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Sample records for mononuclear cells pbmcs

  1. HCV RNA in peripheral blood mononuclear cells (PBMCs) as a ...

    African Journals Online (AJOL)

    Abdel Fatah Fahmy Hanno

    2013-06-27

    PBMCs) of patients with chronic HCV infection, and explore the ... Methods: Twenty-five patients with chronic viral hepatitis C were included. ... ment course, patients who were found to have positive PCR test for ..... consequences.

  2. THE IMMUNOMODULATORY EFFECTS OF PROBIOTIC BACTERIA ON PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS OF ALLERGIC PATIENTS

    Directory of Open Access Journals (Sweden)

    Somaya M. El Sheikh

    2014-01-01

    Full Text Available Allergic diseases represent major health burden. An allergic reaction is characterized by a disrupted T- helper 1⁄T-helper 2 balance toward a preferential allergen specifically induced TH2 cytokine profile, causing allergic inflammation Probiotic bacteria have various benificial effects in many pathologic situation. Studies have shown that the bacteria present in the intestinal micro flora play a role in the TH1/TH2 balance and its modulation can promote the control of infectious and immune processes. Testing the effects of probiotic bacteria on TH1/TH2 cytokine production by peripheral blood mononuclear cells of allergic patients and control subjects. This study included 24 patients allergic to date pollen and 16 healthy control subjects. PBMC of both groups were separated and cultured for 72 h with date pollen allergen (home-made in the presence or absence of Lactobacillus rhamnosus ATCC 7469 (Living and dead and C- phycocyanin (extracted from Spirulina platensis. The cell culture supernatants were collected to measure Interlukin 4 and Interferon gamma by quantitative ELISA. Incubation of PBMCs of allergic patients with living Lactobacillus rhamnosus ATCC 7469 showed marked reduction in IL4 production (median IL4 concentarion = 3.9 pg. compared to PBMCs callenged with pollen alone (mediam IL4 conentration = 52.6 pg. When PBMC were incubated with living Lactobacillus rhamnosus in absence of allergen significant increase in and IFNγ (median concentration = 42.75 pg. was obtained, compared to PBMC challenged with allergen alone (median = 22.8 pg. When PBMCs incubated with heat killed Lactobacillus rhamnosus either in presence or absence of the offending allergen, marked reduction in IL4 production was obtained (median = 10.6, 3.6 pg respectively compared to PBMC incubated with allergen alone (median = 52.6 pg. When PBMCs incubated with dead Lactobacillus rhamnosus, marked increase in IFNγ production

  3. Generation of human iPSC line GRX-MCiPS4F-A2 from adult peripheral blood mononuclear cells (PBMCs with Spanish genetic background

    Directory of Open Access Journals (Sweden)

    Sonia Cabrera

    2015-09-01

    Full Text Available We have generated iPSCs from peripheral blood mononuclear cells (PBMCs of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

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

    Science.gov (United States)

    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

  5. Human peripheral blood mononuclear cells (PBMCs) from smokers release higher levels of IL-1-like cytokines after exposure to combustion-generated ultrafine particles

    Science.gov (United States)

    De Falco, Gianluigi; Terlizzi, Michela; Sirignano, Mariano; Commodo, Mario; D’Anna, Andrea; Aquino, Rita P.; Pinto, Aldo; Sorrentino, Rosalinda

    2017-01-01

    Ultrafine particles (UFP) generated by combustion processes are often associated with adverse health effects. However, little is known about the inflammatory processes generated by UFP that may underlie their toxicological activity. Murine macrophages (J774.1 cells) and human peripheral blood mononuclear cells (PBMCs) were used to evaluate the molecular mechanism underlying the pro-inflammatory activity of UFP. The addition of soot particles to J774.1 cells induced a concentration-dependent release of IL-1α, IL-1β and IL-33 This effect was not associated with cell death and, in contrast to literature, was pronounced at very low concentrations (5–100 pg/ml). Similarly, UFP induced the release of IL-1α, IL-18 and IL-33 by PBMCs. However, this effect was solely observed in PBMCs obtained from smokers, as the PBMCs from non-smokers instead released higher levels of IL-10. The release of these cytokines after UFP exposure was caspase-1- and NLRP3 inflammasome-dependent in PBMCs from healthy smokers, whereas IL-1α release was calpain-dependent. These results show that UFP at very low concentrations are able to give rise to an inflammatory process that is responsible for IL-1α, IL-18 and IL-33 release, which is pronounced in PBMCs from smokers, confirming that these individuals are especially susceptible to inflammatory-based airway diseases once exposed to air pollution. PMID:28223692

  6. 人扁桃体和外周血中B细胞亚群、表型和功能的比较%Comparison of subpopulations, phenotypes and functions of human B cells in tonsillar mononuclear cells and PBMCs

    Institute of Scientific and Technical Information of China (English)

    冯敛; 李祖望; 李华斌; 吴长有

    2011-01-01

    目的:比较观察人扁桃体和外周血中B细胞的亚群分布、表型及功能特征.方法:分离人扁桃体单个核细胞和外周血PBMCs,流式细胞术检测其B细胞表面分子的表达;将人扁桃体单个核细胞和外周血PBMCs进行体外培养后,ELISA法检测培养上清中免疫球蛋白(Ig)含量.结果:流式细胞术检测结果表明,与PBMCs中B细胞亚群不同,扁桃体单个核细胞中含有较高比例的生发中心细胞(GC cells),且初始B细胞(naive B cells)比例较低.与PBMCs相比,扁桃体单个核细胞中CD19+CD38(high)CD20+浆细胞的比例较高,但两者B细胞表面IgM和IgG表达的差异并不显著(P>0.05).扁桃体B细胞上表达CD69的水平明显高于PBMCs中B细胞,而CD25在扁桃体及PBMCs中B细胞上均不表达或表达甚微.ELISA检测结果表明,未经刺激的扁桃体单个核细胞产生IgG的含量高于PBMCs,IgA和IgM含量与PBMCs相比,差异不显著.结论:人扁桃体和外周血中B细胞亚群分布及功能特征均存在一定差别,为进一步研究B细胞如何在淋巴组织中发挥免疫功能提供了实验依据.%In this study, we aimed to compare the subpopulations, phenotypes and functions of human B cells in tonsillar mononuclear cells and PBMCs. Firstly, tonsillar mononuclear cells and PBMCs were isolated and the surface markers on B cells were detected by flow cytometry (FCM). Tonsillar mononuclear cells and PBMCs were then cultured for 7 days, and the production of immunoglobulins in supernatants was tested by ELISA. FCM analysis revealed that increased frequencies of germinal center cells (GC cells) were existed in tonsillar B cell subsets compared with that in PBMCs, and the proportion of naive B cells was lower in tonsillar B cells than that in PBMCs. Different from what observed in PBMCs, higher frequencies of CD19+CD38highCD20+/- plasma cells were existed in tonsillar mononuclear cells, but the expression of IgM and IgG on tonsillar B cells were similar

  7. Paeoniflorin suppresses inflammatory response in imiquimod-induced psoriasis-like mice and peripheral blood mononuclear cells (PBMCs) from psoriasis patients.

    Science.gov (United States)

    Chen, Tao; Fu, Li-Xin; Zhang, Li-Wen; Yin, Bin; Zhou, Pei-Mei; Cao, Na; Lu, Yong-Hong

    2016-08-01

    Psoriasis is one of the most common immune-mediated chronic inflammatory skin disorders, characterized by hyperproliferation of keratinocytes, dilation and growth of dermal capillary vasculature, and cellular infiltration of T cells, dendritic cells (DCs), and neutrophils. Paeoniflorin (PF), the principal component of total glucosides of paeony (TGP), displays anti-inflammatory and antioxidant properties in several animal models. In this study, we investigated the anti-inflammatory effects and mechanisms of PF in imiquimod (IMQ)-induced psoriasis-like mouse model. The effects of PF on inflammatory cytokine expression in peripheral blood mononuclear cells (PBMCs) from patients with psoriasis vulgaris were also observed. Our results indicated that PF effectively attenuated the clinical and histopathologic changes in IMQ-induced psoriasis-like mouse model. Furthermore, PF reduced the infiltration of T cells, CD11c(+)DCs, and neutrophils in lesional skin. In addition, PF also significantly decreased the mRNA expression of inflammatory cytokines, such as IL-17, INF-γ, IL-6, and TNF-α, in IMQ-induced psoriasis-like mouse model and PBMCs from patients with psoriasis vulgaris. Hence, our data suggest that PF can inhibit leukocyte infiltration and decrease the expression of inflammatory cytokines such as IL-17, INF-γ, IL-6, and TNF-α. PF might be a candidate drug for the treatment of psoriasis.

  8. AMPA, NMDA and kainate glutamate receptor subunits are expressed in human peripheral blood mononuclear cells (PBMCs) where the expression of GluK4 is altered by pregnancy and GluN2D by depression in pregnant women.

    Science.gov (United States)

    Bhandage, Amol K; Jin, Zhe; Hellgren, Charlotte; Korol, Sergiy V; Nowak, Krzysztof; Williamsson, Louise; Sundström-Poromaa, Inger; Birnir, Bryndis

    2017-04-15

    The amino acid glutamate opens cation permeable ion channels, the iGlu receptors. These ion channels are abundantly expressed in the mammalian brain where glutamate is the main excitatory neurotransmitter. The neurotransmitters and their receptors are being increasingly detected in the cells of immune system. Here we examined the expression of the 18 known subunits of the iGlu receptors families; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA) and delta in human peripheral blood mononuclear cells (PBMCs). We compared the expression of the subunits between four groups: men, non-pregnant women, healthy pregnant women and depressed pregnant women. Out of 18 subunits of the iGlu receptors, mRNAs for 11 subunits were detected in PBMCs from men and non-pregnant women; AMPA: GluA3, GluA4, kainate: GluK2, GluK4, GluK5, NMDA: GluN1, GluN2C, GluN2D, GluN3A, GluN3B, and delta: GluD1. In the healthy and the depressed pregnant women, in addition, the delta GluD2 subunit was identified. The mRNAs for GluK4, GluK5, GluN2C and GluN2D were expressed at a higher level than other subunits. Gender, pregnancy or depression during pregnancy altered the expression of GluA3, GluK4, GluN2D, GluN3B and GluD1 iGlu subunit mRNAs. The greatest changes recorded were the lower GluA3 and GluK4 mRNA levels in pregnant women and the higher GluN2D mRNA level in healthy but not in depressed pregnant women as compared to non-pregnant individuals. Using subunit specific antibodies, the GluK4, GluK5, GluN1, GluN2C and GluN2D subunit proteins were identified in the PBMCs. The results show expression of specific iGlu receptor subunit in the PBMCs and support the idea of physiology-driven changes of iGlu receptors subtypes in the immune cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Immune Modulation in Normal Human Peripheral Blood Mononuclear Cells (PBMCs) (Lymphocytes) in Response to Benzofuran-2-Carboxylic Acid Derivative KMEG during Spaceflight

    Science.gov (United States)

    Okoro, Elvis; Mann, Vivek; Ellis, Ivory; Mansoor, Elvedina; Olamigoke, Loretta; Marriott, Karla Sue; Denkins, Pamela; Williams, Willie; Sundaresan, Alamelu

    2017-08-01

    Microgravity and radiation exposure during space flight have been widely reported to induce the suppression of normal immune system function, and increase the risk of cancer development in humans. These findings pose a serious risk to manned space missions. Interestingly, recent studies have shown that benzofuran-2-carboxylic acid derivatives can inhibit the progression of some of these devastating effects on earth and in modeled microgravity. However, these studies had not assessed the impacts of benzofuran-2- carboxylic acid and its derivatives on global gene expression under spaceflight conditions. In this study, the ability of a specific benzofuran-2-carboxylic acid derivative (KMEG) to confer protection from radiation and restore normal immune function was investigated following exposure to space flight conditions on the ISS. Normal human peripheral blood mononuclear cells (lymphocytes) treated with 10 µ g/ml of KMEG together with untreated control samples were flown on Nanoracks hardware on Spacex-3 flight. The Samples were returned one month later and gene expression was analyzed. A 1g-ground control experiment was performed in parallel at the Kennedy spaceflight center. The first overall subtractive unrestricted analysis revealed 78 genes, which were differentially expressed in space flight KMEG, untreated lymphocytes as compared to the corresponding ground controls. However, in KMEG-treated space flight lymphocytes, there was an increased expression of a group of genes that mediate increased transcription, translation and innate immune system mediating functions of lymphocytes as compared to KMEG-untreated samples. Analysis of genes related to T cell proliferation in spaceflight KMEG-treated lymphocytes compared to 1g-ground KMEG- treated lymphocytes revealed six T cell proliferation and signaling genes to be significantly upregulated (p mitochondria from the accumulation of oxidatively damaged membrane proteins. Overall, our analysis indicates that KMEG

  10. Transcriptome analysis reveals common differential and global gene expression profiles in bluetongue virus serotype 16 (BTV-16 infected peripheral blood mononuclear cells (PBMCs in sheep and goats

    Directory of Open Access Journals (Sweden)

    Anjali Singh

    2017-03-01

    Full Text Available Bluetongue is an economically important infectious, arthropod borne viral disease of domestic and wild ruminants, caused by Bluetongue virus (BTV. Sheep are considered the most susceptible hosts, while cattle, buffalo and goats serve as reservoirs. The viral pathogenesis of BTV resulting in presence or absence of clinical disease among different hosts is not clearly understood. In the present study, transcriptome of sheep and goats peripheral blood mononuclear cells infected with BTV-16 was explored. The differentially expressed genes (DEGs identified were found to be significantly enriched for immune system processes - NFκB signaling, MAPK signaling, Ras signaling, NOD signaling, RIG signaling, TNF signaling, TLR signaling, JAK-STAT signaling and VEGF signaling pathways. Greater numbers of DEGs were found to be involved in immune system processes in goats than in sheep. Interestingly, the DEHC (differentially expressed highly connected gene network was found to be dense in goats than in sheep. Majority of the DEHC genes in the network were upregulated in goats but down-regulated in sheep. The network of differentially expressed immune genes with the other genes further confirmed these findings. Interferon stimulated genes - IFIT1 (ISG56, IFIT2 (ISG54 and IFIT3 (ISG60 responsible for antiviral state in the host were found to be upregulated in both the species. STAT2 was the TF commonly identified to co-regulate the DEGs, with its network showing genes that are downregulated in sheep but upregulated in goats. The genes dysregulated and the networks perturbed in the present study indicate host variability with a positive shift in immune response to BTV in goats than in sheep.

  11. Immune Modulation in Normal Human Peripheral Blood Mononuclear Cells (PBMCs) (Lymphocytes) in Response to Benzofuran-2-Carboxylic Acid Derivative KMEG during Spaceflight

    Science.gov (United States)

    Okoro, Elvis; Mann, Vivek; Ellis, Ivory; Mansoor, Elvedina; Olamigoke, Loretta; Marriott, Karla Sue; Denkins, Pamela; Williams, Willie; Sundaresan, Alamelu

    2017-07-01

    Microgravity and radiation exposure during space flight have been widely reported to induce the suppression of normal immune system function, and increase the risk of cancer development in humans. These findings pose a serious risk to manned space missions. Interestingly, recent studies have shown that benzofuran-2-carboxylic acid derivatives can inhibit the progression of some of these devastating effects on earth and in modeled microgravity. However, these studies had not assessed the impacts of benzofuran-2- carboxylic acid and its derivatives on global gene expression under spaceflight conditions. In this study, the ability of a specific benzofuran-2-carboxylic acid derivative (KMEG) to confer protection from radiation and restore normal immune function was investigated following exposure to space flight conditions on the ISS. Normal human peripheral blood mononuclear cells (lymphocytes) treated with 10 µ g/ml of KMEG together with untreated control samples were flown on Nanoracks hardware on Spacex-3 flight. The Samples were returned one month later and gene expression was analyzed. A 1g-ground control experiment was performed in parallel at the Kennedy spaceflight center. The first overall subtractive unrestricted analysis revealed 78 genes, which were differentially expressed in space flight KMEG, untreated lymphocytes as compared to the corresponding ground controls. However, in KMEG-treated space flight lymphocytes, there was an increased expression of a group of genes that mediate increased transcription, translation and innate immune system mediating functions of lymphocytes as compared to KMEG-untreated samples. Analysis of genes related to T cell proliferation in spaceflight KMEG-treated lymphocytes compared to 1g-ground KMEG- treated lymphocytes revealed six T cell proliferation and signaling genes to be significantly upregulated (p C-myc related proliferation, promote antiapoptotic activity and protects mitochondria from the accumulation of

  12. Isolation of Cultured Endothelial Progenitor Cells in vitro from PBMCs and CD133~+ Enriched Cells

    Institute of Scientific and Technical Information of China (English)

    郑伟红; 万亚峰; 马小鹏; 李兴睿; 杨志芳; 殷茜; 易继林

    2010-01-01

    Two isolation methods for sorting of endothelial progenitor cells(EPCs):from peripheral blood mononuclear cells(PBMCs)and CD133+ enriched cells were compared,by defining the cell morphology,phenotype,reproductive activities and function in vitro,to provide a reference for clinical application of EPCs.PBMCs from healthy subjects were used either directly for cell culture or for CD133+ sorting.The two groups of cells were cultured in complete medium 199(M199)for 7 to 14 days and the phenotypes of EPCs were an...

  13. Peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus me-diate the adhesion of T lymphocytes to endothelial ceils%SLE患者外周血单一核细胞介导T淋巴细胞对血管内皮细胞黏附的实验研究

    Institute of Scientific and Technical Information of China (English)

    杨骥; 储以微; 李明

    2009-01-01

    目的 探讨活动期SLE患者外周血单一核细胞(PBMC)是否介导了T淋巴细胞对血管内皮细胞的黏附.方法 体外培养人脐静脉内皮细胞(HUVEC),活动期SLE PBMC培养上清液预刺激48 h,通过RT-PCR检测PBMC培养上清液诱导HUVEC黏附分子的表达;划痕实验检测PBMC培养上清液诱导内皮细胞运动迁移能力的改变;HUVEC与T细胞共培养,研究PBMC培养上清液预刺激是否影响T细胞对HUVEC的黏附.结果 HUVEC经过活动期SLE PBMC培养上清刺激后,内皮细胞黏附分子ICAM-1mRNA,VCAM-1 mRNA和E-钙黏蛋白mRNA表达明显增加,而IL-17抗体能明显抑制黏附分子的表达.黏附分子表达增加介导了内皮细胞运动能力增加,介导T细胞对内皮细胞的黏附,而IL-17中和抗体能抑制T细胞对内皮细胞的黏附和抑制内皮细胞的运动迁移.结论 活动期SLE PBMC培养上清液可以通过诱导血管内皮细胞黏附分子表达,黏附T细胞,介导狼疮血管炎的发生.%Objective To observe whether peripheral blood mononuclear cells (PBMCs) from patients with active systemic lupus erythematosus (SLE) can induce the adhesion of T lymphocytes to endothelial cells. Methods Sera and PBMCs were obtained from patients with active SLE and normal human controls. PBMCs were cultivated and culture supematants were harvested. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro with or without the presence of the sera or culture super-natants of PBMCs. Some cells were pretreated with the antibody to IL-17 before the treatment with the sera or supematants. After another 48-hour culture, RT-PCR and real-time PCR were used to detect the mRNA expressions of adhesion molecules, including intercellular adhesion moleeule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-cadherin in HUVECs, wound healing assay to estimate the motility of HUVECs. Additionally, T lymphocytes were added to HUVECs 48 hours after stimulation with the sera or

  14. Tissue-Related Hypoxia Attenuates Proinflammatory Effects of Allogeneic PBMCs on Adipose-Derived Stromal Cells In Vitro

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    Polina I. Bobyleva

    2016-01-01

    Full Text Available Human adipose tissue-stromal derived cells (ASCs are considered a perspective tool for regenerative medicine. Depending on the application mode ASC/allogeneic immune cell interaction can occur in the systemic circulation under plenty high concentrations of O2 and in target tissues at lower O2 levels. Here we examined the effects of allogeneic PHA-stimulated peripheral blood mononuclear cells (PBMCs on ASCs under ambient (20% oxygen and “physiological” hypoxia (5% O2. As revealed with microarray analysis ASCs under 20% O2 were more affected by activated PBMCs, which was manifested in differential expression of more than 300 genes, whereas under 5% O2 only 140 genes were changed. Altered gene pattern was only partly overlapped at different O2 conditions. Under O2 ASCs retained their proliferative and differentiative capacities, mesenchymal phenotype, and intracellular organelle’ state. ASCs were proinflammatory activated on transcription level that was confirmed by their ability to suppress activation and proliferation of mitogen-stimulated PBMCs. ASC/PBMCs interaction resulted in anti-inflammatory shift of paracrine mediators in conditioning medium with significant increase of immunosuppressive LIF level. Our data indicated that under both ambient and tissue-related O2 ASCs possessed immunosuppressive potential and maintained functional activity. Under “physiological” hypoxia ASCs were less susceptible to “priming” by allogeneic mitogen-activated PBMCs.

  15. Study of the inhibition by polymorphonuclear leukocytes of TNF-α release from human mononuclear cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ The present study was undertaken to investigate the effect of human PMNs on the production of TNF-α by the human peripheral blood mononuclear cells (PBMCs) and to elucidate its tentative mechanism.

  16. Controlled exposure to diesel exhaust and traffic noise - Effects on oxidative stress and activation in mononuclear blood cells

    DEFF Research Database (Denmark)

    Hemmingsen, Jette Gjerke; Møller, Peter; Jantzen, Kim

    2015-01-01

    unaltered in peripheral blood mononuclear cells (PBMCs). No significant differences in DNA damage levels, measured by the comet assay, were observed after DE exposure, whereas exposure to high noise levels was associated with significantly increased levels of hOGG1-sensitive sites in PBMCs. Urinary levels...

  17. Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Maynard, Scott; Keijzers, Guido; Gram, Martin

    2013-01-01

    . Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production...

  18. Leptin induces the phagocytosis and protective immune response in Leishmania donovani infected THP-1 cell line and human PBMCs.

    Science.gov (United States)

    Dayakar, Alti; Chandrasekaran, Sambamurthy; Veronica, Jalaja; Maurya, Radheshyam

    2016-01-01

    Visceral leishmaniasis (VL) is an infectious disease responsible for several deaths in malnourished children due to impaired cell-mediated immunity, which is accompanied by low circulating leptin levels. The cytokine function of leptin is implicated for several immune regulation activities such as hematopoiesis, angiogenesis, innate and adaptive immunity. Its deficiency associated with polarization of Th2 response, which coincides with VL pathogenesis. To determine the cytokine role of leptin in case of experimental VL, we tested the leptin associated Th1/Th2 type cytokine profile at mRNA level from Leishmania donovani infected human monocytic leukemia cell line (THP-1) and peripheral blood mononuclear cells (PBMCs). We also tested the effect of leptin on macrophages activation (viz. studying the phosphorylation of signaling moieties), phagocytic activity and intracellular reactive oxygen species (ROS) production during infection. We observed that leptin induced Th1 specific response by upregulation of IL-1α, IL-1β, IL-8 and TNF-α in THP-1 and IFN-γ, IL-12 and IL-2 in PBMCs. We also observed the downregulation of Th2 type cytokine i.e. IL-10 in THP-1 and unaltered expression of cytokines i.e. TGF-β, IL-10 and IL-4 in PBMCs. In addition, leptin stimulates the macrophages by inducing phosphorylation of Erk1/2 and Akt which are usually dephosphorylated in L. donovani infection. In concordance, leptin also induces the macrophage phagocytic activity by enhancing the intracellular ROS generation which helps in phagolysosome formation and oxidative killing of the parasite. In compilation, leptin is able to maintain the defensive environment against L. donovani infection through the classical macrophage activity.

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

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    Yukari Komuta

    2016-06-01

    Full Text Available 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.

  20. Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged...

  1. Peripheral blood mononuclear cells as a model to study the response of energy homeostasis-related genes to acute changes in feeding conditions

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Keijer, J.; Palou, A.

    2010-01-01

    Peripheral blood mononuclear cells (PBMCs) are readily accessible biological material and a potential tissue source to discover novel biomarkers of response to environmental exposures including nutrition. We analyzed whether PBMCs could reflect molecular changes that take place in response to differ

  2. Peripheral blood mononuclear cells as a model to study the response of energy homeostasis-related genes to acute changes in feeding conditions

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Keijer, J.; Palou, A.

    2010-01-01

    Peripheral blood mononuclear cells (PBMCs) are readily accessible biological material and a potential tissue source to discover novel biomarkers of response to environmental exposures including nutrition. We analyzed whether PBMCs could reflect molecular changes that take place in response to differ

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

    Science.gov (United States)

    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.

  4. The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F;

    2000-01-01

    A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma...... cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse...... an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found...

  5. Circulating angiogenic cells can be derived from cryopreserved peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Tanja Sofrenovic

    Full Text Available BACKGROUND: Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs. METHODS: PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw or 28 (late thaw days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential. RESULTS: The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34(+VEGFR2(+CD133(+ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle. CONCLUSION: Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34(+VEGFR2(+CD133(+ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.

  6. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

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    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  7. Study of the inhibition by polymorphonuclear leukocytes of TNF-α release from human mononuclear cells and its mechanism

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The present study was undertaken to investigate the effect of human PMNs on the production of TNF-α by the human peripheral blood mononuclear cells (PBMCs) and to elucidate its tentative mechanism. Human PMNs and PBMCs were isolated from the venous blood of healthy donors by dextran sedimentation and density gradient centrifugation. In the presence of lipopolysaccharide (LPS), PMNs and PBMCs were cocultured at the ratio of 2:1 for 20 h and the concentration of TNF-α in the supernatant was measured by enzyme-linked immunosorbent assay. The binding rate of monocytes with the fluorescein isothiocyanate-labeled LPS (FITC-LPS) and the mean surface fluorescence intensity of monocytes were analyzed by flow cytometry. Results showed that PMNs were capable of inhibiting the TNF-α release from PBMCs (P<0.05). PMNs suppressed the TNF-α release from PBMCs by 45% on average when PMNs and PBMCs cocultured at the ratio of 2:1. Paraformaldehyde-fixed PMNs still demonstrated the same inhibition (P<0.05),which proved that the inhibition was dependent on cell-to-cell contact and suggested that effector molecules responsible for this effect existed on the cell surface of PMNs. In the presence of PMNs, the binding rate of monocytes with the FITC-LPS and the mean surface fluorescence intensity of monocytes were not affected compared with PBMCs alone (P>0.05). As incubation time was prolonged, the binding of FITC-LPS to monocytes increased (P<0.05). Thus PMNs did not block the binding of LPS with monocytes. It was concluded that PMNs suppressed the TNF-α release from PBMCs via cell-to-cell interaction. In a cell-contact dependent manner, PMNs might interfere with the signal transduction pathway through which LPS activated PBMCs, thus attenuating the response of PBMCs to LPS and downregulating the TNF-α release.

  8. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...... strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found...... that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic...

  9. General anesthesia-associatedDNA damage in peripheral blood mononuclear cells of surgical patients

    Institute of Scientific and Technical Information of China (English)

    Wang Haiyan; Zhou Qi; Fu Huo

    2011-01-01

    Objective:To evaluate retrospectively the effect of general anesthesia onDNA damage in the blood mononuclear cells (PBMCs) of surgical patients in order to provide evidence for a better nursing care during the procedure.Methods: Clinical charts of76 patients who underwent operation under general anesthesia and76 healthy control subjects with documented results of DNA damage extent inPBMCs from the single-cell gel electrophoresis(SCGE) or comet assay and serum contents of superoxide dismutase(SOD) and malondialdehyde(MDA)from biochemical analyses were reviewed. The percentage of comet PBMCs and tailDNAand serum contents of SOD and MAD were analyzed by student t-test.Results: Compared with healthy control subjects, generally anesthetized surgical patients had significantly higher % cometPBMCs and % tail DNA(P<0.05) and significantly lower serum concentrations ofSOD (P<0.05) and significantly higher serum concentrations ofMAD (P<0.05). Compared with levels before general anesthesia in surgical patients, % cometPBMCs, % tailDNA, and serum levels ofMADwere significantly higher (P<0.05 or0.01), and serum levels ofSOD were significantly lower (P<0.05), after general anesthesia.Conclusions: General anesthesia during surgery causes a certain degree of hypoxia and PBMC damage. Particular attention should be paid to monitoring and maintenance of blood oxygen saturation in patients undergoing surgery under general anesthesia.

  10. Elevated peripheral blood mononuclear cell-derived superoxide production in healthy young black men.

    Science.gov (United States)

    Deo, Shekhar H; Holwerda, Seth W; Keller, David M; Fadel, Paul J

    2015-03-01

    Several studies have demonstrated that blacks exhibit elevations in systemic oxidative stress. However, the source(s) and mechanism(s) contributing to the elevation in oxidative stress remain unclear. Given that peripheral blood mononuclear cells (PBMCs) can be a major source of NADPH oxidase-derived superoxide production, we tested the hypothesis that young black men demonstrate greater superoxide production and NADPH oxidase expression in PBMCs compared with whites. PBMCs were freshly isolated from whole blood in young normotensive black (n = 18) and white (n = 16) men. Intracellular superoxide production in PBMCs was measured using dihydroethidium fluorescence, protein expression of NADPH oxidase subunits, gp91(phox) (membranous) and p47(phox) (cytosolic) in PBMCs were assessed using Western blot analysis, and plasma protein carbonyls were measured as a marker of systemic oxidative stress. Black men showed elevated intracellular superoxide production (4.3 ± 0.5 vs. 2.0 ± 0.6 relative fluorescence units; black men vs. white men, P superoxide production or NADPH oxidase subunit protein expression. These findings indicate that black men exhibit greater resting PBMC-derived superoxide production and an upregulation of the NADPH oxidase pathway with a possible contribution to increases in systemic oxidative stress.

  11. Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type 1 isolation from vertically infected pediatric patients.

    Science.gov (United States)

    Paul, M O; Tetali, S; Pahwa, S

    1994-05-01

    In this study, we examined variables related to human immunodeficiency virus (HIV) isolation utilizing samples from 51 HIV-infected (153 plasma and 122 peripheral blood mononuclear cell [PBMC] samples) and 57 uninfected (182 plasma and 163 PBMC samples) infants. Our chief observation was that cryopreservation of donor PBMCs does not significantly alter their sensitivity or specificity for isolation of HIV from patient PBMCs or plasma.

  12. Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

    Directory of Open Access Journals (Sweden)

    Niina Hopper

    Full Text Available This study characterized peripheral blood mononuclear cells (PBMC in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC's were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1 MSC alone; (2 MSCs and PBMCs at a ratio of 20:1; (3 MSCs and PBMC at a ratio of 2:1 and (4 PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006. However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%. This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007, BMP6 10.7-fold (p = 0.0004, GDF5 2.0-fold (p = 0.002 and COL1 5.0-fold (p = 0.046. The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key

  13. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

    OpenAIRE

    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 ...

  14. Dietary exposure to benzoxazinoids enhances bacteria-induced monokine responses by peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Damgaard, Dres; Jensen, Bettina Margrethe; Palarasah, Yaseelan

    2015-01-01

    -out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production...... of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A 3-wk diet with high BX content enhanced IL-1β responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 h of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation......SCOPE: To examine potentially immunomodulating effects of dietary benzoxazinoids (BXs), present in cereal grains. METHODS AND RESULTS: Nineteen healthy volunteers were randomly distributed into two groups, who received diets with high or low content of BXs for 3 wk. After a week's wash...

  15. Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Kadoya, Masatoshi; Yamamoto, Aihiro; Hamaguchi, Masahide; Obayashi, Hiroshi; Mizushima, Katsura; Ohta, Mitsuhiro; Seno, Takahiro; Oda, Ryo; Fujiwara, Hiroyoshi; Kohno, Masataka; Kawahito, Yutaka

    2014-06-06

    Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14(+) peripheral blood mononuclear cells (CD14(+) PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14(+) PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14(+) PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Increased expression of the 20S proteasome in peripheral blood mononuclear cells of type 2 diabetic patients

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Objective To investigate the dynamic expression of the 20S proteasome in peripheral blood mononuclear cells(PBMCs)of type 2 diabetic patients without vascular complications.Methods PBMCs were prepared from 30 type 2 diabetic patients and 30 nondiabetic controls.The general indexes including weight,height and blood pressure were recorded.Fasting plasma glucose,fasting plasma insulin and glycosylated hemoglobin were measured.The protein level of the 20S proteasome was measured by Western blotting.The mRNA exp...

  17. Regulatory activity of azabisphosphonate-capped dendrimers on human CD4+ T cell proliferation enhances ex-vivo expansion of NK cells from PBMCs for immunotherapy

    Directory of Open Access Journals (Sweden)

    Caminade Anne-Marie

    2009-09-01

    Full Text Available Abstract Background Adoptive cell therapy with allogenic NK cells constitutes a promising approach for the treatment of certain malignancies. Such strategies are currently limited by the requirement of an efficient protocol for NK cell expansion. We have developed a method using synthetic nanosized phosphonate-capped dendrimers allowing such expansion. We are showing here that this is due to a specific inhibitory activity towards CD4+ T cell which could lead to further medical applications of this dendrimer. Methods Mononuclear cells from human peripheral blood were used to investigate the immunomodulatory effects of nanosized phosphonate-capped dendrimers on interleukin-2 driven CD4+T cell expansion. Proliferation status was investigated using flow cytometry analysis of CFSE dilution and PI incorporation experiments. Magnetic bead cell sorting was used to address activity towards individual or mixed cell sub-populations. We performed equilibrium binding assay to assess the interaction of fluorescent dendrimers with pure CD4+ T cells. Results Phosphonate-capped dendrimers are inhibiting the activation, and therefore the proliferation; of CD4+ T cells in IL-2 stimulated PBMCs, without affecting their viability. This allows a rapid enrichment of NK cells and further expansion. We found that dendrimer acts directly on T cells, as their regulatory property is maintained when stimulating purified CD4+ T cells with anti-CD3/CD28 microbeads. Performing equilibrium binding assays using a fluorescent analogue, we show that the phosphonate capped-dendrimers are specifically interacting with purified CD4+ T cells. Ultimately, we found that our protocol prevents the IL-2 related expansion of regulatory T cells that would be deleterious for the activity of infused NK cells. Conclusion High yield expansion of NK cells from human PBMCs by phosphonate-capped dendrimers and IL-2 occurs through the specific inhibition of the CD4+ lymphocyte compartment. Given the

  18. The Impact of Glyphosate, Its Metabolites and Impurities on Viability, ATP Level and Morphological changes in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Kwiatkowska, Marta; Jarosiewicz, Paweł; Michałowicz, Jaromir; Koter-Michalak, Maria; Huras, Bogumiła; Bukowska, Bożena

    2016-01-01

    The toxicity of herbicides to animals and human is an issue of worldwide concern. The present study has been undertaken to assess toxic effect of widely used pesticide—glyphosate, its metabolites: aminomethylphosphonic acid (AMPA) and methylphosphonic acid and its impurities: N-(phosphonomethyl)iminodiacetic acid (PMIDA), N-methylglyphosate, hydroxymethylphosphonic acid and bis-(phosphonomethyl)amine on human peripheral blood mononuclear cells (PBMCs). We have evaluated the effect of those compounds on viability, ATP level, size (FSC-A parameter) and granulation (SSC-A parameter) of the cells studied. Human peripheral blood mononuclear cells were exposed to different concentrations of glyphosate, its metabolites and impurities (0.01–10 mM) for 4 and 24 h. It was found that investigated compounds caused statistically significant decrease in viability and ATP level of PBMCs. The strongest changes in cell viability and ATP level were observed after 24 h incubation of PBMCs with bis-(phosphonomethyl)amine, and particularly PMIDA. Moreover, all studied compounds changed cell granularity, while PMIDA and bis-(phosphonomethyl)amine altered PBMCs size. It may be concluded that bis-(phosphonomethyl)amine, and PMIDA caused a slightly stronger damage to PBMCs than did glyphosate. Changes in the parameters studied in PBMCs were observed only at high concentrations of the compounds examined, which clearly shows that they may occur in this cell type only as a result of acute poisoning of human organism with these substances. PMID:27280764

  19. Equine colostral carbohydrates reduce lipopolysaccharide-induced inflammatory responses in equine peripheral blood mononuclear cells.

    Science.gov (United States)

    Vendrig, J C; Coffeng, L E; Fink-Gremmels, J

    2012-12-01

    Increasing evidence suggests that reactions to lipopolysaccharide (LPS), particularly in the gut, can be partly or completely mitigated by colostrum- and milk-derived oligosaccharides. Confirmation of this hypothesis could lead to the development of new therapeutic concepts. To demonstrate the influence of equine colostral carbohydrates on the inflammatory response in an in vitro model with equine peripheral blood mononuclear cells (PBMCs). Carbohydrates were extracted from mare colostrum, and then evaluated for their influence on LPS-induced inflammatory responses in PBMCs isolated from the same mares, mRNA expression of tumour necrosis factor-alpha, interleukin-6 and interleukin-10 was measured as well as the protein levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10). Equine colostral carbohydrates significantly reduced LPS-induced TNF-alpha protein at both times measured and significantly reduced LPS-induced TNF-alpha, IL-6 and IL-10 mRNA expression by PBMCs. Moreover, cell viability significantly increased in the presence of high concentrations of colostral carbohydrates. Carbohydrates derived from equine colostrum reduce LPS-induced inflammatory responses of equine PBMCs. Colostrum and milk-derived carbohydrates are promising candidates for new concepts in preventive and regenerative medicine.

  20. Telomerase Activity in Peripheral Blood Mononuclear Cells from Senile Patients with Pneumonia

    Institute of Scientific and Technical Information of China (English)

    LIU Jian; ZHOU Zhen; LIU Xiaoqing

    2006-01-01

    To investigate the changes of the activity of telomerase in peripheral blood mononuclear cells (PBMCs) from senile patients with pneumonia, the telomerase activity was examined before and after the stimulation of phytohemagglutinin-M (PHA-M) in PBMCs from 10 control subjects (group A), 12 non-senile patients with pneumonia (group B) and 9 senile patients with pneumonia (group C). Also observed was the proliferative response of these PBMCs to PHA-M. The results showed that, both with or without the stimulation of PHA-M, the values of telomerase activity in PBMCs from group C patients (A values: pre-stimulation, 0.43±0.04; post-stimulation, 0.63±0.03) were significantly lower than those in PBMCs from both group A patients (A values: prestimulation, 0.65±0.05;post-stimulation, 1.26±0.13;P<0.001, respectively) and group B patients (A values: pre-stimulation, 0.63±0.03; post-stimulation, 0.93±0.03;P<0.05, respectively). The results of MTT test showed that the proliferative activity of PBMCs in group C patients (A value: 0.35±0.03) was also significantly lower than that in group A patients (A value:0. 55±0.04; P<0.05) and group B patients (A value: 0.46±0.03;P<0.05). These results indicate that the telomerase activity decreases in senile patients with pneumonia, which may be one of the mechanisms for the weakened immune function in those patients.

  1. Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue.

    Science.gov (United States)

    Perdomo-Celis, Federico; Salgado, Doris M; Castañeda, Diana M; Narváez, Carlos F

    2016-05-01

    Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-γ]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r = 0.74; P dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P ≤ 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3(+) CD8(+) amine-positive cells was found before cryopreservation (P = 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-γ-producing CD3(+) cells after stimulation compared with healthy controls and convalescent-phase patients (P ≤ 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho = -0.56; P = 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs.

  2. Bacillus Calmette-Guérin enhances production and secretion of type IV collagenases in peripheral blood mononuclear cells.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Solaleh Emamgholipour

    2016-01-01

    Full Text Available 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.

  4. In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors

    DEFF Research Database (Denmark)

    Glue, C; Millner, A; Bødtger, Uffe;

    2002-01-01

    was to investigate the in vitro effect of metabolites of phthalate plastisizers, such as whether an adjuvant effect is paralleled by changes of the cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells (PBMCs) from allergics and non-allergics. The toxicity monitored by cell...... determined using Quantitative Competitive RT-PCR. PBMCs from allergics and non-allergics were incubated with monophthalate 220 microg/ml) for up to 48 h and cytokine expression (IL-4, IL-5, IFN-gamma) was measured using real-time PCR. The cytotoxic level of monophthalates is 20-200 microg/ml, depending...

  5. Peripheral blood mononuclear cell traffic plays a crucial role in mother-to-infant transmission of hepatitis B virus.

    Science.gov (United States)

    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.

  6. Peripheral blood mononuclear cells from field cattle immune to bovine viral diarrhea virus (BVDV) are permissive in vitro to BVDV.

    Science.gov (United States)

    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.

  7. Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander;

    2013-01-01

    single-strand breaks (SSBs) and double-strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity...... in a study population consisting of 216 individuals from a population-based sample of twins aged 40-77 years. Age in this range did not seem to have any effect on the SSB parameters. However, γ-H2AX response and DSB repair capacity decreased with increasing age, although the associations did not reach...

  8. Medroxyprogesterone acetate increases HIV-1 infection of unstimulated peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Sampah, Maame Efua S; Laird, Gregory M; Blankson, Joel N; Siliciano, Robert F; Coleman, Jenell S

    2015-06-19

    Several observational studies suggest that medroxyprogesterone acetate (MPA) injectable contraceptives may increase a woman's risk of sexual HIV-1 acquisition. In-vitro studies are conflicting, mainly due to differences in the type of progestin studied or activation status of the primary cells. We sought to determine whether MPA increases infection of unstimulated peripheral blood mononuclear cells (PBMCs). Freshly isolated PBMCs from normal blood donors were treated with physiologic MPA concentrations ranging from 0.003 to 5 ng/ml and infected with GFP-tagged R5-tropic or X4-tropic HIV-1 pseudoviruses by spinoculation. The infection was limited to a single cycle. Cells were stained with CD3, CD8 and CD14. Infection was quantified as the percentage of GFP cells by flow cytometry. Absolute infection was greater among unstimulated MPA-treated CD3⁺CD8⁻ T cells vs. untreated cells across MPA concentrations of 0.003-3 ng/ml using R5 (P  0.5). The CD3⁺CD8⁻ T-cell population of MPA-treated unstimulated PBMCs were more susceptible to HIV-1 infection than untreated cells. The increased infection was partly due to monocytes and was lost when PBMC were exogenously stimulated. These data provide confirmation of a biological association between MPA exposure and increased susceptibility to HIV-1 infection, particularly among women who inject drugs.

  9. Acute modulation of cytokine gene expression in bovine peripheral blood mononuclear cells (PBMCs) by endogenous cortisol

    Science.gov (United States)

    Cortisol suppresses many aspects of immune function. However, recent publications suggest acute cortisol exposure may actually enhance immune function (Dhabhar. 2009. Neuroimmunomod. 16:300). The objective of this study was to determine the influence of acute increases in endogenous cortisol on expr...

  10. Derivation of autism spectrum disorder-specific induced pluripotent stem cells from peripheral blood mononuclear cells.

    Science.gov (United States)

    DeRosa, Brooke A; Van Baaren, Jessica M; Dubey, Gaurav K; Lee, Joycelyn M; Cuccaro, Michael L; Vance, Jeffery M; Pericak-Vance, Margaret A; Dykxhoorn, Derek M

    2012-05-10

    Induced pluripotent stem cells (iPSCs) hold tremendous potential both as a biological tool to uncover the pathophysiology of disease by creating relevant cell models and as a source of stem cells for cell-based therapeutic applications. Typically, iPSCs have been derived by the transgenic overexpression of transcription factors associated with progenitor cell or stem cell function in fibroblasts derived from skin biopsies. However, the need for skin punch biopsies to derive fibroblasts for reprogramming can present a barrier to study participation among certain populations of individuals, including children with autism spectrum disorders (ASDs). In addition, the acquisition of skin punch biopsies in non-clinic settings presents a challenge. One potential mechanism to avoid these limitations would be the use of peripheral blood mononuclear cells (PBMCs) as the source of the cells for reprogramming. In this article we describe, for the first time, the derivation of iPSC lines from PBMCs isolated from the whole blood of autistic children, and their subsequent differentiation in GABAergic neurons.

  11. The DNA methylome of human peripheral blood mononuclear cells.

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    Yingrui Li

    Full Text Available DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand, we report a comprehensive (92.62% methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE. These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.

  12. Controlled meal frequency without caloric restriction alters peripheral blood mononuclear cell cytokine production

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    Longo Dan L

    2011-03-01

    Full Text Available Abstract Background Intermittent fasting (IF improves healthy lifespan in animals by a mechanism involving reduced oxidative damage and increased resistance to stress. However, no studies have evaluated the impact of controlled meal frequency on immune responses in human subjects. Objective A study was conducted to establish the effects of controlled diets with different meal frequencies, but similar daily energy intakes, on cytokine production in healthy male and female subjects. Design In a crossover study design with an intervening washout period, healthy normal weight middle-age male and female subjects (n = 15 were maintained for 2 months on controlled on-site one meal per day (OMD or three meals per day (TMD isocaloric diets. Serum samples and peripheral blood mononuclear cells (PBMCs culture supernatants from subjects were analyzed for the presence of inflammatory markers using a multiplex assay. Results There were no significant differences in the inflammatory markers in the serum of subjects on the OMD or TMD diets. There was an increase in the capacity of PBMCs to produce cytokines in subjects during the first month on the OMD or TMD diets. Lower levels of TNF-α, IL-17, MCP-1 and MIP-1β were produced by PBMCs from subjects on the OMD versus TMD diet. Conclusions PBMCs of subjects on controlled diets exhibit hypersensitivities to cellular stimulation suggesting that stress associated with altered eating behavior might affect cytokine production by immune cells upon stimulation. Moreover, stimulated PBMCs derived from healthy individuals on a reduced meal frequency diet respond with a reduced capability to produce cytokines.

  13. Effects of aspirin-triggered resolvin D1 on peripheral blood mononuclear cells from patients with Chagas' heart disease.

    Science.gov (United States)

    Ogata, Haline; Teixeira, Maxelle Martins; Sousa, Rodrigo Cunha de; Silva, Marcos Vinícius da; Correia, Dalmo; Rodrigues Junior, Virmondes; Levy, Bruce David; Rogério, Alexandre de Paula

    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.

  14. Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis

    DEFF Research Database (Denmark)

    Oturai, D B; Søndergaard, H B; Börnsen, L;

    2016-01-01

    Quantitative real-time PCR (qPCR) involves the need of a proper standard for normalizing the gene expression data. Different studies have shown the validity of reference genes to vary greatly depending on tissue, cell subsets and experimental context. This study aimed at the identification...... of suitable reference genes for qPCR studies using different peripheral blood cell subsets (whole blood (WB) cells, peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD4(+) T cells, CD8(+) T cells, NK cells, monocytes, B cells and dendritic cells) from healthy controls (HC), patients with relapsing...... stable combination for analyses of cell subsets between HC and RRMS patients, while the combination of UBC and YWHAZ was superior for analysis of cell subsets between HC, RRMS and RRMS-IFN-β groups. GAPDH was generally unsuitable for blood cell subset studies in multiple sclerosis. In conclusion, we...

  15. Pancreatic Cancer Cell Lines Can Induce Prostaglandin E2 Production from Human Blood Mononuclear Cells

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    Svitlana P. Grekova

    2011-01-01

    Full Text Available Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2 in the pathogenesis of a wide range of malignancies. The protumorigenic properties of COX-2 are generally thought to be mediated by its product, PGE2, which is shown to promote tumor spread and growth by multiple mechanisms but most importantly through modulation of the local immune response in the tumor. Pancreatic tumor cells produce various amounts of PGE2, some of them being even deficient in COX enzymes or other PGE2 synthases. Here we describe that, beside pancreatic tumor cells or stromal fibroblasts, human peripheral blood mononuclear cells can also produce PGE2 upon coculture with pancreatic cancer cells. Stimulating of cellular cPLA2 within PBMCs by secreted factors, presumably sPLA2, from tumor cells appeared crucial, while the direct contact between PBMCs and PDACs seemed to be dispensable for this effect. Our data is emphasizing the complex interactions participating in the formation of the tolerogenic immune milieu within pancreatic tumors.

  16. Metabolic Profiling of Human Peripheral Blood Mononuclear Cells: Influence of Vitamin D Status and Gender

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    Magdalena Stepien

    2014-04-01

    Full Text Available Metabolic profiling of peripheral blood mononuclear cells (PBMC could serve as a less invasive and more direct alternative to tissue biopsies or serum in metabolomic research. We conducted two exploratory independent studies in order to characterise PBMC’s metabolomic profile following short-term vitamin D3 supplementation and to determine gender effects. In the first study, eight healthy males and females aged 40–65 y were randomly selected for profiling of PBMCs after receiving either 15 µg of vitamin D3 or placebo for four weeks. In the second study, twenty younger healthy males and females were studied. Cell metabolites were extracted and deproteinised using methanol/chloroform/water method and analysed by GC-MS. Higher vitamin D status had no effect on the fatty acid profile of PBMCs, but inflammatory biomarkers and adipokines correlated positively with stearic acid levels. In the second study, no gender-specific metabolites were identified. Valine, leucine and aspartic acid were identified as potential BMI-sensitive amino acids. Larger studies are needed to confirm the influence of BMI on these parameters. This work clearly demonstrates the utility of metabolomics profiling of PBMCs and paves the way for future applications of metabolomics in identifying metabolic profiles of blood cells as a measure for dietary intakes or physiological status.

  17. Hepatitis G Viral RNA Co-infection in Plasma and Peripheral Blood Mononuclear Cells in Patients with Hepatitis C

    Institute of Scientific and Technical Information of China (English)

    LI Shuli; ZENG Linglan; LUO Duande; LIU Wei; GUO Jingsong; YANG Xiaoming

    2001-01-01

    The incidence of the co-infection of hepatitis G virus (HGV) and hepatitis C virus(HCV) and its clinical implication was investigated and the difference in the positive rate of HGV RNA and HCV RNA between plasma and peripheral blood mononuclear cells (PBMCs) observed. By using reverse transcriptase polymerase chain reaction (RT-PCR) assay, HCV-RNA and HGV-RNA in plasma and PBMCs of 72 patients with hepatitis C was detected. It was showed that HGV RNA was positive in plasma of 11 patients, in PBMCs of 15 patients, and simultaneously in both of plasma and PBMCs of 10 patients with the co-infection rate being 22.2 %. Nine patients were both HGV RNA and HCV RNA positive in plasma, 11 patients were both HGV RNA and HCV RNA positive in PBMC, and 6 patients were both HGV RNA and HCV RNA positive in both plasma and PBMC with the positive rate being 12.4 %, 15.3 % and 8.3 % respectively. The positive rate of both HGV RNA and HCV RNA in PBMCs was higher than in plasma. It was concluded that the HGV co-infection rate in the patients with hepatitis C was 22. 2 %. Simultaneous examination of plasma and PBMC can improve clinically detectable rate.

  18. Increases of microRNA let-7e in peripheral blood mononuclear cells in Hashimoto's disease.

    Science.gov (United States)

    Kagawa, Tomoya; Watanabe, Mikio; Inoue, Naoya; Otsu, Hiroshi; Saeki, Minori; Katsumata, Yuka; Takuse, Yukina; Iwatani, Yoshinori

    2016-04-25

    MicroRNA (miRNA) is a family of non-coding RNAs that have important roles in various vital functions. It has been reported that let-7e, a miRNA, may be involved in the regulation of interleukin (IL)-10 production. The purpose of this study was to evaluate the role of let-7e as a regulator of IL-10 production in the pathological processes of autoimmune thyroid diseases (AITDs). We evaluated the association between let-7e expression and intracellular expression of IL-10 in the peripheral blood mononuclear cells (PBMCs) collected from 11 healthy volunteers. Then we investigated the expression levels of let-7e in the PBMCs of 50 patients with Graves' disease (GD), 42 patients with Hashimoto's disease (HD) and 28 healthy controls. We found negative correlations between the expression level of let-7e and IL-10 messengerRNA (mRNA) and between the expression level of let-7e and proportion of IL-10(+) cells in stimulated PBMCs from healthy volunteers (r = -0.44, p = 0.0267 and r = -0.49, p = 0.0166, respectively). The expression levels of let-7e were significantly increased in HD patients compared with those in GD patients and healthy volunteers (p = 0.0003 and p = 0.0011, respectively). let-7e may be associated with the pathogenesis of HD through the regulation of intracellular IL-10 expression.

  19. Molecular epidemiology of different hepatitis C genotypes in serum and peripheral blood mononuclear cells in jahrom city of iran.

    Science.gov (United States)

    Ashrafi Hafez, Asghar; Baharlou, Rasoul; Mousavi Nasab, Seyed Dawood; Ahmadi Vasmehjani, Abbas; Shayestehpour, Mohammad; Joharinia, Negar; Ahmadi, Nayeb Ali

    2014-05-01

    The Hepatitis C Virus (HCV) is considered essentially hepatotropic, yet the virus compartments have also been found in important extra hepatic sites. Detection of HCV RNA in extra hepatic reservoirs such as peripheral blood mononuclear cells (PBMCs) is important for determining disease progression and treatment effectiveness. The present study aimed to determine different HCV genotypes in patients' plasma and PBMC specimens, in Jahrom city of Iran. Blood samples of 137 patients with established HCV were collected at the Honari clinic. These patients were anti-HCV and plasma HCV RNA positive. After plasma RNA extraction and obtaining a pellet of approximately 3-5 × 10(6) PBMCs, Real-time PCR was performed, using specific-genotype primers. Finally, data analysis was done by the Statistical Package for Social Sciences (SPSS) software. Subtype 3 was the most common genotype in plasma (57.7%) and PBMCs (51.1%). Subtype 1a was detected in 36.5% and 30.7% of plasma samples and PBMCs, respectively whereas subtype 4 was not detected in any of the cases. There was a genotype difference between plasma and PBMCs of 12.4% of patients. In four patients no genotype was detected in their plasma but genotype 3 was detected in the PBMCs. It is suggested that determination of the target genotype by plasma subtyping for choosing the proper antiviral therapy is essential but may result in therapy failure. HCV genotyping in PBMC samples, along with plasma specimens, might be more beneficial. Therefore determining the HCV genotype in PBMCs, before beginning the therapy is useful due to the possibility of occult infection detection.

  20. Relationship of glucocorticoid receptor expression in peripheral blood mononuclear cells and the cochlea of guinea pigs and effects of dexamethasone administration.

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    Ling Lu

    Full Text Available BACKGROUND: Glucocorticoids (GCs are widely used to treat sudden sensorineural hearing loss (SSNHL and significantly improve hearing. However, GC insensitivity has been observed in some patients of SSNHL. OBJECTIVE: To study the correlation between GR expression in peripheral blood mononuclear cells (PBMCs and in the cochlea of guinea pigs at mRNA and protein levels. METHODS: One group of guinea pigs received dexamethasone (10 mg/kg/day intraperitoneally for 7 consecutive days (dexamethasone group, and another group of guinea pigs received normal saline (control group. Real time PCR and Western blotting were used to detect the expression of GR mRNA and GR protein in PBMCs and the cochleae. RESULTS: The GR mRNA and GR protein were detected in both PBMCs and the cochlear tissue of guinea pigs. GR mRNA and GR protein levels in PBMCs were positively correlated with those in the cochlea. The expression of GR mRNA and GR protein was significantly increased in the dexamethasone group compared to the control group. CONCLUSIONS: Levels of GR mRNA and GR protein in the PBMCs were positively correlated with those in the cochlea of guinea pigs. Systemic dexamethasone treatment can significantly up-regulate GR expression in PBMCs and in the cochlea. Measurement of the GR level in PBMCs could be used as an indicator of GR level in the cochlea.

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

    Science.gov (United States)

    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 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.

  2. Bisphenol A and its analogs induce morphological and biochemical alterations in human peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    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.

  3. In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

    Science.gov (United States)

    Turin, Lauretta; Lucchini, Barbara; Bronzo, Valerio; Luzzago, Camilla

    2012-11-01

    The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

  4. Mononuclear Cells and Vascular Repair in HHT

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    Calinda eDingenouts

    2015-03-01

    Full Text Available Hereditary hemorrhagic telangiectasia (HHT or Rendu-Osler-Weber disease is a rare genetic vascular disorder known for its endothelial dysplasia causing arteriovenous malformations and severe bleedings. HHT-1 and HHT-2 are the most prevalent variants and are caused by heterozygous mutations in endoglin and ALK1, respectively. An undervalued aspect of the disease is that HHT patients experience persistent inflammation. Although endothelial and mural cells have been the main research focus trying to unravel the mechanism behind the disease, wound healing is a process with a delicate balance between inflammatory and vascular cells. Inflammatory cells are part of the mononuclear cells (MNCs fraction, and can, next to eliciting an immune response, also have angiogenic potential. This biphasic effect of MNC can hold a promising mechanism to further elucidate treatment strategies for HHT patients. Before MNC are able to contribute to repair, they need to home to and retain in ischemic and damaged tissue. Directed migration (homing of mononuclear cells following tissue damage is regulated by the stromal cell derived factor 1 (SDF1. MNCs that express the C-X-C chemokine receptor 4 (CXCR4 migrate towards the tightly regulated gradient of SDF1. This directed migration of monocytes and lymphocytes can be inhibited by dipeptidyl peptidase 4 (DPP4. Interestingly, MNC of HHT patients express elevated levels of DPP4 and show impaired homing towards damaged tissue. Impaired homing capacity of the MNCs might therefore contribute to the impaired angiogenesis and tissue repair observed in HHT patients. This review summarizes recent studies regarding the role of MNCs in the etiology of HHT and vascular repair, and evaluates the efficacy of DPP4 inhibition in tissue integrity and repair.

  5. The Gene Expression Patterns of Peripheral Blood Mononuclear Cells in Patients with Systemic Lupus Erythematosus

    Institute of Scientific and Technical Information of China (English)

    LI Shouxin; JIANG Wei; HUANG Rui; WANG Xiaohui; LIU Wen; SHEN Shouyin

    2007-01-01

    This study examined the gene expression patterns of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE) by using serial analysis of gene expression (SAGE) technology. Following the construction of serial analysis of gene expression (SAGE) library of PBMCs collected from 3 cases of familial SLE patients, a large scale of tag sequencing was performed. The data extracted from sequencing files was analyzed with SAGE 2000 V 4.5 software.The top 30 expressed genes of SLE patients were uploaded to http://david.niaid.nih. gov/david/ease.htm and the functional classification of genes was obtained. The differences among those expressed gene were analyzed by Chi-square tests. The results showed that a total of 1286 unique SAGE tags were identified from 1814 individual SAGE tags. Among the 1286 unique tags, 86.8% had single copy, and only 0.2% tags had more than 20 copies. And 68.4% of the tags matched known expressed sequences, 41.1% of which matched more than one known expressed sequence. About 31.6% of the tags had no match and could represent potentially novel genes. Approximately one third of the top 30 genes were ribosomal protein, and the rest were genes related to metabolism or with unknown functions. Eight tags were found to express differentially in SAGE library of SLE patients. This study draws a profile of gene expression patterns of PBMCs in patients with SLE. Comparison of SAGE database from PBMCs between normal individuals and SLE patients will help us to better understand the pathogenesis of SLE.

  6. West Nile Virus Challenge Alters the Transcription Profiles of Innate Immune genes in Rabbit Peripheral Blood Mononuclear Cells

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    Muhammad Jasim eUddin

    2015-12-01

    Full Text Available 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 2h, 6h, 12h and 24h 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 highest expression seen for TLR3, 4 and 6. TLRs-signalling 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 9 transcripts suggests the involvement of oxidative stress and apoptosis in WNV stimulated rabbit PBMCs, respectively. The level of WNVNSW2011 RNA increased at 24h 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 & 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 infection study.

  7. Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells.

    Science.gov (United States)

    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.

  8. DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    Kwiatkowska, Marta; Reszka, Edyta; Woźniak, Katarzyna; Jabłońska, Ewa; Michałowicz, Jaromir; Bukowska, Bożena

    2017-07-01

    Glyphosate is a very important herbicide that is widely used in the agriculture, and thus the exposure of humans to this substance and its metabolites has been noted. The purpose of this study was to assess DNA damage (determination of single and double strand-breaks by the comet assay) as well as to evaluate DNA methylation (global DNA methylation and methylation of p16 (CDKN2A) and p53 (TP53) promoter regions) in human peripheral blood mononuclear cells (PBMCs) exposed to glyphosate. PBMCs were incubated with the compound studied at concentrations ranging from 0.1 to 10 mM for 24 h. The study has shown that glyphosate induced DNA lesions, which were effectively repaired. However, PBMCs were unable to repair completely DNA damage induced by glyphosate. We also observed a decrease in global DNA methylation level at 0.25 mM of glyphosate. Glyphosate at 0.25 mM and 0.5 mM increased p53 promoter methylation, while it did not induce statistically significant changes in methylation of p16 promoter. To sum up, we have shown for the first time that glyphosate (at high concentrations from 0.5 to 10 mM) may induce DNA damage in leucocytes such as PBMCs and cause DNA methylation in human cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  10. Capsaicin Modulates the Immune Cross Talk Between Human Mononuclears and Cells from Two Colon Carcinoma Lines.

    Science.gov (United States)

    Bessler, Hanna; Djaldetti, Meir

    2017-01-01

    Capsaicin, the pungent alkaloid of the chili peppers, has gained a worldwide reputation. In addition to its culinary assets, capsaicin possesses analgesic, anti-inflammatory, antimicrobial, and even carcinopreventive properties. Considering the linkage between chronic inflammation and tumorigenesis, the aim of the study was to evaluate the role of capsaicin in the immune interplay between human peripheral blood mononuclear cells (PBMCs) and HT-29 or RKO cells from human colon carcinoma lines. PBMCs were incubated for 24 hours with either HT-29 or RKO cells and concentrations of capsaicin ranging between 10 and 200 µM. Subsequently, the generation of the following cytokines was examined: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-γ, IL-1ra, and IL-10. Capsaicin caused a concentration-dependent inhibition of colon cancer cells proliferation but had no effect on PBMC viability. 200 µM of capsaicin suppressed the production of all cytokines tested. At lower concentrations, the secretion of TNF-α, IL-1β, IFN-γ, IL-10, and IL-1ra was inhibited concentration-dependently, whereas that of IL-6 was stimulated. Capsaicin causes a concentration-dependent alteration of the immune balance between PBMC and colon carcinoma cells expressed as an inhibited generation of inflammatory cytokines. These findings indicate the existence of an additional immunomodulatory mechanism by which this alkaloid may prevent tumor development.

  11. Matrix metalloproteinase-9 and its natural inhibitor TIMP-1 expressed or secreted by peripheral blood mononuclear cells from patients with systemic lupus erythematosus.

    Science.gov (United States)

    Matache, Cristiana; Stefanescu, Maria; Dragomir, Cristina; Tanaseanu, Stefanita; Onu, Adrian; Ofiteru, Augustin; Szegli, Geza

    2003-06-01

    Matrix metalloproteinase-9 (MMP-9) was involved in inflammation and immune system dysfunctions. Besides immunologic abnormalities, systemic lupus erythematosus (SLE) also presents chronic inflammatory components. Therefore, a role of MMP-9 in SLE pathology might be supposed. To verify this hypothesis, SLE patients and healthy donors were compared for the MMP-9 and MMP-9 mRNA levels in peripheral blood mononuclear cells (PBMCs), the spontaneous secretion of MMP-9 and TIMP-1 and the MMP-9 activity. Thus, we found that fresh PBMCs from SLE patients expressed a significantly higher activity of MMP-9 and spontaneously released higher levels of MMP-9, as compared to healthy donors, while the secreted TIMP-1 level was the same for both groups. When the patients were sub-grouped based on disease status, the most increased pro-MMP-9 activity inside the PBMCs was identified for relapse SLE sub-group. A similar observation for SLE patients with positive serum fibrinogen was found. Following culture, the PBMCs from remission SLE patients secreted significantly higher MMP-9 level, than the PBMCs from relapse SLE patients. PBMCs from relapse SLE patients secreted the highest levels of TIMP-1, although this difference was not statistically significant. Taken together, these observations suggested the multiple roles of MMP-9 and TIMP-1 in progress of inflammation and tissue damage and/or in repair, depending on clinical stages of SLE.

  12. Optimization of lentiviral vector transduction into peripheral blood mononuclear cells in combination with the fibronectin fragment CH-296 stimulation.

    Science.gov (United States)

    Chono, Hideto; Goto, Yumi; Yamakawa, Satoko; Tanaka, Shinya; Tosaka, Yasuhiro; Nukaya, Ikuei; Mineno, Junichi

    2011-03-01

    Large scale T-cell expansion and efficient gene transduction are required for adoptive T-cell gene therapy. Based on our previous observations, human peripheral blood mononuclear cells (PBMCs) can be expanded efficiently while conserving a naïve phenotype by stimulating with both recombinant human fibronectin fragment (CH-296) and anti-CD3 monoclonal antibodies. In this article, we explored the possibility of using this co-stimulation method to generate engineered T cells using lentiviral vector. Human PBMCs were stimulated with anti-CD3 together with immobilized CH-296 or anti-CD28 antibody as well as anti-CD3/anti-CD28 conjugated beads and transduced with lentiviral vector simultaneously. Co-stimulation with CH-296 gave superior transduction efficiency than with anti-CD28. Next, PBMCs were stimulated and transduced with anti-CD3/CH-296 or with anti-CD3/CD28 beads. T-cell expansion, gene transfer efficiencies and immunophenotypes were analysed. Stimulation with anti-CD3/CH-296 resulted in more than 10-times higher cell expansion and higher gene transfer efficiency with conservation of the naïve phenotype compared with anti-CD3/CD28 stimulation method. Thus, lentiviral transduction with anti-CD3/CH-296 co-stimulation is an efficient way to generate large numbers of genetically modified T cells and may be suitable for many gene therapy protocols that use adoptive T-cell transfer therapy.

  13. Study on the relationship between level of CD58 expression in peripheral blood mononuclear cell and severity of HBV infection

    Institute of Scientific and Technical Information of China (English)

    XIE Ming; WANG Xiang-ling; JI Yu-qiang; LI Jie; MENG Zhao-jun; SHI Lin; YUAN Yu-kang

    2005-01-01

    Background As one of the intercellular adhesion molecules, CD58 plays important roles in promotion of the adhesion between T cells and target cells, hyperplasia, activation of T cells and natural killer cells, and balance between Th1 and Th2. We studied the relationship between the levels of CD58 expression in peripheral blood mononuclear cells (PBMCs) and severity of HBV infection. Methods The levels of CD58 mRNA in PBMCs were detected using quantitative reverse transcription PCR. The percentage of CD58 positive cells was detected by flow cytometry in patients and healthy controls. Results The levels of CD58 mRNA and the percentage of CD58 positive cells in patients infected with HBV were significantly higher than that in the control. Based on severity of HBV infection, the patients were classified into four groups. The expression of CD58 increased significantly in an order from mild chronic, moderate chronic, severe chronic to severe hepatitis groups. The levels of CD58 mRNA and the percentage of CD58 positive cells in PBMCs from patients with HBV infection were both positively correlated with serum levels of ALT and AST.Conclusion The level of CD58 expression is related with the severity of HBV infection and the degree of liver damage.

  14. Immune effects of cocoa procyanidin oligomers on peripheral blood mononuclear cells.

    Science.gov (United States)

    Kenny, Thomas P; Keen, Carl L; Schmitz, Harold H; Gershwin, M Eric

    2007-02-01

    There has been considerable work on the relationships between nutrition and the immune response, particularly on studies that have focused on adaptive responses. There is increasing recognition of the importance of innate immunity in host protection and initiation of cytokine networks. In this study, we examined the effect of select cocoa flavanols and procyanidins on innate responses in vitro. Peripheral blood mono-nuclear cells (PBMCs), as well as purified monocytes and CD4 and CD8 T cells, were isolated from healthy volunteers and cultured in the presence of cocoa flavanol fractions that differ from another by the degree of flavanol polymerization: short-chain flavanol fraction (SCFF), monomers to pentamers; and long-chain flavanol fraction (LCFF), hexamers to decamers. Parallel investigations were also done with highly purified flavanol monomers and procyanidin dimers. The isolated cells were then challenged with lipopolysaccharide (LPS) with quantitation of activation using CD69 and CD83 expression and analysis of secreted tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-10, and granulocyte macrophage colony-stimulating factor (GM-CSF). The chain length of flavanol fractions had a significant effect on cytokine release from both unstimulated and LPS-stimulated PBMCs. For example, there was a striking increase of LPS-induced synthesis of IL-1beta, IL-6, IL-10, and TNF-alpha in the presence of LCFF. LCFF and SCFF, in the absence of LPS, stimulated the production of GM-CSF. In addition, LCFF and SCFF increased expression of the B cell markers CD69 and CD83. There were also unique differential responses in the mononuclear cell populations studied. We conclude that the oligomers are potent stimulators of both the innate immune system and early events in adaptive immunity.

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

    Science.gov (United States)

    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.

  16. [Effect of airborne fungi on interleukin-4 mRNA expression in the peripheral blood mononuclear cells of rats].

    Science.gov (United States)

    Kan, H; Song, W; Jiang, R

    2001-09-01

    The effect of indoor airborne fungi on interleukin-4 (IL-4) mRNA expression in the peripheral blood mononuclear cells (PBMCs) of rats was studied by inoculating via trachea with fungi suspension. A simplified competitive RT-PCR method was used to measure the IL-4 mRNA expression in plasma. The results showed that interleukin-4 expression was much higher in the affected groups than that in the control one. It suggested that IL-4 might play an important role in the sensitization progress of fungi.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Effects of estradiol and progesterone on the proinflammatory cytokine production by mononuclear cells from patients with chronic hepatitis C

    Institute of Scientific and Technical Information of China (English)

    Ying Yuan; Katsuyoshi Tamaki; Masayuki Shono; Tetsuji Takayama; Ichiro Shimizu; Mi Shen; Eriko Aoyagi; Hidetaka Takenaka; Tatuzo Itagaki; Marl Urata; Katsutaka Sannomiya; Nao Kohno

    2008-01-01

    AIM: To investigate the effects of estradiol (E2) and progesterone on the unstimulated and oxidative stressstimulated production of tumor necrosis factor (TNF)-α,interleukin (IL)-1β, IL-8, and macrophage chemotactic protein (ICP)-1 by peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C and healthy controls.METHODS: The PBMCs were separated from agematched 72 males and 71 females with and without chronic hepatitis C, who were divided into two groups based on a mean menopausal age of 50 years. Oxidative stress was induced by hydrogen peroxide in the cells incubated in serum-free media. CytokJnes in the culture supernatant were measured by an enzyme-linked immunosorbent assay.RESULTS: The highest levels of the spontaneous production of TNF-α, IL-1β, IL-8, and MCP-1 by the unstimulated PBMCs were in the older male patients with chronic hepatitis C and the lowest levels were in the premenopausal female-healthy controls. E2 inhibited the cytokine production by the unstimulated PBMCs from the older male and post-menopausal female patients, which was further stimulated by progesterone. The exposure to hydrogen peroxide in the PBMCs from the younger male and pre-menopausal female healthy subjects induced the production of cytokines. The change rates of the hydrogen peroxide-stimulated cytokine production were suppressed by E2 and enhanced by progesterone.CONCLUSION: These findings suggest that E2 may play a favorable role in the course of persistent liver injury by preventing the accumulation of monocytes-macrophages and by inhibiting proinflammatory cytokine production,whereas progesterone may counteract the favorable E2 effects.

  20. A method for biomarker measurements in peripheral blood mononuclear cells isolated from anxious and depressed mice: β-arrestin 1 protein levels in depression and treatment

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    Indira eMENDEZ-DAVID

    2013-09-01

    Full Text Available A limited number of biomarkers in the central and peripheral systems which are known may be useful for diagnosing major depressive disorders and predicting the effectiveness of antidepressant treatments. Since 60% of depressed patients do not respond adequately to medication or are resistant to antidepressants, it is imperative to delineate more accurate biomarkers. Recent clinical studies suggest that β-arrestin 1 levels in human mononuclear leukocytes may be an efficient biomarker. If potential biomarkers such as β-arrestin 1 could be assessed from a source such as peripheral blood cells, then they could be easily monitored and used to predict therapeutic responses. However, no previous studies have measured β-arrestin 1 levels in peripheral blood mononuclear cells (PBMCs in anxious-depressive rodents.This study aimed to develop a method to detect β-arrestin protein levels through immunoblot analyses of mouse PBMCs isolated from whole blood. In order to validate the approach, β-arrestin levels were then compared in naïve, anxious/depressed mice, and anxious/depressed mice treated treated with a selective serotonin reuptake inhibitor (SSRI; fluoxetine, 18 mg/kg/day in the drinking water. The results demonstrated that mouse whole blood collected by submandibular bleeding permitted isolation of enough PBMCs to assess circulating proteins such as β-arrestin 1. β-arrestin 1 levels were successfully measured in healthy human subject and naïve mouse PBMCs. Interestingly, PBMCs from anxious/depressed mice showed significantly reduced β-arrestin 1 levels. These decreased β-arrestin 1 expression levels were restored to normal levels with chronic fluoxetine treatment. The results suggest that isolation of PBMCs from mice by submandibular bleeding is a useful technique to screen putative biomarkers of the pathophysiology of mood disorders and the response to antidepressants. In addition, these results confirm that β-arrestin 1 is a potential

  1. Increased Responsiveness to Toll-Like Receptor 4 Stimulation in Peripheral Blood Mononuclear Cells from Patients with Recent Onset Rheumatoid Arthritis

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    M. L. Kowalski

    2008-01-01

    Full Text Available Background. Cell signaling via Toll-like receptors (TLRs leads to synovial inflammation in rheumatoid arthritis (RA. We aimed to assess effects of TLR2 and TLR4 stimulation on proinflammatory cytokine production by peripheral blood mononuclear cells (PBMCs from patients with recent-onset RA, osteoarthrosis (OA, and healthy control (HC. Methods. PBMCs were stimulated with LPS, biglycan and cytokine mix. Cytokines were analyzed in supernatants with ELISA. Expression of toll-like receptors mRNA in leukocytes was analyzed using real-time qPCR. Results. PBMCs from RA patients spontaneously produced less IL-6 and TNFα than cells from OA and HC subjects. LPS increased cytokines' production in all groups. In RA patients increase was dramatic (30 to 48-fold and 17 to 31-fold, for respective cytokines compared to moderate (2 to 8-fold in other groups. LPS induced 15-HETE generation in PBMCs from RA (mean 251% and OA patients (mean 43%, although only in OA group, the increase was significant. TLR2 and TLR4 gene expressions decreased in response to cytokine mix, while LPS enhanced TLR2 expression in HC and depressed TLR4 expression in OA patients. Conclusion. PBMCs from recent-onset RA patients are overresponsive to stimulation with bacterial lipopolysaccharide. TLR expression is differentially regulated in healthy and arthritic subjects.

  2. Cytotoxic effect of wine polyphenolic extracts and resveratrol against human carcinoma cells and normal peripheral blood mononuclear cells.

    Science.gov (United States)

    Matić, Ivana; Zizak, Zeljko; Simonović, Mladen; Simonović, Branislav; Godevac, Dejan; Savikin, Katarina; Juranić, Zorica

    2010-08-01

    Red and white wine polyphenols have been reported to provide substantial health benefits. In this study, the cytotoxic activity of red and white wine polyphenolic extracts and of resveratrol was evaluated against different cancer cell lines--human cervix adenocarcinoma HeLa, human breast adenocarcinoma MDA-MB-361, and human breast carcinoma MDA-MB-453--and normal human peripheral blood mononuclear cells (PBMCs). Qualitative and quantitative compositions of wine polyphenolic extracts obtained by fractional vacuum distillation of corresponding wines were determined using spectrophotometric methods and high-performance liquid chromatography with diode array detection and liquid chromatography with electrospray ionization-time of flight mass spectrometry analysis. It was demonstrated that wine polyphenolic extracts and resveratrol exerted higher cytotoxic activity against HeLa and MDA-MB-453 cells in comparison to MDA-MB-361 cells and unstimulated and stimulated PBMCs. Furthermore, white wine polyphenolic extract exhibited a significantly higher antiproliferative action on cancer cell lines than red wine extract. The presence of condensed or fragmented nuclei in HeLa cells, pretreated with extract of white wine and stained with a mixture of acridine orange and ethidium bromide, pointed to the morphological signs of apoptosis. In addition, HeLa cells in late stages of apoptosis or secondary necrosis were also observed. Results from our study suggest that polyphenolic extracts from red and white wine may have anticarcinogenic potential.

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

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    Claire Jones

    Full Text Available 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.

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

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    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.

  5. 10th NTES Conference: Nickel and Arsenic Compounds Alter the Epigenome of Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Brocato, Jason; Costa, Max

    2015-01-01

    The mechanisms that underlie metal carcinogenesis are the subject of intense investigation; however, data from in vitro and in vivo studies are starting to piece together a story that implicates epigenetics as a key player. Data from our lab has shown that nickel compounds inhibit dioxygenase enzymes by displacing iron in the active site. Arsenic is hypothesized to inhibit these enzymes by diminishing ascorbate levels--an important co-factor for dioxygenases. Inhibition of histone demethylase dioxygenases can increase histone methylation levels, which also may affect gene expression. Recently, our lab conducted a series of investigations in human subjects exposed to high levels of nickel or arsenic compounds. Global levels of histone modifications in peripheral blood mononuclear cells (PBMCs) from exposed subjects were compared to low environmentally exposed controls. Results showed that nickel increased H3K4me3 and decreased H3K9me2 globally. Arsenic increased H3K9me2 and decreased H3K9ac globally. Other histone modifications affected by arsenic were sex-dependent. Nickel affected the expression of 2756 genes in human PBMCs and many of the genes were involved in immune and carcinogenic pathways. This review will describe data from our lab that demonstrates for the first time that nickel and arsenic compounds affect global levels of histone modifications and gene expression in exposed human populations.

  6. microRNA expression profile of peripheral blood mononuclear cells of Klinefelter syndrome.

    Science.gov (United States)

    Sui, Weiguo; Ou, Minglin; Chen, Jiejing; Li, Huan; Lin, Hua; Zhang, Yue; Li, Wuxian; Xue, Wen; Tang, Donge; Gong, Weiwei; Zhang, Ruohan; Li, Fengyan; Dai, Yong

    2012-11-01

    microRNAs are a type of small non-coding RNAs which play important roles in post-transcriptional gene regulation, and the characterization of microRNA expression profiling in peripheral blood mononuclear cells (PBMCs) from patients with Klinefelter syndrome requires further investigation. In this study, PBMCs were obtained from patients with Klinefelter syndrome and normal controls. After preparation of small RNA libraries, the two groups of samples were sequenced simultaneously using next generation high-throughput sequencing technology, and novel and known microRNAs were analyzed. A total of 9,772,392 and 9,717,633 small RNA reads were obtained; 8,014,466 (82.01%) and 8,104,423 (83.40%) genome-matched reads, 64 and 49 novel microRNAs were identified in the library of Klinefelter syndrome and the library of healthy controls, respectively. There were 71 known microRNAs with differential expression levels between the two libraries. Clustering of over-represented gene ontology (GO) classes in predicted targets of novel microRNAs in the Klinefelter syndrome library showed that the most significant GO terms were genes involved in the endomembrane system, nucleotide binding and kinase activity. Our data revealed that there are a large number of microRNAs deregulated in PBMCs taken from patients with Klinefelter syndrome, of which certain novel and known microRNAs may be involved in the pathological process of Klinefelter syndrome. Further studies are necessary to determine the roles of microRNAs in the pathological process of Klinefelter syndrome in the future.

  7. Imbalance of Th17 and Treg in peripheral blood mononuclear cells of active tuberculosis patients

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    Jie Luo

    Full Text Available Abstract Objective: Maintaining a right balance between Th17 and Treg might be critical to the immunopathogenesis of active tuberculosis (TB. This study aimed to assess whether the Th17/Treg balance is altered in active TB patients. Methods: 250 study subjects (90 active TB patients, 80 latent TB subjects, and 80 healthy controls were recruited for the study. The expression of Th17 and Treg in peripheral blood mononuclear cells (PBMCs in the 250 subjects was investigated by flow cytometry. Plasma levels of cytokines IL-17 and IL-10, which are related to Th17 and Treg, respectively, were determined by ELISA. Results: The percentages of Th17 and Treg in PBMCs from active TB patients were significantly higher than those from latent TB or control groups (Th17: 4.31 ± 1.35% vs. 1.58 ± 0.71% or 1.15 ± 0.49%, p < 0.05; Treg: 11.44 ± 2.69% vs. 7.54 ± 1.56% or 4.10 ± 0.99%, p < 0.05. The expression of IL-17 and IL-10 was significantly increased in active TB patients in comparison to that in latent TB or control groups (IL-17: 16.85 ± 9.68 vs. 7.23 ± 5.19 or 8.21 ± 5.51 pg/mL, p < 0.05; IL-10: 28.70 ± 11.27 vs. 20.25 ± 8.57 or 13.94 ± 9.00 pg/mL, p < 0.05. Conclusions: Our study demonstrated an altered balance of Treg/Th17 in active TB patients, with higher percentages of Th17 and Treg in PBMCs. Further research on this imbalance may offer a new direction for TB treatment.

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

    Science.gov (United States)

    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

    2015-10-01

    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. 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). 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. 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.

  9. Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

    Institute of Scientific and Technical Information of China (English)

    Chang-Zheng Ke; Yue Chen; Zuo-Jiong Gong; Zhong-Ji Meng; Li Liu; Ze-Jiu Ren; Zuo-Hua Zhou

    2006-01-01

    AIM: To study the dynamic changes of hepatits B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy.METHODS: A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine amonotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n = 42) and control group (n = 30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment.RESULTS: In the treatment group, HBV DNA became negative both in serum and in PBMC, of 38 and 25 out of 42 cases respectively during the 48 Wk oflamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P < 0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC.CONCLUSION: Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs.The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum.

  10. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

    Science.gov (United States)

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido

    2012-08-30

    Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards.

  11. Ethanol suppression of peripheral blood mononuclear cell trafficking across brain endothelial cells in immunodeficiency virus infection

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    Lola C Hudson

    2010-01-01

    Full Text Available Lola C Hudson1, Brenda A Colby1, Rick B Meeker21Department of Molecular Biosciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; 2Department of Neurology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USAAbstract: Earlier studies suggested that the combination of alcohol use and immunodeficiency virus infection resulted in more severe neurologic disease than either condition individually. These deleterious interactions could be due to increased immune cell and virus trafficking or may result from interactions between ethanol and human immunodeficiency virus (HIV-associated toxicity within the brain. To determine the extent to which increased trafficking played a role, we examined the effect of ethanol on the migration of different peripheral blood mononuclear cell (PBMCs subsets across a brain endothelial cell monolayer. We utilized combinations of feline brain endothelial cells with astrocytes, and/or microglia with either acute exposure to 0.08 g/dL ethanol, a combination of ethanol and feline immunodeficiency virus (FIV, or FIV alone. Adherence of PBMCs to endothelium was increased in all combinations of cells with the addition of ethanol. Despite increased PBMC adhesion with ethanol treatment, transmigration of B cells, monocytes, CD4 T cells and CD8 T cells was not increased and was actually decreased in the presence of astrocytes. Expression of three common adhesion molecules, intercellular adhesion molecule-1 (ICAM1, ICAM2, and vascular cell adhesion molecule, was unchanged or slightly decreased by ethanol. This indicated that although adherence is increased by ethanol it is not due to an increased expression of adhesion molecules. RANTES, MIP1α, MIP1β, and MCP-1 mRNA expression was also studied in brain endothelial cells, astrocytes and microglia by reverse transcriptase-polymerase chain reaction. Ethanol treatment of astrocytes resulted in modest changes of

  12. Ellagic Acid, a Dietary Polyphenol, Inhibits Tautomerase Activity of Human Macrophage Migration Inhibitory Factor and Its Pro-inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Sarkar, Souvik; Siddiqui, Asim A; Mazumder, Somnath; De, Rudranil; Saha, Shubhra J; Banerjee, Chinmoy; Iqbal, Mohd S; Adhikari, Susanta; Alam, Athar; Roy, Siddhartha; Bandyopadhyay, Uday

    2015-05-27

    Ellagic acid (EA), a phenolic lactone, inhibited tautomerase activity of human macrophage migration inhibitory factor (MIF) noncompetitively (Ki = 1.97 ± 0.7 μM). The binding of EA to MIF was determined by following the quenching of tryptophan fluorescence. We synthesized several EA derivatives, and their structure-activity relationship studies indicated that the planar conjugated lactone moiety of EA was essential for MIF inhibition. MIF induces nuclear translocation of NF-κB and chemotaxis of peripheral blood mononuclear cells (PBMCs) to promote inflammation. We were interested in evaluating the effect of EA on nuclear translocation of NF-κB and chemotactic activity in human PBMCs in the presence of MIF. The results showed that EA inhibited MIF-induced NF-κB nuclear translocation in PBMCs, as evident from confocal immunofluorescence microscopic data. EA also inhibited MIF-mediated chemotaxis of PBMCs. Thus, we report MIF-inhibitory activity of EA and inhibition of MIF-mediated proinflammatory responses in PBMCs by EA.

  13. Paracrine Factors from Irradiated Peripheral Blood Mononuclear Cells Improve Skin Regeneration and Angiogenesis in a Porcine Burn Model.

    Science.gov (United States)

    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

    2016-04-29

    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.

  14. Preventive activity of olive oil phenolic compounds on alkene epoxides induced oxidative DNA damage on human peripheral blood mononuclear cells.

    Science.gov (United States)

    Fuccelli, Raffaela; Sepporta, Maria Vittoria; Rosignoli, Patrizia; Morozzi, Guido; Servili, Maurizio; Fabiani, Roberto

    2014-01-01

    The aim of this study was to investigate the ability of epoxides of styrene (styrene-7,8-oxide; SO) and 1,3-butadiene (3,4-epoxy-1-butene; 1,2:3,4:-diepoxybutane) to cause oxidative stress and oxidative DNA damage on human peripheral blood mononuclear cells (PBMCs) and whether a complex mixture of olive oil phenols (OOPE) could prevent these effects. The DNA damage was measured by the single-cell gel electrophoresis (SCGE; comet assay). We found that the DNA damage induced by alkene epoxides could be prevented by N-acetyl-cysteine (10 mM) and catalase (100 U/ml). Alkene epoxides caused a significant (P DNA glycosylase (FPG)- and Endonuclease III (ENDO III)-sensitive sites in PBMCs, demonstrating the presence of oxidized bases. OOPE (1 μg of total phenols/ml) was able to prevent the alkene epoxide induced DNA damage both after 2 and 24 h of incubation. In addition, OOPE completely inhibited the SO-induced intracellular peroxide accumulation in PBMCs and prevented the oxidative DNA damage induced by SO, as evidenced by the disappearance of both FPG- and ENDO III-sensitive sites. This is the first study demonstrating the ability of OOPE to prevent the DNA damage induced by alkene epoxides providing additional information about the chemopreventive properties of olive oil.

  15. Increased expression of Siglec-1 on peripheral blood monocytes and its role in mononuclear cell reactivity to autoantigen in rheumatoid arthritis.

    Science.gov (United States)

    Xiong, Yi-Song; Cheng, Yue; Lin, Qiu-Shui; Wu, Ai-Lin; Yu, Juan; Li, Chang; Sun, Yi; Zhong, Ren-Qian; Wu, Li-Juan

    2014-02-01

    Elevated expression of Siglec-1 on circulating monocytes has been reported in some inflammatory and autoimmune diseases, but its expression and role in RA has not been elucidated. The aims of this study were to determine the expression of Siglec-1 in peripheral blood and to explore its role in mononuclear cell reactivity to autoantigen in RA. Siglec-1 protein and mRNA levels in 42 RA patients, 39 OA patients, 28 SLE patients and 42 normal controls were determined by flow cytometry and quantitative RT-PCR, respectively. In addition, 10 patients with active RA received DMARDs for 12 weeks and the frequencies of Siglec-1-positive cells and the 28-joint DAS (DAS28) were assessed before and after therapy. Furthermore, TNF-α, IFN-γ and type II collagen were used to up-regulate Siglec-1. Peripheral blood mononuclear cells (PBMCs) from different groups were stimulated with mitogens or antigens and cell proliferation and cytokine production were determined. The protein and mRNA levels of Siglec-1 on PBMCs and monocytes in RA patients were significantly higher than those in OA patients and healthy controls. Moreover, the expression of Siglec-1 protein on PBMCs was positively correlated with DAS28, ESR, high-sensitivity CRP and IgM-RF, but not with anti-CCP antibody. Interestingly, Siglec-1 expression was decreased in parallel with the decrease in the DAS28 after 12 weeks of anti-rheumatic treatment. Furthermore, TNF-α, IFN-γ and type II collagen can up-regulate Siglec-1 in PBMCs. Elevated PBMC proliferation and proinflammatory cytokine production to collagen stimulation in RA patients decreased when Siglec-1 was inhibited by anti-Siglec-1 antibodies. Elevated Siglec-1 expression in PBMCs and monocytes can potentially serve as a biomarker for monitoring disease activity in RA. Siglec-1 may also play a proinflammatory role in stimulating lymphocyte proliferation and activation in RA.

  16. The Role of Innate Immune Cells in the Response of Heat-Treated Mycobacterium tuberculosis (M.tb) Antigens Stimulating PBMCs

    Institute of Scientific and Technical Information of China (English)

    ChaoWang; JunLi; HuaixinZheng; HaimingWei; RuijunZhang; BaiqingLi

    2004-01-01

    The proliferation response of γδT cells to the antigen from heat-treated Mycobacterium tuberculosis H37Ra (M.tb Ag) was used as a good model in γδT cell research. From preliminary research it is found that activated NK cells positively elevated γδT cells proliferation after simulating PBMCs with M.tb Ag. To investigate different behaviors of NK cells, γδNKT cells, γδT cells and relationships between these cell subsets, activation and proliferation of different cell subsets of PBMCs in response to M.tb Ag were analyzed. We demonstrated that NK cells, γδNKT cells and γδT cells could be activated after stimulation with M.tb Ag. γδNKT cells and γδT cells proliferated while the number of NK cells decreased after 11 day-simulation with M.tb Ag. Meanwhile, at the early time of stimulation the cytotoxicity of PBMCs was enhanced. Cellular & Molecular Immunology.2004;1(6):467-470.

  17. The Role of Innate Immune Cells in the Response of Heat-Treated Mycobacterium tuberculosis (M.tb) Antigens Stimulating PBMCs

    Institute of Scientific and Technical Information of China (English)

    Chao Wang; Jun Li; Huaixin Zheng; Haiming Wei; Ruijun Zhang; Baiqing Li; Zhigang Tian

    2004-01-01

    The proliferation response of γδT cells to the antigen from heat-treated Mycobacterium tuberculosis H37Ra (M.tb Ag) was used as a good model in γδT cell research. From preliminary research it is found that activated NK cells positively elevated γδT cells proliferation after simulating PBMCs with M. tb Ag. To investigate different behaviors of NK cells, γδNKT cells, γδT cells and relationships between these cell subsets, activation and proliferation of different cell subsets of PBMCs in response to M. tb Ag were analyzed. We demonstrated that NK cells, γδNKT cells and γδT cells could be activated after stimulation with M. tb Ag. γδNKT cells and γδT cells proliferated while the number of NK cells decreased after 11 day-simulation with M. tb Ag. Meanwhile, at the early time of stimulation the cytotoxicity of PBMCs was enhanced. Cellular & Molecular Immunology.2004;1(6):467-470.

  18. The clinical significance of EBV DNA in the plasma and peripheral blood mononuclear cells of patients with or without EBV diseases.

    Science.gov (United States)

    Kanakry, Jennifer A; Hegde, Aparna M; Durand, Christine M; Massie, Allan B; Greer, Amy E; Ambinder, Richard F; Valsamakis, Alexandra

    2016-04-21

    Epstein-Barr virus (EBV) is a ubiquitous virus that establishes a latent infection within the host and in some cases can lead to the development of EBV-associated lymphomas, lymphoproliferative disorders, hemophagocytic lymphohistiocytosis, solid tumors, and other diseases. We studied the clinical significance of detecting EBV DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of 2146 patients who had blood specimens sent to the Johns Hopkins Hospital clinical laboratory for viral quantitative real-time polymerase chain reaction assay over a 5-year period. Within this largely immunocompromised and hospitalized cohort, 535 patients (25%) had EBV detected in plasma or PBMCs. When EBV was detected in the absence of an EBV(+)disease (n = 402), it was present only in PBMCs in 69% of cases. Immunocompromised patients were less likely to have EBV in plasma than in PBMCs in the absence of EBV(+)disease. In patients with active, systemic EBV(+)diseases (n = 105), EBV was detected in plasma in 99% of cases but detected in PBMCs in only 54%. Across a range of copy number cutoffs, EBV in plasma had higher specificity and sensitivity for EBV(+)disease as compared with EBV in PBMCs. EBV copy number in plasma distinguished untreated, EBV(+)lymphoma from EBV(+)lymphoma in remission and EBV(-)lymphoma, and also distinguished untreated, EBV(+)posttransplantation lymphoproliferative disorder (PTLD) from EBV(+)PTLD in remission and EBV(-)PTLD. EBV copy number quantification is a useful diagnostic marker across the spectrum of EBV(+)diseases, even among immunocompromised patients, with plasma specimens more indicative of EBV(+)disease than PBMCs.

  19. Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up.

    Directory of Open Access Journals (Sweden)

    Arnaud François

    Full Text Available Recent findings indicate that microglia in Alzheimer's disease (AD is senescent whereas peripheral blood mononuclear cells (PBMCs could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1-42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA, and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3 were quantified by western blot and cytokines (Interleukin (IL-1β, Tumor necrosis Factor (TNF-α and IL-6 by Luminex X-MAP® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.

  20. Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India.

    Science.gov (United States)

    Kishore, Amit; Sodhi, Monika; Kumari, Parvesh; Mohanty, A K; Sadana, D K; Kapila, Neha; Khate, K; Shandilya, Umesh; Kataria, R S; Mukesh, M

    2014-09-01

    Circulating leukocytes can be used as an effective model to understand the heat stress response of different cattle types and buffaloes. This investigation aimed to determine the temporal profile of HSPs (HSP40, HSP60, HSP70, and HSP90) expression in circulating peripheral blood mononuclear cells (PBMCs) of Murrah buffaloes, Holstein-Friesian (HF), and Sahiwal cows in response to sublethal heat shock at 42 °C. The viability data indicated HF PBMCs to be the most affected to the heat shock, whereas Sahiwal PBMCs were least affected, indicating its better survivability during the heat stress condition. The qRT-PCR expression data showed significant increase in mRNA expression of the analyzed HSPs genes after heat stimuli to the PBMCs under in vitro condition. In each case, the HSPs were most upregulated at 2 h after the heat stress. Among the HSPs, HSP70 was relatively more expressed followed by HSP60 indicating the action of molecular chaperones to stabilize the native conformation of proteins. However, PBMCs from different cattle types and buffaloes showed difference in the extent of transcriptional response. The level of expression of HSPs throughout the time period of heat stress was highest in buffaloes, followed by HF and Sahiwal cows. The higher abundance of HSP70 mRNA at each time point after heat stress showed prolonged effect of heat stress in HF PBMCs. The data presented here provided initial evidence of transcriptional differences in PBMCs of different cattle types and buffaloes and warrant further research.

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

    Science.gov (United States)

    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 (pporcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (pporcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time.

  2. Response to rhinovirus infection by human airway epithelial cells and peripheral blood mononuclear cells in an in vitro two-chamber tissue culture system.

    Directory of Open Access Journals (Sweden)

    Devi Rajan

    Full Text Available Human rhinovirus (HRV infections are associated with the common cold, occasionally with more serious lower respiratory tract illnesses, and frequently with asthma exacerbations. The clinical features of HRV infection and its association with asthma exacerbation suggest that some HRV disease results from virus-induced host immune responses to infection. To study the HRV-infection-induced host responses and the contribution of these responses to disease, we have developed an in vitro model of HRV infection of human airway epithelial cells (Calu-3 cells and subsequent exposure of human peripheral blood mononuclear cells (PBMCs to these infected cells in a two-chamber trans-well tissue culture system. Using this model, we studied HRV 14 (species B and HRV 16 (species A induced cytokine and chemokine responses with PBMCs from four healthy adults. Infection of Calu-3 cells with either virus induced HRV-associated increases in FGF-Basic, IL-15, IL-6, IL-28A, ENA-78 and IP-10. The addition of PBMCs to HRV 14-infected cells gave significant increases in MIP-1β, IL-28A, MCP-2, and IFN-α as compared with mock-infected cells. Interestingly, ENA-78 levels were reduced in HRV 14 infected cells that were exposed to PBMCs. Addition of PBMCs to HRV 16-infected cells did not induce MIP-1β, IL-28A and IFN-α efficiently nor did it decrease ENA-78 levels. Our results demonstrate a clear difference between HRV 14 and HRV 16 and the source of PBMCs, in up or down regulation of several cytokines including those that are linked to airway inflammation. Such differences might be one of the reasons for variation in disease associated with different HRV species including variation in their link to asthma exacerbations as suggested by other studies. Further study of immune responses associated with different HRVs and PBMCs from different patient groups, and the mechanisms leading to these differences, should help characterize pathogenesis of HRV disease and generate

  3. Increased interleukin-23 receptor+ T cells in peripheral blood mononuclear cells of patients with systemic lupus erythematosus

    Science.gov (United States)

    2010-01-01

    Introduction Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies and immune complex deposition in various organs. Aberrations in the T lymphocyte compartment and dysregulated cytokine production are key features of SLE pathogenesis and disease progression. Recently, the role of the interleukin (IL)-17/IL-23 axis in the pathogenesis of SLE has been reported. IL-23 and IL-23R are essential for expansion of pathogenic IL-17-producing T lymphocytes and have been shown to be important in the pathogenesis of lupus in animal models. Methods In this study, the expression of IL-23R and IL-17 in CD4+ and CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMCs) of SLE patients and control subjects were examined by flow cytometry. Twenty-nine SLE patients and 10 control subjects were recruited in this study. Patients were divided into active and inactive groups based on the SLE disease activity index (SLEDAI). As another disease control population, five psoriatic patients were recruited in this study. Results Percentages of both IL23R+ CD4+ and IL-23R+ CD8+ T cell subsets were significantly higher in freshly isolated PBMCs from both groups of SLE patients compared to control subjects (P = 0.0021 and P = 0.0006, respectively). In addition, this difference was maintained after ex vivo stimulation with plate-bound anti-CD3/CD28 antibodies (P = 0.007 and P = 0.0019, respectively). When the fold increase in IL-17+ T cells after ex vivo stimulation for three days was compared between patients and controls, SLE patients exhibited significantly higher increases in CD4+ IL-17+ and CD8+ IL-17+ T cells, suggesting that PBMCs from SLE patients promoted the expansion of IL-17-producing T cells upon stimulation more vigorously than control PBMCs. These trends were not observed in psoriasis patients. The correlations between IL-23R+ T cells and IL-17+ T cells and IL-23R+ CD8+ T cells and SLEDAI scores in patients were also found

  4. Application of the comet assay for investigation of oxidative DNA damage in equine peripheral blood mononuclear cells.

    Science.gov (United States)

    Marlin, David J; Johnson, Lucy; Kingston, Demelza A; Smith, Nicola C; Deaton, Chris M; Mann, Sarah; Heaton, Paul; Van Vugt, Fenneke; Saunders, Kelly; Kydd, Julia; Harris, Pat A

    2004-08-01

    Oxidative stress occurs when antioxidant defense mechanisms are overwhelmed by free radicals and may lead to DNA damage, which has been implicated in processes such as aging and diseases such as cancer. The two main techniques presently used to quantify DNA damage are measurement of 8-hydroxydeoxyguanosine and the Comet assay (also known as single-cell gel electrophoresis). The aim of this study was to apply the comet assay to equine peripheral blood mononuclear cells (PBMCs) and identify two conditions in which we hypothesized that oxidative DNA damage would be increased in PBMCs: aging and equine recurrent airway obstruction (RAO, a condition similar to human asthma). The images obtained were similar to those previously published for humans, cats, and dogs. The optimum concentration of H(2)O(2) to estimate susceptibility to exogenous damage was 50 microM. Mean intraassay coefficients of variation were 4.7 and 9.7% for endogenous and exogenous tail-DNA quantities, respectively, and 7.3 and 8.3%, respectively, for interassay coefficients. There was no significant difference in either endogenous or exogenous percentages of tail DNA for samples collected from six ponies on three consecutive days. There was no significant difference in endogenous, exogenous, or exogenous (corrected for endogenous) oxidative DNA damage between mature and aged ponies. However, young pony foals had significantly less endogenous DNA damage than mature or aged ponies (P < 0.05). RAO-affected horses without airway inflammation (i.e., in clinical remission) had significantly greater endogenous damage compared with non-RAO-affected control animals (P = 0.009). There was a significant correlation between endogenous percentage of tail DNA in PBMCs and red blood cell hemolysate glutathione concentration (r = 0.720; P < 0.001). In conclusion, the comet assay appears to be suitable for investigating DNA damage in equine PBMCs.

  5. Use of accelerator mass spectrometry to measure the pharmacokinetics and peripheral blood mononuclear cell concentrations of zidovudine.

    Science.gov (United States)

    Vuong, Le T; Ruckle, Jon L; Blood, Arlin B; Reid, Michael J; Wasnich, Richard D; Synal, Hans-Arno; Dueker, Stephen R

    2008-07-01

    The remarkable sensitivity of accelerator mass spectrometry (AMS) is finding many new applications in pharmacology. In this study AMS was used to measure [(14)C]-Zidovudine (ZDV) concentrations at the drug's site of action (peripheral blood mononuclear cells, PBMCs) following a dose of 520 ng (less than one-millionth of the standard daily dose) to a healthy volunteer. In addition, the pharmacokinetics of this microdose were determined and compared to previously published parameters for therapeutic doses. Microdose ZDV pharmacokinetic parameters fell within reported 95% confidence intervals or standard deviations of most previously published values for therapeutic doses. Blood, urine, stool, saliva, and isolated PBMCs were collected periodically through 96 h postdose and analyzed for ZDV and metabolite concentrations. The results showed that ZDV is rapidly absorbed and eliminated, has one major metabolite, and is sequestered in PBMCs. (14)C mass balance assessments indicated a significant portion of ZDV remained after 96 h with a much prolonged elimination half-life. Results of this study demonstrate the usefulness of microdosing and AMS as a tool for studying the pharmacokinetic characteristics, including PBMC concentrations, of ZDV and underscore the value of AMS as a tool with which to perform pharmacokinetic and mass balance studies using trace amounts of radiolabeled compound.

  6. Potential antidepressant and resilience mechanism revealed by metabolomic study on peripheral blood mononuclear cells of stress resilient rats.

    Science.gov (United States)

    Li, Juan; Zhang, Shu-Xiao; Wang, Wei; Cheng, Ke; Guo, Hua; Rao, Cheng-Long; Yang, De-Yu; He, Yong; Zou, De-Zhi; Han, Yu; Zhao, Li-Bo; Li, Peng-Fei; Xie, Peng

    2017-03-01

    Resilience is an active coping response to stress, which plays a very important role in major depressive disorder study. The molecular mechanisms underlying such resilience are poorly understood. Peripheral blood mononuclear cells (PBMCs) were promising objects in unveiling the underlying pathogenesis of resilience. Hereby we carried out successive study on PBMCs metabolomics in resilient rats of chronic unpredictable mild stress (CUMS) model. A gas chromatography-mass spectrometry (GC-MS) metabolomic approach coupled with principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to detect differential metabolites in PBMCs of resilient rats. Ingenuity Pathways Analysis (IPA) was applied for pathway analysis. A set of differential metabolites including Malic acid, Ornithine, l-Lysine, Stigmasterol, Oleic acid, γ-Tocopherol, Adenosine and N-acetyl-d-glucosamine were significantly altered in resilient rats, meanwhile promoting antidepressant research. As revealed by IPA that aberrant energy metabolism, HIFα signaling, neurotransmitter, O-GlcNAcylation and cAMP signaling cascade in peripheral might be evolved in the pathogenesis of coping mechanism. The GC-MS based metabolomics may contribute to better understanding of resilience, as well as shedding light on antidepressant discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever.

    Science.gov (United States)

    Durbin, Anna P; Vargas, Maria José; Wanionek, Kimberli; Hammond, Samantha N; Gordon, Aubree; Rocha, Crisanta; Balmaseda, Angel; Harris, Eva

    2008-07-05

    In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14(+), CD32(+), CD86(+), CD11c(+)). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis.

  8. Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation.

    Science.gov (United States)

    Nishad, S; Ghosh, Anu

    2016-02-01

    Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50-500 mGy) have been strongly debated even as researchers continue to search for elusive 'radiation signatures' in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after 'ex vivo' exposure with two radiation doses (300 mGy and 1 Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ± 1.5-fold (p ≤ 0.05), with only three proteins showing ≥ 2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78 KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300 mGy and 48.3% for 1 Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Influenza a virus induces an immediate cytotoxic activity in all major subsets of peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Sanda Sturlan

    Full Text Available BACKGROUND: A replication defective influenza A vaccine virus (delNS1 virus was developed. Its attenuation is due to potent stimulation of the innate immune system by the virus. Since the innate immune system can also target cancer cells, we reasoned that delNS1 virus induced immune-stimulation should also lead to the induction of innate cytotoxic effects towards cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMCs, isolated CD56+, CD3+, CD14+ and CD19+ subsets and different combinations of the above subsets were stimulated by delNS1, wild type (wt virus or heat inactivated virus and co-cultured with tumor cell lines in the presence or absence of antibodies against the interferon system. Stimulation of PBMCs by the delNS1 virus effectively induced cytotoxicity against different cancer cell lines. Surprisingly, virus induced cytotoxicity was exerted by all major subtypes of PBMCs including CD56+, CD3+, CD14+ and CD19+ cells. Virus induced cytotoxicity in CD3+, CD14+ and CD19+ cells was dependent on virus replication, whereas virus induced cytotoxicity in CD56+ cells was only dependent on the binding of the virus. Virus induced cytotoxicity of isolated cell cultures of CD14+, CD19+ or CD56+ cells could be partially blocked by antibodies against type I and type II (IFN interferon. In contrast, virus induced cytotoxicity in the complete PBMC preparation could not be inhibited by blocking type I or type II IFN, indicating a redundant system of activation in whole blood. CONCLUSIONS/SIGNIFICANCE: Our data suggest that apart from their well known specialized functions all main subsets of peripheral blood cells also initially exert a cytotoxic effect upon virus stimulation. This closely links the innate immune system to the adaptive immune response and renders delNS1 virus a potential therapeutic tool for viro-immunotherapy of cancer.

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

    Science.gov (United States)

    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.

  11. Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event.

    Science.gov (United States)

    Laye, Matthew J; Solomon, Thomas P J; Karstoft, Kristian; Pedersen, Karin K; Nielsen, Susanne D; Pedersen, Bente K

    2012-03-01

    Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects (n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 (P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4(+) or CD8(+) cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.

  12. Peripheral blood mononuclear cells from rat model of pleurisy: The effects of hesperidin on ectoenzymes activity, apoptosis, cell cycle and reactive oxygen species production.

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    Adefegha, Stephen Adeniyi; Leal, Daniela Bitencourt Rosa; Doleski, Pedro Henrique; Ledur, Pauline Christ; Ecker, Assis

    2017-07-01

    The present study investigates the effect of hesperidin; a flavonone commonly found in citrus fruits, on the ectoenzymes (ectonucleotidase and ecto-adenosine deaminase) activity, cell viability, apoptosis, cell cycle arrest and reactive oxygen species production in peripheral blood mononuclear cells (PBMCs) from rat model of pleurisy. Wistar rats were pretreated with either saline or hesperidin (80mg/kg) by oral gavage for 21days and injected intrapleurally with 2% carrageenan or saline on the 22nd day. PBMCs were subsequently prepared after 4h of carrageenan induction. The results revealed that hesperidin may exhibit its anti-inflammatory effects through possible modulation of ectonucleotidase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) activities, reduction of intracellular reactive oxygen species, prevention of DNA damage and modulation of apoptosis as well as activation of cell cycle arrest. This study suggests some possible underlying anti-inflammatory mechanisms of hesperidin on PBMCs in acute inflammatory condition. Furthermore, hesperidin may minimize oxidative injury mediated pleurisy in rat. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Corzo, Gerardo; Espino-Solis, Gerardo Pavel

    2017-03-01

    A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1β, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and β were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1β, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology.

  14. Defective production of interferon-γ and tumour necrosis factor-α by AIDS mononuclear cells after in vitro exposure to Rhodococcus equi

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    S. Delia

    1995-01-01

    Full Text Available The production of interferon-γ and tumour necrosis factor-α was evaluated in the peripheral blood mononuclear cells (PBMCs from healthy donors and AIDS patients after Rhodococcus equi infection in vitro. PBMCs from healthy donors secreted elevated levels of IFN-γ and TNF-α when challenged in vitro with killed R. equi, whereas the release of both cytokines was impaired in supernatant cultures from AIDS patients. We conclude that the failure of IFN-γ generation in AIDS patients in response to R. equi is not antigen-specific but it may reflect the global impairment of T-cell function. In such patients, however, the infection with R. equi, a facultative intracellular pathogen which survives and replicates within macrophages, may be responsible for the impairment in the TNF-α release, possibly enhancing the HIV-induced macrophage dysftmction.

  15. Allergen challenge of peripheral blood mononuclear cells from patients with seasonal allergic rhinitis increases IL-17RB, which regulates basophil apoptosis and degranulation.

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    Wang, H; Mobini, R; Fang, Y; Barrenäs, F; Zhang, H; Xiang, Z; Benson, M

    2010-08-01

    Previously, expression profiling has been used to analyse allergen-challenged T-helper type 2 cells, nasal biopsies and nasal fluid cells from patients with seasonal allergic rhinitis (SAR). Allergen-challenged peripheral blood mononuclear cells (PBMCs) provide a human in vitro model of how antigen-presenting cells, CD4+ T cells and effector cells such as basophils interact in allergic inflammation. To identify novel genes and pathways in allergen-challenged PBMCs from patients with SAR using gene expression profiling and functional studies. PBMCs from 11 patients with SAR and 23 healthy controls were analysed with gene expression profiling. mRNA expression of IL17RB in basophils was evaluated using quantitative real-time PCR. Membrane protein expression and apoptosis of basophils were examined by flow cytometry. Degranulation of basophils was assessed by measuring beta-hexosaminidase release. Cytokine release was measured using ELISA. Gene expression microarray analysis of allergen-challenged PBMCs showed that 209 out of 44000 genes were differentially expressed in patients compared with controls. IL17RB was the gene whose expression increased most in patients (P<0.0001). FACS analysis of PBMCs showed, for the first time, that basophils express IL-17RB. Following allergen challenge, IL-17RB protein increased significantly on basophils from patients compared with controls (P<0.05). IL-3 significantly increased both mRNA and protein expressions of IL17RB. Activation of IL-17RB by its ligand, IL-25, inhibited apoptosis of basophils. Moreover, IgE-mediated degranulation was enhanced by IL-25. Increased expression of IL-17RB on allergen-challenged basophil is regulated by IL-3, inhibits apoptosis and promotes IgE-mediated degranulation of basophils.

  16. Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves.

    Science.gov (United States)

    Ai, Ye; Sanders, Claire K; Marrone, Babetta L

    2013-10-01

    A microfluidic device was developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. In this device, two identical surface acoustic waves (SAWs) generated by interdigital transducers (IDTs) propagated toward a microchannel, which accordingly built up a standing surface acoustic wave (SSAW) field across the channel. A numerical model, coupling a piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SSAW-based particle manipulation. It was found that the pressure nodes across the channel were individual planes perpendicular to the solid substrate. In the separation experiments, two side sheath flows hydrodynamically focused the injected particle or cell mixtures into a very narrow stream along the centerline. Particles flowing through the SSAW field experienced an acoustic radiation force that highly depends on the particle properties. As a result, dissimilar particles or cells were laterally attracted toward the pressure nodes at different magnitudes, and were eventually switched to different outlets. Two types of fluorescent microspheres with different sizes were successfully separated using the developed device. In addition, Escherichia coli bacteria premixed in peripheral blood mononuclear cells (PBMCs) were also efficiently isolated using the SSAW-base separation technique. Flow cytometric analysis on the collected samples found that the purity of separated E. coli bacteria was 95.65%.

  17. Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

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    Lucette A Cysique

    Full Text Available To determine the contribution of peripheral blood mononuclear cells' (PBMCs HIV DNA levels to HIV-associated dementia (HAD and non-demented HIV-associated neurocognitive disorders (HAND in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART.Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points.At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03. Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05. Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC. Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04, and higher in those with no ART treatment during HIV infection 1st year (p = .03. Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03, and motor-coordination (p = .02-.12 to a lesser extent.PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

  18. Short-chain fatty acids produced by synbiotic mixtures in skim milk differentially regulate proliferation and cytokine production in peripheral blood mononuclear cells.

    Science.gov (United States)

    Asarat, M; Apostolopoulos, V; Vasiljevic, T; Donkor, O

    2015-01-01

    Short chain fatty acids (SCFAs) are major products of prebiotic fermentation and confer human health benefits such as immune-regulation. In this study, reconstituted skim milk supplemented with prebiotics (RSMP) including inulin, hi-maize or β-glucan was fermented by probiotic strains of Lactobacillus spp. and Bifidobacteria spp. After 24 h of fermentation, probiotics growth and SCFAs production were investigated and the produced SCFAs were extracted. Inulin and Lactobacillus rhamnosus GG ATCC 53013 (LGG) combination released highest concentrations of SCFAs compared to LGG and hi-maize or β-glucan. Extracted SCFAs were then used for in vitro immune modulation study in human peripheral blood mononuclear cells (PBMCs). In lipopolysaccharide (LPS)-stimulated PBMCs, SCFAs particularly butyrate down-regulated tumor necrosis factor alpha, interleukin (IL)-12, interferon gamma (IFN-γ) and transforming growth factor beta-1 (TGF-β1), and up-regulated IL-4, IL-10, while no significant effect was noted in non-LPS-stimulated PBMCs. The results indicate that SCFAs regulated cytokine milieu in LPS-stimulated PBMCs to anti-inflammatory cytokines.

  19. Transcriptional profiling of peripheral blood mononuclear cells in pancreatic cancer patients identifies novel genes with potential diagnostic utility.

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    Michael J Baine

    Full Text Available BACKGROUND: It is well known that many malignancies, including pancreatic cancer (PC, possess the ability to evade the immune system by indirectly downregulating the mononuclear cell machinery necessary to launch an effective immune response. This knowledge, in conjunction with the fact that the trancriptome of peripheral blood mononuclear cells has been shown to be altered in the context of many diseases, including renal cell carcinoma, lead us to study if any such alteration in gene expression exists in PC as it may have diagnostic utility. METHODS AND FINDINGS: PBMC samples from 26 PC patients and 33 matched healthy controls were analyzed by whole genome cDNA microarray. Three hundred eighty-three genes were found to be significantly different between PC and healthy controls, with 65 having at least a 1.5 fold change in expression. Pathway analysis revealed that many of these genes fell into pathways responsible for hematopoietic differentiation, cytokine signaling, and natural killer (NK cell and CD8+ T-cell cytotoxic response. Unsupervised hierarchical clustering analysis identified an eight-gene predictor set, consisting of SSBP2, Ube2b-rs1, CA5B, F5, TBC1D8, ANXA3, ARG1, and ADAMTS20, that could distinguish PC patients from healthy controls with an accuracy of 79% in a blinded subset of samples from treatment naïve patients, giving a sensitivity of 83% and a specificity of 75%. CONCLUSIONS: In summary, we report the first in-depth comparison of global gene expression profiles of PBMCs between PC patients and healthy controls. We have also identified a gene predictor set that can potentially be developed further for use in diagnostic algorithms in PC. Future directions of this research should include analysis of PBMC expression profiles in patients with chronic pancreatitis as well as increasing the number of early-stage patients to assess the utility of PBMCs in the early diagnosis of PC.

  20. Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Maynard, Scott; Keijzers, Guido; Gram, Martin;

    2013-01-01

    . Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production......, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters....... However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects...

  1. Comparison of the immunosuppressive effects of dexamethasone, flunixin meglumine and meloxicam on the in vitro response of calf peripheral blood mononuclear cells.

    Science.gov (United States)

    Maeda, Yousuke; Tanaka, Rie; Ohtsuka, Hiromichi; Matsuda, Keiici; Tanabe, Taishi; Oikawa, Masaaki

    2011-07-01

    This study compared the immunosuppressive effects of dexamethasone (DEX), flunixin meglumine (FLU) and meloxicam (MEL) on the peripheral blood mononuclear cells (PBMCs) of seven healthy Holstein calves in vitro. DEX significantly inhibited lymphocyte proliferation and expression of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 messenger RNA (mRNA) in comparison with FLU and MEL. FLU and MEL dose-dependently inhibited lymphocyte proliferation, but did not significantly reduce mRNA expression. Our in vitro study indicates that steroidal anti-inflammatory drugs (SAIDs) as well as nonsteroidal anti-inflammatory drugs (NSAIDs) have immunosuppressive effects on calf PBMCs. These findings are important for assessing the indications and complications of NSAIDs in calves.

  2. Cytokine/chemokine secretion and proteomic identification of upregulated annexin A1 from peripheral blood mononuclear cells cocultured with the liver fluke Opisthorchis viverrini.

    Science.gov (United States)

    Hongsrichan, Nuttanan; Intuyod, Kitti; Pinlaor, Porntip; Khoontawad, Jarinya; Yongvanit, Puangrat; Wongkham, Chaisiri; Roytrakul, Sittiruk; Pinlaor, Somchai

    2014-05-01

    We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) cocultured with adult liver flukes (Opisthorchis viverrini) for 6 to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increases in secretion of proinflammatory cytokines, costimulating protein, adhesion molecules, and chemotactic chemokines relative to untreated controls. In contrast, secretion of the CD40 ligand, interleukin 16, and macrophage inflammatory protein 1β decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Annexin A1 (ANXA1) was selected for further study, and immunoblotting showed upregulation of ANXA1 expression in PBMCs after 12 and 24 h coculture with liver flukes. In an in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, which are in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation, and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host defense against O. viverrini infection and tissue resolution of inflammation.

  3. Cord blood versus age 5 mononuclear cell proliferation on IgE and asthma

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    Perera Frederica

    2010-08-01

    Full Text Available Abstract Background Fetal immune responses following exposure of mothers to allergens during pregnancy may influence the subsequent risk of childhood asthma. However, the association of allergen-induced cord blood mononuclear cell (CBMC proliferation and cytokine production with later allergic immune responses and asthma has been controversial. Our objective was to compare indoor allergen-induced CBMC with age 5 peripheral blood mononuclear cell (PBMC proliferation and determine which may be associated with age 5 allergic immune responses and asthma in an inner city cohort. Methods As part of an ongoing cohort study of the Columbia Center for Children's Environmental Health (CCCEH, CBMCs and age 5 PBMCs were cultured with cockroach, mouse, and dust mite protein extracts. CBMC proliferation and cytokine (IL-5 and IFN-γ responses, and age 5 PBMC proliferation responses, were compared to anti-cockroach, anti-mouse, and anti-dust mite IgE levels, wheeze, cough, eczema and asthma. Results Correlations between CBMC and age 5 PBMC proliferation in response to cockroach, mouse, and dust mite antigens were nonsignificant. Cockroach-, mouse-, and dust mite-induced CBMC proliferation and cytokine responses were not associated with allergen-specific IgE at ages 2, 3, and 5, or with asthma and eczema at age 5. However, after adjusting for potential confounders, age 5 cockroach-induced PBMC proliferation was associated with anti-cockroach IgE, total IgE, and asthma (p Conclusion In contrast to allergen-induced CBMC proliferation, age 5 cockroach-induced PBMC proliferation was associated with age 5 specific and total IgE, and asthma, in an inner-city cohort where cockroach allergens are prevalent and exposure can be high.

  4. Human peripheral blood mononuclear cells exhibit heterogeneous CD52 expression levels and show differential sensitivity to alemtuzumab mediated cytolysis.

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    Sambasiva P Rao

    Full Text Available Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs display the highest number while natural killer (NK cells, plasmacytoid dendritic cells (pDCs and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.

  5. Opposing effects of low molecular weight heparins on the release of inflammatory cytokines from peripheral blood mononuclear cells of asthmatics.

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    Madhur D Shastri

    Full Text Available T-cell-mediated inflammatory cytokines, such as interleukin (IL-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α, play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs, widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs of asthmatic subjects to identify the specific components responsible for the effects.PBMCs from asthmatic subjects (consist of ~75% of T-cells were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release.Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units, were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units, were associated with the stimulatory effect of dalteparin.Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two

  6. Shared signatures of social stress and aging in peripheral blood mononuclear cell gene expression profiles.

    Science.gov (United States)

    Snyder-Mackler, Noah; Somel, Mehmet; Tung, Jenny

    2014-10-01

    Chronic social stress is a predictor of both aging-related disease and mortality risk. Hence, chronic stress has been hypothesized to directly exacerbate the process of physiological aging. Here, we evaluated this hypothesis at the level of gene regulation. We compared two data sets of genome-wide gene expression levels in peripheral blood mononuclear cells (PBMCs): one that captured aging effects and another that focused on chronic social stress. Overall, we found that the direction, although not necessarily the magnitude, of significant gene expression changes tends to be shared between the two data sets. This overlap was observable at three levels: (i) individual genes; (ii) general functional categories of genes; and (iii) molecular pathways implicated in aging. However, we also found evidence that heterogeneity in PBMC composition limits the power to detect more extensive similarities, suggesting that our findings reflect an underestimate of the degree to which age and social stress influence gene regulation in parallel. Cell type-specific data on gene regulation will be important to overcome this limitation in the future studies.

  7. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

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    Katika, Madhumohan R. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Hendriksen, Peter J.M. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Shao, Jia [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Loveren, Henk van [Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Peijnenburg, Ad, E-mail: ad.peijnenburg@wur.nl [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands)

    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

  8. Co-culture of primary human tumor hepatocytes from patients with hepatocellular carcinoma with autologous peripheral blood mononuclear cells: study of their in vitro immunological interactions

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    Doumba Polyxeni P

    2013-01-01

    Full Text Available Abstract Background Many studies have suggested that the immune response may play a crucial role in the progression of hepatocellular carcinoma (HCC. Therefore, our aim was to establish a (i functional culture of primary human tumor hepatocytes and non-tumor from patients with hepatocellular carcinoma (HCC and (ii a co-culture system of HCC and non-HCC hepatocytes with autologous peripheral blood mononuclear cells (PBMCs in order to study in vitro cell-to-cell interactions. Methods Tumor (HCC and non-tumor (non-HCC hepatocytes were isolated from the liver resection specimens of 11 patients operated for HCC, while PBMCs were retrieved immediately prior to surgery. Four biopsies were obtained from patients with no liver disease who had surgery for non malignant tumor (normal hepatocytes. Hepatocytes were either cultured alone (monoculture or co-cultured with PBMCs. Flow cytometry measurements for MHC class II expression, apoptosis, necrosis and viability (7AAD were performed 24 h, 48 h and 72 h in co-culture and monocultures. Results HCC and non-HCC hepatocytes exhibited increased MHC-II expression at 48h and 72h in co-culture with PBMCs as compared to monoculture, with MHC II-expressing HCC hepatocytes showing increased viability at 72 h. PBMCs showed increased MHC-II expression (activation in co-culture with HCC as compared to non-HCC hepatocytes at all time points. Moreover, CD8+ T cells had significantly increased apoptosis and necrosis at 48h in co-culture with HCC hepatocytes as compared to monocultures. Interestingly, MHC-II expression on both HCC and non-HCC hepatocytes in co-culture was positively correlated with the respective activated CD8+ T cells. Conclusions We have established an in vitro co-culture model to study interactions between autologous PBMCs and primary HCC and non-HCC hepatocytes. This direct interaction leads to increased antigen presenting ability of HCC hepatocytes, activation of PBMCs with a concomitant apoptosis of

  9. No oxidative stress or DNA damage in peripheral blood mononuclear cells after exposure to particles from urban street air in overweight elderly

    DEFF Research Database (Denmark)

    Hemmingsen, Jette Gjerke; Jantzen, Kim; Møller, Peter

    2015-01-01

    Exposure to traffic-related particulate matter (PM) has been associated with increased risk of lung disease, cancer and cardiovascular disease especially in elderly and overweight subjects. The proposed mechanisms involve intracellular production of reactive oxygen species (ROS), inflammation...... particle-filtered or sham-filtered air from a busy street with number of concentrations and PM2.5 levels of 1800/cm(3) versus 23 000/cm(3) and 3 µg/m(3) versus 24 µg/m(3), respectively. Peripheral blood mononuclear cells (PBMCs) were collected and assayed for production of ROS with and without ex vivo...

  10. Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Maynard, Scott; Keijzers, Guido; Gram, Martin;

    2013-01-01

    Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality....... Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production...

  11. Effects of a healthy Nordic diet on gene expression changes in peripheral blood mononuclear cells in response to an oral glucose tolerance test in subjects with metabolic syndrome

    DEFF Research Database (Denmark)

    Leder, Lena; Kolehmainen, Marjukka; Narverud, Ingunn

    2016-01-01

    BACKGROUND: Diet has a great impact on the risk of developing features of metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), and cardiovascular diseases (CVD). We evaluated whether a long-term healthy Nordic diet (ND) can modify the expression of inflammation and lipid metabolism......-related genes in peripheral blood mononuclear cells (PBMCs) during a 2-h oral glucose tolerance test (OGTT) in individuals with MetS. METHODS: A Nordic multicenter randomized dietary study included subjects (n = 213) with MetS, randomized to a ND group or a control diet (CD) group applying an isocaloric study...

  12. Prion protein expression and processing in human mononuclear cells: the impact of the codon 129 prion gene polymorphism.

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    Christiane Segarra

    Full Text Available BACKGROUND: So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD, thought to result from the Bovine Spongiform Encephalopathy (BSE prion agent, have shown Methionine-Methionine (M/M homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs from three blood donor populations with Methionine-Methionine (M/M, Valine-Valine (V/V and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism. CONCLUSIONS/SIGNIFICANCE: These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.

  13. Pro-inflammatory action of MIF in acute myocardial infarction via activation of peripheral blood mononuclear cells.

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    David A White

    Full Text Available OBJECTIVES: Macrophage migration inhibitory factor (MIF, a pro-inflammatory cytokine, has been implicated in the pathogenesis of multiple inflammatory disorders. We determined changes in circulating MIF levels, explored the cellular source of MIF, and studied the role of MIF in mediating inflammatory responses following acute myocardial infarction (MI. METHODS AND RESULTS: We recruited 15 patients with MI, 10 patients with stable angina and 10 healthy volunteers and measured temporal changes of MIF in plasma. Expression of MIF, matrix metalloproteinase-9 (MMP-9 and interleukin-6 (IL-6 in cultured peripheral blood mononuclear cells (PBMCs and the media were measured by ELISA or real-time PCR. Compared to controls, plasma levels of MIF and IL-6 were significantly elevated at admission and 72 h post-MI. In contrast, expression of MIF, MMP-9 and IL-6 by PBMCs from MI patients was unchanged at admission, but significantly increased at 72 h. Addition of MIF activated cultured PBMCs by upregulating expression of inflammatory molecules and also synergistically enhanced stimulatory action of IL-1β which were inhibited by anti-MIF interventions. In a mouse MI model we observed similar changes in circulating MIF as seen in patients, with reciprocal significant increases in plasma MIF and reduction of MIF content in the infarct myocardium at 3 h after MI. MIF content in the infarct myocardium was restored at 72 h post-MI and was associated with robust macrophage infiltration. Further, anti-MIF intervention significantly reduced inflammatory cell infiltration and expression of monocyte chemoattractant protein-1 at 24 h and incidence of cardiac rupture in mice post-MI. CONCLUSION: MI leads to a rapid release of MIF from the myocardium into circulation. Subsequently MIF facilitates PBMC production of pro-inflammatory mediators and myocardial inflammatory infiltration. Attenuation of these events, and post-MI cardiac rupture, by anti-MIF interventions suggests

  14. Perturbations in the apoptotic pathway and mitochondrial network dynamics in peripheral blood mononuclear cells from bipolar disorder patients.

    Science.gov (United States)

    Scaini, G; Fries, G R; Valvassori, S S; Zeni, C P; Zunta-Soares, G; Berk, M; Soares, J C; Quevedo, J

    2017-05-02

    Bipolar disorder (BD) is a severe psychiatric disorder characterized by phasic changes of mood and can be associated with progressive structural brain change and cognitive decline. The numbers and sizes of glia and neurons are reduced in several brain areas, suggesting the involvement of apoptosis in the pathophysiology of BD. Because the changes in mitochondrial dynamics are closely related with the early process of apoptosis and the specific processes of apoptosis and mitochondrial dynamics in BD have not been fully elucidated, we measured the apoptotic pathway and the expression of mitochondrial fission/fusion proteins from BD patients and healthy controls. We recruited 16 patients with BD type I and sixteen well-matched healthy controls and investigated protein levels of several pro-apoptotic and anti-apoptotic factors, as well as the expression of mitochondrial fission/fusion proteins in peripheral blood mononuclear cells (PBMCs). Our results showed that the levels of the anti-apoptotic proteins Bcl-xL, survivin and Bcl-xL/Bak dimer were significantly decreased, while active caspase-3 protein levels were significantly increased in PBMCs from BD patients. Moreover, we observed the downregulation of the mitochondrial fusion-related proteins Mfn2 and Opa1 and the upregulation of the fission protein Fis1 in PBMCs from BD patients, both in terms of gene expression and protein levels. We also showed a significantly decrease in the citrate synthase activity. Finally, we found a positive correlation between Mfn2 and Opa1 with mitochondrial content markers, as well as a negative correlation between mitochondrial fission/fusion proteins and apoptotic markers. Overall, data reported here are consistent with the working hypothesis that apoptosis may contribute to cellular dysfunction, brain volume loss and progressive cognitive in BD. Moreover, we show an important relationship between mitochondrial dynamics and the cell death pathway activation in BD patients, supporting

  15. Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-07-22

    DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4 > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

  16. Anti-Sonic hedgehog blocking antibody enhances killing effect of PBMCs on cervical carcinoma HeLa cells%Sonic hedgehog阻断抗体增强外周血单个核细胞抗宫颈癌HeLa细胞的作用

    Institute of Scientific and Technical Information of China (English)

    孙艳; 黄莉霞; 黄文浩; 黄斌

    2011-01-01

    AIM: To investigate the effect of anti - Sonic hedgehog( Shh ) blocking antibody on the killing effect of peripheral blood mononuclear cells( PBMCs ) on cervical carcinoma HeLa cells. METHODS: The expression levels of Shh and Shh signaling molecules in HeLa cells were detected by immunocytochemistry and RT - PCR. PBMCs from health peoples were isolated by the method of Ficoll density gradient centrifugation, and then co - cultured with HeLa cells in vitro. The expression of CD3, CD69 and CD71 was assayed by flew cytometry. The concentrations of IFN - 7, IL - 10 and IL - 4 in culture supernatants were detected by ELISA. The killing effect of PBMCs on HeLa cells was observed under microscope. RESULTS: Shh and Shh signaling molecules were expressed in HeLa cells. The level of Shh expression didn t change significantly in the 6th passage of HeLa cells. CD3 + cells were increased in the co - culture system. The expression of CD69 and CD 71, and the secretion of IFN - 7 were increased, while the secretion of IL - 10 was decreased in the co - culture system treated with anti - Shh blocking antibody. Anti - Shh blocking antibody has no effect on the secretion of IL -4. The killing effect of PBMCs on HeLa cells was strengthened by anti - Shh blocking antibody. CONCLUSION : Anti - Shh blocking antibody promotes the activation of PBMCs and enhances the killing effect of PBMCs on cervical carcinoma HeLa cells.%目的:探讨Sonic hedgehog(Shh)阻断抗体对外周血单个核细胞(PBMCs)抗宫颈癌HeLa细胞作用的影响.方法:免疫细胞化学技术和RT-PCR法检测Shh及其信号分子在HeLa 细胞中的表达;Ficoll密度梯度离心法分离正常人PBMCs,并与HeLa细胞建立共培养体系;于共培养体系中加入Shh阻断抗体,流式细胞术检测CD3、CD69和CD71分子表达;ELISA法检测细胞因子IFN-γ、IL-10和IL-4分泌;显微镜观察PBMCs 对HeLa细胞的杀伤.结果:HeLa细胞表达Shh及其信号分子,HeLa细胞传代6次对Shh的表达水

  17. Gene expression profiles in peripheral blood mononuclear cells of SARS patients

    Institute of Scientific and Technical Information of China (English)

    Shi-Yan Yu; Yun-Wen Hu; Xiao-Ying Liu; Wei Xiong; Zhi-Tong Zhou; Zheng-Hong Yuan

    2005-01-01

    AIM: To investigate the role of inflammatory and anti-viral genes in the pathogenesis of SARS.METHODS: cDNA microarrays were used to screen the gene expression profiles of peripheral blood mononuclear cells (PBMCs) in two SARS patients (one in the acute severe phase and the other in the convalescent phase)and a healthy donor. In addition, real-time qualitative PCR was also performed to verify the reproducibility of the microarray results. The data were further analyzed.RESULTS: Many inflammatory and anti-viral genes were differentially expressed in SARS patients. Compared to the healthy control or the convalescent case, plenty of pro-inflammatory cytokines such as IL-1, TNF-α, IL-8, and MAPK signaling pathway were significantly upregulated in the acute severe case. However, anti-inflammatory agents such as IL-4 receptor, IL-13 receptor, IL-1Ra,and TNF-α-induced proteins 3 and 6 also increased dramatically in the acute severe case. On the contrary, a lot of IFN-stimulated genes like PKR, GBP-1 and 2, CXCL-10and 11, and JAK/STAT signal pathway were downregulated in the acute severe case compared to the convalescent case.CONCLUSION: Gene expression in SARS patients mirrors a host state of inflammation and anti-viral immunity at the transcription level, and understanding of gene expression profiles may make contribution to further studies of the SARS pathogenesis.

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

    Science.gov (United States)

    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|, p93% 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 individuals at risk of developing cardiomyopathy. PMID:26919708

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

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    Nisha Jain Garg

    2016-02-01

    Full Text Available 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|, p93% 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 individuals at risk of developing cardiomyopathy.

  20. Methotrexate-related response on human peripheral blood mononuclear cells may be modulated by the Ala16Val-SOD2 gene polymorphism.

    Science.gov (United States)

    Barbisan, Fernanda; Motta, Jéssica de Rosso; Trott, Alexis; Azzolin, Verônica; Dornelles, Eduardo Bortoluzzi; Marcon, Matheus; Algarve, Thaís Doeler; Duarte, Marta Maria Medeiros Frescura; Mostardeiro, Clarice Pinheiro; Unfer, Taís Cristina; Schott, Karen Lilian; da Cruz, Ivana Beatrice Mânica

    2014-01-01

    Methotrexate (MTX) is a folic acid antagonist used in high doses as an anti-cancer treatment and in low doses for the treatment of some autoimmune diseases. MTX use has been linked to oxidative imbalance, which may cause multi-organ toxicities that can be attenuated by antioxidant supplementation. Despite the oxidative effect of MTX, the influence of antioxidant gene polymorphisms on MTX toxicity is not well studied. Therefore, we analyzed here whether a genetic imbalance of the manganese-dependent superoxide dismutase (SOD2) gene could have some impact on the MTX cytotoxic response. An in vitro study using human peripheral blood mononuclear cells (PBMCs) obtained from carriers with different Ala16Val-SOD2 genotypes (AA, VV and AV) was carried out, and the effect on cell viability and proliferation was analyzed, as well as the effect on oxidative, inflammatory and apoptotic markers. AA-PBMCs that present higher SOD2 efficiencies were more resistance to high MTX doses (10 and 100 µM) than were the VV and AV genotypes. Both lipoperoxidation and ROS levels increased significantly in PBMCs exposed to MTX independent of Ala16Val-SOD2 genotypes, whereas increased protein carbonylation was observed only in PBMCs from V allele carriers. The AA-PBMCs exposed to MTX showed decreasing SOD2 activity, but a concomitant up regulation of the SOD2 gene was observed. A significant increase in glutathione peroxidase (GPX) levels was observed in all PBMCs exposed to MTX. However, this effect was more intense in AA-PBMCs. Caspase-8 and -3 levels were increased in cells exposed to MTX, but the modulation of these genes, as well as that of the Bax and Bcl-2 genes involved in the apoptosis pathway, presented a modulation that was dependent on the SOD2 genotype. MTX at a concentration of 10 µM also increased inflammatory cytokines (IL-1β, IL-6, TNFα and Igγ) and decreased the level of IL-10 anti-inflammatory cytokine, independent of SOD2 genetic background. The results suggest that

  1. Methotrexate-related response on human peripheral blood mononuclear cells may be modulated by the Ala16Val-SOD2 gene polymorphism.

    Directory of Open Access Journals (Sweden)

    Fernanda Barbisan

    Full Text Available Methotrexate (MTX is a folic acid antagonist used in high doses as an anti-cancer treatment and in low doses for the treatment of some autoimmune diseases. MTX use has been linked to oxidative imbalance, which may cause multi-organ toxicities that can be attenuated by antioxidant supplementation. Despite the oxidative effect of MTX, the influence of antioxidant gene polymorphisms on MTX toxicity is not well studied. Therefore, we analyzed here whether a genetic imbalance of the manganese-dependent superoxide dismutase (SOD2 gene could have some impact on the MTX cytotoxic response. An in vitro study using human peripheral blood mononuclear cells (PBMCs obtained from carriers with different Ala16Val-SOD2 genotypes (AA, VV and AV was carried out, and the effect on cell viability and proliferation was analyzed, as well as the effect on oxidative, inflammatory and apoptotic markers. AA-PBMCs that present higher SOD2 efficiencies were more resistance to high MTX doses (10 and 100 µM than were the VV and AV genotypes. Both lipoperoxidation and ROS levels increased significantly in PBMCs exposed to MTX independent of Ala16Val-SOD2 genotypes, whereas increased protein carbonylation was observed only in PBMCs from V allele carriers. The AA-PBMCs exposed to MTX showed decreasing SOD2 activity, but a concomitant up regulation of the SOD2 gene was observed. A significant increase in glutathione peroxidase (GPX levels was observed in all PBMCs exposed to MTX. However, this effect was more intense in AA-PBMCs. Caspase-8 and -3 levels were increased in cells exposed to MTX, but the modulation of these genes, as well as that of the Bax and Bcl-2 genes involved in the apoptosis pathway, presented a modulation that was dependent on the SOD2 genotype. MTX at a concentration of 10 µM also increased inflammatory cytokines (IL-1β, IL-6, TNFα and Igγ and decreased the level of IL-10 anti-inflammatory cytokine, independent of SOD2 genetic background. The results

  2. Effect of 900 MHz Electromagnetic Radiation on the Induction of ROS in Human Peripheral Blood Mononuclear Cells

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    Mortazavi S.M.J.

    2015-09-01

    Full Text Available 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.

  3. Increased Calpain Correlates with Th1 Cytokine Profile in PBMCs from MS Patients

    Science.gov (United States)

    Imam, Sarah A.; Guyton, Mary K.; Haque, Azizul; Vandenbark, Arthur; Tyor, William R.; Ray, Swapan K.; Banik, Naren L.

    2007-01-01

    Multiple sclerosis (MS) is a devastating autoimmune demyelinating disease of the CNS. This study investigated whether expression and activity of the calcium-activated protease calpain correlated with Th1/Th2 dysregulation in MS patients during states of relapse and remission. Calpain expression and activity were significantly increased in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to controls, with the highest expression and activity noted during relapse. Th1 cytokines were highest and Th2 cytokines were lowest in MS patients during relapse. Treatment with calpain inhibitor, calpeptin, decreased Th1 cytokines in PBMCs from MS patients. Calpain inhibitor also reduced degradation of myelin basic protein (MBP) by inhibiting the calpain secreted from MBP-specific T cells. Taken together, these results suggested calpain involvement in Th1/Th2 dysregulation in MS patients. PMID:17765980

  4. Medroxyprogesterone acetate alters Mycobacterium bovis BCG-induced cytokine production in peripheral blood mononuclear cells of contraceptive users.

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    Léanie Kleynhans

    Full Text Available Most individuals latently infected with Mycobacterium tuberculosis (M.tb contain the infection by a balance of effector and regulatory immune responses. This balance can be influenced by steroid hormones such as glucocorticoids. The widely used contraceptive medroxyprogesterone acetate (MPA possesses glucocorticoid activity. We investigated the effect of this hormone on immune responses to BCG in household contacts of active TB patients. Multiplex bead array analysis revealed that MPA demonstrated both glucocorticoid and progestogenic properties at saturating and pharmacological concentrations in peripheral blood mononuclear cells (PBMCs and suppressed antigen specific cytokine production. Furthermore we showed that PBMCs from women using MPA produced significantly lower levels of IL-1α, IL-12p40, IL-10, IL-13 and G-CSF in response to BCG which corresponded with lower numbers of circulating monocytes observed in these women. Our research study is the first to show that MPA impacts on infections outside the genital tract due to a systemic effect on immune function. Therefore MPA use could alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult or adolescent women in the future.

  5. Adenosine A2A Receptor and IL-10 in Peripheral Blood Mononuclear Cells of Patients with Mild Cognitive Impairment

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    Beatrice Arosio

    2011-01-01

    Full Text Available Adenosine suppresses immune responses through the A2A receptor (A2AR. This study investigated the interleukin 10 (IL-10 genetic profile and the expression of A2AR in peripheral blood mononuclear cells (PBMCs of patients with mild cognitive impairment (MCI, Alzheimer disease (AD, and age-matched controls to verify, if they may help distinguish different forms of cognitive decline. We analyzed the IL-10 genotype and the expression of A2AR in 41 subjects with AD, 10 with amnestic MCI (a-MCI, 49 with multiple cognitive domain MCI (mcd-MCI, and 46 controls. There was a significant linear increase in A2AR mRNA levels and A2AR density from mcd-MCI to a-MCI, with intermediate levels being found in AD. The IL-10 AA genotype frequency was 67% in a-MCI, 46% in AD, 35% in mcd-MCI, and 20% in controls. These data suggest that the assessment of the IL-10 genotype and the expression of A2AR in PBMCs may be a valuable means of differentiating between a-MCI and mcd-MCI.

  6. Gene expression profiling in human peripheral blood mononuclear cells using high-density filter-based cDNA microarrays.

    Science.gov (United States)

    Walker, J; Rigley, K

    2000-05-26

    Microarray technology has provided the ability to analyse the expression profiles for thousands of genes in parallel. The need for highly specialised equipment to use certain types of microarrays has restricted the application of this technology to a small number of dedicated laboratories. High-density filter-based cDNA microarrays provide a low-cost option for performing high-throughput gene expression analysis. We have used a model system in which filter-based cDNA microarrays representing over 4000 known human genes were used to monitor the kinetics of gene expression in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemagluttinin (PHA). Using software-based cluster analysis, we identified 104 genes that altered in expression levels in response to PHA stimulation of PBMCs and showed that there was a considerable overlap between genes with similar temporal expression profiles and similar functional roles. Comparison of microarray quantitation with quantitative PCR showed almost identical expression profiles for a number of genes. Coupled with the fact that our findings are in agreement with a large number of independent observations, we conclude that the use of filter-based cDNA microarrays is a valid and accurate method for high-throughput gene expression profiling.

  7. Relationship between leptin concentration and body fat with peripheral blood mononuclear cells cytokines among obese and overweight adults.

    Science.gov (United States)

    Zarrati, M; Salehi, E; Razmpoosh, E; Shoormasti, R S; Hosseinzadeh-Attar, M J; Shidfar, F

    2017-02-01

    Overweight and obesity has been suggested to be well correlated with altered levels of pro-inflammatory cytokines. The purpose of this study is to assess the relationship of body fat mass (BFM), body fat percentage (BFP) and leptin levels with peripheral blood mononuclear cells (PBMCs) cytokines among obese and overweight adults. Eighty-two overweight and obese individuals were divided into two BMI-category groups (BMI leptin and anthropometric indices were measured and PBMCs were cultured. Mean of leptin concentrations were 23.14 ± 4.07 and 28.25 ± 4.35 pg/ml among individuals with BMI leptin (P leptin (P Leptin had a positive significant correlation with IFN-γ (p = 0.03, r = 0.251). Regarding these results, we proved that BFP, BFM and leptin levels have significant correlations with some PBMC cytokines. Focusing on such strategies may lead to promises for alleviating obesity and its co-morbidities.

  8. Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry

    Science.gov (United States)

    Kip, A.E.; Rosing, H.; Hillebrand, M.J.X.; Castro, M.M.; Gomez, M.A.; Schellens, J.H.M.; Beijnen, J.H.; Dorlo, T.P.C.

    2015-01-01

    Phagocytes, the physiological compartment in which Leishmania parasites reside, are the main site of action of the drug miltefosine, but the intracellular pharmacokinetics of miltefosine remain unexplored. We developed a bioanalytical method to quantify miltefosine in human peripheral blood mononuclear cells (PBMCs), expanding from an existing high performance liquid chromatography-tandem mass spectrometry method for the quantification of miltefosine in plasma. The method introduced deuterated miltefosine as an internal standard. Miltefosine was extracted from PBMC pellets by addition of 62.5% methanol. Supernatant was collected, evaporated and reconstituted in plasma. Chromatographic separation was performed on a reversed phase C18 column and detection with a triple-quadrupole mass spectrometer. Miltefosine was quantified using plasma calibration standards ranging from 4 to 1000 ng/mL. This method was validated with respect to its PBMC matrix effect, selectivity, recovery and stability. No matrix effect could be observed from the PBMC content (ranging from 0.17 to 26.3 × 106 PBMCs) reconstituted in plasma, as quality control samples were within 3.0% of the nominal concentration (precision less than 7.7%). At the lower limit of quantitation of 4 ng/mL plasma, corresponding to 0.12 ng/106 PBMCs in a typical clinical sample, measured concentrations were within 8.6% of the nominal value. Recovery showed to be reproducible as adding additional pre-treatment steps did not increase the recovery with more than 9%. This method was successfully applied to measure intracellular miltefosine concentrations in PBMC samples from six cutaneous leishmaniasis patients up to one month post-treatment. PMID:26160472

  9. CRP and TNF-α  Induce PAPP-A Expression in Human Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Weiping Li

    2012-01-01

    Full Text Available Objective. The effects of C-reactive protein (CRP and tumor necrosis factor-α (TNF-α on pregnancy-associated plasma protein-A (PAPP-A expression in human peripheral blood mononuclear cells (PBMCs require further investigation. Methods. The PAPP-A levels in culture supernatants, PAPP-A mRNA expression, and cellular PAPP-A expression were measured in human PBMCs isolated from fresh blood donations provided by 6 healthy volunteers (4 donations per volunteer. Analyses were conducted by ultrasensitive ELISA, western blotting, and RT-PCR following stimulation with CRP or TNF-α cytokines. Results. PAPP-A mRNA and protein levels after CRP stimulation peaked at 24 hours, whereas peak PAPP-A mRNA and protein levels were achieved after TNF-α stimulation at only 2 and 8 hours, respectively. These findings indicate the dose-dependent effect of CRP and TNF-α stimulation. Actinomycin D treatment completely prevented CRP and TNF-α induction of PAPP-A mRNA and protein expression. Additionally, nuclear factor- (NF- κB inhibitor (BAY11-7082 potently inhibited both CRP and TNF-α stimulated PAPP-A mRNA and protein expression. Conclusions. Human PBMCs are capable of expressing PAPP-A in vitro, expression that may be regulated by CRP and TNF-α through the NF-κB pathway. This mechanism may play a significant role in the observed increase of serum PAPP-A levels in acute coronary syndrome (ACS.

  10. Standardized Serum-Free Cryomedia Maintain Peripheral Blood Mononuclear Cell Viability, Recovery, and Antigen-Specific T-Cell Response Compared to Fetal Calf Serum-Based Medium

    Science.gov (United States)

    Schulz, Julia C.; Kemp-Kamke, Beatrice; Zimmermann, Heiko; von Briesen, Hagen

    2011-01-01

    The ability to analyze cryopreserved peripheral blood mononuclear cells (PBMCs) from biobanks for antigen-specific T-cell immunity is necessary to evaluate responses to immune-based therapies. Comprehensive studies have demonstrated that the quality of frozen PBMCs is critical and the maintenance of cell viability and functionality by using appropriate cryopreservation techniques is a key to the successful outcome of assays using PBMCs. Different cryomedia additives affect cell viability. The most common additive is fetal calf serum (FCS), although it is widely known that each FCS lot has to be tested before usage to prevent nonspecific stimulation of T-cells. Also, shipping of samples containing FCS is critical because of many import restrictions. Often, dimethyl sulfoxide (DMSO) is added as a cryoprotectant. However, DMSO concentration has to be reduced significantly because of its toxic effect on cells at room temperature. Therefore, we have developed freezing approaches to minimize cytotoxicity of cryoprotectants and maintain T-cell functionality. We compared different additives to the widely used FCS and found bovine serum albumin fraction V to be an appropriate substitute for the potentially immune-modulating FCS. We also found that DMSO concentration can be reduced by the addition of hydroxyethyl starch. Using our serum-free cryomedia, the PBMC recovery was more than 83% and the PBMC viability was more than 98%. Also, the T-cell functionality measured by enzyme-linked immunospot (ELISpot) was optimal after cryopreservation with our new cryomedia. On the basis of our experimental results, we could finally design 2 different, fully working cryomedia that are standardized, serum free, and manufactured under GMP conditions. PMID:21977240

  11. Expression of VSTM1-v2 Is Increased in Peripheral Blood Mononuclear Cells from Patients with Rheumatoid Arthritis and Is Correlated with Disease Activity.

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    Dashan Wang

    Full Text Available Rheumatoid arthritis (RA is a chronic, systematic autoimmune disease that mainly affects joints and bones. Although the precise etiology is still unknown, Th17 cell is being recognized as an important mediator in pathogenesis of RA. VSTM1-v2 is a novel cytokine which has recently been reported to promote the differentiation of Th17 cells. This study is performed to study whether VSTM1-v2 can be recognized as a biomarker of RA, and is correlated to IL-17 expression. We obtained peripheral blood mononuclear cells (PBMCs from 40 patients with RA and 40 age- and sex-matched healthy controls by standard Ficoll-Paque Plus density centrifugation. The mRNA expression levels of VSTM1-v2 and IL-17A in PBMCs were detected by real time-PCR. Disease activity parameters of RA were measured by routine methods. Our results showed that VSTM1-v2 mRNA expression in PBMCs from RA patients was significantly increased in comparison of that in healthy individuals. The VSTM1-v2 mRNA expression level was positively correlated with IL-17A mRNA expression level, DAS28, CRP and ESR, but was not correlated to RF, Anti-CCP or ANA. VSTM1-v2 might be a biomarker of RA and a novel factor in the pathogenesis of RA.

  12. Recombinant human T-cell leukemia virus types 1 and 2 Tax proteins induce high levels of CC-chemokines and downregulate CCR5 in human peripheral blood mononuclear cells.

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    Barrios, Christy S; Abuerreish, Muna; Lairmore, Michael D; Castillo, Laura; Giam, Chou-Zen; Beilke, Mark A

    2011-12-01

    Human T-cell leukemia viruses types 1 (HTLV-1) and 2 (HTLV-2) produce key transcriptional regulatory gene products, known as Tax1 and Tax2, respectively. Tax1 and Tax2 transactivate multiple host genes involved in cellular immune responses within the cellular microenvironment, including induction of genes encoding expression of CC-chemokines. It is speculated that HTLV Tax proteins may act as immune modulators. In this study, recombinant Tax1 and Tax2 proteins were tested for their effects on the viability of cultured peripheral blood mononuclear cells (PBMCs), and their ability to induce expression of CC-chemokines and to downregulate the level of CCR5 expression in PBMCs. PBMCs obtained from uninfected donors were cultured in a range of Tax1 and Tax2 concentrations (10-100 pM), and supernatant fluids were harvested at multiple time points for quantitative determinations of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5. Treatment of PBMCs with Tax1 and Tax2 proteins (100 pM) resulted in a significant increase in viability over a 7-d period compared to controls (pCCR5-positive cells compared to those of uninfected donors and from mock-treated lymphocytes, respectively (p<0.05). These results suggest that Tax1 and Tax2 could promote innate immunity in the extracellular environment during HTLV-1 and HTLV-2 infections via CC-chemokine ligands and receptors.

  13. Evaluation of the effect of 2,4-dichlorophenol on oxidative parameters and viability of human blood mononuclear cells (in vitro).

    Science.gov (United States)

    Bukowska, B; Wieteska, P; Kwiatkowska, M; Sicińska, P; Michalowicz, J

    2016-07-01

    2,4-Dichlorophenol (2,4-DCP) is formed in drinking water as a result of its chlorination, and it is created in the environment during transformation of various xenobiotics such as triclosan or herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The molecular mechanism depicting the action of phenolic compounds on nucleated blood cells has been insufficiently studied, and therefore, we have assessed the effect of 2,4-DCP on the structure and viability of human peripheral blood mononuclear cells (PBMCs). We have evaluated necrotic, apoptotic, and morphological changes (alterations in the size and granulation) in PBMCs incubated with 2,4-DCP in the concentration ranging from 10 to 500 µg mL(-1) for 4 h at 37°C. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, lipid peroxidation, and protein carbonylation in the incubated cells. We have noted that 2,4-DCP increased ROS formation and lipid peroxidation (from 10 µg mL(-1)) and oxidized proteins (from 50 µg mL(-1)) in PBMCs. The compound studied also provoked apoptotic (from 50 µg mL(-1)), necrotic (from 100 µg mL(-1)) and alterations in the size and granulation (from 50 µg mL(-1)) in the incubated cells. The analysis of quinolinium 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethyl-ammonio)-propyl]-diiodide/propidium iodide staining revealed that 2,4-DCP (50-250 µg mL(-1)) more strongly increased the number of apoptotic than necrotic cells, which suggests that this cell death type is mainly provoked by this compound in PBMCs. The observed changes were caused by relatively high concentrations of 2,4-DCP, which cannot influence human organism during environmental exposure and thus may only occur as a result of acute or subacute poisoning with this compound.

  14. The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes

    Science.gov (United States)

    Jonitz-Heincke, Anika; Tillmann, Jenny; Klinder, Annett; Krueger, Simone; Kretzer, Jan Philippe; Høl, Paul Johan; Paulus, Alexander C.; Bader, Rainer

    2017-01-01

    Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs). Thus, we exposed cells with a mixture of 200 µg/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-α, MCP-1, MMP1, TIMP1) were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions. PMID:28773099

  15. Characteristics of spontaneously proliferating mononuclear cells in rheumatoid arthritis.

    Science.gov (United States)

    Froebel, K; Dickson, R; Lewis, D; Jasani, M K; Sturrock, R D

    1984-10-01

    The phenomenon of increased spontaneous incorporation of 3H-thymidine (3H-TdR) into peripheral blood mononuclear cells in rheumatoid arthritis (RA) has been investigated. The activity was found to be short lived and affected less than 1% of cells. Using a Percoll density gradient we identified two populations of active cells. RA patients with active synovitis and increased 3H-TdR incorporation in the low density population of cells have higher overall 3H-TdR incorporation than normal controls and patients with inactive RA. The low density cell population is enriched for Ia+ cells. The data are consistent with raised spontaneous 3H-TdR incorporation being due to an in-vivo cell mediated immune response.

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

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    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.

  17. A novel and simple method for generation of human dendritic cells from unfractionated peripheral blood mononuclear cells within 2 days: its application for induction of HIV-1-reactive CD4(+) T cells in the hu-PBL SCID mice.

    Science.gov (United States)

    Kodama, Akira; Tanaka, Reiko; Saito, Mineki; Ansari, Aftab A; Tanaka, Yuetsu

    2013-01-01

    Because dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. Generally, monocyte-derived DCs (MDDCs) were generated from purified monocytes by multiple steps of time-consuming physical manipulations for an extended period cultivation. In this study, we developed a novel, simple and rapid method for the generation of type-1 helper T cell (Th1)-stimulating human DCs directly from bulk peripheral blood mononuclear cells (PBMCs). PBMCs were cultivated in the presence of 20 ng/ml of granulocyte-macrophage colony-stimulating factor, 20 ng/ml of interleukin-4 (IL-4) and 1,000 U/ml of interferon-β for 24 h followed by 24 h maturation with a cytokine cocktail containing 10 ng/ml of tumor necrosis factor-α (TNF-α), 10 ng/ml of IL-1β and 1 μg/ml of prostaglandin E2. The phenotype and biological activity of these new DCs for induction of allogeneic T cell proliferation and cytokine production were comparable to those of the MDDCs. Importantly, these new DCs pulsed with inactivated HIV-1 could generated HIV-1-reactive CD4(+) T cell responses in humanized mice reconstituted with autologous PBMCs from HIV-1-negative donors. This simple and quick method for generation of functional DCs will be useful for future studies on DC-mediated immunotherapies.

  18. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells.

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    Bakkenist, Christopher J; Czambel, R Kenneth; Hershberger, Pamela A; Tawbi, Hussein; Beumer, Jan H; Schmitz, John C

    2015-01-01

    Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

  19. A flow cytometry technique to study intracellular signals NF-κB and STAT3 in peripheral blood mononuclear cells

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    Chavarin Patricia

    2007-07-01

    Full Text Available Abstract Background Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-κB (nuclear factor kappaB and STAT (signal transducer and activator of transcription families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-κB and STAT rely on specific ELISAs, Western Blots and – most recently described – flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. Results The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells. To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1β, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA; the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-κB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. Conclusion In response to IL1β, or IL10, the levels of phosphorylated NF-κB and STAT3 – respectively – increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages – but, interestingly, not T-lymphocytes (in the context of PBMCs – responded significantly to sCD40L by increasing phosphorylated NF-κB.

  20. Fetal hemoglobin accumulation in vitro. Effect of adherent mononuclear cells.

    OpenAIRE

    Javid, J; Pettis, P K

    1983-01-01

    In clonal cultures of erythroid burst-forming units (BFU-E) obtained from blood, the accumulation of fetal and adult hemoglobins (Hb F and Hb A) was measured by radioligand immunoassay. Inclusion of adherent mononuclear cells in the culture promoted a striking increase in the relative amount of Hb F in each of 44 experiments with 14 donors. In two-thirds of the instances, this was accounted for by a selective increase in the absolute amount of Hb F. The differential effect on Hb F and Hb A ac...

  1. Erythroid-specific transcriptional changes in PBMCs from pulmonary hypertension patients.

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    Chris Cheadle

    Full Text Available BACKGROUND: Gene expression profiling of peripheral blood mononuclear cells (PBMCs is a powerful tool for the identification of surrogate markers involved in disease processes. The hypothesis tested in this study was that chronic exposure of PBMCs to a hypertensive environment in remodeled pulmonary vessels would be reflected by specific transcriptional changes in these cells. METHODOLOGY/PRINCIPAL FINDINGS: The transcript profiles of PBMCs from 30 idiopathic pulmonary arterial hypertension patients (IPAH, 19 patients with systemic sclerosis without pulmonary hypertension (SSc, 42 scleroderma-associated pulmonary arterial hypertensio patients (SSc-PAH, and 8 patients with SSc complicated by interstitial lung disease and pulmonary hypertension (SSc-PH-ILD were compared to the gene expression profiles of PBMCs from 41 healthy individuals. Multiple gene expression signatures were identified which could distinguish various disease groups from controls. One of these signatures, specific for erythrocyte maturation, is enriched specifically in patients with PH. This association was validated in multiple published datasets. The erythropoiesis signature was strongly correlated with hemodynamic measures of increasing disease severity in IPAH patients. No significant correlation of the same type was noted for SSc-PAH patients, this despite a clear signature enrichment within this group overall. These findings suggest an association of the erythropoiesis signature in PBMCs from patients with PH with a variable presentation among different subtypes of disease. CONCLUSIONS/SIGNIFICANCE: In PH, the expansion of immature red blood cell precursors may constitute a response to the increasingly hypoxic conditions prevalent in this syndrome. A correlation of this erythrocyte signature with more severe hypertension cases may provide an important biomarker of disease progression.

  2. Down-regulation of miR-181a can reduce heat stress damage in PBMCs of Holstein cows.

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    Chen, Kun-Lin; Fu, Yuan-Yuan; Shi, Min-Yan; Li, Hui-Xia

    2016-09-01

    Heat stress can weaken the immune system and even increase livestock's susceptibility to disease. MicroRNA (miR) is short non-coding RNA that functions in post-transcriptional regulation of gene expression and some phenotypes. Our recent study found that miR-181a is highly expressed in the serum of heat-stressed Holstein cows, but the potential function of miR-181a is still not clarified. In this study, peripheral blood mononuclear cells (PBMCs), isolated from Holstein cows' peripheral blood, were used to investigate the effects of miR-181a inhibitor on heat stress damage. Our results showed that significant apoptosis and oxidative damage were induced by heat stress in PBMCs. However, with apoptosis, the levels of reactive oxygen species (ROS) and content of malondialdehyde (MDA) were reduced, while the content of glutathione (GSH) and the activity of superoxide dismutase (SOD) were increased even under heat stress conditions after transfecting miR-181a inhibitors to PBMCs. Meanwhile, mRNA expression of bax and caspase-3 was significantly decreased, but mRNA expression of bcl-2 was increased in transfected PBMCs. In conclusion, our results demonstrated that down-regulation of miR-181a can reduce heat stress damage in PBMCs of Holstein cows.

  3. High concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of human peripheral blood mononuclear cells via mTORC1 activation.

    Science.gov (United States)

    Zhenyukh, Olha; Civantos, Esther; Ruiz-Ortega, Marta; Sánchez, Maria Soledad; Vázquez, Clotilde; Peiró, Concepción; Egido, Jesús; Mas, Sebastián

    2017-03-01

    Leucine, isoleucine and valine are essential aminoacids termed branched-chain amino acids (BCAA) due to its aliphatic side-chain. In several pathological and physiological conditions increased BCAA plasma concentrations have been described. Elevated BCAA levels predict insulin resistance development. Moreover, BCAA levels higher than 2mmol/L are neurotoxic by inducing microglial activation in maple syrup urine disease. However, there are no studies about the direct effects of BCAA in circulating cells. We have explored whether BCAA could promote oxidative stress and pro-inflammatory status in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. In cultured PBMCs, 10mmol/L BCAA increased the production of reactive oxygen species (ROS) via both NADPH oxidase and the mitochondria, and activated Akt-mTOR signalling. By using several inhibitors and activators of these molecular pathways we have described that mTOR activation by BCAA is linked to ROS production and mitochondrial dysfunction. BCAA stimulated the activation of the redox-sensitive transcription factor NF-κB, which resulted in the release of pro-inflammatory molecules, such as interleukin-6, tumor necrosis factor-α, intracellular adhesion molecule-1 or CD40L, and the migration of PBMCs. In conclusion, elevated BCAA blood levels can promote the activation of circulating PBMCs, by a mechanism that involving ROS production and NF-κB pathway activation. These data suggest that high concentrations of BCAA could exert deleterious effects on circulating blood cells and therefore contribute to the pro-inflammatory and oxidative status observed in several pathophysiological conditions.

  4. Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells.

    Science.gov (United States)

    Maynard, Scott; Keijzers, Guido; Gram, Martin; Desler, Claus; Bendix, Laila; Budtz-Jørgensen, Esben; Molbo, Drude; Croteau, Deborah L; Osler, Merete; Stevnsner, Tinna; Rasmussen, Lene Juel; Dela, Flemming; Avlund, Kirsten; Bohr, Vilhelm A

    2013-11-01

    Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality. Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters. However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects of mitochondrial oxidative phosphorylation capacity in PBMCs, but may have other underlying cellular dysfunctions that contribute to dNTP imbalance and altered ROS production.

  5. Aerobic training increases the expression of adiponectin receptor genes in the peripheral blood mononuclear cells of young men

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    SH Lee

    2015-09-01

    Full Text Available Little is known about the effect of exercise training on the expression of adiponectin receptor genes in peripheral blood mononuclear cells (PBMCs. In this study, we investigated the effects of aerobic training on the expression of AdipoR1 and AidpoR2 mRNAs in PBMCs, whole body insulin sensitivity, and circulating adiponectins in men. Thirty young men were randomly assigned to either a control (n=15 or an exercise (n=15 group. Subjects assigned to the exercise group underwent a 12-week jogging and/or running programme on a motor-driven treadmill at an intensity of 60%-75% of the age-based maximum heart rate with duration of 40 minutes per session and a frequency of 5 days per week. Two-way mixed ANOVA with repeated measures was used to test any significant time-by-group interaction effects for the measured variables at p=0.05. We found significant time-by-group interaction effects for waist circumference (p=0.001, VO2 max (p<0.001, fasting insulin (p=0.016, homeostasis model assessment for insulin resistance (HOMA-IR (p=0.010, area under the curve (AUC for insulin response during the 75-g oral glucose tolerance test (p=0.002, high-molecular weight (HMW adiponectin (p=0.016, and the PBMC mRNA levels of AdipoR1 (p<0.001 and AdipoR2 (p=0.001. The exercise group had significantly increased mRNA levels of AdipoR1 and AdipoR2 in PBMCs, along with increased whole body insulin sensitivity and HMW adiponectin, decreased waist circumference, and increased VO2 max compared with the control group. In summary, the current findings suggest that exercise training modulates the expression of AdipoR1 and AdipoR2 mRNAs in PBMCs, implying that manipulation of the expression of these genes could be a potential surrogate for lifestyle intervention-mediated improvements of whole body insulin sensitivity and glucose homeostasis.

  6. Members of the Candida parapsilosis Complex and Candida albicans are Differentially Recognized by Human Peripheral Blood Mononuclear Cells.

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    Estrada-Mata, Eine; Navarro-Arias, María J; Pérez-García, Luis A; Mellado-Mojica, Erika; López, Mercedes G; Csonka, Katalin; Gacser, Attila; Mora-Montes, Héctor M

    2015-01-01

    The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high morbility and mortality rates, and are considered as relevant as those caused by Candida albicans. Since the fungal cell wall is the first point of contact with the host cells, here we performed a comparison of this organelle in members of the C. parapsilosis complex, and its relevance during interaction with human peripheral blood mononuclear cells (PBMCs). We found that the wall of the C. parapsilosis complex members is similar in composition, but differs to that from C. albicans, with less mannan content and more β-glucan and porosity levels. Furthermore, lectin-based analysis showed increased chitin and β1,3-glucan exposure at the surface of C. parapsilosis sensu lato when compared to C. albicans. Yeast cells of members of the C. parapsilosis complex stimulated more cytokine production by human PBMCs than C. albicans cells; and this significantly changed upon removal of O-linked mannans, indicating this wall component plays a significant role in cytokine stimulation by C. parapsilosis sensu lato. When inner wall components were exposed on the wall surface, C. parapsilosis sensu stricto and C. metapsilosis, but not C. orthopsilosis, stimulated higher cytokine production. Moreover, we found a strong dependency on β1,3-glucan recognition for the members of the C. parapsilosis complex, but not for live C. albicans cells; whereas TLR4 was required for TNFα production by the three members of the complex, and stimulation of IL-6 by C. orthopsilosis. Mannose receptor had a significant role during TNFα and IL-1β stimulation by members of the complex. Finally, we demonstrated that purified N- and O-mannans from either C. parapsilosis sensu lato or C. albicans are capable to block the recognition of these pathogens by human PBMCs. Together; our results suggest that the innate immune recognition of the members of the C. parapsilosis complex is differential

  7. Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells.

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    Giovanni Nardo

    Full Text Available BACKGROUND: Amyotrophic lateral sclerosis (ALS is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments. METHODOLOGY/PRINCIPAL FINDINGS: We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC, a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%, and from patients with neurological disorders that may resemble ALS (91%, between two levels of disease severity (90%, and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing. CONCLUSIONS/SIGNIFICANCE: Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.

  8. Amyotrophic lateral sclerosis multiprotein biomarkers in peripheral blood mononuclear cells.

    Science.gov (United States)

    Nardo, Giovanni; Pozzi, Silvia; Pignataro, Mauro; Lauranzano, Eliana; Spano, Giorgia; Garbelli, Silvia; Mantovani, Stefania; Marinou, Kalliopi; Papetti, Laura; Monteforte, Marta; Torri, Valter; Paris, Luca; Bazzoni, Gianfranco; Lunetta, Christian; Corbo, Massimo; Mora, Gabriele; Bendotti, Caterina; Bonetto, Valentina

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal progressive motor neuron disease, for which there are still no diagnostic/prognostic test and therapy. Specific molecular biomarkers are urgently needed to facilitate clinical studies and speed up the development of effective treatments. We used a two-dimensional difference in gel electrophoresis approach to identify in easily accessible clinical samples, peripheral blood mononuclear cells (PBMC), a panel of protein biomarkers that are closely associated with ALS. Validations and a longitudinal study were performed by immunoassays on a selected number of proteins. The same proteins were also measured in PBMC and spinal cord of a G93A SOD1 transgenic rat model. We identified combinations of protein biomarkers that can distinguish, with high discriminatory power, ALS patients from healthy controls (98%), and from patients with neurological disorders that may resemble ALS (91%), between two levels of disease severity (90%), and a number of translational biomarkers, that link responses between human and animal model. We demonstrated that TDP-43, cyclophilin A and ERp57 associate with disease progression in a longitudinal study. Moreover, the protein profile changes detected in peripheral blood mononuclear cells of ALS patients are suggestive of possible intracellular pathogenic mechanisms such as endoplasmic reticulum stress, nitrative stress, disturbances in redox regulation and RNA processing. Our results indicate that PBMC multiprotein biomarkers could contribute to determine amyotrophic lateral sclerosis diagnosis, differential diagnosis, disease severity and progression, and may help to elucidate pathogenic mechanisms.

  9. Autologous Intravenous Mononuclear Stem Cell Therapy in Chronic Ischemic Stroke

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    Bhasin A

    2012-01-01

    Full Text Available Background: The regenerative potential of brain has led to emerging therapies that can cure clinico-motor deficits after neurological diseases. Bone marrow mononuclear cell therapy is a great hope to mankind as these cells are feasible, multipotent and aid in neurofunctional gains in Stroke patients. Aims: This study evaluates safety, feasibility and efficacy of autologous mononuclear (MNC stem cell transplantation in patients with chronic ischemic stroke (CIS using clinical scores and functional imaging (fMRI and DTI. Design: Non randomised controlled observational study Study: Twenty four (n=24 CIS patients were recruited with the inclusion criteria as: 3 months–2years of stroke onset, hand muscle power (MRC grade at least 2; Brunnstrom stage of recovery: II-IV; NIHSS of 4-15, comprehendible. Fugl Meyer, modified Barthel Index (mBI and functional imaging parameters were used for assessment at baseline, 8 weeks and at 24 weeks. Twelve patients were administered with mean 54.6 million cells intravenously followed by 8 weeks of physiotherapy. Twelve patients served as controls. All patients were followed up at 24 weeks. Outcomes: The laboratory and radiological outcome measures were within normal limits in MNC group. Only mBI showed statistically significant improvement at 24 weeks (p<0.05 whereas the mean FM, MRC, Ashworth tone scores in the MNC group were high as compared to control group. There was an increased number of cluster activation of Brodmann areas BA 4, BA 6 post stem cell infusion compared to controls indicating neural plasticity. Cell therapy is safe and feasible which may facilitate restoration of function in CIS.

  10. Immunohistochemical demonstration of lysozyme in normal, reactive and neoplastic cells of the mononuclear phagocyte system.

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    Motoi,Makoto

    1984-04-01

    Full Text Available Using the peroxidase antiperoxidase (PAP method, lysozyme (LZM was shown to exist in normal, reactive and neoplastic cells belonging to the mononuclear phagocyte system (MPS, but was not detected in histiocytosis X cells. Immunostaining for cytoplasmic LZM by the PAP method is useful for identification of mononuclear phagocytes and for diagnosis of the diseases in which these cells participate.

  11. Downregulation of TIM-3 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.Z. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Huang, W.Y.; Qiao, Y.; Chen, Y.; Du, S.Y.; Chen, D.; Yu, S. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Liu, N. [Department of Nephrology, First Affiliated Hospital, China Medical University, Shenyang (China); Dou, L.Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Jiang, Y. [Central Laboratory, First Affiliated Hospital, China Medical University, Shenyang (China); Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang (China); Department of Dermatology, First Affiliated Hospital, China Medical University, Shenyang (China)

    2014-10-17

    The T-cell immunoglobulin and mucin domain (TIM) family is associated with autoimmune diseases, but its expression level in the immune cells of systemic lupus erythematosus (SLE) patients is not known. The aim of this study was to investigate whether the expression of TIM-3 mRNA is associated with pathogenesis of SLE. Quantitative real-time reverse transcription-polymerase chain reaction analysis (qRT-PCR) was used to determine TIM-1, TIM-3, and TIM-4 mRNA expression in peripheral blood mononuclear cells (PBMCs) from 132 patients with SLE and 62 healthy controls. The PBMC surface protein expression of TIMs in PBMCs from 20 SLE patients and 15 healthy controls was assayed by flow cytometry. Only TIM-3 mRNA expression decreased significantly in SLE patients compared with healthy controls (P<0.001). No significant differences in TIM family protein expression were observed in leukocytes from SLE patients and healthy controls (P>0.05). SLE patients with lupus nephritis (LN) had a significantly lower expression of TIM-3 mRNA than those without LN (P=0.001). There was no significant difference in the expression of TIM-3 mRNA within different classes of LN (P>0.05). Correlation of TIM-3 mRNA expression with serum IgA was highly significant (r=0.425, P=0.004), but was weakly correlated with total serum protein (r{sub s}=0.283, P=0.049) and serum albumin (r{sub s}=0.297, P=0.047). TIM-3 mRNA expression was weakly correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; r{sub s}=-0.272, P=0.032). Our results suggest that below-normal expression of TIM-3 mRNA in PBMC may be involved in the pathogenesis of SLE.

  12. Lol p I-induced IL-4 and IFN-gamma production by peripheral blood mononuclear cells of atopic and nonatopic subjects during and out of the pollen season.

    Science.gov (United States)

    Gagnon, R; Akoum, A; Hébert, J

    1993-04-01

    The reciprocal effects of IL-4 and IFN-gamma on IgE synthesis have been well established. It has also been shown that these two lymphokines are secreted by different subsets of CD4+ T cells (TH1 and TH2), and that TH2 helper T lymphocytes could be involved in the pathophysiology of allergic diseases. But little is known about the effects of an allergen on the profile of lymphokine synthesis by human peripheral blood mononuclear cells (PBMCs) of allergic and nonallergic subjects. We studied the production of IL-4 and IFN-gamma by PBMCs of atopic and nonatopic donors after in vitro stimulation by the group 1 allergen from Lolium perenne pollen (Lol p I), during and out of the grass pollen season. On natural exposure to pollen, Lol p I-induced IL-4 production was observed only with atopic donors (6 of 8), whereas the synthesis of IFN-gamma was observed for all nonatopic donors (7 of 7) and most allergic patients (5 of 7). At the time of the study, higher amounts of IFN-gamma were produced by PBMCs of nonatopic donors than by PBMCs of atopic patients. Out of the pollen season the production of IL-4 was not observed either by atopic (n = 11) or by nonatopic subjects (n = 5). On the other hand, IFN-gamma was produced by PBMCs of most subjects (atopic, 10 of 11; nonatopic, 5 of 5), but at the time of the study no difference was observed between the two groups. These results show that Lol p I induces different profiles of IL-4 and IFN-gamma production by PBMCs of atopic and nonatopic subjects. In atopic subjects this profile of lymphokine synthesis is influenced by the natural exposure to pollen, which is in keeping with the seasonal rise of IgE antibodies.

  13. Use of cryopreserved peripheral mononuclear blood cells in biomonitoring

    DEFF Research Database (Denmark)

    Risom, Lotte; Knudsen, Lisbeth E.

    1999-01-01

    This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood c....... We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis (UDS) in PMBC incubated with either autologous plasma or fetal bovine serum (FBS). Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of B....../T-lymphocytes and monocytes was measured in phytohemaglutinin (PHA)-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant...

  14. Malachite green toxicity assessed on Asian catfish primary cultures of peripheral blood mononuclear cells by a proteomic analysis.

    Science.gov (United States)

    Pierrard, Marie-Aline; Kestemont, Patrick; Delaive, Edouard; Dieu, Marc; Raes, Martine; Silvestre, Frédéric

    2012-06-15

    The potential genotoxic and carcinogenic properties reported for malachite green (MG) and the frequent detection of MG residues in fish and fish products, despite the ban of MG, have recently generated great concern. Additional toxicological data are required for a better understanding of the mechanism of action and a more comprehensive risk assessment for the exposure of fish to this fungicide. To date, the use of fish peripheral blood mononuclear cells (PBMCs) has not been exploited as a tool in the assessment of the toxicity of chemicals. However, PBMCs are exposed to toxicants and can be easily collected by blood sampling. The present study aims at better understanding the effects of MG by a proteomic analysis of primary cultured PBMC from the Asian catfish, Pangasianodon hypophthalmus, exposed to MG. The two lowest concentrations of 1 and 10 ppb were selected based on the MTS (water soluble tetrazolium salts) cytotoxicity test. Using a proteomic analysis (2D-DIGE), we showed that 109 proteins displayed significant changes in abundance in PBMC exposed during 48 h to MG. Most of these proteins were successfully identified by nano LC-MS/MS and validated through the Peptide and Protein Prophet of Scaffold™ software, but only 19 different proteins were considered corresponding to a single identification per spot. Our data suggest that low concentrations of MG could affect the mitochondrial metabolic functions, impair some signal transduction cascades and normal cell division, stimulate DNA repair and disorganize the cytoskeleton. Altogether, these results confirm that the mitochondrion is a target of MG toxicity. Further studies on the identified proteins are needed to better understand the mechanisms of MG toxicity in fish produced for human consumption.

  15. Three-Color Flow Cytometry Detection of Intracellular Cytokines in Peripheral Blood Mononuclear Cells: Comparative Analysis of Phorbol Myristate Acetate-Ionomycin and Phytohemagglutinin Stimulation

    Science.gov (United States)

    Baran, Jarołsaw; Kowalczyk, Danuta; Ożóg, Mariola; Zembala, Marek

    2001-01-01

    The assessment of intracellular cytokines at the single-cell level by flow cytometry has recently become a potent tool in many areas of cell biology and in defining the role of cytokines in various human diseases. Three-color flow cytometry for detection of intracellular cytokines combined with simultaneous determination of lymphocytes (CD3+ and CD4+) or monocytes (CD33+ and CD14+) was used for comparison of phytohemagglutinin (PHA)-and phorbol myristate acetate (PMA)-ionomycin-induced production of intracellular cytokines in peripheral blood mononuclear cells (PBMCs) of healthy donors. We found that the number of PBMCs stained for tumor necrosis factor alpha and gamma interferon after 6 h of activation was higher when PMA-ionomycin was used for stimulation, while the frequencies of cells positive for interleukin 4 (IL-4) were similar for both stimulators. However, PMA-ionomycin stimulation caused prominent alterations of cell morphology and membrane expression of CD4 and CD14. In contrast, PHA did not cause downregulation of surface markers and resulted in less pronounced alterations in both forward and side scatter signals during flow cytometry analysis. Moreover, during 48 h of culture PHA stimulated tumor necrosis factor beta and IL-10 production, which was not observed when PMA-ionomycin was used. We conclude that the use of PHA for cell activation may limit in vitro artifacts and allow more precise analysis of intracellular cytokine production in various disease states. PMID:11238213

  16. Functional and Pharmacological Analysis of Cardiomyocytes Differentiated from Human Peripheral Blood Mononuclear-Derived Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Michael Riedel

    2014-07-01

    Full Text Available 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.

  17. Functional and pharmacological analysis of cardiomyocytes differentiated from human peripheral blood mononuclear-derived pluripotent stem cells.

    Science.gov (United States)

    Riedel, Michael; Jou, Chuanchau J; Lai, Shuping; Lux, Robert L; Moreno, Alonso P; Spitzer, Kenneth W; Christians, Elizabeth; Tristani-Firouzi, Martin; Benjamin, Ivor J

    2014-07-08

    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.

  18. Autologous Bone Marrow Mononuclear Cells Intrathecal Transplantation in Chronic Stroke

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2014-01-01

    Full Text Available Cell therapy is being widely explored in the management of stroke and has demonstrated great potential. It has been shown to assist in the remodeling of the central nervous system by inducing neurorestorative effect through the process of angiogenesis, neurogenesis, and reduction of glial scar formation. In this study, the effect of intrathecal administration of autologous bone marrow mononuclear cells (BMMNCs is analyzed on the recovery process of patients with chronic stroke. 24 patients diagnosed with chronic stroke were administered cell therapy, followed by multidisciplinary neurorehabilitation. They were assessed on functional independence measure (FIM objectively, along with assessment of standing and walking balance, ambulation, and hand functions. Out of 24 patients, 12 improved in ambulation, 10 in hand functions, 6 in standing balance, and 9 in walking balance. Further factor analysis was done. Patients of the younger groups showed higher percentage of improvement in all the areas. Patients who underwent cell therapy within 2 years after the stroke showed better changes. Ischemic type of stroke had better recovery than the hemorrhagic stroke. This study demonstrates the potential of autologous BMMNCs intrathecal transplantation in improving the prognosis of functional recovery in chronic stage of stroke. Further clinical trials are recommended. This trial is registered with NCT02065778.

  19. Differential expression of microRNA in peripheral blood mononuclear cells as specific biomarker for major depressive disorder patients.

    Science.gov (United States)

    Fan, Hui-min; Sun, Xin-yang; Guo, Wei; Zhong, Ai-fang; Niu, Wei; Zhao, Lin; Dai, Yun-hua; Guo, Zhong-min; Zhang, Li-yi; Lu, Jim

    2014-12-01

    Currently, diagnosis and treatment of major depressive disorder (MDD) are based on the patients' description of symptoms, mental status examinations, and clinical behavioral observations, which increases the chance of misdiagnosis. There is a serious need to find a practical biomarker for the proper diagnosis of MDD. This study aimed to explore the possibility of microRNA (miRNA) in peripheral blood mononuclear cells (PBMCs) as specific blood-based biomarker for MDD patients. By using an Affymetrix array that covers 723 human miRNAs, we identified 26 miRNAs with significant changes in expression in PBMCs of MDD patients. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in a larger cohort of 81 MDD patients and 46 healthy controls confirmed that the expression levels of 5 miRNAs (miRNA-26b, miRNA-1972, miRNA-4485, miRNA-4498, and miRNA-4743) were up-regulated. By receiver operating characteristic (ROC) curve analysis, the combining area under the ROC curve (AUC) of these five miRNAs was 0.636 [95% confidence interval (CI): 0.58-0.90]. MiRNA target gene prediction and functional annotation analysis showed that there was a significant enrichment in several pathways associated with nervous system and brain functions, supporting the hypothesis that differentially-regulated miRNAs may be involved in mechanism underlying development of MDD. We conclude that altered expression of miRNAs in PMBCs might be involved in multiple stages of MDD pathogenesis, and thus might be able to serve as specific biomarker for diagnosis of MDD.

  20. Correlation analyses revealed global microRNA-mRNA expression associations in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wang, Lan; Zhu, Jiang; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Xiao-Wei; Xia, Wei; Xie, Fang-Fei; He, Pei; Bing, Peng-Fei; Qiu, Ying-Hua; Lin, Xiang; Lu, Xin; Zhang, Lei; Yi, Neng-Jun; Zhang, Yong-Hong; Lei, Shu-Feng

    2017-09-06

    MicroRNAs (miRNAs) can regulate gene expression through binding to complementary sites in the 3'-untranslated regions of target mRNAs, which will lead to existence of correlation in expression between miRNA and mRNA. However, the miRNA-mRNA correlation patterns are complex and remain largely unclear yet. To establish the global correlation patterns in human peripheral blood mononuclear cells (PBMCs), multiple miRNA-mRNA correlation analyses and expression quantitative trait locus (eQTL) analysis were conducted in this study. We predicted and achieved 861 miRNA-mRNA pairs (65 miRNAs, 412 mRNAs) using multiple bioinformatics programs, and found global negative miRNA-mRNA correlations in PBMC from all 46 study subjects. Among the 861 pairs of correlations, 19.5% were significant (P correlation network was complex and highlighted key miRNAs/genes in PBMC. Some miRNAs, such as hsa-miR-29a, hsa-miR-148a, regulate a cluster of target genes. Some genes, e.g., TNRC6A, are regulated by multiple miRNAs. The identified genes tend to be enriched in molecular functions of DNA and RNA binding, and biological processes such as protein transport, regulation of translation and chromatin modification. The results provided a global view of the miRNA-mRNA expression correlation profile in human PBMCs, which would facilitate in-depth investigation of biological functions of key miRNAs/mRNAs and better understanding of the pathogenesis underlying PBMC-related diseases.

  1. Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

    Directory of Open Access Journals (Sweden)

    Shuei-Kuen Tseng

    2014-09-01

    Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

  2. 4-1BB gene expression in peripheral blood mononuclear cells from orthotopic liver transplant recipients with graft acceptance

    Institute of Scientific and Technical Information of China (English)

    万云乐; 郑树森; 贾长库; 杨家印; 金晓凌; 赵志成

    2003-01-01

    Objective To investigate the gene expression of 4-1BB in peripheral blood mononuclear cells (PBMCs) and the possible significance of the 4-1BB pathway after clinical orthotopic liver transplantation (OLT). Methods 4-1BB mRNA levels in PBMCs from 22 OLT patients were analyzed by RT-PCR. 4-1BB protein expressed on the surface of CD4+ and CD8+ T cells were detected by flow cytometry, and visualized with direct immunofluorescence and confocal fluorescence microscopy. Patients with primary liver cancer (PLC) and healthy volunteers served as controls. Six cases of recently performed liver transplantation were also observed in this study.Results 4-1BB mRNA was detected in PBMCs from both liver transplant patients with long-term graft acceptance (22 cases) and from transplant patients on day 1 to day 3 post-transplantation (6 cases), but was not found in PBMCs from transplant patients on day 7 to day 30 post-transplantation (6 cases). 4-1BB mRNA was also not found in samples from 8 of the healthy controls and 7 of the PLC patients, though very low expression was detected in the other 4 healthy volunteers and 6 PLC patients. Simultaneously, 4-1BB protein was expressed at nearly undetectable levels on CD4+ and CD8+ T cells from healthy controls, PLC patients, as well as OLT patients within the first month post-transplantation (6 cases). However, 4-1BB expression was found on the surface of CD4+ and CD8+ T cells from liver transplant patients with long-term graft acceptance. Direct immunofluorescent staining and confocal fluorescence microscopy clearly revealed evidence of 4-1BB protein on cell membranes of CD4+ and CD8+ T cells from liver transplant patients with long-term graft acceptance. Simultaneously, a significantly higher percentage of CD3+ CD25+ T cells were found in liver transplant patients with long-term graft acceptance group as compared with the healthy control group (P<0.05). The expression of 4-1BB protein on T cells did not correlate with the survival

  3. Senescence-Related Changes in Gene Expression of Peripheral Blood Mononuclear Cells from Octo/Nonagenarians Compared to Their Offspring

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    Amirah Abdul Rahman

    2013-01-01

    Full Text Available Mechanisms determining both functional rate of decline and the time of onset in aging remain elusive. Studies of the aging process especially those involving the comparison of long-lived individuals and young controls are fairly limited. Therefore, this research aims to determine the differential gene expression profile in related individuals from villages in Pahang, Malaysia. Genome-wide microarray analysis of 18 samples of peripheral blood mononuclear cells (PBMCs from two groups: octo/nonagenarians (80–99 years old and their offspring (50.2 ± 4.0 years old revealed that 477 transcripts were age-induced and 335 transcripts were age-repressed with fold changes ≥1.2 in octo/nonagenarians compared to offspring. Interestingly, changes in gene expression were associated with increased capacity for apoptosis (BAK1, cell cycle regulation (CDKN1B, metabolic process (LRPAP1, insulin action (IGF2R, and increased immune and inflammatory response (IL27RA, whereas response to stress (HSPA8, damage stimulus (XRCC6, and chromatin remodelling (TINF2 pathways were downregulated in octo/nonagenarians. These results suggested that systemic telomere maintenance, metabolism, cell signalling, and redox regulation may be important for individuals to maintain their healthy state with advancing age and that these processes play an important role in the determination of the healthy life-span.

  4. Influence of Irradiated Peripheral Blood Mononuclear Cells on Both Ex Vivo Proliferation of Human Natural Killer Cells and Change in Cellular Property

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    María Delso-Vallejo

    2017-07-01

    Full Text Available Clinical studies with adoptive immunotherapy using allogeneic natural killer (NK cells showed feasibility, but also limitation regarding the transfused absolute cell numbers. First promising results with peripheral blood mononuclear cells (PBMCs as feeder cells to improve the final cell number need further optimization and investigation of the unknown controlling mechanism in the cross-talk to NK cells. We investigated the influence of irradiated autologous PBMCs to boost NK cell proliferation in the presence of OKT3 and IL-2. Our findings demonstrate a requirement for receptor–ligand interactions between feeders and NK cells to produce soluble factors that can sustain NK cell proliferation. Thus, both physical contact between feeder and NK cells, and soluble factors produced in consequence, are required to fully enhance NK cell ex vivo proliferation. This occurred with an indispensable role of the cross-talk between T cells, monocytes, and NK cells, while B cells had no further influence in supporting NK cell proliferation under these co-culture conditions. Moreover, gene expression analysis of highly proliferating and non-proliferating NK cells revealed important phenotypic changes on 5-day cultured NK cells. Actively proliferating NK cells have reduced Siglec-7 and -9 expression compared with non-proliferating and resting NK cells (day 0, independently of the presence of feeder cells. Interestingly, proliferating NK cells cultured with feeder cells contained increased frequencies of cells expressing RANKL, B7-H3, and HLA class II molecules, particularly HLA-DR, compared with resting NK cells or expanded with IL-2 only. A subset of HLA-DR expressing NK cells, co-expressing RANKL, and B7-H3 corresponded to the most proliferative population under the established co-culture conditions. Our results highlight the importance of the crosstalk between T cells, monocytes, and NK cells in autologous feeder cell-based ex vivo NK cell expansion

  5. Telomere Length in Peripheral Blood Mononuclear Cells of Patients on Chronic Hemodialysis Is Related With Telomerase Activity and Treatment Duration.

    Science.gov (United States)

    Stefanidis, Ioannis; Voliotis, Georgios; Papanikolaou, Vassilios; Chronopoulou, Ioanna; Eleftheriadis, Theodoros; Kowald, Axel; Zintzaras, Elias; Tsezou, Aspasia

    2015-09-01

    Telomere shortening to a critical limit is associated with replicative senescence. This process is prevented by the enzyme telomerase. Oxidative stress and chronic inflammation are factors accelerating telomere loss. Chronic hemodialysis, typically accompanied by oxidative stress and inflammation, may be also associated with replicative senescence. To test this hypothesis, we determined telomere length and telomerase activity in peripheral blood mononuclear cells (PBMCs) in a cross-sectional study. Hemodialysis patients at the University Hospital Larissa and healthy controls were studied. Telomere length was determined by the TeloTAGGG Telomere Length Assay and telomerase activity by Telomerase PCR-ELISA (Roche Diagnostics GmbH, Mannheim, Germany). We enrolled 43 hemodialysis patients (17 females; age 65.0 ± 12.7 years) and 23 controls (six females; age 62.1 ± 15.7 years). Between the two groups, there was no difference in telomere length (6.95 ± 3.25 vs. 7.31 ± 1.96 kb; P = 0.244) or in telomerase activity (1.82 ± 2.91 vs. 2.71 ± 3.0; P = 0.085). Telomere length correlated inversely with vintage of hemodialysis (r = -0.332, P = 0.030). In hemodialysis patients, positive telomerase activity correlated with telomere length (r = 0.443, P = 0.030). Only age, and neither telomere length nor telomerase activity, was an independent survival predictor (hazard ratio 1.116, 95% confidence interval 1.009-1.234, P = 0.033). In this study, telomere length and telomerase activity in PBMCs are not altered in hemodialysis patients compared with healthy controls. Long duration of hemodialysis treatment is associated with telomere shortening and positive telomerase activity with an increased telomere length in PBMCs of hemodialysis patients. The underlying mechanism and clinical implications of our findings require further investigation.

  6. Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

    Science.gov (United States)

    Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

    2017-04-01

    Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

  7. Absence of peripheral blood mononuclear cells priming in hemodialysis patients

    Directory of Open Access Journals (Sweden)

    Santos B.C.

    2003-01-01

    Full Text Available As a consequence of the proinflammatory environment occurring in dialytic patients, cytokine overproduction has been implicated in hemodialysis co-morbidity. However, there are discrepancies among the various studies that have analyzed TNF-alpha synthesis and the presence of peripheral blood mononuclear cell (PBMC priming in this clinical setting. We measured bioactive cytokine by the L929 cell bioassay, and evaluated PBMC TNF-alpha production by 32 hemodialysis patients (HP and 51 controls. No difference in TNF-alpha secretion was observed between controls and HP (859 ± 141 vs 697 ± 130 U/10(6 cells. Lipopolysaccharide (5 µg/ml did not induce any further TNF-alpha release, showing no PBMC priming. Paraformaldehyde-fixed HP PBMC were not cytotoxic to L929 cells, suggesting the absence of membrane-anchored TNF-alpha. Cycloheximide inhibited PBMC cytotoxicity in HP and controls, indicating lack of a PBMC TNF-alpha pool, and dependence on de novo cytokine synthesis. Actinomycin D reduced TNF-alpha production in HP, but had no effect on controls. Therefore, our data imply that TNF-alpha production is an intrinsic activity of normal PBMC and is not altered in HP. Moreover, TNF-alpha is a product of de novo synthesis by PBMC and is not constitutively expressed on HP cell membranes. The effect of actinomycin D suggests a putative tighter control of TNF-alpha mRNA turnover in HP. This increased dependence on TNF-alpha RNA transcription in HP may reflect an adaptive response to hemodialysis stimuli.

  8. A module of human peripheral blood mononuclear cell transcriptional network containing primitive and differentiation markers is related to specific cardiovascular health variables.

    Science.gov (United States)

    Moldovan, Leni; Anghelina, Mirela; Kantor, Taylor; Jones, Desiree; Ramadan, Enass; Xiang, Yang; Huang, Kun; Kolipaka, Arunark; Malarkey, William; Ghasemzadeh, Nima; Mohler, Peter J; Quyyumi, Arshed; Moldovan, Nicanor I

    2014-01-01

    Peripheral blood mononuclear cells (PBMCs), including rare circulating stem and progenitor cells (CSPCs), have important yet poorly understood roles in the maintenance and repair of blood vessels and perfused organs. Our hypothesis was that the identities and functions of CSPCs in cardiovascular health could be ascertained by analyzing the patterns of their co-expressed markers in unselected PBMC samples. Because gene microarrays had failed to detect many stem cell-associated genes, we performed quantitative real-time PCR to measure the expression of 45 primitive and tissue differentiation markers in PBMCs from healthy and hypertensive human subjects. We compared these expression levels to the subjects' demographic and cardiovascular risk factors, including vascular stiffness. The tested marker genes were expressed in all of samples and organized in hierarchical transcriptional network modules, constructed by a bottom-up approach. An index of gene expression in one of these modules (metagene), defined as the average standardized relative copy numbers of 15 pluripotency and cardiovascular differentiation markers, was negatively correlated (all ptranscriptional network. Furthermore, the coordinated gene expression in these modules can be linked to cardiovascular risk factors and subclinical cardiovascular disease; thus, this measure may be useful for their diagnosis and prognosis.

  9. Influence of the invasion of peripheral blood mononuclear cells by hepatitis B virus on immune response of the patients with chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    XING Tong-jing; ZHANG Lian; HOU Jin-lin; ZHANG Ming-xia; YANG Jie; LUO Kang-xian

    2001-01-01

    To explore the influence of HBV invasion into peripheral blood mononuclear cells (PBMC) on the immune response of patients with chronic hepatitis B. Methods: The cytokine levels in the culture supernatant of PBMC from 56 patients with chronic hepatitis B were determined by ELISA, and PCR was employed to amplify the HBV DNA. Results: The levels of IFN-γ in patients with hepatitis B was lower than thoset of the control, but the difference was not statistically significant, while the levels of IL-4 were significantly higher than those of the control (P<0.01). The serum levels of HBV DNA were negatively correlated with that of IFN-y in culture supernatants of PBMC. Thirty-five patients positive of HBV DNA in the PBMCs were identified from 56 patients with hepatitis B,and their IFN-γ level proved to be significantly different. Conclusions: Th2 cell-mediated immune response is predominant in chronic hepatitis B which is associated with the chronicity of HBV infection. HBV invasion into the PBMCs may affect Th1 and Th2 cell-mediated immune response of the patients with chronic hepatitis B.

  10. Tax posttranslational modifications and interaction with calreticulin in MT-2 cells and human peripheral blood mononuclear cells of human T cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

    Science.gov (United States)

    Medina, Fernando; Quintremil, Sebastian; Alberti, Carolina; Barriga, Andres; Cartier, Luis; Puente, Javier; Ramírez, Eugenio; Ferreira, Arturo; Tanaka, Yuetsu; Valenzuela, Maria Antonieta

    2014-04-01

    The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

  11. Are Peripheral Blood Mononuclear Cells Derived from Patients with Certain Myopathies Suitable for Personalized Drug Screening?

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    Andriy V. Shatillo

    2014-12-01

    Full Text Available Background: Limb girdle muscular dystrophies (LGMDs and several other disorders which share their specific phenotype are rare, predominantly hereditary conditions with no curative treatment. Differential diagnosis of these myopathies is quite challenging and expensive in many cases. Therefore, a significant proportion of patients remains undiagnosed and untreated for a long time. At the same time there is a huge amount of drugs and supplements potentially able to modify the course of some of these muscular dystrophies. That is why a simple empirical approach able to define a patient’s reaction to a specific compound seems rational. Because most common basic pathogenetic mechanisms for these quite different disorders increase the vulnerability of muscle cells (or decrease ability for reparation during mechanical stress, we propose a simple, noninvasive and inexpensive approach for individualized drug screening based on the drug’s influence on the mechanical vulnerability of peripheral blood mononuclear cells (PBMC. Methods: PBMC derived from 8 patients with Duchenne muscular dystrophy (DMD, 2 patients with LGMD2A, 1 patient with LGMD2B, 1 with MERRF syndrome, 1 with facioscapulohumeral muscular dystrophy (FSHD and 13 matched control subjects were irradiated by ultrasound in the presence of several compounds (lisinopril, vitamin D3, prednisolon, tocopherol, topiramate, glutargin, α-lipoic acid, essentiale, and physiological solution. Then viability indexes of the samples were detected by citotoxic assays based on vital dye (neutral red and resazurin metabolism. Results: In cytotoxicity tests with active transport of neutral red into PBMC derived from DMD patients, the cells showed signs of destruction at 1.06±0.52 minutes of ultrasounding compared to 1.75±0.6 minutes in control. PBMCs from patients with other myopathies have either normal or decreased resistance to ultrasound. The addition of tocopherol significantly changes the PBMC

  12. Assessment of 188Re marked anti MHC class Ⅱ antibody by peripheral blood mononuclear cells stimulated by donor alloantigen

    Institute of Scientific and Technical Information of China (English)

    DING Guo-ping; CAO Li-ping; LIU Jie; LIU Da-ren; QUE Ri-sheng; ZHU Lin-hua; ZHOU Yi-ming; MAO Ke-jie; HU Jun-an

    2011-01-01

    Background Previous studies showed that anti MHC-Ⅱ monoclone antibody (MAb) only had partial inhibiting effect of alloreactive mixed lymphocyte reaction (MLR) in vitro and it was unsteady and non-persistent. The aim of this research was to determine whether radioactive isotope 188Re marked MHC-Ⅱ antibody could benefit the allograft acceptance in transplantation as compared to normal MHC-Ⅱ antibody.Methods 188Re was incorporated to 2E9/13F(ab')2 which is against swine MHC class Ⅱ antigen (MAb-188Re). Porcine peripheral blood mononuclear (PBMC) cells were examined for proliferation and cytokine mRNA expression after stimulation with MHC-Ⅱ MAb or MAb-188Re.Results The proliferative response of recipient PBMCs in mixed lymphocyte reaction (MLR) to donor alloantigen showed that the stimulation index of MAb-188Re group was significantly lower than the MHC-Ⅱ MAb group and control (P<0.05). mRNA expression of interleukin 2, interferon Y and tumor necrosis factor α (type 1 cytokines) was lower in MAb-188Re group than the MHC-Ⅱ MAb group, while interleukin 10 (type 2 cytokines) was higher in MAb-188Re group in the first 24 hours.Conclusion MAb-188Re could help the graft acceptance by inhibiting T cell proliferation, lowering the expression of type 1 cytokines and elevating the type 2 cytokines produced by PBMC.

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

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    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

  14. MicroRNA Expression in Alzheimer Blood Mononuclear Cells

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    Hyman M. Schipper

    2007-01-01

    Full Text Available Various coding genes representing multiple functional categories are downregulated in blood mononuclear cells (BMC of patients with sporadic Alzheimer disease (AD. Noncoding microRNAs (miRNA regulate gene expression by degrading messages or inhibiting translation. Using BMC as a paradigm for the study of systemic alterations in AD, we investigated whether peripheral miRNA expression is altered in this condition. MicroRNA levels were assessed using the microRNA microarray (MMChip containing 462 human miRNA, and the results validated by real time PCR. Sixteen AD patients and sixteen normal elderly controls (NEC were matched for ethnicity, age, gender and education. The expression of several BMC miRNAs was found to increase in AD relative to NEC levels, and may differ between AD subjects bearing one or two APOE4 alleles. As compared to NEC, miRNAs signifi cantly upregulated in AD subjects and confi rmed by qPCR were miR-34a and 181b. Predicted target genes downregulated in Alzheimer BMC that correlated with the upregulated miRNAs were largely represented in the functional categories of Transcription/Translation and Synaptic Activity. Several miRNAs targeting the same genes were within the functional category of Injury response/Redox homeostasis. Taken together, induction of microRNA expression in BMC may contribute to the aberrant systemic decline in mRNA levels in sporadic AD.

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

    OpenAIRE

    2010-01-01

    Abstract: 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 ...

  16. Hypoxanthine Guanine Phosphoribosyl Transferase Is the Most Stable Reference Gene for Gene Expression Analysis by Quantitative PCR in Peripheral Blood Mononuclear Cells from Women with the Polycystic Ovary Syndrome

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    Milutinović Danijela Vojnović

    2014-09-01

    Full Text Available Background: The polycystic ovary syndrome (PCOS is a frequent endocrine disorder that affects women of reproductive age. As the syndrome is strongly associated with obesity, it is of interest to examine the gene expression diffe rences that accompany its development and the associ a ted metabolic disturbances. Real-time RT PCR is a standard method for studying changes in gene expression. However, to obtain accurate and reliable results, validation of reference genes is obligatory. The aim of this study was to identify a suitable reference for the normalization of gene expression in peripheral blood mononuclear cells (PBMCs from obese and normal-weight women with PCOS.

  17. Tryptamine and dimethyltryptamine inhibit indoleamine 2,3 dioxygenase and increase the tumor-reactive effect of peripheral blood mononuclear cells.

    Science.gov (United States)

    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. Copyright © 2013 John Wiley & Sons, Ltd.

  18. Manutenção da viabilidade das células mononucleares de sangue periférico humano em extratos e formulações de própolis - doi: 10.4025/actascihealthsci.v34i1.8954 Maintenance of human peripheral blood mononuclear cell viability in propolis extracts and formulations - doi: 10.4025/actascihealthsci.v34i1.8954

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    Selma Lucy Franco

    2011-12-01

    Full Text Available Neste estudo comparou-se a viabilidade das células mononucleares de sangue periférico (PBMCs humano quando mantidas em diferentes extratos e formulações de própolis. As PBMCs  (106 cels mL-1, provenientes de doadores saudáveis (n=5, foram estocadas a 20ºC nas diferentes soluções de própolis, assim como em solução salina balanceada de Hank’s (HBSS, utilizada como controle do experimento. A viabilidade celular foi determinada pelo método de exclusão com azul de Tripan. Quando incubadas por 1h, apenas dois extratos, denominados A70D e D70D, apresentaram desempenho satisfatório na manutenção da viabilidade celular, semelhante (p > 0,05 ao controle HBSS e diferindo estatisticamente (p  In this study a comparison was made of human mononuclear cell (PBMCs viability, when cells were kept in different propolis extracts and formulations. PBMCs (106 cell mL-1, obtained from healthy donors (n=5, were incubated at 20ºC in the different propolis solutions, as well as in Hank’s balanced salt solution (HBSS, used as experimental control. The cell viability was analyzed by Trypan blue exclusion assay. When incubated for 1h, only two extracts, denominated A70D and D70D, showed appropriate results for maintaining cell viability. A70D and D70D showed better viability (p 0.05 from the HBSS control. A70D and D70D were tested in five dilutions in propylene glycol, over 24h, with analysis at 0, 30 minutes, 1, 3, 6, 10 and 24h. The most concentrated fractions showed the worst performance (p

  19. Protective Effect of Yinhua Miyanling Tablet on Lipopolysaccharide-Induced Inflammation through Suppression of NLRP3/Caspase-1 Inflammasome in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Sai, Jingying; Zheng, Jingtong; Liu, Chuangui; Lu, Yanjiao; Wang, Guoqiang; Wang, Ting; Guan, Xuewa; Chen, Fang; Fang, Keyong; Zhang, Chao; Lu, Junying; Zhang, Xiaotian; Zhu, Hailin

    2016-01-01

    Yinhua Miyanling Tablet (YMT), the Chinese formula, has long been administrated in clinical practice for the treatment of acute pyelonephritis and acute urocystitis. In the current study, we aimed to investigate the anti-inflammatory effect of YMT in vitro and to evaluate the association between anti-inflammation and innate immune response. Human peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation and then were stimulated by Lipopolysaccharide (LPS). The differential gene expression of inflammation-related genes after drug administration was assessed using PCR array, and the protein levels of differential genes were measured by ELISA and Western blot. The result showed that YMT significantly inhibited the expression of NLRP3, Caspase-1, and the downstream cytokine IL-1β and suppressed the production of inflammatory mediators TNF-α, IL-6, IL-10, and MCP-1 in a dose-dependent manner compared to the LPS group (P diseases.

  20. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro

    Science.gov (United States)

    2013-03-11

    Bacillus cereus group of bacteria, are attributed to poly- γ-D-glutamate acid (PGA) capsule, lethal toxin (LT) and edema toxin (ET) [10-12]. These toxins...M, Hellman M, Muhie S, et al. (2013) Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro...author and source are credited. Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Exposed to Bacillus anthracis in vitro Rasha

  1. Phenotyping of peripheral blood mononuclear cells of patients with advanced heavily pre-treated adenocarcinoma of the stomach and gastro-esophageal junction.

    Science.gov (United States)

    Kuehnle, Marie-Cristine; Attig, Sebastian; Britten, Cedrik M; Schulze-Bergkamen, Henning; Lordick, Florian; von Wichert, Goetz; Thuss-Patience, Peter; Stein, Alexander; Schuler, Martin; Bassermann, Florian; Sahin, Ugur; Türeci, Ozlem

    2014-12-01

    Immunotherapeutic approaches are emerging as promising new treatment options for patients with solid cancers. The host immune system in cancer patients is dysfunctional due to a number of reasons. The level of immunosuppression is variable at the time of diagnosis and depends on the particular cancer entity, stage, and prior anti-cancer therapies. For many cancer entities, the immune alterations of the respective patient population have not been further characterized even though a patient's immunophenotype may be prognostic for the course of the disease or predictive for clinical/biological response to immunotherapy. In this study, we used flow cytometry to determine the phenotype of peripheral blood mononuclear cells (PBMCs) from 30 patients with heavily pre-treated, advanced adenocarcinoma of the stomach and gastro-esophageal junction. The frequencies and activation status of relevant immune effector populations were determined in PBMCs and compared to those of healthy individuals. This report provides comprehensive immune phenotyping data of a patient population with a high medical need.

  2. Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM).

    Science.gov (United States)

    Palmirotta, Raffaele; De Marchis, Maria Laura; Ludovici, Giorgia; Leone, Barbara; Savonarola, Annalisa; Ialongo, Cristiano; Spila, Antonella; De Angelis, Francesco; Ferroni, Patrizia; Della-Morte, David; Guadagni, Fiorella

    2012-07-19

    Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).
Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.

  3. Phosphorylation of Histone H2A.X in Peripheral Blood Mononuclear Cells May Be a Useful Marker for Monitoring Cardiometabolic Risk in Nondiabetic Individuals

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    So Ra Yoon

    2017-01-01

    Full Text Available Phosphorylation of H2A.X (serine 139 in the histone H2A family located in the downstream of the DNA damage kinase signaling cascade is an important indicator of DNA damage. Recently, phosphorylation of H2A.X was proposed as a sensitive biomarker of aging. This study investigated if phosphorylation of H2A.X in peripheral blood mononuclear cells (PBMCs is associated with cardiometabolic risk in nondiabetic individuals. Basic parameters and oxidative stress/inflammatory markers were measured in nondiabetic healthy Koreans (n=119. Phosphorylation of H2A.X was measured randomly among the study subjects using a flow cytometer. According to the number of metabolic syndrome risk factor (MetS-RF, the study subjects were subdivided into “super healthy” (MetS−RF=0, n=71 and “MetS-risk” (MetS−RF≥1, n=48 groups. Phosphorylation of H2A.X in PBMCs (percentages and mean fluorescence intensity was significantly higher in the MetS-risk group than in the super healthy group after adjusting for age, sex, cigarette smoking, and alcohol consumption. Phosphorylated H2A.X was positively correlated with the number of MetS-RF as well as waist circumference, blood pressures, triglyceride, HbA1C, oxidized LDL, high sensitivity C-reactive protein, tumor necrosis factor-alpha, and alanine aminotransferase after the adjustment. The present study suggested that phosphorylated H2A.X in circulating PBMCs measured by flow cytometer may be a useful marker for monitoring cardiometabolic risk in nondiabetic individuals.

  4. Fully automated analysis of chemically induced γH2AX foci in human peripheral blood mononuclear cells by indirect immunofluorescence.

    Science.gov (United States)

    Willitzki, Annika; Lorenz, Sebastian; Hiemann, Rico; Guttek, Karina; Goihl, Alexander; Hartig, Roland; Conrad, Karsten; Feist, Eugen; Sack, Ulrich; Schierack, Peter; Heiserich, Lisa; Eberle, Caroline; Peters, Vanessa; Roggenbuck, Dirk; Reinhold, Dirk

    2013-11-01

    Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose- and time-dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage.

  5. Naive Treg-like CCR7(+) mononuclear cells indicate unfavorable prognosis in hepatocellular carcinoma.

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    Shi, Jie-Yi; Duan, Meng; Sun, Qi-Man; Yang, Liuxiao; Wang, Zhi-Chao; Mynbaev, Ospan A; He, Yi-Feng; Wang, Ling-Yan; Zhou, Jian; Tang, Qi-Qun; Cao, Ya; Fan, Jia; Wang, Xiao-Ying; Gao, Qiang

    2016-07-01

    Chemokine receptor-like 1 (CCRL1) has the potential in creating a low level of CCL19 and CCL21 to hinder CCR7(+) cell tracking to tumor tissue. Previously, we found a tumor suppressive role of CCRL1 by impairing CCR7-related chemotaxis of tumor cells in human hepatocellular carcinoma (HCC). Here, we reported a contribution of CCR7(+) mononuclear cells in the tumor microenvironment to the progression of disease. Immunohistochemistry was used to investigate the distribution and clinical significance of CCR7(+) cells in a cohort of 240 HCC patients. Furthermore, the phenotype, composition, and functional status of CCR7(+) cells were determined by flow cytometry, immunofluorescence, and in vitro co-culture assays. We found that CCR7(+) mononuclear cells were dispersed around tumor tissue and negatively related to tumoral expression of CCRL1 (P CCR7(+) mononuclear cells positively correlated with the absence of tumor capsule, vascular invasion, and poor differentiation (P CCR7(+) mononuclear cells was significantly associated with worse survival and increased recurrence. We found that CCR7(+) mononuclear cells featured a naive Treg-like phenotype (CD45RA(+)CD25(+)FOXP3(+)) and possessed tumor-promoting potential by producing TGF-β1. Moreover, CCR7(+) cells were also composed of several immunocytes, a third of which were CD8(+) T cells. CCR7(+) Treg-like cells facilitate tumor growth and indicate unfavorable prognosis in HCC patients, but fortunately, their tracking to tumor tissue is under the control of CCRL1.

  6. 银屑病患者外周血单个核细胞对自身角质形成细胞的促生长作用%Psoriatic peripheral blood mononuclear cells stimulate the proliferation of epidermal keratinocytes in autologous mixed culture reaction

    Institute of Scientific and Technical Information of China (English)

    王刚; 刘玉峰

    2001-01-01

    目的了解银屑病患者外周血单个核细胞(PBMCs)对自体表皮角质形成细胞(KCs)增生的作用. 方法分离3例银屑病患者的PBMCs,经30 Gy钴照射后与来自同一患者的%AIM To learn the effect of psoriatic peripheral blood mononuclear cells (PBMC) on the proliferation of autologous epidermal keratinocytes. METHODS Peripheral blood mononuclear cells were isolated from 3 patients with psoriasis vulgaris. After irradiating in Cobalt gamma ray of 30 Gy, the cells were cocultured with psoriatic epidermal keratinocytes that were obtained from the same patient. The changes of keratinocyte proliferation were detected by 3H-TdR incorporation assay. RESULTS Keratinocytes involved and uninvolved in Psoriatic underwent a significant proliferation response to autologous peripheral blood mononuclear cells in the mixed cultures. CONCLUSION Interaction of keratinocytes with infiltrated mononuclear cells in epidermis may induce the hyperproliferation of the keratinocytes and thus play an important role in the pathogenesis of psoriasis.

  7. Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo.

    Science.gov (United States)

    Liu, Qian; Guan, Liping; Huang, Bing; Li, Weihua; Su, Qiao; Yu, Minbin; Xu, Xiaoping; Luo, Ting; Lin, Shaochun; Sun, Xuerong; Chen, Mengfei; Chen, Xigu

    2011-06-01

    Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate

  8. Photoperiodic induced melatonin regulates immunity and expression pattern of melatonin receptor MT1 in spleen and bone marrow mononuclear cells of male golden hamster.

    Science.gov (United States)

    Vishwas, Dipanshu Kumar; Haldar, Chandana

    2013-11-05

    The pineal gland transduces day length information into chemical signal of melatonin that ultimately translates photic stimulus into season-specific immune responses to promote survival of individual from incidence of opportunistic diseases. To date, the immune adjustments being a result of photoperiodic exposures for any nocturnal seasonally breeding rodent have not been systematically examined. Therefore, we evaluated the humoral and cell mediated immune responses of photoperiodically entrained male golden hamsters. Short day induced melatonin increased the immune parameters such as spleen mass, total leukocyte (TLC) and lymphocyte count (LC), proliferation of splenocytes, peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMNCs) along with serum IL-2 and anti-Keyhole Limpet Hemocyanin (KLH) IgG production when compared with long day experienced hamsters. Short term melatonin treatment (for two weeks) to long day hamsters enhanced to some extent the proliferation of splenocytes, PBMC and TLC/LC. We also localized the melatonin membrane receptor MT1 in spleen and BMMNCs that strongly supported our western blot analysis for the expression of MT1 in spleen suggesting that different photoperiodically induced circulatory melatonin is responsible for the immunomodulation. Therefore, photoperiod can influence the peripheral melatonin level to improve immune responses of hamsters according to season for better survival.

  9. Induction of RET dependent and independent pro-inflammatory programs in human peripheral blood mononuclear cells from Hirschsprung patients.

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    Marta Rusmini

    Full Text Available Hirschsprung disease (HSCR is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes and adaptive (B and T lymphocytes human peripheral blood mononuclear cells (PBMCs and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1 trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1 and cytokines (IL-1β, IL-6 and IL-8 and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα. Although at different levels, the modulation of these "RET-dependent genes" occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these "RET-independent genes", there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of

  10. Infected peripheral blood mononuclear cells transmit latent varicella zoster virus infection to the guinea pig enteric nervous system.

    Science.gov (United States)

    Gan, Lin; Wang, Mingli; Chen, Jason J; Gershon, Michael D; Gershon, Anne A

    2014-10-01

    Latent wild-type (WT) and vaccine (vOka) varicella zoster virus (VZV) are found in the human enteric nervous system (ENS). VZV also infects guinea pig enteric neurons in vitro, establishes latency and can be reactivated. We therefore determined whether lymphocytes infected in vitro with VZV secrete infectious virions and can transfer infection in vivo to the ENS of recipient guinea pigs. T lymphocytes (CD3-immunoreactive) were preferentially infected following co-culture of guinea pig or human peripheral blood mononuclear cells with VZV-infected HELF. VZV proliferated in the infected T cells and expressed immediate early and late VZV genes. Electron microscopy confirmed that VZV-infected T cells produced encapsulated virions. Extracellular virus, however, was pleomorphic, suggesting degradation occurred prior to release, which was confirmed by the failure of VZV-infected T cells to secrete infectious virions. Intravenous injection of WT- or vOka-infected PBMCs, nevertheless, transmitted VZV to recipient animals (guinea pig > human lymphocytes). Two days post-inoculation, lung and liver, but not gut, contained DNA and transcripts encoding ORFs 4, 40, 66 and 67. Twenty-eight days after infection, gut contained DNA and transcripts encoding ORFs 4 and 66 but neither DNA nor transcripts could any longer be found in lung or liver. In situ hybridization revealed VZV DNA in enteric neurons, which also expressed ORF63p (but not ORF68p) immunoreactivity. Observations suggest that VZV infects T cells, which can transfer VZV to and establish latency in enteric neurons in vivo. Guinea pigs may be useful for studies of VZV pathogenesis in the ENS.

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

    Science.gov (United States)

    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.

  12. Activation of expression in peripheral blood mononuclear cells in Sonic hedgehog pathway%外周血单个核细胞在Sonic hedgehog信号通路中的活化表达

    Institute of Scientific and Technical Information of China (English)

    赵乾焜; 梁森; 闫慧明

    2015-01-01

    Objective To discuss the expression of Sonic hedgehog blocking antibody (Shh Ab) in peripheral blood mononuclear cells(PBMCs) of anti-gastric cancer cells. Methods Healthy human PBM-Cs were centrifugated by Ficoll density gradient (GES-1 cells were treated by nitrite amides) and gastric cancer cells were co-cultured with MC. To observe the expression and semi-quantitative analysis of Shh and Gli-1 by RT-PCR, Shh blocking antibodies were added in the co-culture system, and the expression of CD3, CD5, CD69 was assayed by flow cytometry. Results RT-PCR showed that Gli-1mRNA in MC+Shh Ab cell group was 0.284 5±0.002 5, lower than the MC cell group 0.516 7 ± 0.010 9 (P<0.05). Flow cytometry showed Shh blocking antibodies promoted the expression of CD3 and CD69. Shh Ab enhanced cell killing PBMCs for MC. Conclusion Shh Ab can promote activation of PBMCs, and enhances anti-carcinogenic effect of nitrite amides.%目的探讨Sonic hedgehog阻断抗体(Shh Ab)对外周血单个核细胞(PBMCs)抗胃癌MC细胞作用的表达。方法 Ficoll密度梯度离心法分离正常人PBMCs,并与胃癌MC细胞(GES-1细胞经亚硝酰胺类化合物处理)建立共培养体系;RT-PCR观察Shh、Gli-1基因的表达并进行半定量数据分析;于共培养体系中加入Shh阻断抗体,流式细胞术检测CD3、CD5、CD69分子表达。结果RT-PCR结果显示Gli-1mRNA在MC+Shh Ab细胞组值为0.2845±0.0025,低于MC 细胞组的0.5167±0.0109(P<0.05);流式细胞检测Shh Ab可促进CD3、CD69分子表达,对CD5分子没有显著影响;Shh Ab增强PBMCs对MC细胞的杀伤。结论 Shh Ab可促进PBMCs化,增强PBMCs抗亚硝酰胺类化合物致癌机制的作用。

  13. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA

    Directory of Open Access Journals (Sweden)

    Cinar Mehmet Ulas

    2013-02-01

    Full Text Available Abstract Background As an in vitro model porcine peripheral blood mononuclear cells (PBMCs is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P  Conclusion There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder. In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram

  14. Manutenção da viabilidade das células mononucleares de sangue periférico humano em extratos e formulações de própolis = Maintenance of human peripheral blood mononuclear cell viability in propolis extracts and formulations

    Directory of Open Access Journals (Sweden)

    Ana Regina Casaroto

    2012-01-01

    Full Text Available Neste estudo comparou-se a viabilidade das células mononucleares de sangue periférico (PBMCs humano quando mantidas em diferentes extratos e formulações de própolis. As PBMCs (106 cels mL-1, provenientes de doadores saudáveis (n=5, foram estocadas a 20ºC nas diferentes soluções de própolis, assim como em solução salina balanceada de Hank’s (HBSS, utilizada como controle do experimento. A viabilidade celular foi determinada pelo método de exclusão com azul de Tripan. Quando incubadas por 1h, apenas dois extratos, denominados A70D e D70D, apresentaram desempenho satisfatório na manutenção da viabilidade celular, semelhante (p > 0,05 ao controle HBSS e diferindo estatisticamente (p In this study a comparison was made of human mononuclear cell (PBMCs viability, when cells were kept in different propolis extracts and formulations. PBMCs (106 cell mL-1, obtained from healthy donors (n=5, were incubated at 20ºC in the different propolis solutions, as well as in Hank’s balanced salt solution (HBSS, used as experimental control. The cell viability was analyzed by Trypan blue exclusion assay. When incubated for 1h, only two extracts, denominated A70D and D70D, showed appropriate results for maintaining cell viability. A70D and D70D showed better viability (p 0.05 from the HBSS control. A70D and D70D were tested in five dilutions in propylene glycol, over 24h, with analysis at 0, 30 minutes, 1, 3, 6, 10 and 24h. The most concentrated fractions showed the worst performance (p < 0.05 in comparison with their original extracts, which remained close to 80% viability over 24h. Reduction in viability proportional to increase in concentration of formulations was observed. The results suggest that propolis solutions in appropriate concentrations may be used in future studies on alternatives to mediums routinely used in dental practice for storing avulsed teeth.

  15. A meta-analysis of public microarray data identifies gene regulatory pathways deregulated in peripheral blood mononuclear cells from individuals with Systemic Lupus Erythematosus compared to those without.

    Science.gov (United States)

    Kröger, Wendy; Mapiye, Darlington; Entfellner, Jean-Baka Domelevo; Tiffin, Nicki

    2016-11-15

    Systemic Lupus Erythematosus (SLE) is a complex, multi-systemic, autoimmune disease for which the underlying aetiological mechanisms are poorly understood. The genetic and molecular processes underlying lupus have been extensively investigated using a variety of -omics approaches, including genome-wide association studies, candidate gene studies and microarray experiments of differential gene expression in lupus samples compared to controls. This study analyses a combination of existing microarray data sets to identify differentially regulated genetic pathways that are dysregulated in human peripheral blood mononuclear cells from SLE patients compared to unaffected controls. Two statistical approaches, quantile discretisation and scaling, are used to combine publicly available expression microarray datasets and perform a meta-analysis of differentially expressed genes. Differentially expressed genes implicated in interferon signaling were identified by the meta-analysis, in agreement with the findings of the individual studies that generated the datasets used. In contrast to the individual studies, however, the meta-analysis and subsequent pathway analysis additionally highlighted TLR signaling, oxidative phosphorylation and diapedesis and adhesion regulatory networks as being differentially regulated in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to controls. Our analysis demonstrates that it is possible to derive additional information from publicly available expression data using meta-analysis techniques, which is particularly relevant to research into rare diseases where sample numbers can be limiting.

  16. Effect of chronic low dose natural radiation in human peripheral blood mononuclear cells: Evaluation of DNA damage and repair using the alkaline comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, P.R. Vivek, E-mail: prvkumar06@gmail.com [Low Level Radiation Research Laboratory, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, IRE Campus, Beach Road, Kollam 691 001, Kerala (India); Seshadri, M. [Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India); Jaikrishan, G. [Low Level Radiation Research Laboratory, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, IRE Campus, Beach Road, Kollam 691 001, Kerala (India); Das, Birajalaxmi [Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India)

    2015-05-15

    Highlights: • Effect of chronic low dose natural radiation in radio adaptive response studied. • PBMCs of subjects from NLNRA and HLNRA were challenged with gamma radiation. • DNA damage and repair in PBMCs was compared using the alkaline comet assay. • Significant reduction in DNA damage in subjects of high dose group from HLNRA noted. • Probable induction of an in vivo radio adaptive response in subjects from HLNRA. - Abstract: This study investigates whether peripheral blood mononuclear cells (PBMCs) from inhabitants of Kerala in southwest India, exposed to chronic low dose natural radiation in vivo (>1 mSv year{sup −1}), respond with a radioadaptive response to a challenging dose of gamma radiation. Toward this goal, PBMCs isolated from 77 subjects from high-level natural radiation areas (HLNRA) and 37 subjects from a nearby normal level natural radiation area (NLNRA) were challenged with 2 Gy and 4 Gy gamma radiation. Subjects from HLNRA were classified based on the mean annual effective dose received, into low dose group (LDG) and high dose group (HDG) with mean annual effective doses of 2.69 mSv (N = 43, range 1.07 mSv year{sup −1} to 5.55 mSv year{sup −1}) and 9.62 mSv (N = 34, range 6.07 mSv year{sup −1} to17.41 mSv year{sup −1}), respectively. DNA strand breaks and repair kinetics (at 7 min, 15 min and 30 min after 4 Gy) were evaluated using the alkaline single cell gel electrophoresis (comet) assay. Initial levels of DNA strand breaks observed after either a 2 Gy or a 4 Gy challenging dose were significantly lower in subjects of the HDG from HLNRA compared to subjects of NLNRA (2 Gy, P = 0.01; 4 Gy, P = 0.02) and LDG (2 Gy P = 0.01; 4 Gy, P = 0.05). Subjects of HDG from HLNRA showed enhanced rejoining of DNA strand breaks (HDG/NLNRA, P = 0.06) during the early stage of repair (within 7 min). However at later times a similar rate of rejoining of strand breaks was observed across the groups (HDG, LDG and NLNRA). Preliminary results from

  17. Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction.

    Science.gov (United States)

    Paludo, F J O; Bristot, I J; Alho, C S; Gelain, D P; Moreira, J C F

    2014-02-01

    Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.

  18. Hypomethylation of long interspersed nucleotide element-1 in peripheral mononuclear cells of juvenile systemic lupus erythematosus patients in China.

    Science.gov (United States)

    Huang, Xiaolan; Su, Gaixiu; Wang, Zhen; Shangguan, Shaofang; Cui, Xiaodai; Zhu, Jia; Kang, Min; Li, Shengnan; Zhang, Ting; Wu, Fengqi; Wang, Li

    2014-03-01

    Methylation abnormalities in T lymphocytes have been reported to correlate with systemic lupus erythematosus (SLE). Previous studies identified hypomethylation in the promoter of several genes linked to SLE. Long interspersed nucleotide element-1 (LINE-1) constitutes 17-25% of the human genome, and LINE-1 hypomethylation has been reported in SLE. Limited information is available regarding LINE-1 methylation in juvenile SLE (JSLE). Methylation levels of LINE-1 in peripheral blood mononuclear cells (PBMCs) from 59 JSLE and 47 control samples were examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total homocysteine (tHcy) concentrations in plasma were measured by immunoassay. Significant hypomethylation of LINE-1 was observed in PBMCs from JSLE patients (60.93% in cases compared with 62.88% in controls, P = 0.001). Significant LINE-1 hypomethylation was observed in active SLE compared to controls (60.66% vs. 62.88%, P = 0.001). According to other clinical parameters, a significant correlation was found between LINE-1 methylation levels and the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2000) of the cases (r = -0.285, P = 0.032). The risk of JSLE increased with decreasing levels of LINE-1 methylation, with an odds ratio of 14.5 (95% CI: 2.8-75.6, P = 0.002). Cases had significantly higher plasma concentrations of tHcy than controls (15.11 vs. 11.02 μmol/L, P = 0.028); the correlation between LINE-1 methylation levels and tHcy was significant (r = -0.4, P = 0.013). Correlations between methylation levels of LINE-1 and complement component 3 were significant (r = 0.317, P = 0.044; r = 0.387, P = 0.031, in total JSLE and active JSLE, respectively). Hypomethylation of LINE-1 is associated with risk of JSLE, and LINE-1 methylation levels were related to disease activity and clinical manifestations. The correlation between tHcy levels and LINE-1 methylation was significant. © 2013 Asia Pacific League of

  19. Shorter telomeres in peripheral blood mononuclear cells from older persons with sarcopenia: results from an exploratory study

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    Emanuele eMarzetti

    2014-08-01

    Full Text Available Background. Telomere shortening in peripheral blood mononuclear cells (PBMCs has been associated with biological age and several chronic degenerative diseases. However, the relationship between telomere length and sarcopenia, a hallmark of the aging process, is unknown. The aim of the present study was therefore to determine whether PBMC telomeres obtained from sarcopenic older persons were shorter relative to non-sarcopenic peers. We further explored if PBMC telomere length was associated with frailty, a major clinical correlate of sarcopenia.Methods. Analyses were conducted in 142 persons aged >/= 65 years referred to a geriatric outpatient clinic (University Hospital. The presence of sarcopenia was established according to the European Working Group on Sarcopenia in Older People criteria, with bioelectrical impedance analysis used for muscle mass estimation. The frailty status was determined by both the Fried’s criteria (physical frailty, PF and a modified Rockwood’s frailty index (FI. Telomere length was measured in PBMCs by quantitative real-time polymerase chain reaction according to the Telomere/Single copy gene ratio (T/S method.Results. Among 142 outpatients (mean age 75.0 ± 6.5 years, 59.2% women, sarcopenia was diagnosed in 23 individuals (19.3%. The PF phenotype was detected in 74 participants (52.1%. The average FI score was 0.46 ± 0.17. PBMC telomeres were shorter in sarcopenic subjects (T/S = 0.21; 95% CI: 0.18 – 0.24 relative to non-sarcopenic individuals (T/S = 0.26; 95%: CI: 0.24 – 0.28; p = 0.01, independent of age, gender, smoking habit, or comorbidity. No significant associations were determined between telomere length and either PF or FI.Conclusion. PBMC telomere length, expressed as T/S values, is shorter in older outpatients with sarcopenia. The cross-sectional assessment of PBMC telomere length is not sufficient at capturing the complex, multidimensional syndrome of frailty.

  20. Transcriptomic Response of Porcine PBMCs to Vaccination with Tetanus Toxoid as a Model Antigen

    Science.gov (United States)

    Adler, Marcel; Murani, Eduard; Brunner, Ronald; Ponsuksili, Siriluck; Wimmers, Klaus

    2013-01-01

    The aim of the present study was to characterize in vivo genome-wide transcriptional responses to immune stimulation in order to get insight into the resulting changes of allocation of resources. Vaccination with tetanus toxoid was used as a model for a mixed Th1 and Th2 immune response in pig. Expression profiles of PBMCs (peripheral blood mononuclear cells) before and at 12 time points over a period of four weeks after initial and booster vaccination at day 14 were studied by use of Affymetrix GeneChip microarrays and Ingenuity Pathway Analysis (IPA). The transcriptome data in total comprised more than 5000 genes with different transcript abundances (DE-genes). Within the single time stages the numbers of DE-genes were between several hundred and more than 1000. Ingenuity Pathway Analysis mainly revealed canonical pathways of cellular immune response and cytokine signaling as well as a broad range of processes in cellular and organismal growth, proliferation and development, cell signaling, biosynthesis and metabolism. Significant changes in the expression profiles of PBMCs already occurred very early after immune stimulation. At two hours after the first vaccination 679 DE-genes corresponding to 110 canonical pathways of cytokine signaling, cellular immune response and other multiple cellular functions were found. Immune competence and global disease resistance are heritable but difficult to measure and to address by breeding. Besides QTL mapping of immune traits gene expression profiling facilitates the detection of functional gene networks and thus functional candidate genes. PMID:23536793

  1. Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever

    National Research Council Canada - National Science Library

    Sasaki, K; Yamagishi, N; Kizaki, K; Sasaki, K; Devkota, B; Hashizume, K

    2014-01-01

    .... Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes...

  2. Studies of biological properties of Uncaria tomentosa extracts on human blood mononuclear cells.

    Science.gov (United States)

    Bors, Milena; Michałowicz, Jaromir; Pilarski, Radosław; Sicińska, Paulina; Gulewicz, Krzysztof; Bukowska, Bożena

    2012-08-01

    Uncaria tomentosa (Willd.) DC is a lignified climbing plant from South and Central America, which (under the name of "vilcacora" or "cat's claw") has become highly popular in many countries due to its proven immunostimmulatory and anti-inflammatory activities and also with respect to its anticancer and antioxidative effects. There are insufficient data on the mechanism of U. tomentosa action on normal blood mononuclear cells. The aim of the study was to analyze the impact of ethanol and aqueous extracts from bark and leaves of Uncaria tomentosa on the structure and function of human mononuclear cells and to find out whether the kind of extractant used modulates biological activity of the extracts studied. Plant material consisted of four different extracts: (1) ethanol extract from leaves, (2) aqueous extract from leaves, (3) ethanol extract from bark and (4) aqueous extract from bark. The effect of these extracts on protein damage as well as on free-radical formation in human peripheral blood mononuclear cells was analyzed. Moreover, changes in viability, size, and granularity as well as apoptotic alterations in human blood mononuclear cells exposed to U. tomentosa extracts were investigated. The oxidative changes were observed in mononuclear blood cells exposed to both ethanol and aqueous extracts obtained from bark and leaves. Moreover, in the cells studied the extracts from U. tomentosa induced apoptosis and a decrease in viability of mononuclear blood cells, with the exception of aqueous extract from leaves. Additionally, no statistically significant changes in the cell size were observed both for aqueous extracts from leaves and bark. Changes in the blood mononuclear cell granularity were observed at 250 μg/mL for all extracts examined. The strongest changes were observed for the ethanol extract of the bark, which increased cell granularity at 50 μg/mL and changed cell size at 100 μg/mL. The conducted research showed differences in biological activity

  3. RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with disease progression in dengue patients.

    Science.gov (United States)

    Banerjee, Arup; Shukla, Shweta; Pandey, Abhay Deep; Goswami, Saptamita; Bandyopadhyay, Bhaswati; Ramachandran, Vishnampettai; Das, Shukla; Malhotra, Arjun; Agarwal, Amitesh; Adhikari, Srima; Rahman, Mehebubar; Chatterjee, Shatakshee; Bhattacharya, Nemai; Basu, Nandita; Pandey, Priyanka; Sood, Vikas; Vrati, Sudhanshu

    2017-08-01

    Patients infected with Dengue virus usually present a mild, self-limiting febrile dengue infection (DI) that occasionally leads to a potentially lethal complication, called the severe dengue (DS). The ability to identify the prognostic markers of DS could allow an improved disease intervention and management. To identify the transcriptional signatures associated with the dengue disease progression, we carried out the high-throughput sequencing of the RNA isolated from the peripheral blood mononuclear cells (PBMCs) of the dengue patients of varying severity and compared with that in the patients with other febrile illnesses (OFIs) or the healthy controls. The transcriptional signatures that discriminated the DS patients from OFI and DI patients were broadly related to the pathways involving glycine, serine, and threonine metabolisms, extracellular matrix organization, ubiquitination, and cytokines and inflammatory response. Several upregulated genes in the inflammatory process (MPO, DEFA4, ELANE, AUZ1, CTSG, OLFM4, SLC16A14, and CRISP3) that were associated with the dengue disease progression are known to facilitate leukocyte-mediated migration, and neutrophil activation and degranulation process. High activity of MPO and ELANE in the plasma samples of the follow-up and recovered dengue patients, as well as and the presence of a larger amount of cell-free dsDNA in the DS patients, suggested an association of neutrophil-mediated immunity with dengue disease progression. Careful monitoring of some of these gene transcripts, and control of the activity of proteins encoded by them, may have a great translational significance for the prognosis and management of the dengue patients. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Conditioning causes an increase in glucose transporter-4 levels in mononuclear cells in sled dogs.

    Science.gov (United States)

    Schnurr, Theresia M; Reynolds, Arleigh J; Gustafson, Sally J; Duffy, Lawrence K; Dunlap, Kriya L

    2014-10-01

    This study was designed to investigate the effects of physical conditioning on the expression of the insulin sensitive glucose transporter-4 protein (GLUT4) on mononuclear cells and HOMA-IR levels in dogs and compared to results reported in human skeletal muscle and the skeletal muscle of rodent models. Blood was sampled from conditioned dogs (n = 8) and sedentary dogs (n = 8). The conditioned dogs were exercised four months prior the experiment and were following a uniform training protocol, whereas the sedentary dogs were not. GLUT4 expression in mononuclear cells and plasma insulin levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA). Blood glucose levels were determined using blood plasma. HOMA-IR was calculated using plasma insulin and blood glucose levels using the linear approximation formula. Our results indicate that the state of conditioning had a significant effect on the GLUT4 expression at the surface of mononuclear cells. HOMA-IR was also affected by conditioning in dogs. GLUT4 levels in mononuclear cells of sled dogs were inversely correlated with the homeostasis model assessment of insulin sensitivity. This study demonstrates that conditioning increases GLUT4 levels in mononuclear cells of sled dogs as it has been previously reported in skeletal muscle. Our results support the potential of white blood cells as a proxy tissue for studying insulin signaling and may lead to development of a minimally invasive and direct marker of insulin resistance. This may be the first report of GLUT4 in mononuclear cells in response to exercise and measured with ELISA.

  5. INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.

    Science.gov (United States)

    Franklin, Ashley D; Crosier, Adrienne E; Vansandt, Lindsey M; Mattson, Elliot; Xiao, Zhengguo

    2015-06-01

    Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of cheetahs (Acinonyx jubatus ; n=3) and stimulated with lipopolysaccharides (LPS) to induce the production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 for establishment of cross-reactivity between these cheetah cytokines and feline-specific cytokine antibodies provided in commercially available Feline DuoSet® ELISA kits (R&D Systems, Inc., Minneapolis, Minnesota 55413, USA). This study found that feline-specific cytokine antibodies bind specifically to cheetah proinflammatory cytokines TNF-α, IL-1β, and IL-6 from cell culture supernatants. The assays also revealed that cheetah PBMCs produce a measurable, cell concentration-dependent increase in proinflammatory cytokine production after LPS stimulation. To enable the use of these kits, which are designed for cell culture supernatants for analyzing cytokine concentrations in cheetah serum, percent recovery and parallelism of feline cytokine standards in cheetah serum were also evaluated. Cytokine concentrations in cheetah serum were approximated based on the use of domestic cat standards in the absence of cheetah standard material. In all cases (for cytokines TNF-α, IL-1β, and IL-6), percent recovery increased as the serum sample dilution increased, though percent recovery varied between cytokines at a given dilution factor. A 1:2 dilution of serum resulted in approximately 45, 82, and 7% recovery of TNF-α, IL-1β, and IL-6 standards, respectively. Adequate parallelism was observed across a large range of cytokine concentrations for TNF-α and IL-1β; however, a significant departure from parallelism was observed between the IL-6 standard and the serum samples (P=0.004). Therefore, based on our results, the Feline DuoSet ELISA (R&D Systems, Inc.) kits are valid assays for the measurement of TNF-α and IL-1β in cheetah serum but should not be used for accurate measurement of IL-6.

  6. Effect of Blood Component Coatings of Enosseal Implants on Proliferation and Synthetic Activity of Human Osteoblasts and Cytokine Production of Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Hulejova, Hana; Bartova, Jirina; Riedel, Tomas; Pesakova, Vlasta

    2016-01-01

    The study monitored in vitro early response of connective tissue cells and immunocompetent cells to enosseal implant materials coated by different blood components (serum, activated plasma, and plasma/platelets) to evaluate human osteoblast proliferation and synthetic activity and inflammatory response presented as a cytokine profile of peripheral blood mononuclear cells (PBMCs) under conditions imitating the situation upon implantation. The cells were cultivated on coated Ti-plasma-sprayed (Ti-PS), Ti-etched (Ti-Etch), Ti-hydroxyapatite (Ti-HA), and ZrO2 surfaces. The plasma/platelets coating supported osteoblast proliferation only on osteoconductive Ti-HA and Ti-Etch whereas activated plasma enhanced proliferation on all surfaces. Differentiation (BAP) and IL-8 production remained unchanged or decreased irrespective of the coating and surface; only the serum and plasma/platelets-coated ZrO2 exhibited higher BAP and IL-8 expression. RANKL production increased on serum and activated plasma coatings. PBMCs produced especially cytokines playing role in inflammatory phase of wound healing, that is, IL-6, GRO-α, GRO, ENA-78, IL-8, GM-CSF, EGF, and MCP-1. Cytokine profiles were comparable for all tested surfaces; only ENA-78, IL-8, GM-CSF, and MCP-1 expression depended on materials and coatings. The activated plasma coating led to uniformed surfaces and represented a favorable treatment especially for bioinert Ti-PS and ZrO2 whereas all coatings had no distinctive effect on bioactive Ti-HA and Ti-Etch. PMID:27651560

  7. Changes in some pro-and anti-inflammatory cytokines produced by bovine peripheral blood mononuclear cells following foot and mouth disease vaccination

    Directory of Open Access Journals (Sweden)

    N. Delirezh

    2016-09-01

    Full Text Available Interleukin (IL-17 is exclusively produced by CD4 helper T-cells upon activation. It most often acts as a pro-inflammatory cytokine, which stimulates the release of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF. In this study, we studied the in-vitro IL-17 response to specific antigens and a variety of mitogens and compared the IL-17 response to IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-γ responses. We used a foot and mouth disease (FMD vaccine as specific antigens and mitogens (phytohemagglutinin [PHA], pokeweed mitogen [PWM], and concanavalin A [Con A] to stimulate peripheral blood mononuclear cells (PBMCs of vaccinated calves. Cell culture supernatant was harvested and analyzed for cytokines, using commercially available bovine ELISA kits. The mitogens induced a significant increase in IL-17 production. IL-17 was produced at high levels in response to the T cell-stimulated mitogens, PHA, and Con A, and at low levels in response to PWM mitogens. In contrast, level of the produced IL-17 cytokines in response to the FMDV antigens was lower as compared to those produced by mitogens. The FMDV antigens and mitogens significantly increased IL-17 production. There was not a correlation between IL-17 production and type-1 cytokine, IFN-γ, and IL-2, while there was a correlation between type-2 cytokine, IL-4, and IL-5 at either cytokine level produced by PBMCs stimulated by FMDV antigens. Moreover, there was an interaction between IL-17 and IL-6, that is, as IL-6 cytokine level elevated or diminished, IL-17 cytokine level increased or decreased, as well.

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

    NARCIS (Netherlands)

    Jeurink, P.V.; Lull Noguera, C.; Savelkoul, H.F.J.; Wichers, H.J.

    2008-01-01

    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 (Agaric

  9. Intracoronary infusion of mononuclear cells after PCI-treated myocardial infarction and arrhythmogenesis : is it safe?

    NARCIS (Netherlands)

    Robbers, L. F. H. J.; Nijveldt, R.; Beek, A. M.; Kemme, M. J. B.; Delewi, R.; Hirsch, Alexander; van der Laan, A. M.; van der Vleuten, P. A.; Piek, J. J.; Zijlstra, F.; van Rossum, A. C.

    2012-01-01

    To reduce long-term morbidity after revascularised acute myocardial infarction, different therapeutic strategies have been investigated. Cell therapy with mononuclear cells from bone marrow (BMMC) or peripheral blood (PBMC) has been proposed to attenuate the adverse processes of remodelling and subs

  10. Mononuclear cells in subcutaneous haemorrhage with special consideration of myeloid percursor cells.

    Science.gov (United States)

    Oehmichen, M; Windisch, A; Meissner, C

    2000-10-01

    Various hematogenous markers were used to differentiate and quantify the types of mononuclear cells present in subcutaneous haemorrhages. Fifty samples of subcutaneous bleeding with a survival time of a few minutes to more than 48 hours were studied. The various cell types were detected using the following stains: Naphthol AS-D chloracetate esterase for myeloid cells, including mast cells; (alpha1-antichymotrypsin for monocytes/macrophages; UCHL1 for T-lymphocytes; and L26 for B lymphocytes. The percentage of monocytes/macrophages was found to increase in dependence on survival time, whereas T-lymphocytes declined. Within minutes of injury neutrophilic granulocytes had emigrated into the surrounding tissue and mast cell degranulation had occurred within the haemorrhagic zone. Esterase-positive mononuclear cells, namely metamyelocytes, were detected within minutes after injury and were still present after survival times exceeding 48 hours; however, no dependence on survival time or cause of death was found. Although the increasing number of monocytes/ macrophages and T-lymphocytes was expected, the sometimes high percentage of myeloid precursor cells within the wound were surprising. Possible explanations for this phenomenon are discussed.

  11.  The impact of IL18 gene polymorphisms on mRNA levels and interleukin-18 release by peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Violetta Dziedziejko

    2012-06-01

    Full Text Available  Introduction:Interleukin-18 (IL-18 is a pleiotropic cytokine playing an important role as a modulator of immune responses, found to play a role in pathogenesis of numerous inflammatory-associated disorders. In the present study a potential association between 7 common single-nucleotide polymorphisms (SNPs spanning the whole IL18 gene, gene expression and the release of IL-18 from the stimulated peripheral blood mononuclear cells (PBMCs was investigated.Materials/Methods:PBMCs were isolated from peripheral blood of 29 healthy volunteers, genotyped for the presence of IL18 SNPs: rs1946518: A>C, rs187238: G>C, rs360718: A>C, rs360722: C>T, rs360721: C>G, rs549908: T>G, and rs5744292: A>G. IL-18 concentration and IL18 mRNA levels were investigated after incubation of cells for 48 h with different stimulants (PHA, LPS, and anti-CD3/CD28 antibodies.Results:After treatment with LPS and antibodies IL-18 concentrations were significantly lower in rs1946518AA homozygotes than in C allele carriers. When differences in IL18 mRNA levels between non-stimulated and stimulated cells were analyzed, significantly decreased gene expression was noted in rs1946518 AA homozygotes (as compared with C allele carriers in samples treated with PHA and LPS. Similar trends were observed in the case of rs187238 SNP; however, the differences reached statistical significance only after PHA treatment.Conclusions:Our study supports the role of rs1946518 (-607A>C and rs187238 (-137G>C SNPs as genetic determinants of the observed variability in IL18 expression.

  12. Differential miRNA expressions in peripheral blood mononuclear cells for diagnosis of lung cancer.

    Science.gov (United States)

    Ma, Jie; Lin, Yanli; Zhan, Min; Mann, Dean L; Stass, Sanford A; Jiang, Feng

    2015-10-01

    Tremendous efforts have been made to develop cancer biomarkers by detecting circulating extracellular miRNAs directly released from tumors. Yet, none of the cell-free biomarkers has been accepted to be used for early detection of non-small cell lung cancer (NSCLC). Peripheral blood mononucleated cells (PBMCs) act as the first line of defense against malignancy in immune system, their dysfunction may occur as an early event in cancer immunogenicity or immune evasion. We proposed to investigate whether analysis of miRNA expressions of PBMCs has diagnostic value for NSCLC. We first used a microarray to analyze PBMCs of 16 stage I NSCLC patients and 16 cancer-free smokers, and identified seven PBMC miRNAs with a significantly altered expression level in NSCLC patients. In a training set of 84 NSCLC patients and 69 cancer-free smokers, a panel of two miRNAs (miRs-19b-3p and -29b-3p) were developed from the seven PBMC miRNAs, producing 72.62% sensitivity and 82.61% specificity in identifying NSCLC. Furthermore, the miRNAs could identify squamous cell lung carcinoma (SCC), a major type of NSCLC, with 80.00% sensitivity and 89.86% specificity. The expression levels of the miRNAs were independent of disease stage. In a testing set of 56 NSCLC patients and 46 controls, the performance of the biomarkers was reproducibly confirmed. The study presents the first in-depth analysis of PBMC miRNA profile of NSCLC patients. The assessment of PBMC miRNAs may provide a new diagnostic approach for the early detection of NSCLC.

  13. Probiotic Bio-Three induces Th1 and anti-inflammatory effects in PBMC and dendritic cells

    Institute of Scientific and Technical Information of China (English)

    Man-Chin; Hua; Tzou-Yien; Lin; Chien-Chang; Chen

    2010-01-01

    AIM:To investigate the immune response of peripheral blood mononuclear cells(PBMCs)and dendritic cells (DCs)that were stimulated by probiotic preparations. METHODS:PBMCs were isolated,cultured,and stimulated with Bio-Three(a mixture of Bacillus mesentericus, Clostridium butyricum and Enterococcus faecalis;105, 10 6 and 10 7 CFU/mL for 24 h).Cytokine production of (1)circulating PBMCs;(2)PBMCs stimulated by probiotic preparation;(3)monocyte-derived DCs;and(4)DC andT cell co-culture was determined by enzyme-l...

  14. Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer

    DEFF Research Database (Denmark)

    Hermann, G G; Petersen, K R; Steven, K

    1990-01-01

    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......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 51Cr-release assays. The PBMC subsets...

  15. A module of human peripheral blood mononuclear cell transcriptional network containing primitive and differentiation markers is related to specific cardiovascular health variables.

    Directory of Open Access Journals (Sweden)

    Leni Moldovan

    Full Text Available Peripheral blood mononuclear cells (PBMCs, including rare circulating stem and progenitor cells (CSPCs, have important yet poorly understood roles in the maintenance and repair of blood vessels and perfused organs. Our hypothesis was that the identities and functions of CSPCs in cardiovascular health could be ascertained by analyzing the patterns of their co-expressed markers in unselected PBMC samples. Because gene microarrays had failed to detect many stem cell-associated genes, we performed quantitative real-time PCR to measure the expression of 45 primitive and tissue differentiation markers in PBMCs from healthy and hypertensive human subjects. We compared these expression levels to the subjects' demographic and cardiovascular risk factors, including vascular stiffness. The tested marker genes were expressed in all of samples and organized in hierarchical transcriptional network modules, constructed by a bottom-up approach. An index of gene expression in one of these modules (metagene, defined as the average standardized relative copy numbers of 15 pluripotency and cardiovascular differentiation markers, was negatively correlated (all p<0.03 with age (R2 = -0.23, vascular stiffness (R2 = -0.24, and central aortic pressure (R2 = -0.19 and positively correlated with body mass index (R2 = 0.72, in women. The co-expression of three neovascular markers was validated at the single-cell level using mRNA in situ hybridization and immunocytochemistry. The overall gene expression in this cardiovascular module was reduced by 72±22% in the patients compared with controls. However, the compactness of both modules was increased in the patients' samples, which was reflected in reduced dispersion of their nodes' degrees of connectivity, suggesting a more primitive character of the patients' CSPCs. In conclusion, our results show that the relationship between CSPCs and vascular function is encoded in modules of the PBMCs transcriptional

  16. Effect of sesamin against cytokine production from influenza type A H1N1-induced peripheral blood mononuclear cells: computational and experimental studies.

    Science.gov (United States)

    Fanhchaksai, Kanda; Kodchakorn, Kanchanok; Pothacharoen, Peraphan; Kongtawelert, Prachya

    2016-01-01

    In 2009, swine flu (H1N1) had spread significantly to levels that threatened pandemic influenza. There have been many treatments that have arisen for patients since the WHO first reported the disease. Although some progress in controlling influenza has taken place during the last few years, the disease is not yet under control. The development of new and less expensive anti-influenza drugs is still needed. Here, we show that sesamin from the seeds of the Thai medicinal plant Sesamum indicum has anti-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) induced by 2009 influenza virus type A H1N1. In this study, the combinatorial screening method combined with the computational approach was applied to investigate the new molecular binding structures of sesamin against the 2009 influenza virus type A H1N1 (p09N1) crystallized structure. Experimental methods were applied to propose the mechanisms of sesamin against cytokine production from H1N1-induced human PBMC model. The molecular dynamics simulation of sesamin binding with the p09N1 crystallized structure showed new molecular binding structures at ARG118, ILE222, ARG224, and TYR406, and it has been proposed that sesamin could potentially be used to produce anti-H1N1 compounds. Furthermore, the mechanisms of sesamin against cytokine production from influenza type A H1N1-induced PBMCs by ELISA and signaling transduction showed that sesamin exhibits the ability to inhibit proinflammatory cytokines, IL-1β and TNF-α, and to enhance the activity of the immune cell cytokine IL-2 via downregulating the phosphorylated JNK, p38, and ERK1/2 MAPK signaling pathways. This information might very well be useful in the prevention and treatment of immune-induced inflammatory disorders.

  17. A hitchhiker's guide to myeloid cell subsets: practical implementation of a novel mononuclear phagocyte classification system

    Directory of Open Access Journals (Sweden)

    Martin eGuilliams

    2015-08-01

    Full Text Available 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.

  18. [Production of mature red blood cell by using peripheral blood mononuclear cells].

    Science.gov (United States)

    Jia, Yan-Jun; Liu, Jiang; Zhang, Ke-Ying; Shang, Xiao-Yan; Li, Wei; Wang, Li-Jun; Liu, Na; Wang, Lin; Cui, Shuang; Ni, Lei; Zhao, Bo-Tao; Wang, Dong-Mei; Gao, Song-Ming; Zhang, Zhi-Xin

    2014-10-01

    Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.

  19. Nuclear thyroid hormone receptor binding in human mononuclear blood cells after goitre resection

    DEFF Research Database (Denmark)

    Kvetny, J; Matzen, L E; Blichert-Toft, M

    1989-01-01

    Nuclear thyroxine and triiodothyronine receptor-binding in human mononuclear blood cells were examined in 14 euthyroid persons prior to and 1, 6, 24 and 53 weeks after goitre resection. One week after resection decreased serum T3 from 1.47 nmol/l to 1.14 nmol/l (P less than 0.05), FT4I from 103 a...

  20. Slc27a2 expression in peripheral blood mononuclear cells as a marker for overweight development

    NARCIS (Netherlands)

    Caimari, A.; Oliver, P.; Rodenburg, W.; Keijer, J.; Palou, A.

    2010-01-01

    Background: Peripheral blood mononuclear cells (PBMC) can be collected easily and repeatedly. Their potential use to reflect the individual's biological status is increasingly explored. Obesity is becoming the most common health problem of the 21st century, being dietary intake an important determin

  1. Interleukin-8 transcripts in mononuclear cells determine impaired graft function after kidney transplantation

    DEFF Research Database (Denmark)

    Borst, Christoffer; Xia, Shengqiang; Bistrup, Claus

    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 ...

  2. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

    2014-01-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred ...

  3. Generation of induced pluripotent stem cells with high efficiency from human umbilical cord blood mononuclear cells.

    Science.gov (United States)

    Wang, Juan; Gu, Qi; Hao, Jie; Bai, Donghui; Liu, Lei; Zhao, Xiaoyang; Liu, Zhonghua; Wang, Liu; Zhou, Qi

    2013-10-01

    Human induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. Generating iPSCs from immunologically immature newborn umbilical cord blood mononuclear cells (UCBMCs) is of great significance. Here we report generation of human iPSCs with great efficiency from UCBMCs using a dox-inducible lentiviral system carrying four Yamanaka factors. We generated these cells by optimizing the existing iPSC induction protocol. The UCBMC-derived iPSCs (UCB-iPSCs) have characteristics that are identical to pluripotent human embryonic stem cells (hESCs). This study highlights the use of UCBMCs to generate highly functional human iPSCs that could accelerate the development of cell-based regenerative therapy for patients suffering from various diseases.

  4. Vigorous, but differential mononuclear cell response of cirrhotic patients to bacterial ligands

    Institute of Scientific and Technical Information of China (English)

    Varenka J Barbero-Becerra; María Concepción Gutiérrez-Ruiz; Carmen Maldonado-Bernal; Félix I Téllez-Avila; Roberto Alfaro-Lara; Florencia Vargas-Vorácková

    2011-01-01

    AIM: To study the role of gram-positive and gram-negative bacteria in the pathogenesis of liver injury, specifically the activation of inflammatory mediators. METHODS: Peripheral blood mononuclear cells of 20 out-patients were studied, 10 of them with cirrhosis. Peripheral blood mononuclear cells were isolated and exposed to lipopolysaccharide or lipoteichoic acid. CD14, Toll-like receptor 2 and 4 expression was determined by flow cytometry, and tumor necrosis factor (TNF) α, interleukin (IL)-1β, IL-6, IL-12 and IL-10 secretion in supernatants was determined by ELISA. RESULTS: Higher CD14, Toll-like receptor 2 and 4 expression was observed in peripheral blood mononuclear cells from cirrhotic patients, (P < 0.01, P < 0.006, P < 0.111) respectively. Lipopolysaccharide and lipoteichoic acid induced a further increase in CD14 expression (P < 0.111 lipopolysaccharide, P < 0.013 lipoteichoic acid), and a decrease in Toll-like receptor 2 (P < 0.008 lipopolysaccharide, P < 0.008 lipoteichoic acid) and Toll-like receptor 4 (P < 0.008 lipopolysaccharide, P < 0.028 lipoteichoic acid) expression. With the exception of TNFα, absolute cytokine secretion of peripheral blood mononuclear cells was lower in cirrhotic patients under nonexposure conditions (P < 0.070 IL-6, P < 0.009 IL-1β, P < 0.022 IL-12). Once exposed to lipopolysaccharide or lipoteichoic acid, absolute cytokine secretion of peripheral blood mononuclear cells was similar in cirrhotic and non-cirrhotic patients, determining a more vigorous response in the former (P < 0.005 TNFα, IL-1β, IL-6, IL-2 and IL-10 lipopolysaccharide; P < 0.037 TNFα; P < 0.006 IL-1β; P < 0.005 IL-6; P < 0.007 IL-12; P < 0.014 IL-10 lipoteichoic acid). Response of peripheral blood mononuclear cells was more intense after lipopolysaccharide than after lipoteichoic acid exposure. CONCLUSION: Peripheral blood mononuclear cells of cirrhotic patients are able to respond to a sudden bacterial ligand exposure, particularly lipopolysaccharide

  5. Expression of CCR5 in peripheral blood mononuclear cells in patients with psoriasis%银屑病患者外周血单个核细胞CCR5的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    黄远忠; 董正蓉; 林伯盛; 马丹晓

    2011-01-01

    目的 研究银屑病患者外周血单个核细胞趋化因子受体5(CCR5)的表达及其与银屑病皮损面积和严重程度指数(PASI)的关系.方法 分离35例中重度寻常型银屑病患者及30例健康对照者外周血单个核细胞(PBMCs),用RT-PCR法测定PBMCs培养前后CCR5 mRNA的表达水平,免疫荧光标记一流式细胞仪检测CCR5阳性细胞比率.结果 治疗前银屑病患者外周血单个核细胞中CCR5 mRNA表达水平明显高于治疗后及健康对照组,并与PASI呈正相关(r=0.516,P<0.05).结论 CCR5可能通过活化与趋化单个核细胞到银屑病皮损而参与了银屑病的发病.%Objective To investigate the expression of CCR5 in the peripheral blood mononuclear cells (PBMCs) in patients with psoriasis and the correlation between CCR5 expression and psoriasis area and severity index (PASI).Methods PBMCs were isolated from 35 moderate to severe psoriasis patients and 30 health control. Expression of CCR5 mRNA and the rate of CCR5 positive cells on PBMC before and after culture was respectively detected by RT-PCR and immunofluorescence marker flow cytometry. Results CCR5 mRNA level in PBMCs from untreated psoriasis patients was higher than those in treated patients and normal controls. CCR5 mRNA expression was positively correlated with PASI (r=0.516,P<0.05). Conclusion Our study suggests that CCR5 may be involved in the pathogenesis of the progression of psoriasis by activating and aggregating mononuclear cells into psoriasis lesions.

  6. Expression and Significance of Toll-like Receptor 2, 4 of Peripheral Blood Mononuclear Cells in Acute Abdomen Patients Associated with Systemic Inflammatory Response Syndrome

    Institute of Scientific and Technical Information of China (English)

    XIONG Jing; WANG Yang; ZHU Zhonghua; LIU Jianshe

    2006-01-01

    The changes of Toll-like receptor (TLR) 2, 4 of peripheral blood mononuclear cells (PBMCs) in the acute abdomen patients associated with systemic inflammatory response syndrome (SIRS) and their potential significance were explored. A clinical study was performed on 103 acute abdomen patients in whom 65 were associated with SIRS. Forty healthy individuals served as normal controls. The mRNA expression of TLR2, 4 was detected by RT-PCR, and the expression of TNF-αand IL-6 by ELISA. The level of plasma endotoxin, hospital stay and mortality were measured. It was found that the endotoxin level was increased to varying degrees in all the acute abdomen patients, and the endotoxin level was and hospital stay longer in SIRS group than in non-SIRS group (P<0.01).TLR2 mRNA, TLR4 mRNA, IL-6 and TNF-α could be detected with low value in normal controls,but they were up-regulated markedly on the 1 st day after admission. Then TLR4 mRNA, IL-6 and TNF-α were decreased gradually, but TLR2 mRNA maintained at a high level till the 5th day. These indexes above in SIRS group were higher than those in non-SIRS group (P<0.01). The results of correlation analysis revealed the expression of TLR2, 4 mRNA was positively correlated with the levels of TNF-α and IL-6, and the hospital stay. The results of Logistic regression demonstrated that overexpression of TLR2, 4 mRNA might result in higher risk of multiple organ dysfunction syndrome (MODS). It was concluded that in the acute abdomen patients associated with SIRS, the expression of TLR2, 4 in PBMCs was increased markedly, suggesting that TLR might play an important role in the pathogenesis of acute abdomen associated with SIRS.

  7. Peripheral blood mononuclear cells as a source to detect markers of homeostatic alterations caused by the intake of diets with an unbalanced macronutrient composition.

    Science.gov (United States)

    Díaz-Rúa, Rubén; Keijer, Jaap; Caimari, Antoni; van Schothorst, Evert M; Palou, Andreu; Oliver, Paula

    2015-04-01

    Peripheral blood mononuclear cells (PBMCs) are accessible in humans, and their gene expression pattern was shown to reflect overall physiological response of the body to a specific stimulus, such as diet. We aimed to study the impact of sustained intake (4months) of diets with an unbalanced macronutrient proportion (rich in fat or protein) administered isocalorically to a balanced control diet, as physiological stressors on PBMC whole-genome gene expression in rats, to better understand the effects of these diets on metabolism and health and to identify biomarkers of nutritional imbalance. Dietary macronutrient composition (mainly increased protein content) altered PBMC gene expression, with genes involved in immune response being the most affected. Intake of a high-fat (HF) diet decreased the expression of genes related to antigen recognition/presentation, whereas the high-protein (HP) diet increased the expression of these genes and of genes involved in cytokine signaling and immune system maturation/activation. Key energy homeostasis genes (mainly related to lipid metabolism) were also affected, reflecting an adaptive response to the diets. Moreover, HF diet feeding impaired expression of genes involved in redox balance regulation. Finally, we identified a common gene expression signature of 7 genes whose expression changed in the same direction in response to the intake of both diets. These genes, individually or together, constitute a potential risk marker of diet macronutrient imbalance. In conclusion, we newly show that gene expression analysis in PBMCs allows for detection of diet-induced physiological deviations that distinguish from a diet with a proper and equilibrated macronutrient composition. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Effects of selenium on peripheral blood mononuclear cell membrane fiuidity,interleukin-2 production and interleukin-2 receptor expression in patients with chronic hepatitis

    Institute of Scientific and Technical Information of China (English)

    Shui-Xiang He; Bing Wu; Xin-Ming Chang; Hong-Xia Li; Wen Qiao

    2004-01-01

    AIM: To study the effect of selenium on peripheral blood mononuclear cell (PBMC) membrane fluidity and immune function in patients with chronic hepatitis.METHODS: PBMCs were pretreated with selenium (1.156x 10-7 mol/L) for 6 h in vitro or extracted directly from patients after administration of selenium-yeast continuously for 8-12 wk (200 μg/d), and then exposed to Con-A for 48 h. The membrane fluidity, interleukin-2 (IL-2) production and interleukin-2 receptor (IL-2R) expression in PBMCs and malondialdehyde (MDA) concentration in medium and lipid peroxide (LPO) in plasma were determined.RESULTS: The PBMC membrane fluidity, IL-2 production and IL-2R expression in patients with chronic hepatitis were significantly lower than those in healthy blood donators (particle adhesive degree R, 0.17±0.01 vs0.14±0.01,P<0.01; IL-2, 40.26±9.55 vs72.96±11.36, P<0.01; IL-2R,31.05±5.09 vs 60.58±10.56, P<0.01), and the MDA concentration in medium in patients with chronic hepatitis was significantly higher than that in healthy blood donators (1.44±0.08 vs0.93±0.08, P<0.01). Both in vitro and in vivo administration of selenium could reverse the above parameters.CONCLUSION: Supplement of selenium can suppress lipid peroxidation, and improve PBMC membrane fluidity and immune function in patients with chronic hepatitis.

  9. Genotoxic and cytostatic effects of 6-pentadecyl salicylic anacardic acid in transformed cell lines and peripheral blood mononuclear cells.

    Science.gov (United States)

    Alam-Escamilla, David; Estrada-Muñiz, Elizabet; Solís-Villegas, Erik; Elizondo, Guillermo; Vega, Libia

    2015-01-01

    In Mexico, as in many other countries, traditional medicine is used for the treatment of several diseases. In particular, Amphipterygium adstringens infusion is used for gastritis, gastric ulcers, and gastric cancer. Extracts from this tree have microbicidal effects against Helicobacter pylori, an important risk factor for gastric cancer development. Anacardic acids are constituents of A. adstringens, and 6-pentadecyl salicylic acid (6-PSA) is the most abundant. However, there is a lack of information regarding the effects of 6-PSA on cancer cells. Therefore, we investigated whether 6-PSA has differential effects on the induction of genotoxicity, cytostaticity, and apoptosis in normal human peripheral blood mononucleated cells (PBMCs), bone marrow polychromatic erythrocytes of Balb/c mice, and human transformed cell lines derived from both gastric cancer (AGS cells) and leukaemia (K562 cells). Treatment with 6-PSA (30-150 μM) reduced the viability of AGS and K562 cells together with a moderate, but significant, increase in the frequency of micronucleated cells and the induction of DNA breakage (Comet Assay). Moreover, 6-PSA increased the apoptosis rate in both the AGS and K562 cell lines in a caspase 8-dependent manner. In contrast, neither cytotoxicity nor genotoxicity were observed in PBMCs or bone marrow polychromatic erythrocytes of Balb/c mice after treatment with low doses of 6-PSA (0.2-2.0 mg/Kg). Instead, 6-PSA treatment resulted in the inhibition of PBMC proliferation, which was reversible after the compound was removed. Additionally, 6-PSA treatments (2-20 mg/Kg) increased the frequency of mature polychromatic erythrocytes in the bone marrow, suggesting a possible effect on the differentiation process of immune cells. The present results indicate that 6-PSA induces cytotoxicity and moderate genotoxicity, together with an increase in the apoptosis rate, in a caspase 8-dependent manner in gastric cancer cells. In contrast, a low toxicity was observed when

  10. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W

    1999-01-01

    Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS...

  11. A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

    DEFF Research Database (Denmark)

    Moesby, Lise; Jensen, S; Hansen, E W;

    1999-01-01

    Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS...

  12. Multiparametric analyses of human PBMCs loaded ex vivo with a candidate idiotype vaccine for HCV-related lymphoproliferative disorders.

    Directory of Open Access Journals (Sweden)

    Annacarmen Petrizzo

    Full Text Available Hepatitis C virus (HCV has been identified as one of the major risk factors for type II mixed cryoglobulinemia (MC, during the clinical evolution of chronic hepatitis, which may lead to development of B cell non-Hodgkin's lymphoma (NHL. We have previously shown that the candidate idiotype vaccine, based on the IGKV3-20 light chain protein, is able to induce activation and maturation of circulating antigen presenting cells (APCs in both HCV-positive and HCV-negative healthy control subjects, with production of Th2-type cytokines. Here, the effect of the recombinant IGKV3-20 protein on human peripheral blood mononuclear cells (PBMCs from HCV-positive subjects, with known blood levels of cryoglobulins, is shown via gene expression profiling analysis combined to multiparameter flow cytometry and multiplex analyses of cytokines.

  13. Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues.

    Science.gov (United States)

    Scoville, Steven D; Keller, Karen A; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A; Freud, Aharon G

    2015-07-30

    We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method.

  14. Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

    Science.gov (United States)

    Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

    2015-01-01

    We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

  15. [Influence of Opiate Abuse on Expression of Toll-like Receptor 9 in Peripheral Blood Mononuclear Cells of HIV-1-Infected Individuals].

    Science.gov (United States)

    Pan, Peijiang; Wei, Fumei; Jiang, Junjun; Liang, Bingyu; Huang, Jiegang; Liao, Yanyan; Su, Jinming; Li, Yu; Yang, Xiaoyi; Chen, Hui; Ye, Li; Liang, Hao

    2015-03-01

    The aim of this study was to investigate the influence of opiate abuse on the expression of Toll-like receptor 9 (TLR9) in the peripheral blood mononuclear cells (PBMCs) of HIV-1-infected patients and to elucidate possible mechanisms involved in the enhancement of HIV-1 replication by opiate abuse. A total of 200 participants were enrolled in the study by random selection from methadone treatment centers and voluntary HIV counseling and testing centers in the cities of Nanning, Liuzhou, and Qinzhou. These participants included 50 HIV-positive opiate abusers (Opiates HIV(+) group), 50 HIV-negative opiate abusers (Opiates HIV(-) group), 50 HIV-positive subjects who were not opiate abusers (Non-opiates HIV (+) group), and 50 HIV-negative subjects who were not opiate abusers (Control group). PBMCs were isolated from the peripheral blood samples from the subjects and the expression levels of TLR9 mRNA and protein were determined by q-PCR and western blot respectively. There was no significant difference among the four groups in age, gender, nationality, domicile, marital status, educational background or duration of drug abuse (P > 0.05). The median viral loads of the Opiates HIV(+) were significantly higher than those of the Non-Opiates HIV(+) groups (4.450 x 10(3) and 3.977 x 10(3) copies/mL respectively, P Opiates HIV(+), Non-Opiates HIV(+), Opiates HIV(-) and Control groups were (2.13 +/- 1.59) x 10(-3), (3.66 +/- 2.22) x 10(-3), (1.96 +/- 1.42) x 10(-3) and (7.66 +/- 4.87) x 10(-3), respectively. The expression of TLR9 mRNA was significantly lower in both HIV-1-infected and -uninfected groups of opiate abusers compared with groups of non-abusers (P Opiates HIV(+) group and the Opiates HIV(-) group (P > 0.05). However, in the non-opiate groups, the expression levels of TLR9 mRNA in the HIV(+) group were significantly lower than that of the control group (POpiates HIV(+), Non-Opiates HIV(+), and Opiates HIV(-) groups compared to the control group. These results

  16. Expression of matrix metalloproteinase-1 mRNA in peripheral blood mononuclear cells of systemic lupus erythematosus patients and its relationship with atherosclerosis

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-ying; BAO Shu-meng; SHOU Wei-ling; LUAN Hai-xia; ZHANG Yang; FENG Xue; TONG Da-wei; ZHANG Shu-lan; HU Chao-jun; ZENG Xiao-feng; LI Yong-zhe

    2009-01-01

    Background Matrix metalloproteinase-1(MMP-1)plays an important role in atherosclerosis.This study was to examine expression of MMP-1 mRNA in peripheral blood mononuclear cells(PBMCs)of patients with systemic lupus erythematosus(SLE),and to explore its relationship with atherosclerosis in SLE.Methods Fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)was used to examine the expression of MMP-1 mRNA in PBMCs in 80 SLE patients,including 39 prone to atherosclerosis(Group A)and 41 unprone to atherosclerosis(Group B).Meanwhile,30 patients who were free of cardiovascular diseases and 30 healthy individuals were selected as disease and normal control group(Groups C and D).The changes of MMP-1 gene expression were analyzed by differences of cycle threshold(△Ct),with the following formula:△Ct = Ct_(target) gene-Ct_(reference) gene.Results The expression level of MMP-1 mRNA in Group A was significantly higher than that of group B(△Ct=8.64±2.43 vs △Ct=12.09±2.26,t=6.588,P<0.01).The expression level of MMP-1 mRNA of SLE patients was significantly higher than that of Group C(△Ct=10.41±2.90 vs △Ct=12.29±2.51,t=-3.135,P<0.01)and Group D(△Ct=10.41±2.90 vs △Ct=12.48±1.69,t=3.675,P<0.01).Conclusions In comparison to disease and control group,expression of MMP-1 mRNA in PBMCs of SLE patients was significantly elevated,and significant difference of MMP-1 mRNA expression was also found between SLE patients prone and unprone to atherosclerosis,indicating that expression of MMP-1 mRNA may be correlated with the pathogenesis and activity of atherosclerosis in SLE.

  17. mRNA Expression of Chemokine Receptors on Peripheral Blood Mononuclear Cells and Correlation with Clinical Features in Systemic Lupus Erythematosus Patients

    Institute of Scientific and Technical Information of China (English)

    Yu-mei Li; Zhi-qiang Chen; Xu Yao; Ai-zhen Yang; An-sheng Li; Dong-ming Liu; Juan-qin Gong

    2010-01-01

    Objective To investigate the expressions of chemokine receptors and interleukin (1L) receptors on the peripheral blood mononuclear cells (PBMCs) from systemic lupus erythematosus (SLE) patients and their correlations with clinical features as well as SLE disease activity index (SLEDAI).Methods The mRNA expressions of ehemokine receptors and IL receptors on PBMCs of 93 SLE patients and 30 healthy controls were detected by reverse transcription-polymerase chain reaction, including CCR2, CCR3, CCR4, CCRS, CCR6, CCR8, CXCR3, CXCRS, CX3CR1, XCR1, IL-4R, and IL-10R. The clinical features of SLE patients were recorded. The correlations of chemokine receptors and IL receptors mRNA expressions with clinical features as well as SLEDAI were assayed using linear regression analysis.Results The level of CCR5 mRNA in SLE patients (including active and inactive SLE) was signifi-cantly higher than that in healthy controls (P0.05). CX3CR1 mRNA expression significantly increased from healthy control to inactive SLE to active SLE in sequence. The others (except for CCR8, CXCR3, and IL-10R) in active SLE patients were significantly higher than those in both inactive SLE patients and healthy controls (all P<0.05). There were positive correlations between SLEDAI and CCR2 (r=0.424, t=4.313, P<0.001), CCR3 (r=0.518, t=5.410, P<0.001), CCR4 (r=0.376, t=3.851, P<0.001), CCR6 (r=0.457, t=4.513, P<0.001), CXCR5 (r=0.455, t=4.629, P<0.001), CX3CR1 (r=0.445, t=4.523, P<0.001), as well as XCR1 (r=0.540, t=5.445, P<0.001). And CCR5 mRNA expression level was positively correlated with IL-4R mRNA (r=0.313, t=2.353, P<0.05). The patients with myositis and cutaneous vasculitis simultaneously showed lower levels of CCR5 and CX3CR1, and CCR5 expression was negatively correlated with the scores of SLEDAI in SLE cases accompanied by photosensitivity (r=0.426, t=-2.155, P<0.05).Conclusion Increased expressions of CCR5 and CX3CR1 on PBMCs may be indicators in clinical survey for SLE.

  18. Modulation of adhesion molecules by cholesterol-lowering therapy in mononuclear cells from hypercholesterolemic patients.

    Science.gov (United States)

    Cerda, Alvaro; Rodrigues, Alice Cristina; Alves, Camila; Genvigir, Fabiana Dalla Vecchia; Fajardo, Cristina Moreno; Dorea, Egidio Lima; Gusukuma, Maria Cecilia; Pinto, Gelba Almeida; Hirata, Mario Hiroyuki; Hirata, Rosario Dominguez Crespo

    2015-08-01

    Cholesterol-lowering therapy has been related with several pleiotropic effects including anti-inflammatory action in vascular endothelium; however, their influence on monocyte adhesion molecules is poorly described. To investigate the effect of inhibitors of synthesis (statins) and absorption (ezetimibe) of cholesterol on expression of adhesion molecules L-selectin, PSGL-1, VLA-4, LFA-1, and Mac-1 in mononuclear cells in vivo and in vitro using THP-1 cells. The influence of simvastatin (10 mg/day), ezetimibe (10 mg/day), and their combination (10 mg each/day) on mRNA expression of adhesion molecules was analyzed in peripheral blood mononuclear cells (PBMC) from hypercholesterolemics. The effects of atorvastatin, simvastatin, and ezetimibe on mRNA and protein expression of adhesion molecules were also evaluated in THP-1 cells. Simvastatin/ezetimibe combination, but not the monotherapies, reduced the mRNA expression of the PSGL-1, LFA-1, and Mac-1 genes in PBMC from hypercholesterolemics. Total and LDL cholesterol in serum correlated with PSGL-1 mRNA expression, whereas HDL cholesterol negatively correlated with mRNA levels of L-selectin and VLA-4 genes (P molecules in PBMC from hypercholesterolemics and THP-1 cells. Simvastatin/ezetimibe combination gives more benefits by reducing to a larger extent the expression of adhesion molecules in mononuclear cells. © 2015 John Wiley & Sons Ltd.

  19. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  20. Effects of carvedilol on oxidative stress in polymorphonuclear and mononuclear cells in patients with essential hypertension.

    Science.gov (United States)

    Yasunari, Kenichi; Maeda, Kensaku; Nakamura, Munehiro; Watanabe, Takanori; Yoshikawa, Junichi; Asada, Akira

    2004-04-01

    To compare the effects of carvedilol and propranolol on oxidative stress in leukocytes and C-reactive protein levels in patients with hypertension. Sixty hypertensive patients were randomly assigned to carvedilol (20 mg; n = 30) or propranolol (60 mg; n = 30) for 6 months. Thirty normotensive subjects who were given placebo served as controls. Oxidative stress in polymorphonuclear cells and mononuclear cells were measured by gated flow cytometry. C-reactive protein levels were measured by immunonephelometric assay. Oxidative stress in polymorphonuclear cells and mononuclear cells was increased significantly in hypertensive patients compared with in normotensive controls. After 6 months of treatment, carvedilol decreased oxidative stress significantly in polymorphonuclear cells by a mean of 45 arbitrary units (95% confidence interval [CI]: 32 to 59 arbitrary units; P <0.001) and propranolol decreased oxidative stress significantly by 20 arbitrary units (95% CI: 7 to 33 arbitrary units; P <0.003; P = 0.001 for difference between treatments). Carvedilol also decreased oxidative stress significantly in mononuclear cells by 23 arbitrary units (95% CI: 15 to 31 arbitrary units; P <0.001), whereas propranolol decreased oxidative stress by 2 arbitrary units (95% CI: 7 to 12 arbitrary units; P = 0.62; P = 0.002 for difference between treatments). Carvedilol decreased C-reactive protein levels significantly by a median of 0.073 mg/dL (interquartile range, 0.034 to 0.112 mg/dL; P <0.001), whereas propranolol decreased levels by 0.012 mg/dL (interquartile range, 0.009 to 0.032 mg/dL; P = 0.26; P = 0.003 for difference between treatments). These findings suggest that carvedilol inhibits oxidative stress in polymorphonuclear and mononuclear cells, as well as lowers C-reactive protein levels, to a greater extent than does propranolol in hypertensive patients.

  1. Dynamic acquisition of HTLV-1 tax protein by mononuclear phagocytes: Role in neurologic disease.

    Science.gov (United States)

    Matsuura, Eiji; Enose-Akahata, Yoshimi; Yao, Karen; Oh, Unsong; Tanaka, Yuetsu; Takashima, Hiroshi; Jacobson, Steven

    2017-03-15

    Pathology of HTLV-1 associated myelopathy/Tropical spastic paraparesis (HAM/TSP) is believed to be the result of "bystander damage" involving effector CD8 (+) T lymphocytes (CTLs) killing of virus infected cells. But the specific cellular events leading up to tissue injury are still unclear. Here, we developed the Microscopy Imaging of Cytotoxic T lymphocyte assay with Fluorescence emission (MI-CaFé), an optimized visualization analysis to explore the interactions between CTLs and virus infected or viral antigen presenting target cells. Various cell-to-cell formations can be observed and our results demonstrate elevated frequencies of CTL-target cell conjugates in HAM/TSP patient PBMCs compared to control PBMCs. Furthermore, HTLV-1 Tax protein expression can be localized at the cell-cell junctions and also tracked moving from an infected cell to a CD14 (+) mononuclear phagocyte (MP). Activation of CD14 (+) MPs in HAM/TSP patient PBMCs and antigenic presentation of HTLV-1 Tax by MPs can be inferred by their spontaneous cytotoxicity after 18h of in vitro culture. Given that CD4 (+) T lymphocytes are the primary reservoirs of HTLV-1 and MPs are scavenger cells responsible for pathogen clearance, spontaneous cytotoxicity against MPs in HAM/TSP PBMCs suggests a mechanism of chronic inflammation, secondary to low level of persistent virus infection within the central nervous system. Published by Elsevier B.V.

  2. Adhesion of subsets of human blood mononuclear cells to porcine endothelial cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cellular immune response is a major barrier to xenotransplantation, and cell adhesion is the first step in intercellular recognition. Flow-cytometric adhesion assay has been used to investigate the differential adhesions of monocyte (Mo), natural killer cell (NK) and T lymphocyte (T) present within human peripheral blood mononuclear cells (PBMC) to porcine aortic endothelial cells (PAEC), and to demonstrate the effect of human interferon-γ(hIFN-γ) or/and tumor necrosis factor-α (hTNF-α) pretreatment of PAEC on their adhesiveness for different PBMC subsets. The preferential sequence for PBMC subset binding to resting PAEC is Mo, NK and T cells, among which T cells show the slightest adherence; hTNF-α can act across the species, and augment Mo, NK and T cell adhesion ratios by 40%, 110% and 3 times, respectively. These results confirm at the cell level that host Mo and NK cells are major participants in the cellular xenograft rejection, thereby, providing a prerequisite for further studying the human Mo/NK-PAEC interactive mechanisms.

  3. Tax secretion from peripheral blood mononuclear cells and Tax detection in plasma of patients with human T-lymphotropic virus-type 1-associated myelopathy/tropical spastic paraparesis and asymptomatic carriers.

    Science.gov (United States)

    Medina, Fernando; Quintremil, Sebastián; Alberti, Carolina; Godoy, Fabián; Pando, María E; Bustamante, Andrés; Barriga, Andrés; Cartier, Luis; Puente, Javier; Tanaka, Yuetsu; Valenzuela, María A; Ramírez, Eugenio

    2016-03-01

    Human T-lymphotropic virus-type 1 (HTLV-1) is the etiologic agent of the neurologic disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Tax viral protein plays a critical role in viral pathogenesis. Previous studies suggested that extracellular Tax might involve cytokine-like extracellular effects. We evaluated Tax secretion in 18 h-ex vivo peripheral blood mononuclear cells (PBMCs) cultures from 15 HAM/TSP patients and 15 asymptomatic carriers. Futhermore, Tax plasma level was evaluated from other 12 HAM/TSP patients and 10 asymptomatic carriers. Proviral load and mRNA encoding Tax were quantified by PCR and real-time RT-PCR, respectively. Intracellular Tax in CD4(+)CD25(+) cells occurred in 100% and 86.7% of HAM/TSP patients and asymptomatic carriers, respectively. Percentage of CD4(+)CD25(+) Tax+, proviral load and mRNA encoding Tax were significantly higher in HAM/TSP patients. Western blot analyses showed higher secretion levels of ubiquitinated Tax in HAM/TSP patients than in asymptomatic carriers. In HTLV-1-infected subjects, Western blot of plasma Tax showed higher levels in HAM/TSP patients than in asymptomatic carriers, whereas no Tax was found in non-infected subjects. Immunoprecipitated plasma Tax resolved on SDS-PAGE gave two major bands of 57 and 48 kDa allowing identification of Tax and Ubiquitin peptides by mass spectrometry. Relative percentage of either CD4(+)CD25(+) Tax+ cells, or Tax protein released from PBMCs, or plasma Tax, correlates neither with tax mRNA nor with proviral load. This fact could be explained by a complex regulation of Tax expression. Tax secreted from PBMCs or present in plasma could potentially become a biomarker to distinguish between HAM/TSP patients and asymptomatic carriers.

  4. Protective effects of curcumin on methylglyoxal-induced oxidative DMA damage and cell injury in human mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Wen-hsiung CHAN; Hsin-jung WU

    2006-01-01

    Aim: To examine the effect of curcumin on oxidative DNA damage and cell apoptosis and injury caused by the reaction of methylglyoxal(MG) with amino acids. Methods: We used DNA strand breaks to examine the effect of curcumin on oxidative DNA damage. In addition, reactive oxygen species(ROS) formation occurs in MG-treated mononuclear cells, so the effect of curcumin on ROS generation was measured using 2',7'-dichlorofluorescin diacetate(DCF-DA) as the detection reagent. Moreover, the impact effects of curcumin on MG-induced cell apoptosis and ROS injury were analyzed by TUNEL and ELISA assay. The collagen I attachment ability of mononuclear cells was examined by trypan blue staining. Results: Our results revealed that curcumin prevented MG/lysine-induced oxidative stress and DNA damage. Curcumin also inhibited MG-induced apoptosis and generation of ROS in mononuclear cells. MG-treated mononuclear cells displayed a lower degree of attachment to collagen (the major component of the vessel wall subendo-thelium), whereas cells pretreated with curcumin before MG treatment exhibited restored affinities for collagen. Conclusion: These results demonstrated that oxidative stress plays a role in MG-induced cell injury and alterations in attachment ability, and that curcumin blocks these effects by virtue of its antioxidant properties.

  5. Effect of malaria components on blood mononuclear cells involved in immune response.

    Science.gov (United States)

    Punsawad, Chuchard

    2013-09-01

    During malaria infection, elevated levels of pro-inflammatory mediators and nitric oxide production have been associated with pathogenesis and disease severity. Previous in vitro and in vivo studies have proposed that both Plasmodium falciparum hemozoin and glycosylphosphatidylinositols are able to modulate blood mononuclear cells, contributing to stimulation of signal transduction and downstream regulation of the NF-κB signaling pathway, and subsequently leading to the production of pro-inflammatory cytokines, chemokines, and nitric oxide. The present review summarizes the published in vitro and in vivo studies that have investigated the mechanism of intracellular signal transduction and activation of the NF-κB signaling pathway in blood mononuclear cells after being inducted by Plasmodium falciparum malaria components. Particular attention is paid to hemozoin and glycosylphosphatidylinositols which reflect the important mechanism of signaling pathways involved in immune response.

  6. Effect of malaria components on blood mononuclear cells involved in immune response

    Institute of Scientific and Technical Information of China (English)

    Chuchard Punsawad

    2013-01-01

    During malaria infection, elevated levels of pro-inflammatory mediators and nitric oxide production have been associated with pathogenesis and disease severity. Previous in vitro and in vivo studies have proposed that both Plasmodium falciparum hemozoin and glycosylphosphatidylinositols are able to modulate blood mononuclear cells, contributing to stimulation of signal transduction and downstream regulation of the NF-κB signaling pathway, and subsequently leading to the production of pro-inflammatory cytokines, chemokines, and nitric oxide. The present review summarizes the published in vitro and in vivo studies that have investigated the mechanism of intracellular signal transduction and activation of the NF-κB signaling pathway in blood mononuclear cells after being inducted by Plasmodium falciparum malaria components. Particular attention is paid to hemozoin and glycosylphosphatidylinositols which reflect the important mechanism of signaling pathways involved in immune response.

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

    Science.gov (United States)

    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.

  8. Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells.

    Science.gov (United States)

    Montes de Oca, Pável; Macotela, Yazmín; Nava, Gabriel; López-Barrera, Fernando; de la Escalera, Gonzalo Martínez; Clapp, Carmen

    2005-05-01

    Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1beta. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

  9. MHC-unrestricted lysis of MUC1-expressing cells by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wright, Stephen E; Rewers-Felkins, Kathleen A; Quinlin, Imelda S; Fogler, William E; Phillips, Catherine A; Townsend, Mary; Robinson, William; Philip, Ramila

    2008-01-01

    Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

  10. Treatment with at Homeopathic Complex Medication Modulates Mononuclear Bone Marrow Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Beatriz Cesar

    2011-01-01

    Full Text Available A homeopathic complex medication (HCM, with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells. Mice were our biological model used in this research. We now report in vivo immunophenotyping of total bone marrow cells and ex vivo effects of the medication on mononuclear cell differentiation at different times. Cells were examined by light microscopy and cytokine levels were measured in vitro. After in vivo treatment with HCM, a pool of cells from the new marrow microenvironment was analyzed by flow cytometry to detect any trend in cell alteration. The results showed decreases, mainly, in CD11b and TER-119 markers compared with controls. Mononuclear cells were used to analyze the effects of ex vivo HCM treatment and the number of cells showing ring nuclei, niche cells and activated macrophages increased in culture, even in the absence of macrophage colony-stimulating factor. Cytokines favoring stromal cell survival and differentiation in culture were induced in vitro. Thus, we observe that HCM is immunomodulatory, either alone or in association with other products.

  11. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng;

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold pe...

  12. Intra-arterial Infusion of Autologous Bone Marrow Mononuclear Stem Cells in Subacute Ischemic Stroke Patients.

    Science.gov (United States)

    Ghali, Azza Abass; Yousef, Mohamed Khalil; Ragab, Osama AbdAllah; ElZamarany, Enas Arafa

    2016-01-01

    Based on many preclinical and small clinical trials, stem cells can help stroke patient with the possibility of replacing the cells and supporting the remaining cells. The aim of this study was to evaluate the safety and feasibility of bone marrow mononuclear (BMMN) stem cell transplantation in subacute ischemic stroke patients. Thirty-nine (n = 39) patients with subacute ischemic cerebral infarct due to large artery occlusion in the middle cerebral artery (MCA) territory were recruited. They were distributed into two groups: first group (n = 21) served as an experimental group, which received intra-arterial (IA) mononuclear stem cells (bone marrow-derived mononuclear cell), while the other group (n = 18) served as a control group. All the patients were evaluated clinically by National Institutes of Health Stroke Scale, modified Rankin Scale, Barthel Index, modified and standardized Arabic version of the Comprehensive Aphasia Test, and radiological for 12 months. The stem cell-treated group showed better improvement, but it was not significant when compared with the non-treated group. The volume of infarction changes at the end of the study was non-significant between both the groups. There was no, or minimal, adverse reactions in stem cell-treated group. The study results suggest that autologous BMMN stem cell IA transplantation in subacute MCA ischemic stroke patients is safe with very minimal hazards, but no significant improvement of motor, language disturbance, or infarction volume was detected in stem cell-treated group compared with the non-treated group.

  13. miRNA-146a, miRNA-155 and JNK expression levels in peripheral blood mononuclear cells according to grade of knee osteoarthritis.

    Science.gov (United States)

    Soyocak, Ahu; Kurt, Hulyam; Ozgen, Merih; Turgut Cosan, Didem; Colak, Ertugrul; Gunes, Hasan Veysi

    2017-09-05

    Osteoarthritis (OA) is the most common joint disease characterized by joint pain and a progressive loss of articular cartilage. OA known as a non-inflammatory disease. Despite this the recent studies are shown synovitis and low inflammation to have a role in OA pathophysiology. The aim of this study to determine the roles of a potential therapeutic targets miRNA-146a, miRNA-155 and JNK expression levels in OA patients. Peripheral mononuclear blood cells (PBMCs) were extracted from OA patients and healthy subjects. The expression levels of miRNA-146a, miRNA-155 and JNK were quantified using by real-time PCR assay. According to study results a statistically significant increase was observed only in miRNA-155 expression level (p=0,039). However, miRNA-146a and miRNA-155 expressions increased in the progressive stages (grade 3 and grade 4) in OA patients. Our data suggests that correlation of miRNAs regulating and signal pathways can play an important role in OA pathogenesis and disease progression. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Differential expression of oxidative stress and inflammation related genes in peripheral blood mononuclear cells in response to a low-calorie diet: a nutrigenomics study.

    Science.gov (United States)

    Crujeiras, Ana B; Parra, Dolores; Milagro, Fermín I; Goyenechea, Estibaliz; Larrarte, Eider; Margareto, Javier; Martínez, J Alfredo

    2008-12-01

    Nutrigenomics is a new application of omics technologies in nutritional science. Nutrigenomics aims to identify molecular markers of diet-related diseases and mechanisms of interindividual variability in response to food. The aim of this study was to evaluate peripheral blood mononuclear cells (PBMC) as a model system and readily available source of RNA to discern gene expression signatures in relation to personalized therapy of obesity. PBMC were collected from obese men before and after an 8-week low-calorie diet (LCD) to lose weight. Changes in gene expression before and after the LCD were initially screened using a DNA-microarray platform and validated by qRT-PCR. Global gene expression analysis identified 385 differentially expressed transcripts after the LCD. Further analyses showed a decrease in some specific oxidative stress and inflammation genes. Interestingly, expression of these genes was directly related to body weight, while a lower IL8 gene expression was associated with higher fat mass decrease. Collectively, these observations suggest that PBMCs are a suitable RNA source and model system to perform nutrigenomics studies related to obesity and development of personalized dietary treatments. IL8 gene expression warrant further research as a putative novel biomarker of changes in body fat percentage in response to an LCD.

  15. The Peripheral Blood Mononuclear Cell Count Is Associated With Bone Health in Elderly Men: A Cross-Sectional Population-Based Study.

    Science.gov (United States)

    Lin, Xianfeng; Yu, Hejun; Zhao, Chenchen; Qian, Yu; Hong, Dun; Huang, Kangmao; Mo, Jian; Qin, An; Fang, Xiangqian; Fan, Shunwu

    2016-04-01

    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) blood pressure (SBP) count is significantly associated with bone loss in elderly men and the exact mechanism requires further clarification.

  16. Expression of Caspase-1 Gene Transcript Variant mRNA in Peripheral Blood Mononuclear Cells of Patients with Primary Gout in Different TCM Syndromes

    Science.gov (United States)

    Dang, Wan-Tai; Xu, Dan; Xie, Wen-Guang; Zhou, Jing-Guo

    2015-01-01

    A large number of studies have shown that cysteinyl aspartate specific protease-1 (CASP1) played an important role in the inflammatory response of primary gout, but the decreased expression of different CASP1 transcript variant could inhibit the activation of IL-1β. Our study mainly analyzed the expression level and function of CASP1 gene transcript variant mRNA in peripheral blood mononuclear cells of patients with gout in different TCM syndromes. The expression of CASP1 gene transcript variant and IL-1β mRNA in PBMCs were detected in patients with PG [acute phase (AP: 44 cases); nonacute phase (NAP: 52 cases)] and healthy controls (HC: 30 cases) by reverse transcription-polymerase chain reaction and/or real-time quantitative polymerase chain reaction. The expressions of plasma IL-1β in patients with PG and HC were detected by enzyme-linked immunosorbent assay. Dysregulated expression of the CASP1 gene and its transcript variant, plasma proinflammatory cytokines in all patients with primary gout in different TCM syndromes, correlation analysis showed that there was negative correlation between the expression of CASP1-gamma gene transcript variant mRNA and IL-1β protein in APPG group. The study suggested that CASP1 gene and its transcript variant may play a critical role in the inflammatory response of patients with PG in different phases and TCM syndromes. PMID:26557856

  17. Expression of Caspase-1 Gene Transcript Variant mRNA in Peripheral Blood Mononuclear Cells of Patients with Primary Gout in Different TCM Syndromes

    Directory of Open Access Journals (Sweden)

    Wan-Tai Dang

    2015-01-01

    Full Text Available A large number of studies have shown that cysteinyl aspartate specific protease-1 (CASP1 played an important role in the inflammatory response of primary gout, but the decreased expression of different CASP1 transcript variant could inhibit the activation of IL-1β. Our study mainly analyzed the expression level and function of CASP1 gene transcript variant mRNA in peripheral blood mononuclear cells of patients with gout in different TCM syndromes. The expression of CASP1 gene transcript variant and IL-1β mRNA in PBMCs were detected in patients with PG [acute phase (AP: 44 cases; nonacute phase (NAP: 52 cases] and healthy controls (HC: 30 cases by reverse transcription-polymerase chain reaction and/or real-time quantitative polymerase chain reaction. The expressions of plasma IL-1β in patients with PG and HC were detected by enzyme-linked immunosorbent assay. Dysregulated expression of the CASP1 gene and its transcript variant, plasma proinflammatory cytokines in all patients with primary gout in different TCM syndromes, correlation analysis showed that there was negative correlation between the expression of CASP1-gamma gene transcript variant mRNA and IL-1β protein in APPG group. The study suggested that CASP1 gene and its transcript variant may play a critical role in the inflammatory response of patients with PG in different phases and TCM syndromes.

  18. Sex differences in cyclosporine pharmacokinetics and ABCB1 gene expression in mononuclear blood cells in African American and Caucasian renal transplant recipients.

    Science.gov (United States)

    Tornatore, Kathleen M; Brazeau, Daniel; Dole, Kiran; Danison, Ryan; Wilding, Gregory; Leca, Nicolae; Gundroo, Aijaz; Gillis, Kathryn; Zack, Julia; DiFrancesco, Robin; Venuto, Rocco C

    2013-10-01

    Cyclosporine exhibits pharmacokinetic and pharmacodynamic variability in renal transplant recipients (RTR) attributed to P-glycoprotein (P-gp), an ABCB1 efflux transporter that influences bioavailability and intracellular distribution. Data on race and sex influences on P-gp in RTR are lacking. We investigated sex and race influences on cyclosporine pharmacokinetics and ABCB1 gene expression in peripheral blood mononuclear cells (PBMC). Fifty-four female and male African American and Caucasian stable RTR receiving cyclosporine and mycophenolic acid completed a 12-hour study. ABCB1 gene expression was assessed in PBMCs pre-dose and 4 hours after cyclosporine. Statistical analysis used mixed effects models on transformed, normalized ABCB1 expression and cyclosporine pharmacokinetics. Sex and race differences were observed for the dose-normalized area under the concentration curve (AUC0-12 /Dose) [P = .0004], apparent clearance [P = .0004] and clearance/body mass index (CL/BMI) [P = .027] with slowest clearance and greatest drug exposure in females. Sex and race differences were found pre-dose and 4 hours for ABCB1 [P cyclosporine clearance and lower ABCB1 gene expression in PBMC suggesting reduced efflux activity and greater intracellular drug exposure. © The Author(s) 2013.

  19. Aggressive periodontitis and chronic arthritis: blood mononuclear cell gene expression and plasma protein levels of cytokines and cytokine inhibitors

    DEFF Research Database (Denmark)

    Sørensen, Lars K; Havemose-Poulsen, Anne; Bendtzen, Klaus

    2009-01-01

    BACKGROUND: Cytokines and cytokine inhibitors have been associated with many immunoinflammatory diseases. In the present study, we examined whether peripheral blood mononuclear cell (PBMC) gene expression mirrors the corresponding plasma levels of clinically important pro- and anti-inflammatory c......BACKGROUND: Cytokines and cytokine inhibitors have been associated with many immunoinflammatory diseases. In the present study, we examined whether peripheral blood mononuclear cell (PBMC) gene expression mirrors the corresponding plasma levels of clinically important pro- and anti...

  20. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect...

  1. Validation of using gene expression in mononuclear cells as a marker for hepatic cholesterol metabolism

    Directory of Open Access Journals (Sweden)

    Dutta Amrita

    2006-08-01

    Full Text Available Abstract HMG-CoA reductase and the LDL receptor are ubiquitously expressed in major tissues. Since the liver plays a major role in regulating circulating LDL, it is usually of interest to measure the effects of drug or dietary interventions on these proteins in liver. In humans, peripheral blood mononuclear cells have been used as a surrogate for liver to assess regulation of these genes, although there is concern regarding the validity of this approach. The purpose of this study was to evaluate the relationship between liver and mononuclear cell expression of HMG-CoA reductase and the LDL receptor in guinea pigs, a well established model for human cholesterol and lipoprotein metabolism. We extracted RNA from liver and mononuclear cells of guinea pigs from a previous study where the effects of rapamycin, an immunosuppresant drug used for transplant patients, on lipid metabolism were evaluated. Guinea pigs were assigned to three different diets containing the same amount of fat (15 g/100 g and cholesterol (0.08 g/100 g for a period of 3 weeks. The only difference among diets was the concentration of rapamycin: 0, 0.0028 or 0.028 g/100 g. There were no differences in plasma LDL cholesterol (LDL-C among groups. Values were 78.4 ± 14.3, 65.8 ± 17.2 and 68.4 ± 45.4 mg/dL (P > 0.05 for guinea pigs treated with 0, low or high doses of rapamycin, respectively. The mRNA abundance for the LDL receptor and HMG-CoA reductase was measured both in liver (n = 30 and mononuclear cells (n = 22 using reverse transcriptase PCR. In agreement with the finding of no changes in plasma LDL-C, there were also no differences for the expression of HMG-CoA reductase or the LDL receptor among groups. However, a positive correlation was found between liver and mononuclear cells for both HMG-CoA reductase (r = 0.613, P

  2. Dopamine Agonists Exert Nurr1-inducing Effect in Peripheral Blood Mononuclear Cells of Patients with Parkinson's Disease

    Institute of Scientific and Technical Information of China (English)

    Li-Min Zhang; Cong-Cong Sun; Ming-Shu Mo; Luan Cen; Lei Wei; Fei-Fei Luo; Yi Li

    2015-01-01

    Background:Nurr1 plays an essential role in the development,survival,and function maintenance ofmidbrain dopaminergic (DA) neurons,and it is a potential target for Parkinson's disease (PD).Nurr1 mRNA can be detected in peripheral blood mononuclear cells (PBMCs),but whether there is any association of altered Nurr1 expression in PBMC with the disease and DA drug treatments remains elusive.This study aimed to measure the Nurrl mRNA level in PBMC and evaluate the effect of Nurr1 expression by DA agents in vivo and in vitro.Methods:The mRNA levels of Nurrl in PBMC of four subgroups of 362 PD patients and 193 healthy controls (HCs) using real-time polymerase chain reaction were measured.The nonparametric Mann-Whitney U-test and Kruskal-Wallis test were performed to evaluate the differences between PD and HC,as well as the subgroups of PD.Multivariate linear regression analysis was used to evaluate the independent association of Nurr1 expression with Hoehn and Yahr scale,age,and drug treatments.Besides,the Nurr1 expression in cultured PBMC was measured to determine whether DA agonist pramipexole affects its mRNA level.Results:The relative Nurr1 mRNA levels in DA agonists treated subgroup were significant higher than those in recent-onset cases without any anti-PD treatments (de novo) (P < 0.001) and HC groups (P < 0.010),respectively.Furthermore,the increase in Nurr1 mRNA expression was seen in DA agonist and L-dopa group.Multivariate linear regression showed DA agonists,L-dopa,and DA agonists were independent predictors correlated with Nurrl mRNA expression level in PBMC.In vitro,in the cultured PBMC treated with 10 μmol/L pramipexole,the Nurr1 mRNA levels were significantly increased by 99.61%,71.75%,73.16% in 2,4,and 8 h,respectively (P < 0.001).Conclusions:DA agonists can induce Nurr1 expression in PBMC,and such effect may contribute to DA agonists-mediated neuroprotection on DA neurons.

  3. Differential MicroRNA Analyses of Burkholderia pseudomallei- and Francisella tularensis-Exposed hPBMCs Reveal Potential Biomarkers

    Science.gov (United States)

    Herrera-Galeano, J. Enrique; Frey, Kenneth G.; Schully, Kevin L.; Luu, Truong V.; Pesce, John; Mokashi, Vishwesh P.; Keane-Myers, Andrea M.; Bishop-Lilly, Kimberly A.

    2017-01-01

    Increasing evidence that microRNAs (miRNAs) play important roles in the immune response against infectious agents suggests that miRNA might be exploitable as signatures of exposure to specific infectious agents. In order to identify potential early miRNA biomarkers of bacterial infections, human peripheral blood mononuclear cells (hPBMCs) were exposed to two select agents, Burkholderia pseudomallei K96243 and Francisella tularensis SHU S4, as well as to the nonpathogenic control Escherichia coli DH5α. RNA samples were harvested at three early time points, 30, 60, and 120 minutes postexposure, then sequenced. RNAseq analyses identified 87 miRNAs to be differentially expressed (DE) in a linear fashion. Of these, 31 miRNAs were tested using the miScript miRNA qPCR assay. Through RNAseq identification and qPCR validation, we identified differentially expressed miRNA species that may be involved in the early response to bacterial infections. Based upon its upregulation at early time points postexposure in two different individuals, hsa-mir-30c-5p is a miRNA species that could be studied further as a potential biomarker for exposure to these gram-negative intracellular pathogens. Gene ontology functional analyses demonstrated that programmed cell death is the first ranking biological process associated with miRNAs that are upregulated in F. tularensis-exposed hPBMCs. PMID:28791299

  4. Umbelliprenin is Potentially Toxic Against the HT29, CT26, MCF-7, 4T1, A172, and GL26 Cell Lines, Potentially Harmful Against Bone Marrow-Derived Stem Cells, and Non-Toxic Against Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Rashidi, Mohsen; Ziai, Seyed Ali; Moini Zanjani, Taraneh; Khalilnezhad, Ahad; Jamshidi, Hamidreza; Amani, Davar

    2016-01-01

    Background Resistance to chemotherapy is a growing concern, thus natural anticancer agents are drawing the attention of many scientists and clinicians. One natural anticancer agent, umbelliprenin, is a coumarin produced by many species of Ferula. Objectives We aimed to examine the inhibitory effect of umbelliprenin on human and mouse bone marrow-derived stem cells (BMDSCs), peripheral blood mononuclear cells (PBMCs), and different cancer cell lines. Materials and Methods In this in vitro experimental study, the HT29, CT26, MCF-7, 4T1, A172, and GL26 cancer cells and human and mouse BMDSCs and PBMCs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), incubated at 37°C for 24 hours in a 5% CO2 atmosphere, and then were treated with different concentrations of umbelliprenin dissolved in dimethyl sulfoxide (DMSO) (3, 6, 12, 25, 50, 100, and 200 µg/mL) for 24, 48, and 72 hours at 37°C. Each experiment was performed in triplicate. Finally, the cell survival rate was assessed by MTT assay. The IC50 values were calculated based on the log values using GraphPad Prism version 5 software for windows (La Jolla CA, USA) and were expressed as mean ± SEM. Results Umbelliprenin inhibited the cancer cells in a concentration-dependent (P 0.05). The most sensitive and resistant cell lines at the 24-hour incubation time were 4T1 (IC50, 30.9 ± 3.1 µg/mL) and A172 (IC50, 51.9 ± 6.7 µg/mL); at the 48-hour incubation time: 4T1 (IC50, 30.6 ± 2.6 µg/mL) and CT26 (IC50, 53.2 ± 3.6 µg/mL); and at the 72-hour incubation time: HT29 (IC50, 37.1 ± 1.4 µg/mL) and 4T1 (IC50, 62.2 ± 4.8 µg/mL). Both human and mouse BMDSCs showed the highest resistance at the 24-hour incubation time (IC50s, 254.7 ± 21 and 204.4 ± 4.5 µg/mL, respectively) and the highest sensitivity at the 72-hour incubation time (IC50s, 120.4 ± 5 and 159.0 ± 7.3 µg/mL, respectively). The PBMCs of both human and mouse origin revealed very strong resistance to the studied

  5. Cell type-specific responses of peripheral blood mononuclear cells to silver nanoparticles.

    Science.gov (United States)

    Greulich, C; Diendorf, J; Gessmann, J; Simon, T; Habijan, T; Eggeler, G; Schildhauer, T A; Epple, M; Köller, M

    2011-09-01

    Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30μgml(-1) silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15μgml(-1) was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25μgml(-1) and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure.

  6. Role of peripheral blood mononuclear cell transportation from mother to baby in HBV intrauterine infection.

    Science.gov (United States)

    Shao, Qingliang; Zhao, Xiaxia; Yao Li, M D

    2013-12-01

    We aimed to investigate the role of peripheral blood mononuclear cell transportation from mother to baby in hepatitis B virus (HBV) intrauterine infection. Thirty HBsAg-positive pregnant women in the second trimester and their aborted fetuses were included in this study. Enzyme-linked-immunosorbent-assay was utilized to detect HBsAg in the peripheral blood of pregnant women and the femoral vein blood of their aborted fetuses. HBV-DNA in serum and peripheral blood mononuclear cells (PBMC) and GSTM1 alleles of pregnant women and their aborted fetuses were detected by nested polymerase chain reaction (PCR) and seminested PCR, respectively. We also examined the location of placenta HBsAg and HBcAb using immunohistochemical staining. The expression of placenta HBV-DNA was detected by in situ hybridization. For the 30 aborted fetuses, the HBV intrauterine infection rate was 43.33%. The HBV-positive rates of HBsAg in peripheral blood, serum, and PBMC were 10% (3/30), 23.33% (7/30), and 33.33% (10/30), respectively. Maternal-fetal PBMC transport was significantly positively correlated with fetal PBMC HBV-DNA (P = 0.004). Meanwhile, the rates of HBV infection gradually decreased from the maternal side to the fetus side of placenta (decidual cells > trophoblastic cells > villous mesenchymal cells > villous capillary endothelial cells). However, no significant correlation between placenta HBV infection and HBV intrauterine infection was observed (P = 0.410). HBV intrauterine infection was primarily due to peripheral blood mononuclear cell maternal-fetal transportation in the second trimester in pregnant women.

  7. Natural killer cell subsets and receptor expression in peripheral blood mononuclear cells of a healthy Korean population: Reference range, influence of age and sex, and correlation between NK cell receptors and cytotoxicity.

    Science.gov (United States)

    Phan, Minh-Trang; Chun, Sejong; Kim, Sun-Hee; Ali, Alaa Kassim; Lee, Seung-Hwan; Kim, Seokho; Kim, Soo-Hyun; Cho, Duck

    2017-02-01

    The purpose of this study was to identify CD56(bright) and CD56(dim) natural killer (NK) cell subsets and analyze their receptors expression in a healthy Korean population, and to determine whether receptor expression correlates with age, sex, and cytotoxicity. We performed multicolor flow cytometry assays to analyze the expression of various NK cell receptors (CD16, NKG2A, NKG2C, NKG2D, CD57, DNAM-1, CD8a, CD62L, NKp30, and NKp46) on both CD3(-)/CD56(dim) and CD3(-)/CD56(bright) NK cells in whole-blood samples from 122 healthy donors. The expression of these receptors was compared according to age (60years, n=27) and gender (male, n=61, female, n=61). NK cell cytotoxicity assays were performed with peripheral blood mononuclear cells (PBMCs) from 18 individuals. The results were compared to the expression levels of NKp30 and NKp46 receptors. A normal reference range for NK cell receptor expression in two NK cell subsets was established. NKp46 and NKG2D expression gradually decreased with age (pcell proportion and numbers, frequencies of CD56(dim) cells, and CD57 expression increased with age (pcell proportion and numbers, frequencies of CD56(dim) cells, and CD57 expression compared to those of women (pcell frequency (r=-0.42, pcell cytotoxicity was found to positively correlate with NCR expression (p=0.02), but not NK cell proportion (p=0.80). We have established a profile of NK cell surface receptors for a Korean population, and revealed that age and gender have an effect on the expression of NK cell receptors in the population. Our data might explain why neither NK cell numbers nor proportions correlate with NK cell cytotoxicity. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  8. Usefulness of liver infiltrating CD86-positive mononuclear cells for diagnosis of autoimmune hepatitis

    Institute of Scientific and Technical Information of China (English)

    Kazutaka Kurokohchi; Shigeki Kuriyama; Tsutomu Masaki; Takashi Himoto; Akihiro Deguchi; Seiji Nakai; Asahiro Morishita; Hirohito Yoneyama; Yasuhiko Kimura; Seishiro Watanabe

    2006-01-01

    AIM: Although the pathogenic mechanism underlying autoimmune hepatitis (AIH) remains unclear, the immune system is thought to be critical for the progression of the disease. Cellular immune responses may be linked to the hepatocellular damage in AIH. Recently, much attention has been focused on the critical functions of costimulatory molecules expressed on mononuclear cells in the generation of effective T cell-mediated immune responses. Analysis of costimulatory molecule expressed on mononuclear cells from the patients with AIH may give us insight into the pathogenic mechanism of hepatocellular damage in AIH.METHODS: Peripheral blood mononuclear cells (PBMC)were taken from the patients with AIH (34 cases) and healthy controls (25 cases). Liver infiltrating mononuclear cells (LIMCs) were taken from the patients with AIH (18 cases), the patient with chronic hepatitis C (CH-C) (13 cases) and the patients with fatty liver (2 cases).Using flow cytometry, the cells were analyzed for the expression of costimulatory molecules, such as CD80,CD86, and CD152 (CTLA-4). The results were compared with clinical data such as the level of gammaglobulin,histological grade, presence or absence of corticosteroids administration and the response to corticosteroids.RESULTS: The levels of CD80+, CD86+ and CD152+PBMC were significantly reduced in the patients with AIH as compared with healthy controls. By contrast,those cells were significantly higher in LTMC than in PBMC of the patients with AIH. Especially, the level of CD86+ LIMC showed a marked increase irrespective of the degree of disease activity in the patients with ATH,although CD86+ cells were rarely present in PBMC. The levels of CD86+ cells were present in significantly higher frequency in patients with AIH than in the patients with CH-C. Furthermore, the patients with AIH with high levels of CD86+ LIMC showed good responses to corticosteroids, whereas 2 cases of AIH with low levels of CD86+ LIMC did not respond well

  9. Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients.

    Science.gov (United States)

    Pochard, Pierre; Gosset, Philippe; Grangette, Corinne; Andre, Claude; Tonnel, André-Bernard; Pestel, Joël; Mercenier, Annick

    2002-10-01

    Among factors potentially involved in the increased prevalence of allergic diseases, modification of the intestinal bacteria flora or lack of bacterial stimulation during childhood has been proposed. Lactic acid bacteria (LAB) present in fermented foods or belonging to the natural intestinal microflora were shown to exert beneficial effects on human health. Recent reports have indicated their capacity to reduce allergic symptoms. The purpose of this investigation was to determine the effect of LAB on the production of type 2 cytokines, which characterize allergic diseases. PBMCs from patients allergic to house dust mite versus those from healthy donors were stimulated for 48 hours with the related Dermatophagoides pteronyssinus allergen or with a staphylococcal superantigen. The effect of LAB preincubation was assessed by measuring the type 2 cytokine production by means of specific ELISA. The tested gram-positive LAB were shown to inhibit the secretion of T(H)2 cytokines (IL-4 and IL-5). This effect was dose dependent and was observed irrespective of the LAB strain used. No significant inhibition was induced by the control, gram-negative Escherichia coli TG1. Interestingly, LAB reduced the T(H)2 cytokine production from allergic PBMCs specifically restimulated with the related allergen. The inhibition mechanism was shown to be dependent on antigen-presenting cells (ie, monocytes) and on the involvement of IL-12 and IFN-gamma. The tested LAB strains were demonstrated to exhibit an anti-T(H)2 activity, and thus different strains of this family might be useful in the prevention of allergic diseases.

  10. A novel and simple method for the generation of functional human dendritic cells from unfractionated peripheral blood mononuclear cells within 2 days: its application for induction of HIV-1-reactive CD4+ T cells in the hu-PBL SCID mice

    Directory of Open Access Journals (Sweden)

    Akira eKodama

    2013-09-01

    Full Text Available Because dendritic cells (DCs play a critical role in the regulation of adaptive immune responses, they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. Generally, monocyte-derived DCs (MDDCs were generated from purified monocytes by multiple steps of time-consuming physical manipulations for an extended period cultivation. In this study, we developed a novel, simple and rapid method for the generation of type-1 helper T cell (Th1-stimulating human DCs directly from bulk peripheral blood mononuclear cells (PBMCs. PBMCs were cultivated in the presence of 20 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng/ml of interleukin-4 (IL-4 and 1,000 U/ml of interferon-β (IFN-β for 24 hours followed by 24 hour maturation with a cytokine cocktail containing 10 ng/ml of tumor necrosis factor-α (TNF-α, 10 ng/ml of IL-1β and 1 μg/ml of prostaglandin E2 (PGE2. The phenotype and biological activity of these new DCs for induction of allogeneic T cell proliferation and cytokine production were comparable to those of the MDDCs. Importantly, these new DCs pulsed with inactivated HIV-1 could generated HIV-1-reactive CD4+ T cell responses in humanized mice reconstituted with autologous PBMCs from HIV-1-negative donors. This simple and quick method for generation of functional DCs will be useful for future studies on DC-mediated immunotherapies.

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

    Science.gov (United States)

    Yu, Nan; Yan, Wenjie; Yin, Tailang; 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O. [Univ. of Alberta, Edmonton (Canada)] [and others

    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.

  13. Outcomes of autologous bone marrow mononuclear cells for cerebral palsy: an open label uncontrolled clinical trial.

    Science.gov (United States)

    Nguyen, Liem Thanh; Nguyen, Anh Tuan; Vu, Chinh Duy; Ngo, Doan V; Bui, Anh V

    2017-04-12

    Stem cell therapy has emerged as a promising method for improving motor function of patients with cerebral palsy. The aim of this study is to assess the safety and effectiveness of autologous bone marrow mononuclear stem cell transplantation in patients with cerebral palsy related to oxygen deprivation. An open label uncontrolled clinical trial was carried out at Vinmec International Hospital. The intervention consisted of two administrations of stem cells, the first at baseline and the second 3 months later. Improvement was monitored at 3 months and 6 months after the first administration of stem cells, using the Gross Motor Function Measure (GMFM) and Modified Ashworth Score which measures muscle tone. No severe complications were recorded during the study. After transplantation, 12 patients encountered fever without infections and 9 patients experienced vomiting which was easily managed with medications. Gross motor function was markedly improved 3 months or 6 months after stem cell transplantation than at baseline. The post-transplantation GMFM-88 total score, each of its domains and the GMFM-66 percentile were all significantly higher (p-value  0.05). Autologous bone marrow mononuclear cell transplantation appears to be a safe and effective therapy for patients with cerebral palsy. ClinicalTrials.gov Identifier: NCT02569775 . Retrospectively registered on October 15, 2015.

  14. Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC and EBV-transformed lymphocytes from the same donor

    Directory of Open Access Journals (Sweden)

    Delgrosso Kathleen

    2011-09-01

    Full Text Available Abstract Background The creation of lymphoblastoid cell lines (LCLs through Epstein-Barr virus (EBV transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings. Results We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs. Conclusions By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.

  15. Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

    DEFF Research Database (Denmark)

    Søndergaard, S R; Essen, M V; Schjerling, P

    2002-01-01

    The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin...... infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed...... mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells...

  16. Obesity alters the expression profile of clock genes in peripheral blood mononuclear cells

    Science.gov (United States)

    Tahira, Kazunobu; Fukuda, Noboru; Aoyama, Takahiko; Tsunemi, Akiko; Matsumoto, Siroh; Nagura, Chinami; Matsumoto, Taro; Soma, Masayoshi; Shimba, Shigeki; Matsumoto, Yoshiaki

    2011-01-01

    Introduction The aim of this study was to investigate the association between the variation in expression profile of clock genes and obesity using peripheral blood mononuclear (PMN) cells. Material and methods The subjects comprised 10 obese patients and 10 healthy volunteers. Blood was collected at different time-points during the day and levels of blood sugar, IRI, adiponectin and leptin were determined. Peripheral blood mononuclear cells were sampled, and expression levels of brain and muscle Arnt-like protein-1 (BMAL1), Period (PER)1, PER2, Cryptochrome (CRY)1, CRY2, and REV-ERBα mRNA were quantified. Results During the day, the expression levels of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells of the obese group were all significantly higher compared to those in the non-obese group. In addition, expression of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells increased between 12:00 and 21:00 in the obese group. In PMN cells of both groups, PER1 gene expression showed a bimodal pattern, with high expression at 9:00 and 18:00. Conclusions Differences were observed in the expression profile variation of clock genes between the obese and non-obese groups. This study reveals the differences in clock gene expression profiles between obese and non-obese subjects, with evidence for two distinct chronotypes, and suggests a contribution of these chronotypes to fat accumulation in humans. PMID:22328874

  17. Decreased Expression of Innate Immunity-Related Genes in Peripheral Blood Mononuclear Cells from Patients with IgG4-Related Disease.

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    Akio Nakajima

    Full Text Available IgG4-related disease (IgG4-RD is a new clinical entity of unknown etiology characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Although aberrancies in acquired immune system functions, including increases in Th2 and Treg cytokines observed in patients with IgG4-RD, its true etiology remains unclear. To investigate the pathogenesis of IgG4-RD, this study compared the expression of genes related to innate immunity in patients with IgG4-RD and healthy controls.Peripheral blood mononuclear cells (PBMCs were obtained from patients with IgG4-RD before and after steroid therapy and from healthy controls. Total RNA was extracted and DNA microarray analysis was performed in two IgG4-RD patients to screen for genes showing changes in expression. Candidate genes were validated by real-time RT-PCR in 27 patients with IgG4-RD and 13 healthy controls.DNA microarray analysis identified 21 genes that showed a greater than 3-fold difference in expression between IgG4-RD patients and healthy controls and 30 genes that showed a greater than 3-fold change in IgG4-RD patients following steroid therapy. Candidate genes related to innate immunity, including those encoding Charcot-Leyden crystal protein (CLC, membrane-spanning 4-domain subfamily A member 3 (MS4A3, defensin alpha (DEFA 3 and 4, and interleukin-8 receptors (IL8R, were validated by real-time RT-PCR. Expression of all genes was significantly lower in IgG4-RD patients than in healthy controls. Steroid therapy significantly increased the expression of DEFA3, DEFA4 and MS4A3, but had no effect on the expression of CLC, IL8RA and IL8RB.The expression of genes related to allergy or innate immunity, including CLC, MS4A3, DEFA3, DEFA4, IL8RA and IL8RB, was lower in PBMCs from patients with IgG4-RD than from healthy controls. Although there is the limitation in the number of patients applied in DNA microarray, impaired expression of genes related to innate immunity may be

  18. Effect of chronic low dose natural radiation in human peripheral blood mononuclear cells: Evaluation of DNA damage and repair using the alkaline comet assay.

    Science.gov (United States)

    Kumar, P R Vivek; Seshadri, M; Jaikrishan, G; Das, Birajalaxmi

    2015-05-01

    This study investigates whether peripheral blood mononuclear cells (PBMCs) from inhabitants of Kerala in southwest India, exposed to chronic low dose natural radiation in vivo (>1 mSv year(-1)), respond with a radioadaptive response to a challenging dose of gamma radiation. Toward this goal, PBMCs isolated from 77 subjects from high-level natural radiation areas (HLNRA) and 37 subjects from a nearby normal level natural radiation area (NLNRA) were challenged with 2 Gy and 4 Gy gamma radiation. Subjects from HLNRA were classified based on the mean annual effective dose received, into low dose group (LDG) and high dose group (HDG) with mean annual effective doses of 2.69 mSv (N=43, range 1.07 mSv year(-1) to 5.55 mSv year(-1)) and 9.62 mSv (N = 34, range 6.07 mSv year(-1) to 17.41 mSv year(-1)), respectively. DNA strand breaks and repair kinetics (at 7 min, 15 min and 30 min after 4 Gy) were evaluated using the alkaline single cell gel electrophoresis (comet) assay. Initial levels of DNA strand breaks observed after either a 2 Gy or a 4 Gy challenging dose were significantly lower in subjects of the HDG from HLNRA compared to subjects of NLNRA (2 Gy, P = 0.01; 4 Gy, P = 0.02) and LDG (2 Gy P = 0.01; 4 Gy, P=0.05). Subjects of HDG from HLNRA showed enhanced rejoining of DNA strand breaks (HDG/NLNRA, P = 0.06) during the early stage of repair (within 7 min). However at later times a similar rate of rejoining of strand breaks was observed across the groups (HDG, LDG and NLNRA). Preliminary results from our study suggest in vivo chronic low-level natural radiation provides an initial exposure that allows an adaptation to a subsequent higher radiation exposure, perhaps through improving DNA repair via an unknown mechanism. Therefore, further investigations would be necessary in this population to understand the biological and health effects of chronic low-level natural radiation exposures. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Prevention of diabetic microangiopathy by prophylactic transplant of mobilized peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Bin ZHOU; Xiao-cang CAO; Zhi-hong FANG; Cui-lin ZHENG; Zhi-bo HAN; He REN; Man-chiu POON; Zhong-chao HAN

    2007-01-01

    Aim: To investigate whether the prophylactic local delivery of mobilized periph-eral blood mononuclear cells (M-PBMNC) could prevent peripheral microangio-pathy in diabetic nude mice. Methods: Diabetic nude mice were induced with intraperitoneal injections of streptozotocin. With the time course of diabetes, we detected the capillary and arteriole density of mice adductor muscles by immuno-histopathy. In situ apoptosis was detected by using TdT-mediated dUTP nick end labeling (TUNEL) methods. M-PBMNC were labeled and locally delivered to the adductor muscles. Mononuclear cells were also isolated and cultured in vitro for the detection and counting of endothelial progenitor cells(EPC). Results: Rarefication of capillaries and arterioles, enhanced apoptosis in adductor muscles,and reduced circulating EPC in diabetic nude mice. Prophylactic local delivery of M-PBMNC halted the progression of microvascular rarefaction in hind-limb skel-etal muscles by inhibiting apoptosis. We detected the survival, migration and incorporation of transplanted M-PBMNC into the murine vasculature in vivo. In addition, more EPC were available from M-PBMNC than non-mobilized cells.Conclusion: These results suggested that the prophylactic local delivery of M-PBMNC may represent a novel approach for the treatment of microvascular complications in diabetics.

  20. 军团菌体外刺激对人PBMC TLR2 mRNA表达影响%Effects of Legionella on expression of TLR2 mRNA in human peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    高向东; 张帆; 王素萍; 史晓红; 王伟刚; 王芳芳; 董洁敏; 雷智

    2013-01-01

    目的 探讨嗜肺军团菌活菌刺激对人外周血单个核细胞(PBMC)表达TLR2 mRNA的影响.方法 采用不同浓度的嗜肺军团菌活菌悬液刺激体外培养的健康人PBMC,收集细胞运用RT-PCR方法测定TLR2mRNA的表达水平.结果 析因分析结果显示,时间的主效应有统计学意义(F=26.06,P<0.05);时间与浓度的交互作用有统计学意义(F=11.39,P <0.05);24 h时,MOI 1 、MOI 10刺激组TLR2 mRNA的表达量均高于对照组(P<0.05),呈现随菌液浓度升高表达量也逐渐升高,48 h时,各组之间TLR2 mRNA的表达量差异虽无统计学意义(P>0.05),但表达量随浓度升高呈现下降趋势,72 h时,各组之间TLR2 mRNA的表达量差异有统计学意义(P<0.05),但随菌液浓度升高表达量反而降低.结论 用军团菌体外刺激人PBMC,其TLR2mRNA的表达在一定范围内呈一定的时效与量效关系,可为今后的研究提供有益的时间和剂量参考.%Objective To examine the expression of TLR2 in human peripheral blood mononuclear cells (PBMCs) stimulatedby Legionella. Methods Human peripheral blood mononuclear cells ( POMCs) were treated with different concentrations of Legionella(2×106/ml,2×107/ml) ,and real timel reverse transcriptase PCR(RT-PCR) was used to detect TLR2 mRNA expression. Results Factorial analyses showed that leading effect of time had statistical significance (F = 26. 06,P < 0. 05) and there was an interaction between the time and concentration ( F = 11. 39, P < 0. 05). In 24 hours,the expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml and 2×107/ml Legionella were obviously higher than TLR2 mRNA expression in control group. In 48 hours,there was no difference between stimulated group and control group. In 72 hours,the expressions of TLR2 mRNA of PBMCs stimulated with 2×106/ml and 2×107/ml Legionella , were obviously lower than TLR2 mRNA expression in the control group. When the PBMCs were stimulated at the level of 2×106/ml and 2×107/ml

  1. HIV protease inhibitors do not cause the accumulation of prelamin A in PBMCs from patients receiving first line therapy: the ANRS EP45 "aging" study.

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    Sophie Perrin

    Full Text Available BACKGROUND: The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes. METHODS: 35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years containing boosted protease inhibitors (PI/r (comprising lopinavir/ritonavir in 65% of patients were recruited together with 49 seronegative age- and sex-matched control subjects (http://clinicaltrials.gov/, NCT01038999. In more than 88% of patients, the viral load was 500/mm³. Prelamin A processing in peripheral blood mononuclear cells (PBMCs from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L. RESULTS: Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L but not in plasma (60-80 g/L or in medium supplemented with BSA (40 g/L, both of which contain a high protein concentration. CONCLUSIONS: Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells. ClinicalTrials.gov NCT01038999 http://clinicaltrials.gov/ct2/show/NCT01038999.

  2. High Insulin and Leptin Increase Resistin and Inflammatory Cytokine Production from Human Mononuclear Cells

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    Panayoula C. Tsiotra

    2013-01-01

    Full Text Available Resistin and the proinflammatory cytokines, such as TNF-α, IL-6, and IL-1β, produced by adipocytes, and macrophages, are considered to be important modulators of chronic inflammation contributing to the development of obesity and atherosclerosis. Human monocyte-enriched mononuclear cells, from ten healthy individuals, were exposed to high concentrations of insulin, leptin, and glucose (alone or in combination for 24 hours in vitro. Resistin, TNF-α, IL-6, and IL-1β production was examined and compared to that in untreated cells. High insulin and leptin concentrations significantly upregulated resistin and the cytokines. The subsequent addition of high glucose significantly upregulated resistin and TNF-α mRNA and protein secretion, while it did not have any effect on IL-6 or IL-1β production. By comparison, exposure to dexamethasone reduced TNF-α, IL-6, and IL-1β production, while at this time point it increased resistin protein secretion. These data suggest that the expression of resistin, TNF-α, IL-6, and IL-1β from human mononuclear cells, might be enhanced by the hyperinsulinemia and hyperleptinemia and possibly by the hyperglycemia in metabolic diseases as obesity, type 2 diabetes, and atherosclerosis. Therefore, the above increased production may contribute to detrimental effects of their increased adipocyte-derived circulating levels on systemic inflammation, insulin sensitivity, and endothelial function of these patients.

  3. Beryllium alters lipopolysaccharide-mediated intracellular phosphorylation and cytokine release in human peripheral blood mononuclear cells.

    Science.gov (United States)

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

    2009-12-01

    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 found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1beta is enhanced. In addition, 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 1beta 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.

  4. Immunological and molecular biological identification of a true case of T-hairy cell leukaemia

    DEFF Research Database (Denmark)

    Demeter, J; Pálóczi, K; Földi, J

    1990-01-01

    A hairy cell leukaemia (HCL) patient is presented in whom the peripheral blood mononuclear cells (PBMCs) carried suppressor T-cell markers (CD3+, CD2+, CD8+/CD4-, CD38+). Analysis of genomic DNA of PBMNC showed the presence of a monoclonal population of T cells, the T-cell receptor (TCR) beta-cha...

  5. The Effects of Royal Jelly on In-Vitro Cytotoxicity of K562 Cells and Peripheral Blood Mononuclear Cells

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    SE Hosseini

    2014-02-01

    Full Text Available Abstract Background & aim: Royal jelly, secreted by worker bees, has different biological activities on cells and tissues. The aim of this study was to evaluate the effects of royal jelly on peripheral blood mononuclear cells and on the tumor category of K562 cell line. Methods: In the present experimental study, three subjects were selected separately with three repetitions. K562 (104 cells and PBMC (105 cells with different concentrations of royal jelly (5, 10, 25, 50 and 100 mg/ml were cultured under standard conditions for 48 and 72 h separately. The fatality rate on PBMC cells and K562 cancer cells was evaluated by using MTT (Tetrazolium Dye-Reduction Assay. The number of viable cells in PBMC that were exposed for 48 hours with Royal Jelly was evaluated by trypan blue staining. Data were analyzed by ANOVA. Results: The royal jelly had no cytotoxicity effect on PBMC cells but at concentration of 50 and 100 mg/mL the cytotoxicity effect were observed on k562 cells whereas, at 10 and 25 mg/ml the number of PBMC viable cells increased. Conclusion: Due to the lack of lethality of royal jelly on PBMC cells and PBMC cell viability and an increase in the fatality rate of cancer cells in the future, royal jelly can be used as a potential candidate for treatment of leukemia. Keywords: Royal jelly, K562, peripheral blood mononuclear cell

  6. Leptin enhances the release of cytokines by peripheral blood mononuclear cells from acute multiple sclerosis patients

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To explore the effect of leptin on cytokine production by PBMCs obtained from MS patients either in acute (relapse) or in stable (nonrelapse) phase of disease. Methods PBMCs were collected from 25 untreated acute MS patients, 11 stable MS patients and 20 healthy controls. PBMCs were cultured either with RPMI-1640 alone or with leptin (1.25 nmol/ml), phytohemagglutinin (PHA) ( 100 μg/ml), and leptin + PHA. 72 h later the supernate of the culture medium were collected and stored at -70℃. The pro-inflammatory cytokine (IFN-γ) concentration were determined using an enzyme-linked immunosorbent assay ( ELISA), and the anti-inflammatory cytokine (IL-4) concentration were investigated by radioimmunity methods. Results Our data showed that leptin induced IFN-γproduction by PBMCs of patients in an acute phase of disease but not in a stable phase or in healthy controls. Moreover, we found that PHA induced IL-4 production by PBMCs of patients in an acute phase of disease, but leptin inhibited this ability of PHA. Conclusion Leptin can affect on pro- and anti-inflammatory cytokine production by PBMCs collected from MS patients, may be this connected with leptin increase the susceptiveness of MS.

  7. Therapeutic angiogenesis in Buerger's disease (thromboangiitis obliterans) patients with critical limb ischemia by autologous transplantation of bone marrow mononuclear cells.

    Science.gov (United States)

    Motukuru, Vishnu; Suresh, Kalkunte R; Vivekanand, Vivekanand; Raj, Sumanth; Girija, K R

    2008-12-01

    Peripheral arterial disease is a significant problem worldwide. In developing countries such as India, the increased incidence of smoking and other forms of nicotine intake has resulted in a large proportion of young individuals with Buerger's disease. The results of surgical and endovascular treatment for this condition have not been very rewarding. Hence, we focused on providing alternative therapies. Neovascularization by autologous bone marrow mononuclear cell transplantation is being tried as an alternative therapeutic option. We have reviewed our series of patients who underwent autologous bone marrow mononuclear cell transplantation during the last 2 years. We enrolled 38 patients who were chosen to undergo autologous bone marrow mononuclear cell transplantation for nonreconstructible Buerger's disease. We injected the bone marrow mononuclear cells into the calf muscles of the affected limbs in 36 patients. We monitored ulcer healing, ankle-brachial index (ABI), and transcutaneous oximetry (TcPo(2)) level. No procedurally related complications occurred, although one injected sample of bone marrow aspirate later revealed infestation with Strongyloides stercoralis. Two patients were seropositive on the Venereal Disease Research Laboratory test and were not injected with the bone marrow mononuclear cells. Three patients (12%) underwent major amputations disease who have critical limb ischemia.

  8. Conversion of mononuclear cells from human umbilical cord blood into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fang-ting; FANG Jia-zhi; YU Jie; WAN Hui-juan; YE Jing; LONG Xia; YIN Mei-jun; HUANG Chun-qiao

    2006-01-01

    Objective:To evaluate the differentiation of human umbilical cord blood cells into hepatocyte-like cells. Methods: Mononuclear cells (MNCs) derived from human umbilical cord blood were isolated using Ficoll. The experiment was derived into 3 categories: (1) MNCs co-cultured with 50 mg minced liver tissue separated by a trans-well membrane and then collected at 0 h,24 h,48 h and 72 h; (2) MNCs cultured along supplemented with 100 ml/L FBS, 100 μ/ml penicillin, 100 μg/ml streptomycin, 4. 7 μg/ml linoleic acid, 1×ITS, 10-4 mol/L L-Ascorbic acid 2-P and a combination of FGF4 (100 ng/ml) and HGF (20 ng/Ml). Cells were then collected at 0 d and 16 d to examine the expression profile of hepatocyte correlating markers; (3) 0.2-0.3 ml of MNCs with a cell density of 2×107/ml were transplanted into prepared recipient mice [n= 12, injected with 0.4 ml/kg (20%) CCl4 and 150 ng/kg 5-fluorouracil (5-Fu) prior the transplant 24 h and 48 h, respectively] via injection through tail vein. Mice were sacrificed 4 weeks after transplantation. The hepatocyte correlating mRNAs and proteins were determined by RTPCR, immunohistochemical analysis and immunoflurence technique. Results: (1) After 72 h, a number of glycogen positive stained cells were observed with MNCs co-cultured with damaged mouse liver tissues.The expression of hepatocyte markers, human albumin (ALB), α-fetal protein (AFP) and human GATA4 Mrna and proteins were detected by RT-PCR and immunohistochemistry as well. For the confirmation,the DNA sequencing of PCR products was performed. In control groups, MNCs co-cultured with normal mouse hepatocytes or MNCs cultured alone, all markers remained negative. (2) In growth factor supplemented culture system, MNCs developed into larger volume with richer cytoplasm and binucleation after 16 d. Positive expression of ALB, AFP, CK18 and CK19 Mrna were detected with RT-PCR, and ALB positive staining was observed by immunocytochemistry as well. In contrast, MNCs cultured without

  9. Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

    Directory of Open Access Journals (Sweden)

    Zhong-bin LI

    2012-06-01

    Full Text Available Objective  To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods  A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results  HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions  During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

  10. 外周血单核细胞激活效应在原发性高血压患者丹参酮干预过程中的反应%Inhibitory effect of tanshinone on the activation of peripheral blood mononuclear cells in patients with essential hypertension

    Institute of Scientific and Technical Information of China (English)

    占成业; 陶秀良; 周代星; 郑智

    2007-01-01

    BACKGROUND: Preactivation of peripheral blood mononuclear cells (PBMCS) is one of the most important eerly events and facilitating factor for the formation of atherosclerosis. Tanshinone is a lipolytic component extracted from traditional Chinese medicine of denshen, it has definite anti-atherosclerotic effect.OBJECTTVE: To analyze whether PBMCS preactivation existed at early essential hypertension, and investigate the effects of tanshinone on inhibiting the PBMCS activation cultured in vitro by detecting the adhesion and excretory activities of PBMCS.DESTGN: A case-controlled analysis.SETTING: Department of Emergency and Research Room of Traditional Chinese Medicine, Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.PARTTCTPANTS: Thirty patients with untreated essential hypertension or with withdrawal from antihypertensives for at least 2 weeks were selected from the Department of Cardiology, Tongji Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology from January 2003 to October 2004, including 16 males and 14 females, aged (44.6±7.4) years, body mass index of (26.2±4.5) kg/m2, average disease course of (38.5±16.9) months.Informed contents were obtained from all the subjects. Their hypertension was grade Ⅰ-Ⅱ according to the diagnostic standards for hypertension by WHO/ISH in 1999. Secondary hypertension, organic heart disease, hyperglyceridemia,diabetes mellitus, liver and kidney dysfunction, heart, brain, kidney, vessel and other target damaged induced by infection and other clinical conditions and hypertension were excluded by history, physical examination and assistant examination.Another 30 healthy physical examinees with normal blood pressure were enrolled as the normal control group. Human umbilical vein endothelial cells (Species Reserving Center of Wuhan University); Tanshinone injection (Yaan Sanjiu Pharmaceutical, Co., Ltd., batch number: 020724);METHODS:

  11. Autologous bone marrow mononuclear cell transplantation in Duchenne muscular dystrophy - a case report.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Paranjape, Amruta; Bhagawanani, Khushboo; Gokulchandran, Nandini; Badhe, Prerna

    2014-01-01

    Male, 9 FINAL DIAGNOSIS: Duchenne muscular dystrophy Symptoms: Hyporeflexia • hypotonia • weaknes of lower limbs - Clinical Procedure: - Specialty: Neurology. Congenital defects/diseases. Duchenne muscular dystrophy (DMD) is a fatal, genetic, progressive, degenerating muscle disorder. Current treatment options are palliative. Newer options of cellular therapy promise to alter the disease process. Preclinical studies have successfully tested myogenic, neurogenic potential and dystrophin expression of bone marrow mononuclear cells. We treated a 9-year-old boy suffering from DMD with serial autologous bone marrow mononuclear cell transplantations followed by multidisciplinary rehabilitation. Brooke-Vignos score was 10 and he was wheelchair-bound. Over 36 months, gradual progressive improvement was noticed in muscle strength, ambulation with assistive devices, fine motor movements, Brooke-Vignos score, and functional independence measure score. Nine months after the transplantation, electromyography findings showed development of new normal motor unit potentials of the vastus medialis muscle. Magnetic resonance imaging scan of musculoskeletal systems showed no increase in fatty infiltration. This case report provides early investigative findings or the restorative effects of cellular therapy in DMD.

  12. PSP activates monocytes in resting human peripheral blood mononuclear cells: immunomodulatory implications for cancer treatment.

    Science.gov (United States)

    Sekhon, Bhagwant Kaur; Sze, Daniel Man-Yuen; Chan, Wing Keung; Fan, Kei; Li, George Qian; Moore, Douglas Edwin; Roubin, Rebecca Heidi

    2013-06-15

    Polysaccharopeptide (PSP), from Coriolus versicolor, has been used as an adjuvant to chemotherapy, and has demonstrated anti-tumor and immunomodulating effects. However its mechanism remains unknown. To elucidate how PSP affects immune populations, we compared PSP treatments both with and without prior incubation in phytohaemagglutinin (PHA) - a process commonly used in immune population experimentation. We first standardised a capillary electrophoresis fingerprinting technique for PSP identification and characterisation. We then established the proliferative capability of PSP on various immune populations in peripheral blood mononuclear cells, using flow cytometry, without prior PHA treatment. It was found that PSP significantly increased the number of monocytes (CD14(+)/CD16(-)) compared to controls without PHA. This increase in monocytes was confirmed using another antibody panel of CD14 and MHCII. In contrast, proliferations of T-cells, NK, and B-cells were not significantly changed by PSP. Thus, stimulating monocyte/macrophage function with PSP could be an effective therapeutic intervention in targeting tumors.

  13. Lipopolysaccharide and silica-stimulated mononuclear cell prostaglandin production in ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Neville A. Punchard

    2000-01-01

    Full Text Available Basal, lipopolysaccharide (LPS and silica-stimulated prostaglandin (PG production were compared between peripheral blood mononuclear cells (PBMNC from UC patients and healthy subjects (HS. Basal and LPS-stimulated PBMNC PGI2, but not PGE2, production was greater in UC. LPS stimulated both PGE2 and PGI2 by PBMNC from HS and UC patients. Silica stimulated production of both PGs by cells from HS but only PGE2 by cells from UC patients. The differences in responses to silica and LPS may result from differences in activation of NFκB or, alternatively, prior sensitisation to one of these agents. That PBMNC PGE2 production is not increased in UC, as it is in Crohn’s disease, suggests that there are differences in PBMNC behaviour between these two diseases.

  14. Production of cytokines by mononuclear cells of hypertrophic adenoids in children with otitis media with effusion.

    Science.gov (United States)

    Zelazowska-Rutkowska, Beata; Ilendo, Elzbieta; Skotnicka, Bozena; Wysocka, Jolanta; Kasprzycka, Edwina

    2012-01-01

    Hypertrophic adenoids with otitis media with effusion is a common infectious disease and present a serious otological problem in children. Cytokines, potent inflammatory mediators, play important role in the initiation of immunological response in otitis media. Adenoids excised due to hypertrophy with or without chronic otitis media with effusion were used to isolate mononuclear cells. Secretion of cytokines by non-stimulated and PHA-stimulated cells was determined by specific ELISAs. We found a significant increase in the production of IL-5 and TNF-α secreted by adenoidal cells of children with otitis media with effusion compared to group with hypertrophic adenoids. No differences were found in the secretion of IL-8, IL-6, and IL-10 between these two groups of patients. Our results suggest a difference between the immunological responses in the course of hypertrophic adenoids with otitis media as compared to hypertrophic adenoids.

  15. Ganoderic acid C1 isolated from the anti-asthma formula, ASHMI™ suppresses TNF-α production by mouse macrophages and peripheral blood mononuclear cells from asthma patients.

    Science.gov (United States)

    Liu, Changda; Yang, Nan; Song, Ying; Wang, Lixin; Zi, Jiachen; Zhang, Shuwei; Dunkin, David; Busse, Paula; Weir, David; Tversky, Jody; Miller, Rachel L; Goldfarb, Joseph; Zhan, Jixun; Li, Xiu-Min

    2015-08-01

    Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs-Ganoderma lucidum (G. lucidum), Sophora flavescens Ait (S. flavescens) and Glycyrrhiza uralensis Fischer (G. uralensis), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum, but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)-triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases.

  16. Transplantation of autologous bone marrow mononuclear cells for patients with lower limb ischemia

    Institute of Scientific and Technical Information of China (English)

    GU Yong-quan; LI Xue-feng; YU Heng-xi; CUI Shi-jun; WANG Zhong-gao; ZHANG Jian; GUO Lian-rui; QI Li-xing; ZHANG Shu-wen; XU Juan; LI Jian-xin; LUO Tao; JI Bing-xin

    2008-01-01

    Background Many treatment options for lower limb ischemia are difficult to apply for the patients with poor arterial outflow or with poor general conditions.The effect of medical treatment alone is far from ideal.especially in patients with diabetic foot.A high level amputation is inevitable in these patients.This study aimed to explore the effect of transplantation of autologous bone marrow mononuclear cells on the treatment of lower limb ischemia and to compare the effect of intra-artedal transplantation with that of intra-muscular transplantation.Methods In this clinical trial,32 patients with lower limb ischemia were divided into two groups.Group 1 (16 patients with 18 affected limbs) received transplantation of autologous bone marrow mononuclear cells by intra-muscular injection into the affected limbs;and group 2(16 patients with 17 affected limbs)received transplantation of autologous bone marrow mononucJear cells by intra-arterial injection into the affected limbs.Rest pain,coldness,ankle/brachial index (ABI),claudication,transcutaneous oxygen pressure(tcPO2)and angiography(15 limbs of 14 patients)were evaluated before and after the mononuclear cell transplantation to determine the effect of the treatment.Results Two patients died from heart failure.The improvement of rest pain was seen in 76.5%(13/17)of group 1 and 93.3%(14/15)of group 2.The improvement of coldness was 100%in both groups.The increase of ABI was 44.4%(8/18)in group 1 and 41.2%(7,17)in group 2.The value of tcPO2 increased to 20 mmHg or more in 20 limbs.Nine of 15 limbs which underwent angiography showed rich collaterals.Limb salvage rate was 83.3%(15,18)in group 1 and 94.1%(16/17)in group 2.There was no statistically significant difference in the effectiveness of the treatment between the two groups.Conclusions Transplantation of autologous bone marrow mononucJear cells is a simple,safe and effective method for the treatment of lower limb ischemia,and the two approaches for the implantation

  17. [Preliminary study on autologous bone marrow mononuclear cells transplantation for lower limb chronic venous ulcer].

    Science.gov (United States)

    Huang, Wen; Wang, Liwei; Tan, Bin; Zhang, Guozhen; Zhao, Yu; Ren, Guosheng

    2011-05-01

    To investigate the effectiveness of autologous bone marrow mononuclear cells transplantation on lower limb chronic venous ulcer. Between May 2009 and September 2010, 17 patients with lower limb chronic venous ulcer were treated with autologous bone marrow mononuclear cells transplantation (transplantation group) and 10 patients treated without cells transplantation served as control group. In the transplantation group, there were 9 males and 8 females with age of (33.3 +/- 6.1) years, including 11 cases of simple great saphenous vein varicosity and 6 cases of chronic venous insufficiency; the area of ulcer was (4.39 +/- 2.46) cm2; and the duration of ulcer ranged from 3 months to 6 years. In the control group, there were 4 males and 6 females with age of (39.2 +/- 10.3) years, including 7 cases of simple great saphenous vein varicosity and 3 cases of chronic venous insufficiency; and the area of ulcer was (5.51 +/- 2.63) cm2; and the duration of ulcer ranged from 3 months to 2 years. All patients in both groups were classified as C6 according to clinical etiology anatomy pathophysiology (CEAP) classification. No significant difference was found in the general data between 2 groups (P > 0.05). The healing process of ulcer was observed. The granulation tissue was harvested for HE staining before operation and at 3 days after operation in the transplantation group. The microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression of ulcer granulation tissue were observed. In the transplantation group, ulcer healing was accelerated; complete healing was observed in 15 cases, partial healing in 1 case, and no healing in 1 case with the median healing time of 22 days. However, in the control group, the healing process was slower; complete healing of ulcer was observed in 7 cases and no healing in 3 cases with the median healing time of 57.5 days. There was significant difference in the healing time between 2 groups (Z = 0.001 4, P = 0.0027). HE

  18. Avidin inhibits PHA-induced human peripheral blood mononuclear cell proliferation

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    Cicia Firakania

    2016-04-01

    Full Text Available Background: Cell proliferation occurs not only in normal but also in cancer cells. Most of cell proliferation inhibition can be done by inhibiting the DNA synthesis, notably by intervening the formation of purine or pyrimidine. In purine de novo synthesis, it was assumed that biotin plays a role as a coenzyme in carboxylation reaction, one of the pivotal steps in the purine de novo pathways. The aim of this study was to see the avidin potency to bind biotin and inhibit mitosis.Methods: Peripheral blood mononuclear cell (PBMC was cultured in RPMI-1640 medium and stimulated by phytohemagglutinin (PHA in the presence or absence of interleukin-2 (IL-2, with or without avidin. The effect of avidin addition was observed at 24, 48, and 72 hours for cell proliferation, viability, and cell cycle. Statistical analysis was done by one-way ANOVA.Results: Avidin inhibited cell proliferation and viability in culture under stimulation by PHA with and without IL-2. Cell cycle analysis showed that avidin arrested the progression of PBMC after 72 hours of culture. Most cells were found in G0/G1 phase.Conclusion: Inhibition of biotin utilization by avidin binding can halt cell proliferation.

  19. Human umbilical cord blood mononuclear cell transplantation for delayed encephalopathy after carbon monoxide intoxication

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    Gong D

    2013-08-01

    Full Text Available Dianrong Gong,1 Haiyan Yu,1 Weihua Wang,2 Haixin Yang,1 Fabin Han1,21Department of Neurology, 2Centre for Stem Cells and Regenerative Medicine, Liaocheng People's Hospital, The Affiliated Liaocheng Hospital, Taishan Medical University, Shandong, People's Republic of ChinaAbstract: Stem cell transplantation is one of the potential treatments for neurological disorders. Since human umbilical cord stem cells have been shown to provide neuroprotection and promote neural regeneration, we have attempted to transplant the human umbilical cord blood mononuclear cells (hUCB-MNCs to treat patients with delayed encephalopathy after carbon monoxide intoxication (DEACOI. The hUCB-MNCs were isolated from fresh umbilical cord blood and were given to patients subarachnoidally. Physical examinations, mini-mental state examination scores, and computed tomography scans were used to evaluate the improvement of symptoms, signs, and pathological changes of the patient's brain before and after hUCB-MNC transplantation. A total of 12 patients with DEACOI were treated with hUCB-MNCs in this study. We found that most of the patients have shown significant improvements in movement, behavior, and cognitive function, and improved brain images in 1–4 months from the first transplantation of hUCB-MNCs. None of these patients have been observed to have any severe adverse effects. Our study suggests that the hUCB-MNC transplantation may be a safe and effective treatment for DEACOI. Further studies and clinical trials with more cases, using more systematic scoring methods, are needed to evaluate brain structural and functional improvements in patients with DEACOI after hUCB-MNC therapy.Keywords: human umbilical cord blood mononuclear cells, transplantation, delayed encephalopathy after carbon monoxide intoxication, MMSE

  20. Association of poly(ADP-ribose) polymerase activity in circulating mononuclear cells with myocardial dysfunction in patients with septic shock

    Institute of Scientific and Technical Information of China (English)

    Li Li; Hu Bangchuan; Gong Shijin; Yu Yihua; Dai Haiwen; Yan Jing

    2014-01-01

    Background Severe sepsis and septic shock are the leading causes of morbidity and mortality in hospitalized patients.This study aimed to investigate the association of poly(ADP-ribose) polymerase-1 (PARP-1) activity in circulating mononuclear cells with myocardial dysfunction in patients with septic shock.Methods A total of 64 patients with septic shock were divided into the survival group (n=41) and the nonsurvival group (n=23) according to mortality at 28 days after enrollments.PARP-1 activity in circulating mononuclear cells,brain natriuretic peptide,Acute Physiology and Chronic Health Evaluation Ⅱ score,the cardiac index (CI),the cardiac function index (CFI),global ejection fraction (GEF),and the left ventricular contractility index (dp/dt max) were measured after admission to the intensive care unit.Results PARP-1 activity in circulating mononuclear cells of nonsurvival patients with septic shock was significantly higher than that in survival patients.PARP-1 activity in circulating mononuclear cells was strongly,negatively correlated with the CI,the CFI,GEE and dp/dt max.Multiple Logistic regression analysis showed that PARP-1 activity in circulating mononuclear cells was an independent risk factor of myocardial dysfunction.The optimal cutoff point of PARP-1 activity for predicting 28-day mortality was 942 nmol/L with a sensibility of 78.2% and specificity of 65.1%.Conclusion PARP-1 activity in circulating mononuclear cells is significantly associated with myocardial dysfunction and may have prognostic value in patients with septic shock.

  1. Down-regulation of increased TRAF6 expression in the peripheral mononuclear cells of patients with primary Sjögren's syndrome by an EBV-EBER1-specific synthetic single-stranded complementary DNA molecule.

    Science.gov (United States)

    Sipka, Sándor; Zilahi, Erika; Papp, Gábor; Chen, Ji-Qing; Nagy, Andrea; Hegyi, Katalin; Kónya, József; Zeher, Margit

    2017-05-01

    We described earlier a simultaneously increased that the increased expression of miRNA-146a/b was accompanied by an increase in the expression of and TRAF6 and a decrease in the expression of IRAK1 genes in the peripheral mononuclear cells (PBMCs) of patients with primary Sjogren's syndrome (pSS) patients. Recently, the expression of EBV encoded. RNA (EBER) was published in the B cells of salivary glands of in pSS. In the present study, we applied an EBV-EBER1 specific synthetic single stranded complementary DNA molecule (EBV-EBER1-cDNA) to test whether any EBER1 related effect exists also in PBMCs of pSS patients. In the PBMCs of pSS patients and healthy controls, we investigated in vitro the effects of a synthetic single stranded EBV-EBER1-cDNA molecule, synthetic double-stranded (ds)RNA polyinosinic-polycytidylic acid [poly (I:C)] and polyadenylic acid potassium salt poly-adenylic acid [poly-(A)] on the expression of TRAF6 gene tested by qRTPCR. The release of interferon -α was detected by ELISA. EBV-EBER1-cDNA resulted in a significant reduction in the expression of TRAF6 in the cells of patients, but in the healthy controls not, whereas the treatments with poly (I:C) and poly-(A) could not reduce the TRAF6 over-expression. No release of EBER1 could be observed in the culture supernatants of patients with pSS. Only the treatment with poly (I:C) resulted in a significant increase of interferon -α release, and only in the heathy controls. No release of EBER1 molecules took place during the culturing of cells. EBV-EBER- cDNA acted functionally on the cells of patients only. These findings give a further evidence of the linkage between EBV and pSS, furthermore, they show the possible role of EBV-EBER1 in the induction of increased TRAF6 expression in the peripheral B cells of Sjögren's patients. © 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

  2. DNA damage in peripheral blood mononuclear cells and neutrophils of dairy cows during the transition period

    Directory of Open Access Journals (Sweden)

    S. Oikawa

    2012-06-01

    Full Text Available This study was designed to investigate the apoptotic process in peripheral blood mononuclear cells (PBMC and polymorphonuclear neutrophil leukocytes (PMN in dairy cattle during the transition period. Blood samples were collected from 4 dairy cattle at 3 weeks before the expected parturition (wk -3, parturition (wk 0 and 3 weeks after parturition (wk +3. The DNA damage of PBMC and PMN was evaluated based on the comet assay using visual scoring (arbitrary units. Undamaged DNA remained within the core (score 0 and the broken DNA migrated from the core towards the anode forming the tail of a comet (scores 1-4. Significantly higher scores in PBMC at wk 0 and wk +3 were observed compared with those in PMN although there were no significant changes of scores in either cell type during the experimental period. It is suggested that the apoptotic rate of PBMC is accelerated compared with that of PMC during the transition period.

  3. Correlation of TLR2 and TLR4 expressions in peripheral blood mononuclear cells to Th1- and Th2-type immune responses in children with henoch-schönlein purpura.

    Science.gov (United States)

    Chang, Hong; Zhang, Qiu-Ye; Lin, Yi; Cheng, Na; Zhang, Shou-Qing

    2015-01-01

    We discussed the correlation of TLR2 (Toll-like receptor) and TLR4 expressions in peripheral blood mononuclear cells (PBMCs) to Th1- and Th2-type immune responses in children with Henoch-Schönlein Purpura (HSP). The role of TLR2 and TLR4 in the pathogenesis of HSP was analyzed. Sixty-four HSP children treated at our hospital from October 2011 to November 2012 were enrolled and divided into NHSPN group (complicated by renal impairment, 36 cases) and HSPN group (not complicated by renal impairment, 28 cases). In the meantime, 30 normal children receiving physical examination at our hospital were recruited as controls. Peripheral blood T cell subgroups and TLR2 and TLR4 expressions in PBMCs were detected by using flow cytometry; relative expression levels of TLR2 and TLR4 mRNA in PBMCs by real-time quantitative fluorescence PCR, and plasma levels of IFN-γ, IL-4 and IL-6 by ELISA method. Relative expression levels of TLR2 and TLR4 mRNAs in PBMCs and TLR2 and TLR4 protein expressions in children with HSP were significantly higher than those of the controls (P<0.01). The relative expression levels of TLR2 and TLR4 mRNAs in PBMCs and TLR2 and TLR4 protein expressions in HSPN group were obviously higher than those in NHSPN group (P<0.05; P<0.01; P<0.01; P<0.01); CD3(+) T cells and CD3(+)CD4(+) T cells in HSP group were significantly decreased, while CD3(+)CD8(+) T cells and CD3(+)HLADR(+) T activated cells were considerably increased (P<0.01); The plasma levels of IL-4 and IL-6 in HSP group were significantly higher than those of the normal controls (P<0.01, P<0.01); IFN-γ level in the former was much lower than in the control group (P<0.05); IFN-γ/IL-4 ratio in the former was also lower than that in the control (P<0.01); TLR2 and TLR4 expressions in HSP group showed significantly positive correlation with the plasma levels of IL-4 and IL-6 (P<0.01, P<0.05; P<0.01, P<0.01) and significantly negative correlation with IFN-γ/IL-4 ratio (P<0.01; P<0.01). TLR2 and TLR4

  4. Therapeutic polypeptides based on HBV core 18-27 epitope can induce CD8+ CTL-mediated cytotoxicity in HLA-A2+ human PBMCs

    Institute of Scientific and Technical Information of China (English)

    Tong-Dong Shi; Yu-Zhang Wu; Zheng-Cai Jia; Li-Yun Zou; Wei Zhou

    2004-01-01

    AIM: To explore how to improve the immunogenicity of HBcAg CTL epitope based polypeptides and to trigger an HBV-specific HLA I-restricted CD8+ T cell response in vitro.METHODS: A new panel of mimetic therapeutic peptides based on the immunodominant B cell epitope of HBV PreS218-24 region, the CTL epitope of HBcAg18-27 and the universal T helper epitope of tetanus toxoid (TT) 830-843was designed using computerized molecular design method and synthesized by Merrifield's solid-phase peptide synthesis.Their immunological properties of stimulating activation and proliferation of lymphocytes, of inducing TH1 polarization,CD8+ T cell magnification and HBV-specific CD8+ CTL mediated cytotoxicity were investigatedin vitro using HLAA2+ human peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic hepatitis B patients.RESULTS: Results demonstrated that the therapeutic polypeptides based on immunodominant HBcAg18-27 CTL,PreS2 B- and universal TH epitopes could stimulate the activation and proliferation of lymphocytes, induce specifically and effectively CD8+ T cell expansion and vigorous HBVspecific CTL-mediated cytotoxicity in human PBMCs.CONCLUSION: It indicated that the introduction of immunodominant T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of short CTL epitopes in vitro.

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

    Science.gov (United States)

    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

  6. Productive infection of human peripheral blood mononuclear cells by feline immunodeficiency virus: implications for vector development.

    Science.gov (United States)

    Johnston, J; Power, C

    1999-03-01

    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 10(1.3) to 10(2.1) 50% tissue culture infective doses/10(6) 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.

  7. Analysis of quantum dot fluorescence stability in primary blood mononuclear cells.

    Science.gov (United States)

    Summers, Huw D; Holton, Mark D; Rees, Paul; Williams, Paul M; Thornton, Catherine A

    2010-10-01

    A quantitative assessment of fluorescence signal generation and persistence in blood cells, measured at multiple points over a time course, is presented. Quantum dots (QDs) are inorganic fluorophores that are photostable and nonmetabolized and so can provide quantitative measures of cell biology over multiple cell generations. However, if the potential of these nanoparticles for long-term reporting is to be realized, an understanding of the stability of their fluorescence in living cells is essential. CdTe/ZnS and CdSe/ZnS core/shell dots with peak emission wavelengths of 705 nm and 585 nm, respectively, were loaded, via endocytosis into mononuclear cells extracted from primary blood and flow cytometry used to measure the average fluorescence intensity per cell within populations >10⁴. Time-based study showed a saturation-limited uptake of QDs with a characteristic time of 20 min and a maximum fluorescence signal that is linearly proportional to dot solution concentration. The fluorescence signal decreases after attachment and internalization within cells and is accurately described by a biexponential decay with a rapid initial decay followed by a much slower signal loss with characteristic times of 435 and 7,000 min respectively. Comparison with control samples indicates that interaction with the culture media is a major contributory factor to the initial signal decay. These results provide phenomenological descriptions of the evolving QD fluorescence within live cells with associated analytical equations that allow quantitative assessment of QD-based assays.

  8. The photodynamic effect of Victoria blue BO on peripheral blood mononuclear and leukemic cells

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    Fiedorowicz, M. [Hugo Kollatay Univ. of Agriculture, Krakow (Poland); Pituch-Noworolska, A.; Zembala, M. [Polish-American Children`s Hospital, Krakow (Poland). Dept. of Clinical Immunology

    1997-05-01

    The photodynamic effect of Victoria blue BO (VB-BO) and photoirradiation on peripheral blood mononuclear cells was studied. The cells were preincubated with VB-BO followed by photoirradiation and overnight culture. The highest percentage of dead cells (propidium iodide assay in flow cyctometry) was seen in the monocyte population. The lymphocytes showed a lower sensitivity to VB-BO photodynamic action than the monocytes (12% vs 80% of PI-positive cells). The effect of VB-BO and phototreatment on lymphocyte function was studied using a mitogen-induced proliferation assay. A decrease of mitogen response was observed. The VB-BO and photoirradiation were also used on leukemic cells. The leukemic cells from acute myeloid leukemia and B precursors leukemia were sensitive to VB-BO photodynamic action. The high VB-BO sensitivity of monocytes and leukemic cells (myeloid and lymphoid B derived) suggests possible application of VB-BO for selective depletion of monocytes or sensitive leukemic cells. (author).

  9. Suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus.

    Science.gov (United States)

    Ruiz Silva, Mariana; Aguilar Briseño, José A; Upasani, Vinit; van der Ende-Metselaar, Heidi; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2017-06-01

    Dengue and chikungunya are viral diseases transmitted to humans by infected Aedes spp. mosquitoes. With an estimated 390 million infected people per year dengue virus (DENV) currently causes the most prevalent arboviral disease. During the last decade chikungunya virus (CHIKV) has caused large outbreaks and has expanded its territory causing millions of cases in Asia, Africa and America. The viruses share a common mosquito vector and during the acute phase cause similar flu-like symptoms that can proceed to more severe or debilitating symptoms. The growing overlap in the geographical distribution of these mosquito-borne infections has led to an upsurge in reported cases of DENV/CHIKV co-infections. Unfortunately, at present we have little understanding of consequences of the co-infections to the human host. The overall aim of this study was to define viral replication dynamics and the innate immune signature involved in concurrent DENV and CHIKV infections in human peripheral blood mononuclear cells (PBMCs). We demonstrate that concomitant infection resulted in a significant reduction of CHIKV progeny and moderate enhancement of DENV production. Remarkably, the inhibitory effect of DENV on CHIKV infection occurred independently of DENV replication. Furthermore, changes in type I IFN, IL-6, IL-8, TNF-α, MCP-1 and IP-10 production were observed during concomitant infections. Notably, co-infections led to a significant increase in the levels of TNF-α and IL-6, cytokines that are widely considered to play a crucial role in the early pathogenesis of both viral diseases. In conclusion, our study reveals the interplay of DENV/CHIKV during concomitant infection and provides a framework to investigate viral interaction during co-infections.

  10. Suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus.

    Directory of Open Access Journals (Sweden)

    Mariana Ruiz Silva

    2017-06-01

    Full Text Available Dengue and chikungunya are viral diseases transmitted to humans by infected Aedes spp. mosquitoes. With an estimated 390 million infected people per year dengue virus (DENV currently causes the most prevalent arboviral disease. During the last decade chikungunya virus (CHIKV has caused large outbreaks and has expanded its territory causing millions of cases in Asia, Africa and America. The viruses share a common mosquito vector and during the acute phase cause similar flu-like symptoms that can proceed to more severe or debilitating symptoms. The growing overlap in the geographical distribution of these mosquito-borne infections has led to an upsurge in reported cases of DENV/CHIKV co-infections. Unfortunately, at present we have little understanding of consequences of the co-infections to the human host. The overall aim of this study was to define viral replication dynamics and the innate immune signature involved in concurrent DENV and CHIKV infections in human peripheral blood mononuclear cells (PBMCs. We demonstrate that concomitant infection resulted in a significant reduction of CHIKV progeny and moderate enhancement of DENV production. Remarkably, the inhibitory effect of DENV on CHIKV infection occurred independently of DENV replication. Furthermore, changes in type I IFN, IL-6, IL-8, TNF-α, MCP-1 and IP-10 production were observed during concomitant infections. Notably, co-infections led to a significant increase in the levels of TNF-α and IL-6, cytokines that are widely considered to play a crucial role in the early pathogenesis of both viral diseases. In conclusion, our study reveals the interplay of DENV/CHIKV during concomitant infection and provides a framework to investigate viral interaction during co-infections.

  11. Human Neutrophil Elastase Induce Interleukin-10 Expression in Peripheral Blood Mononuclear Cells through Protein Kinase C Theta/Delta and Phospholipase Pathways

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    Jin Kawata

    2016-02-01

    Full Text Available Objective: Neutrophils have an important role in the rapid innate immune response, and the release or active secretion of elastase from neutrophils is linked to various inflammatory responses. Purpose of this study was to determine how the human neutrophil elastase affects the interleukin-10 (IL-10 response in peripheral blood mononuclear cells (PBMC. Materials and Methods: In this prospective study, changes in IL-10 messenger RNA (mRNA and protein expression levels in monocytes derived from human PBMCs were investigated after stimulation with human neutrophil elastase (HNE. A set of inhibitors was used for examining the pathways for IL-10 production induced by HNE. Results: Reverse transcription polymerase chain reaction (RT-PCR showed that stimulation with HNE upregulated IL-10 mRNA expression by monocytes, while the enzyme-linked immunosorbent assay (ELISA revealed an increase of IL-10 protein level in the culture medium. A phospholipase C inhibitor (U73122 partially blunted the induction of IL-10 mRNA expression by HNE, while IL-10 mRNA expression was significantly reduced by a protein kinase C (PKC inhibitor (Rottlerin. A calcium chelator (3,4,5-trimethoxybenzoic acid 8-(diethylaminooctyl ester: TMB-8 inhibited the response of IL-10 mRNA to stimulation by HNE. In addition, pretreatment with a broad-spectrum PKC inhibitor (Ro-318425 partly blocked the response to HNE. Finally, an inhibitor of PKC theta/delta abolished the increased level of IL-10 mRNA expression. Conclusion: These results indicate that HNE mainly upregulates IL-10 mRNA expression and protein production in moncytes via a novel PKC theta/delta, although partially via the conventional PKC pathway.

  12. Toxicity to the normal hemocytes by ALA-PDT for the ex vivo purging of hematopoietic stem cell grafts

    Institute of Scientific and Technical Information of China (English)

    张宝琴; 张镇西; 苗丽霞; 谭璐; 肖谧; 徐霞

    2008-01-01

    Objective To study the toxic effects of 5-amionlevulinic acid-based photodynamic therapy (ALA-PDT) on human peripheral blood mononuclear cells (PBMCs), cord blood mononuclear cells (CBMCs) and peripheral blood stem cells (PBSCs), and furthermore, to understand the possible causes of this response. Methods We used MTT assay to detect the survival rate of PBMCs, CBMCs and PBSCs after treated by ALA-PDT under the optimum experiment conditions with U937 as control; Annexin V-FITC/PI was used to detect the patte...

  13. Do androgen deprivation drugs affect the immune cross-talk between mononuclear and prostate cancer cells?

    Science.gov (United States)

    Salman, Hertzel; Bergman, Michael; Blumberger, Naava; Djaldetti, Meir; Bessler, Hanna

    2014-02-01

    The aim of the study was to examine the effect of androgen deprivation drugs, i.e. leuprolide and bicalutamide on the immune cross-talk between human peripheral blood mononuclear cells (PBMC) and cells from PC-3 and LNCaP human prostate cancer lines. PBMC, PC-3 and LNCaP were separately incubated without and with two androgen-deprivation drugs, i.e. leuprolide and bicalutamide, and the secretion of IL-1β, IL-6, IL-1ra and IL-10 was examined. In addition, the effect of both drugs on the production of those cytokines was carried out after 24 hours incubation of PBMC with both types of cancer cells. Leuprolide or bicalutamide did not affect the production of the cytokines by PBMC or by the prostate cancer cells from the two lines. Incubation of PBMC with PC-3 or LNCaP cells caused increased production of IL-1β, IL-6 and IL-10 as compared with PBMC incubated without malignant cells. While 10(-7) M and 10(-8) M of leuprolide caused a decreased secretion of IL-1β by PBMC previously incubated with prostate cancer cells without the drug, bicalutamide did not affect this PBMC activity at any drug concentration. This observation suggests the existence of an additional mechanism explaining the effect of androgen deprivation therapy in prostate cancer patients.

  14. Binding of toxic-shock-syndrome toxin-1 to human peripheral blood mononuclear cells

    Energy Technology Data Exchange (ETDEWEB)

    Poindexter, N.J.; Schlievert, P.M.

    1987-07-01

    Toxic-shock-syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus and associated with toxic shock syndrome, functions in vitro as both a lymphoproliferative and immunosuppressive protein for human peripheral blood mononuclear cells (PBMs). We analyzed TSST-1-target cell interactions by receptor-ligand binding analyses. In competitive binding experiments, 2 X 10(5) human PBMs or purified cell populations were incubated in the presence of small amounts of (5-50 ng) of /sup 125/I-labeled TSST-1 and increasing amounts of unlabeled TSST-1 (25-10,000 ng). Data were analyzed by the method of Scatchard. Toxin-specific receptors were shown to exist on T lymphocytes within the PBM population. T4+ cells had 27.5 X 10(6) receptors per cell, and T8+ cells had 9 X 10(6) receptors per cell. T4+ and T8+ receptors had dissociation constants of 2.58 X 10(-8) M and 1.8 X 10(-8) M, respectively. These studies confirm earlier work showing that TSST-1 causes the functional activation of a population of T lymphocytes involved in suppression of immunoglobulin responses.

  15. Chemokine receptor expression on the surface of peripheral blood mononuclear cells in Chagas disease.

    Science.gov (United States)

    Talvani, Andre; Rocha, Manoel O C; Ribeiro, Antonio L; Correa-Oliveira, Rodrigo; Teixeira, Mauro M

    2004-01-15

    We evaluated the expression of chemokine receptors (CCR1, CCR2, CCR5, and CXCR4) on the surface of peripheral blood mononuclear cells obtained from patients with chronic chagasic cardiomyopathy (CCC) and noninfected individuals. Only CCR5 and CXCR4 expression was different on the surface of the subsets (CD4, CD8, and CD14) evaluated. Patients with mild CCC had elevated leukocyte expression of CCR5, compared with noninfected individuals or those with severe disease. CXCR4 expression was lower on leukocytes from patients with severe CCC. The differential expression of both receptors on leukocytes of patients with CCC was consistent and clearly correlated with the degree of heart function such that the lower the heart function, the lower the expression of either CCR5 or CXCR4. These results highlight the possible participation of the chemokine system in early forms of chagasic cardiomyopathy and the relevance of heart failure-induced remodeling in modifying immune parameters in infected individuals.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J;

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of ... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

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

    Science.gov (United States)

    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.

  19. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J;

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of TNF-alpha suppression...... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  20. High-Throughput Analysis of the T Cell Receptor Beta Chain Repertoire in PBMCs from Chronic Hepatitis B Patients with HBeAg Seroconversion

    Directory of Open Access Journals (Sweden)

    Yachao Qu

    2016-01-01

    Full Text Available T lymphocytes are the most important immune cells that affect both the development and treatment of hepatitis B. We used high-throughput sequencing to determine the diversity in the V and J regions of the TCRβ chain in 4 chronic hepatitis B patients before and after HBeAg seroconversion. Here, we demonstrate that the 4 patients expressed Vβ12-4 at the highest frequencies of 10.6%, 9.2%, 17.5%, and 7.5%, and Vβ28 was the second most common, with frequencies of 7.8%, 6.7%, 5.3%, and 10.9%, respectively. No significant changes were observed following seroconversion. With regard to the Jβ gene, Jβ2-1 was the most commonly expressed in the 4 patients at frequencies of 5.8%, 6.5%, 11.3%, and 7.3%, respectively. Analysis of the V-J region genes revealed several differences, including significant increases in the expression levels of V7-2-01-J2-1, V12-4-J1-1, and V28-1-J1-5 and a decrease in that of V19-01-J2-3. These results illustrate the presence of biased TCRVβ and Jβ gene expression in the chronic hepatitis B patients. TRBVβ12-4, Vβ28, Jβ2-1, V7-2-01-J2-1, V12-4-J1-1, and V28-1-J1-5 may be associated with the development and treatment of CHB.

  1. High-Throughput Analysis of the T Cell Receptor Beta Chain Repertoire in PBMCs from Chronic Hepatitis B Patients with HBeAg Seroconversion

    Science.gov (United States)

    Huang, Yong; Liu, Di; Huang, Yinuo; Zhang, Zhiyi

    2016-01-01

    T lymphocytes are the most important immune cells that affect both the development and treatment of hepatitis B. We used high-throughput sequencing to determine the diversity in the V and J regions of the TCRβ chain in 4 chronic hepatitis B patients before and after HBeAg seroconversion. Here, we demonstrate that the 4 patients expressed Vβ12-4 at the highest frequencies of 10.6%, 9.2%, 17.5%, and 7.5%, and Vβ28 was the second most common, with frequencies of 7.8%, 6.7%, 5.3%, and 10.9%, respectively. No significant changes were observed following seroconversion. With regard to the Jβ gene, Jβ2-1 was the most commonly expressed in the 4 patients at frequencies of 5.8%, 6.5%, 11.3%, and 7.3%, respectively. Analysis of the V-J region genes revealed several differences, including significant increases in the expression levels of V7-2-01-J2-1, V12-4-J1-1, and V28-1-J1-5 and a decrease in that of V19-01-J2-3. These results illustrate the presence of biased TCRVβ and Jβ gene expression in the chronic hepatitis B patients. TRBVβ12-4, Vβ28, Jβ2-1, V7-2-01-J2-1, V12-4-J1-1, and V28-1-J1-5 may be associated with the development and treatment of CHB. PMID:27818694

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

    Science.gov (United States)

    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.

  3. Peripheral blood mononuclear cell gene expression in healthy adults rapidly transported to high altitude

    Directory of Open Access Journals (Sweden)

    Herman NM

    2014-12-01

    Full Text Available Nicole M Herman,1 Diane E Grill,2 Paul J Anderson,1 Andrew D Miller,1 Jacob B Johnson,1 Kathy A O’Malley,1 Maile L Ceridon Richert,1 Bruce D Johnson1 1Department of Cardiovascular Diseases, 2Department of Biostatistics, Mayo Clinic Rochester, MN, USA Abstract: Although mechanisms of high altitude illness have been studied extensively, the processes behind the development of these conditions are still unclear. Few genome-wide studies on rapid exposure to high altitude have been performed. Each year, scientists and support workers are transferred by plane from McMurdo Station in Antarctica (sea level to the Amundsen-Scott South Pole Station at 2,835 meters. This uniform and rapid transfer to altitude provides a unique opportunity to study the effects of hypobaric hypoxia on gene expression that may help illustrate the body's adaptations to these conditions. We hypothesized that an extensive number of genes would change with rapid exposure to altitude and further expected that these genes would correspond to inflammatory pathways proposed as a mechanism in development of acute mountain sickness. Peripheral venous blood samples were drawn from 98 healthy subjects at sea level and again on day two at altitude. Microarray analysis was performed on these samples. In total, 1,118 probe sets with significant P-values and fold changes (90% upregulated were identified and entered into MetaCore™ software. Several pathways, including oxidative phosphorylation, cytoskeleton remodeling, and platelet aggregation, were significantly represented by the data set and all were upregulated. Many genes changed expression, and the vast majority of these increased. Increased metabolism in peripheral blood mononuclear cells suggests increased inflammatory activity. Keywords: peripheral blood mononuclear cells, microarray, gene expression, acute mountain sickness

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

    Science.gov (United States)

    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 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 attenuates in vitro T-cell and monocyte activation. This

  5. Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells.

    Directory of Open Access Journals (Sweden)

    Gyri T Haugland

    Full Text Available Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (<5 µm, mononuclear blood cells from Atlantic salmon (Salmo salar L. not previously characterized. In order to identify them, we have performed morphological, gene expression, flow cytometry, cytochemical, ultrastructural and functional analyses. Interestingly, they highly express the gene encoding CD83, the most characteristic cell surface marker for dendritic cells in mammals, and MHC class II limited to professional antigen presenting cells. They did not express genes nor did they have cell markers for B-cells, T-cells, monocytes/macrophages or neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.

  6. Principles of bone marrow processing and progenitor cell/mononuclear cell concentrate collection in a continuous flow blood cell separation system.

    Science.gov (United States)

    Hester, J P; Rondón, G; Huh, Y O; Lauppe, M J; Champlin, R E; Deisseroth, A B

    1995-08-01

    The application of continuous flow apheresis technology to processing bone marrow for collection of the mononuclear progenitor cell population appears to follow the same principles as collection of mononuclear cells from peripheral blood. Unlike peripheral blood, however, where mobilization of cells from extravascular sites during the procedures contributes significantly to the final cell yield, the entire quantity of progenitor cells available for recovery from marrow is present in the original marrow when it is pooled. The process then becomes one of attempting optimal recovery of the cells of interest while excluding contaminating erythrocytes and cells of the myeloid series. This study reports the development of a protocol for recovery of MNC, CD33+, CD34+, and CD34+/DR- cells from harvested marrow for autologous and allogeneic transplants using a continuous flow blood cell separator, the variables influencing the recovery of the cells of interest and the clinical response to infusion of the processed cells.

  7. Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

    Science.gov (United States)

    Quintana-Bustamante, Oscar; Segovia, Jose C

    2016-01-01

    Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

  8. One-stage focal cartilage defect treatment with bone marrow mononuclear cells and chondrocytes leads to better macroscopic cartilage regeneration compared to microfracture in goats

    NARCIS (Netherlands)

    Bekkers, J.E.J.; Creemers, L.B.; Tsuchida, A.I.; van Rijen, M.H.P.; Custers, R.J.H.; Dhert, W.J.A.; Saris, Daniël B.F.

    2013-01-01

    Objective The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work

  9. Cpt1a gene expression in peripheral blood mononuclear cells as an early biomarker of diet-related metabolic alterations

    KAUST Repository

    Diaz-Rua, Ruben

    2016-11-23

    Background: Research on biomarkers that provide early information about the development of future metabolic alterations is an emerging discipline. Gene expression analysis in peripheral blood mononuclear cells (PBMC) is a promising tool to identify subjects at risk of developing diet-related diseases.

  10. Quantitative analysis of antiretroviral drugs in lysates of peripheral blood mononuclear cells using MALDI-triple quadrupole mass spectrometry.

    NARCIS (Netherlands)

    Kampen, JJ van; Burgers, P.C.; Gruters, R.A.; Osterhaus, A.D.; Groot, R. de; Luider, T.M.; Volmer, D.A.

    2008-01-01

    We report here on the use of a prototype matrix-assisted laser desorption/ionization (MALDI)-triple quadrupole mass spectrometer for quantitative analysis of six antiretroviral drugs in lysates of peripheral blood mononuclear cells (PBMC). Of the five investigated MALDI matrixes, 2,5-dihydroxybenzoi

  11. Distinct types of fibrocyte can differentiate from mononuclear cells in the presence and absence of serum.

    Directory of Open Access Journals (Sweden)

    S John Curnow

    Full Text Available BACKGROUND: Fibrocytes are bone-marrow derived cells, expressing both haematopoietic and stromal cell markers, which contribute to tissue repair as well as pathological fibrosis. The differentiation of fibrocytes remains poorly characterised and this has limited understanding of their biology and function. In particular two methods are used to generate fibrocytes in vitro that differ fundamentally by the presence or absence of serum. METHODOLOGY/PRINCIPAL FINDINGS: We show here that fibrocytes grown in the absence of serum (SF differentiate more efficiently from peripheral blood mononuclear cells than CD14(+ monocytes, and respond to serum by losing their spindle-shaped fibrocyte morphology. Although fibrocytes generated in the presence of serum (SC express the same range of markers, they differentiate more efficiently from CD14(+ monocytes and do not change their morphology in response to serum. Transcriptional analysis revealed that both types of fibrocyte are distinct from each other, fibroblasts and additional monocyte-derived progeny. The gene pathways that differ significantly between SF and SC fibrocytes include those involved in cell migration, immune responses and response to wounding. CONCLUSIONS/SIGNIFICANCE: These data show that SF and SC fibrocytes are distinct but related cell types, and suggest that they will play different roles during tissue repair and fibrosis where changes in serum proteins may occur.

  12. Autologous bone marrow mononuclear cells in ischemic cerebrovascular accident paves way for neurorestoration: a case report.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Nagrajan, Anjana; Gokulchandran, Nandini; Badhe, Prerna; Paranjape, Amruta; Biju, Hema

    2014-01-01

    In response to acute ischemic stroke, large numbers of bone marrow stem cells mobilize spontaneously in peripheral blood that home onto the site of ischemia activating the penumbra. But with chronicity, the numbers of mobilized cells decrease, reducing the degree and rate of recovery. Cellular therapy has been explored as a new avenue to restore the repair process in the chronic stage. A 67-year-old Indian male with a chronic right middle cerebral artery ischemic stroke had residual left hemiparesis despite standard management. Recovery was slow and partial resulting in dependence to carry out activities of daily living. Our aim was to enhance the speed of recovery process by providing an increased number of stem cells to the site of injury. We administered autologous bone marrow mononuclear cells intrathecally alongwith rehabilitation and regular follow up. The striking fact was that the hand functions, which are the most challenging deficits, showed significant recovery. Functional Independence Measure scores and quality of life improved. This could be attributed to the neural tissue restoration. We hypothesize that cell therapy may be safe, novel and appealing treatment for chronic ischemic stroke. Further controlled trials are indicated to advance the concept of Neurorestoration.

  13. Autologous Bone Marrow Mononuclear Cells in Ischemic Cerebrovascular Accident Paves Way for Neurorestoration: A Case Report

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2014-01-01

    Full Text Available In response to acute ischemic stroke, large numbers of bone marrow stem cells mobilize spontaneously in peripheral blood that home onto the site of ischemia activating the penumbra. But with chronicity, the numbers of mobilized cells decrease, reducing the degree and rate of recovery. Cellular therapy has been explored as a new avenue to restore the repair process in the chronic stage. A 67-year-old Indian male with a chronic right middle cerebral artery ischemic stroke had residual left hemiparesis despite standard management. Recovery was slow and partial resulting in dependence to carry out activities of daily living. Our aim was to enhance the speed of recovery process by providing an increased number of stem cells to the site of injury. We administered autologous bone marrow mononuclear cells intrathecally alongwith rehabilitation and regular follow up. The striking fact was that the hand functions, which are the most challenging deficits, showed significant recovery. Functional Independence Measure scores and quality of life improved. This could be attributed to the neural tissue restoration. We hypothesize that cell therapy may be safe, novel and appealing treatment for chronic ischemic stroke. Further controlled trials are indicated to advance the concept of Neurorestoration.

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

    Science.gov (United States)

    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

  15. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Haller, D.; Brinz, S.

    2004-01-01

    To further investigate the interaction between human mononuclear leucocytes [peripheral blood mononuclear cells (PBMC)] and enterocytes, the effect of a confluent layer of differentiated CACO-2 cells on cytokine kinetics during challenge with bacteria in a compartmentalized coculture model...... cells when leucocytes were stimulated directly with bacteria. This suppression was not paralleled by changes in the production of IL-10, IL-6 and transforming growth factor (TGF)-beta. When the bacteria were applied apically to the CACO-2 cell layer, the production of TNF-alpha, IL-12, IL-1beta, IL-8...... analysis revealed that IL-8 gene expression was equally induced in both CACO-2 and PBMC after apical stimulation with bacteria. Of note, bacteria-stimulated CACO-2 cells produced little or no cytokines in the absence of leucocytes, supporting the concept of leucocyte-epithelial cell cross...

  16. Paracrine Mechanisms of Intravenous Bone Marrow-Derived Mononuclear Stem Cells in Chronic Ischemic Stroke.

    Science.gov (United States)

    Bhasin, Ashu; Srivastava, M V Padma; Mohanty, Sujata; Vivekanandhan, Sivasubramaniam; Sharma, Sakshi; Kumaran, Senthil; Bhatia, Rohit

    2016-01-01

    The emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration. The fate of stem cells is determined by their niche, consisting of surrounding cells and the secreted trophic growth factors. This interim report evaluates the safety, feasibility and efficacy (if any) of bone marrow-derived mononuclear stem cells (BM-MNC) in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF) and brain-derived neurotrophic growth factor (BDNF). Twenty stroke patients and 20 age-matched healthy controls were recruited with the following inclusion criteria: 3 months to 1.5 years from the index event, Medical Research Council (MRC) grade of hand muscles of at least 2, Brunnstrom stage 2-5, conscious, and comprehendible. They were randomized to one group receiving autologous BM-MNC (mean 60-70 million) and to another group receiving saline infusion (placebo). All patients were administered a neuromotor rehabilitation regime for 8 weeks. Clinical assessments [Fugl Meyer scale (FM), modified Barthel index (mBI), MRC grade, Ashworth tone scale] were carried out and serum VEGF and BDNF levels were assessed at baseline and at 8 weeks. No serious adverse events were observed during the study. There was no statistically significant clinical improvement between the groups (FM: 95% CI 15.2-5.35, p = 0.25; mBI: 95% CI 14.3-4.5, p = 0.31). VEGF and BDNF expression was found to be greater in group 1 compared to group 2 (VEGF: 442.1 vs. 400.3 pg/ml, p = 0.67; BDNF: 21.3 vs. 19.5 ng/ml) without any statistically significant difference. Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients. The released growth factors (VEGF and BDNF) in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime. © 2016 The Author(s) Published by S. Karger AG, Basel.

  17. Paracrine Mechanisms of Intravenous Bone Marrow-Derived Mononuclear Stem Cells in Chronic Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Ashu Bhasin

    2016-10-01

    Full Text Available Background: The emerging role of stem cell technology and transplantation has helped scientists to study their potential role in neural repair and regeneration. The fate of stem cells is determined by their niche, consisting of surrounding cells and the secreted trophic growth factors. This interim report evaluates the safety, feasibility and efficacy (if any of bone marrow-derived mononuclear stem cells (BM-MNC in chronic ischemic stroke by studying the release of serum vascular endothelial growth factor (VEGF and brain-derived neurotrophic growth factor (BDNF. Methods: Twenty stroke patients and 20 age-matched healthy controls were recruited with the following inclusion criteria: 3 months to 1.5 years from the index event, Medical Research Council (MRC grade of hand muscles of at least 2, Brunnstrom stage 2-5, conscious, and comprehendible. They were randomized to one group receiving autologous BM-MNC (mean 60-70 million and to another group receiving saline infusion (placebo. All patients were administered a neuromotor rehabilitation regime for 8 weeks. Clinical assessments [Fugl Meyer scale (FM, modified Barthel index (mBI, MRC grade, Ashworth tone scale] were carried out and serum VEGF and BDNF levels were assessed at baseline and at 8 weeks. Results: No serious adverse events were observed during the study. There was no statistically significant clinical improvement between the groups (FM: 95% CI 15.2-5.35, p = 0.25; mBI: 95% CI 14.3-4.5, p = 0.31. VEGF and BDNF expression was found to be greater in group 1 compared to group 2 (VEGF: 442.1 vs. 400.3 pg/ml, p = 0.67; BDNF: 21.3 vs. 19.5 ng/ml without any statistically significant difference. Conclusion: Autologous mononuclear stem cell infusion is safe and tolerable by chronic ischemic stroke patients. The released growth factors (VEGF and BDNF in the microenvironment could be due to the paracrine hypothesis of stem cell niche and neurorehabilitation regime.

  18. Nipah virus infects specific subsets of porcine peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Beata Stachowiak

    Full Text Available Nipah virus (NiV, a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC in the viremic spread of the virus in the swine host. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8- cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166 highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.

  19. Periurethral muscle-derived mononuclear cell injection improves urethral sphincter restoration in rats.

    Science.gov (United States)

    Turco, Marcelo Pitelli; de Souza, Alex Balduino; de Campos Sousa, Isida; Fratini, Paula; Veras, Mariana Matera; Rodrigues, Marcio Nogueira; de Bessa, José; Brolio, Marina Pandolphi; Leite, Katia Ramos Moreira; Bruschini, Homero; Srougi, Miguel; Miglino, Maria Angélica; Gomes, Cristiano Mendes

    2017-03-27

    Investigate the effect of a novel cell-based therapy with skeletal muscle-derived mononuclear cells (SMDMCs) in a rat model of stress urinary incontinence. Male Wistar-Kyoto rats' hind limb muscles were enzymatically dissociated, and SMDMCs were isolated without needing expansion. The cell population was characterized. Twenty female rats underwent urethrolysis. One week later, 10 rats received periurethral injection of 10(6) cells (SMDMC group), and 10 rats received saline injections (Saline group). Ten rats underwent sham surgery (Sham group). Four weeks after injection, animals were euthanized and the urethra was removed. The incorporation of SMDMCs in the female urethra was evaluated with fluorescence in situ hybridization for the detection of Y-chromosomes. Hematoxylin and eosin, Masson's trichrome staining, and immunohistochemistry for actin and myosin were performed. The muscle/connective tissue, actin and myosin ratios were calculated. Morphological evaluation of the urethral diameters and fractional areas of the lumen, mucosa, and muscular layer was performed. SMDMCs population was consistent with the presence of muscle cells, muscle satellite cells, perivascular cells, muscle progenitor cells, and endothelial cells. SMDMCs were incorporated into the urethra. A significant decrease in the muscle/connective tissue ratio was observed in the Saline group compared with the SMDMC and Sham groups. The proportions of actin and myosin were significantly decreased in the Saline group. No differences were observed in the morphometric parameters. SDMSC were incorporated into the rat urethra and promoted histological recovery of the damaged urethral sphincter, resulting in decreased connective tissue deposition and increased muscle content. © 2017 Wiley Periodicals, Inc.

  20. Cytokine profile, proliferation and phosphorylation of ERK1/2 and Akt in circulating mononuclear cells from individuals during the chronic intestinal phase of Schistosomiasis mansoni infection

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    Oliveira-Prado Roberta

    2012-12-01

    Full Text Available Abstract Background The immune response to Schistosoma mansoni is characterized by a granulomatous reaction around the parasite eggs that are trapped in the host liver, and this reaction modulates the immune response during the chronic phase of the disease. The typical peripheral blood mononuclear cell (PBMC response of patients during the chronic intestinal phase of infection is characterized by a decreased response to an S. mansoni soluble egg antigen. To obtain a greater understanding of Schistosoma infections, this study investigated the effects of the soluble egg antigen (SEA and soluble adult worm antigen (SWAP of S. mansoni on cellular proliferation, cytokine production, and ERK1/2 and Akt phosphorylation in PBMCs from infected (XTO and egg-negative (NI individuals living in the same endemic area. Methods The activation status was evaluated by cell immunophenotypic staining (cytometry. The cell proliferation assay was by CFSE method. Cytokine detection assay (Th1 and Th2 was by Cytometric Bead and Array phosphorylation status was by ELISA. Results The XTO, NI and BD (blood donor individuals from an area not endemic for schistosomiasis were compared. The CD4+ T lymphocyte proliferation rate was lower in the XTO group, but not the NI group, after SEA stimulation compared to the BD group. The CD8+ T cell proliferation rate was lower in the XTO group in the unstimulated cultures and after both SEA and SWAP stimulation compared to the BD group. Cytokine analysis after either SEA or SWAP stimulation showed a balanced cytokine pattern in the XTO and NI groups. ERK1/2 and Akt phosphorylation were only marginally detected in all groups; however, a decrease in ERK 1/2 phosphorylation was observed in the SWAP-stimulated XTO group compared to both the NI and BD groups. Conclusions The data indicate that SEA-stimulated CD4+ T cells from infected patients have a lower proliferation rate than the same cells from the NI group. Furthermore, we observed

  1. Effects of a healthy Nordic diet on gene expression changes in peripheral blood mononuclear cells in response to an oral glucose tolerance test in subjects with metabolic syndrome: a SYSDIET sub-study.

    Science.gov (United States)

    Leder, Lena; Kolehmainen, Marjukka; Narverud, Ingunn; Dahlman, Ingrid; Myhrstad, Mari C W; de Mello, Vanessa D; Paananen, Jussi; Carlberg, Carsten; Schwab, Ursula; Herzig, Karl-Heinz; Cloetens, Lieselotte; Storm, Matilda Ulmius; Hukkanen, Janne; Savolainen, Markku J; Rosqvist, Fredrik; Hermansen, Kjeld; Dragsted, Lars O; Gunnarsdottir, Ingibjörg; Thorsdottir, Inga; Risérus, Ulf; Åkesson, Björn; Thoresen, Magne; Arner, Peter; Poutanen, Kaisa S; Uusitupa, Matti; Holven, Kirsten B; Ulven, Stine M

    2016-01-01

    Diet has a great impact on the risk of developing features of metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), and cardiovascular diseases (CVD). We evaluated whether a long-term healthy Nordic diet (ND) can modify the expression of inflammation and lipid metabolism-related genes in peripheral blood mononuclear cells (PBMCs) during a 2-h oral glucose tolerance test (OGTT) in individuals with MetS. A Nordic multicenter randomized dietary study included subjects (n = 213) with MetS, randomized to a ND group or a control diet (CD) group applying an isocaloric study protocol. In this sub-study, we included subjects (n = 89) from three Nordic centers: Kuopio (n = 26), Lund (n = 30), and Oulu (n = 33) with a maximum weight change of ±4 kg, high-sensitivity C-reactive protein concentration ≤10 mg L(-1), and baseline body mass index expression analysis was measured by quantitative real-time polymerase chain reaction (qPCR). We analyzed the mRNA expression changes of 44 genes before and after a 2hOGTT at the beginning and the end of the intervention. The healthy ND significantly down-regulated the expression of toll-like receptor 4 (TLR4), interleukin 18 (IL18), and thrombospondin receptor (CD36) mRNA transcripts and significantly up-regulated the expression of peroxisome proliferator-activated receptor delta (PPARD) mRNA transcript after the 2hOGTT compared to the CD. A healthy ND is able to modify the gene expression in PBMCs after a 2hOGTT. However, more studies are needed to clarify the biological and clinical relevance of these findings.

  2. Co-Administration of Chenopodium Album Allergens and CpG Oligodeoxy-nucleotides Effects on Peripheral Blood Mononuclear Cells of Patients with Allergic Rhinitis Treated with Intranasal Corticosteroids

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    Shokrollah Farrokhi

    2011-06-01

    Full Text Available Allergic Rhinitis (AR is one of the most common chronic diseases in the developed countries. This study was performed to investigate the effect of CpG-ODN in alteration of T-helper (Th1/Th2 balance of patients with AR treated with intranasal corticosteroids (INCs and antihistamines. Peripheral blood mononuclear cells (PBMCs of 20 patients with AR were isolated before and after 45 days therapy.Cytokine production (IL-4, IL-10, IL-13, IFN-γ and specific Ch.a IgE in response to CpG co- administration  of  natural  chenopodium  album  (CpG/Ch.a  or  recombinant  Ch.a  (CpG/rCh.a allergen were investigated in supernatants.of cultured PBMCs using ELISA Intracellular IL-10 was also assessed in CD4+ cells using flow cytometry. Significant increase in production of IFN-γ and IL-10 and decrease in production of IL-4 were found in supernatants of cultured PBMCs activated with CPG/ch.a and CPG/rch.a. of both CpG/Ch.a and CpG/rCh.a compared to allergens alone, before and after therapy.After therapy, IFN-γ production with CpG/Ch.a was significantly increased in comparison with before (237 vs. 44 pg/ml, p=0.001. IFN-γ and IL-10 production with CpG/rCh.a was significantly increased after therapy compared to before (407.6 vs. 109 pg/ml, p=0.01 for IFN-γ; 171.7 vs. 52.6 pg/ml, p=0.008  for  IL-10,  whilst  IL-4  was  significantly decreased (2.1  vs.  5.8  pg/ml,  p=0.02. Intracellular IL-10 expression was also significantly increased in response to either CpG/Ch.a or CpG/rCh.a that showed intracellular assay could be more sensitive than ELISA. Also, treatment with intranasal corticosteroids and antihistamines could enhance this CpG effect, in vitro.

  3. Phenotypes of lung mononuclear phagocytes in HIV seronegative tuberculosis patients: evidence for new recruitment and cell activation

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    José R Lapa e Silva

    1996-06-01

    Full Text Available Mycobacterium tuberculosis preferentially resides in mononuclear phagocytes. The mechanisms by which mononuclear phagocytes keep M. tuberculosis in check or by which the microbe evades control to cause disease remain poorly understood. As an initial effort to delineate these mechanisms, we examined by immunostaining the phenotype of mononuclear phagocytes obtained from lungs of patients with active tuberculosis. From August 1994 to March 1995, consecutive patients who had an abnormal chest X-ray, no demostrable acid-fast bacilli in sputum specimens and required a diagnostic bronchoalveolar lavage (BAL were enrolled. Of the 39 patients enrolled, 21 had microbiologically diagnosed tuberculosis. Thirteen of the 21 tuberculosis patients were either HIV seronegative (n = 12 or had no risk factor for HIV and constituted the tuberculosis group. For comparison, M. tuberculosis negative patients who had BAL samples taken during this time (n = 9 or normal healthy volunteers (n = 3 served as control group. Compared to the control group, the tuberculosis group had significantly higher proportion of cells expressing markers of young monocytes (UCHM1 and RFD7, a marker for phagocytic cells, and increased expression of HLA-DR, a marker of cell activation. In addition, tuberculosis group had significantly higher proportion of cells expressing dendritic cell marker (RFD1 and epithelioid cell marker (RFD9. These data suggest that despite recruitment of monocytes probably from the peripheral blood and local cell activation, host defense of the resident lung cells is insufficient to control M. tuberculosis.

  4. Mononuclear cell therapy reverts cuff-induced thrombosis in apolipoprotein E-deficient mice

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    Lima Leandro C F

    2012-07-01

    Full Text Available Abstract Background Stem/progenitor cell-based therapy has successfully been used as a novel therapeutic strategy for vascular diseases triggered by endothelial dysfunction. The aim of this study was to investigate the effects of mononuclear cell (MNC therapy in situ on carotid cuff-induced occlusive thrombus in the apolipoprotein E knockout (apoE-/- mouse. Methods Spleen-derived MNCs were isolated from green fluorescent protein (GFP-transgenic mice for cell treatment. A cuff-induced thrombus model was produced by placing a nonconstrictive silastic collar around the left common carotid artery in 20-week-old female apoE-/- mice. After 10 days, the cuff was removed, and the animals received in situ MNCs (Cuff-MNC or vehicle (Cuff-Vehicle and were compared with sham-operated animals (Sham. Results The histological analysis showed that the MNC treatment reverted occlusive thrombus formation compared to the vehicle and the vessel lumen area to that observed in the Sham group (MNC, 50 ± 4; Vehicle, 20 ± 4; Sham, 55 ± 2 x103 μm2; p -/- mice. Conclusion In situ short-term MNC therapy was able to revert cuff-induced occlusive thrombi in the carotid arteries of apoE-/- mice, possibly through the homing of EPCs, reduction of oxidative stress and decreased apoptosis.

  5. Hemorrhage activates NF-kappa B in murine lung mononuclear cells in vivo.

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    Shenkar, R; Schwartz, M D; Terada, L S; Repine, J E; McCord, J; Abraham, E

    1996-05-01

    Hemorrhage rapidly increases the expression of proinflammatory and immunoregulatory cytokines in the lungs. Binding elements for the nuclear transcriptional regulatory factors (NF)-kappa B and NF-IL6 (C/EBP beta) are present in the promoter regions of multiple cytokine genes, including those whose expression is increased after blood loss. In the present experiments, we found increased activation in vivo of NF-kappa B in lung mononuclear cells, but not in splenocytes, taken from mice 1 h after hemorrhage. In contrast, hemorrhage did not activate NF-IL6 in lung cells or splenocytes. Inhibition of xanthine oxidase by prior feeding of a tungsten-enriched diet prevented hemorrhage-induced activation in lung cells of NF-kappa B. Incubating splenocytes in vitro with xanthine oxidase activated NF-kappa B but not NF-IL6. Xanthine oxidase-induced activation of NF-kappa B was inhibited by manganese superoxide dismutase, but not by catalase. These results suggest that xanthine oxidase-mediated superoxide anion-dependent activation of NF-kappa B occurs in vivo and in vitro. This mechanism may contribute to increased lung cytokine responses after hemorrhage.

  6. Transplantation of mobilized peripheral blood mononuclear cells for peripheral arterial occlusive disease of the lower extremity

    Institute of Scientific and Technical Information of China (English)

    Xiaofeng YANG; Yanxiang WU; Hongmei WANG; Yifeng XU; Bo XU; Xin LU; Yibin ZANG; Fa WANG; Yue ZHANG

    2006-01-01

    Objectives To assess the clinical efficacy, safety, and feasibility of autologous transplantation of mobilized peripheral blood mononuclear cells (PBMNCs) for patients with peripheral arterial occlusive disease (PAOD) of the lower extremity. Methods A total of 152 patients with PAOD of the lower extremity were enrolled into this non-controlled observational study from November 2003 to March 2006. All patients received subcutaneous injections of recombinant human granulocyte colony-stimulating factor (G-CSF, 450600 μg/day) for 5 days in order to mobilize stem/progenitor cells; their PBMNCs were collected and transplanted by multiple intramuscular injections into ischemic limbs. Patients were followed up for at least 12 weeks. Results At 12 weeks, primarymanifestations,including lower limb pain and coldness, were significantly improved in 137 (90.1%) of the patients; limb ulcers improved or healed in 46 (86.8%) of the 53 patients, while 25 of the 48 (47.9%) patients with limb gangrene remained steady or improved. Ankle-brachial index (ABI) improved in 33 (22%) of the cases, and TcPO2 increased in 45 (30%) of the cases. Angiography before treatment, and at 12 weeks after treatment, was performed in 10 of the patients and showed formation of new collateral vessels. No severe adverse effects or complications specifically related to cell transplantation were observed. Conclusion Autologous transplantation of G-CSF-mobilized PBMNCs might be a safe and effective treatment for lower limb ischemic disorder.(J Geriatr Cardiol 2006; 3:178-80.)

  7. Effect of Static Magnetic Fields on the Peripheral Blood Mononuclear-like Cells

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    Godina-Nava, J. J.; García-Cantú, R.; Cañedo, L.; Rodríguez-Segura, M. A.; Serrano, G.; Alvarado-Alvarez, R.; Toledo-Ramos, F.

    2002-08-01

    In this article the role of static magnetic fields (SMF) in the generation of Ca2+ currents in peripheral blood mononuclear-like cells (PBMLC) is described. Using the sensitivity of Ca2+ channels and pumps to membrane potential and ion concentration we propose a method which uses the conductivity as a dynamical coefficient in the Onsager's reciprocity relations, and the dynamics of the calcium ions described by the electrodiffusion equation deduced by Pelce. The enhanced influx of calcium ion in PBMLC was studied parametrizing the static magnetic fields effects on the conductivity by the coefficients γ, ρ and κ. The parametrization was made according to the symmetry properties of Onsager's reciprocity relations using the most simple expressions. As an example we used available experimental data over chromaffin cell and employing physical considerations concerning to PBMLC, an order of magnitude for the value of ρ ≈ O(-10-5 mol/(Vm2s)), κ = 0, γ ≈ O(-10-3 mol/(VT2m2s) was obtained. The γ parameter was found graphically. With this parametrization, the time to induce calcium current in the cell was always less than the situation without magnetic field application.

  8. Paracrine Effects of Bone Marrow Mononuclear Cells in Survival and Cytokine Expression after 90% Partial Hepatectomy

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    Carlos Oscar Kieling

    2017-01-01

    Full Text Available Acute liver failure is a complex and fatal disease. Cell-based therapies are a promising alternative therapeutic approach for liver failure due to relatively simple technique and lower cost. The use of semipermeable microcapsules has become an interesting tool for evaluating paracrine effects in vivo. In this study, we aimed to assess the paracrine effects of bone marrow mononuclear cells (BMMC encapsulated in sodium alginate to treat acute liver failure in an animal model of 90% partial hepatectomy (90% PH. Encapsulated BMMC were able to increase 10-day survival without enhancing liver regeneration markers. Gene expression of Il-6 and Il-10 in the remnant liver was markedly reduced at 6 h after 90% PH in animals receiving encapsulated BMMC compared to controls. This difference, however, was neither reflected by changes in the number of CD68+ cells nor by serum levels of IL6. On the other hand, treated animals presented increased caspase activity and gene expression in the liver. Taken together, these results suggest that BMMC regulate immune response and promote apoptosis in the liver after 90% PH by paracrine factors. These changes ultimately may be related to the higher survival observed in treated animals, suggesting that BMMC may be a promising alternative to treat acute liver failure.

  9. Impact of age on the efficacy of bone marrow mononuclear cell transplantation in experimental stroke

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    Wagner Daniel-Christoph

    2012-08-01

    Full Text Available Abstract Bone marrow-derived mononuclear cells (BM MNC have been effectively used to treat experimental stroke. Most of the preclinical trials have been performed in young and healthy laboratory animals, even though age and hypertension are major risk factors for stroke. To determine the influence of age on the properties of BM MNCs after cerebral ischemia, we compared the efficacy of aged and young BM MNC in an in vitro model of cerebral hypoxia and in an adapted in vivo model of stroke. Human BM MNCs were obtained from healthy young or aged donors and either co-cultured with rat hippocampal slices exposed to oxygen glucose deprivation (OGD, or transplanted intravenously 24 h after permanent middle cerebral artery occlusion in aged (18 months spontaneously hypertensive rats (SHR. Efficacy was examined by quantification of hippocampal cell death, or respectively, by neurofunctional tests and MR investigations. Co-cultivation with young, but not with aged BM MNCs significantly reduced the hippocampal cell death after OGD. Transplantation of both young and old BM MNCs did not reduce functional deficits or ischemic lesion volume after stroke in aged SHR. These results suggest a significant impact of age on the therapeutic efficacy of BM MNCs after cerebral ischemia.

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

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    Alok Sharma

    2015-07-01

    Full Text Available 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.

  11. Reduction of cytokine release of blood and bronchoalveolar mononuclear cells by ambroxol.

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    Pfeifer, S; Zissel, G; Kienast, K; Müller-Quernheim, J

    1997-03-24

    Ambroxol is a mucolytic agent frequently used in the treatment of chronic bronchitis. It has been reported, following clinical and in-vitro studies, that ambroxol exhibits an anti-inflammatory action. This capability was investigated by activating bronchoalveolar lavage cells and peripheral blood mononuclear cells in-vitro to elicit the release of tumor necrosis factor alpha, interleukin-2 and interferon gamma, whilst simultaneously exposing them to varying pharmacological concentrations of ambroxol (10, 1, and 0.1 microM). After 24 h it was observed that the isolated tissue-culture supernatants showed a dose-dependent reduction in the concentration of the tested cytokines; 10 microM (12 to 37% reduction) and 1 microM to (6 to 27% reduction). At 0.1 microM, a significant reduction could only be observed in the release of interleukin-2 by bronchoalveolar lavage cells. These results demonstrate, that ambroxol exhibits anti-inflammatory actions in concentrations achievable in vivo.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  14. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    Science.gov (United States)

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; preceptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor gamma

  15. Transplantation of mononuclear cells from bone marrow in a rat model of Huntington’s disease

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    Serrano T

    2016-12-01

    Full Text Available Teresa Serrano,1 Paula Pierozan,2 Esteban Alberti,1 Lisette Blanco,1 Karelys de la Cuétara Bernal,1 María E González,1 Nancy Pavón,1 Lourdes Lorigados,1 María A Robinson-Agramonte,1 Jorge A Bergado1 1International Center for Neurological Restoration (CIREN, La Habana, Cuba; 2Department of Biochemistry, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil Abstract: This article investigates the possible effects of transplantation of mononuclear bone marrow cells (mBMCs to ameliorate or prevent the behavioral impairments and the cellular damage observed in a quinolinic acid (QA model of Huntington’s disease. mBMCs were isolated using a standard procedure and implanted within the QA-lesioned striatum. Behavior was explored using motor (beam test and memory (object recognition and Morris water maze tests. Morphology was evaluated using conventional histology (cresyl violet, bisbenzimide (to evaluate cell vitality, and immunohystochemistry to identify neurons or glia. mBMC-transplanted animals showed improvements in motor coordination (beam test. Regarding memory, object recognition was significantly improved in transplanted animals, while spatial memory (Morris water maze test was not severely affected by QA and, therefore, the results after transplantation were significant only in the probe-trial retention test. In samples taken from the animals that participated in the behavioral tests, a preserved morphology of striatal neurons and a reduced glial reaction indicated a possible neuroprotective effect of the transplanted mBMCs. A parallel study confirmed that the transplanted mBMCs have a long survival period (1 year follow-up. The results presented confirm the possibility that mBMC transplantation may be a viable therapeutic option for Huntington’s disease. Keywords: mononuclear bone marrow cells, Huntington’s disease, quinolinic acid, transplant, Fluoro-Jade C

  16. Rac1 regulates the NLRP3 inflammasome which mediates IL-1beta production in Chlamydophila pneumoniae infected human mononuclear cells.

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    Julia Eitel

    Full Text Available Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1β, a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1β protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1β production dependent on a NLRP3 inflammasome-mediated processing of proIL-1β which is controlled by Rac1.

  17. Autologous transplantation of bone marrow mononuclear cells improved heart function after myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    Guo-sheng LIN; Jing-jun L(U); Xue-jun JIANG; Xiao-yan LI; Geng-shan LI

    2004-01-01

    AIM: To investigate whether autologous transplantation of adult stem cells could improve post-infarcted heart function. METHODS: Bone marrow mononuclear cells (MNCs) were isolated from adult rabbits' tibias after coronary ligation. These cells were exposed to 5-azacytidine 10 μmol/L for 24 h on the third day of culture. After being labeled with bromodeoxyuridine (BrdU), the cells were auto-transplanted into bordering zone of the infarcted area at 2 weeks after injury. The animals were killed at 3 days, 2 weeks, 1 month, and 2 months after transplantation,respectively. The left ventricular functions, capillary density, and cardiac nerve density were measured and the differentiation of the engrafted cells was determined by immunostaining. RESULTS: BrdU-labeled MNCs were well aligned with the host cardiomyocytes. Parts of them were incorporated into capillary and arteriolar vessel walls. In addition to inducing angiogenic ligands (basic fibroblast growth factor, vascular endothelial growth factor) and imflammation cytokines (interleukin 1-β) during the early period of MNCs implantation, MNCs induced 2.0-fold increase in capillary density as well. Moreover, GAP43-positive and TH-positive nerve density were markedly higher in the MNCs-treated groups than that in the non-treated hearts. Left ventricular ejection fraction,LV+dp/dt and LV-dp/dtmax were 47 %, 67 %, and 55 % in MNCs-treated heart respectively, which was higher than that of the control heart, whereas left ventricular end-diastolic volume, left ventricular end-diastolic diameter,and left ventricular end-diastolic pressure were 45 %, 22 %, and 50 % respectively in MNCs-treated heart, which was lower than that of the control heart at 2 months after cell transplantation. CONCLUSION: Autologous transplantation of MNCs induced angiogenesis and nerve sprouting and improved left ventricular diastolic function.

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

    Institute of Scientific and Technical Information of China (English)

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

    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 (t = 0.2145, P = 0.0281) and histologic grade (t = 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.

  19. TRAV gene expression in PBMCs and TILs in patients with breast cancer analyzed by a DNA melting curve (FQ-PCR) technique for TCR α chain CDR3 spectratyping.

    Science.gov (United States)

    He, X Y; Yang, W M; Tang, W T; Ma, R; Sun, Y P; Wang, P; Yao, X S

    2012-01-01

    To explore the expression of the TRAV gene in peripheral blood mononuclear cells (PBMCs) and in tumor-infiltrating lymphocytes (TILs) in the patients with breast cancer using a DNA melting curve (FQ-PCR) technique for T cell receptor (TCR) alpha chain CDR3 spectratyping. Peripheral blood samples and tissue samples were obtained from thirty breast cancer patients. Total RNA was extracted from PBMCs and tumor tissues and then reverse transcribed into cDNA. FQ-PCR was used to amplify the human TCR alpha chain CDR3 region with the primers to the TRAV and TRAC genes. TCR alpha chain CDR3 spectratyping and partial CDR3 sequencing were used to determine use of TRAV gene product in T cell responses. TCR alpha CDR3 spectratyping showed preferential usage of certain TRAV genes in the PBMCs and TILs of all patients with breast cancer. The frequencies of TRAV1.1, TRAV9, and TRAV29 exceeded 30% in PBMCs and the frequencies of TRAV1.1 and TRAV22 exceeded 30% in TILs. More than three quarters of the patients (23/30) overexpressed the same gene in both PBMCs and TILs; for example, patient-1 highly expressed TRAV9 in the PBMCs and TILs. Patients with positive or negative tumor markers of estrogen receptor (ER), progesterone receptor (PR), pS2, C-erbB-2, nm23, P53, and Ki-67 showed no significant common TRAV gene expression, but some TRAV gene preferential usage frequencies exceeded 20%. For example, five of seven patients positive for ER had high levels of expression of TRAV1.1 and TRAV3. Finally, the amino acid sequence of TCR CDR3 region showed some common motifs in some of the patients. TRAV gene expression was complex and diverse in the patients with breast cancer. The TRAV gene usage may be closely related to the diversity of breast tumor antigens and the differential immune responses observed in individual patients. Research into the immunological mechanism of T cells may provide guidance for individual T cell-directed therapy for breast cancer.

  20. Effects of Malnutrition on Neutrophil/Mononuclear Cell Apoptotic Functions in Children with Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Cakir, Fatma Betul; Berrak, Su Gülsün; Aydogan, Gonul; Tulunay, Aysin; Timur, Cetin; Canpolat, Cengiz; Eksioglu Demiralp, Emel

    2017-04-01

    Recent studies claim that apoptosis may explain immune dysfunction observed in malnutrition. The objective of this study was to determine the effect of malnutrition on apoptotic functions of phagocytic cells in acute lymphoblastic leukemia (ALL). Twenty-eight ALL patients (13 with malnutrition) and thirty controls were enrolled. Neutrophil and mononuclear cell apoptosis of ALL patients and the control group were studied on admission before chemotherapy and repeated at a minimum of three months after induction of chemotherapy or when the nutritional status of leukemic children improved. The apoptotic functions of both ALL groups on admission were significantly lower than those of the control group. The apoptotic functions were lower in ALL patients with malnutrition than those in ALL patients without malnutrition, but this was not statistically significant. The repeated apoptotic functions of both ALL groups were increased to similar values with the control group. This increase was found to be statistically significant. The apoptotic functions in ALL patients were not found to be affected by malnutrition. However, after dietary intervention, increased apoptotic functions in both ALL patient groups deserve mentioning. Dietary intervention should always be recommended as malnutrition or cachexia leads to multiple complications. Enhanced apoptosis might originate also from remission state of cancer.

  1. Immunomodulatory activity of Semecarpus anacardium extract in mononuclear cells of normal individuals and rheumatoid arthritis patients.

    Science.gov (United States)

    Singh, Divya; Aggarwal, Amita; Mathias, Amrita; Naik, Sita

    2006-12-06

    Semecarpus anacardium (SA) Linn. (family Anacardiaceae), is a plant well-known for its medicinal value in Ayurveda. The nut extracts of this plant have been traditionally used as antihelminthic, anti-fungal, anti-carcinogenic and in the treatment of nervous debilities and arthritis. In this study we have evaluated crude ethanolic extract of SA nuts for its anti-inflammatory activities in vitro using peripheral blood and synovial fluid mononuclear cells of healthy individuals and rheumatoid arthritis (RA) patients. SA extract inhibited the spontaneous and LPS induced production of proinflammatory cytokines IL-1beta and IL-12p40 but had no effect on TNF-alpha and IL-6 production, both at protein and mRNA level. The crude extract also suppressed LPS induced nuclear translocation of transcription factors, NF-kappaB and AP-1; the inhibition of NF-kappaB was through the inhibition of IkappaBalpha phosphorylation. The extract also suppressed LPS activated nitric oxide production in mouse macrophage cell line, RAW 264.7. Our results for the first time show that SA extract can inhibit proinflammatory cytokine production and demonstrate its mechanism of action.

  2. A clinical study of autologous bone marrow mononuclear cells for cerebral palsy patients: a new frontier.

    Science.gov (United States)

    Sharma, Alok; Sane, Hemangi; Gokulchandran, Nandini; Kulkarni, Pooja; Gandhi, Sushant; Sundaram, Jyothi; Paranjape, Amruta; Shetty, Akshata; Bhagwanani, Khushboo; Biju, Hema; Badhe, Prerna

    2015-01-01

    Cerebral palsy is a nonprogressive heterogeneous group of neurological disorders with a growing rate of prevalence. Recently, cellular therapy is emerging as a potential novel treatment strategy for cerebral palsy. The various mechanisms by which cellular therapy works include neuroprotection, immunomodulation, neurorestoration, and neurogenesis. We conducted an open label, nonrandomized study on 40 cases of cerebral palsy with an aim of evaluating the benefit of cellular therapy in combination with rehabilitation. These cases were administered autologous bone marrow mononuclear cells intrathecally. The follow-up was carried out at 1 week, 3 months, and 6 months after the intervention. Adverse events of the treatment were also monitored in this duration. Overall, at six months, 95% of patients showed improvements. The study population was further divided into diplegic, quadriplegic, and miscellaneous group of cerebral palsy. On statistical analysis, a significant association was established between the symptomatic improvements and cell therapy in diplegic and quadriplegic cerebral palsy. PET-CT scan done in 6 patients showed metabolic improvements in areas of the brain correlating to clinical improvements. The results of this study demonstrate that cellular therapy may accelerate the development, reduce disability, and improve the quality of life of patients with cerebral palsy.

  3. A Clinical Study of Autologous Bone Marrow Mononuclear Cells for Cerebral Palsy Patients: A New Frontier

    Directory of Open Access Journals (Sweden)

    Alok Sharma

    2015-01-01

    Full Text Available Cerebral palsy is a nonprogressive heterogeneous group of neurological disorders with a growing rate of prevalence. Recently, cellular therapy is emerging as a potential novel treatment strategy for cerebral palsy. The various mechanisms by which cellular therapy works include neuroprotection, immunomodulation, neurorestoration, and neurogenesis. We conducted an open label, nonrandomized study on 40 cases of cerebral palsy with an aim of evaluating the benefit of cellular therapy in combination with rehabilitation. These cases were administered autologous bone marrow mononuclear cells intrathecally. The follow-up was carried out at 1 week, 3 months, and 6 months after the intervention. Adverse events of the treatment were also monitored in this duration. Overall, at six months, 95% of patients showed improvements. The study population was further divided into diplegic, quadriplegic, and miscellaneous group of cerebral palsy. On statistical analysis, a significant association was established between the symptomatic improvements and cell therapy in diplegic and quadriplegic cerebral palsy. PET-CT scan done in 6 patients showed metabolic improvements in areas of the brain correlating to clinical improvements. The results of this study demonstrate that cellular therapy may accelerate the development, reduce disability, and improve the quality of life of patients with cerebral palsy.

  4. Effect of spirulina on the secretion of cytokines from peripheral blood mononuclear cells.

    Science.gov (United States)

    Mao, T K; VAN DE Water, J; Gershwin, M E

    2000-01-01

    ABSTRACT The purpose of this study was to evaluate the immunomodulatory activity of Spirulina, a bluegreen alga used as a food supplement. The effects of Spirulina on the secretion of three cytokines from unstimulated and stimulated human peripheral blood mononuclear cells (PBMC) were examined. In resting PBMC, Spirulina stimulated secretion of interleukin (IL)-1beta, IL-4, and interferon (IFN)-gamma to nearly 2.0, 3.3, and 13.6 times basal levels, respectively. Spirulina induced levels of IFN-gamma (229 +/- 104 pg/ml) that were comparable to those seen after phytohemagglutinin (PHA) stimulation (476 +/- 121 pg/ml). However, it was much less mitogenic than PHA (13.1 +/- 6.9 pg/ml) with respect to the induction of IL-4 secretion (0.34 +/- 0.1 pg/ml). In PHA-stimulated cells, Spirulina enhanced secretion of IL-1beta, IL-4, and IFN-beta by 2.9, 4.0., and 1.6 times, respectively. Although Spirulina stimulates several cytokines, it is clearly more effective in the generation of a Thl-type response. This in vitro study offers additional data for consideration of the potential therapeutic benefits of Spirulina.

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

    Science.gov (United States)

    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.

  6. Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse.

    Science.gov (United States)

    González, L O; Pidal, I; Junquera, S; Corte, M D; Vázquez, J; Rodríguez, J C; Lamelas, M L; Merino, A M; García-Muñiz, J L; Vizoso, F J

    2007-10-08

    An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissular inhibitors of metalloproteinase (TIMP)-1, -2 and -3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast were performed. To identify specific groups of tumours with distinct expression profiles the data were analysed by unsupervised hierarchical cluster analysis by each cellular type. We did not find well-defined cluster of cases for tumour cells or fibroblastic cells. However, for mononuclear inflammatory cells the dendogram shows a first-order division of the tumours into two distinct MMP/TIMP molecular profiles, designated group 1 (n=89) and group 2 (n=42). Matrix metalloproteinase-7, -9, -11, -13 and -14, and TIMP-1 and -2, were identified as showing significant high expression in group 2 compared with group 1. Multivariate analysis demonstrated that clustering for mononuclear inflammatory cells was the most potent independent factor associated with distant relapse-free survival (group 2: 5.6 (3.5-9.6), P<0.001). We identify a phenotype of mononuclear inflammatory cells infiltrating tumours, which is associated with the development of distant metastasis. Therefore, this finding suggests that these host inflammatory cells could be a possible target for inhibition of metastasis.

  7. The Study of Chlamydia Pneumoniae DNA in the Peripheral Blood Mononuclear Cell of Coronary Heart Disease

    Institute of Scientific and Technical Information of China (English)

    Li Tao; Xu Xiang Guang; Zhang Guo Liang; Fang Weihua

    2004-01-01

    Objectives To detection of chlamydia pneumoniae (Cpn) DNA in the circulating mononuclear cell fractions of coronary heart disease and to investigate the association between infection with chlamydia pneumoniae and coronary heart disease (CHD) and prospectively whether blood -based nested polymerase chain reaction ( nPCR ) is useful in identifying Cpn infection. Methods The peripheral blood mononuclear cell (PBMC) Cpn DNA was examined using nPCR technique and confirmed by electrophoresis in 150 patients with CHD. Select 55 patients with clinical suspected CHD but angiography result are normal as control group (CG). Then we conducted a prospective , randomized, double - blind, placebo -controlled study of 6 months of azithromycin and placebo treatment in CHD group. Patients with Cpn DNA positive were then randomized to receive azithromycin or placebo. After treatment blood sample were collected for repeated measurement . Results Chlamydia pneumoniae DNA was detected in 49(32.7% ) of 150persons with CHD and in 1 ( 1.8% ) of 55 persons with control group,odds ratio 26.2, 95% confidence interva13.52 - 194.98. The positivity rates of nPCR in CHD groups were higher than those in control group. 16 cases (29. 1% ) in latent coronary heart diseases(LCHD) group , 19 cases (39.6%) in unstable angina(UAP) group ,and 14 cases (29.9%) in acute myocardial infarction (AMI)group were Cpn positive by nPCR. There were no significant difference among in AMIUAP and LCHD group. There were significiant difference in Cpn DNA negative rates after the azithromycin and the placebo treatment. Conclusions Chlamydia pneumoniae is present in PBMC of a significant proportion of persons with CHD. The potential role of chlamydia pneumoniae in coronary atherosclerosis may therefore be more related to acceleration of disease or systemic effects by persistent infection than to sudden initiation of progressive coronary artery disease by acute infection. The detection of Cpn DNA in PBMC with nPCR may be

  8. Use of human umbilical cord blood mononuclear cells to prevent perinatal brain injury: a preclinical study.

    Science.gov (United States)

    Dalous, Jérémie; Pansiot, Julien; Pham, Hoa; Chatel, Paul; Nadaradja, Céline; D'Agostino, Irene; Vottier, Gaëlle; Schwendimann, Leslie; Vanneaux, Valérie; Charriaut-Marlangue, Christiane; Titomanlio, Luigi; Gressens, Pierre; Larghero, Jérôme; Baud, Olivier

    2013-01-01

    Cerebral palsy (CP) is the most frequent neurological disorder associated with perinatal injury of the developing brain. Major brain lesions associated with CP are white matter damage (WMD) in preterm infants and cortico-subcortical lesions in term newborns. Cell therapy is considered promising for the repair of brain damage. Human umbilical cord blood mononuclear cells (hUCB-MNCs) are a rich source of various stem cells that could be of interest in repairing perinatal brain damage. Our goal was to investigate the potential of hUCB-MNCs to prevent or repair brain lesions in an animal model of excitotoxic brain injury. We induced neonatal brain lesions using intracranial injections of ibotenate, a glutamate agonist, in 5-day-old rat pups. hUCB-MNCs were injected either intraperitoneally (i.p.) or intravenously (i.v.) soon or 24 h after ibotenate injection, and their neurological effects were assessed using histology and immunohistochemistry. hUCB-MNCs injected i.p. did not reach the systemic circulation but high amounts induced a significant systemic inflammatory response and increased the WMD induced by the excitotoxic insult. This effect was associated with a significant 40% increase in microglial activation around the white matter lesion. hUCB-MNCs injected i.v. soon or 24 h after the excitotoxic insult did not affect lesion size, microglial activation, astroglial cell density, or cell proliferation within the developing white matter or cortical plate at any concentration used. We demonstrated that hUCB-MNCs could not integrate into the developing brain or promote subsequent repair in most conditions tested. We found that the intraperitoneal injection of high amounts of hUCB-MNCs aggravated WMD and was associated with systemic inflammation.

  9. Cytokine production by bone marrow mononuclear cells in inherited bone marrow failure syndromes.

    Science.gov (United States)

    Matsui, Ken; Giri, Neelam; Alter, Blanche P; Pinto, Ligia A

    2013-10-01

    Fanconi anaemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anaemia (DBA), and Shwachman-Diamond syndrome (SDS) are characterized by the progressive development of bone marrow failure. Overproduction of tumour necrosis factor-α (TNF-α) from activated bone marrow T-cells has been proposed as a mechanism of FA-related aplasia. Whether such overproduction occurs in the other syndromes is unknown. We conducted a comparative study on bone marrow mononuclear cells to examine the cellular subset composition and cytokine production. We found lower proportions of haematopoietic stem cells in FA, DC, and SDS, and a lower proportion of monocytes in FA, DC, and DBA compared with controls. The T- and B-lymphocyte proportions were similar to controls, except for low B-cells in DC. We did not observe overproduction of TNF-α or IFN-γ by T-cells in any patients. Induction levels of TNF-α, interleukin (IL)-6, IL-1β, IL-10, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor in monocytes stimulated with high-dose lipopolysaccharide (LPS) were similar at 4 h but lower at 24 h when compared to controls. Unexpectedly, patient samples showed a trend toward higher cytokine level in response to low-dose (0·001 μg/ml) LPS. Increased sensitivity to LPS may have clinical implications and could contribute to the development of pancytopenia by creating a chronic subclinical inflammatory micro-environment in the bone marrow. © Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

  10. Validation of a quantitative real-time PCR assay for HTLV-1 proviral load in peripheral blood mononuclear cells.

    Science.gov (United States)

    Rosadas, Carolina; Cabral-Castro, Mauro Jorge; Vicente, Ana Carolina Paulo; Peralta, José Mauro; Puccioni-Sohler, Marzia

    2013-11-01

    The objective of this study was to validate a TaqMan real-time PCR assay for HTLV-1 proviral load detection in peripheral blood mononuclear cells. TARL-2 cells were used to generate a standard curve. Peripheral blood mononuclear cell gDNA from 27 seropositive and 23 seronegative samples was analyzed. The sensitivity, specificity, accuracy, precision, dynamic range of the standard curve and qPCR efficiency were evaluated. All of the positive samples amplified the target gene. All of the negative samples amplified only the control gene (β-actin). The assay presented 100% specificity and sensibility. The intra- and inter-assay variability was 2.4% and 2.2%, respectively. The qPCR efficiency, slope and correlation coefficients (r2) were all acceptable. The limit of detection was 1 copy/rxn. This assay can reliably quantify HTLV-1 proviral load.

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

    Science.gov (United States)

    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. Published by Elsevier B.V.

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

    Directory of Open Access Journals (Sweden)

    Han-Kyu Lee

    2016-03-01

    Full Text Available 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.

  13. Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis

    Science.gov (United States)

    Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

    2017-01-01

    Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1

  14. 鼻咽癌外周血单个核细胞和血浆中EB病毒DNA水平变化及与放化疗疗效的关系%The Dynamic Change of Peripheral Blood Mononuclear Cell and Plasma Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma and Correlation with the Radiotherapy and Chemotherapy Curative Effect

    Institute of Scientific and Technical Information of China (English)

    陈岩松; 陈燕; 林莺莺; 郑瑜宏

    2016-01-01

    Objective To compare the levels of peripheral blood mononuclear cell and plasma Epstein-barr virus DNA in patients with nasopharyngeal carcinoma (NPC) before and after radiotherapy and chemotherapy, and to discuss the correla-tion between the levels of EBV DNA and the short-term curative effect of the radiochemotherapy. Methods Real-time fluo-rescent quantitative PCR method were determined in 2015 July to November in fujian province tumor hospital radiotherapy was treated 75 cases of nasopharyngeal carcinoma patients before and after the radiation and chemotherapy peripheral blood mononuclear cells and plasma EBV DNA level.Compare the concentrations before and after therapy in CR、PR、SD、PD groups. The relationship was analyzed between plasma and PBMCs EBV DNA. Results CR, PR of peripheral blood mononuclear cells and plasma EBV DNA before and after treatment decreased significantly, the difference was statistically significant (P0.05). Peripheral blood mononuclear cells and plasma EBV DNA overall detection rate of 78.7%, 53.3%, obvious difference (P0.05)。外周血单个核细胞和血浆EBV DNA总体检出率为78.7%、53.3%,差异有统计学意义(P<0.05);两种类型标本EBV DNA结果呈正相关(r=0.481,P=0.00)。结论外周血单个核细胞和血浆EBV DNA检测可作为评价鼻咽癌放化疗近期疗效的指标。

  15. Effect of dietary fat saturation and cholesterol on low density lipoprotein degradation by mononuclear cells of Cebus monkeys.

    Science.gov (United States)

    Kuo, P C; Rudd, M A; Nicolosi, R; Loscalzo, J

    1989-01-01

    The mechanism by which dietary unsaturated fatty acids lower low density lipoprotein (LDL) cholesterol is unknown. Unsaturated fatty acids incorporated into the cell membrane can increase membrane fluidity and, as a result, dramatically alter membrane-dependent cell functions. Therefore, we examined the effect of long-term dietary consumption of corn oil and coconut oil with and without cholesterol in amounts equivalent to those of a typical Western diet on the degradation of human LDL by peripheral blood mononuclear cells in Cebus albifrons monkeys. Cellular LDL degradation was dramatically enhanced in the mononuclear cells isolated from animals fed corn oil in comparison with those from animals fed coconut oil. The addition of cholesterol to the diets resulted in a slight attenuation of LDL degradation in the corn oil group while no effect was noted in the coconut oil group. Crossover LDL binding and degradation experiments with LDL isolated from animals fed corn oil diets and coconut oil diets demonstrated increased binding and degradation of LDL in mononuclear cells from animals fed corn oil diets. Enhanced mononuclear cell LDL degradation was accompanied by increased cellular cis-unsaturated fatty acyl content, increased membrane fluidity, and decreased plasma cholesterol. Increased cellular cis-unsaturated fatty acyl content with its concomitant increase in membrane fluidity mirrored the dietary lipid profile of the host animal. A linear relationship was observed between cellular LDL degradation and both cellular cis-unsaturated fatty acyl content and membrane fluidity. These observations parallel results noted in whole-animal LDL catabolic studies with these same animals described elsewhere. These data suggest a novel mechanism by which dietary unsaturated fatty acids exert their LDL-lowering effect.

  16. Marked reduction of AKT1 expression and deregulation of AKT1-associated pathways in peripheral blood mononuclear cells of schizophrenia patients.

    Directory of Open Access Journals (Sweden)

    Nico J M van Beveren

    Full Text Available BACKGROUND: Recent studies have suggested that deregulated AKT1 signaling is associated with schizophrenia. We hypothesized that if this is indeed the case, we should observe both decreased AKT1 expression as well as deregulation of AKT1 regulated pathways in Peripheral Blood Mononuclear Cells (PBMCs of schizophrenia patients. OBJECTIVES: To examine PBMC expression levels of AKT1 in schizophrenia patients versus controls, and to examine whether functional biological processes in which AKT1 plays an important role are deregulated in schizophrenia patients. METHODS/RESULTS: A case-control study, investigating whole-genome PBMC gene expression in male, recent onset (<5 years schizophrenia patients (N = 41 as compared to controls (N = 29. Genes, differentially expressed between patients and controls were identified using ANOVA with Benjamini-Hochberg correction (false discovery rate (FDR = 0.05. Functional aspects of the deregulated set of genes were investigated with the Ingenuity Pathway Analysis (IPA Software Tool. We found significantly decreased PBMC expression of AKT1 (p<0.001, t = -4.25 in the patients. AKT1 expression was decreased in antipsychotic-free or -naive patients (N = 11, in florid psychotic (N = 20 and in remitted (N = 21 patients. A total of 1224 genes were differentially expressed between patients and controls (FDR = 0.05. Functional analysis of the entire deregulated gene set indicated deregulated canonical pathways involved in a large number of cellular processes: immune system, cell adhesion and neuronal guidance, neurotrophins and (neural growth factors, oxidative stress and glucose metabolism, and apoptosis and cell-cycle regulation. Many of these processes are associated with AKT1. CONCLUSIONS: We show significantly decreased PBMC gene expression of AKT1 in male, recent-onset schizophrenia patients. Our observations suggest that decreased PBMC AKT1 expression is a stable trait in recent onset

  17. Proteomic analysis of Fasciola hepatica excretory and secretory products (FhESPs) involved in interacting with host PBMCs and cytokines by shotgun LC-MS/MS.

    Science.gov (United States)

    Liu, Qing; Huang, Si-Yang; Yue, Dong-Mei; Wang, Jin-Lei; Wang, Yujian; Li, Xiangrui; Zhu, Xing-Quan

    2017-02-01

    Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates.

  18. Mechanisms of pancreatic islet cell destruction. Dose-dependent cytotoxic effect of soluble blood mononuclear cell mediators on isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    contents of insulin and glucagon in a dose-dependent manner. A maximal effect on islet function was obtained with supernatant concentrations down to 5%. Supernatants of mononuclear cells stimulated with tuberculin were more potent than supernatants produced by lectin stimulation. Culture medium......Supernatants of peripheral blood mononuclear cells from healthy human donors stimulated with recall antigen (purified protein derivative of tuberculin) or lectin (phytohaemagglutinin) markedly inhibited the insulin release from isolated human and rat islets of Langerhans, and decreased rat islet...... reconstituted with tuberculin or phytohaemagglutinin did not impair islet function. Electron microscopy demonstrated that supernatants were cytotoxic to islet cells. The cytotoxic mononuclear cell mediator(s) was non-dialysable, sensitive to heating to 56 degrees C, labile even when stored at -70 degrees C...

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

    Science.gov (United States)

    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

  20. Characterization of Bone Marrow Mononuclear Cells on Biomaterials for Bone Tissue Engineering In Vitro

    Directory of Open Access Journals (Sweden)

    Dirk Henrich

    2015-01-01

    Full Text Available 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.

  1. Response of porcine peripheral blood mononuclear cells to CpG-containing oligodeoxynucleotide

    DEFF Research Database (Denmark)

    Kamstrup, Søren; Verthelyi, D.; Klinman, D.M.

    2001-01-01

    Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found in bacter......Exposure to bacterial DNA generates a "danger signal" that stimulates cellular elements of the mammalian immune system to proliferate and/or secrete cytokines. Stimulation is critically dependent on hexameric motifs that contain an unmethylated CpG dinucleotide: these are commonly found...... in bacterial but not vertebrate DNA. Different motifs are optimally stimulatory in different species. This work examines whether oligodeoxynucleotides (ODNs) containing CpG motifs stimulate peripheral blood mononuclear cells from pigs. Results show that pigs respond to CpG ODN by proliferating and secreting IL......-6, IL-12 and TNF-alpha. By screening a large panel (>100) of ODNs, the palindromic hexamer 'ATCGAT' was identified as being optimally active in all animals examined (N = 10). These findings are the first to establish the immunostimulatory activity of CpG ODN in pigs, and suggest that the therapeutic...

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

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    Coignoul Freddy

    2007-09-01

    Full Text Available Abstract 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.

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

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    Alok Sharma

    2013-01-01

    Full Text Available 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.

  4. Acción protectora de la melatonina sobre células mononucleares de sangre periférica humana sometidas a radiación gamma Co60

    Directory of Open Access Journals (Sweden)

    Martha Mesa-Villanueva

    2009-04-01

    Full Text Available Protective action of melatonin on human peripheral blood mononuclear cells exposed to gamma radiation Co60. Objective: toevaluate the protective effect of melatonin on peripheral blood mononuclear cells (PBMCs exposed in vitro to ionizing radiation. Materialsand methods: PBMCs drawn from healthy volunteers were incubated with 0, 1x10-5, 1x10-6 y 1x10-7 M melatonin for 10 minutes beforebeing exposed to gamma radiation (300 cGy; Co60 source. Afterwards, DNA damage was evaluated with the comet assay. Results:PBMCs pretreated with melatonin showed comet tails shorter than those without the hormone treatment, as well as a lower percentage ofcells with severe DNA damage. Conclusion: melatonin doses of 1x10-5, 1x10-6 and 1x10-7 M provide in vitro protection to PBMCs fromDNA damage (single strand breaks and alkali-labile sites induced by gamma radiation (300 cGy; Co60 source.

  5. Bone Marrow Mononuclear Cell Transplantation Restores Inflammatory Balance of Cytokines after ST Segment Elevation Myocardial Infarction.

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    Kirsi Alestalo

    Full Text Available Acute myocardial infarction (AMI launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI.Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection.Twenty-six patients (control group, n = 12; BMMNC group, n = 14 from the previously reported FINCELL study (n = 80 were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups (Kendall's tau, control 0.6; BMMNC 0.7. At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group (Kendall's tau, control 0.3; BMMNC 0.7.BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI.

  6. The autologous bone marrow mononuclear cell transplantation by intracoronary route treat patients with severe heart failure after myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    高连如

    2006-01-01

    Objective To investigate the chronic effects of intracoronary autologous bone marrow mononuclear cell (BM-MNCs) transplantation in patients with refractory heart failure (RIHF) after myocardial infarction. Methods Thirty patients with RIHF (LVEF<40%) were enrolled in this nonrandomized study, autologous BM-MNCs (5.0±0.7)×107 were transplanted with via infarct-related coronary artery in 16 patients and 14 patients received

  7. Observation on the safety:clinical trail on intracoronary autologus bone marrow mononuclear cells transplantation for acute myocardiol infarction

    Institute of Scientific and Technical Information of China (English)

    姚康

    2006-01-01

    Objective To investigate the safety of autologous bone marrow mononuclear cell (BM-MNCs) transplantation by intracoronary infusion in patients with acute myocardial infarction (AMI). Methods One hundred and eighty-four patients with AMI treated with percutaneous coronary intervention (PCI) were randomized in a 1:1 way to either intracoronary transplantation of autologous BM-MNCs (n =92) right after PCI or to sodium chloride concluding heparin (controlled, n=92) via a micro

  8. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, L.P. [Programa de Pós-Graduação em Neurociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Iglesias, D. [Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Nicola, F.C. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Steffens, D. [Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Valentim, L.; Witczak, A.; Zanatta, G. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Achaval, M. [Departamento de Ciências Morfológicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Pranke, P. [Laboratório de Hematologia e Células-Tronco, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Netto, C.A. [Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil)

    2011-12-23

    Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old) were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a) 1 h after surgery, into the injury site at a concentration of 5 x 10{sup 6} cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group); b) into the cisterna magna, 9 days after lesion at a concentration of 5 x 10{sup 6} cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group). The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day). The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05). The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

  9. Transplantation of mononuclear cells from human umbilical cord blood promotes functional recovery after traumatic spinal cord injury in Wistar rats

    Directory of Open Access Journals (Sweden)

    L.P. Rodrigues

    2012-01-01

    Full Text Available Cell transplantation is a promising experimental treatment for spinal cord injury. The aim of the present study was to evaluate the efficacy of mononuclear cells from human umbilical cord blood in promoting functional recovery when transplanted after a contusion spinal cord injury. Female Wistar rats (12 weeks old were submitted to spinal injury with a MASCIS impactor and divided into 4 groups: control, surgical control, spinal cord injury, and one cell-treated lesion group. Mononuclear cells from umbilical cord blood of human male neonates were transplanted in two experiments: a 1 h after surgery, into the injury site at a concentration of 5 x 10(6 cells diluted in 10 µL 0.9% NaCl (N = 8-10 per group; b into the cisterna magna, 9 days after lesion at a concentration of 5 x 10(6 cells diluted in 150 µL 0.9% NaCl (N = 12-14 per group. The transplanted animals were immunosuppressed with cyclosporin-A (10 mg/kg per day. The BBB scale was used to evaluate motor behavior and the injury site was analyzed with immunofluorescent markers to label human transplanted cells, oligodendrocytes, neurons, and astrocytes. Spinal cord injury rats had 25% loss of cord tissue and cell treatment did not affect lesion extension. Transplanted cells survived in the injured area for 6 weeks after the procedure and both transplanted groups showed better motor recovery than the untreated ones (P < 0.05. The transplantation of mononuclear cells from human umbilical cord blood promoted functional recovery with no evidence of cell differentiation.

  10. Responses of synovial fluid and peripheral blood mononuclear cells to bacterial antigens and autologous antigen presenting cells.

    Science.gov (United States)

    Klasen, I S; Melief, M J; Swaak, T J; Severijnen, A J; Hazenberg, M P

    1993-01-01

    The specificity of T cells in the inflamed joints of patients with rheumatoid arthritis (RA) has been the subject of much study. Bacterial antigens are suspect in the aetiology of rheumatic diseases. The responsiveness of the mononuclear cell fraction of peripheral blood and synovial fluid of patients with RA and of patients with rheumatic diseases other than RA to bacterial antigens such as cell wall fragments of the anaerobic intestinal flora, cell wall fragments of Streptococcus pyogenes, intestinal flora derived peptidoglycan polysaccharide complexes, the 65 kilodalton protein of Mycobacterium tuberculosis, and muramyldipeptide was investigated. No significant difference in response was found to all these bacterial antigens in the synovial fluid of patients with RA compared with the responses in patients with other rheumatic diseases. The highest responsiveness in the synovial fluid of the patients with RA was to the streptococcal cell wall fragments and to the 65 kilodalton protein. Higher responses to several bacterial antigens in the synovial fluid of patients with RA were found compared with peripheral blood from the same patient group. The antigen presenting cell population of the synovial fluid in patients with RA and the patients with other rheumatic diseases was found to be stimulatory for autologous peripheral blood T cells even in the absence of antigen. This suggests an important role for the synovial antigen presenting cell in the aetiology of inflammatory joint diseases. PMID:8447692

  11. Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells.

    Science.gov (United States)

    Haugland, Gyri T; Jordal, Ann-Elise O; Wergeland, Heidrun I

    2012-01-01

    Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type.

  12. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.

    Science.gov (United States)

    Dowey, Sarah N; Huang, Xiaosong; Chou, Bin-Kuan; Ye, Zhaohui; Cheng, Linzhao

    2012-11-01

    Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

  13. Microarray analysis of MicroRNA expression in peripheral blood mononuclear cells of critically ill patients with influenza A (H1N1)

    Science.gov (United States)

    2013-01-01

    Background With concerns about the disastrous health and economic consequences caused by the influenza pandemic, comprehensively understanding the global host response to influenza virus infection is urgent. The role of microRNA (miRNA) has recently been highlighted in pathogen-host interactions. However, the precise role of miRNAs in the pathogenesis of influenza virus infection in humans, especially in critically ill patients is still unclear. Methods We identified cellular miRNAs involved in the host response to influenza virus infection by performing comprehensive miRNA profiling in peripheral blood mononuclear cells (PBMCs) from critically ill patients with swine-origin influenza pandemic H1N1 (2009) virus infection via miRNA microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays. Receiver operator characteristic (ROC) curve analysis was conducted and area under the ROC curve (AUC) was calculated to evaluate the diagnostic accuracy of severe H1N1 influenza virus infection. Furthermore, an integrative network of miRNA-mediated host-influenza virus protein interactions was constructed by integrating the predicted and validated miRNA-gene interaction data with influenza virus and host-protein-protein interaction information using Cytoscape software. Moreover, several hub genes in the network were selected and validated by qRT-PCR. Results Forty-one significantly differentially expressed miRNAs were found by miRNA microarray; nine were selected and validated by qRT-PCR. QRT-PCR assay and ROC curve analyses revealed that miR-31, miR-29a and miR-148a all had significant potential diagnostic value for critically ill patients infected with H1N1 influenza virus, which yielded AUC of 0.9510, 0.8951 and 0.8811, respectively. We subsequently constructed an integrative network of miRNA-mediated host-influenza virus protein interactions, wherein we found that miRNAs are involved in regulating important pathways, such as mitogen

  14. Expression of EB virus in peripheral blood mononuclear cells in children with systemic lupus erythematosus%EB病毒在系统性红斑狼疮患儿外周血单个核细胞中表达的意义

    Institute of Scientific and Technical Information of China (English)

    丁艳; 何小解; 廖旺; 杨慧兰; 向伟; 党西强; 易著文

    2015-01-01

    Objective To discuss the role of EB virus (EBV)in the pathogenesis of systemic lupus erythematosus(SLE) in children through investigating the copies of EBV DNA and expression of EBV genes in peripheral blood mononuclear cells(PBMCs).Methods (1)PBMCs were isolated from 30 patients with SLE and 12 healthy normal controls respectively and DNA was extracted from PBMCs.(2) PBMCs were co-cultured with EBV for 12 days and RNA was extracted from PBMCs.(3)Real-time fluorescence quantitative PCR(Real-time PCR) was applied to detect the copies of EBV DNA in PBMCs.(4)Reverse transcription PCR was applied to detect expression of EBV genes.Results (1) Compared with the healthy control group [(40.1 ± 11.6) copies/μg],a significant increase of EBV DNA copies was observed in SLE group[(658.6 ± 183.6) copies/μg] (P <0.05).The EBV DNA copies in the active SLE group [(785.2 ± 179.2) copies/μg] were significantly higher than those in the non-active SLE group [(586.0 ± 193.1) copies/μg] (P < 0.05).(2)There was no correlation between EBV DNA copies and systemic lupus erythematosus disease activity index (r =0.03,P > 0.05).(3) After PBMCs got co-cultured with EBV,expression of latent EBV genes and lytic genes were both increased in the patients and healthy controls.The latent EBV genes including latent membrane protein 1 (LMP1),LMP2,EBV nuclear antigen 1 and the lytic genes including BCRF1,BLLF1 were all increased significantly in the patients compared with the healthy controls (all P < 0.05).Conclusions There is a significant increase of EBV DNA copies and aberrant expression of EBV genes in SLE patients,which suggests that EBV may contribute to the pathogenesis of SLE.%目的 通过研究EB病毒(EBV)在系统性红斑狼疮(SLE)患儿外周血单个核细胞(PBMCs)中DNA拷贝数及EBV基因的表达,探讨EBV在儿童SLE发病中的作用.方法 1.分离30例SLE患儿和12例健康对照儿童PBMCs,提取DNA.2.用B95.8 EBV株与提取的PBMCs共同培养12 d,提取培养前后的PBMCs

  15. Peripheral blood mononuclear cells: a potential source of homeostatic imbalance markers associated with obesity development.

    Science.gov (United States)

    Oliver, Paula; Reynés, Bàrbara; Caimari, Antoni; Palou, Andreu

    2013-04-01

    Peripheral blood mononuclear cells (PBMC) have a great potential for nutrition and obesity studies. PBMC reflect the nutritional response of key organs involved in energy homeostasis maintenance, which is altered in the obese state. Here, we aimed to determine the usefulness of PBMC as a source of early markers of obesity. To that purpose, we analysed whether PBMC could reflect the insensitivity to changes in feeding conditions associated with obesity during the development of this pathology. Expression of key genes central to energy metabolism was measured by Q-PCR in PBMC samples of normoweight (control) and cafeteria-fed (obese) rats in feeding, fasting and refeeding conditions. Samples were obtained monthly from 2 (beginning of cafeteria diet-feeding) to 6 months of age. In general terms, expression of genes related to fatty acid synthesis (Fasn, Srebp1) and adipogenesis (Pparg) decreased with fasting and increased with refeeding. Conversely, the expression of a key gene regulating beta-oxidation (Cpt1a) and the gene for an orexigenic neuropeptide (Npy)-in accordance with their metabolic role-increased with fasting and decreased with refeeding. This expression pattern disappeared in obese rats, in which insensitivity to feeding conditions was observed after only 1 month of cafeteria diet-feeding. Thus, during development, PBMC accurately reflect nutritional regulation of energy homeostasic genes and the insensitivity to feeding associated with obesity, even in the earlier stages with a low degree of overweight. For this reason, this set of blood cells could constitute a potential source of biomarkers of early homeostatic imbalance which would be useful in nutrition studies that could help prevent the occurrence of obesity.

  16. Chemokine regulation in response to beryllium exposure in human peripheral blood mononuclear and dendritic cells.

    Science.gov (United States)

    Hong-Geller, Elizabeth; Pardington, Paige E; Cary, Robert B; Sauer, Nancy N; Gupta, Goutam

    2006-02-01

    Exposure to beryllium (Be) induces a delayed-type hypersensitivity immune reaction in the lungs of susceptible individuals, which leads to the onset of Be sensitivity and Chronic Beryllium Disease (CBD). Although some mechanistic aspects of CBD have begun to be characterized, very little is known about the molecular mechanisms by which Be activates the host immune response. To gain insight into the cellular response to Be exposure, we have performed global microarray analysis using a mixture of peripheral blood mononuclear and dendritic cells (PBMC/DCs) from a non-CBD source to identify genes that are specifically upregulated in response to BeSO(4) stimulation, compared to a control metal salt, Al(2)(SO(4))(3). We identified a number of upregulated immunomodulatory genes, including several chemokines in the MIP-1 and GRO families. Using PBMC/DCs from three different donors, we demonstrate that BeSO(4) stimulation generally exhibits an increased rate of both chemokine mRNA transcription and release compared to Al(2)(SO(4))(3) exposure, although variations among the individual donors do exist. We show that MIP-1 alpha and MIP-1 beta neutralizing antibodies can partially inhibit the ability of BeSO(4) to stimulate cell migration of PBMC/DCs in vitro. Finally, incubation of PBMC/DCs with BeSO(4) altered the binding of the transcription factor RUNX to the MIP-1 alpha promoter consensus sequence, indicating that Be can regulate chemokine gene activation. Taken together, these results suggest a model in which Be stimulation of PBMC/DCs can modulate the expression and release of different chemokines, leading to the migration of lymphocytes to the lung and the formation of a localized environment for development of Be disease in susceptible individuals.

  17. Micro-Raman spectroscopy of single leukemic cells

    Institute of Scientific and Technical Information of China (English)

    Changmei Cai; Rong Chen; Juqiang Lin; Yongzeng Li; Shangyuan Feng

    2008-01-01

    The Raman spectra from leukemic cell line (HL60) and normal human peripheral blood mononuclear cells (PBMCs) are obtained by confocal micro-Raman spectroscopy using near-infrared laser (785 nm) excitation. The scanning range is from 500 to 2000 cm-1. The two average Raman spectra of normal PBMCs and carcinoma cells have clear differences because their structure and amount of nucleic acid, protein, and other major molecules are changed. The spectra are also compared and analyzed by principal component analysis (PCA) to demonstrate the two distinct clusters of normal and transformed cells. The sensitivity of this technique for identifying transformed cells is 100%.

  18. Histamine and histamine-receptor antagonists modify gene expression and biosynthesis of interferon gamma in peripheral human blood mononuclear cells and in CD19-depleted cell subsets

    NARCIS (Netherlands)

    Horváth, B V; Szalai, C; Mándi, Y; László, V; Radvány, Z; Darvas, Z; Falus, A

    1999-01-01

    The effect of histamine and histamine antagonists was examined on gene expression and biosynthesis of bacterial endotoxin (LPS) induced interferon gamma (IFNgamma) both in human peripheral mononuclear cells (PMBC) and in T-cell enriched fractions. We found, that histamine inhibited the LPS induced t

  19. HIV-Specific Antibody-Dependent Cellular Cytotoxicity (ADCC) - Mediating Antibodies Decline while NK Cell Function Increases during Antiretroviral Therapy (ART)

    DEFF Research Database (Denmark)

    Jensen, Sanne Skov; Fomsgaard, Anders; Borggren, Marie

    2015-01-01

    the ability of effector cells and antibodies to mediate ADCC separately and in combination using the ADCC-PanToxiLux assay. The ability of the peripheral blood mononuclear cells (PBMCs) to mediate ADCC was significantly higher in individuals who had been treated with ART before seroconversion, compared...

  20. 1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis

    NARCIS (Netherlands)

    E.M. Colin (Edgar); P. Asmawidjaja (Patrick); J.P. van Hamburg (Jan Piet); A.M.C. Mus (Adriana); M. van Driel (Marjolein); J.M.W. Hazes (Mieke); J.P.T.M. van Leeuwen (Hans); E.W. Lubberts (Erik)

    2010-01-01

    textabstractObjective. To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D3(1,25[OH]2D3) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. Methods. Peripheral blood mononuclear cells (PBMCs) and CD4+CD4

  1. Intravenous autologous bone marrow mononuclear stem cell therapy for ischemic stroke: a multicentric, randomized trial.

    Science.gov (United States)

    Prasad, Kameshwar; Sharma, Alka; Garg, Ajay; Mohanty, Sujata; Bhatnagar, Shinjini; Johri, Sharat; Singh, Kunwar Karni; Nair, Velu; Sarkar, Ravi Shankar; Gorthi, Sankar Prasad; Hassan, Kaukab Maqbool; Prabhakar, Sudesh; Marwaha, Neelam; Khandelwal, Niranjan; Misra, Usha Kant; Kalita, Jayantee; Nityanand, Soniya

    2014-12-01

    Pilot studies have suggested benefit from intravenous administration of bone marrow mononuclear stem cells (BMSCs) in stroke. We explored the efficacy and safety of autologous BMSCs in subacute ischemic stroke. This was a phase II, multicenter, parallel group, randomized trial with blinded outcome assessment that included 120 patients. Patients with subacute ischemic stroke were randomly assigned to the arm that received intravenous infusion of autologous BMSCs or to control arm. Coprimary clinical efficacy outcomes were Barthel Index score and modified Rankin scale at day 180. Secondary outcomes were change in infarct volume, National Institute of Health Stroke Scale (NIHSS) at day 90 and 180. Main safety outcomes were adverse events, any new area of (18)fluorodeoxyglucose positron emission tomography uptake in any body part over 365 days. Fifty-eight patients received a mean of 280.75 million BMSCs at median of 18.5 days after stroke onset. There was no significant difference between BMSCs arm and control arm in the Barthel Index score (63.1 versus 63.6; P=0.92), modified Rankin scale shift analysis (P=0.53) or score >3 (47.5% versus 49.2%; P=0.85), NIHSS score (6.3 versus 7.0; P=0.53), change in infarct volume (-11.1 versus -7.36; P=0.63) at day 180. Adverse events were also similar in the 2 arms, and no patient showed any new area of (18)fluorodeoxyglucose uptake. With the methods used, results of this hitherto first randomized controlled trial indicate that intravenous infusion of BMSCs is safe, but there is no beneficial effect of treatment on stroke outcome. URLs: http://ctri.nic.in/Clinicaltrials and http://www.clinicaltrials.gov. Unique identifiers: CTRI-ROVCTRI/2008/091/0004 and NCT0150177. © 2014 American Heart Association, Inc.

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

    Science.gov (United States)

    Tsunoda, Fumiyoshi; Lamon-Fava, Stefania; Asztalos, Bela F; Iyer, Lakshmanan K; Richardson, Kris; Schaefer, Ernst J

    2015-08-01

    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 (PBMC). Subjects were sampled at baseline and six weeks after receiving either: olive oil 6.0 g/day (n = 16), EPA 1.8 g/day (n = 16), or DHA 1.8 g/day (n = 18). PBMC were subjected to gene expression analysis by microarray with key findings confirmed by quantitative real-time polymerase chain reaction (Q-PCR). Plasma phospholipid EPA increased 3 fold in the EPA group, and DHA increased 63% in the DHA group (both p expression in the following pathways: 1) interferon signaling, 2) receptor recognition of bacteria and viruses, 3) G protein signaling, glycolysis and glycolytic shunting, 4) S-adenosyl-l-methionine biosynthesis, and 5) cAMP-mediated signaling including cAMP responsive element protein 1 (CREB1), as well as many other individual genes including hypoxia inducible factor 1, α subunit (HIF1A). The findings for CREB1 and HIF1A were confirmed by Q-PCR analysis. Our data indicate that EPA supplementation was associated with significant effects on gene expression involving the interferon pathway as well as down-regulation of CREB1 and HIF1A, which may relate to its beneficial effect on CVD risk reduction. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris.

    Science.gov (United States)

    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.

  4. Contribution of mononuclear bone marrow cells to carbon tetrachloride-induced liver fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    Bao-Qiang Cao; Ji-Zong Lin; Yue-Si Zhong; Shao-Bin Huang; Nan Lin; Zhao-Feng Tang; Rui Chen; Peng Xiang; Rui-Yun Xu

    2007-01-01

    AIM:To study the inhibitory effect of mononuclear bone marrow cell (BMC) transplantation on carbon tetrachloride (CCIt) -induced liver fibrosis in rats.METHODS:Rat liver fibrosis models were induced by CCN and alcohol administration. After 8 wk,twenty rats were randomly allocated into treatment group (n = 10) and control group (n = 10). BMC were infused into the rats in treatment group via the portal vein,while heparinized saline was infused in control group. CCU was hypodermically injected into the rats twice a week for 4 wk. At the end of wk 12,all rats were humanely sacrificed. Liver samples were taken and stained with HE or Masson trichrome. The general conditions,liver fibrSsis (hydroxyproline and collagen fibre) and liver pathological grades in rats were evaluated.± 128.8μg/g in treatment group,and 596.0 ± 341. 8μg/g in control group.The percentage of collagen fibre was 3.75% ± 0.98% in treatment group and 5.02% ± 0.44% in control group.There was a significant difference berween the two groups (P<0.05).Liver pathological grade decreased from grade Ⅳ to grade Ⅲ partially in treatment group (P<0.05) with no obvious improvement in control group (P<0.05).There was a significant difference between treatment group and control group(P<0.05).CONCLUSION: Transplantation of BMC can improve liver fibrosis due to chronic liver injury in rats.

  5. Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

    Science.gov (United States)

    de Roos, Baukje; Duthie, Susan J; Polley, Abigael C J; Mulholland, Francis; Bouwman, Freek G; Heim, Carolin; Rucklidge, Garry J; Johnson, Ian T; Mariman, Edwin C; Daniel, Hannelore; Elliott, Ruan M

    2008-06-01

    This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual matching improved the CV with on average between 1 and 16%. The average between laboratory CV for each of the matched spots after automatic matching ranged between 4 and 54% for depleted plasma proteins, between 5 and 60% for platelet proteins, and between 18 and 70% for PBMC proteins. This variation must be considered when designing sufficiently powered studies that use proteomics tools for biomarker discovery. The use of tricine in the running buffer for the second dimension appears to enhance the resolution of proteins especially in the high molecular weight range.

  6. Bone marrow mononuclear cell therapy in ischaemic stroke: a systematic review.

    Science.gov (United States)

    Kumar, A; Prasad, M; Jali, V P; Pandit, A K; Misra, S; Kumar, P; Chakravarty, K; Kathuria, P; Gulati, A

    2017-05-01

    Bone marrow mononuclear cell (BM-MNC) therapy has emerged as a potential therapy for the treatment of stroke. We performed a systematic review of published studies using BM-MNC therapy in patients with ischaemic stroke (IS). Literature was searched using MEDLINE, PubMed, EMBASE, Trip Database, Cochrane library and clinicaltrial.gov to identify studies on BM-MNC therapy in IS till June, 2016. Data were extracted independently by two reviewers. STATA version 13 was used for carrying out meta-analysis. We included non-randomized open-label, single-arm and non-randomized comparative studies or randomized controlled trials (RCTs) if BM-MNCs were used to treat patients with IS in any phase after the index stroke. One randomized trial, two non-randomized comparative trials and four single-arm open-label trials (total seven studies) involving 227 subjects (137 patients and 90 controls) were included in the systematic review and meta-analysis. The pooled proportion for favourable clinical outcome (modified Rankin Scale score ≤2) in six studies involving 122 subjects was 29% (95% CI 0.16-0.43) who were exposed to BM-MNCs and pooled proportion for favourable clinical outcome of 69 subjects (taken from two trials) who did not receive BM-MNCs was 20% (95% CI 0.12-0.32). The pooled difference in the safety outcomes was not significant between both the groups. Our systematic review suggests that BM-MNC therapy is safe up to 1 year post-intervention and is feasible; however, its efficacy in the case of IS patients is debatable. Well-designed randomized controlled trials are required to provide more information on the efficacy of BM-MNC transplantation in patients with IS. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: implications for immune cell biobanking.

    Directory of Open Access Journals (Sweden)

    Anita Posevitz-Fejfár

    Full Text Available Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum, biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9: at a distal location (shipped overnight and in the central laboratory (processed immediately. PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might

  8. Immunostimulatory acivity of Calophyllum brasiliense, Ipomoea pes-caprae and Matayba elaeagnoides demonstrated by human peripheral blood mononuclear cells proliferation.

    Science.gov (United States)

    Philippi, Marina Elisa; Duarte, Bruna Momm; Da Silva, Carolina Vieira; De Souza, Michel Thomaz; Niero, Rivaldo; Cechinel Filho, Valdir; Bueno, Edneia Casagranda

    2010-01-01

    This study evaluates the effect of methanol extracts of three Brazilian medicinal plants on in vitro proliferation of human mononuclear cells. Lymphoproliferation assay was carried out by incubating human peripheral blood mononuclear cells from healthy donors (1 x 10(6) cells/mL) with extracts of Calophyllum brasiliense (roots), Ipomoea pes-caprae (whole plant) and Matayba elaeagnoides (bark), both at 10, 50, 100 and 200 microg/mL, alone or with phytohemagglutinin (PHA, 5 microg/mL), in 96-well microplates at 37 degrees C with 5% CO2, for 72 h. The quantification of cell proliferation assay was performed by blue tetrazolium (MTT) reduction with reading at 540 nm. Cells incubated with only the culture medium were used as negative control for cell proliferation, while the positive control consisted of cells and PHA. The results suggest that the extracts of all three studied plants induce T lymphocyte proliferation. I. pes-caprae showed immunostimulatory activity three times higher than the C. brasiliense extract, while that of the M. elaeagnoides extract was 1.5 times higher. The results demonstrate immunostimulatory effects of these three plants, therefore the continuity of these studies is recommended, in order to determine the active principles.

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

    Science.gov (United States)

    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),