear cell (PBMC) cytokine responses to Schistosoma mansoni adult worm antigen (SWA) when stimulated alone or enriched with autologous eosinophils. Production of the Th-2 type cytokines interleukin (IL)-4, IL-5 and IL-13 was lower (P = 0·017, 0·018 and <0·001, respectively) in PBMC + eosinophil cultures than in PBMC-only cultures stimulated with SWA. Substantial levels of IL-13, IL-10, interferon gamma and tumour necrosis factor alpha were recorded in cultures of eosinophils, but none of these cytokines showed significant association with the observed eosinophil-induced drop in cytokine responses of PBMC. Transwell experiments suggested that the observed effect is due to soluble mediators that downmodulate production of Th-2 type cytokines. This study shows that eosinophils may down-modulate schistosome-specific Th-2 type cytokine responses in S. mansoni-infected individuals. The mechanism of this immune modulation remains to be elucidated. PMID:27169695

  15. Probiotic supplement consumption alters cytokine production from peripheral blood mononuclear cells: a preliminary study using healthy individuals.

    PubMed

    Hepburn, N J; Garaiova, I; Williams, E A; Michael, D R; Plummer, S

    2013-12-01

    The objective of this study was to examine the effect of daily probiotic supplementation upon the immune profile of healthy participants by the assessment of ex vivo cytokine production. Twenty healthy adult volunteers received a multi-strain probiotic supplement consisting of two strains of Lactobacillus acidophilus (CUL60 and CUL21), Bifidobacterium lactis (CUL34) and Bifidobacterium bifidum (CUL20) and fructooligosaccharide for 12 weeks. Blood samples were collected at baseline, 6 and 12 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured ex vivo in the presence or absence of lipopolysaccharide and cytokine production was assessed. Postintervention, a significant decrease in the production of interleukin-6 and interleukin-1β was apparent when PBMCs were incubated in the presence of lipopolysaccharide, whilst a significant increase in IL-10 and transforning growth factor-β production was seen when the cells were incubated without an additional stimulus. This preliminary study demonstrates the potential of a multi-strain probiotic supplement to alter the immune response as demonstrated by changes in ex vivo cytokine production. Such results demonstrate the potential benefit of probiotic supplementation for healthy individuals and warrants further investigation. PMID:24311314

  16. Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells.

    PubMed

    Montano, Marco Aurélio Echart; da Cruz, Ivana Beatrice Mânica; Duarte, Marta Maria Medeiros Frescura; Krewer, Cristina da Costa; da Rocha, Maria Izabel de Ugalde Marques; Mânica-Cattani, Maria Fernanda; Soares, Felix Alexandre Antunes; Rosa, Guilherme; Maris, Angélica Francesca; Battiston, Francielle Garghetti; Trott, Alexis; Lera, Juan Pablo Barrio

    2012-10-01

    Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question. PMID:22688013

  17. Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model

    PubMed Central

    Hacker, Stefan; Mittermayr, Rainer; Nickl, Stefanie; Haider, Thomas; Lebherz-Eichinger, Diana; Beer, Lucian; Mitterbauer, Andreas; Leiss, Harald; Zimmermann, Matthias; Schweiger, Thomas; Keibl, Claudia; Hofbauer, Helmut; Gabriel, Christian; Pavone-Gyöngyösi, Mariann; Redl, Heinz; Tschachler, Erwin; Mildner, Michael; Ankersmit, Hendrik Jan

    2016-01-01

    Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (SecPBMC), apoptotic PBMCs (Apo-SecPBMC), or controls. The wounds treated with Apo-SecPBMC had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-SecPBMC had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-SecPBMC significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting. PMID:27125302

  18. Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model.

    PubMed

    Hacker, Stefan; Mittermayr, Rainer; Nickl, Stefanie; Haider, Thomas; Lebherz-Eichinger, Diana; Beer, Lucian; Mitterbauer, Andreas; Leiss, Harald; Zimmermann, Matthias; Schweiger, Thomas; Keibl, Claudia; Hofbauer, Helmut; Gabriel, Christian; Pavone-Gyöngyösi, Mariann; Redl, Heinz; Tschachler, Erwin; Mildner, Michael; Ankersmit, Hendrik Jan

    2016-01-01

    Burn wounds pose a serious threat to patients and often require surgical treatment. Skin grafting aims to achieve wound closure but requires a well-vascularized wound bed. The secretome of peripheral blood mononuclear cells (PBMCs) has been shown to improve wound healing and angiogenesis. We hypothesized that topical application of the PBMC secretome would improve the quality of regenerating skin, increase angiogenesis, and reduce scar formation after burn injury and skin grafting in a porcine model. Full-thickness burn injuries were created on the back of female pigs. Necrotic areas were excised and the wounds were covered with split-thickness mesh skin grafts. Wounds were treated repeatedly with either the secretome of cultured PBMCs (Sec(PBMC)), apoptotic PBMCs (Apo-Sec(PBMC)), or controls. The wounds treated with Apo-Sec(PBMC) had an increased epidermal thickness, higher number of rete ridges, and more advanced epidermal differentiation than controls. The samples treated with Apo-Sec(PBMC) had a two-fold increase in CD31+ cells, indicating more angiogenesis. These data suggest that the repeated application of Apo-Sec(PBMC) significantly improves epidermal thickness, angiogenesis, and skin quality in a porcine model of burn injury and skin grafting. PMID:27125302

  19. Clinical outcome of autologous mononuclear cells transplantation for spinal cord injury: a systematic review and meta-analysis

    PubMed Central

    Aghayan, Hamid Reza; Arjmand, Babak; Yaghoubi, Mohsen; Moradi-Lakeh, Maziar; Kashani, Homa; Shokraneh, Farhad

    2014-01-01

    Background: Cell-based therapies can be used to treat neurological diseases and spinal cord injuries. The aim of this study was to assess the clinical outcome of bone marrow derived mononuclear cells (BM-MNCs) transplantation in patients with spinal cord injuries. Methods: Following a systematic review to detect clinical intervention studies, a meta-analysis was done for pooling data to estimate the outcome of BM-MNCs transplantation. The percentage of the patients with improved ASIA scale from one grade to a higher grade was defined as the main outcome. By considering the study design and outcome measurement, two reviewers independently extracted the data. Results: Eight relevant primary studies were found; seven qualified studies, with a combined total of 328 patients were assessed by meta-analysis, including 314 ASIA-A, 13 ASIA-B, 94 cervical, 227 thoracic and 60 acute injuries. The percentage of the patients’ improvement was tested by meta-analysis through random and fixed models. The overall percentage of all patients’ improved ASIA scale after a one- year follow-up (95% CIs) was 43 (0.27-0.59). Conclusion: Data from published trials revealed that encouraging results were achieved by autologous BMMNCs for the treatment of spinal cord injury. However, the number of clinical trials included in the systematic review was too limited to reach a definite conclusion. More qualified clinical trials with standardized methods are needed to truly justify the outcome of this therapeutic modality in SCI patients. PMID:25678991

  20. Age-Related Disease Association of Endogenous γ-H2AX Foci in Mononuclear Cells Derived from Leukapheresis

    PubMed Central

    Schurman, Shepherd H.; Dunn, Christopher A.; Greaves, Rebecca; Yu, Binbing; Ferrucci, Luigi; Croteau, Deborah L.; Seidman, Michael M.; Bohr, Vilhelm A.

    2012-01-01

    The phosphorylated form of histone H2AX (γ-H2AX) forms immunohistochemically detectable foci at DNA double strand breaks. In peripheral blood mononuclear cells (PBMCs) derived from leukapheresis from patients enrolled in the Baltimore Longitudinal Study of Aging, γ-H2AX foci increased in a linear fashion with regards to age, peaking at ∼57 years. The relationship between the frequency of γ-H2AX foci and age-related pathologies was assessed. We found a statistically significant (p = 0.023) 50% increase in foci in PBMCs derived from patients with a known history of vitamin D deficiency. In addition, there were trends toward increased γ-H2AX foci in patients with cataracts (34% increase, p<0.10) and in sleep apnea patients (44%, p<0.10). Among patients ≥57 y/o, we found a significant (p = 0.037) 36% increase in the number of γ-H2AX foci/cell for patients with hypertension compared to non-hypertensive patients. Our results support a role for increased DNA damage in the morbidity of age-related diseases. γ -H2AX may be a biomarker for human morbidity in age-related diseases. PMID:23029205

  1. Synaptic plasticity and sensory-motor improvement following fibrin sealant dorsal root reimplantation and mononuclear cell therapy

    PubMed Central

    Benitez, Suzana U.; Barbizan, Roberta; Spejo, Aline B.; Ferreira, Rui S.; Barraviera, Benedito; Góes, Alfredo M.; de Oliveira, Alexandre L. R.

    2014-01-01

    Root lesions may affect both dorsal and ventral roots. However, due to the possibility of generating further inflammation and neuropathic pain, surgical procedures do not prioritize the repair of the afferent component. The loss of such sensorial input directly disturbs the spinal circuits thus affecting the functionality of the injuried limb. The present study evaluated the motor and sensory improvement following dorsal root reimplantation with fibrin sealant (FS) plus bone marrow mononuclear cells (MC) after dorsal rhizotomy. MC were used to enhance the repair process. We also analyzed changes in the glial response and synaptic circuits within the spinal cord. Female Lewis rats (6–8 weeks old) were divided in three groups: rhizotomy (RZ group), rhizotomy repaired with FS (RZ+FS group) and rhizotomy repaired with FS and MC (RZ+FS+MC group). The behavioral tests electronic von-Frey and Walking track test were carried out. For immunohistochemistry we used markers to detect different synapse profiles as well as glial reaction. The behavioral results showed a significant decrease in sensory and motor function after lesion. The reimplantation decreased glial reaction and improved synaptic plasticity of afferent inputs. Cell therapy further enhanced the rewiring process. In addition, both reimplanted groups presented twice as much motor control compared to the non-treated group. In conclusion, the reimplantation with FS and MC is efficient and may be considered an approach to improve sensory-motor recovery following dorsal rhizotomy. PMID:25249946

  2. Lawsonia intracellularis-specific interferon γ gene expression by peripheral blood mononuclear cells in vaccinated and naturally infected foals.

    PubMed

    Pusterla, Nicola; Mapes, Samantha; Gebhart, Connie

    2012-05-01

    The cell-mediated immune response to Lawsonia intracellularis, the agent of equine proliferative enteropathy (EPE), was investigated in vaccinated and naturally infected foals. Interferon (IFN)-γ gene expression was determined in peripheral blood mononuclear cells collected from vaccinated (n=6) and control foals (n=6) every 30 days for 180 days following first vaccine administration, and from clinically affected foals (n=16) within 7-10 days of diagnosing EPE. Seroconversion (immunoperoxidase monolayer assay titer ≥60) occurred in 5/6 vaccinated foals between 60 and 90 days following the first vaccine administration and these foals remained seropositive for the remaining study period. IFN-γ gene expression in all vaccinated foals was significantly higher (P<0.05) on days 60-180 following first vaccine administration compared to IFN-γ gene expression in control foals. When IFN-γ gene transcription was compared between naturally infected and vaccinated foals, a significant difference (P<0.05) was observed only for day 0. PMID:21689957

  3. Docosahexaenoic acid (DHA) and docosapentaenoic acid (DPAn-6) algal oils reduce inflammatory mediators in human peripheral mononuclear cells in vitro and paw edema in vivo.

    PubMed

    Nauroth, Julie M; Liu, Ying Chun; Van Elswyk, Mary; Bell, Rebecca; Hall, Eileen Bailey; Chung, Gloria; Arterburn, Linda M

    2010-05-01

    The anti-inflammatory activity associated with fish oil has been ascribed to the long-chain polyunsaturated fatty acids (LC-PUFA), predominantly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Here we examined the anti-inflammatory effects of two DHA-rich algal oils, which contain little EPA, and determined the contribution of the constituent fatty acids, particularly DHA and docosapentaenoic acid (DPAn-6). In vitro, lipopolysaccharide (LPS)-stimulated Interleukin-1 beta (IL-1beta) and Tumor Necrosis Factor-alpha (TNF-alpha) secretion in human peripheral blood mononuclear cells (PBMC) was inhibited with apparent relative potencies of DPAn-6 (most potent) > DHA > EPA. In addition, DPAn-6 decreased intracellular levels of cyclooxygenase-2 (COX-2) and was a potent inhibitor of pro-inflammatory prostaglandin E2 (PGE2) production. DHA/DPAn-6-rich DHA-S (DHA-S) algal oil was more effective at reducing edema in rats than DHA-rich DHA-T (DHA-T), suggesting that DPAn-6 has anti-inflammatory properties. Further in vivo analyses demonstrated that feeding DPAn-6 alone, provided as an ethyl ester, reduced paw edema to an extent approaching that of indomethacin and enhanced the anti-inflammatory activity of DHA when given in combination. Together, these results demonstrate that DPAn-6 has anti-inflammatory activity and enhances the effect of DHA in vitro and in vivo. Thus, DHA-S algal oil may have potential for use in anti-inflammatory applications. PMID:20364438

  4. Fc receptor-bearing peripheral blood mononuclear cells in breast cancer patients: a possible marker of tumour burden and prognosis.

    PubMed Central

    Bray, J; McPherson, T A

    1981-01-01

    Indirect immunofluorescence was used to identify and quantitate peripheral blood mononuclear (PBM) cells possessing high avidity Fc receptors in 105 patients upon referral to the breast cancer clinic at the Cross Cancer Institute. The cell detected was shown to be a non-adherent PBM, probably belonging to the T or null cell population. The mean percentage +/- 2 standard deviations of PBM-positive cells in 75 patients with no disease or benign breast disease was 5.3 +/- ;6.7, and this was significantly (P less than 0.001) less than the percentage found for 31 patients with breast cancer. The percentage of PBM-positive cells correlated directly with tumour burden in patient with small (less than or equal to 5 cm) tumours without regional node or extranodal metastases (5/13 had greater than or equal to 12% positive PBM) and in those with small tumours plus regional node metastases, but without extranodal metastases (8/10 had greater than or equal to 12% positive PBM). This correlation was less, however, in patients with large tumours (greater than 5 cm), and in those with extranodal metastases (4/8 had greater than or equal to 12% positive PBM), and in patients tested postoperatively (1/13 had greater than or equal to 12% positive PBM) even though 6!13 had regional node metastases at the time of surgery. Thus, this relatively simple assay, which can be done on peripheral blood samples, may turn out to be useful in patients with breast cancer as a prognostic marker insofar as it may be an indirect indicator of tumour burden preoperatively. If so, it may lead to a more aggressive postoperative adjuvant therapy approach to the subpopulation of node-negative PBM-positive breast cancer patients than is currently used for node-negative patients. PMID:7035033

  5. In Vitro Infection of Human Peripheral Blood Mononuclear Cells by GB Virus C/Hepatitis G Virus

    PubMed Central

    Fogeda, Marta; Navas, Sonia; Martín, Julio; Casqueiro, Mercedes; Rodríguez, Elena; Arocena, Carlos; Carreño, Vicente

    1999-01-01

    GB virus C (GBV-C), also known as hepatitis G virus, is a recently discovered flavivirus-like RNA agent with unclear pathogenic implications. To investigate whether human peripheral blood mononuclear cells (PBMC) are susceptible to in vitro GBV-C infection, we have incubated PBMC from four healthy blood donors with a human GBV-C RNA-positive serum. By means of (i) strand-specific reverse transcription-PCR, cloning, and sequencing; (ii) sucrose ultracentrifugation and RNase sensitivity assays; (iii) fluorescent in situ hybridization; and (iv) Western blot analysis, it has been demonstrated that GBV-C is able to infect in vitro cells and replicate for as long as 30 days under the conditions developed in our cell culture system. The concentration of GBV-C RNA increased during the second and third weeks of culture. The titers of the genomic strand were 10 times higher than the titers of the antigenomic strand. In addition, the same predominant GBV-C sequence was found in all PBMC cultures and in the in vivo-GBV-C-infected PBMC isolated from the donor of the inoculum. GBV-C-specific fluorescent in situ hybridization signals were confined to the cytoplasm of cells at different times during the culture period. Finally, evidence obtained by sucrose ultracentrifugation, RNase sensitivity assays, and Western blot analysis of the culture supernatants suggests that viral particles are released from in vitro-GBV-C-infected PBMC. In conclusion, our study has demonstrated, for the first time, GBV-C replication in human lymphoid cells under experimental in vitro infection conditions. PMID:10196301

  6. Co-stimulation with TLR7/8 and TLR9 agonists induce down-regulation of innate immune responses in sheep blood mononuclear and B cells.

    PubMed

    Booth, Jayaum S; Buza, Joram J; Potter, Andrew; Babiuk, Lorne A; Mutwiri, George K

    2010-05-01

    Toll-like receptors (TLRs) play an important role in the activation of innate and adaptive immune responses. Stimulation with multiple TLR agonists may result in synergistic, complimentary or inhibitory effects on innate immune responses. In this study, we investigated the effects of co-stimulation of sheep peripheral blood mononuclear cells (PBMC) and B cells with agonists for TLR3, 4, 7/8 and 9. Sheep PBMC stimulated with either CpG (TLR9 agonist) or RNA oligoribonucleotides ([ORNs], TLR7/8 agonist) exhibited significant IL-12 production, but only CpG induced IFNalpha, IgM and proliferative responses. In contrast, poly(I:C) (TLR3 agonist) and LPS (TLR4 agonist) did not induce any of these responses. Interestingly, we observed that co-stimulation of PBMC with CpG+ORN or CpG+imiquimod (another TLR7/8 agonist) resulted in significant reduction in CpG-induced IFNalpha production, B cell proliferation and IgM responses. Pre-incubation of cells with CpG prior to exposure of the cells to imiquimod resulted in similar inhibitory responses indicating that the down-regulatory mechanisms are not associated with competition for cellular uptake or for receptors of the two agonists. Sheep B cells constitutively expressed TLR7, TLR8 and TLR9 mRNA transcripts, suggesting a possible role of TLR cross-talk in the down-regulatory mechanisms. Down-regulation of responses by co-stimulation with closely related TLRs may be a regulatory mechanism by which the host prevents overstimulation of innate immune responses. PMID:20051250

  7. Genome-Wide Profiling of RNA from Dried Blood Spots: Convergence with Bioinformatic Results Derived from Whole Venous Blood and Peripheral Blood Mononuclear Cells.

    PubMed

    McDade, Thomas W; M Ross, Kharah; L Fried, Ruby; Arevalo, Jesusa M G; Ma, Jeffrey; Miller, Gregory E; Cole, Steve W

    2016-01-01

    Genome-wide transcriptional profiling has emerged as a powerful tool for analyzing biological mechanisms underlying social gradients in health, but utilization in population-based studies has been hampered by logistical constraints and costs associated with venipuncture blood sampling. Dried blood spots (DBS) provide a minimally invasive, low-cost alternative to venipuncture, and in this article we evaluate how closely the substantive results from DBS transcriptional profiling correspond to those derived from parallel analyses of gold-standard venous blood samples (PAXgene whole blood and peripheral blood mononuclear cells [PBMC]). Analyses focused on differences in gene expression between African-Americans and Caucasians in a community sample of 82 healthy adults (age 18-70 years; mean 35). Across 19,679 named gene transcripts, DBS-derived values correlated r = .85 with both PAXgene and PBMC values. Results from bioinformatics analyses of gene expression derived from DBS samples were concordant with PAXgene and PBMC samples in identifying increased Type I interferon signaling and up-regulated activity of monocytes and natural killer (NK) cells in African-Americans compared to Caucasian participants. These findings demonstrate the feasibility of DBS in field-based studies of gene expression and encourage future studies of human transcriptome dynamics in larger, more representative samples than are possible with clinic- or lab-based research designs. PMID:27337553

  8. Investigating the specific core genetic-and-epigenetic networks of cellular mechanisms involved in human aging in peripheral blood mononuclear cells

    PubMed Central

    Li, Cheng-Wei; Wang, Wen-Hsin; Chen, Bor-Sen

    2016-01-01

    Aging is an inevitable part of life for humans, and slowing down the aging process has become a main focus of human endeavor. Here, we applied a systems biology approach to construct protein-protein interaction networks, gene regulatory networks, and epigenetic networks, i.e. genetic and epigenetic networks (GENs), of elderly individuals and young controls. We then compared these GENs to extract aging mechanisms using microarray data in peripheral blood mononuclear cells, microRNA (miRNA) data, and database mining. The core GENs of elderly individuals and young controls were obtained by applying principal network projection to GENs based on Principal Component Analysis. By comparing the core networks, we identified that to overcome the accumulated mutation of genes in the aging process the transcription factor JUN can be activated by stress signals, including the MAPK signaling, T-cell receptor signaling, and neurotrophin signaling pathways through DNA methylation of BTG3, G0S2, and AP2B1 and the regulations of mir-223 let-7d, and mir-130a. We also address the aging mechanisms in old men and women. Furthermore, we proposed that drugs designed to target these DNA methylated genes or miRNAs may delay aging. A multiple drug combination comprising phenylalanine, cholesterol, and palbociclib was finally designed for delaying the aging process. PMID:26895224

  9. Effects of Lycium barbarum Polysaccharides on Apoptosis, Cellular Adhesion, and Oxidative Damage in Bone Marrow Mononuclear Cells of Mice Exposed to Ionizing Radiation Injury

    PubMed Central

    Zhou, Jing; Pang, Hua; Li, Wenbo; Liu, Qiong; Xu, Lu; Liu, Qian; Liu, Ying

    2016-01-01

    Lycium barbarum has been used for more than 2500 years as a traditional herb and food in China. We investigated the effects of Lycium barbarum polysaccharides (LBP) on apoptosis, oxidative damage, and expression of adhesion molecules in bone marrow mononuclear cells (BMNC) of mice injured by ionizing radiation. Kunming mice were exposed to X-rays; then mice in the LBP groups were continuously injected with various concentrations of LBP intraperitoneally for 14 days. Mice in the control group were continuously injected with normal saline (NS) by the same route for 14 days. A normal group was set up. After 1, 7, and 14 days of treatment, mice were killed and BMNC were extracted. Cell cycle, apoptosis, and the expression of adhesion molecules CD44 and CD49d were detected by flow cytometry. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were identified by colorimetric analyses. LBP significantly decreased the percentage of G0/G1 phase, apoptosis, MDA level, and expression of CD44 and CD49d and distinctly increased the activity of SOD. LBP showed a protective effect on BMNC against ionizing radiation-induced apoptosis and oxidative damage and altered the expression of adhesion molecule. PMID:27314019

  10. Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method.

    PubMed

    Eberlein, B; Baur, A; Neundorfer, M; Jahn, G

    1991-05-01

    Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method. PMID:1909827

  11. Male Microchimerism at High Levels in Peripheral Blood Mononuclear Cells from Women with End Stage Renal Disease before Kidney Transplantation

    PubMed Central

    Albano, Laetitia; Rak, Justyna M.; Azzouz, Doua F.; Cassuto-Viguier, Elisabeth; Gugenheim, Jean; Lambert, Nathalie C.

    2012-01-01

    Patients with end stage renal diseases (ESRD) are generally tested for donor chimerism after kidney transplantation for tolerance mechanism purposes. But, to our knowledge, no data are available on natural and/or iatrogenic microchimerism (Mc), deriving from pregnancy and/or blood transfusion, acquired prior to transplantation. In this context, we tested the prevalence of male Mc using a real time PCR assay for DYS14, a Y-chromosome specific sequence, in peripheral blood mononuclear cells (PBMC) from 55 women with ESRD, prior to their first kidney transplantation, and compared them with results from 82 healthy women. Male Mc was also quantified in 5 native kidney biopsies obtained two to four years prior to blood testing and in PBMC from 8 women collected after female kidney transplantation, several years after the initial blood testing. Women with ESRD showed statistically higher frequencies (62%) and quantities (98 genome equivalent cells per million of host cells, gEq/M) of male Mc in their PBMC than healthy women (16% and 0.3 gEq/M, p<0.00001 and p = 0.0005 respectively). Male Mc was increased in women with ESRD whether they had or not a history of male pregnancy and/or of blood transfusion. Three out of five renal biopsies obtained a few years prior to the blood test also contained Mc, but no correlation could be established between earlier Mc in a kidney and later presence in PBMC. Finally, several years after female kidney transplantation, male Mc was totally cleared from PBMC in all women tested but one. This intriguing and striking initial result of natural and iatrogenic male Mc persistence in peripheral blood from women with ESRD raises several hypotheses for the possible role of these cells in renal diseases. Further studies are needed to elucidate mechanisms of recruitment and persistence of Mc in women with ESRD. PMID:22403639

  12. Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity

    PubMed Central

    Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

    2016-01-01

    Background Dopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown. Methods In a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥0.80 m in women and ≥0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36. Results CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO. Conclusions Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome. PMID:26808524

  13. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease

    PubMed Central

    Lyra, Andre Castro; Soares, Milena Botelho Pereira; da Silva, Luiz Flavio Maia; Fortes, Marcos Fraga; Silva, André Goyanna Pinheiro; Mota, Augusto César de Andrade; Oliveira, Sheilla A; Braga, Eduardo Lorens; de Carvalho, Wilson Andrade; Genser, Bernd; dos Santos, Ricardo Ribeiro; Lyra, Luiz Guilherme Costa

    2007-01-01

    AIM: To evaluate the safety and feasibility of bone marrow cell (BMC) transplantation in patients with chronic liver disease on the waiting list for liver transplantation. METHODS: Ten patients (eight males) with chronic liver disease were enrolled to receive infusion of autologous bone marrow-derived cells. Seven patients were classified as Child-Pugh B and three as Child-Pugh C. Baseline assessment included complete clinical and laboratory evaluation and abdominal MRI. Approximately 50 mL of bone marrow aspirate was prepared by centrifugation in a ficoll-hypaque gradient. At least of 100 millions of mononuclear-enriched BMCs were infused into the hepatic artery using the routine technique for arterial chemoembolization for liver tumors. Patients were followed up for adverse events up to 4 mo. RESULTS: The median age of the patients was 52 years (range 24-70 years). All patients were discharged 48 h after BMC infusion. Two patients complained of mild pain at the bone marrow needle puncture site. No other complications or specific side effects related to the procedure were observed. Bilirubin levels were lower at 1 (2.19 ± 0.9) and 4 mo (2.10 ± 1.0) after cell transplantation that baseline levels (2.78 ± 1.2). Albumin levels 4 mo after BMC infusion (3.73 ± 0.5) were higher than baseline levels (3.47 ± 0.5). International normalized ratio (INR) decreased from 1.48 (SD = 0.23) to 1.43 (SD = 0.23) one month after cell transplantation. CONCLUSION: BMC infusion into hepatic artery of patients with advanced chronic liver disease is safe and feasible. In addition, a decrease in mean serum bilirubin and INR levels and an increase in albumin levels are observed. Our data warrant further studies in order to evaluate the effect of BMC transplantation in patients with advanced chronic liver disease. PMID:17373741

  14. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases

    PubMed Central

    Kjaergaard, Christian H.; Qayyum, Munzarin F.; Wong, Shaun D.; Xu, Feng; Hemsworth, Glyn R.; Walton, Daniel J.; Young, Nigel A.; Davies, Gideon J.; Walton, Paul H.; Johansen, Katja Salomon; Hodgson, Keith O.; Hedman, Britt; Solomon, Edward I.

    2014-01-01

    Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9–11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy. PMID:24889637

  15. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

    PubMed

    Kjaergaard, Christian H; Qayyum, Munzarin F; Wong, Shaun D; Xu, Feng; Hemsworth, Glyn R; Walton, Daniel J; Young, Nigel A; Davies, Gideon J; Walton, Paul H; Johansen, Katja Salomon; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I

    2014-06-17

    Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy. PMID:24889637

  16. mRNA profiles of cytokine receptors in unstimulated peripheral blood mononuclear cells from patients with chronic idiopathic urticaria.

    PubMed

    Gao, Jianming; Yang, Aizhen; Chen, Min; Li, Ansheng; Yao, Xu; Li, Yumei; Xie, Shihai; Yang, Xueyuan; Zhong, Liansheng; Chen, Zhiqiang

    2011-03-01

    This present study was aimed to investigate the roles of the receptors of Th1/Th2 cytokines and chemokines in the pathogenesis of chronic idiopathic urticaria (CIU). Thirty patients with CIU, 30 patients with dermographism and 30 healthy controls were randomly enrolled. Reverse transcription-PCR (RT-PCR) was used to analyze the mRNA of cytokine receptors in peripheral blood mononuclear cells (PBMCs). The mRNA levels of tumor necrosis factor receptor (TNFR), interferon-γ receptor (IFN-γR), and interleukin-10 receptor (IL-10R) were statistically increased in the CIU group (P < 0.05), while IL-2R, IL-4R, IL-6R, and IL-13R showed no significant differences between the CIU and other groups. The mRNA levels of CCR3 and CCR6 were statistically increased in the CIU group (P < 0.05). The toll-like receptor 2 (TLR2) mRNA level was significantly lower in the CIU group than the healthy control group (P < 0.05). These findings indicate that the regulation of mRNA of TNFR, IFN-γR, IL-10R, CCR3, CCR6 and TLR2 may be involved in the pathogenesis of CIU. PMID:23554682

  17. Mitochondrial respiratory dysfunctions of blood mononuclear cells link with cardiac disturbance in patients with early-stage heart failure

    PubMed Central

    Li, Peng; Wang, Bin; Sun, Fang; Li, Yingsha; Li, Qiang; Lang, Hongmei; Zhao, Zhigang; Gao, Peng; Zhao, Yu; Shang, Qianhui; Liu, Daoyan; Zhu, Zhiming

    2015-01-01

    Patients with cardiometabolic risk factors and asymptomatic cardiac hypertrophy are hallmarks of early-stage heart failure (HF). We hypothesized that mitochondrial respiratory dysfunctions of peripheral blood mononuclear cells (PBMCs) may be associated with inflammation and oxidative stress in early-stage HF patients complicated with cardiometabolic risk factors. Totally 49 subjects were enrolled with 25 early-stage HF patients (stages A and B) having cardiac hypertrophy and dysfunction and 24 healthy controls. It showed that excessive inflammation and reduced antioxidant capacity were closely associated with cardiac abnormalities in early-stage HF patients. Furthermore, the values of mitochondrial respiratory functional parameters R, CIOXPHOS, CIIOXPHOS, CI+IIOXPHOS, CI+IIETS and CIIETS were significantly lowered in early-stage HF patients. Interestingly, these respiratory parameters were correlated with inflammation and antioxidant capacity in participants. Finally, cardiometabolic risk factors such as salt intake and blood pressure were related to the mitochondrial respiratory dysfunctions, which were further validated by in vitro experiments. Our study indicated that cardiometabolic risk factor-mediated mitochondrial respiratory dysfunctions of PBMCs link with the cellular inflammation / oxidative stress and cardiac disturbance in early-stage HF. PMID:26018291

  18. Oral coenzyme Q10 supplementation improves clinical symptoms and recovers pathologic alterations in blood mononuclear cells in a fibromyalgia patient.

    PubMed

    Cordero, Mario D; Cotán, David; del-Pozo-Martín, Yaiza; Carrión, Angel M; de Miguel, Manuel; Bullón, Pedro; Sánchez-Alcazar, José Antonio

    2012-01-01

    Fibromyalgia (FM) is a chronic pain syndrome with unknown etiology. Recent studies have shown evidence demonstrating that mitochondrial dysfunction and oxidative stress may have a role in the pathophysiology of FM. Coenzyme Q10 (CoQ10) is an essential electron carrier in the mitochondrial respiratory chain and a strong antioxidant. Low CoQ10 levels have been detected in patients with FM, and a significant decrease of clinical symptoms has been reported after oral CoQ10 supplementation. In this report, we show the effect of CoQ10 treatment on clinical symptoms, blood mononuclear cells, and mitochondrial and oxidative stress markers from a woman with FM. After CoQ10 treatment, the patient reported a significant improvement of clinical symptoms. At the cellular level, CoQ10 treatment restored mitochondrial dysfunction and the mtDNA copy number, decreased oxidative stress, and increased mitochondrial biogenesis. Our results suggest that CoQ10 could be an alternative therapeutic approach for FM. PMID:22898267

  19. Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus

    PubMed Central

    Xu, Yuan-Yong; Liu, Hui-Hui; Zhong, Yan-Wei; Liu, Chang; Wang, Yong; Jia, Lei-Li; Qiao, Fei; Li, Xin-Xin; Zhang, Chuan-Fu; Li, Shen-Long; Li, Peng; Song, Hong-Bin; Li, Qiao

    2015-01-01

    The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus. PMID:25678845

  20. Variability of gemcitabine accumulation and its relationship to expression of nucleoside transporters in peripheral blood mononuclear cells.

    PubMed

    Choi, Min-Koo

    2012-05-01

    The concentrative nucleoside transporter CNT1 and equilibrated nucleoside transporter ENT1 mediate the cellular uptake of naturally occurring pyrimidine and purine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs, thereby regulating drug responses or toxicity at the target site. The objectives of this study were to analyze interindividual variations in the cellular accumulation of gemcitabine and to examine the correlation between the uptake of gemcitabine and expression levels of CNT1 and ENT1 transporters. Gemcitabine was a substrate for both CNT1 and ENT1 with higher affinity to CNT1 than to ENT1. The difference in gemcitabine uptake was 4.8-fold in peripheral blood mononuclear cells (PBMCs) from 10 subjects. Among these, the CNT1- and ENT1-mediated uptake of gemcitabine was 14.3- and 16.5-folds, respectively. CNT1-mediated gemcitabine uptake showed a higher correlation with the CNT1 expression level than did ENT1-mediated uptake with ENT1 expression level. In conclusion, CNT1 seemed to be a major contributing factor to gemcitabine uptake in PBMCs and showed 14.3-fold inter-individual variations. However, ENT1-mediated uptake of gemcitabine might compensate for the total uptake of gemcitabine; therefore, the variation in the apparent accumulation of gemcitabine was smaller than that of the individual transporters. PMID:22644860

  1. Cholesterol Transporters ABCA1 and ABCG1 Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Metabolic Syndrome

    PubMed Central

    Tavoosi, Zahra; Moradi-Sardareh, Hemen; Saidijam, Massoud; Yadegarazari, Reza; Borzuei, Shiva; Soltanian, Alireza; Goodarzi, Mohammad Taghi

    2015-01-01

    ABCA1 and ABCG1 genes encode the cholesterol transporter proteins that play a key role in cholesterol and phospholipids homeostasis. This study was aimed at evaluating and comparing ABCA1 and ABCG1 genes expression in metabolic syndrome patients and healthy individuals. This case-control study was performed on 36 patients with metabolic syndrome and the same number of healthy individuals in Hamadan (west of Iran) during 2013-2014. Total RNA was extracted from mononuclear cells and purified using RNeasy Mini Kit column. The expression of ABCA1 and ABCG1 genes was performed by qRT-PCR. Lipid profile and fasting blood glucose were measured using colorimetric procedures. ABCG1 expression in metabolic syndrome patients was significantly lower (about 75%) compared to that of control group, while for ABCA1 expression, there was no significant difference between the two studied groups. Comparison of other parameters such as HDL-C, FBS, BMI, waist circumference, and systolic and diastolic blood pressure between metabolic syndrome patients and healthy individuals showed significant differences (P < 0.05). Decrease in ABCG1 expression in metabolic syndrome patients compared to healthy individuals suggests that hyperglycemia, related metabolites, and hyperlipidemia over the transporter capacity resulted in decreased expression of ABCG1. Absence of a significant change in ABCA1 gene expression between two groups can indicate a different regulation mechanism for ABCA1 expression. PMID:26788366

  2. Expression levels of seven candidate genes in human peripheral blood mononuclear cells and their association with preeclampsia

    PubMed Central

    Garza-Veloz, I.; Carrillo-Sanchez, K.; Martinez-Gaytan, V.; Cortes-Flores, R.; Ochoa-Torres, M. A.; Guerrero, G. G.; Rodriguez-Sanchez, I. P.; Cancela-Murrieta, C. O.; Zamudio-Osuna, M.; Badillo-Almaraz, J. I.; Castruita-De la Rosa, C.

    2014-01-01

    Objective To evaluate the peripheral blood mononuclear cell (PBMC) expression levels of hemeoxygenase 1 (HMOX-1), superoxide dismutase 1 (SOD-1), vascular endothelial growth factor A (VEGF-A), transforming growth factor beta 1 (TGF-β1), interleukin (IL)-6, IL-15 and AdipoQ genes to study their association with preeclampsia (PE). Methods A total of 177 pregnant women were recruited: 108 cases and 69 controls. Quantification of gene expression was measured by quantitative real-time polymerase chain reaction (PCR) using TaqMan probes. Results Underexpression of VEGF-A and TGF-β1 was a constant in most of the cases (80.91% and 76.36%, respectively) and their expression was associated with onset and/or severity of disease (p values < 0.05). IL-6, IL-15 and AdipoQ, showed low or no expression in PBMC samples evaluated. Conclusion PBMC underexpression of VEGF-A and TGF-β1 is a hallmark of PE in the study population. PMID:24295154

  3. Transcriptional profiling of left ventricle and peripheral blood mononuclear cells in a rat model of postinfarction heart failure

    PubMed Central

    2013-01-01

    Background Myocardial infarction (MI) often results in left ventricular (LV) remodeling followed by heart failure (HF). It is of great clinical importance to understand the molecular mechanisms that trigger transition from compensated LV injury to HF and to identify relevant diagnostic biomarkers. The aim of this study was to investigate gene expression in the LV and to evaluate their reflection in peripheral blood mononuclear cells (PBMCs). Methods MI was induced in rats by ligation of the proximal left coronary artery. Rats with small, moderate, and large MI size were included into the experiment two months after the operation. The development of heart failure was estimated by echocardiography and catheterization. Microarrays were used to compare the LV and PBMCs transcriptomes of control and experimental animals. Results Only rats with a large MI developed extensive LV remodeling and heart failure. 840 transcripts were altered in LV of failing hearts, and especially numerous were those associated with the extracellular matrix. In contrast, no significant gene expression changes were seen in LVs of rats with moderate or small MI that had compensated LV injury. We showed that ceruloplasmin was similarly overexpressed in the heart and blood in response to HF, whereas downregulation of tetraspanin 12 was significant only in the PBMCs. Conclusion A large size of infarcted area is critical for progression of LV remodeling and HF development, associated with altered gene expression in the heart. Ceruloplasmin and tetraspanin 12 are potential convenient markers in readily obtainable PBMCs. PMID:24206753

  4. Hepatitis C virus RNA detection in serum and peripheral blood mononuclear cells of patients with hepatitis C

    PubMed Central

    Zhou, Ping; Cai, Qing; Chen, You-Chun; Zhang, Mu-Sen; Guan, Jian; Li, Xiao-Juan

    1997-01-01

    AIM: To investigate the existence and clinical significance of hepatitis C virus (HCV) RNA in the serum and peripheral blood mononuclear cells (PBMC) of patients with hepatitis C. METHODS: HCV RNA was detected by nested polymerase chain reaction (Nested PCR) in serum and in PBMC of 46 patients with acute hepatitis C (AHC) and in 42 patients with chronic hepatitis C (CHC). RESULTS: The positive rate of HCV RNA in PBMC of patients with CHC was markedly higher than that of patients with AHC (P < 0.01). The positive rates of HCV RNA in serum of patients with AHC and CHC and in PBMC of patients with CHC were significantly higher than those of anti-HCV positive patients with normal alanine aminotransferase (ALT) levels (P < 0.01). HCV RNA was negative in the serum of two patients, but could be detected in PBMC. In 12 patients, anti HCV was negative while HCV RNA was positive in serum. CONCLUSION: (1) detection of serum HCV RNA by nested PCR might be helpful in the early diagnosis of anti-HCV negative hepatitis C; (2) liver damage in patients with hepatitis C might be correlated with HCV-viremia; (3) infection of PBMC by HCV might play an important role in chronic liver damage in patients with HCV and in the chronicity of its clinical course; and (4) PBMC might be considered as a “reservoir” for HCV. PMID:27041960

  5. Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

    PubMed

    Gupta, V; Mishra, N; Pateriya, A; Behera, S P; Rajukumar, K

    2014-01-01

    The aim of this study was to determine the in vitro permissivity of peripheral blood mononuclear cells (PBMCs) from bovine viral diarrhea virus (BVDV)-immune field cattle to homologous and heterologous BVDVs. PBMCs from seventeen BVDV-naïve and sixteen BVDV-immune animals were infected with noncytopathic BVDV-1 or BVDV-2. The immune status of cattle was indicated by the presence of virus neutralizing antibodies, while viral load of PBMCs was determined by real-time RT-PCR. The results revealed that the PBMCs from naïve or immune animals were permissive to either BVDV-1 or BVDV-2, but the viral load was significantly higher for the naïve than for the immune animals. Furthermore, the load of homologous virus in PBMCs from immune animals was lower than that of heterologous virus. Our results provide evidence that the PBMCs from BVDV-immune cattle in field are susceptible to reinfection with homologous or heterologous BVDV, albeit to a lower extent in the former case. PMID:24957715

  6. Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis.

    PubMed

    Choi, E-W; Shin, I-S; Bhang, D-H; Lee, D-H; Bae, B-K; Kang, M-S; Kim, D-Y; Hwang, C-Y; Lee, C-W; Youn, H-Y

    2006-10-01

    To elucidate the hormonal change and alteration in cytokine expression in peripheral blood mononuclear cells (PBMC) during the early stage of autoimmune thyroiditis, we have developed a canine model of this disease, in which normal dogs were immunized with bovine thyroglobulin (Tg) and/or canine thyroid extract. Serum samples were collected weekly, anti-canine Tg antibody was measured by enzyme-linked immunosorbent assay (ELISA) and thyroid stimulating hormone (TSH) and total T4 levels by radioimmunoassay. We also assayed T lymphocyte proliferation in response to Tg, as well as measuring cytokine mRNA by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). All six dogs immunized with bovine Tg had both canine Tg autoantibody and anti-T4 antibody. When the sample from the highest TgAA titre time-point was compared with baseline the expression of mRNA encoding the Th1-type cytokine such as interferon (IFN)-gamma, interleukin (IL)-18 and IL-15 was increased during the development of autoimmune thyroiditis. Expression of the Th2-type cytokine, IL-6 showed minimal change and IL-4 expression was not detected in any of the samples. Expression of the T suppressive cytokine, IL-10 and transforming growth factor (TGF)-beta was increased in the presence of antigen stimulation. These findings suggest that, although autoimmune thyroiditis is an organ-specific autoimmune disease, systemic cytokine mRNA expression is also changed. PMID:16968404

  7. In vitro production of tumour necrosis factor and prostaglandin E2 by peripheral blood mononuclear cells from tuberculosis patients.

    PubMed Central

    Cadranel, J; Philippe, C; Perez, J; Milleron, B; Akoun, G; Ardaillou, R; Baud, L

    1990-01-01

    We investigated the production of tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) by peripheral blood mononuclear cells (PBMC) from tuberculosis patients and healthy controls. PBMC from tuberculosis patients generated constitutively more TNF-alpha than did control PBMC. This production was significantly higher for patients with high-grade fever and cachexia. The increase of TNF-alpha production by PBMC from tuberculosis patients was associated with a comparatively weaker elevation of PGE2 synthesis which did not parallel fever or weight loss. In vitro treatment of control PBMC with the tuberculin purified protein derivative (PPD) promoted an increased TNF-alpha production which was similar to that of untreated PBMC from tuberculosis patients. Thus, the increased TNF-alpha production in tuberculosis could be explained by the in vivo exposure of PBMC to mycobacterial antigens. In contrast, the concentration of PGE2 was weaker in the medium of untreated PBMC from tuberculosis patients than in the medium of PPD-treated control PBMC, suggesting that PGE2 synthesis by PBMC was limited in tuberculosis by unidentified factors. PMID:2387094

  8. Complex effects of vitamin E and vitamin C supplementation on in vitro neonatal mononuclear cell responses to allergens.

    PubMed

    Wassall, Heather J H; Devereux, Graham; Seaton, Anthony; Barker, Robert N

    2013-09-01

    Low maternal dietary vitamin E (but not vitamin C) intake during pregnancy has been associated with increased in vitro cord blood mononuclear cell (CBMC) proliferative responses, childhood wheezing and asthma. We investigated whether these associations reflect direct effects of vitamin E by investigating the effects of supplementing CBMC cultures with physiological concentrations of vitamin E. CBMC from seventy neonates were cultured supplemented with either nothing, α-tocopherol or ascorbic acid. Proliferative, IFN-γ, IL-4, IL-10 and TGF-β responses were measured. In general, vitamin E supplementation was associated with a trend for reduced proliferative responses after stimulation with antigens and house dust mite, and with increased proliferation after stimulation with timothy grass allergen. There was a trend for CBMC cultures to exhibit decreased secretion of IFN-γ, IL-10 and IL-4. Supplementation with vitamin C had no effect on CBMC proliferation, but increased IFN-γ and IL-4 production, and decreased IL-10 production. In conclusion, in vitro vitamin E and C supplementation of CBMC modifies neonatal immune function, but not in a manner predicted by observational epidemiological studies. The observed associations between vitamin E and childhood respiratory disease are complex, and the nature and form of nutritional intervention need to be carefully considered before inclusion in trials. PMID:24067384

  9. Peripheral Blood Mononuclear Cell Gene Expression Remains Broadly Altered Years after Successful Interferon-Based Hepatitis C Virus Treatment

    PubMed Central

    Waldron, Paul Ravi; Holodniy, Mark

    2015-01-01

    Background. Inflammatory gene expression in peripheral blood mononuclear cells (PBMCs) is altered in chronic Hepatitis C Virus (HCV) infection. Duration of changes after pegylated interferon- (peg-IFN-) based HCV treatment is unclear. Methods. PBMC mRNA expression of 184 inflammatory response genes was analyzed (nCounter GX Human Inflammation Kit, Nanostring) from peg-IFN treatment nonresponders (NR, n = 18), sustained virologic responders (SVR, n = 22), and spontaneous clearers (SC, n = 15). Logistic regression was used for comparison. Results. Median time from last treatment was 2 and 2.7 years in SVR and NR, respectively (p = NS). Mean mRNA counts were significantly different for 42 and 29 genes comparing SVR to SC patients and NR to SC, respectively, and no genes comparing SVR to NR. Differential expression of 24 genes was significantly different in both SVR and NR groups compared to SC. Among these 24 acute and chronic inflammatory cascade genes, significant upregulation was noted for proinflammatory transcription regulators Fos, CEBPB, and MyD88 in SVR and NR compared to SC. HDAC4 was significantly downregulated in SVR and NR compared to the SC group. Conclusions. PBMC inflammatory gene expression patterns in SVR resemble NR more than SC patients. A generalized inflammatory response persists in PBMCs long after successful peg-IFN treatment for HCV infection. PMID:26568966

  10. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce inflammation and apoptosis in porcine peripheral blood mononuclear cells in vitro.

    PubMed

    Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Shao, Guoqing

    2015-01-30

    Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (EP), a disease that causes considerable economic losss in swine industry. Lipid-associated membrane proteins (LAMPs) of mycoplasma play important roles in causing mycoplasma diseases. The present study explores the pathogenic mechanisms of M. hyopneumoniae LAMPs by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of peripheral blood mononuclear cells (PBMCs) of pig. LAMP treatment inhibited the growth of PBMCs. Up-regulation of cytokines, such as IL-6 and IL-1β, as well as increased production of nitric oxide (NO) and superoxide anion were all detected in the supernatant of LAMPs-treated PBMCs. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMPs of M. hyopneumoniae induced a time-dependent apoptosis in lymphocyts and monocytes from PBMCs, which was blocked by NOS inhibitor or antioxidant. In addition, LAMPs induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in PBMCs. These findings demonstrated that M. hyopneumoniae LAMPs induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of PBMCs in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, as well as caspase activation. PMID:25481242