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Sample records for human cells peripheral

  1. Glucocorticoids entrain molecular clock components in human peripheral cells.

    Science.gov (United States)

    Cuesta, Marc; Cermakian, Nicolas; Boivin, Diane B

    2015-04-01

    In humans, shift work induces a desynchronization between the circadian system and the outside world, which contributes to shift work-associated medical disorders. Using a simulated night shift experiment, we previously showed that 3 d of bright light at night fully synchronize the central clock to the inverted sleep schedule, whereas the peripheral clocks located in peripheral blood mononuclear cells (PBMCs) took longer to reset. This underlines the need for testing the effects of synchronizers on both the central and peripheral clocks. Glucocorticoids display circadian rhythms controlled by the central clock and are thought to act as synchronizers of rodent peripheral clocks. In the present study, we tested whether the human central and peripheral clocks were sensitive to exogenous glucocorticoids (Cortef) administered in the late afternoon. We showed that 20 mg Cortef taken orally acutely increased PER1 expression in PBMC peripheral clocks. After 6 d of Cortef administration, the phases of central markers were not affected, whereas those of PER2-3 and BMAL1 expression in PBMCs were shifted by ∼ 9.5-11.5 h. These results demonstrate, for the first time, that human peripheral clocks are entrained by glucocorticoids. Importantly, they suggest innovative interventions for shift workers and jet-lag travelers, combining synchronizing agents for the central and peripheral clocks.

  2. 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...

  3. 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.

  4. 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.

  5. 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

  6. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

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    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  7. 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...

  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. 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.

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

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    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.

  11. kappa+lambda+ dual receptor B cells are present in the human peripheral repertoire

    OpenAIRE

    1995-01-01

    It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. T...

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

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    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.

  13. Generation of human induced pluripotent stem cells from peripheral blood using the STEMCCA lentiviral vector.

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    Sommer, Andreia Gianotti; Rozelle, Sarah S; Sullivan, Spencer; Mills, Jason A; Park, Seon-Mi; Smith, Brenden W; Iyer, Amulya M; French, Deborah L; Kotton, Darrell N; Gadue, Paul; Murphy, George J; Mostoslavsky, Gustavo

    2012-10-31

    Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)(1-4). Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies(5). We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors(6). These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs(7-9). In one study, a Tet inducible version of the STEMCCA vector was employed(9), which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies. Here we demonstrate a protocol for the generation of human iPSCs from

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

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    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.

  15. 2-Methoxyestradiol induce the conversion of human peripheral blood memory B lymphocytes into plasma cells.

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    Cayer, Marie-Pierre; Drouin, Mathieu; Proulx, Maryse; Jung, Daniel

    2010-04-15

    2-Methoxyestradiol (2ME), an end-metabolite of 17beta-estradiol, is an antiproliferative agent that is currently being tested in clinical trials for cancer treatment. We hereby report that sub-cytotoxic concentrations of 2ME influence the in vitro proliferation of human peripheral blood B lymphocytes. More surprisingly, we have observed that 2ME induces the conversion of CD138(-) B lymphocytes into CD138(+) cells of phenotype similar to immunoglobulin (Ig)-secreting plasma cells. Normal human B lymphocytes expressing CD138 increased in response to 2ME in a dose-dependent fashion, from 2% at baseline up to 31% in cells cultured in the presence of 0.75 microM 2ME. Moreover, most of the converted cells were also CD27(+) and secreted high levels of IgG (151 microg/10(6)cells/24h). IEF studies revealed that conversion occurred in a polyclonal manner. We then exploited this effect of 2ME to gain further insights into the molecular mechanisms that govern changes in transcription factors involved in plasma cells differentiation. Plasma cells generated by 2ME treatment of normal human B lymphocytes expressed elevated levels of IRF4 and reduced levels of Pax5 and Bcl-6. Similarly, levels of XBP-1 and Blimp-1 transcripts were increased. Our results suggest that the differentiation of peripheral blood B lymphocytes into plasma cells requires a similar modulation of transcription factors expression that for tonsil and bone marrow B lymphocytes.

  16. Characterization of T-bet and eomes in peripheral human immune cells.

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    Knox, James J; Cosma, Gabriela L; Betts, Michael R; McLane, Laura M

    2014-01-01

    The T-box transcription factors T-bet and Eomesodermin (Eomes) have been well defined as key drivers of immune cell development and cytolytic function. While the majority of studies have defined the roles of these factors in the context of murine T-cells, recent results have revealed that T-bet, and possibly Eomes, are expressed in other immune cell subsets. To date, the expression patterns of these factors in subsets of human peripheral blood mononuclear cells beyond T-cells remain relatively uncharacterized. In this study, we used multiparametric flow cytometry to characterize T-bet and Eomes expression in major human blood cell subsets, including total CD4(+) and CD8(+) T-cells, γδ T-cells, invariant NKT cells, natural killer cells, B-cells, and dendritic cells. Our studies identified novel cell subsets that express T-bet and Eomes and raise implications for their possible functions in the context of other human immune cell subsets besides their well-known roles in T-cells.

  17. A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

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    Schmetzer, Oliver; Valentin, Patricia; Smorodchenko, Anna; Domenis, Rossana; Gri, Giorgia; Siebenhaar, Frank; Metz, Martin; Maurer, Marcus

    2014-11-01

    The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs.

  18. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

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    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J., E-mail: tokare@niehs.nih.gov

    2015-07-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

  19. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

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    Fernández, Esteban R; Olivera, Gabriela C; Quebrada Palacio, Luz P; González, Mariela N; Hernandez-Vasquez, Yolanda; Sirena, Natalia María; Morán, María L; Ledesma Patiño, Oscar S; Postan, Miriam

    2014-01-01

    Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  20. Altered distribution of peripheral blood memory B cells in humans chronically infected with Trypanosoma cruzi.

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    Esteban R Fernández

    Full Text Available Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.

  1. Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

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    Sayers Ian

    2009-07-01

    Full Text Available Abstract Background Expression of the urokinase plasminogen activator receptor (UPAR has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC, airway smooth muscle (HASM and peripheral cells. Results Using Rapid Amplification of cDNA Ends (RACE a conserved transcription start site (-42 to -77 relative to ATG was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression, including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein. Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts, whereas classical exon 7 (encoding the GPI-linked protein was preferentially expressed in peripheral cells (~80% of transcripts, often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role. Conclusion We have

  2. Effects of peripheral benzodiazepine receptor ligands on proliferation and differentiation of human mesenchymal stem cells.

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    Lee, D H; Kang, S K; Lee, R H; Ryu, J M; Park, H Y; Choi, H S; Bae, Y C; Suh, K T; Kim, Y K; Jung, Jin Sup

    2004-01-01

    The peripheral benzodiazepine receptor (PBR) has been known to have many functions such as a role in cell proliferation, cell differentiation, steroidogenesis, calcium flow, cellular respiration, cellular immunity, malignancy, and apoptosis. However, the presence of PBR has not been examined in mesenchymal stem cells. In this study, we demonstrated the expression of PBR in human bone marrow stromal cells (hBMSCs) and human adipose stromal cells (hATSCs) by RT-PCR and immunocytochemistry. To determine the roles of PBR in cellular functions of human mesenchymal stem cells (hMSCs), effects of diazepam, PK11195, and Ro5-4864 were examined. Adipose differentiation of hMSCs was decreased by high concentration of PBR ligands (50 microM), whereas it was increased by low concentrations of PBR ligands (<10 microM). PBR ligands showed a biphasic effect on glycerol-3-phosphate dehydrogenase (GPDH) activity. High concentration of PBR ligands (from 25 to 75 microM) inhibited proliferation of hMSCs. However, clonazepam, which does not have an affinity to PBR, did not affect adipose differentiation and proliferation of hMSCs. The PBR ligands did not induce cell death in hMSCs. PK11195 (50 microM) and Ro5-5864 (50 microM) induced cell cycle arrest in the G(2)/M phase. These results indicate that PBR ligands play roles in adipose differentiation and proliferation of hMSCs.

  3. CD1d-restricted peripheral T cell lymphoma in mice and humans

    Science.gov (United States)

    Bachy, Emmanuel; Urb, Mirjam; Chandra, Shilpi; Robinot, Rémy; Bricard, Gabriel; de Bernard, Simon; Traverse-Glehen, Alexandra; Gazzo, Sophie; Blond, Olivier; Khurana, Archana; Baseggio, Lucile; Heavican, Tayla; Ffrench, Martine; Crispatzu, Giuliano; Mondière, Paul; Schrader, Alexandra; Taillardet, Morgan; Thaunat, Olivier; Martin, Nadine; Dalle, Stéphane; Le Garff-Tavernier, Magali; Salles, Gilles; Lachuer, Joel; Hermine, Olivier; Asnafi, Vahid; Roussel, Mikael; Lamy, Thierry; Herling, Marco; Iqbal, Javeed; Buffat, Laurent; Marche, Patrice N.; Gaulard, Philippe; Kronenberg, Mitchell; Defrance, Thierry

    2016-01-01

    Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia, known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans. PMID:27069116

  4. Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes

    Institute of Scientific and Technical Information of China (English)

    TANG Ling-ling; ZHANG Zhe; ZHENG Jie-sheng; SHENG Ji-fang; LIU Ke-zhou

    2005-01-01

    Objective: This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. Methods: PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFα) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFα or lipopolysaccharide (LPS) stimulations for 24 h. Results: After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD 1 a, CD80 and CD86, features of DCs. TNFα treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. Conclusion: This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

  5. Growth and differentiation of eosinophils from human peripheral blood CD 34+ cells.

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    Shalit, M

    1997-01-01

    Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte and neutrophil colonies. IL5 alone did not support colony growth. In contrast GM-CSF and IL3 alone or together supported the generation of more than 50% eosinophil colonies. Addition of IL5 increased the fraction of eosinophil colonies to over 70%. Under the best conditions (IL3 + GM-CSF + IL5), the addition of interferon-a or LPS inhibited colony growth by 51% and 58%, respectively. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5 responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF, washed, and plated with IL5 alone. Only when progenitors were grown at least 3 days, could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/104 cells plated at day 3 and 134 colonies/104 cells at day 7). Growth of CD34+ in liquid culture for 28 days in the presence of IL3, GM-CSF and IL5 resulted in almost 250 fold increase in cell number, yielding a population of 83% maturing eosinophils. We used our culture system and the sensitive technique of RT-PCR to analyze the kinetics of production of mRNA transcripts encoding several eosinophil proteins. Freshly isolated CD34+ cells contained no eosinophil granule protein transcripts and barely detectable amounts of some oxidase protein transcripts. At day 3 of culture no cells recognizable by histochemical staining as eosinophils could be detected, but transcripts for all five eosinophil granule proteins were present. These transcripts increased

  6. 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.

  7. Derivation of induced pluripotent stem cells from human peripheral blood T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Matthew E Brown

    Full Text Available Induced pluripotent stem cells (iPSCs hold enormous potential for the development of personalized in vitro disease models, genomic health analyses, and autologous cell therapy. Here we describe the generation of T lymphocyte-derived iPSCs from small, clinically advantageous volumes of non-mobilized peripheral blood. These T-cell derived iPSCs ("TiPS" retain a normal karyotype and genetic identity to the donor. They share common characteristics with human embryonic stem cells (hESCs with respect to morphology, pluripotency-associated marker expression and capacity to generate neurons, cardiomyocytes, and hematopoietic progenitor cells. Additionally, they retain their characteristic T-cell receptor (TCR gene rearrangements, a property which could be exploited for iPSC clone tracking and T-cell development studies. Reprogramming T-cells procured in a minimally invasive manner can be used to characterize and expand donor specific iPSCs, and control their differentiation into specific lineages.

  8. Dissecting Interferon-Induced Transcriptional Programs in Human Peripheral Blood Cells

    Science.gov (United States)

    Waddell, Simon J.; Popper, Stephen J.; Rubins, Kathleen H.; Griffiths, Michael J.; Brown, Patrick O.; Levin, Michael; Relman, David A.

    2010-01-01

    Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons α, β, ω and γ, IL12 and TNFα; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNγ stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNγ and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFα stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNγ, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings. PMID:20339534

  9. Human umbilical cord mesenchymal stem cells promote peripheral nerve repairvia paracrine mechanisms

    Institute of Scientific and Technical Information of China (English)

    Zhi-yuan Guo; Xun Sun; Xiao-long Xu; Qing Zhao; Jiang Peng; Yu Wang

    2015-01-01

    Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the para-crine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These ifndings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

  10. Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

    Directory of Open Access Journals (Sweden)

    Zhi-yuan Guo

    2015-01-01

    Full Text Available Human umbilical cord-derived mesenchymal stem cells (hUCMSCs represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

  11. Chronic Inorganic Arsenic Exposure In Vitro Induces a Cancer Cell Phenotype in Human Peripheral Lung Epithelial Cells

    Science.gov (United States)

    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J.

    2015-01-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. PMID:25804888

  12. 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.

  13. 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.

  14. 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.

  15. Peripheral giant cell granuloma

    Directory of Open Access Journals (Sweden)

    Padam Narayan Tandon

    2012-01-01

    Full Text Available Peripheral giant cell granuloma or the so-called "giant cell epulis" is the most common oral giant cell lesion. It normally presents as a soft tissue purplish-red nodule consisting of multinucleated giant cells in a background of mononuclear stromal cells and extravasated red blood cells. This lesion probably does not represent a true neoplasm, but rather may be reactive in nature, believed to be stimulated by local irritation or trauma, but the cause is not certainly known. This article reports a case of peripheral giant cell granuloma arising at the maxillary anterior region in a 22-year-old female patient. The lesion was completely excised to the periosteum level and there is no residual or recurrent swelling or bony defect apparent in the area of biopsy after a follow-up period of 6 months.

  16. 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.

  17. 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 ...

  18. 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

  19. A dendritic cell-based assay for measuring memory T cells specific to dengue envelope proteins in human peripheral blood.

    Science.gov (United States)

    Sun, Peifang; Beckett, Charmagne; Danko, Janine; Burgess, Timothy; Liang, Zhaodong; Kochel, Tadeusz; Porter, Kevin

    2011-05-01

    Dengue envelope (E) protein is a dominant immune inducer and E protein-based vaccines elicited partial to complete protection in non-human primates. To study the immunogenicity of these vaccines in humans, an enzyme linked immunospot (ELISPOT) assay for measuring interferon gamma (IFN-γ) production was developed. Cells from two subject groups, based on dengue-exposure, were selected for assay development. The unique feature of the IFN-γ ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells. IFN-γ production, ranging from 53-513 spot forming units per million peripheral blood mononuclear cells (PBMCs), was observed in dengue-exposed subjects as compared to 0-45 IFN-γ spot forming units in dengue-unexposed subjects. Further, both CD4(+) and CD8(+) T cells, and cells bearing CD45RO memory marker, were the major sources of IFN-γ production. The assay allowed quantification of E-specific IFN-γ-secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Results suggested that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.

  20. 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.

  1. 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...

  2. PerioGlasU acts on human stem cells isolated from peripheral blood

    Directory of Open Access Journals (Sweden)

    Vincenzo Sollazzo

    2010-01-01

    Conclusion : PG has a differentiation effect on mesenchymal stem cells derived from peripheral blood. The obtained results can be relevant to better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

  3. Xenotransplantation of human adipose-derived stem cells in the regeneration of a rabbit peripheral nerve.

    Science.gov (United States)

    Lasso, J M; Pérez Cano, R; Castro, Y; Arenas, L; García, J; Fernández-Santos, M E

    2015-12-01

    Adipose tissue-derived mesenchymal stem cells (AdMSCs) are useful in the regeneration of neural tissues. Furthermore, xenotransplantation of human adipose tissue-derived mesenchymal stem cells (hAdMSCs) into animal models has already been tested and the results encouraged us to study peripheral nerve regeneration in rabbits, in order to test the feasibility of a xenotransplantation of hAdMSCs. To promote end-to-end nerve fiber contacts of a 4-cm gap in the peroneal nerve of white New Zealand rabbits, an autologous vein conduit was used and three groups of animals were evaluated. In Group I, the gap was repaired with a vein conduit refilled with fibrin. Group II was similar, but the animals were treated with cyclosporine A. In Group III, a fibrin scaffold with hAdMSCs was placed inside the autologous vein conduit, and animals were treated with cyclosporine A. Neurofilament immunohistochemistry results showed 100% nerve regeneration at the vein guidance channel 90 days after the surgery in the hAdMSC-transplanted group but lesser neural regeneration in the neurofilaments of groups I and II. The analysis of variance (ANOVA) test showed statistically significant differences among all groups (p nerve regeneration through a vein conduit that connected a 4-cm gap created at the peroneal nerve of rabbits. Animals treated with hAdMSCs presented negative inflammatory response at the regenerated nerve gaps, but it was demonstrated that hAdMSCs were incorporated to the new nerve creating neural tissue and endothelial cells. However, hAdMSCs required immunosuppression with cyclosporine A to achieve axonal regeneration. Copyright © 2015 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  4. Antigenotoxic Properties of Agaricus blazei against Hydrogen Peroxide in Human Peripheral Blood Cells

    Science.gov (United States)

    Borozan, Sunčica; Topalović, Dijana; Ciptasari, Ummi; Bajić, Vladan

    2017-01-01

    The ability of Agaricus blazei mushroom in its dried and powdered mycelial form was evaluated for its antigenotoxic properties for the first time. Antigenotoxic effects in human peripheral blood cells against H2O2-induced DNA damage were examined in pretreatment and posttreatment protocol by comet assay. The results showed better antigenotoxic properties of Agaricus blazei on the interventional level, respectively, after treatment. Agaricus blazei in concentration of 250 μg/mL after treatment was most efficient in regard to its action against DNA damage. The evaluation of repair kinetics showed decrease in H2O2 induced DNA damage 15 min after the application of A. blazei, reaching the maximum potency after 30 min. Analysis of antioxidant properties of Agaricus blazei revealed strong •OH scavenging properties and moderate reducing power, while its DPPH scavenging ability was weak. In regard to our findings, we can conclude that our preliminary results demonstrated antigenotoxic properties of Agaricus blazei and its strong •OH scavenging ability. Mechanisms underlying its properties should be further evaluated in in vivo studies.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Efforts of the human immune system to maintain the peripheral CD8+ T cell compartment after childhood thymectomy.

    Science.gov (United States)

    Zlamy, Manuela; Almanzar, Giovanni; Parson, Walther; Schmidt, Christian; Leierer, Johannes; Weinberger, Birgit; Jeller, Verena; Unsinn, Karin; Eyrich, Matthias; Würzner, Reinhard; Prelog, Martina

    2016-01-01

    Homeostatic mechanisms to maintain the T cell compartment diversity indicate an ongoing process of thymic activity and peripheral T cell renewal during human life. These processes are expected to be accelerated after childhood thymectomy and by the influence of cytomegalovirus (CMV) inducing a prematurely aged immune system. The study aimed to investigate proportional changes and replicative history of CD8+ T cells, of recent thymic emigrants (RTEs) and CD103+ T cells (mostly gut-experienced) and the role of Interleukin-(IL)-7 and IL-7 receptor (CD127)-expressing T cells in thymectomized patients compared to young and old healthy controls. Decreased proportions of naive and CD31 + CD8+ T cells were demonstrated after thymectomy, with higher proliferative activity of CD127-expressing T cells and significantly shorter relative telomere lengths (RTLs) and lower T cell receptor excision circles (TRECs). Increased circulating CD103+ T cells and a skewed T cell receptor (TCR) repertoire were found after thymectomy similar to elderly persons. Naive T cells were influenced by age at thymectomy and further decreased by CMV. After childhood thymectomy, the immune system demonstrated constant efforts of the peripheral CD8+ T cell compartment to maintain homeostasis. Supposedly it tries to fill the void of RTEs by peripheral T cell proliferation, by at least partly IL-7-mediated mechanisms and by proportional increase of circulating CD103+ T cells, reminiscent of immune aging in elderly. Although other findings were less significant compared to healthy elderly, early thymectomy demonstrated immunological alterations of CD8+ T cells which mimic features of premature immunosenescence in humans.

  7. 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.

  8. 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.

  9. Recombinant human thrombopoietin augments mobilization of peripheral blood progenitor cells for autologous transplantation.

    Science.gov (United States)

    Linker, Charles; Anderlini, Paolo; Herzig, Roger; Christiansen, Neal; Somlo, George; Bensinger, William; Fay, Joseph; Lynch, Joseph P; Goodnough, Lawrence T; Ashby, Mark; Benyunes, Mark C; Jones, Dennie V; Yang, Timothy A; Miller, Langdon L; Weaver, Charles

    2003-06-01

    This study assessed the ability of various schedules of recombinant human thrombopoietin (rhTPO) to enhance mobilization of peripheral blood progenitor cells (PBPCs) in 134 patients with cancer undergoing high-dose chemotherapy and autologous PBPC transplantation. Patients received the study drug on days 1, 3, and 5 before initiation of granulocyte colony-stimulating factor (G-CSF) 10 microg/kg/day on day 5 and pheresis starting on day 9. Randomly assigned treatments on days 1, 3, and 5 were: group 1 (n=27) placebo, placebo, rhTPO 1.5 microg/kg; group 2 (n=27) rhTPO 1.5 microg/kg, placebo, placebo; groups 3 (n=28) and 4 (n=22) rhTPO 0.5 microg/kg on all 3 treatment days; and group 5 (n=30) placebo on all 3 treatment days. After high-dose chemotherapy and PBPC transplantation, groups 1 through 4 received rhTPO 1.5 microg/kg days 0, +2, +4, and +6 with either G-CSF 5 microg/kg/day (groups 1-3) or granulocyte-macrophage colony-stimulating factor 250 microg/m(2)/day (group 4). Group 5 received placebo plus G-CSF 5 microg/kg/day. The addition of rhTPO to G-CSF increased median CD34+ cell yield/pheresis in cohorts in which rhTPO was started before day 5, with higher yields in groups 2 (2.67 x 10(6)/kg) and groups 3 and 4 (3.10 x 10(6)/kg) than in group 1 (1.86 x 10(6)/kg) or group 5 (1.65 x 10(6)/kg) (P=.006 across groups). Comparing rhTPO to placebo, higher percentages of patients achieved the minimum yield of CD34+ > or =2 x 10(6)/kg (92% v 75%; P=.050) as well as the target yield of CD34+ > or =5 x 10(6)/kg (73% v 46%; P= .041). rhTPO-treated patients required fewer phereses to achieve minimum (P= .011) and target (P= .015) CD34+ cell values. rhTPO given after transplantation did not speed platelet recovery. No neutralizing antibodies were observed. We conclude that rhTPO can safely enhance mobilization of PBPC, reduce the number of leukapheresis, and allow more patients to meet minimal cell yield requirements to receive high-dose chemotherapy with PBPC

  10. Antitumor activities of human dendritic cells derived from peripheral and cord blood

    Institute of Scientific and Technical Information of China (English)

    Jin-Kun Zhang; Jun Li; Hai-Bin Chen; Jin-Lun Sun; Yao-Juan Qu; Juan-Juan Lu

    2002-01-01

    AIM: To observe the biological specialization of humanperipheral blood dendritic cells (DC) and cord blood derivedDC and its effects on effector cells killing humanhepatocarcinoma cell line BEL-7402 in vitro.METHODS: The DC biological characteristics were detectedwith immunohistochemical and MTT assay. Two antitumorexperimental groups are: peripheral blood DC and cordblood DC groups. Peripheral blood DC groups used LAKcells as the effector cells and BEL-7402 as target cells, whilecord blood DC groups used CTL induced by tumor antigentwice pulsed DC as effector cells and BEL-7402 as targetcells, additional peripheral blood DC and cord blood DC areadded to observe its stimulating activities to effector cells.The effector's cytotoxicity to tumor cells were detected withneutral red colorimetric assay at two effector/target ratios of5:1 and 10: 1.RESULTS: Peripheral blood DC and cord blood DC highlyexpressed HLA-ABC, HLA-DR, HLA-DQ, CD54 and S-100protein. The stimulating activities to lymphocyteproliferation were compared between experimental groups(DC added) and control group (no DC added). In sixexperiment subgroups, the DC/lymphocyte ratio wassequentially 0.25: 100, 0.5: 100, 1: 100, 2: 100, 4: 100 and 8:100, A values(x± s) were 0.75396± 0.009, 0.84916± 0.010,0.90894± 0.012, 0.98371 ± 0.007, 1.01299 ± 0.006 and 1.20384± 0.006 in peripheral blood DC groups and 0.77650 ± 0.005,0.83008± 0.007, 0.92725 ± 0.007, 1.05990 ± 0.010, 1.15583 ±0.011, 1.22983 ± 0.011 in cord blood DC groups. A value was0.59517 ± 0.005 in control group. The stimulating activitieswere higher in experimental groups than in control group ( P< 0.01 ), which were increased when the DC concentrationwas enlarged ( P < 0.01 ). Two differently derived DCs hadthe same phenotypes and similar stimulating activities ( P >0.05). In peripheral blood DC groups, the cytotoxicity (x ±s) of the LD groups (experimental groups) and L groups(control group) was 58.16% ± 2.03% (5: 1), 46.18% ±2

  11. 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

  12. Erythropoietic Potential of CD34+ Hematopoietic Stem Cells from Human Cord Blood and G-CSF-Mobilized Peripheral Blood

    Directory of Open Access Journals (Sweden)

    Honglian Jin

    2014-01-01

    Full Text Available Red blood cell (RBC supply for transfusion has been severely constrained by the limited availability of donor blood and the emergence of infection and contamination issues. Alternatively, hematopoietic stem cells (HSCs from human organs have been increasingly considered as safe and effective blood source. Several methods have been studied to obtain mature RBCs from CD34+ hematopoietic stem cells via in vitro culture. Among them, human cord blood (CB and granulocyte colony-stimulating factor-mobilized adult peripheral blood (mPB are common adult stem cells used for allogeneic transplantation. Our present study focuses on comparing CB- and mPB-derived stem cells in differentiation from CD34+ cells into mature RBCs. By using CD34+ cells from cord blood and G-CSF mobilized peripheral blood, we showed in vitro RBC generation of artificial red blood cells. Our results demonstrate that CB- and mPB-derived CD34+ hematopoietic stem cells have similar characteristics when cultured under the same conditions, but differ considerably with respect to expression levels of various genes and hemoglobin development. This study is the first to compare the characteristics of CB- and mPB-derived erythrocytes. The results support the idea that CB and mPB, despite some similarities, possess different erythropoietic potentials in in vitro culture systems.

  13. Transcriptional Activity of Human Endogenous Retroviruses in Human Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Emanuela Balestrieri

    2015-01-01

    Full Text Available Human endogenous retroviruses (HERVs have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individual’s life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease.

  14. [Gene transfer in human hematopoietic stem cells isolated from peripheral blood].

    Science.gov (United States)

    Mannoni, P

    1996-01-01

    To insert a new genetic information by gene transfer into haemopoietic stem cells would result in expression of the transgene in progenitors and progeny of cell blood lineages. If successfull, such an approach would open interesting prospectives in the field of experimental research and in the possibility to treat genetic defects affecting blood lineages such as immune deficiencies (ADA, SCID, AIDS) or enzymes defects. Moreover progenitors could be engineered to become more resistant to chemotherapy or oncogenic process. Many parameters and technical problems are still involved in this issue, including identification, isolation and selection of the most primitive progenitors, and search for the most efficient vectors to insert new genes into the target cells. So far retroviral vectors have been shown to be the most effective but search for better vectors are still underway. Peripheral blood stem cells isolated from patients stimulated by cytokines and/or chemotherapy appear interesting target cells for genetic manipulations aimed to correct an acquired or genetic defect.

  15. Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype.

    Science.gov (United States)

    Weiss, Tamara; Taschner-Mandl, Sabine; Bileck, Andrea; Slany, Astrid; Kromp, Florian; Rifatbegovic, Fikret; Frech, Christian; Windhager, Reinhard; Kitzinger, Hugo; Tzou, Chieh-Han; Ambros, Peter F; Gerner, Christopher; Ambros, Inge M

    2016-12-01

    The remarkable feature of Schwann cells (SCs) to transform into a repair phenotype turned the spotlight on this powerful cell type. SCs provide the regenerative environment for axonal re-growth after peripheral nerve injury (PNI) and play a vital role in differentiation of neuroblastic tumors into a benign subtype of neuroblastoma, a tumor originating from neural crest-derived neuroblasts. Hence, understanding their mode-of-action is of utmost interest for new approaches in regenerative medicine, but also for neuroblastoma therapy. However, literature on human SCs is scarce and it is unknown to which extent human SC cultures reflect the SC repair phenotype developing after PNI in patients. We performed high-resolution proteome profiling and RNA-sequencing on highly enriched human SC and fibroblast cultures, control and ex vivo degenerated nerve explants to identify novel molecules and functional processes active in repair SCs. In fact, we found cultured SCs and degenerated nerves to share a similar repair SC-associated expression signature, including the upregulation of JUN, as well as two prominent functions, i.e., myelin debris clearance and antigen presentation via MHCII. In addition to myelin degradation, cultured SCs were capable of actively taking up cell-extrinsic components in functional phagocytosis and co-cultivation assays. Moreover, in cultured SCs and degenerated nerve tissue MHCII was upregulated at the cellular level along with high expression of chemoattractants and co-inhibitory rather than -stimulatory molecules. These results demonstrate human SC cultures to execute an inherent program of nerve repair and support two novel repair SC functions, debris clearance via phagocytosis-related mechanisms and type II immune-regulation. GLIA 2016;64:2133-2153. © 2016 The Authors. Glia Published by Wiley Periodicals, Inc.

  16. Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

    Science.gov (United States)

    Fuerstenau-Sharp, Maya; Zimmermann, Martina E; Stark, Klaus; Jentsch, Nico; Klingenstein, Melanie; Drzymalski, Marzena; Wagner, Stefan; Maier, Lars S; Hehr, Ute; Baessler, Andrea; Fischer, Marcus; Hengstenberg, Christian

    2015-01-01

    Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

  17. Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Maya Fuerstenau-Sharp

    Full Text Available Induced pluripotent stem (iPS cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V. In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

  18. Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

    Science.gov (United States)

    Kishino, Yoshikazu; Seki, Tomohisa; Fujita, Jun; Yuasa, Shinsuke; Tohyama, Shugo; Kunitomi, Akira; Tabei, Ryota; Nakajima, Kazuaki; Okada, Marina; Hirano, Akinori; Kanazawa, Hideaki; Fukuda, Keiichi

    2014-01-01

    Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

  19. Radiolabeling human peripheral blood stem cells for positron emission tomography (PET imaging in young rhesus monkeys.

    Directory of Open Access Journals (Sweden)

    Alice F Tarantal

    Full Text Available These studies focused on a new radiolabeling technique with copper ((64Cu and zirconium ((89Zr for positron emission tomography (PET imaging using a CD45 antibody. Synthesis of (64Cu-CD45 and (89Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC was assessed in vitro (viability, population doubling times, colony forming units. hPBSC viability was maintained as the dose of (64Cu-TETA-CD45 increased from 0 (92% to 160 µCi/mL (76%, p>0.05. Radiolabeling efficiency was not significantly increased with concentrations of (64Cu-TETA-CD45 >20 µCi/mL (p>0.50. Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p0.05, and a trend towards increased radiolabeling efficiency was noted as the dose of (89Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0-40 µCi/mL (p<0.05. A greater than 2,000 fold-increase in the level of (89Zr-Df-CD45 labeling efficiency was observed when compared to (64Cu-TETA-CD45. Similar to (64Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05 (growth, colony formation. Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×10(6 hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI, then 0.3 mCi of (89Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that (89Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high.

  20. Human peripheral blood B-Cell compartments: A crossroad in B-cell traffic

    NARCIS (Netherlands)

    M. Perez-Andres; B. Paiva; W.G. Nieto (Wendy); A. Caraux; A. Schmitz; J. Almeida (Julia); R.F. Vogt; G.E. Marti; A.C. Rawstron; M.C. van Zelm (Menno); J.J.M. van Dongen (Jacques); H.E. Johnsen (Hans); B. Klein (Binie); A. Orfao (Alberto)

    2010-01-01

    textabstractA relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lympho

  1. Human Peripheral Blood B-Cell Compartments : A Crossroad in B-Cell Traffic

    NARCIS (Netherlands)

    Perez-Andres, M.; Paiva, B.; Nieto, W. G.; Caraux, A.; Schmitz, A.; Almeida, J.; Vogt, R. F.; Marti, G. E.; Rawstron, A. C.; Van Zelm, M. C.; Van Dongen, J. J. M.; Johnsen, H. E.; Klein, B.; Orfao, A.

    2010-01-01

    A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues.

  2. Human Peripheral Blood B-Cell Compartments : A Crossroad in B-Cell Traffic

    NARCIS (Netherlands)

    Perez-Andres, M.; Paiva, B.; Nieto, W. G.; Caraux, A.; Schmitz, A.; Almeida, J.; Vogt, R. F.; Marti, G. E.; Rawstron, A. C.; Van Zelm, M. C.; Van Dongen, J. J. M.; Johnsen, H. E.; Klein, B.; Orfao, A.

    2010-01-01

    A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues.

  3. Peripheral T-Cell Lymphoma

    Science.gov (United States)

    Getting the Facts Peripheral T-Cell Lymphoma Overview Lymphoma is the most common blood cancer. The two main forms of lymphoma are Hodgkin lymphoma and ... develop into lymphomas: B-lymphocytes (B-cells) and T-lymphocytes (T-cells). Peripheral T-cell lymphoma (PTCL) ...

  4. Human peripheral blood-born hematosphere as a niche for hematopoietic stem cell expansion

    Institute of Scientific and Technical Information of China (English)

    Jin Hur; Eun Ju Lee; Hyun-Jai Cho; Hyun-Jae Kang; Byung-Hee Oh; Young-Bae Park; Hyo-Soo Kim; Jonghanne Park; Sang Eun Lee; Chang-Hwan Yoon; Jae Hee Jang; Ji Min Yang; Tae-Kyu Lee; Jae-Il Choi; Han-Mo Yang

    2011-01-01

    @@ Dear Editor, Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) derived from the adult peripheral blood has been widely used in the treatment of various hematological diseases [1].However,the small number of circulating HSPC is the major limitation and necessitates additional interventions such as G-CSF mobilization and leukapheresis.There have been several attempts to overcome the limitation with ex vivo expansion of HSPC.These strategies are largely based on supplementation of one or more "stem cell niche components"such as supporting-cells,growth factors,extracellular matrix (ECM) or physicochemical microenvironment in the bone marrow [2].Spheroid culture methods of stem ceils from different tissues have been successfully used for expansion of cardiac and neural stem cells.These spheres sensitize target stem cells to growth factors and provide sufficient cell-to-cell and cell-to-matrix contacts,mimicking the in vivo stem cell niche [3,4].Here we asked whether spheroid culture of blood mononuclear cells (MNCs) would potentiate the expansion of circulating blood HSPC.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

  7. Peripheral CD4+ T cell cytokine responses following human challenge and re-challenge with Campylobacter jejuni.

    Science.gov (United States)

    Fimlaid, Kelly A; Lindow, Janet C; Tribble, David R; Bunn, Janice Y; Maue, Alexander C; Kirkpatrick, Beth D

    2014-01-01

    Campylobacter jejuni is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to C. jejuni infection is limited. A previous human challenge model has shown that C. jejuni elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which C. jejuni-naïve subjects were challenged and re-challenged with C. jejuni CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4+ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4+ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.

  8. Peripheral CD4+ T cell cytokine responses following human challenge and re-challenge with Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Kelly A Fimlaid

    Full Text Available Campylobacter jejuni is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to C. jejuni infection is limited. A previous human challenge model has shown that C. jejuni elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which C. jejuni-naïve subjects were challenged and re-challenged with C. jejuni CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4+ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4+ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.

  9. Relation between clinical mature and immature lymphocyte cells in human peripheral blood and their spatial label free scattering patterns

    Science.gov (United States)

    Zhang, Lu; Zhao, Xin; Zhang, Zhenxi; Zhao, Hong; Chen, Wei; Yuan, Li

    2016-07-01

    A single living cell's light scattering pattern (LSP) in the horizontal plane, which has been denoted as the cell's "2D fingerprint," may provide a powerful label-free detection tool in clinical applications. We have recently studied the LSP in spatial scattering planes, denoted as the cell's "3D fingerprint," for mature and immature lymphocyte cells in human peripheral blood. The effects of membrane size, morphology, and the existence of the nucleus on the spatial LSP are discussed. In order to distinguish clinical label-free mature and immature lymphocytes, the special features of the spatial LSP are studied by statistical method in both the spatial and frequency domains. Spatial LSP provides rich information on the cell's morphology and contents, which can distinguish mature from immature lymphocyte cells and hence ultimately it may be a useful label-free technique for clinical leukemia diagnosis.

  10. Stimulated human peripheral T cells produce high amounts of IL-35 protein in a proliferation-dependent manner.

    Science.gov (United States)

    Guttek, Karina; Reinhold, Dirk

    2013-10-01

    The p35 subunit of IL-12 and the Epstein-Barr virus-induced gene 3 (EBI3) have been shown to form a heterodimeric cytokine, named interleukin-35 (IL-35). Recently, mRNA expression of both IL-12p35 and EBI3 was clearly shown in stimulated human T effector cells. Here, we investigated the production of IL-35 protein in human anti-CD3/CD28-stimulated pan T cells as well as T cell subpopulations using a specific human IL-35 ELISA system. We measured high concentrations of IL-35 (up to 3 ng/ml) in cell culture supernatants of stimulated pan T cells as well as CD4(+), CD8(+) and CD4(+)CD25(-) T cell subpopulations at 72 h after stimulation. Very low amounts of IL-35, in the range of 100pg/ml, were detectable in supernatants of resting T cells. These observations could be confirmed using a dot-blot assay for IL-12p35 and EBI3. High concentrations of IL-35 could be also measured in cell culture supernatants of both, resting and stimulated CD4(+)CD25(+) T cells. In order to learn more about the regulation of IL-35 production, we studied the effect of dexamethasone, cyclosporine A and rapamycin on IL-35 production of anti-CD3/CD28-stimulated human pan T cells as well as CD4(+) and CD8(+) T cell subpopulations. All three drugs significantly suppressed IL-35 production of these cells in a proliferation-dependent manner. In summary, we could show that stimulated human peripheral blood T cells of healthy donors produce high amounts of IL-35 protein. However, the biological function of this cytokine remains to be elucidated.

  11. KANK1 inhibits cell growth by inducing apoptosis though regulating CXXC5 in human malignant peripheral nerve sheath tumors

    Science.gov (United States)

    Cui, Zhibin; Shen, Yingjia; Chen, Kenny H.; Mittal, Suresh K.; Yang, Jer-Yen; Zhang, GuangJun

    2017-01-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are a type of rare sarcomas with a poor prognosis due to its highly invasive nature and limited treatment options. Currently there is no targeted-cancer therapy for this type of malignancy. Thus, it is important to identify more cancer driver genes that may serve as targets of cancer therapy. Through comparative oncogenomics, we have found that KANK1 was a candidate tumor suppressor gene (TSG) for human MPNSTs. Although KANK1 is known as a cytoskeleton regulator, its tumorigenic function in MPNSTs remains largely unknown. In this study, we report that restoration of KANK1 in human MPNST cells inhibits cell growth both in human cell culture and xenograft mice by increasing apoptosis. Consistently, knockdown of KANK1 in neurofibroma cells promoted cell growth. Using RNA-seq analysis, we identified CXXC5 and other apoptosis-related genes, and demonstrated that CXXC5 is regulated by KANK1. Knockdown of CXXC5 was found to diminish KANK1-induced apoptosis in MPNST cells. Thus, KANK1 inhibits MPNST cell growth though CXXC5 mediated apoptosis. Our results suggest that KANK1 may function as a tumor suppressor in human MPNSTs, and thus it may be useful for targeted therapy. PMID:28067315

  12. 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.

  13. Homeostatic 'bystander' proliferation of human peripheral blood B cells in response to polyclonal T-cell stimulation in vitro.

    Science.gov (United States)

    Jasiulewicz, Aleksandra; Lisowska, Katarzyna A; Pietruczuk, Krzysztof; Frąckowiak, Joanna; Fulop, Tamas; Witkowski, Jacek M

    2015-11-01

    The mechanisms of maintenance of adequate numbers of B lymphocytes and of protective levels of immunoglobulins in the absence of antigenic (re)stimulation remain not fully understood. Meanwhile, our results presented here show that both peripheral blood naive and memory B cells can be activated strongly and non-specifically (in a mitogen-like fashion) in 5-day in vitro cultures of anti-CD3- or concanavalin A (Con A)-stimulated peripheral blood mononuclear cells of healthy people. This polyclonal, bystander activation of the B cells includes multiple divisions of most of them (assessed here by the flow cytometric technique of dividing cell tracking) and significant antibody [immunoglobulin M (IgM) and IgG] secretion. Observed proliferation of the CD19(+) B cells depends on contact with stimulated T helper (Th) cells (via CD40-CD40L interaction) and on the response of B cells to secreted interleukins IL-5, IL-10 and IL-4, and is correlated with the levels of these Th-derived molecules, while it does not involve the ligation of the BCR/CD19 complex. We suggest that the effect might reflect the situation occurring in vivo as the homeostatic proliferation of otherwise non-stimulated, peripheral B lymphocytes, providing an always ready pool for efficient antibody production to any new (or cognate) antigen challenge.

  14. Members of the Candida parapsilosis complex and Candida albicans are differentially recognized by human peripheral blood mononuclear cells

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    Eine eEstrada-Mata

    2016-01-01

    Full Text Available The systemic infections caused by members of the Candida parapsilosis complex are currently associated to high mobility 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. 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 peripheral blood mononuclear cells 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 TNF 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 peripheral blood mononuclear cells. Together; our results suggest that the innate immune

  15. Expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin on dendritic cells generated from human peripheral blood monocytes

    Institute of Scientific and Technical Information of China (English)

    Jun Li; Zhi-Hua Feng; Guang-Yu Li; Dan-Lei Mou; Qing-He Nie

    2006-01-01

    AIM: To generate dendritic cells (DCs) from human peripheral blood and to detect the expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN; CD209) for the further study of DC-SIGN in hepatitis C virus (HCV) transmission.METHODS: Peripheral blood monocytes were isolated from blood of healthy individuals by Ficoll-Hypaque sedimentation and cultured in complete medium containing rhGM-CSF and rhIL-4. Cells were cultured for seven days, with cytokine addition every two days to obtain immature DCs. Characteristics of the cultured cells were observed under light and scanning microscope, and the expression of DC-SIGN was detected by immunofluorescence staining.RESULTS: After seven-day culture, a large number of cells with typical characteristics of DCs appeared. Their characteristics were observed under light and scanning electron microscope. These cells had a variety of cell shapes such as those of bipolar elongate cells, elaborate stellate cells and DCs. DC-SIGN was detected by immunofluorescence staining and its expression level on cultivated dendritic cells was high.CONCLUSION: DCs with a high expression of DC-SIGN can be generated from human peripheral blood monocytes in complete medium containing rhGM-CSF and rhIL-4.

  16. The effects of low-dose methotrexate on thymidylate synthetase activity in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Hornung, N; Stengaard-Pedersen, K.; Ehrnrooth, E

    2001-01-01

    ) and dihydrofolate reductase essential for DNA synthesis. This study investigated the effects of low-dose MTX on TS activity and proliferation in human peripheral blood mononuclear cells (PBMC). METHODS: The MTX concentrations in our experiments were chosen according to the plasma concentrations measured in 8 RA......, there was no difference between TS activity measured before and after MTX intake in 6 RA patients on long-term MTX treatment. CONCLUSION: We show that low concentrations of MTX inhibit TS activity in vitro. An in vivo effect cannot, however, be proven given our study design. The role of these in vitro findings...

  17. In vitro response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells exposed to {sup 60}Co at single fraction

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Lidia Maria; Campos, Tarcisio Passos Ribeiro de [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Engenharia Nuclear]. E-mail: lidia.andrade@unifenas.br; Leite, M.F. [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Fisiologia e Biofisica; Goes, A.M. [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia

    2005-10-15

    Radiotherapy using gamma rays is a common modality of breast cancer treatment. The aim of this research is to investigate the biological response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells (PBMC) exposed in vitro to {sup 60} Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min{sup -1} rate. Cells were irradiated at room temperature by the Theratron 80 radiotherapy system. Biological response was evaluated through cellular viability using MTT assay and nucleus damages visualized by Propidium Iodide assay and electrophoresis agarose gel after gamma irradiation. Nucleus damages induced by {sup 60} Co irradiation were compared to damage caused by cell exposure to 10% methanol. The 50 Gy dose of irradiation did not stimulate nucleus damages at the same level as that affected by 10% methanol induction in the MDAMB-231. Further studies are necessary to understand these mechanisms in the MDAMB-231 human breast carcinoma cell line.(author)

  18. Peripheral dentinogenic ghost cell tumor

    Directory of Open Access Journals (Sweden)

    Sushant S Kamat

    2013-01-01

    Full Text Available Dentinogenic ghost cell tumors (DGCT are uncommon lesions mainly with rare peripheral types. This report presents a case of peripheral DGCT on the left side of the mandibular alveolar ridge of a heavy smoker, a 68-year-old man, with main presenting feature as a mild pain. Submandibular lymphadenopathy and radiological "saucerization" were evident. Differential diagnosis included fibroma, neurofibroma, peripheral ameloblastoma, peripheral odontogenic fibroma, and peripheral giant cell granuloma. Histologically, ameloblastoma-like epithelial elements were seen in association with grouped ghost cells. Proliferating polyhedral cells and stellate reticulum-like cells with various densities were spread over a wide range of the field. The lesion was curetted and after 2 years of follow up, it did not recur.

  19. Inhibitory Effects of Berberine on the Activation and Cell Cycle Progression of Human Peripheral Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    Lihui Xu; Yi Liu; Xianhui He

    2005-01-01

    The immunosuppressive property of berberine, an isoquinoline alkaloid, has been well documented, but the mechanism of its action on lymphocytes has not been completely elucidated. The present study is to investigate the effect of berberine on the activation and proliferation of lymphocytes, in particular T lymphocytes. Whole peripheral blood from healthy donors was stimulated with phytohemagglutinin (PHA) alone or phorbol dibutyrate (PDB) plus ionomycin, and the expression of CD69 and CD25 on T lymphocytes was evaluated with flow cytometry.The distribution of cell cycles and cell viability were analyzed by staining with propidium iodide (PI) and 7-aminoactinomycin D (7-AAD), respectively. The results showed that 100 μmol/L and 50 μmol/L of berberine significantly inhibited CD69 expression on T cells stimulated with PDB plus ionomycin or PHA, whereas the effect of 25 μmol/L berberine was not significant. As the incubation time increased, the extent of inhibition decreased.Similarly, the expression of CD25 was also reduced by berberine in a dose-dependent manner over the concentration range of 25-100 μmol/L. Besides, this alkaloid could block lymphocyte cell cycle progression from G0/G1 phase to S and G2/M phase without phase specificity. Moreover, analysis following 7-AAD staining revealed that berberine had no significant cytotoxicity on lymphocytes. Taken together, berberine significantly inhibits the expression of activation antigens on T lymphocytes and also blocks the progression of cell cycles of lymphocytes,suggesting that berberine may exert immunosuppressive effect through inhibiting the activation and proliferation of T cells.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. In-depth immunophenotyping data of IL-6R on the human peripheral regulatory T cell (Treg compartment

    Directory of Open Access Journals (Sweden)

    Ricardo C. Ferreira

    2017-06-01

    Full Text Available We provide in this paper a detailed characterization of the human peripheral CD4+ CD127lowCD25+ regulatory T cell (Treg compartment, with a particular emphasis in defining the population expressing higher levels of the IL-6 receptor (IL-6R. We provide a description of the phenotype of this population by assessing both the surface expression by flow cytometry as well as their transcriptional profile and functional features. In addition, we also present functional data describing the responsiveness of these subsets to IL-6 signalling in vitro and to IL-2 in vivo. The data presented in this paper support the research article “Human IL-6RhiTIGIT− CD4+CD127lowCD25+ T cells display potent in vitro suppressive capacity and a distinct Th17 profile” (Ferreira RC et al., 2017; doi: 10.1016/j.clim.2017.03.002 [1].

  2. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    Energy Technology Data Exchange (ETDEWEB)

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul [School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor (Malaysia); Rohaya, M. A. W. [Department of Orthodontics, Faculty of Dentistry, Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur (Malaysia)

    2013-11-27

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

  3. Altered Cytokine Production By Specific Human Peripheral Blood Cell Subsets Immediately Following Spaceflight

    Science.gov (United States)

    Crucian, Brian E.; Cubbage, Michael L.; Sams, Clarence F.

    1999-01-01

    In this study, we have attempted to combine standard immunological assays with the cellular resolving power of the flow cytometer to positively identify the specific cell types involved in spaceflight-induced immune alterations. We have obtained whole blood samples from 27 astronauts collected at three timepoints (L-10, R+0 and R+3) surrounding four recent space shuttle missions. The duration of these missions ranged from 10 to 18 days. Assays performed included serum/urine cortisol, comprehensive subset phenotyping, assessment of cellular activation markers and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following spaceflight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated trends towards a decreased percentage of T cells and an increased percentage of B cells. Nearly all of the astronauts exhibited an increased CD4:CD8 ratio, which was dramatic in some individuals. Assessment of memory (CD45RA+) vs. naive (CD45RO+) CD4+ T cell subsets was more ambiguous, with subjects tending to group more as a flight crew. All subjects from one mission demonstrated an increased CD45RA:CD45RO ratio, while all subjects from another Mission demonstrated a decreased ratio. While no significant trend was seen in the monocyte population as defined by scatter, a decreased percentage of the CD14+ CD16+ monocyte subset was seen following spaceflight in all subjects tested. In general, most of the cellular changes described above which were assessed at R+O and compared to L-10 trended to pre-flight levels by R+3. Although no significant differences were seen in the expression of the cellular activation markers CD69 and CD25 following exposure to microgravity, significant alterations were seen in cytokine production in response to mitogenic activation for specific subsets. T cell (CD3+) production of IL-2 was significantly decreased

  4. Engineering human peripheral blood stem cell grafts that are depleted of naïve T cells and retain functional pathogen-specific memory T cells.

    Science.gov (United States)

    Bleakley, Marie; Heimfeld, Shelly; Jones, Lori A; Turtle, Cameron; Krause, Diane; Riddell, Stanley R; Shlomchik, Warren

    2014-05-01

    Graft-versus-host disease (GVHD) is a frequent major complication of allogeneic hematopoietic cell transplantation (HCT). Approaches that selectively deplete T cells that cause GVHD from allogeneic stem cell grafts and preserve T cells specific for pathogens may improve HCT outcomes. It has been hypothesized that the majority of T cells that can cause GVHD reside within the naïve T cell (TN) subset, and previous studies performed in mouse models and with human cells in vitro support this hypothesis. As a prelude to translating these findings to the clinic, we developed and evaluated a novel 2-step clinically compliant procedure for manipulating peripheral blood stem cells (PBSC) to remove TN, preserve CD34(+) hematopoietic stem cells, and provide for a fixed dose of memory T cells (TM) that includes T cells with specificity for common opportunistic pathogens encountered after HCT. Our studies demonstrate effective and reproducible performance of the immunomagnetic cell selection procedure for depleting TN. Moreover, after cell processing, the CD45RA-depleted PBSC products are enriched for CD4(+) and CD8(+) TM with a central memory phenotype and contain TM cells that are capable of proliferating and producing effector cytokines in response to opportunistic pathogens. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Comparison of complement-dependent cytotoxicity and indirect immunofluorescence for enumeration of T-cell subpopulations in human peripheral blood.

    Science.gov (United States)

    Gratama, J W; Schuurman, R K; Van Leeuwen, A; Jansen, J; Oljans, P; Tanke, H J; Van Rood, J J

    1983-11-11

    The monitoring of T-lymphocyte subsets of recipients of organ grafts enables studies on immune reconstitution (after bone-marrow transplantation) and may predict graft rejection (after kidney transplantation). Quantitation of human peripheral T-lymphocyte subsets from healthy volunteers and from recipients of a bone-marrow graft by a complement dependent cytotoxicity (CDC) assay, based on the use of propidium iodide, and by an indirect immunofluorescence (IIF) technique has been compared using the monoclonal antibodies OKT3, OKT4 and OKT8. Except for OKT3 in healthy individuals--for which no significant difference was found between CDC and IIF--CDC detected significantly more cells of each subset than IIF. Furthermore, the CDC results indicated the presence of low numbers of OKT4+8+ cells in the peripheral blood of healthy individuals and--with higher numbers--following marrow transplantation. Results of depletion experiments, obtained by fluorescence activated cell sorting (FACS) for either OKT4 or OKT8, supported this conclusion. OKT4/OKT8 ratios were calculated from enumerations by the CDC assay and by the IIF assay and found to be linearly related, both in healthy persons and in marrow-graft recipients. Thus, the CDC assay is a reliable method for monitoring T-cell subsets, allowing detection of lymphocytes carrying low densities of membrane determinants.

  7. 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.

  8. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Musarrat, Javed

    2012-12-01

    Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka=1.2×10(4)M(-1)) and binding capacity (n=1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow cytometry analysis of annexin V(-)/7-AAD(+) stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor-DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and

  9. 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.

  10. Modulation of Human Peripheral Blood Mononuclear Cell Signaling by Medicinal Cannabinoids

    Science.gov (United States)

    Utomo, Wesley K.; de Vries, Marjan; Braat, Henri; Bruno, Marco J.; Parikh, Kaushal; Comalada, Mònica; Peppelenbosch, Maikel P.; van Goor, Harry; Fuhler, Gwenny M.

    2017-01-01

    Medical marijuana is increasingly prescribed as an analgesic for a growing number of indications, amongst which terminal cancer and multiple sclerosis. However, the mechanistic aspects and properties of cannabis remain remarkably poorly characterized. In this study we aimed to investigate the immune-cell modulatory properties of medical cannabis. Healthy volunteers were asked to ingest medical cannabis, and kinome profiling was used to generate comprehensive descriptions of the cannabis challenge on inflammatory signal transduction in the peripheral blood of these volunteers. Results were related to both short term and long term effects in patients experimentally treated with a medical marijuana preparation for suffering from abdominal pain as a result of chronic pancreatitis or other causes. The results reveal an immunosuppressive effect of cannabinoid preparations via deactivation of signaling through the pro-inflammatory p38 MAP kinase and mTOR pathways and a concomitant deactivation of the pro-mitogenic ERK pathway. However, long term cannabis exposure in two patients resulted in reversal of this effect. While these data provide a powerful mechanistic rationale for the clinical use of medical marijuana in inflammatory and oncological disease, caution may be advised with sustained use of such preparations. PMID:28174520

  11. Human T cell priming assay: depletion of peripheral blood lymphocytes in CD25(+) cells improves the in vitro detection of weak allergen-specific T cells.

    Science.gov (United States)

    Vocanson, Marc; Achachi, Amine; Mutez, Virginie; Cluzel-Tailhardat, Magalie; Varlet, Béatrice Le; Rozières, Aurore; Fournier, Philippe; Nicolas, Jean-François

    2014-01-01

    To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25(+) Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1a(neg) DCs, CD25(+) T cells, and CD56(+) regulatory cells.CD4(+)CD25(+)FoxP3(+) Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25(+) cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56(+) cells exerted an additional control of T cell priming since co-depletion of both CD56(+) and CD25(+) cells improved the magnitude of chemical-specific T cell activation. Finally, CD1a(low) MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1a(high) MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25(+) and CD56(+) cells, use of CD1a(high) MDDC, and nanoparticle encapsulation of allergens, it was

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  13. Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

    Directory of Open Access Journals (Sweden)

    Mabel B. Esteves

    2005-06-01

    Full Text Available Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100nM ouabain to cultures of peripheral blood lymphocytes activated with 5µg/ml phytohemagglutinin (PHA did not modify the increased expression of the Fas receptor or its ligand FasL induced by the mitogen. However, treatment with ouabain potentiated apoptosis induced by an anti-Fas agonist antibody. A synergy between ouabain and PHA was also observed with regard to plasma membrane depolarization. PHA per se did not induce dissipation of mitochondrial membrane potential but when cells were also exposed to ouabain a marked depolarization could be observed, and this was a late event. It is possible that the inhibitory effect of ouabain on activated peripheral blood lymphocytes involves the potentiation of some of the steps of the apoptotic process and reflects an exacerbation of the mechanism of activation-induced cell death.Quando linfócitos são ativados por lectinas mitogênicas apresentam mudanças do potencial de membrana, elevação das concentrações citoplasmáticas de cálcio, proliferação e/ou morte celular induzida por ativação (AICD. Concentrações baixas de ouabaína (um inibidor da Na,K-ATPase suprimem a proliferação induzida por mitógenos e aumentam a morte celular. Para entender os mecanismos envolvidos, uma série de parâmetros foram avaliados usando sondas fluorescentes e citometria de fluxo. A adição de 100nM de ouabaína para culturas de linfócitos de sangue periférico ativadas por fitohemaglutinina (PHA não modificou o aumento de expressão do receptor Fas ou de

  14. 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.

  15. Cordyceps sinensis: Genotoxic Potential in Human Peripheral Blood Cells and Antigenotoxic Properties Against Hydrogen Peroxide by Comet Assay.

    Science.gov (United States)

    Vasiljevic, Jovana D; Zivkovic, Lada P; Cabarkapa, Andrea M; Bajic, Vladan P; Djelic, Ninoslav J; Spremo-Potparevic, Biljana M

    2016-06-01

    Context • Cordyceps sinensis (C sinensis) is a well-known, traditional, Chinese medicinal mushroom, valued for its beneficial properties for human health. C sinensis has been reported to have immunomodulatory, anticancer, antiaging, antioxidant and anti-inflammatory activity. Despite potential medicinal benefits, no previously published reports are available about the genotoxicity or antigenotoxicity of C sinensis, as detected by comet assay. Objective • The objective of the study was to evaluate both the genotoxic and antigenotoxic potential of an extract of C sinensis (CS extract) in human peripheral blood cells. Design • The research team designed a pilot study. Setting •The study was conducted at the Center for Biological Research, University of Belgrade, in Belgrade, Serbia. Participants • Participants were 6 healthy individuals (2 males and 4 females), between the ages of 20 and 45 y, recruited on a voluntary basis, who provided heparinized, peripheral blood samples. Intervention • Four concentrations of the CS extract-125 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL-were used in the treatment of tested blood cells from the blood samples. Three independent procedures were performed: (1) a genotoxicity assessment, (2) an antigenotoxicity assessment for pretreatment of human cells with the CS extract prior to their exposure to hydrogen peroxide (H2O2) (ie, an evaluation of the benefits of the CS extract as a preventive agent); and (3) posttreatment of human cells with the CS extract after their exposure to H2O2 (ie, an evaluation of the benefits of the CS extract as an interventional agent). Outcome Measures • Cells were graded by eye inspection into 5 classes, depending on the extent of DNA damage, representing: (1) class A-undamaged cells with no tail (95%).Results • The CS extract proved to be nongenotoxic because no induced DNA damage was detected at all tested concentrations. For the antigenotoxicity assessment of the pretreatment with

  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. 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.

  19. 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.

  20. Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood

    DEFF Research Database (Denmark)

    Rådinger, Madeleine; Jensen, Bettina M; Kuehn, Hye Sun;

    2010-01-01

    Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been...... conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell-driven disease, human mast cell models may have greater relevance. Recently, a number of systems have been developed...... to allow investigators to readily obtain sufficient quantities of human mast cells to conduct these studies. These mast cells release the appropriate suite of inflammatory mediators in response to known mast cell activators including antigen. These systems have also been employed to examine the signaling...

  1. Expansion of Natural Killer Cells in Peripheral Blood in a Japanese Elderly with Human T-Cell Lymphotropic Virus Type 1-Related Skin Lesions

    Directory of Open Access Journals (Sweden)

    Shinsaku Imashuku

    2014-01-01

    Full Text Available Natural killer (NK cells were proposed to play an important role in the pathogenesis of human T-cell lymphotropic virus type 1- (HTLV-1- associated neurologic disease. Our patient was a 77-year-old Japanese man, who had been treated for infective dermatitis associated with HTLV-1 for nearly 10 years. When referred to us, he had facial eczema/edema as well as extensive dermatitis at the neck/upper chest and nuchal area/upper back regions. Dermal lesions had CD3+CD4+ cells, but no NK cells. Flow cytometry of his peripheral blood showed a phenotype of CD2+ (97%, CD3+ (17%, CD4+ (12%, CD7+ (94%, CD8+ (6%, CD11c+ (70%, CD16+ (82%, CD19+ (0%, CD20+ (0%, CD56+ (67%, HLA-DR+ (68%, and NKp46+ (36%. Absolute numbers of CD56+NK cells in the peripheral blood were in a range of 986/μL–1,270/μL. The expanded NK cells in the peripheral blood are considered to be reactive, to maintain the confinement of the HTLV-1-positive CD4+ cells in the skin, and to prevent the progression of the disease.

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

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    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.

  3. 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.

  4. Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy.

    Science.gov (United States)

    Hu, Mingqian; Wang, Jiongkun; Cai, Jiye; Wu, Yangzhe; Wang, Xiaoping

    2008-09-12

    To date, nanoscale imaging of the morphological changes and adhesion force of CD4(+) T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4(+) T cells. The AFM images revealed that the volume of activated CD4(+) T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4(+) T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.

  5. Evidence from human and animal studies: Pathological roles of CD8+ T cells in autoimmune peripheral neuropathies

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    Mu eYang

    2015-10-01

    Full Text Available Autoimmune peripheral neuropathies such as Guillain Barre Syndrome (GBS and chronic inflammatory demyelinating polyneuropathy (CIDP affect millions of people worldwide. Despite significant advances in understanding the pathology, the molecular and cellular mechanisms of immune-mediated neuropathies remain elusive. T lymphocytes definitely play an important role in disease pathogenesis and CD4+ T cells have been the main area of research for decades. This is partly due to the fact that the most frequent animal model to study autoimmune peripheral neuropathy is experimental allergic neuritis (EAN. As it is induced commonly by immunization with peripheral nerve proteins, EAN is driven mainly by CD4+ T cells. However, similarly to what has been reported for patients suffering from multiple sclerosis, a significant body of evidence indicate that CD8+ T cells may play a pathogenic role in GBS and CIDP disease development and/or progression. Here, we summarize clinical studies pertaining to the presence and potential role of CD8+ T cells in autoimmune peripheral neuropathy. We also discuss the findings from our most recent studies using a transgenic mouse line (L31 mice in which the T cell co-stimulator molecule B7.2 (CD86 is constitutively expressed in antigen presenting cells of the nervous tissues. L31 mice spontaneously develop peripheral neuropathy, and CD8+ T cells are found accumulating in peripheral nerves of symptomatic animals. Interestingly, depletion of CD4+ T cells accelerates disease onset and increases disease prevalence. Finally, we point out some unanswered questions for future research to dissect the critical roles of CD8+ T cells in autoimmune peripheral neuropathies.

  6. Human Flt3L generates dendritic cells from canine peripheral blood precursors: implications for a dog glioma clinical trial.

    Directory of Open Access Journals (Sweden)

    Weidong Xiong

    Full Text Available BACKGROUND: Glioblastoma multiforme (GBM is the most common primary brain tumor in adults and carries a dismal prognosis. We have developed a conditional cytotoxic/immunotherapeutic approach using adenoviral vectors (Ads encoding the immunostimulatory cytokine, human soluble fms-like tyrosine kinase 3 ligand (hsFlt3L and the conditional cytotoxic molecule, i.e., Herpes Simplex Type 1- thymide kinase (TK. This therapy triggers an anti-tumor immune response that leads to tumor regression and anti-tumor immunological memory in intracranial rodent cancer models. We aim to test the efficacy of this immunotherapy in dogs bearing spontaneous GBM. In view of the controversy regarding the effect of human cytokines on dog immune cells, and considering that the efficacy of this treatment depends on hsFlt3L-stimulated dendritic cells (DCs, in the present work we tested the ability of Ad-encoded hsFlt3L to generate DCs from dog peripheral blood and compared its effects with canine IL-4 and GM-CSF. METHODOLOGY/PRINCIPAL FINDINGS: Our results demonstrate that hsFlT3L expressed form an Ad vector, generated DCs from peripheral blood cultures with very similar morphological and phenotypic characteristics to canine IL-4 and GM-CSF-cultured DCs. These include phagocytic activity and expression of CD11c, MHCII, CD80 and CD14. Maturation of DCs cultured under both conditions resulted in increased secretion of IL-6, TNF-alpha and IFN-gamma. Importantly, hsFlt3L-derived antigen presenting cells showed allostimulatory potential highlighting their ability to present antigen to T cells and elicit their proliferation. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that hsFlt3L induces the proliferation of canine DCs and support its use in upcoming clinical trials for canine GBM. Our data further support the translation of hsFlt3L to be used for dendritic cells' vaccination and gene therapeutic approaches from rodent models to canine patients and its future

  7. In Vitro Infection of Human Peripheral Blood Mononuclear Cells by GB Virus C/Hepatitis G Virus

    Science.gov (United States)

    Fogeda, Marta; Navas, Sonia; Martín, Julio; Casqueiro, Mercedes; Rodríguez, Elena; Arocena, Carlos; Carreño, Vicente

    1999-01-01

    GB virus C (GBV-C), also known as hepatitis G virus, is a recently discovered flavivirus-like RNA agent with unclear pathogenic implications. To investigate whether human peripheral blood mononuclear cells (PBMC) are susceptible to in vitro GBV-C infection, we have incubated PBMC from four healthy blood donors with a human GBV-C RNA-positive serum. By means of (i) strand-specific reverse transcription-PCR, cloning, and sequencing; (ii) sucrose ultracentrifugation and RNase sensitivity assays; (iii) fluorescent in situ hybridization; and (iv) Western blot analysis, it has been demonstrated that GBV-C is able to infect in vitro cells and replicate for as long as 30 days under the conditions developed in our cell culture system. The concentration of GBV-C RNA increased during the second and third weeks of culture. The titers of the genomic strand were 10 times higher than the titers of the antigenomic strand. In addition, the same predominant GBV-C sequence was found in all PBMC cultures and in the in vivo-GBV-C-infected PBMC isolated from the donor of the inoculum. GBV-C-specific fluorescent in situ hybridization signals were confined to the cytoplasm of cells at different times during the culture period. Finally, evidence obtained by sucrose ultracentrifugation, RNase sensitivity assays, and Western blot analysis of the culture supernatants suggests that viral particles are released from in vitro-GBV-C-infected PBMC. In conclusion, our study has demonstrated, for the first time, GBV-C replication in human lymphoid cells under experimental in vitro infection conditions. PMID:10196301

  8. In vitro detection of contact allergens: development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells.

    Science.gov (United States)

    Reuter, Hendrik; Spieker, Jochem; Gerlach, Silke; Engels, Ursula; Pape, Wolfgang; Kolbe, Ludger; Schmucker, Robert; Wenck, Horst; Diembeck, Walter; Wittern, Klaus-Peter; Reisinger, Kerstin; Schepky, Andreas G

    2011-02-01

    Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

  9. Effect of advanced glycosylation end products on activity of protein kinase C in human peripheral blood mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objectives TO investigate the effect of advanced glycosylation end products (AGEs) on the activity of protein kinese C (PKC) in human peripheral bloodmononuclear Cells (PBMC) and to observe whether aminoguanidine (AG) can influence the effect of AGEs. Methods After PBMC were isoiated from human peripheral blood and incubated with different concentrations of AGEs-BSA for various periods, total PKC activity in PBMC was determined by measuring the incorporation of 32P from [γ-32P] ATP=into a special substrate using Prornega PKC assay kit. Results AGEs-BSA increased the total PKC activity in PBMC from 83.43±6.57 pmol/min/mg protein to 116.8±13.82 pmol/min/mg protein with a peak at 15 min.AGEs-BSA also increased the total PKC activity in a concentration-dependent manner from 83.1±6.4 pmol/min/mg protein(control) to 119.1±13.3 pmol/min/mg protein (control vs AGEs-BSA 400 mg/L, P<0.01). Furthermore, AGEs-BSA induced an elevation of PKC activity in a glycosylating time-related manner,from 80.9±8.2 (control) to 118.3±11.5 pmol/min/mg protein (glycasytation for 12 wk, P<0.01). The total PKC activity stimulated by AGEs-BSA pretreated with AG (100, 200 mg/L) was markedly lower than that of AGEs-BSA group not pretreated with AG ( P<0.05, P<0.01). Conclusions AGEs-BSA increased the total PKC activity in PBMC in a concentration and incubation time dependent manner. The ability of AGEs-B.SA to stimulate PKC activity was markedly decreased by pretreatment of AGEs-BSA with AG.

  10. Comparative Study of Regulatory T Cell Function of Human CD25+CD4+ T Cells from Thymocytes, Cord Blood, and Adult Peripheral Blood

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    Chikao Morimoto

    2008-09-01

    Full Text Available CD25+CD4+ regulatory T cells suppress T cell activation and regulate multiple immune reactions in in vitro and in vivo studies. To define the regulatory function of human CD25+CD4+ T cells at various stages of maturity, we investigated in detail the functional differences of CD25+CD4+ T cells from thymocytes, cord blood (CB, and adult peripheral blood (APB. CB CD25+CD4+ T cells displayed low-FOXP3 protein expression level and had no suppressive activity. In contrast, CD25+CD4+ T cells from thymocytes or APB expressed high expression level of FOXP3 protein associated with significant suppressive activity. Although CB CD25+CD4+ T cells exhibited no suppressive activity, striking suppressive activity was observed following expansion in culture associated with increased FOXP3 expression and a shift from the CD45RA+ to the CD45RA− phenotype. These functional differences in CD25+CD4+ T cells from Thy, CB, and APB hence suggest a pathway of maturation for Treg in the peripheral immune system.

  11. Down-regulation of human leukocyte antigens class I on peripheral T lymphocytes and NK cells from subjects in region of high-incidence gastrointestinal tumor

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhi-mian; LI Ying-jie; GUAN Xiao; YANG Xiao-yun; GAO Xi-mei; YANG Xiao-jing; WANG Li-shui; ZOU Xiong

    2011-01-01

    Background Many types of human tumors can suppress the immune system to enhance their survival. Loss or down-regulation of human leukocyte antigens (HLA) class I on tumors is considered to be a major mechanism of tumor immune escape. Our previous studies found that HLA class I on peripheral-blood mononuclear cells was significantly lower in gastric cancer patients. The present study made an analysis of HLA class I expression on peripheral-blood T lymphocytes and NK cells from subjects of Lijiadian village, a village with high-incidence gastrointestinal tumor. Methods A total of 181 villagers from Lijiadian village and 153 normal controls from the Department of Health Examination Center were enrolled in this study. Using a multi-tumor markers detection system, these villagers were divided into two groups: high-risk group (tumor markers positive group) and low-risk group (tumor markers negative group). The percentage of T lymphocytes and NK cells and levels of HLA class I on their surface were determined in these subjects by flow cytometry.Results Percentages of T lymphocytes and NK cells in peripheral-blood mononuclear cells did not vary with age. The expression level of HLA class I on peripheral T lymphocytes and NK cells was not affected by age or gender, but was significantly down-regulated in Lijiadian villagers (P<0.05), especially on the surface of NK cells (P<0.01). Compared with the low-risk group, there was a significant reduction of HLA class I on peripheral T lymphocytes (P <0.05) and NK cells (P <0.05) in the high-risk group.Conclusions HLA class I on peripheral T lymphocytes and NK cells may be involved in tumorigenesis and development of gastrointestinal tumor, and understanding their changes in expression may provide new insights into the mechanism of tumor immunity.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  14. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood.

    Science.gov (United States)

    Olivier, Emmanuel; Qiu, Caihong; Bouhassira, Eric E

    2012-08-01

    The current supply of red blood cells expressing rare blood groups is not sufficient to cover all the existing transfusion needs for chronically transfused patients, such as sickle cell disease homozygous carriers, because of alloimmunization. In vitro production of cultured red blood cells is slowly emerging as a possible complement to the existing collection-based red blood cell procurement system. The yield of cultured red blood cells can theoretically be maximized by amplifying the stem, progenitor, or precursor compartment. Here, we combined methods designed to expand these three compartments to optimize the yield of cultured red blood cells and found that exposing CD34(+) cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced the highest yield of erythroid cells. This novel serum-free red blood cell production protocol was efficient on CD34(+) cells derived from human embryonic stem cells, 6-8-week yolk sacs, 16-18-week fetal livers, cord blood, and peripheral blood. The yields of cells obtained with these new protocols were larger by an order of magnitude than the yields observed previously. Globin expression analysis by high-performance liquid chromatography revealed that these expansion protocols generally yielded red blood cells that expressed a globin profile similar to that expected for the developmental age of the CD34(+) cells.

  15. Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults.

    Science.gov (United States)

    Than, S; Oyaizu, N; Tetali, S; Romano, J; Kaplan, M; Pahwa, S

    1997-08-01

    This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.

  16. Interleukin-6 infusion during human endotoxaemia inhibits in vitro release of the urokinase receptor from peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Ostrowski, S R; Plomgaard, P; Fischer, C P

    2005-01-01

    Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro...... in humans. Healthy subjects received intravenous endotoxin injection [high-dose, 2 ng/kg (n=8) and low-dose, 0.06 ng/kg (n=7)], coadministration of 0.06 ng/kg endotoxin and 3 h recombinant human (rh)IL-6 infusion (n=7) or 3 h infusion of rhIL-6 (n=6), rhTNF-alpha (n=6) or NaCl (n=5). Soluble uPAR (su......PAR) was measured by enzyme-linked immunosorbent assay in plasma and supernatants from unstimulated and phytohaemagglutinin and lipopolysaccharide-stimulated peripheral blood mononuclear cell (PBMC) cultures incubated for 24 h. The spontaneous and stimulated uPAR-release from PBMC cultures was enhanced 5 h after...

  17. 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.

  18. Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke.

    Directory of Open Access Journals (Sweden)

    M Z Ratajczak

    2006-06-01

    Full Text Available The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day and 144 hrs (6th day since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM and erythroid (BFU-E-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.

  19. 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.

  20. Both pituitary and placental growth hormone transcripts are expressed in human peripheral blood mononuclear cells (PBMC)

    NARCIS (Netherlands)

    Melen, L; Hennen, G; Dullaart, RPF; Igout, A

    1997-01-01

    The hGH-V gene codes for a variant of human pituitary growth hormone (hGH-N) named placental growth hormone (hPGH). hPGH shares 93% amino acid identity with hGH-N. Until now the hGH-V gene was considered to be exclusively expressed in human placenta, where it replaces maternal circulating hGH-N at t

  1. Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics.

    Science.gov (United States)

    Brosseron, Frederic; May, Caroline; Schoenebeck, Bodo; Tippler, Bettina; Woitalla, Dirk; Kauth, Marion; Brockmann, Kathrin; Meyer, Helmut E; Berg, Daniela; Bufe, Albrecht; Marcus, Katrin

    2012-10-01

    Density gradient centrifugation and magnetic- or fluorescence-activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample. The workflow targeted erythrocytes, monocytes, and T lymphocytes. Pancoll® density gradient centrifugation was used together with subsequent MACS™ isolation. Purity of monocytes and T lymphocytes was controlled by fluorescence-activated cell sorting analysis, and cells were used for carrier-ampholine-based 2D-PAGE to confirm compatibility of the procedure to standard proteomic applications. Gradient centrifugation resulted in an average of 125 μL of packed erythrocytes per milliliter blood. MACS™ sorting reached purities of 90 ± 2% (monocytes) and 93 ± 2% (T lymphocytes), with an average yield of 12 × 10(4) monocytes or T lymphocytes. 2D-PAGE of isolated cells showed well-separated spot patterns. A combined isolation holds substantial advantages especially in clinical studies, as it allows for the comparison of findings not only between individuals, but also between different cell types derived from one donor. Our approach ensured high reproducibility, yields, and purities of cells as required for reliable proteome analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 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

    18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency...

  3. Generation of induced pluripotent stem cells from a small amount of human peripheral blood using a combination of activated T cells and Sendai virus.

    Science.gov (United States)

    Seki, Tomohisa; Yuasa, Shinsuke; Fukuda, Keiichi

    2012-03-15

    Induced pluripotent stem cells (iPSCs) have become important cell sources for genetic disease models, and they have the potential to be cell sources for future clinical therapies. However, invasive tissue sampling reduces the number of candidates who consent to donate cells for iPSC generation. In addition, integrated transgenes can potentially insert at inappropriate points in the genome, and in turn have a direct oncogenic effect. Technical modifications using a combination of activated T cells and a temperature-sensitive mutant of Sendai virus (SeV) can avoid invasive tissue sampling and residual transgene issues in generating iPSCs. Such advances may increase the number of consenting patients for cell donations. Here we present a detailed protocol for the generation of iPSCs from a small amount of human peripheral blood using a combination of activated T cells and mutant SeV encoding human OCT3/4, SOX2, KLF4 and c-MYC; T cell-derived iPSCs can be generated within 1 month of blood sampling.

  4. 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.

  5. Procalcitonin neutralizes bacterial LPS and reduces LPS-induced cytokine release in human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Matera Giovanni

    2012-05-01

    Full Text Available Abstract Background Procalcitonin (PCT is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS, reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT. Results PCT significantly decreased (p Salmonella typhimurium (rough chemotype and Escherichia coli (smooth chemotype. Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC. When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10 and tumor necrosis factor alpha (TNFα by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1 exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours. Conclusions This study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators.

  6. Mechanism and role of MCP-1 upregulation upon chikungunya virus infection in human peripheral blood mononuclear cells

    Science.gov (United States)

    Ruiz Silva, Mariana; van der Ende-Metselaar, Heidi; Mulder, H. Lie; Smit, Jolanda M.; Rodenhuis-Zybert, Izabela A.

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1/CCL2)-mediated migration of monocytes is essential for immunological surveillance of tissues. During chikungunya virus (CHIKV) infection however, excessive production of MCP-1 has been linked to disease pathogenesis. High MCP-1 serum levels are detected during the viremic phase of CHIKV infection and correlate with the virus titre. In vitro CHIKV infection was also shown to stimulate MCP-1 production in whole blood; yet the role and the mechanism of MCP-1 production upon infection of human peripheral blood mononuclear cells remain unknown. Here we found that active CHIKV infection stimulated production of MCP-1 in monocytes. Importantly however, we found that communication with other leukocytes is crucial to yield MCP-1 by monocytes upon CHIKV infection. Indeed, blocking interferon-α/β receptor or the JAK1/JAK2 signalling downstream of the receptor abolished CHIKV-mediated MCP-1 production. Additionally, we show that despite the apparent correlation between IFN type I, CHIKV replication and MCP-1, modulating the levels of the chemokine did not influence CHIKV infection. In summary, our data disclose the complexity of MCP-1 regulation upon CHIKV infection and point to a crucial role of IFNβ in the chemokine secretion. We propose that balance between these soluble factors is imperative for an appropriate host response to CHIKV infection. PMID:27558873

  7. 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.

  8. 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

    18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency...... was observed for the level of hOGG1-sensitive sites, whereas there was no association with the level of strand breaks. The effect of age on oxidatively damaged DNA in women disappeared in multivariate models, which showed robust positive associations between DNA damage and plasma levels of triglycerides...

  9. Bovine colostrum modulates immune activation cascades in human peripheral blood mononuclear cells in vitro

    DEFF Research Database (Denmark)

    Jenny, Marcel; Pedersen, Ninfa R; Hidayat, Budi J

    2010-01-01

    factors and has a long history of use in traditional medicine. In an approach to evaluate the effects of bovine colostrum (BC) on the T-cell/macrophage interplay, we investigated and compared the capacity of BC containing low and high amounts of lactose and lactoferrin to modulate tryptophan degradation...... of lactose present in BC seems to diminish the activity of BC in our test system, since BC with higher amounts of lactose attenuated the stimulatory as well as the suppressive activity of BC....

  10. 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.

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

    DEFF Research Database (Denmark)

    Kampen, G T; Poulsen, L K; Reimert, C M

    1997-01-01

    Histamine releasing factors, i.e. cytokines capable of inducing histamine release from basophils or mast cells, have been suggested to be involved in the pathogenesis of, for example, allergic late-phase reactions. Here we describe a controlled method for production and determination of histamine......). The preparations of HRA induced dose- and Ca2+-dependent histamine release from leukocytes. Supernatants of parallel cultures of unstimulated MNC did not induce histamine release. The HRA was neither due to exogenous histamine releasing compounds (e.g. Con A) nor to residual histamine in the preparations of HRA....... The kinetics of HRA induced histamine release (half-maximal release after > 40 min) were slower and more protracted than those of anti-IgE induced histamine release. However, based on a comparison between HRA induced histamine release from leukocytes and purified (97%) basophils, this did not appear to be due...

  12. 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.

  13. Quercetin protects human peripheral blood mononuclear cells from OTA-induced oxidative stress, genotoxicity, and inflammation.

    Science.gov (United States)

    Periasamy, Ramyaa; Kalal, Iravathy Goud; Krishnaswamy, Rajashree; Viswanadha, VijayaPadma

    2016-07-01

    Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins world wide, and is detrimental to human and animal health. This study evaluated the protective effect of quercetin against OTA-induced cytotoxicity, genotoxicity, and inflammatory response in lymphocytes. Cytotoxicity determined by MTT assay revealed IC20 value of OTA to be 20 µM, which was restored to near control values by pretreatment with quercetin. Oxidative stress parameters such as antioxidant enzymes, LPO and PCC levels indicated that quercetin exerted a protective effect on OTA-induced oxidative stress. Quercetin exerted an antigenotoxic effect on OTA-induced genotoxicity, by significantly reducing the number of structural aberrations in chromosomes and comet parameters like, % olive tail moment from 2.76 ± 0.02 to 0.56 ± 0.02 and % tail DNA from 56.23 ± 2.56 to 12.36 ± 0.56 as determined by comet assay. OTA-induced NO, TNF-α, IL-6, and IL-8 were significantly reduced in the quercetin pretreated samples indicating its anti-inflammatory role. Our results demonstrate for the first time that quercetin exerts a cytoprotective effect against OTA-induced oxidative stress, genotoxicity, and inflammation in lymphocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 855-865, 2016.

  14. Peripheral T-cell lymphoma.

    Science.gov (United States)

    Rosenberg, Benjamin

    2005-12-30

    A 32-year-old man presented with a 5-year history of cutaneous nodules on his head and a diffuse, lichenified eruption. Histopathologic examination showed an atypical lymphocytic infiltrate. Immunophenotyping studies determined that the lymphocyte population to be CD4-positive, with partial loss of CD3 and CD7, and immunogenotyping studies showed a clonal rearrangement of the T-cell receptor. A positron-emission tomography scan showed increased uptake in cervical, axillary, and inguinal lymph nodes. A diagnosis of peripheral T-cell lymphoma was made, and the patient is undergoing chemotherapy.

  15. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector

    Science.gov (United States)

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  16. 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

  17. 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.

  18. 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

  19. Transplantation of Human Dental Pulp-Derived Stem Cells or Differentiated Neuronal Cells from Human Dental Pulp-Derived Stem Cells Identically Enhances Regeneration of the Injured Peripheral Nerve.

    Science.gov (United States)

    Ullah, Imran; Park, Ju-Mi; Kang, Young-Hoon; Byun, June-Ho; Kim, Dae-Geon; Kim, Joo-Heon; Kang, Dong-Ho; Rho, Gyu-Jin; Park, Bong-Wook

    2017-09-01

    Human dental mesenchymal stem cells isolated from the dental follicle, pulp, and root apical papilla of extracted wisdom teeth have been known to exhibit successful and potent neurogenic differentiation capacity. In particular, human dental pulp-derived stem cells (hDPSCs) stand out as the most prominent source for in vitro neuronal differentiation. In this study, to evaluate the in vivo peripheral nerve regeneration potential of hDPSCs and differentiated neuronal cells from DPSCs (DF-DPSCs), a total of 1 × 10(6) hDPSCs or DF-hDPSCs labeled with PKH26 tracking dye and supplemented with fibrin glue scaffold and collagen tubulization were transplanted into the sciatic nerve resection (5-mm gap) of rat models. At 12 weeks after cell transplantation, both hDPSC and DF-hDPSC groups showed notably increased behavioral activities and higher muscle contraction forces compared with those in the non-cell transplanted control group. In immunohistochemical analysis of regenerated nerve specimens, specific markers for angiogenesis, axonal fiber, and myelin sheath increased in both the cell transplantation groups. Pretransplanted labeled PKH26 were also distinctly detected in the regenerated nerve tissues, indicating that transplanted cells were well-preserved and differentiated into nerve cells. Furthermore, no difference was observed in the nerve regeneration potential between the hDPSC and DF-hDPSC transplanted groups. These results demonstrate that dental pulp tissue is an excellent stem cell source for nerve regeneration, and in vivo transplantation of the undifferentiated hDPSCs could exhibit sufficient and excellent peripheral nerve regeneration potential.

  20. Rapid detection of dendritic cell and monocyte disorders using CD4 as a lineage marker of the human peripheral blood antigen presenting cell compartment

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    Laura eJardine

    2013-12-01

    Full Text Available Dendritic cells (DCs and monocytes are critical regulators and effectors of innate and adaptive immune responses. Monocyte expansion has been described in many pathological states while monocyte and DC deficiency syndromes are relatively recent additions to the catalogue of human primary immunodeficiency disorders. Clinically applicable screening tests to diagnose and monitor these conditions are lacking. Conventional strategies for identifying human DCs and monocytes have been based on the use of a lineage gate to exclude lymphocytes, thus preventing simultaneous detection of DCs, monocytes and lymphocyte subsets. Here we demonstrate that CD4 is a reliable lineage marker for the human peripheral blood antigen presenting cell compartment that can be used to identify DCs and monocytes in parallel with lymphocytes. Based on this principle, simple modification of a standard lymphocyte phenotyping assay permits simultaneous enumeration of four lymphocyte and five DC/monocyte populations from a single sample. This approach is applicable to clinical samples and facilitates the diagnosis of DC and monocyte disorders in a wide range of clinical settings, including genetic deficiency, neoplasia and inflammation.

  1. Outcomes of peripheral blood stem cell transplantation in patients from human leukocyte antigen matched or mismatched unrelated donors

    Institute of Scientific and Technical Information of China (English)

    Cao Tingting; Li Yanfen; Wang Quanshun; Li Honghua; Bo Jian; Zhao Yu; Jing Yu

    2014-01-01

    Background Allogeneic peripheral blood stem cell transplantation from unrelated donors (UR-PBSCT) is an alternative treatment for many hematologic diseases due to lack of human leukocyte antigen (HLA)-identical sibling donors.This study aimed to evaluate the impact of the degree of the HLA match on the clinical efficacy of UR-PBSCT.Methods Patients who underwent UR-PBSCT from September 2003 to September 2012 were retrospectively investigated.They were divided into three groups according to high-resolution molecular typing.SPSS version 17.0 was used to analysis and compare the statistics of engraftment,incidence of GVHD,other complications and survival among the groups.Results One hundred and eleven patients received UR-PBSCT,60 of them with an HLA matched donor (10/10),36 of them with a one locus mismatched donor (9/10),and 15 of them with a two loci mismatched donor (8/10).Similar basic characteristics were found in the three groups.No differences were found in engraftment of myeloid cells or platelets in the three groups (P>0.05).Two-year cumulative incidence of relapse,overall survival (OS) and disease-free survival (DFS) among those three groups were similar (P>0.05).The cumulative incidence of 100-day Ⅲ-Ⅳ aGVHD in the HLA matched group and the one HLA locus mismatched group were significantly lower than that in the two HLA loci mismatched group (3.3%,8.6%,and 26.7%,P=0.009).The occurrence rate of new pulmonary infections in the HLA matched group was lower than in the two HLA mismatched groups (26.67%,52.78%,and 41.18%,P=0.035).The cumulative incidence of 100-day and 2-year transplantation related mortality (TRM) in two HLA loci mismatched group was higher than in the HLA matched group and in the one HLA locus mismatched group,(8.4%,11.8% and 33.3%,P=0.016) and (12.3%,18.7% and 47.5%,P=0.002).Conclusions HLA mismatch will not significantly impact the engraftment or 2-year survival after UR-PBSCT,but two mismatched HLA loci may

  2. 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.

  3. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.

    Science.gov (United States)

    Okita, Keisuke; Yamakawa, Tatsuya; Matsumura, Yasuko; Sato, Yoshiko; Amano, Naoki; Watanabe, Akira; Goshima, Naoki; Yamanaka, Shinya

    2013-03-01

    The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αβT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.

  4. A novel,rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+monocytes within 48 hours of in vitro culture

    Institute of Scientific and Technical Information of China (English)

    Xin Guan; Ji-Run Peng; Lan Yuan; Hui Wang; Yu-Hua Wei; Xi-Sheng Leng

    2004-01-01

    AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis factorα (TNFα), interleukin1β (IL-1β), interleukin-6 (IL-6)and prostaglandin E2 (PGE2).One study showed that mature monocyte-derived dendritic cells could be obtained within 48 h ofin vitro culture with the same protocol as standard 7-d culture and referred to as FastDCs. Here we aimed to fuse human hepatocellular carcinoma cell line HCCLM3 cells with mature monocytederived dendritic cells within 48 h ofin vitro culture (FastDC).METHODS: HCCLMl3 cells were cultured in RPMI 1640 with 150 mL/L fetal calf serum (FCS). CD14+monocytes from healthy human peripheral blood were purified with MACS CD14 isolation kit and cultured in six-well plates in fresh complete DC medium containing RPMI-1640, 20 mL/Lheat inactivated human AB serum, 2 mmol/L L-glutamine,100 μg/mL gentamicin, 1000 U/mL GM-CSF and 500 U/mL IL-4 for 24 h, then proinflammatory mediators such as TNFα(1000 U/mL), IL-1β (10 ng/mL), IL-6 (10 ng/mL) and PGE2(1μg/mL) were supplemented for another 24 h, and thus mature FastDCs were generated. HCCLM3 cells and FastDCs were labeled with red fluorescent dye PKH26-GL and green fluorescent dye PKH67-GL respectively. After the red fluorescent-stained HCCLM3 cells were irradiated with 50 Gy, FastDCs and irradiated HCCLM3 cells were fused in 500 mL/L polyethylene glycol(PEG)+100 mL/L dimethyl sulfoxide (DMSO) to generate novel dendritomas. The FastDCs and novel dendritomas were immunostained with antiCD80, anti-CD86, anti-CD83, anti-HLA-DR mAbs and analyzed by fluorescence

  5. Human skin: an independent peripheral endocrine organ.

    Science.gov (United States)

    Zouboulis, C C

    2000-01-01

    The historical picture of the endocrine system as a set of discrete hormone-producing organs has been substituted by organs regarded as organized communities in which the cells emit, receive and coordinate molecular signals from established endocrine organs, other distant sources, their neighbors, and themselves. In this wide sense, the human skin and its tissues are targets as well as producers of hormones. Although the role of hormones in the development of human skin and its capacity to produce and release hormones are well established, little attention has been drawn to the ability of human skin to fulfil the requirements of a classic endocrine organ. Indeed, human skin cells produce insulin-like growth factors and -binding proteins, propiomelanocortin derivatives, catecholamines, steroid hormones and vitamin D from cholesterol, retinoids from diet carotenoids, and eicosanoids from fatty acids. Hormones exert their biological effects on the skin through interaction with high-affinity receptors, such as receptors for peptide hormones, neurotransmitters, steroid hormones and thyroid hormones. In addition, the human skin is able to metabolize hormones and to activate and inactivate them. These steps are overtaken in most cases by different skin cell populations in a coordinated way indicating the endocrine autonomy of the skin. Characteristic examples are the metabolic pathways of the corticotropin-releasing hormone/propiomelanocortin axis, steroidogenesis, vitamin D, and retinoids. Hormones exhibit a wide range of biological activities on the skin, with major effects caused by growth hormone/insulin-like growth factor-1, neuropeptides, sex steroids, glucocorticoids, retinoids, vitamin D, peroxisome proliferator-activated receptor ligands, and eicosanoids. At last, human skin produces hormones which are released in the circulation and are important for functions of the entire organism, such as sex hormones, especially in aged individuals, and insulin-like growth

  6. An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

    NARCIS (Netherlands)

    Vissers, M.; Habets, M.N.; Ahout, I.M.L.; Jans, J.; Jonge, M.I. de; Diavatopoulos, D.A.; Ferwerda, G.

    2013-01-01

    Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a

  7. An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

    NARCIS (Netherlands)

    Vissers, M.; Habets, M.N.; Ahout, I.M.L.; Jans, J.; Jonge, M.I. de; Diavatopoulos, D.A.; Ferwerda, G.

    2013-01-01

    Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology. Here, we describe a pro

  8. Biphasic influence of PGE2 on the resorption activity of osteoclast-like cells derived from human peripheral blood monocytes and mouse RAW264.7 cells.

    Science.gov (United States)

    Lutter, Anne-Helen; Hempel, Ute; Anderer, Ursula; Dieter, Peter

    2016-08-01

    Osteoclasts are large bone-resorbing cells of hematopoietic origin. Their main function is to dissolve the inorganic component hydroxyapatite and to degrade the organic bone matrix. Prostaglandin E2 (PGE2) indirectly affects osteoclasts by stimulating osteoblasts to release factors that influence osteoclast activity. The direct effect of PGE2 on osteoclasts is still controversial. To study the influence of PGE2 on osteoclast activity, human peripheral blood monocytes (hPBMC) and mouse RAW264.7 cells were cultured on osteoblast-derived extracellular matrix. hPBMC and RAW264.7 cells were differentiated by the addition of macrophage colony-stimulation factor and receptor activator of NFκB ligand and treated with PGE2 before and after differentiation induction. The pit area, an indicator of resorption activity, and the activity of tartrate-resistant acid phosphatase were dose-dependently inhibited when PGE2 was present ab initio, whereas the resorption activity remained unchanged when the cells were exposed to PGE2 from day 4 of culture. These results lead to the conclusion that PGE2 treatment inhibits only the differentiation of precursor osteoclasts whereas differentiated osteoclasts are not affected.

  9. 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.

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

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    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

  11. The miRNA and mRNA Signatures of Peripheral Blood Cells in Humans Infected with Trypanosoma brucei gambiense.

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    Smiths Lueong

    Full Text Available Simple, reliable tools for diagnosis of human African Trypanosomiases could ease field surveillance and enhance patient care. In particular, current methods to distinguish patients with (stage II and without (stage I brain involvement require samples of cerebrospinal fluid. We describe here an exploratory study to find out whether miRNAs from peripheral blood leukocytes might be useful in diagnosis of human trypanosomiasis, or for determining the stage of the disease. Using microarrays, we measured miRNAs in samples from Trypanosoma brucei gambiense-infected patients (9 stage I, 10 stage II, 8 seronegative parasite-negative controls and 12 seropositive, but parasite-negative subjects. 8 miRNAs (out of 1205 tested showed significantly lower expression in patients than in seronegative, parasite-negative controls, and 1 showed increased expression. There were no clear differences in miRNAs between patients in different disease stages. The miRNA profiles could not distinguish seropositive, but parasitologically negative samples from controls and results within this group did not correlate with those from the trypanolysis test. Some of the regulated miRNAs, or their predicted mRNA targets, were previously reported changed during other infectious diseases or cancer. We conclude that the changes in miRNA profiles of peripheral blood lymphocytes in human African trypanosomiasis are related to immune activation or inflammation, are probably disease-non-specific, and cannot be used to determine the disease stage. The approach has little promise for diagnostics but might yield information about disease pathology.

  12. Proteomic validation of multifunctional molecules in mesenchymal stem cells derived from human bone marrow, umbilical cord blood and peripheral blood.

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    Jumi Kim

    Full Text Available Mesenchymal stem cells (MSCs are one of the most attractive therapeutic resources in clinical application owing to their multipotent capability, which means that cells can differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle and marrow stroma. Depending on the cellular source, MSCs exhibit different application potentials according to their different in vivo functions, despite similar phenotypic and cytological characteristics. To understand the different molecular conditions that govern the different application or differentiation potential of each MSC according to cellular source, we generated a proteome reference map of MSCs obtained from bone marrow (BM, umbilical cord blood (CB and peripheral blood (PB. We identified approximately 30 differentially regulated (or expressed proteins. Most up-regulated proteins show a cytoskeletal and antioxidant or detoxification role according to their functional involvement. Additionally, these proteins are involved in the increase of cell viability, engraftment and migration in pathological conditions in vivo. In summary, we examined differentially expressed key regulatory factors of MSCs obtained from several cellular sources, demonstrated their differentially expressed proteome profiles and discussed their functional role in specific pathological conditions. With respect to the field of cell therapy, it may be particularly crucial to determine the most suitable cell sources according to target disease.

  13. Morbidly obese human subjects have increased peripheral blood CD4 + T cells with skewing toward a Treg- and Th2-dominated phenotype

    NARCIS (Netherlands)

    K. van der Weerd (Kim); W.A. Dik (Willem); B. Schrijver (Benjamin); D.H. Schweitzer (Dave ); A.W. Langerak (Anton); H.A. Drexhage (Hemmo); R.M. Kiewiet-Kemper (Rosalie); M.O. van Aken (Maarten); A. van Huisstede (Astrid); J.J.M. van Dongen (Jacques); A.J. van der Lelij (Aart Jan); F.J.T. Staal (Frank); P.M. van Hagen (Martin)

    2012-01-01

    textabstractObesity is associated with local T-cell abnormalities in adipose tissue. Systemic obesity-related abnormalities in the peripheral blood T-cell compartment are not well defined. In this study, we investigated the peripheral blood T-cell compartment of morbidly obese and lean subjects. We

  14. The nuclear lamina promotes telomere aggregation and centromere peripheral localization during senescence of human mesenchymal stem cells

    NARCIS (Netherlands)

    Raz, Vered; Vermolen, Bart J.; Garini, Yuval; Onderwater, Jos J.M.; Mommaas-Kienhuis, Mieke A.; Koster, Abraham J.; Young, Ian T.; Tanke, Hans; Dirks, Roeland W.

    2008-01-01

    Ex vivo, human mesenchymal stem cells (hMSCs) undergo spontaneous cellular senescence after a limited number of cell divisions. Intranuclear structures of the nuclear lamina were formed in senescent hMSCs, which are identified by the presence of Hayflick-senescence-associated factors. Notably, spati

  15. Candida albicans and Candida parapsilosis induce different T-cell responses in human peripheral blood mononuclear cells

    NARCIS (Netherlands)

    Toth, A.; Csonka, K.; Jacobs, C.; Vagvolgyi, C.; Nosanchuk, J.D.; Netea, M.G.; Gacser, A.

    2013-01-01

    Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C.

  16. Candida albicans and Candida parapsilosis induce different T-cell responses in human peripheral blood mononuclear cells

    NARCIS (Netherlands)

    Toth, A.; Csonka, K.; Jacobs, C.; Vagvolgyi, C.; Nosanchuk, J.D.; Netea, M.G.; Gacser, A.

    2013-01-01

    Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C. parapsilosis-stimulate

  17. Candida albicans and Candida parapsilosis induce different T-cell responses in human peripheral blood mononuclear cells

    NARCIS (Netherlands)

    Toth, A.; Csonka, K.; Jacobs, C.; Vagvolgyi, C.; Nosanchuk, J.D.; Netea, M.G.; Gacser, A.

    2013-01-01

    Candida parapsilosis is the third most frequent cause of candidemia. Despite its clinical importance, little is known about the human immunological response to C. parapsilosis. In this study, we compared the cytokine responses evoked by Candida albicans and C. parapsilosis. C. parapsilosis-stimulate

  18. Nutrigenomics in human peripheral blood mononuclear cells : the effects of fatty acids on gene expression profiles of human circulating cells as assessed in human intervention studies

    NARCIS (Netherlands)

    Bouwens, M.

    2009-01-01

    Research on the effects of nutrition on the function and health of organs in the human body, such as liver and intestine, is difficult, because for this research organ tissue is needed. Since nutrition research is usually performed in healthy volunteers, this tissue is difficult to obtain. However,

  19. 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.

  20. HCMV infection of humanized mice after transplantation of G-CSF-mobilized peripheral blood stem cells from HCMV-seropositive donors.

    Science.gov (United States)

    Hakki, Morgan; Goldman, Devorah C; Streblow, Daniel N; Hamlin, Kimberly L; Krekylwich, Craig N; Fleming, William H; Nelson, Jay A

    2014-01-01

    Human cytomegalovirus (HCMV) infection, including primary infection resulting from transmission from a seropositive donor to a seronegative recipient (D(+)/R(-)), remains a significant problem in the setting of peripheral blood stem cell transplantation (PBSCT). The lack of a suitable animal model for studying HCMV transmission after PBSCT is a major barrier to understanding this process and, consequently, developing novel interventions to prevent HCMV infection. Our previous work demonstrated that human CD34(+) progenitor cell-engrafted NOD-scid IL2Rγc(null) (NSG) mice support latent HCMV infection after direct inoculation and reactivation after treatment with granulocyte colony-stimulating factor. To more accurately recapitulate HCMV infection in the D(+)/R(-) PBSCT setting, granulocyte colony-stimulating factor-mobilized peripheral blood stem cells from seropositive donors were used to engraft NSG mice. All recipient mice demonstrated evidence of HCMV infection in liver, spleen, and bone marrow. These findings validate the NSG mouse model for studying HCMV transmission during PBSCT.

  1. Alpha Interferon Inhibits Human Herpesvirus 8 (HHV-8) Reactivation in Primary Effusion Lymphoma Cells and Reduces HHV-8 Load in Cultured Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Monini, Paolo; Carlini, Francesca; Stürzl, Michael; Rimessi, Paola; Superti, Fabiana; Franco, Marina; Melucci-Vigo, Gianna; Cafaro, Aurelio; Goletti, Delia; Sgadari, Cecilia; Butto’, Stefano; Leone, Patrizia; Leone, Pasqualina; Chiozzini, Chiara; Barresi, Caterina; Tinari, Antonella; Bonaccorsi, Angela; Capobianchi, Maria R.; Giuliani, Massimo; di Carlo, Aldo; Andreoni, Massimo; Rezza, Giovanni; Ensoli, Barbara

    1999-01-01

    Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi’s sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-α), we analyzed the effects of IFN-α or anti-IFN-α antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-α inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-α inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-α was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-α Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-α Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-α reduced the HHV-8 load in PBMC. Thus, IFN-α appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-α may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation. PMID:10196299

  2. Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

    Science.gov (United States)

    Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

    2016-06-15

    A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies.

  3. Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies

    Directory of Open Access Journals (Sweden)

    Ramu A. Subbramanian

    2012-07-01

    Full Text Available Cryopreserved peripheral blood mononuclear cells (PBMC constitute an important component of immune monitoring studies as they allow for efficient batch- testing of samples as well as for the validation and extension of original studies in the future. In this study, we systematically test the permutations of PBMC thawing practices commonly employed in the field and identify conditions that are high and low risk for the viability of PBMC and their functionality in downstream ELISPOT assays. The study identifies the addition of ice-chilled washing media to thawed cells at the same temperature as being a high risk practice, as it yields significantly lower viability and functionality of recovered PBMC when compared to warming the cryovials to 37 °C and adding a warm washing medium. We found thawed PBMC in cryovials could be kept up to 30 minutes at 37 °C in the presence of DMSO before commencement of washing, which surprisingly identifies exposure to DMSO as a low risk step during the thawing process. This latter finding is of considerable practical relevance since it permits batch-thawing of PBMC in high-throughput immune monitoring environments.

  4. Generation and characterization of the human iPSC line PBMC1-iPS4F1 from adult peripheral blood mononuclear cells

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    Rosa Montes

    2015-11-01

    Full Text Available Here we describe the generation and characterization of the human induced pluripotent stem cell (iPSC line PBMC1-iPS4F1 from peripheral blood mononuclear cells from a healthy female with Spanish background. We used heat sensitive, non-integrative Sendai viruses containing the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc, whose expression was silenced in the established iPSC line. Characterization of the PBMC1-iPS4F1 cell line included analysis of typical pluripotency-associated factors at mRNA and protein level, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation studies.

  5. Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors.

    Science.gov (United States)

    Shalit, M; Sekhsaria, S; Malech, H L

    1995-01-01

    Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5

  6. 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.

  7. Grain and bean lysates improve function of endothelial progenitor cells from human peripheral blood: involvement of the endogenous antioxidant defenses.

    Directory of Open Access Journals (Sweden)

    Daniela Lucchesi

    Full Text Available Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs, the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG, a Triticum Sativum grain powder, and Lady Joy (LJ, a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H2O2. Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2-like 2/ARE (antioxidant response element activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35-0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35-0.7 mg/ml protect EPCs viability against H2O2-induced injury. LG 0.7 and LJ 0.35-0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H2O2. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H2O2, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H2O2-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2 expression; upon H2O2 exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H2O2 exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a

  8. Isolation And Cell Culture of Human Peripheral Blood Fibrocytes%人外周血纤维细胞的分离、培养方法

    Institute of Scientific and Technical Information of China (English)

    崔磊; 李学拥; 李跃军; 李金清; 吕晓星

    2012-01-01

    目的:探索简便易行的外周血纤维细胞体外分离、培养方法极其生物学特征与功能.方法:采用Ficoll密度梯度离心分离法分离成人外周血,所获得的白细胞在一定条件要求下体外培养,采用流式细胞技术、细胞免疫荧光染色等对贴壁生长的成纤维样细胞进行鉴定,在扫描电镜下进一步观察其形态结构.结果:培养至第14天时,外周血纤维细胞开始分化成熟.血液来源的取材、首次换液的时间、细胞的接种密度、血清浓度等多种因素均会对外周血纤维细胞的培养造成影响.免疫荧光染色结果显示培养至12天时CD34和COL Ⅰ均为强阳性表达,继续培养至28天时,血液来源的细胞表面抗原CD34发生明显丢失,免疫荧光染色几乎不能显色;相反COL Ⅰ持续表达阳性,取培养14天的贴壁细胞,经流式细胞仪分析,Col Ⅰ+细胞为81.6%,显示PBFCs不断向成纤维细胞分化的特性.结论:采用密度梯度离心法配合适当的培养条件,成人外周血中存在的前体细胞经体外分离、培养可分化为外用血纤维细胞,并保持其生物学特性.%Objectives: To establish a simple and efficient method for isolating and culturing peripheral blood fibrocytes in vitro, study the functions and relationship between the expression specific molecule makers expression and the morphological characteristics. Methods: Total peripheral blood leukocytes were isolated from human peripheral blood by centrifugation over Ficoll-Paque. Adhered fibroblast-like cells were detected by imrminocytochemis- try, flow cytometry and electron microscopy. Results: Cultured to day 14, peripheral blood mature fiber cells begin to differentiate. The source of blood draw, the first exchange of fluid impact on the culture of the cell inoculation density, serum concentration, and other factors will peripheral blood fibrocytes. Imrnunocytochemical staining of PBFCs showed that after 14 days,CD34+ and COL I

  9. A Long-Gap Peripheral Nerve Injury Therapy Using Human Skeletal Muscle-Derived Stem Cells (Sk-SCs): An Achievement of Significant Morphological, Numerical and Functional Recovery.

    Science.gov (United States)

    Tamaki, Tetsuro; Hirata, Maki; Nakajima, Nobuyuki; Saito, Kosuke; Hashimoto, Hiroyuki; Soeda, Shuichi; Uchiyama, Yoshiyasu; Watanabe, Masahiko

    2016-01-01

    Losses in vital functions of the somatic motor and sensory nervous system are induced by severe long-gap peripheral nerve transection injury. In such cases, autologous nerve grafts are the gold standard treatment, despite the unavoidable sacrifice of other healthy functions, whereas the prognosis is not always favorable. Here, we use human skeletal muscle-derived stem cells (Sk-SCs) to reconstitute the function after long nerve-gap injury. Muscles samples were obtained from the amputated legs from 9 patients following unforeseen accidents. The Sk-SCs were isolated using conditioned collagenase solution, and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN/29+) cells. Cells were separately cultured/expanded under optimal conditions for 2 weeks, then injected into the athymic nude mice sciatic nerve long-gap model (7-mm) bridging an acellular conduit. After 8-12 weeks, active cell engraftment was observed only in the Sk-34 cell transplanted group, showing preferential differentiation into Schwann cells and perineurial/endoneurial cells, as well as formation of the myelin sheath and perineurium/endoneurium surrounding regenerated axons, resulted in 87% of numerical recovery. Differentiation into vascular cell lineage (pericyte and endothelial cells) were also observed. A significant tetanic tension recovery (over 90%) of downstream muscles following electrical stimulation of the sciatic nerve (at upper portion of the gap) was also achieved. In contrast, Sk-DN/29+ cells were completely eliminated during the first 4 weeks, but relatively higher numerical (83% vs. 41% in axon) and functional (80% vs. 60% in tetanus) recovery than control were observed. Noteworthy, significant increase in the formation of vascular networks in the conduit during the early stage (first 2 weeks) of recovery was observed in both groups with the expression of key factors (mRNA and protein levels), suggesting the paracrine effects to angiogenesis. These results suggested that the human Sk

  10. Antioxidant compounds in the seaweed Gelidiella acerosa protects human Peripheral Blood Mononuclear Cells against TCDD induced toxicity.

    Science.gov (United States)

    Ilavarasi, K; Chermakani, P; Arif Nisha, S; Sheeja Malar, D; Pandima Devi, K

    2015-04-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental toxin formed as an unintentional by-product of incomplete combustion. Several therapeutic approaches have evolved to combat its toxicity since it elicits immunotoxicity, neurotoxicity, hepatotoxicity, carcinogenicity and lethality. Search for drugs from natural resources especially from seaweeds has become intense due to their enormous pharmacological potential. Hence, the present study aims at revealing the protective effect of methanolic extract of G. acerosa (MEGA) in Peripheral Blood Mononuclear Cells (PBMC) against TCDD induced toxicity, by assessing the antioxidant, anti-apoptotic and cytoprotective activities. The results of antioxidant assays suggests that MEGA reverted TCDD induced toxicity by causing an alteration in the levels of antioxidant enzymes (Catalase [CAT], Superoxide dismutase [SOD], Glutathione peroxidase [GPx], Glutathione-S-transferase [GST]) and Glutathione [GSH]. The results of lipid peroxidation assay and protein carbonyl content reveal that MEGA protects PBMC from TCDD induced macromolecular damage. MEGA was found to exhibit significant (p TCDD induced oxidative DNA damage. Levels of phase-I detoxification enzymes determined by EROD assay and semi-quantitative RT-PCR showed that TCDD up-regulates the expression of CYP1A1 and upon co-treatment with MEGA, the expression got slightly decreased suggesting its protective role. Preliminary phytochemical analysis demonstrates that the extract is rich in cardiac glycosides and terpenoids. LC-MS analysis revealed the presence of antioxidants including caffeic acid, phytol and mannoheptulose in MEGA, which could be attributed for the observed protective effect against TCDD induced toxicity.

  11. 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...

  12. Human immunodeficiency virus type 1 RNA detection in peripheral blood mononuclear cells by polymerase chain reaction: enhanced sensitivity after mitogenic stimulation.

    Science.gov (United States)

    Tetali, S K; Oyaizu, N; Paul, M; Pahwa, S

    1993-01-01

    The aim of this study was to investigate whether stimulus-induced up-regulation of human immunodeficiency virus type 1 (HIV-1) expression in peripheral blood mononuclear cells (PBMC) could enhance the diagnostic sensitivity of the polymerase chain reaction (PCR). PBMC derived from 11 HIV-1-infected asymptomatic adults were cultured with a stimulus of phytohemagglutinin (PHA) plus phorbol 12-myristate 13-acetate (PMA) for 36 h prior to lysing the cells for PCR. In all 11 patients studied, the intensity of PCR-assisted HIV RNA amplification (RNA-PCR) performed on stimulated cells was significantly (p < 0.001) higher than that obtained on unstimulated cells. A comparison of conventional PCR-assisted DNA amplification (DNA-PCR) with that of RNA-PCR was made on seven patients. The sensitivity of DNA-PCR was also increased by prior stimulation of cells, although not to the same extent as was observed for RNA-PCR. The results of our study indicate that the sensitivity of PCR can be significantly enhanced by prior activation of cells with PHA and PMA.

  13. Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in SCID mice.

    Science.gov (United States)

    Hardwick, M; Rone, J; Han, Z; Haddad, B; Papadopoulos, V

    2001-11-01

    MDA-MB-231 (MDA-231) human breast cancer cells have a high proliferation rate, lack the estrogen receptor, express the intermediate filament vimentin, the hyaluronan receptor CD44, and are able to form tumors in nude mice. The MDA-231 cell line has been used in our laboratory to examine the role of the peripheral-type benzodiazepine receptor (PBR) in the progression of cancer. During these studies 2 populations of MDA-231 cells were subcloned based on the levels of PBR. The subclones proliferated at approximately the same rate, lacked the estrogen receptor, expressed vimentin and CD44, and had the same in vitro chemoinvasive and chemotactic potential. Both restriction fragment length polymorphism and comparative genomic hybridization analyses of genomic DNA from these cells indicated that both subclones are of the same genetic lineage. Only the subclone with high PBR levels, however, was able to form tumors when injected in SCID mice. These data suggest that the ability of MDA-231 cells to form tumors in vivo may depend on the amount of PBR present in the cells.

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

    Directory of Open Access Journals (Sweden)

    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

  15. Human Invariant NKT Cells Induce IL-1β Secretion by Peripheral Blood Monocytes via a P2X7-Independent Pathway.

    Science.gov (United States)

    Felley, Laura E; Sharma, Akshat; Theisen, Erin; Romero-Masters, James C; Sauer, John-Demian; Gumperz, Jenny E

    2016-09-15

    The cytokine IL-1β plays a central role in inflammatory responses that are initiated by microbial challenges, as well as in those that are due to endogenous processes (often called sterile inflammation). IL-1β secretion that occurs independently of microbial stimulation is typically associated with the presence of endogenous alarmins, such as extracellular ATP (an indicator of cytopathic damage). In this study, we show that IL-2-activated human invariant NKT (iNKT) cells stimulate the secretion of IL-1β protein by human peripheral blood monocytes in a manner that requires neither the presence of microbial compounds nor signaling through the extracellular ATP receptor P2X7 Monocyte IL-1β production was specifically induced by iNKT cells, because similarly activated polyclonal autologous T cells did not have this effect. Secretion of IL-1β protein occurred rapidly (within 3-4 h) and required cell contact between the iNKT cells and monocytes. Similar to IL-1β production induced by TLR stimulation, the iNKT-induced pathway appeared to entail a two-step process involving NF-κB signaling and IL1B gene transcription, as well as assembly of the NLRP3 inflammasome and activation of caspase-1. However, in contrast to the classical inflammasome-mediated pathway of IL-1β production, activation of monocytes via P2X7 was dispensable for iNKT-induced IL-1β secretion, and potassium efflux was not required. Moreover, the iNKT-induced effect involved caspase-8 activity, yet it induced little monocyte death. These results suggest that IL-2-activated human iNKT cells induce monocytes to produce IL-1β through a distinctive pathway that does not require the presence of microbial danger signals or alarmins associated with cytopathic damage.

  16. 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.

  17. Oxazolone and ethanol induce colitis in non-obese diabetic-severe combined immunodeficiency interleukin-2Rγnull mice engrafted with human peripheral blood mononuclear cells

    Science.gov (United States)

    Nolte, T; Zadeh-Khorasani, M; Safarov, O; Rueff, F; Gülberg, V; Herbach, N; Wollenberg, A; Mueller, T; Siebeck, M; Wolf, E; Gropp, R

    2013-01-01

    Oxazolone-induced colitis in mice has become a recognized model to study the efficacy of therapeutics targeting the immunological response underlying the development of inflammatory bowel disease. However, this model cannot be used when therapeutics designed to address human targets do not interact with the respective murine counterpart. In this study, we examined the induction of oxazolone mediated colitis in non-obese diabetic-severe combined immunodeficiency interleukin-2Rγnull (NOD-SCID IL2Rγnull) mice engrafted with human peripheral blood mononuclear cells (hPBMC) derived from patients suffering from ulcerative colitis (UC), atopic dermatitis (AD) and healthy volunteers. NOD-SCID IL2Rγ null mice were engrafted with hPBMC followed by challenge with oxazolone or ethanol vehicle. Mice developed the same symptoms as observed previously in immunocompetent mice. The clinical activity score increased and the colon architecture was characterized by the development of oedema, fibrosis, crypt loss and dense infiltration of predominantly T cells into the lamina propria. Fluorescence activated cell sorter (FACS) analysis of lymphocytes in the colon identified natural killer (NK) T cells as a major constituent. In contrast to studies with immunocompetent mice, we observed the same phenotype in the group challenged with ethanol vehicle. The phenotype was most pronounced in mice engrafted with PBMC derived from a patient suffering from UC, suggesting that the immunological history of the donors predisposes the engrafted mice to react to ethanol. The model described here has the potential to study the efficacy of therapeutics targeting human lymphocytes in a model which is more reflective of the human disease. In addition, it might be developed to elucidate molecular mechanisms underlying the disease. PMID:23574330

  18. 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.

  19. Immunologic testing of xeno-derived osteochondral grafts using peripheral blood mononuclear cells from healthy human donors

    Directory of Open Access Journals (Sweden)

    Targoni Oleg S

    2005-06-01

    Full Text Available Abstract Background One means of treating osteoarthritis is with autologous or allogeneic osteochondral grafts. The purpose of this study was to evaluate the innate immunological response in humans toward xeno-derived osteochondral grafts that have been partially or entirely treated by the photooxidation process. Methods The antigens tested included bovine, porcine, ovine and equine osteochondral samples that have been treated in successive steps of photooxidation. ELISPOT assays were used to evaluate the production of IL-1, IL-4, IL-6, IL-10, IL-12 and TNF-α by human monocytes in response to the antigens. Results Results indicated vigorous production of IL-1, IL-6, IL-10 and TNF-α in response to untreated bovine, porcine and equine specimens. This indicates that these samples are perceived as foreign, or stimulatory, by the human monocytes. There was no induction of IL-4 or IL-12, which is required for Th2 and Th1 immunity, respectively. In contrast, the processed bovine, porcine and equine samples did not induce significant activation of cells of the innate immune system. This occurred after the first step in processing (after cleaning in increasing strengths of ethanol. This suggests that the processing steps dramatically, if not completely, negated the immunostimulatory properties of the test sample. The results for the ovine samples indicate a reverse response. Conclusion The findings of the study suggest that photooxidized bovine, porcine or equine samples have the potential to be used as an osteochondral graft. Although the first step in processing reduced the immunological response, photooxidation is still necessary to retain the structure and mechanical integrity of the cartilage, which would allow for immediate joint resurfacing.

  20. Dose response and adaptive response of non-homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to γ radiation.

    Science.gov (United States)

    Shelke, Shridevi; Das, Birajalaxmi

    2015-05-01

    Ionising radiation induces single-strand breaks, double-strand breaks (DSB) and base damages in human cell. DSBs are the most deleterious and if not repaired may lead to genomic instability and cell death. DSB can be repaired through non-homologous end joining (NHEJ) pathway in resting lymphocytes. In this study, NHEJ genes and proteins were studied in irradiated human peripheral blood mononuclear cells (PBMC) at resting stage. Dose-response, time point kinetics and adaptive-response studies were conducted in irradiated PBMC at various end points such as DNA damage quantitation, transcription and protein expression profile. Venous blood samples were collected from 20 random, normal and healthy donors with written informed consent. PBMC was separated and irradiated with various doses between 0.1 and 2.0 Gy ((60)CO-γ source) for dose-response study. Repair kinetics of DNA damage and time point changes in expression of genes and proteins were studied in post-irradiated PBMC at 2.0 Gy at various time points up to 240 min. Adaptive-response study was conducted with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4-h incubation. Our results revealed that Ku70, Ku80, XLF and Ligase IV were significantly upregulated (P Adaptive-response study showed significantly increased expression of the proteins involved in NHEJ, suggesting their role in adaptive response in human PBMC at G0/G1, which has important implications to human health. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. 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

  2. Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages.

    Science.gov (United States)

    Mattana, Antonella; Sanna, Manuela; Cano, Antonella; Delogu, Giuseppe; Erre, Giuseppe; Roberts, Craig W; Henriquez, Fiona L; Fiori, Pier Luigi; Cappuccinelli, Piero

    2016-10-01

    Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.

  3. Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages

    Science.gov (United States)

    Sanna, Manuela; Cano, Antonella; Delogu, Giuseppe; Erre, Giuseppe; Roberts, Craig W.; Henriquez, Fiona L.; Fiori, Pier Luigi; Cappuccinelli, Piero

    2016-01-01

    Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response. PMID:27481240

  4. Vaccenic acid-mediated reduction in cytokine production is independent of c9,t11-CLA in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Jaudszus, Anke; Jahreis, Gerhard; Schlörmann, Wiebke; Fischer, Janine; Kramer, Ronny; Degen, Christian; Rohrer, Carsten; Roth, Alexander; Gabriel, Holger; Barz, Dagmar; Gruen, Michael

    2012-10-01

    The ruminant trans fatty acid vaccenic acid (tVA) favorably alters markers of inflammation. However, it is not yet clear whether these effects are attributed to its endogenous partial conversion to c9,t11-CLA, which is known to possess anti-inflammatory properties. We compared the cytokine reducing potential of tVA to c9,t11-CLA in human T-helper (Th) cells as a main source of cytokine production during inflammation. Secondly, we assessed whether a bioconversion of tVA to c9,t11-CLA via stearoyl-CoA desaturase (SCD) encoded activity takes place in peripheral blood mononuclear cells (PBMC) in order to relate the outcomes of intracellular cytokine measurement to the degree of conversion. TVA reduced the percentage of both IL-2 and TNF-α expressing Th cells significantly, but to a lesser extent compared to c9,t11-CLA, as determined by flow cytometry after alloreactive stimulation of PBMC. Pre-treatment with the selective PPARγ antagonist T0070907 largely re-established the IL-2 and TNF-α positive Th cell population in both tVA and c9,t11-CLA treated cultures. Interestingly, while the portion of tVA dose-dependently increased within the cellular lipid fraction, the initially marginal amount of c9,t11-CLA remained unaltered. However, SCD mRNA although abundantly expressed in PBMC was not regulated by tVA. Conclusively, these results suggest that the cytokine reducing effect of tVA in human T cells is independent of c9,t11-CLA, since no bioconversion occurred. Moreover, the data provide evidence that tVA mechanistically acts in a manner similar to c9,t11-CLA.

  5. Human beta-mannosidase deficiency associated with peripheral neuropathy.

    Science.gov (United States)

    Levade, T; Graber, D; Flurin, V; Delisle, M B; Pieraggi, M T; Testut, M F; Carrière, J P; Salvayre, R

    1994-01-01

    Human beta-mannosidosis is an inherited lysosomal storage disorder described in only seven families. We present a further case in a black African 14-year-old boy with severely deficient beta-mannosidase activity, bilateral thenar and hypothenar amyotrophy, electrophysiologically demonstrable demyelinating peripheral neuropathy, and cytoplasmic vacuolation of skin fibroblasts and lymphoid cells. The clinical and biochemical features of our patient are compared to those of previously reported patients.

  6. Transforming growth factor beta-1 and interleukin-17 gene transcription in peripheral blood mononuclear cells and the human response to infection.

    LENUS (Irish Health Repository)

    White, Mary

    2012-02-01

    INTRODUCTION: The occurrence of severe sepsis may be associated with deficient pro-inflammatory cytokine production. Transforming growth factor beta-1 (TGFbeta-1) predominantly inhibits inflammation and may simultaneously promote IL-17 production. Interleukin-17 (IL-17) is a recently described pro-inflammatory cytokine, which may be important in auto-immunity and infection. We investigated the hypothesis that the onset of sepsis is related to differential TGFbeta-1 and IL-17 gene expression. METHODS: A prospective observational study in a mixed intensive care unit (ICU) and hospital wards in a university hospital. Patients (59) with severe sepsis; 15 patients with gram-negative bacteraemia but without critical illness and 10 healthy controls were assayed for TGFbeta-1, IL-17a, IL-17f, IL-6 and IL-1beta mRNA in peripheral blood mononuclear cells (PBMC) by quantitative real-time PCR and serum protein levels by ELISA. RESULTS: TGFbeta-1 mRNA levels are reduced in patients with bacteraemia and sepsis compared with controls (p=0.02). IL-6 mRNA levels were reduced in bacteraemic patients compared with septic patients and controls (p=0.008). IL-1beta mRNA levels were similar in all groups, IL-17a and IL-17f mRNA levels are not detectable in peripheral blood mononuclear cells. IL-6 protein levels were greater in patients with sepsis than bacteraemic and control patients (p<0.0001). Activated TGFbeta-1 and IL-17 protein levels were similar in all groups. IL-1beta protein was not detectable in the majority of patients. CONCLUSIONS: Down regulation of TGFbeta-1 gene transcription was related to the occurrence of infection but not the onset of sepsis. Interleukin-17 production in PBMC may not be significant in the human host response to infection.

  7. Anti-tumor effects of fusion vaccine prepared by renal cell carcinoma 786-O cell line and peripheral blood dendritic cells of healthy volunteers in vitro and in human immune reconstituted SCID mice.

    Science.gov (United States)

    Hu, Zhi; Liu, Shihui; Mai, Xuancheng; Hu, Zili; Liu, Chuan

    2010-01-01

    Dendritic cells (DC), as professional antigen presenting cells, play the central role in the process of body initiating the anti-tumor immunity, and the study on DC anti-tumor vaccine has become heated in recent years. In this study, we used polyethylene glycol (PEG) to induce renal cell carcinoma (RCC) 786-O cell line fused with peripheral blood DC of healthy volunteers, and discuss the biological characteristics of fusion vaccine and its anti-tumor effects in vitro and in human immune reconstituted SCID mice model of RCC. The study found that PEG could effectively induce cell fusion, and the expressions of CD86 and HLA-DR in fusion vaccine group were significantly up-regulated compared with the DC control group; the secretion of IL-12 was much higher and longer than that of the control; the functions of dendritic cell-tumor fusion vaccine to stimulate the proliferation of allogenic T lymphocytes and to kill RCC786-O cells in vitro were significantly higher than those of the control group, and after the killing, apoptosis body was observed in the target cells; after the injection of fusion vaccine into human immune reconstituted SCID mice model of RCC786-O via vena caudalis, the volume of mice tumor was reduced significantly, proliferation index of tumor cells decreased obviously compared with that of the control group, and more hemorrhage and putrescence focuses presented, accompanying large quantity of lymphocytes soakage. The results of this experimental study shows that fusion vaccine of RCC786-O cell line and DC can significantly stimulate the proliferation of allogenic T cells and specifically inhibit and kill RCC cells in vitro and in vivo, which makes the DC-RCC786-O fusion vaccine a possible new way of effective RCC immunotherapy.

  8. Morbidly obese human subjects have increased peripheral blood CD4+ T cells with skewing toward a Treg- and Th2-dominated phenotype.

    Science.gov (United States)

    van der Weerd, Kim; Dik, Willem A; Schrijver, Benjamin; Schweitzer, Dave H; Langerak, Anton W; Drexhage, Hemmo A; Kiewiet, Rosalie M; van Aken, Maarten O; van Huisstede, Astrid; van Dongen, Jacques J M; van der Lelij, Aart-Jan; Staal, Frank J T; van Hagen, P Martin

    2012-02-01

    Obesity is associated with local T-cell abnormalities in adipose tissue. Systemic obesity-related abnormalities in the peripheral blood T-cell compartment are not well defined. In this study, we investigated the peripheral blood T-cell compartment of morbidly obese and lean subjects. We determined all major T-cell subpopulations via six-color flow cytometry, including CD8+ and CD4+ T cells, CD4+ T-helper (Th) subpopulations, and natural CD4+CD25+FoxP3+ T-regulatory (Treg) cells. Moreover, molecular analyses to assess thymic output, T-cell proliferation (T-cell receptor excision circle analysis), and T-cell receptor-β (TCRB) repertoire (GeneScan analysis) were performed. In addition, we determined plasma levels of proinflammatory cytokines and cytokines associated with Th subpopulations and T-cell proliferation. Morbidly obese subjects had a selective increase in peripheral blood CD4+ naive, memory, natural CD4+CD25+FoxP3+ Treg, and Th2 T cells, whereas CD8+ T cells were normal. CD4+ and CD8+ T-cell proliferation was increased, whereas the TCRB repertoire was not significantly altered. Plasma levels of cytokines CCL5 and IL-7 were elevated. CD4+ T-cell numbers correlated positively with fasting insulin levels. The peripheral blood T-cell compartment of morbidly obese subjects is characterized by increased homeostatic T-cell proliferation to which cytokines IL-7 and CCL5, among others, might contribute. This is associated with increased CD4+ T cells, with skewing toward a Treg- and Th2-dominated phenotype, suggesting a more anti-inflammatory set point.

  9. In vitro response of human peripheral blood mononuclear cells to AISI 316L austenitic stainless steel subjected to nitriding and collagen coating treatments.

    Science.gov (United States)

    Stio, Maria; Martinesi, Maria; Treves, Cristina; Borgioli, Francesca

    2015-02-01

    Surface modification treatments can be used to improve the biocompatibility of austenitic stainless steels. In the present research two different modifications of AISI 316L stainless steel were considered, low temperature nitriding and collagen-I coating, applied as single treatment or in conjunction. Low temperature nitriding produced modified surface layers consisting mainly of S phase, which enhanced corrosion resistance in PBS solution. Biocompatibility was assessed using human peripheral blood mononuclear cells (PBMC) in culture. Proliferation, lactate dehydrogenase (LDH) levels, release of cytokines (TNF-α, IL-1β, IL-12, IL-10), secretion of metalloproteinase (MMP)-9 and its inhibitor TIMP-1, and the gelatinolytic activity of MMP-9 were determined. While the 48-h incubation of PBMC with all the sample types did not negatively influence cell proliferation, LDH and MMP-9 levels, suggesting therefore a good biocompatibility, the release of the pro-inflammatory cytokines was always remarkable when compared to that of control cells. However, in the presence of the nitrided and collagen coated samples, the release of the pro-inflammatory cytokine IL-1β decreased, while that of the anti-inflammatory cytokine IL-10 increased, in comparison with the untreated AISI 316L samples. Our results suggest that some biological parameters were ameliorated by these surface treatments of AISI 316L.

  10. 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.

  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

    2017-08-01

    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

  12. Ruta 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer.

    Science.gov (United States)

    Pathak, Sen; Multani, Asha S; Banerji, Pratip; Banerji, Prasanta

    2003-10-01

    Although conventional chemotherapies are used to treat patients with malignancies, damage to normal cells is problematic. Blood-forming bone marrow cells are the most adversely affected. It is therefore necessary to find alternative agents that can kill cancer cells but have minimal effects on normal cells. We investigated the brain cancer cell-killing activity of a homeopathic medicine, Ruta, isolated from a plant, Ruta graveolens. We treated human brain cancer and HL-60 leukemia cells, normal B-lymphoid cells, and murine melanoma cells in vitro with different concentrations of Ruta in combination with Ca3(PO4)2. Fifteen patients diagnosed with intracranial tumors were treated with Ruta 6 and Ca3(PO4)2. Of these 15 patients, 6 of the 7 glioma patients showed complete regression of tumors. Normal human blood lymphocytes, B-lymphoid cells, and brain cancer cells treated with Ruta in vitro were examined for telomere dynamics, mitotic catastrophe, and apoptosis to understand the possible mechanism of cell-killing, using conventional and molecular cytogenetic techniques. Both in vivo and in vitro results showed induction of survival-signaling pathways in normal lymphocytes and induction of death-signaling pathways in brain cancer cells. Cancer cell death was initiated by telomere erosion and completed through mitotic catastrophe events. We propose that Ruta in combination with Ca3(PO4)2 could be used for effective treatment of brain cancers, particularly glioma.

  13. Transcriptome Analysis of Human Peripheral Blood Mononuclear Cells Exposed to Lassa Virus and to the Attenuated Mopeia/Lassa Reassortant 29 (ML29), a Vaccine Candidate

    Science.gov (United States)

    Zapata, Juan Carlos; Carrion, Ricardo; Patterson, Jean L.; Crasta, Oswald; Zhang, Yan; Mani, Sachin; Jett, Marti; Poonia, Bhawna; Djavani, Mahmoud; White, David M.; Lukashevich, Igor S.; Salvato, Maria S.

    2013-01-01

    Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease. PMID:24069471

  14. Transcriptome analysis of human peripheral blood mononuclear cells exposed to Lassa virus and to the attenuated Mopeia/Lassa reassortant 29 (ML29, a vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Juan Carlos Zapata

    Full Text Available Lassa virus (LASV is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG, as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

  15. Transcriptome analysis of human peripheral blood mononuclear cells exposed to Lassa virus and to the attenuated Mopeia/Lassa reassortant 29 (ML29), a vaccine candidate.

    Science.gov (United States)

    Zapata, Juan Carlos; Carrion, Ricardo; Patterson, Jean L; Crasta, Oswald; Zhang, Yan; Mani, Sachin; Jett, Marti; Poonia, Bhawna; Djavani, Mahmoud; White, David M; Lukashevich, Igor S; Salvato, Maria S

    2013-01-01

    Lassa virus (LASV) is the causative agent of Lassa Fever and is responsible for several hundred thousand infections and thousands of deaths annually in West Africa. LASV and the non-pathogenic Mopeia virus (MOPV) are both rodent-borne African arenaviruses. A live attenuated reassortant of MOPV and LASV, designated ML29, protects rodents and primates from LASV challenge and appears to be more attenuated than MOPV. To gain better insight into LASV-induced pathology and mechanism of attenuation we performed gene expression profiling in human peripheral blood mononuclear cells (PBMC) exposed to LASV and the vaccine candidate ML29. PBMC from healthy human subjects were exposed to either LASV or ML29. Although most PBMC are non-permissive for virus replication, they remain susceptible to signal transduction by virus particles. Total RNA was extracted and global gene expression was evaluated during the first 24 hours using high-density microarrays. Results were validated using RT-PCR, flow cytometry and ELISA. LASV and ML29 elicited differential expression of interferon-stimulated genes (ISG), as well as genes involved in apoptosis, NF-kB signaling and the coagulation pathways. These genes could eventually serve as biomarkers to predict disease outcomes. The remarkable differential expression of thrombomodulin, a key regulator of inflammation and coagulation, suggests its involvement with vascular abnormalities and mortality in Lassa fever disease.

  16. Human peripheral blood mononuclear cell in vitro system to test the efficacy of food bioactive compounds: Effects of polyunsaturated fatty acids and their relation with BMI

    KAUST Repository

    Cifre, Margalida

    2016-11-22

    Scope: To analyse the usefulness of isolated human peripheral blood mononuclear cells (PBMC) to rapidly/easily reflect n-3 long-chain polyunsaturated fatty acid (LCPUFA) effects on lipid metabolism/inflammation gene profile, and evaluate if these effects are body mass index (BMI) dependent. Methods and results: PBMC from normoweight (NW) and overweight/obese (OW/OB) subjects were incubated with physiological doses of docosahexaenoic (DHA), eicosapentaenoic acid (EPA), or their combination. PBMC reflected increased beta-oxidation-like capacity (CPT1A expression) in OW/OB but only after DHA treatment. However, insensitivity to n-3 LCPUFA was evident in OW/OB for lipogenic genes: both PUFA diminished FASN and SREBP1C expression in NW, but no effect was observed for DHA in PBMC from high-BMI subjects. This insensitivity was also evident for inflammation gene profile: all treatments inhibited key inflammatory genes in NW; nevertheless, no effect was observed in OW/OB after DHA treatment, and EPA effect was impaired. SLC27A2, IL6 and TNFα PBMC expression analysis resulted especially interesting to determine obesity-related n-3 LCPUFA insensitivity. Conclusion: A PBMC-based human in vitro system reflects n-3 LCPUFA effects on lipid metabolism/inflammation which is impaired in OW/OB. These results confirm the utility of PBMC ex vivo systems for bioactive-compound screening to promote functional food development and to establish appropriate dietary strategies for obese population.

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

    Science.gov (United States)

    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.

  18. Association between exposure to dioxin-like polychlorinated biphenyls and miR-191 expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Guida, Maurizio; Marra, Maria Luisa; Marra, Marialuisa; Zullo, Fulvio; Guida, Marco; Trifuoggi, Marco; Biffali, Elio; Borra, Marco; De Mieri, Giovanna; D'Alessandro, Raffaella; De Felice, Bruna

    2013-04-30

    Accumulating evidence shows an association between deregulation of miRNAs and exposure to environmental chemicals; miRNAs play a unique regulatory role in gene expression. Among environmental pollutants, dioxins are a family of compounds that are known to have multiple hazardous effects. Also, in utero exposure of the fetus to dioxins has been shown to cause impaired psychomotor development, decreased immune function and skin disease. miR-191 is a microRNA that has been found to be up-regulated by dioxin in hepatocellular carcinoma cells in vitro. Our study provides the first molecular evidence in vivo of a positive relationship between levels of polychlorinated biphenyls (PCBs) and miR-191 expression in human peripheral blood mononuclear cells. miR-191 expression was significantly correlated with blood concentrations of total PCB and, in particular, of 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169, a coplanar congener). Blood concentrations of PCB 169 correlated significantly with miR-191 expression in pregnant women living in a PCB-polluted area, who underwent therapeutic abortion due to fetal malformations. These data suggest that miRNAs could be potential biomarkers to clarify the mechanisms of environmental disease.

  19. 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.

    Science.gov (United States)

    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.

  20. 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

  1. Molecular and stimulus-response profiles illustrate heterogeneity between peripheral and cord blood-derived human mast cells

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Frandsen, Pernille M; Raaby, Ellen M

    2014-01-01

    Different protocols exist for in vitro development of HuMCs from hematopoietic stem cells, which results in distinct mast cells regarding molecular markers and activation patterns. Here, we introduce a SR profile using immunological, neurogenic, and pharmacological stimuli to characterize cellular...... functionality. Mast cells were obtained from three culture protocols using two types of PBdMCs (CD34(+) PBdMC or CD133(+) PBdMC) and one type of CBdMC (CD133(+) CBdMC). We analyzed resting cells for specific mast cell markers at protein and mRNA levels, thereby creating a molecular profile. To characterize...... the SR profile, we stimulated cells with anti-IgE, C3a, C5a, Substance P, or Compound 48/80 and measured the release of histamine and cytokines (IL-10, IL-13, GM-CSF, TNF-α). Molecular profiling revealed that CD133(+) CBdMC expressed less chymase, FcεRIα, and CD203c but more CD117 compared with CD34...

  2. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    Science.gov (United States)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  3. Recombinant human thrombopoietin in combination with granulocyte colony-stimulating factor enhances mobilization of peripheral blood progenitor cells, increases peripheral blood platelet concentration, and accelerates hematopoietic recovery following high-dose chemotherapy.

    Science.gov (United States)

    Somlo, G; Sniecinski, I; ter Veer, A; Longmate, J; Knutson, G; Vuk-Pavlovic, S; Bhatia, R; Chow, W; Leong, L; Morgan, R; Margolin, K; Raschko, J; Shibata, S; Tetef, M; Yen, Y; Forman, S; Jones, D; Ashby, M; Fyfe, G; Hellmann, S; Doroshow, J H

    1999-05-01

    Lineage-specific growth factors mobilize peripheral blood progenitor cells (PBPC) and accelerate hematopoietic recovery after high-dose chemotherapy. Recombinant human thrombopoietin (rhTPO) may further increase the progenitor-cell content and regenerating potential of PBPC products. We evaluated the safety and activity of rhTPO as a PBPC mobilizer in combination with granulocyte colony-stimulating factor (G-CSF) in 29 breast cancer patients treated with high-dose chemotherapy followed by PBPC reinfusion. Initially, patients received escalating single doses of rhTPO intravenously (IV) at 0.6, 1.2, or 2.4 micrograms/kg, on day 1. Subsequent patients received rhTPO 0.6 or 0.3 micrograms/kg on days -3, -1, and 1, or 0.6 micrograms/kg on days -1 and 1. G-CSF, 5 micrograms/kg IV or subcutaneously (SC) twice daily, was started on day 3 and continued through aphereses. Twenty comparable, concurrently and identically treated patients (who were eligible and would have been treated on protocol but for the lack of study opening) mobilized with G-CSF alone served as comparisons. CD34(+) cell yields were substantially higher with the first apheresis following rhTPO and G-CSF versus G-CSF alone: 4.1 x 10(6)/kg (range, 1.3 to 17.6) versus 0.8 x 10(6)/ kg (range, 0.3 to 4.2), P =.0003. The targeted minimum yield of 3 x 10(6) CD34(+) cells/kg was procured following a single apheresis procedure in 61% of the rhTPO and G-CSF-mobilized group versus 10% of G-CSF-mobilized patients (P =.001). In rhTPO and G-CSF mobilized patients, granulocyte (day 8 v 9, P =.0001) and platelet recovery (day 9 v 10, P =.07) were accelerated, and fewer erythrocyte (3 v 4, P =.02) and platelet (4 v 5, P =.02) transfusions were needed compared with G-CSF-mobilized patients. Peripheral blood platelet counts, following rhTPO and G-CSF, were increased by greater than 100% and the platelet content of PBPC products by 60% to 110% on the first and second days of aphereses (P rhTPO at 0.6 microgram/kg. rhTPO is

  4. Cell-Specific Variation in E-Selectin Ligand Expression among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology.

    Science.gov (United States)

    Silva, Mariana; Fung, Ronald Kam Fai; Donnelly, Conor Brian; Videira, Paula Alexandra; Sackstein, Robert

    2017-03-22

    Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. In this study, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and Western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human PBMCs. Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sialyl Lewis X (sLe(X)) and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4(+) and CD8(+) T cells but no binding by B cells. Monocytes prominently present sLe(X) decorations on an array of protein scaffolds, including P-selectin glycoprotein ligand-1, CD43, and CD44 (rendering the E-selectin ligands cutaneous lymphocyte Ag, CD43E, and hematopoietic cell E-selectin/L-selectin ligand, respectively), and B cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLe(X) reveal high transcript levels among circulating monocytes and low levels among circulating B cells, and, commensurately, cell surface α(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLe(X) are abundantly expressed on human monocytes yet are relatively deficient on B cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory

  5. Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation

    Directory of Open Access Journals (Sweden)

    Nadella Kiran S

    2008-06-01

    Full Text Available Abstract Background Adult T-cell leukemia/lymphoma (ATLL is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1; however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated. Results We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1α (MIP-1α, a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was

  6. 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.

  7. 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.

  8. The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors.

    Science.gov (United States)

    Houimel, Mehdi

    2014-08-01

    A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74-92% and 90-96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3-IGKV1, IGHV3-IGVL1, and IGHV3-IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Cell enrichment-free massive ex-vivo expansion of peripheral CD20⁺ B cells via CD40-CD40L signals in non-human primates.

    Science.gov (United States)

    Kim, Jung-Sik; Byun, Nari; Chung, Hyunwoo; Kim, Hyun-Je; Kim, Jong-Min; Chun, Taehoon; Lee, Won-Woo; Park, Chung-Gyu

    2016-04-22

    Non-human primates (NHPs) are valuable as preclinical resources that bridge the gap between basic science and clinical application. B cells from NHPs have been utilized for the development of B-cell targeted drugs and cell-based therapeutic modalities; however, few studies on the ex-vivo expansion of monkey B cells have been reported. In this study, we developed a highly efficient ex-vivo expansion protocol for monkey B cells resulting in 99% purity without the requirement for prior cell-enrichment procedures. To this end, monkey peripheral blood mononuclear cells (PBMCs) were stimulated for 12 days with cells constitutively expressing monkey CD40L in expansion medium optimized for specific and massive expansion of B cells. The B cells expansion rates obtained were 2-5 times higher than those previously reported in humans, with rates ranging from 7.9 to 16.6 fold increase. Moreover, expanded B cells sustained high expression of co-stimulatory molecules including CD83 and CD86 until day 12 of culture, and the simple application of a brief centrifugation resulted in a CD20(+) B cell purity rate of greater than 99%. Furthermore, small amounts of CD3(+)CD20(+)BT-like cells were generated and CD16 was expressed at moderate levels on expanded B cells. Thus, the establishment of this protocol provides a method to produce quantities of homogeneous, mature B cells in numbers sufficient for the in vitro study of B cell immunity as well as for the development of B cell-diagnostic tools and cell-based therapeutic modalities.

  11. Radio-adaptive response of base excision repair genes and proteins in human peripheral blood mononuclear cells exposed to gamma radiation.

    Science.gov (United States)

    Toprani, Sneh M; Das, Birajalaxmi

    2015-09-01

    Radio-adaptive response is a mechanism whereby a low-dose exposure (priming dose) induces resistance to a higher dose (challenging dose) thus significantly reducing its detrimental effects. Radiation-induced DNA damage gets repaired through various DNA repair pathways in human cells depending upon the type of lesion. The base excision repair (BER) pathway repairs radiation-induced base damage, abasic sites and single-strand breaks in cellular DNA. In the present study, an attempt has been made to investigate the involvement of BER genes and proteins in the radio-adaptive response in human resting peripheral blood mononuclear cells (PBMC). Venous blood samples were collected from 20 randomly selected healthy male individuals with written informed consent. PBMC were isolated and irradiated at a priming dose of 0.1 Gy followed 4h later with a challenging dose of 2.0 Gy (primed cells). Quantitation of DNA damage was done using the alkaline comet assay immediately and expression profile of BER genes and proteins were studied 30 min after the challenging dose using real-time quantitative polymerase chain reaction and western blot, respectively. The overall result showed significant (P ≤ 0.05) reduction of DNA damage in terms of percentage of DNA in tail (%T) with a priming dose of 0.1 Gy followed by a challenging dose of 2.0 Gy after 4 h. Twelve individuals showed significant (P ≤ 0.05) reduction in %T whereas eight individuals showed marginal reduction in DNA damage that was not statistically significant. However, at the transcriptional level, BER genes such as APE1, FEN1 and LIGASE1 showed significant (P ≤ 0.05) up-regulation in both groups. Significant (P ≤ 0.05) up-regulation was also observed at the protein level for OGG1, APE1, MBD4, FEN1 and LIGASE1 in primed cells. Up-regulation of some BER genes and proteins such as APE1, FEN1 and LIGASE1 in primed cells of resting PBMC is suggestive of active involvement of the BER pathway in radio-adaptive response.

  12. [A preliminary study on the effects of feto-maternal microchimerism in activated human leukocyte antigen haploidentical mobilized peripheral blood stem cells on treatment of solid tumors].

    Science.gov (United States)

    Cao, Shui; Yu, Jin-pu; Li, Hui; An, Xiu-mei; Xin, Ning; Ren, Xiu-bao

    2009-10-01

    To study the effect of feto-maternal microchimerism in the treatment of activated human leukocyte antigen (HLA) haploidentical mobilized peripheral blood cells against solid tumors. Genomic DNA samples of 25 pairs of HLA haploidentical donors and recipients were extracted. The donor-derived HLA-DRB loci were detected with nested PCR-sequence specific primer (SSP) typing. The mixed lymphocyte proliferation action between the patients and respective donors, the engraftment of donor's cells and the serum levels of Th1/Th2 type of cytokines were measured with MTT, FISH and ELISA method respectively. The survival time of patients with or without feto-maternal microchimerism were compared as well. Using nested PCR-SSP typing, the positive rates of feto-maternal microchimerism in the 25 pairs of HLA haploidentical donors and recipients were 40% in the maternal/children pairs and 0 in the paternal/children pairs. The chimerism positive patients showed less proliferation activity when cocultured with respective donors as compared with unrelated ones (P = 0.03). Only one chimerism positive patient experienced the engraft of donor's cell 3 months after treatment as the donor derived XX chromosome was identified with FISH. When the data of chimerism positive patients were deleted, the serum levels of IFNgamma 1 month after treatment dropped dramatically from 171.4 (26.3 approximately 258.4) ng/L to 29.4 (1.2 approximately 39.9) ng/L. The survival time in chimerism positive patients of the maternal/children pairs was significantly longer than that in chimerism negative patients, which was (31.2 +/- 4.3) months and (11.1 +/- 3.3) months, respectively (P = 0.036). Feto-maternal microchimerism might induce anergy in the HLA haploidentical donors, favor the engraftment of donor's progenitors and maintenance of positive microenvironment and prolong the survival time.

  13. Seasonal variation in vitamin D3 levels is paralleled by changes in the peripheral blood human T cell compartment

    NARCIS (Netherlands)

    Khoo, A.L.; Koenen, H.J.P.M.; Chai, L.Y.; Sweep, F.C.; Netea, M.G.; Ven, A.J.A.M. van der; Joosten, I.

    2012-01-01

    It is well-recognized that vitamin D(3) has immune-modulatory properties and that the variation in ultraviolet (UV) exposure affects vitamin D(3) status. Here, we investigated if and to what extent seasonality of vitamin D(3) levels are associated with changes in T cell numbers and phenotypes. Every

  14. A New Synthetic Compound, 2-OH, Enhances Interleukin-2 and Interferon-γ Gene Expression in Human Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Woan-Fang Tzeng

    2009-07-01

    Full Text Available A new synthetic compound, 6-hydroxy-2-tosylisoquinolin-1(2H-one (2-OH, was selected for immunopharmacological activity tests. The effects of 2-OH on human peripheral blood mononuclear cell (PBMC proliferation were determined by tritiated thymidine uptake. Compared to phytohemagglutinin (PHA; 5 μg/mL stimulation, 2-OH significantly enhanced PBMC proliferation in a dose-dependent manner. The 50% enhancement activity (EC50 for 2-OH was 4.4±0.1 μM. In addition, effects of 2-OH on interleukin-2 (IL-2 and interferon-γ (IFN-γ production in PBMC were determined by enzyme immunoassay. Results demonstrated that 2-OH stimulated IL-2 and IFN-γ production in PBMC. Data from reverse transcription-polymerase chain reaction (RT-PCR and real-time PCR indicated that IL-2 and IFN-γ mRNA expression in PBMC could be induced by 2-OH. Therefore, 2-OH enhanced IL-2 and IFN-γ production in PBMC by modulation their gene expression. We suggest that 2-OH may be an immunomodulatory agent.

  15. Wheat gliadin promotes the interleukin-4-induced IgE production by normal human peripheral mononuclear cells through a redox-dependent mechanism.

    Science.gov (United States)

    Dugas, Bernard; Dugas, Nathalie; Conti, Marc; Calenda, Alphonse; Pino, Paco; Thomas, Yolène; Mazier, Dominique; Vouldoukis, Ioannis

    2003-03-21

    Increased levels of serum IgE have been described in gliadin-intolerant patients; however, biological mechanisms implicated in this immunoglobulin production remained unknown. In this study, we demonstrated that in vitro crude gliadins and gliadin lysates (Glilys) promoted the IL-4-induced IgE production by human peripheral blood mononuclear cells (PBMC), indicating that the biological process related to gliadin intolerance and/or allergy may lead to IgE production in vivo. It was found that crude gliadin and Glilys potentiated, after 13 days of culture in a dose-dependent manner, IL-4-induced IgE production and, to a lesser extent, the IgG production, while they did not affect IgA or IgM productions. This promoting effect of gliadin and Glilys on the IL-4-induced activation of normal human PBMC was also observed on the early release (2 days) of the soluble fraction of CD23, suggesting its possible involvement in IgE potentiation. The promoting effect of crude gliadin and Glilys appeared to be indirect because they did not modify purified B-lymphocytes IgE production after IL-4 and anti-CD40 monoclonal antibody stimulation. In addition, as revealed by luminol-dependent chemiluminescence, we demonstrated that crude gliadin and Glilys promoted a substantial production of free radicals by normal human PBMC, treated or not with IL-4. This redox imbalance associated with an increased IgE production led us to evaluate the effect of pharmacological antioxidants (N-acetyl-cysteine (NAC) and Cu/Zn-superoxide dismutase (SOD1)) on IgE production by human PBMC. The NAC and the intracellularly delivered SOD1 were found to suppress the IL-4+/-crude gliadin or Glilys-induced IgE production by normal human PBMC. Taken together, these data indicated that gliadin specifically enhanced IL-4-induced IgE production by normal human PBMC, probably by the regulation of redox pathways, and that this 'pro-allergenic' effect could be counteracted by natural antioxidants: thiols and

  16. 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

  17. 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

    Directory of Open Access Journals (Sweden)

    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

  18. Temporal induction of pro-inflammatory and regulatory cytokines in human peripheral blood mononuclear cells by Campylobacter jejuni and Campylobacter coli

    Science.gov (United States)

    Kuhnert, Peter

    2017-01-01

    Campylobacter jejuni along with C. coli are major cause of human gastroenteritis worldwide. So far, the human immune response against Campylobacter is not entirely clear. We hypothesize that it is coordinated by an interaction between pro-inflammatory and regulatory cytokines which is influenced by bacterial and host-individual differences. Accordingly, we used peripheral blood mononuclear cells (PBMC) from healthy donors to study the primary systemic immune response to C. jejuni and C. coli. PBMC were stimulated by different strains of C. jejuni and C. coli for three time points (5, 10, 24 hours). The production of the pro-inflammatory (IL-6, IL-8, IFN-γ) and the regulatory (IL-10) cytokines were measured by ELISA. All strains induced higher levels of IL-8 and IL-6 than IFN-γ and IL-10. In contrast to IL-8 and IL-6, IL-10 showed a steeper increase over time. While IFN-γ did not show any further increase between 10 and 24 hours. Interestingly, there was a significant correlation between IL-8 and IL-10 which peaked at 24 hours. Despite the variability of the used bacterial strains, their effect on cytokine production was less pronounced than the inter-person differences. The strongest significant effect of the strain was on the level of IL-10. IL-10 and IL-6 were significantly influenced by strain-person interaction. In conclusion, the systemic immune response to C. coli and C. jejuni is characterized by an early pro-inflammatory reaction with later initiation of regulatory immune response which is influenced mainly by the host, explaining the individual variations in disease severity. Additional work is needed to determine the cellular sources of the produced cytokines as well as the campylobacter molecules that might contribute to this stimulation. PMID:28196097

  19. Expression Profile of Human Fc Receptor-Like 1, 2, and 4 Molecules in Peripheral Blood Mononuclear Cells of Patients with Hashimoto's Thyroiditis and Graves' Disease.

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    Rostamzadeh, D; Dabbaghmanesh, M H; Shabani, M; Hosseini, A; Amirghofran, Z

    2015-08-01

    Recently identified Fc receptor-like (FCRL) molecules are new members of the immunoglobulin superfamily dominantly expressed by B cells. Although FCRL expression patterns have been studied in normal and malignant cells, their biological functions and roles remain to be clearly identified in humans. Research has particularly focused on FCRL gene polymorphisms in autoimmune diseases, however, their involvement in the pathogenesis of autoimmune diseases is an interesting field for investigation. In the present study, we have investigated the gene expression profiles of FCRL1, 2, and 4 in 2 common thyroid diseases, Hashimoto's thyroiditis (HT) and Graves' disease (GD). FCRL1, 2, and 4 expressions were determined in peripheral blood samples of 55 HT patients, 40 GD patients and equal numbers of normal subjects by quantitative real-time PCR. Our results showed downregulation of FCRL1 and upregulation of FCRL2 transcripts in both HT and GD groups compared to healthy counterparts. Overexpression of FCRL4 was observed only in GD patients compared to controls. A significant correlation was observed between all FCRL gene expression levels in HT patients. Only FCRL2 and 4 had a correlation in GD patients. In addition, FCRL1, 2, and 4 gene expressions showed no correlations with the level of anti-thyroid peroxidase antibody (anti-TPO) or anti-thyroglobulin (anti-Tg) antibody from patients' sera. In conclusion, expressions of activating or inhibitory FCRL1, 2, and 4 showed significant alterations in HT and GD patients compared to healthy subjects. © Georg Thieme Verlag KG Stuttgart · New York.

  20. Use of recombinant lentivirus pseudotyped with vesicular stomatitis virus glycoprotein G for efficient generation of human anti-cancer chimeric T cells by transduction of human peripheral blood lymphocytes in vitro

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    Kolokoltsov Andrey A

    2006-02-01

    Full Text Available Abstract Background Genetic redirection of lymphocytes that have been genetically engineered to recognize antigens other than those originally programmed in their germlines is a potentially powerful tool for immunotherapy of cancers and potentially also of persistent viral infections. The basis for this procedure is that both cancers and some viruses have developed strikingly similar mechanisms of evading attacks by host immune mechanisms. To redirect human peripheral blood lymphocytes (PBLs with a chimeric T cell receptor (chTCR so that they recognize a new target requires a high degree of transfection efficiency, a process that is regarded as technically demanding. Results Infection with a retroviral vector carrying a chTCR cassette was shown to transduce 100% of rapidly dividing murine T cells but typically, only ~10% of PBLs could be infected with the same vector. In contrast with other retroviruses, lentiviruses integrate their genomes into non-dividing cells. To increase host cell range, vesicular stomatitis virus G protein was pseudotyped with a lentivirus vector, which resulted in ~100% PBL transduction efficiency. Signaling of PBLs bearing chimeric receptors was shown by specific proliferation on exposure to cells expressing cognate ligand. Further, T-bodies against CEA showed a startling abilty to cause regression of maligant colon tumors in a nude mouse model of human cancer. Conclusion A lentivirus/VSV pseudotyped virus, which does not require replicating cells for integration of its genome, efficiently transduced a high proportion of human PBLs with chTCRs against CEA. PBLs transduced by infection with a lentivirus/VSV pseudotyped vector were able to proliferate specifically in vitro on exposure to CEA-expressing cells and further they had a startling therapeutic effect in a mouse model of human colon cancer.

  1. 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

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

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    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

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

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    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

  4. Scanning electron microscopy of interaction of peripheral blood lymphocytes from colonic cancer patients with human colonic cancer-derived cells; P-4788.

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    Sugihara,Mutsuto

    1979-12-01

    Full Text Available Peripheral blood lymphocytes and the various lymphocyte fractions from patients with cancer of the colon were cultivated with target cells (P-4788 derived from the colon cancer. Changes in the surface ultrastructure during tumor cell destruction were studied by scanning electron microscopy (SEM. P-4788 cells adhering to the coverslip showed various surface activity. The surfaces of some cells were relatively flat; others were smooth or had fine granules. Still other cells were villous, round or had marked blebs. When host lymphocytes were added to the target cells, adhesion of the two cell groups began by many fine projections. After incubation for 6 h, some lymphocytes had adhered to the target cells. Many lymphocytes had adhered to the target tumor cells by 24--48 h incubation. Ultimately the tumor cells became swollen and disrupted. Most lymphocytes adherent to the target cells had few microvilli. Lymphocytes after elimination of phagocytes by carbonyl iron treatment also adhered readily. Some target cells showed adhesion with lymphocytes passed through nylon-wool columns, although the number of lymphocytes adhering was fewer than in the case of lymphocytes not passed through nylon-wool columns. T cells were collected from lymphocytes that form rosettes with SRBC by isolation with NH4Cl. They had markedly elongated microvilli which in places were sparsely scattered and tended to be localized on the side, a finding which suggests loss of cell activity by the time of SEM. Only a few T cells adhered to target cells and they seemed to be T cells without activity. It was thought that there are cytotoxic cells among T cells and that the co-existence of T cells, non-T cells and monocytes caused target cell destruction.

  5. 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.

  6. Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors.

    Science.gov (United States)

    Su, Rui-Jun; Baylink, David J; Neises, Amanda; Kiroyan, Jason B; Meng, Xianmei; Payne, Kimberly J; Tschudy-Seney, Benjamin; Duan, Yuyou; Appleby, Nancy; Kearns-Jonker, Mary; Gridley, Daila S; Wang, Jun; Lau, K-H William; Zhang, Xiao-Bing

    2013-01-01

    The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs) from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB) CD34(+) cells with improved oriP/EBNA1-based episomal vectors (EV) using a strong spleen focus forming virus (SFFV) long terminal repeat (LTR) promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4)-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs) into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-)/CD19(-) cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

  7. Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors.

    Directory of Open Access Journals (Sweden)

    Rui-Jun Su

    Full Text Available The ability to efficiently generate integration-free induced pluripotent stem cells (iPSCs from the most readily available source-peripheral blood-has the potential to expedite the advances of iPSC-based therapies. We have successfully generated integration-free iPSCs from cord blood (CB CD34(+ cells with improved oriP/EBNA1-based episomal vectors (EV using a strong spleen focus forming virus (SFFV long terminal repeat (LTR promoter. Here we show that Yamanaka factors (OCT4, SOX2, MYC, and KLF4-expressing EV can also reprogram adult peripheral blood mononuclear cells (PBMNCs into pluripotency, yet at a very low efficiency. We found that inclusion of BCL-XL increases the reprogramming efficiency by approximately 10-fold. Furthermore, culture of CD3(-/CD19(- cells or T/B cell-depleted MNCs for 4-6 days led to the generation of 20-30 iPSC colonies from 1 ml PB, an efficiency that is substantially higher than previously reported. PB iPSCs express pluripotency markers, form teratomas, and can be induced to differentiate in vitro into mesenchymal stem cells, cardiomyocytes, and hepatocytes. Used together, our optimized factor combination and reprogramming strategy lead to efficient generation of integration-free iPSCs from adult PB. This discovery has potential applications in iPSC banking, disease modeling and regenerative medicine.

  8. PERIPHERAL GIANT CELL GRANULOMA- A CASE REPORT

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    Ashutosh Agrawal

    2013-07-01

    Full Text Available The peripheral giant cell granuloma (PGCG is a benign inflammatory hyperplastictype of lesion of unknown etiology occurring in gingiva or alveolar ridge. It normally presentsas a soft tissue purplish-red nodule consisting of multinucleated giant cells in a background ofmononuclear stromal cells and extravasated red blood cells. A 75 years old male patientreported with a chief complaint of pain and swelling in lower right back region of mouth. Onintra-oral examination the swelling was red, firm and sessile with smooth surface texture. Theorthopantomogram (OPG revealed a well-demarcated radiolucency extending from distalaspect of mandibular canine to mesial aspect of mandibular first molar. The cone beamcomputed tomography also showed the features suggestive of soft tissue lesion causingcupping resorption of mandible. Excisional biopsy was performed under local anaesthesiaand tissue was examined histopathologically. The lesion was diagnosed as PGCG afterthorough clinical, radiologic and histopathologic examination.

  9. Dragon's blood extracts reduce radiation-induced peripheral blood injury and protects human megakaryocyte cells from GM-CSF withdraw-induced apoptosis.

    Science.gov (United States)

    Ran, Yuanyuan; Xu, Bing; Wang, Ran; Gao, Qian; Jia, Qiutian; Hasan, Murtaza; Shan, Shuangquan; Ma, Hong; Dai, Rongji; Deng, Yulin; Qing, Hong

    2016-01-01

    Dragon's blood (DB), a Chinese traditional herb, was shown to have certain protective effects on radiation-induced bone marrow injury due to the presence of several phenolic compounds. The 50% ethanol extracts (DBE) were separated from DB by the methods of alcohol extracting-water precipitating. The protective effects of DBE on hematopoiesis were studied, particularly on megakaryocytes. In this study, we investigated the in vivo radioprotective effects of DBE on hematopoiesis and pathological changes using an irradiated-mouse model. Moreover, the protective effects and potential molecular mechanisms of DBE on megakaryocytopoiesis in vitro were explored in GM-CSF depletion-induced Mo7e cell model. DBE significantly promoted the recovery of peripheral blood cells in irradiated mice. Histology bone marrow confirmed the protective effect of DBE, as shown by an increased number of hematopoietic cells and a reduction of apoptosis. In a megakaryocytic apoptotic model, DBE (50 µg/mL) markedly alleviated GM-CSF withdrawal-induced apoptosis and cell-cycle arrest of Mo7e cells. DBE (50 µg/mL) also significantly decreased the ratio of Bax to Bcl-2 expression, inhibited the active caspase-3 expression. In addition, DBE could induce ERK1/2 phosphorylation in GM-CSF-depleted Mo7e cell, but not Akt. Our data demonstrated that DBE could effectively accelerate the recovery of peripheral blood cells, especially platelet. DBE attenuated cell apoptosis and cell cycle arrest through the decrease of Bax/Bcl-2 ratio and the reduction of active caspase-3 expression. The effect of DBE on Mo7e cells survival and proliferation is likely associated with the activation of ERK, but not Akt. Copyright © 2015 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

  10. Overexpression of cyclooxygenase-2 in malignant peripheral nerve sheath tumor and selective cyclooxygenase-2 inhibitor-induced apoptosis by activating caspases in human malignant peripheral nerve sheath tumor cells.

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    Michiyuki Hakozaki

    Full Text Available BACKGROUND: Cyclooxygenase-2 (COX-2 is a key enzyme in the conversion of arachidonic acid to prostanoids, and its activation is associated with carcinogenesis as well as inflammation. The antitumor effect of selective COX-2 inhibitors has been noted in various malignancies. Malignant peripheral nerve sheath tumor (MPNST is a rare and aggressive soft tissue sarcoma for which effective treatments have not yet been established. The purpose of this study was to investigate a potential therapeutic role of COX-2 in MPNST. METHODS: We evaluated the expression of COX-2 in 44 cases of high-grade MPNST using immunohistochemical staining and compared the staining results with the characteristics and outcome of the patients. We also investigated the antitumor effect of etodolac, a selective COX-2 inhibitor, on MPNST cells in vitro using the MPNST cell line, FMS-1. RESULTS: Overexpression of COX-2 (≥50% positive cells was observed in 29 cases (65.9%, was significantly associated with a poor overall survival (P = 0.0495, and was considered an independent risk factor for a poor outcome by the results of both univariate and multivariate analysis. Etodolac induced apoptosis of FMS-1 cells through the activation of caspase-8, -9, and -3. Moreover, several caspase inhibitors significantly inhibited etodolac-induced apoptosis. CONCLUSIONS: Selective COX-2 inhibitors including etodolac had an antitumor effect on MPNST cells, and their use holds promise as a novel therapeutic strategy for patients with MPNST to improve their prognoses.

  11. Effects of subcutaneous interleukin-2 therapy on phenotype and function of peripheral blood mononuclear cells in human immunodeficiency virus infected patients

    DEFF Research Database (Denmark)

    Aladdin, H; Larsen, C S; Møller, B K

    2000-01-01

    In the context of clinical therapy with recombinant human interleukin-2 (IL-2), we monitored immunological alteration in 10 human immunodeficiency virus type-I (HIV-1)-infected individuals, on stable antiretroviral therapy, who had a CD4+ cell count between 200 and 500 cells/mm3. Subcutaneous IL-2...

  12. 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...

  13. A case report of peripheral giant cell granuloma

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    Kim, Sung Soo; Jung, Yeon Hwa; Cho, Bong Hae; Nah, Kyung Soo [Dept. of Oral and Maxillofacial Radiology, College of Dentistry, Pusan National University, Pusan(Korea, Republic of)

    1997-08-15

    The authors experienced one case of peripheral giant cell granuloma occurred at the gingiva of right maxillary molar in a 12-year-old male patient. The lesion showed amorphous calcification within soft tissue mass which made difficult to differentiate this lesion from peripheral ossifying fibroma and peripheral odontogenic fibroma clinically and radiographically. The final diagnosis was made histologically.

  14. 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.)

  15. Radiation Sensitivity of Human CD34(+) Cells Versus Peripheral Blood T Lymphocytes of Newborns and Adults: DNA Repair and Mutagenic Effects.

    Science.gov (United States)

    Vandevoorde, C; Vral, A; Vandekerckhove, B; Philippé, J; Thierens, H

    2016-06-01

    As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34(+) cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34(+) cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34(+) cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34(+) cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end

  16. Circulating brain derived neurotrophic factor (BDNF) and frequency of BDNF positive T cells in peripheral blood in human ischemic stroke: Effect on outcome.

    Science.gov (United States)

    Chan, Adeline; Yan, Jun; Csurhes, Peter; Greer, Judith; McCombe, Pamela

    2015-09-15

    The aim of this study was to measure the levels of circulating BDNF and the frequency of BDNF-producing T cells after acute ischaemic stroke. Serum BDNF levels were measured by ELISA. Flow cytometry was used to enumerate peripheral blood leukocytes that were labelled with antibodies against markers of T cells, T regulatory cells (Tregs), and intracellular BDNF. There was a slight increase in serum BDNF levels after stroke. There was no overall difference between stroke patients and controls in the frequency of CD4(+) and CD8(+) BDNF(+) cells, although a subgroup of stroke patients showed high frequencies of these cells. However, there was an increase in the percentage of BDNF(+) Treg cells in the CD4(+) population in stroke patients compared to controls. Patients with high percentages of CD4(+) BDNF(+) Treg cells had a better outcome at 6months than those with lower levels. These groups did not differ in age, gender or initial stroke severity. Enhancement of BDNF production after stroke could be a useful means of improving neuroprotection and recovery after stroke.

  17. Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue.

    Directory of Open Access Journals (Sweden)

    Amita Joshi

    Full Text Available Treatment of invasive adenovirus (Ad disease in hematopoietic stem cell transplant (SCT recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977 and late protein hexon (H-892 were compared in peripheral blood (PB and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.

  18. Automated microscopy system for peripheral blood cells

    Science.gov (United States)

    Boev, Sergei F.; Sazonov, Vladimir V.; Kozinets, Gennady I.; Pogorelov, Valery M.; Gusev, Alexander A.; Korobova, Farida V.; Vinogradov, Alexander G.; Verdenskaya, Natalya V.; Ivanova, Irina A.

    2000-11-01

    The report describes the instrument ASPBS (Automated Screening of Peripheral Blood Cells) designed for an automated analysis of dry blood smears. The instrument is based on computer microscopy and uses dry blood smears prepared according to the standard Romanovskii-Giemza procedure. In comparison with the well-known flow cytometry systems, our instrument provides more detailed information and offers an opporunity of visualizing final results. The basic performances of the instrument are given. Software of this instrument is based on digital image processing and image recognition procedures. It is pointed out that the instrument can be used as a fairly universal tool in scientific research, public demonstrations, in medical treatment, and in medical education. The principle used as the basis of the instrument appeared adequate for creating an instrument version serviceable even during space flights where standard manual procedures and flow cytometry systems fail. The benefit of the use of the instrument in clinical laboratories is described.

  19. Augmentation by interleukin-18 of MHC-nonrestricted killer activity of human peripheral blood mononuclear cells in response to interleukin-12.

    Science.gov (United States)

    Singh, S M; Yanagawa, H; Hanibuchi, M; Miki, T; Okamura, H; Sone, S

    2000-01-01

    Interleukin (IL)-18 is a novel cytokine with pleiotropic functions. In the present study, we examined the induction of the killer activity of peripheral blood mononuclear cells (MNC) against lung cancer cell lines upon treatment with IL-18 in combination with IL-12. Cytotoxic activity was measured by standard (51)Cr release assay. IL-18 (100 ng/ml) was found to significantly augment IL-12-induced killer activity in a MHC-nonrestricted manner against allogeneic NK-resistant Daudi cells and lung cancer cell lines: SBC-3, RERF-LC-AI and A549. IL-18 could augment IL-12-induced killer activity both at the optimal as well as suboptimal doses of the latter. However, IL-18 was found to have little effect on the killer activity of MNC induced by optimal or suboptimal dose of IL-2 or IL-15. Treatment of MNC with IL-18 in combination with IL-12 for a period of more than 4 days was observed to optimally induce the killer activity. As for induction of IFN-gamma production by MNC, IL-18 augmented that induced by IL-2 and IL-15, as well as that induced by IL-12. These results show the potential of IL-18 in combination with IL-12 for clinical application in treatment of cancer.

  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. Simultaneous determination of 2',3'-dideoxyinosine and the active metabolite, 2',3'-dideoxyadenosine-5'-triphosphate in human peripheral-blood mononuclear cell by HPLC-MS/MS and the application to cell pharmacokinetics.

    Science.gov (United States)

    Lan, Xu; Mingdao, Lei; Huilin, Guo; Wei, Gan; Lvjiang, Hu; Yan, Zhou; Gang, Li

    2015-10-01

    A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 2',3'-dideoxyinosine (ddI) and the active metabolites, 2',3'-dideoxyadenosine-5'-triphosphate (ddA-TP) in human peripheral-blood mononuclear cell for the first time. The analytes were separated on a HILIC column (100mm×2.1mm, 1.7μm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The cell homogenates sample was prepared by the solid phase extraction. The calibration curves were linear over a concentration range of 0.5-200.0ng/mL for ddI and 0.25-100.0ng/mL for ddA-TP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ddI and support cell pharmacokinetics after the patients with AIDS were orally administrated with ddI and tenofovir disoproxyl fumarate (TDF).

  2. 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

  3. A novel, rapid strategy to form dendritomas from human dendritic cells and hepatocellular carcinoma cell line HCCLM3 cells using mature dendritic cells derived from human peripheral blood CD14+ monocytes within 48 hours of in vitro culture

    OpenAIRE

    Guan, Xin; Peng, Ji-Run; Yuan, Lan; Wang, Hui; Wei, Yu-Hua; Leng, Xi-Sheng

    2004-01-01

    AIM: Dendritomas formed by fusing cancer cells to dendritic cells have already been applied to clinical treatment trial of several types of cancers. Dendritic cells for the fusion in most trials and experiments were from blood monocytes in standard 7-d protocol culture, which requires 5-7 d of culture with granulocyte-macrophage–colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), followed by 2-3 d of activation with a combination of proinflammatory mediators such as tumor necrosis fa...

  4. Expression of CD 68, CD 45 and human leukocyte antigen-DR in central and peripheral giant cell granuloma, giant cell tumor of long bones, and tuberculous granuloma: An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Anoop Kumar

    2015-01-01

    Conclusion: CD 68 and CD 45 expression was found in central giant cell granuloma, peripheral giant cell granuloma and GCT, suggesting the origin from mononuclear phagocyte system and considering their clinical behavior of osteoclast type. High expressivity of HLA-DR in tuberculous granulomas which is an essential factor for presentation of the microbial antigen to CD 4 helper cells thus reassuring the fact that they are up-regulated in response to infection.

  5. CTLA4 blockade broadens the peripheral T cell receptor repertoire

    Science.gov (United States)

    Robert, Lidia; Tsoi, Jennifer; Wang, Xiaoyan; Emerson, Ryan; Homet, Blanca; Chodon, Thinle; Mok, Stephen; Huang, Rong Rong; Cochran, Alistair J.; Comin-Anduix, Begonya; Koya, Richard C.; Graeber, Thomas G.; Robins, Harlan; Ribas, Antoni

    2014-01-01

    Purpose To evaluate the immunomodulatory effects of CTLA-4 blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). Experimental Design We used next generation sequencing to study the complementarity determining region 3 (CDR3) from the rearranged T cell receptor (TCR) variable beta (V-beta) in PBMC of 21 patients, at baseline and 30–60 days after receiving tremelimumab. Results After receiving tremelimumab there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (p=0.01) and for Shannon index diversity (p=0.04). In comparison, serially collected PBMC from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over one year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared to patients without toxicity (p=0.05). No relevant differences were noted between clinical responders and non-responders. Conclusions CTLA4 blockade with tremelimumab diversifies the peripheral T cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system. PMID:24583799

  6. [Histological and immunophenotypical characteristics of peripheral T-cell lymphomas].

    Science.gov (United States)

    Krivolapov, Iu A

    2005-01-01

    Histopathologic features of immunohistochemically confirmed 37 nodal peripheral T-cell lymphomas are described. Unspecified and 10 angioimmunoblastic T-cell lymphomas were analyzed. The most demonstrative histological features of both types of lymphomas were spectrum of small, medium and large lymphoid cells, lymphoid cells with irregular nuclei, presence of clusters of clear cells, arborizing endothelial venules, increased number of histiocytes, eosinophils and plasma cells. Isolated paracortical expantion, compartmentalization of neoplastic infiltrate and large atypical Reed-Stemberg-like cells were occasional findings. Delineation between peripheral T-cell lymphoma, unspecified and angioimmunoblastic T-cell lymphoma needs evaluation of follicular dendritic cell pattern. The results suggest that detection of histopathologic features typical for peripheral T-cell lymphomas gives an opportunity to compose optimal panel for immunotyping which is absolutely necessary.

  7. Peripheral NK cell phenotypes: multiple changing of faces of an adapting, developing cell.

    Science.gov (United States)

    Perussia, Bice; Chen, Yingying; Loza, Matthew J

    2005-02-01

    We have defined the existence of developmental relationships among human peripheral NK cells with distinct phenotypic and functional characteristics. These findings closely parallel the changes that occur in vivo during NK cell development, and in vitro in experimental culture systems supporting NK cell generation from hematopoietic progenitors. These new insights provide a simplified framework to understand NK cell immunobiology and the cellular bases for their roles in innate immunity, initiation and maintenance of immune responses via regulation of adaptive and accessory cell functions, and immune pathologies.

  8. 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.

  9. Consequences of vitamin D receptor gene polymorphisms for growth inhibition of cultured human peripheral blood mononuclear cells by 1, 25-dihydroxyvitamin D3.

    Science.gov (United States)

    Colin, E M; Weel, A E; Uitterlinden, A G; Buurman, C J; Birkenhäger, J C; Pols, H A; van Leeuwen, J P

    2000-02-01

    In the vitamin D receptor (VDR) gene a BsmI restriction fragment length polymorphism (RFLP) in intron 8 and a translational start-site polymorphism, identified as a FokI RFLP, have been described. Crucial for a proper interpretation of these polymorphisms in association studies is the knowledge whether they have direct consequences for 1,25-(OH)2D3 action at cellular level. The present study was designed to assess functional significance of the FokI and BsmI VDR gene polymorphisms in peripheral blood mononuclear cells (PBMC) with a natural occurring VDR genotype for cell growth inhibition by 1,25-(OH)2D3. PBMC of women were isolated, VDR genotyped and in vitro inhibition by 1,25-(OH)2D3 of Phytohemagglutinin (PHA)-stimulated growth of PBMC was examined in relation to VDR genotype. PHA-stimulated growth and maximal growth inhibition were independent of VDR genotype. However, the FF genotype had a significant lower ED50 than the Ff genotype corresponding to an allele dose effect of 0.32 nM per f allele copy (P = 0.0036). For BsmI genotypes no differences in ED50 were observed. The present study demonstrates for the first time in cells with a natural VDR genotype a direct functional consequence of the VDR gene translational start-site polymorphism for the action of 1,25-(OH)2D3. Especially under conditions of vitamin D insufficiency these findings might have clinical implications.

  10. Peripheral nerve extract effects on mesenchymal cells.

    OpenAIRE

    Dietz, F. R.; Mukhopadhyay, B.; Becker, G.; Daniels, K.; Solursh, M

    1996-01-01

    Several common congenital limb disorders are characterized by normal tissue differentiation but abnormal somatic growth. These include: idiopathic clubfoot, idiopathic leg length discrepancy, hemi-atrophy and hemi-hypertrophy. Both clinical and research studies have suggested that peripheral nerves may be important in regulating somatic growth of limb tissues. To investigate the hypothesis that peripheral nerves convey trophic substances to mesenchymal tissues that are involved in the regulat...

  11. Evaluation of genotoxic activity of tenofovir disoproxil fumarate in human peripheral lymphocytes

    OpenAIRE

    Kubra Kurt; Lale Donbak; Ahmet Kayraldiz

    2016-01-01

    Purpose: Antiretroviral drugs used in the treatment of HIV (Human Immunodeficiency Virus) iinfection, treat by preventing the proliferation of HIV in human body. People with HIV have to use this drugs for lifelong because of inability of the drugs to eradicate the viruses. In this study, we investigated the in vitro genotoxic activity of tenofovir disoproxil fumarate one of the antiretroviral drugs, in human peripheral lymphocytes. Material and Methods: The cells were treated with four d...

  12. Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors

    Directory of Open Access Journals (Sweden)

    Wei Wen

    2016-06-01

    Full Text Available We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB with an improved episomal vector (EV system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B. Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1 equimolar expression of OCT4 (O and SOX2 (S, by using a 2A linker; (2 a higher and gradual increase in the MYC (M to KLF4 (K ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

  13. Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

    Science.gov (United States)

    Wen, Wei; Zhang, Jian-Ping; Xu, Jing; Su, Ruijun Jeanna; Neises, Amanda; Ji, Guang-Zhen; Yuan, Weiping; Cheng, Tao; Zhang, Xiao-Bing

    2016-06-14

    We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

  14. 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.

  15. Impaired Pten expression in human malignant peripheral nerve sheath tumours.

    Directory of Open Access Journals (Sweden)

    Maren Bradtmöller

    Full Text Available Malignant peripheral nerve sheath tumours (MPNST are aggressive sarcomas that develop in about 10% of patients with the genetic disease neurofibromatosis type 1 (NF1. Molecular alterations contributing to MPNST formation have only partially been resolved. Here we examined the role of Pten, a key regulator of the Pi3k/Akt/mTOR pathway, in human MPNST and benign neurofibromas. Immunohistochemistry showed that Pten expression was significantly lower in MPNST (n=16 than in neurofibromas (n=16 and normal nervous tissue. To elucidate potential mechanisms for Pten down-regulation or Akt/mTOR activation in MPNST we performed further experiments. Mutation analysis revealed absence of somatic mutations in PTEN (n=31 and PIK3CA (n=38. However, we found frequent PTEN promotor methylation in primary MPNST (11/26 and MPNST cell lines (7/8 but not in benign nerve sheath tumours. PTEN methylation was significantly associated with early metastasis. Moreover, we detected an inverse correlation of Pten-regulating miR-21 and Pten protein levels in MPNST cell lines. The examination of NF1-/- and NF1+/+Schwann cells and fibroblasts showed that Pten expression is not regulated by NF1. To determine the significance of Pten status for treatment with the mTOR inhibitor rapamycin we treated 5 MPNST cell lines with rapamycin. All cell lines were sensitive to rapamycin without a significant correlation to Pten levels. When rapamycin was combined with simvastatin a synergistic anti-proliferative effect was achieved. Taken together we show frequent loss/reduction of Pten expression in MPNST and provide evidence for the involvement of multiple Pten regulating mechanisms.

  16. 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.

  17. 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.

  18. Design, fabrication and perivascular implantation of bioactive scaffolds engineered with human adventitial progenitor cells for stimulation of arteriogenesis in peripheral ischemia.

    Science.gov (United States)

    Carrabba, M; De Maria, C; Oikawa, A; Reni, C; Rodriguez-Arabaolaza, I; Spencer, H; Slater, S; Avolio, E; Dang, Z; Spinetti, G; Madeddu, P; Vozzi, G

    2016-03-24

    Cell therapy represents a promising option for revascularization of ischemic tissues. However, injection of dispersed cells is not optimal to ensure precise homing into the recipient's vasculature. Implantation of cell-engineered scaffolds around the occluded artery may obviate these limitations. Here, we employed the synthetic polymer polycaprolactone for fabrication of 3D woodpile- or channel-shaped scaffolds by a computer-assisted writing system (pressure assisted micro-syringe square), followed by deposition of gelatin (GL) nanofibers by electro-spinning. Scaffolds were then cross-linked with natural (genipin, GP) or synthetic (3-glycidyloxy-propyl-trimethoxy-silane, GPTMS) agents to improve mechanical properties and durability in vivo. The composite scaffolds were next fixed by crown inserts in each well of a multi-well plate and seeded with adventitial progenitor cells (APCs, 3 cell lines in duplicate), which were isolated/expanded from human saphenous vein surgical leftovers. Cell density, alignment, proliferation and viability were assessed 1 week later. Data from in vitro assays showed channel-shaped/GPTMS-crosslinked scaffolds confer APCs with best alignment and survival/growth characteristics. Based on these results, channel-shaped/GPTMS-crosslinked scaffolds with or without APCs were implanted around the femoral artery of mice with unilateral limb ischemia. Perivascular implantation of scaffolds accelerated limb blood flow recovery, as assessed by laser Doppler or fluorescent microspheres, and increased arterial collaterals around the femoral artery and in limb muscles compared with non-implanted controls. Blood flow recovery and perivascular arteriogenesis were additionally incremented by APC-engineered scaffolds. In conclusion, perivascular application of human APC-engineered scaffolds may represent a novel option for targeted delivery of therapeutic cells in patients with critical limb ischemia.

  19. Cancer stem cell-like cells derived from malignant peripheral nerve sheath tumors.

    Directory of Open Access Journals (Sweden)

    Melanie Spyra

    Full Text Available This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST. Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5 × 10(5. These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors.

  20. V(D)J recombinase mediated inter-chromosomal HPRT alterations at cryptic recombination signal sequences in peripheral human T cells.

    Science.gov (United States)

    Messier, Terri L; O'Neill, J Patrick; Finette, Barry A

    2006-08-01

    The V(D)J recombinase enzyme complex is responsible for the development of a diverse immune system by catalyzing intra-molecular rearrangements of immunoglobulin (Ig) and T cell receptor (TCR) genes at specific recombination signal sequences (RSSs). This enzyme complex has also been implicated in mediating pathologic and non-pathologic intra- and inter-molecular genomic rearrangements at cryptic (Psi) RSSs outside the immune system loci in lymphoid cells. We describe here two V(D)J recombinase mediated genomic rearrangements resulting in alterations at the HPRT locus in human T-cells. These are inter-chromosomal insertions in which DNA fragments are inserted at breakpoints generated by V(D)J recombinase cleavage at Psi RSS sites in the HPRT locus at Xq26. In the first, a TCR signal ended segment from chromosome 14q11 is inserted at a Psi RSS in intron 1 of the HPRT locus. In the second, a DNA fragment from 9q22 is integrated between the coding ends generated by a V(D)J recombinase mediated HPRT deletion. Identification of these in vivo V(D)J mediated inter-chromosomal insertions at Psi RSSs in the HPRT gene supports the accumulating evidence that V(D)J recombinase can mediate mutagenic rearrangements in humans with potential pathologic consequences.

  1. PERIPHERAL GIANT CELL GRANULOMAS OF ORAL CAVITY: OUR EXPERIENCE

    Directory of Open Access Journals (Sweden)

    Kamal

    2015-07-01

    Full Text Available Peripheral giant cell granuloma or the so - called “giant cell epulis” is the most common oral giant cell lesion. Peripheral giant cell granuloma (PGCG is an infrequent exophytic lesion of the oral cavity, also known as giant - cell hyperplasia, osteoclastoma, or giant cell reparative granuloma. Lesions vary in appearance from smooth, regularly outlined masses to irregularly shaped, multilobulated protuberances with surface indentations. Ulcerations of the margin are occasionally seen . This lesion probably does not represent a true neoplasm, but rather may be reactive in nature, believed to be stimulated by local irritation or trauma, but the cause is not certainly known. The aim in publishing this study is to present the clinical, histopathological features and treatment of peripheral giant cell granulomas of various sizes in different age groups in jaws

  2. Properties and functions of naive and memory B cells in human peripheral blood%人外周血初始B细胞和记忆性B细胞亚群的特征和功能

    Institute of Scientific and Technical Information of China (English)

    冯敛; 吴长有

    2010-01-01

    B淋巴细胞是免疫系统的重要免疫成份,主要功能是介导体液免疫应答.在人外周血中,按照B淋巴细胞的发育阶段及功能的不同,可将B淋巴细胞分为初始成熟B细胞、记忆B细胞和浆细胞.记忆B细胞又可分为IgM记忆B细胞和类型转换的记忆B细胞.近年来的研究表明,B淋巴细胞的亚群远比人想象中的复杂,因此对人B细胞各亚群的起源、发育和功能进行更深入的研究,将有助于治疗自身免疫性疾病,在慢性感染性疾病的治疗过程中找到新的策略,并指导研发安全有效的疫苗.%B lymphocytes are of important immunological component in immune system which are capable of mediating effective humoral immune response. According to developmental stages and functional differences, there are mainly three subsets of B cells in human peripheral blood including naive mature B cells, memory B cells and plasma cells. Memory B cells can be further divided into IgM memory B cells and switched memory B cells. However, recent studies indicate that B-cell subsets are more complicated than previously thought. Therefore, further understanding should be focused on the origin,development and specific function of B-cell subsets in human, which will be a great benefit to the development of therapeutic strategies to autoimmune and inflammatory diseases as well as to the design of effective and safe vaccines.

  3. Central and peripheral hemodynamics in exercising humans

    DEFF Research Database (Denmark)

    Calbet, J A L; González-Alonso, J; Helge, J W

    2015-01-01

    In humans, arm exercise is known to elicit larger increases in arterial blood pressure (BP) than leg exercise. However, the precise regulation of regional vascular conductances (VC) for the distribution of cardiac output with exercise intensity remains unknown. Hemodynamic responses were assessed...... during incremental upright arm cranking (AC) and leg pedalling (LP) to exhaustion (Wmax ) in nine males. Systemic VC, peak cardiac output (Qpeak ) (indocyanine green) and stroke volume (SV) were 18%, 23%, and 20% lower during AC than LP. The mean BP, the rate-pressure product and the associated...... perfusion pressure to increase O2 delivery, allowing a similar peak VO2 per kg of muscle mass in both extremities. In summary, despite a lower Qpeak during arm cranking the cardiovascular strain is much higher than during leg pedalling. The adjustments of regional conductances during incremental exercise...

  4. Determination of TiO2, ZrO2, and Al2O3 nanoparticles on genotoxic responses in human peripheral blood lymphocytes and cultured embyronic kidney cells.

    Science.gov (United States)

    Demir, Eşref; Burgucu, Durmuş; Turna, Fatma; Aksakal, Sezgin; Kaya, Bülent

    2013-01-01

    In this study a genotoxic evaluation of titanium dioxide (TiO2, 2.3 nm), zirconium oxide (ZrO2, 6 nm), aluminum oxide (Al2O3, 16.7 nm) nanoparticles (NP) and their ionic forms was conducted using human peripheral blood lymphocytes and cultured human embryonic kidney (HEK293) cells by means of a modified alkaline comet assay with/without the formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) enzymes. Modifications to the comet assay by using lesion-specific endonucleases, such as Endo III and Fpg, detect DNA bases with oxidative damage. Both human peripheral blood lymphocytes and cultured embryonic kidney cells were incubated with TiO2, ZrO2, or Al2O3 NP at concentrations of 1, 10, or 100 μg/ml. Our results showed no significant induction in DNA damage by the comet assay with/without the Endo III and Fpg enzymes at all concentrations of ZrO2 and Al2O3. In the case of TiO2 NP only the highest concentration of 100 μg/ml significantly induced a genotoxic response. Data thus indicate that both ZrO2 and Al2O3 NP were not genotoxic in our system and in the case of TiO2 the lowest-observed-adverse-effect level (LOAEL) for genotoxicity was 100 μg/ml. Evidence indicates that these metallic NP are considered safe in light of the fact that no genotoxicity was noted with ZrO2 and Al2O3 and that the highest TiO2 concentration is not environmentally relevant.

  5. 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

  6. 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.

  7. Photoacoustic tomography of small-animal and human peripheral joints

    Science.gov (United States)

    Wang, Xueding; Chamberland, David L.; Fowlkes, J. Brian; Carson, Paul L.; Jamadar, David A.

    2008-02-01

    As an emerging imaging technology that combines the merits of both light and ultrasound, photoacoustic tomography (PAT) holds promise for screening and diagnosis of inflammatory joint diseases such as rheumatoid arthritis. In this study, the feasibility of PAT in imaging small-animal joints and human peripheral joints in a noninvasive manner was explored. Ex vivo rat tail and fresh cadaveric human finger joints were imaged. Based on the intrinsic optical contrast, intra- and extra-articular tissue structures in the joints were visualized successfully. Using light in the near-infrared region, the imaging depth of PAT is sufficient for cross-sectional imaging of a human peripheral joint as a whole organ. PAT, as a novel imaging modality with unique advantages, may contribute significantly to the early diagnosis of inflammatory joint disorders and accurate monitoring of disease progression and response to therapy.

  8. Humanized Chronic Graft-versus-Host Disease in NOD-SCID il2rγ-/- (NSG Mice with G-CSF-Mobilized Peripheral Blood Mononuclear Cells following Cyclophosphamide and Total Body Irradiation.

    Directory of Open Access Journals (Sweden)

    Hisaki Fujii

    Full Text Available Chronic graft-versus-host disease (cGvHD is the major source of late phase morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Humanized acute GvHD (aGvHD in vivo models using NOD-SCID il2rγ-/- (NSG mice are well described and are important tools for investigating pathogenicity of human cells in vivo. However, there have been only few reported humanized cGvHD mouse models. We evaluated if prolonged inflammation driven by low dose G-CSF-mobilized human PBMCs (G-hPBMCs would lead to cGvHD following cyclophosphamide (CTX administration and total body irradiation (TBI in NSG mice. Engraftment was assessed in peripheral blood (PB and in specific target organs by either flow cytometry or immunohistochemistry (IHC. Tissue samples were harvested 56 days post transplantation and were evaluated by a pathologist. Some mice were kept for up to 84 days to evaluate the degree of fibrosis. Mice that received CTX at 20mg/kg did not show aGvHD with stable expansion of human CD45+ CD3+ T-cells in PB (mean; 5.8 to 23.2%. The pathology and fibrosis scores in the lung and the liver were significantly increased with aggregation of T-cells and hCD68+ macrophages. There was a correlation between liver pathology score and the percentage of hCD68+ cells, suggesting the role of macrophage in fibrogenesis in NSG mice. In order to study long-term survival, 6/9 mice who survived more than 56 days showed increased fibrosis in the lung and liver at the endpoint, which suggests the infiltrating hCD68+ macrophages may be pathogenic. It was shown that the combination of CTX and TBI with a low number of G-hPBMCs (1x106 leads to chronic lung and liver inflammation driven by a high infiltration of human macrophage and mature human T cells from the graft, resulting in fibrosis of lung and liver in NSG mice. In conclusion this model may serve as an important pre-clinical model to further current understanding of the roles of human macrophages in cGvHD.

  9. Humanized Chronic Graft-versus-Host Disease in NOD-SCID il2rγ-/- (NSG) Mice with G-CSF-Mobilized Peripheral Blood Mononuclear Cells following Cyclophosphamide and Total Body Irradiation.

    Science.gov (United States)

    Fujii, Hisaki; Luo, Zhi-Juan; Kim, Hye Jin; Newbigging, Susan; Gassas, Adam; Keating, Armand; Egeler, R Maarten

    2015-01-01

    Chronic graft-versus-host disease (cGvHD) is the major source of late phase morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Humanized acute GvHD (aGvHD) in vivo models using NOD-SCID il2rγ-/- (NSG) mice are well described and are important tools for investigating pathogenicity of human cells in vivo. However, there have been only few reported humanized cGvHD mouse models. We evaluated if prolonged inflammation driven by low dose G-CSF-mobilized human PBMCs (G-hPBMCs) would lead to cGvHD following cyclophosphamide (CTX) administration and total body irradiation (TBI) in NSG mice. Engraftment was assessed in peripheral blood (PB) and in specific target organs by either flow cytometry or immunohistochemistry (IHC). Tissue samples were harvested 56 days post transplantation and were evaluated by a pathologist. Some mice were kept for up to 84 days to evaluate the degree of fibrosis. Mice that received CTX at 20mg/kg did not show aGvHD with stable expansion of human CD45+ CD3+ T-cells in PB (mean; 5.8 to 23.2%). The pathology and fibrosis scores in the lung and the liver were significantly increased with aggregation of T-cells and hCD68+ macrophages. There was a correlation between liver pathology score and the percentage of hCD68+ cells, suggesting the role of macrophage in fibrogenesis in NSG mice. In order to study long-term survival, 6/9 mice who survived more than 56 days showed increased fibrosis in the lung and liver at the endpoint, which suggests the infiltrating hCD68+ macrophages may be pathogenic. It was shown that the combination of CTX and TBI with a low number of G-hPBMCs (1x106) leads to chronic lung and liver inflammation driven by a high infiltration of human macrophage and mature human T cells from the graft, resulting in fibrosis of lung and liver in NSG mice. In conclusion this model may serve as an important pre-clinical model to further current understanding of the roles of human macrophages in cGvHD.

  10. Peripheral fat loss and decline in adipogenesis in older humans.

    Science.gov (United States)

    Caso, Giuseppe; McNurlan, Margaret A; Mileva, Izolda; Zemlyak, Alla; Mynarcik, Dennis C; Gelato, Marie C

    2013-03-01

    Aging is associated with a redistribution of body fat including a relative loss of subcutaneous peripheral fat. These changes in body fat can have important clinical consequences since they are linked to increased risk of metabolic complications. The causes and mechanisms of loss of peripheral fat associated with aging are not clear. The aim of this study was to assess whether defects in adipogenesis contribute to fat loss in aging humans, as suggested from animal studies, and to evaluate the role of inflammation on pathogenesis of fat loss. Preadipocytes isolated from subcutaneous peripheral fat of healthy young and elderly subjects were compared in their ability to replicate and differentiate. The results show that both the rate of replication and differentiation of preadipocytes are reduced in older subjects. The reduction in adipogenesis is accompanied by a higher plasma level of the inflammatory marker, soluble tumor necrosis factor receptor 2, and greater release of tumor necrosis factor α from fat tissue. Thus, the gradual relative loss of peripheral fat in aging humans may in part result from a defect in adipogenesis, which may be linked to inflammation and increased release of proinflammatory cytokines from fat tissue. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Activation of the peripheral endocannabinoid system in human obesity.

    Science.gov (United States)

    Engeli, Stefan; Böhnke, Jana; Feldpausch, Mareike; Gorzelniak, Kerstin; Janke, Jürgen; Bátkai, Sándor; Pacher, Pál; Harvey-White, Judy; Luft, Friedrich C; Sharma, Arya M; Jordan, Jens

    2005-10-01

    Obesity is the main risk factor for the development of type 2 diabetes. Activation of the central endocannabinoid system increases food intake and promotes weight gain. Blockade of the cannabinoid type 1 (CB-1) receptor reduces body weight in animals by central and peripheral actions; the role of the peripheral endocannabinoid system in human obesity is now being extensively investigated. We measured circulating endocannabinoid concentrations and studied the expression of CB-1 and the main degrading enzyme, fatty acid amide hydrolase (FAAH), in adipose tissue of lean (n = 20) and obese (n = 20) women and after a 5% weight loss in a second group of women (n = 17). Circulating levels of anandamide and 1/2-arachidonoylglycerol were increased by 35 and 52% in obese compared with lean women (P obese subjects (P endocannabinoids. Circulating endocannabinoids and CB-1 or FAAH expression were not affected by 5% weight loss. The expression of CB-1 and FAAH was increased in mature human adipocytes compared with in preadipocytes and was found in several human tissues. Our findings support the presence of a peripheral endocannabinoid system that is upregulated in human obesity.

  12. Altered effector function of peripheral cytotoxic cells in COPD

    Directory of Open Access Journals (Sweden)

    Corne Jonathan M

    2009-06-01

    Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

  13. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.

    Science.gov (United States)

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-10-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

  14. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

    Science.gov (United States)

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  15. Expression of CD44v6 gene in normal human peripheral blood

    Institute of Scientific and Technical Information of China (English)

    Jian Song; Dong-Sheng Zhang; Jie Zheng

    2005-01-01

    AIM: To investigate if CD44v6 could be used as a molecular marker of cancer progression and metastasis through the detection of CD44v6 gene expression in normal human peripheral blood.METHODS: RNA was extracted from the peripheral blood mononuclear cells of 50 healthy donors, the expression of CD44v6 was investigated using reverse transcriptasepolymerase chain reaction (RT-PCR).RESULTS: CD44v6 mRNA was detected in 58% of healthy volunteers under the proper controls.CONCLUSION: Our results suggest that the measurement of CD44v6 expression in peripheral blood by RT-PCR is not suitable for detection of circulating tumor cells.

  16. Peripheral nerve extract effects on mesenchymal cells.

    Science.gov (United States)

    Dietz, F R; Mukhopadhyay, B; Becker, G; Daniels, K; Solursh, M

    1996-01-01

    Several common congenital limb disorders are characterized by normal tissue differentiation but abnormal somatic growth. These include: idiopathic clubfoot, idiopathic leg length discrepancy, hemi-atrophy and hemi-hypertrophy. Both clinical and research studies have suggested that peripheral nerves may be important in regulating somatic growth of limb tissues. To investigate the hypothesis that peripheral nerves convey trophic substances to mesenchymal tissues that are involved in the regulation of growth, we developed an in vitro assay to assess the effect of fractions of peripheral nerve on myoblast and chondroblast growth and differentiation in a mammalian (rat) system. Whole rat sciatic nerve extract was fractionated by ammonium sulfate precipitation and by affinity chromatography. Concavalin A chromatography resolved whole nerve extract into a glycoprotein and a non-glycoprotein fraction. Serial ammonium sulfate precipitation yielded three pellet fractions designated as 35%, 70%, and 100% pellets; corresponding to ammonium sulfate concentrations of 0 to 35%, 35 to 70%, and 70 to 100% saturation, respectively. Dialyzed solutions of these pellets as well as the fractions from Concavalin A chromatography were assayed for biological activity in micromass cultures of rat limb bud mesenchyme, which allowed assessment of both myoblast and chondroblast stimulation. Stimulation of protein synthesis and myoblast proliferation (as measured by MF20 staining) occurred with both 70% and 100% ammonium sulfate fractions. Stimulation of chondroblasts (as measured by the number of alcian blue staining nodules) occurred with the 35% and 100% fractions. The glycoprotein fraction from the affinity chromatography stimulated protein synthesis and myoblast proliferation and inhibited chondroblast development. Stimulation of chondroblasts was seen with the non-glycoprotein fraction. No effect on protein synthesis, myoblast proliferation or chondroblast proliferation was found in

  17. Comparative expression profile of miRNA and mRNA in primary peripheral blood mononuclear cells infected with human immunodeficiency virus (HIV-1.

    Directory of Open Access Journals (Sweden)

    Ankit Gupta

    Full Text Available Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.

  18. Peripheral giant cell granuloma: A review of 123 cases

    Directory of Open Access Journals (Sweden)

    Niloofar Shadman

    2009-01-01

    Full Text Available Background: Peripheral giant cell granuloma is one of the reactive hyperplastic lesions of the oral cavity, which originates from the periosteum or periodontal membrane following local irritation or chronic trauma. The purpose of this study was to present the clinical characteristics of peripheral gi-ant cell granuloma in a group of Iranian population. Methods: A series of 123 consecutive confirmed cases of peripheral giant cell granuloma after biopsy were evaluated. Age, sex, anatomic location, consistency, etiologic factor, pain and bleeding history, color, surface texture, and pedicle situation were recorded and were analyzed by chi-square test and values were considered to be significant if P < 0.05. Results: Age ranged from 6 to 75 years (mean 33 years. Women affected more than men (M/F 1:1.1. Peripheral giant cell granuloma was seen in the mandible more than in the maxilla and in the anterior region more than in the posterior region. In most cases, lesions were pink, pedunculated and had non-ulcerated surface. In less than half of the cases, there was no history of bleeding and also pain was rarely reported. Calculus was the most common etiologic factor. Conclusion: The results confirmed that the clinical features of peripheral giant cell granuloma in a group of Iranian population are almost similar to those reported by other investigators.

  19. [239Pu and chromosomal aberrations in human peripheral blood lymphocytes].

    Science.gov (United States)

    Okladnikova, N D; Osovets, S V; Kudriavtseva, T I

    2009-01-01

    The genome status in somatic cells was assessed using the chromosomal aberration (CA) test in peripheral blood lymphocytes from 194 plutonium workers exposed to occupational radiation mainly from low-transportable compounds of airborne 230Pu. Pu body burden at the time of cytogenetic study varied from values close to the method sensitivity to values multiply exceeding the permissible level. Standard (routine) methods of peripheral blood lymphocytes cultivation were applied. Chromatid- and chromosomal-type structural changes were estimated. Aberrations were estimated per 100 examined metaphase cells. The quantitative relationship between the CA frequency and Pu body burden and the absorbed dose to the lung was found. Mathematical processing of results was carried out based on the phenomenological model. The results were shown as theoretical and experimental curves. The threshold of the CA yield was 0.43 +/- 0.03 kBq (Pu body burden) and 6.12 +/- 1.20 cGy (absorbed dose to the lung).

  20. Sympathetic reflex control of blood flow in human peripheral tissues

    DEFF Research Database (Denmark)

    Henriksen, O

    1991-01-01

    Sympathetic vasoconstrictor reflexes are essential for the maintenance of arterial blood pressure in upright position. It has been generally believed that supraspinal sympathetic vasoconstrictor reflexes elicited by changes in baroreceptor activity play an important role. Recent studies on human...... sympathetic vasoconstrictor reflexes are blocked. Blood flow has been measure by the local 133Xe-technique. The results indicate the presence of spinal as well as supraspinal sympathetic vasoconstrictor reflexes to human peripheral tissues. Especially is emphasized the presence of a local sympathetic veno...

  1. Liposomes to target peripheral neurons and Schwann cells.

    Directory of Open Access Journals (Sweden)

    Sooyeon Lee

    Full Text Available While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral

  2. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

    Energy Technology Data Exchange (ETDEWEB)

    Bloom, J.W.; Halonen, M.; Yamamura, H.I.

    1988-02-01

    The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and the classical muscarinic antagonist (/sup 3/H)(-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for (/sup 3/H)PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for (/sup 3/H)(-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind (/sup 3/H)PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (/sup 3/H)(-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

  3. Peripheral vasodilatation determines cardiac output in exercising humans

    DEFF Research Database (Denmark)

    Bada, A A; Svendsen, J H; Secher, N H

    2012-01-01

    conditions, because of a parallel decrease in stroke volume (P exercise. Atrial pacing lowered central venous pressure (P ...In dogs, manipulation of heart rate has no effect on the exercise-induced increase in cardiac output. Whether these findings apply to humans remain uncertain, because of the large differences in cardiovascular anatomy and regulation. To investigate the role of heart rate and peripheral...... vasodilatation in the regulation of cardiac output during steady-state exercise, we measured central and peripheral haemodynamics in 10 healthy male subjects, with and without atrial pacing (100–150 beats min(−1)) during: (i) resting conditions, (ii) one-legged knee extensor exercise (24 W) and (iii) femoral...

  4. Dendritic cells in peripheral tolerance and immunity

    DEFF Research Database (Denmark)

    Gad, Monika; Claesson, Mogens Helweg; Pedersen, Anders Elm

    2003-01-01

    Dendritic cells capable of influencing immunity exist as functionally distinct subsets, T cell-tolerizing and T cell-immunizing subsets. The present paper reviews how these subsets of DCs develop, differentiate and function in vivo and in vitro at the cellular and molecular level. In particular...

  5. Steady state peripheral blood provides cells with functional and metabolic characteristics of real hematopoietic stem cells.

    Science.gov (United States)

    Bourdieu, Antonin; Avalon, Maryse; Lapostolle, Véronique; Ismail, Sadek; Mombled, Margaux; Debeissat, Christelle; Guérinet, Marianne; Duchez, Pascale; Chevaleyre, Jean; Vlaski-Lafarge, Marija; Villacreces, Arnaud; Praloran, Vincent; Ivanovic, Zoran; Brunet de la Grange, Philippe

    2018-01-01

    Hematopoietic stem cells (HSCs), which are located in the bone marrow, also circulate in cord and peripheral blood. Despite high availability, HSCs from steady state peripheral blood (SSPB) are little known and not used for research or cell therapy. We thus aimed to characterize and select HSCs from SSPB by a direct approach with a view to delineating their main functional and metabolic properties and the mechanisms responsible for their maintenance. We chose to work on Side Population (SP) cells which are highly enriched in HSCs in mouse, human bone marrow, and cord blood. However, no SP cells from SSBP have as yet been characterized. Here we showed that SP cells from SSPB exhibited a higher proliferative capacity and generated more clonogenic progenitors than non-SP cells in vitro. Furthermore, xenotransplantation studies on immunodeficient mice demonstrated that SP cells are up to 45 times more enriched in cells with engraftment capacity than non-SP cells. From a cell regulation point of view, we showed that SP activity depended on O2 concentrations close to those found in HSC niches, an effect which is dependent on both hypoxia-induced factors HIF-1α and HIF-2α. Moreover SP cells displayed a reduced mitochondrial mass and, in particular, a lower mitochondrial activity compared to non-SP cells, while they exhibited a similar level of glucose incorporation. These results provided evidence that SP cells from SSPB displayed properties of very primitive cells and HSC, thus rendering them an interesting model for research and cell therapy. © 2017 Wiley Periodicals, Inc.

  6. Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells.

    Science.gov (United States)

    Hiwarkar, Prashant; Qasim, Waseem; Ricciardelli, Ida; Gilmour, Kimberly; Quezada, Sergio; Saudemont, Aurore; Amrolia, Persis; Veys, Paul

    2015-12-24

    Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

  7. Two small lymphocyte subpopulations in human peripheral blood. I. Purification and surface marker profiles

    DEFF Research Database (Denmark)

    Hokland, M; Hokland, P; Heron, I

    1978-01-01

    By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form simultan......By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form...... population' was shown to be highly variable as judged by the surface markers applied after 4 days of culture, and it is suggested that Null cells contain a number of immature lymphoid cells that may acquire their surface marker during culture. It is concluded that the methods described for purification...

  8. Induction and identification of rabbit peripheral blood derived dendritic cells

    Science.gov (United States)

    Zhou, Jing; Yang, FuYuan; Chen, WenLi

    2012-03-01

    Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

  9. Optimization of gene transfer into primitive human hematopoietic cells of granulocyte-colony stimulating factor-mobilized peripheral blood using low-dose cytokines and comparison of a gibbon ape leukemia virus versus an RD114-pseudotyped retroviral vector.

    Science.gov (United States)

    van der Loo, Johannes C M; Liu, B L; Goldman, A I; Buckley, S M; Chrudimsky, K S

    2002-07-20

    Primitive human hematopoietic cells in granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) are more difficult to transduce compared to cells from umbilical cord blood. Based on the hypothesis that MPB cells may require different stimulation for efficient retroviral infection, we compared several culture conditions known to induce cycling of primitive hematopoietic cells. MPB-derived CD34(+) cells were stimulated in the presence or absence of the murine fetal liver cell line AFT024 in trans-wells with G-CSF, stem cell factor (SCF), and thrombopoietin (TPO) (G/S/T; 100 ng/ml) or Flt3-L, SCF, interleukin (IL)-7, and TPO (F/S/7/T; 10-20 ng/ml), and transduced using a GaLV-pseudotyped retroviral vector expressing the enhanced green fluorescence protein (eGFP). Compared to cultures without stroma, the presence of AFT024 increased the number of transduced colony-forming cells (CFC) by 3.5-fold (with G/S/T), long-term culture-initiating cells (LTC-IC) by 4.6-fold (with F/S/7/T), and nonobese diabetic/severe immunodeficiency disease (NOD/SCID)-repopulating cells (SRC) by 6.8-fold (with F/S/7/T). Similar numbers of long-term culture-initiating cells (LTC-IC) and SRC could be transduced using AFT024-conditioned medium (AFT-CM) or a defined medium that had been supplemented with factors identified in AFT-CM. Finally, using our best condition based on transduction with the gibbon ape leukemia virus (GaLV)-pseudotyped vector, we demonstrate a 33-fold higher level of gene transfer (p optimized protocol with low doses of cytokines, and transduction with an RD114 compared to a GaLV-pseudotyped retroviral vector, the overall number of transduced cells in NOD/SCID mice could be improved 144-fold, with a gene-transfer efficiency in SRC of 16.3% (13.3-19.9; n = 6).

  10. Genotoxicity test of self-renovated ceramics in primary human peripheral lymphocytes.

    Science.gov (United States)

    Hua, Nan; Zhu, Huifang; Zhuang, Jing; Chen, Liping

    2014-12-01

    Zirconia-based ceramics is widely used in dentistry. Different compositions of ceramics have different features. Our self-renovated ceramics become more machinable without scarifying its dental restoration properties after adjusting ratio of lanthanum phosphate (LaPO4)/yttrium oxide (Y2O3). In order to evaluate its safety, here, we tested its genotoxicity in primary human peripheral lymphocytes. The human lymphocytes cultured on three groups of different ratios of LaPO4/Y2O3 diphase ceramics for 6 days showed little effect of growth inhibition and similar effect of growth trend to the negative control. Furthermore, single-cell gel electrophoresis (comet assay) indicated that there was no significant difference of the value of tail moment between the tested ceramics and negative control, the IPS Empress II (P > 0.05). Our findings implicate that our self-renovated ceramics do not induce DNA damages in human peripheral lymphocytes and support their future clinic application.

  11. Two small lymphocyte subpopulations in human peripheral blood. I. Purification and surface marker profiles

    DEFF Research Database (Denmark)

    Hokland, M; Hokland, P; Heron, I

    1978-01-01

    By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form simultan......By means of simple rosette sedimentation methods two subsets from human peripheral blood lymphocytes have been isolated: (1) (E, Fc)- and (2) (E, Ig)-. The first subset was obtained by centrifuging suspensions of macrophage-depleted PBL in which E and EA rosettes had been allowed to form...... of small subsets of human lymphocytes are effective and easy to perform and might be used to purify cells for functional studies. Udgivelsesdato: 1978-null...

  12. Peripheral canine CD4(+)CD8(+) double-positive T cells - unique amongst others.

    Science.gov (United States)

    von Buttlar, Heiner; Bismarck, Doris; Alber, Gottfried

    2015-12-15

    T lymphocytes co-expressing CD4 and CD8 ("double-positive T cells") are commonly associated with a thymic developmental stage of T cells. Their first description in humans and pigs as extrathymic T cells with a memory phenotype almost 30 years ago came as a surprise. Meanwhile peripheral double-positive T cells have been described in a growing number of different species. In this review we highlight novel data from our very recent studies on canine peripheral double-positive T cells which point to unique features of double-positive T cells in the dog. In contrast to porcine CD4(+)CD8(+) T cells forming a homogenous cellular population based on their expression of CD4 and CD8α, canine CD4(+)CD8(+) T cells can be divided into three different cellular subsets with distinct expression levels of CD4 and CD8α. Double-positive T cells expressing CD8β are present in humans and dogs but absent in swine. Moreover, canine CD4(+)CD8(+) T cells can not only develop from CD4(+) single-positive T cells but also from CD8(+) single-positive T cells. Together, this places canine CD4(+)CD8(+) T cells closer to their human than porcine counterparts since human double-positive T cells also appear to be heterogeneous in their CD4 and CD8α expression and have both CD4(+) and CD8(+) T cells as progenitor cells. However, CD4(+) single-positive T cells are the more potent progenitors for canine double-positive T cells, whereas CD8(+) single-positive T cells are more potent progenitors for human double-positive T cells. Canine double-positive T cells have an activated phenotype and may have as yet unrecognized roles in vivo in immunity to infection or in inflammatory diseases such as chronic infection, autoimmunity, allergy, or cancer.

  13. Relationship between Single Nucleotide Polymorphism in TNF-α Gene Promoter Region and Inhibitory Effects of Triptolide on TNF-α Production in Peripheral Blood Mononuclear Cells of Healthy Humans

    Institute of Scientific and Technical Information of China (English)

    TU Shenghao; CHEN Hongbo; SHENG Dongyun; HU Yonghong; LIU Peilin

    2006-01-01

    The relationship between tumour necrosis factor-α (TNF-α) gene polymorphism and inhibitory effects of triptolide on TNF-α production from peripheral blood mononuclear cells (PBMC)of healthy humans was investigated. Genomic DNA from 41 healthy people was typed for TNF-α-308 polymorphism by allele-specific polymorphism chain reaction (AS-PCR). The TNF-α concentration in the supernatant was measured by ELISA. The results showed that the production of TNF-α from TNF-α -308 non-G/G genotype PBMC was higher than that from TNF-α-308 G/G genotype PBMC after stimulated by LPS. Triptolide could lower the production of TNF-α from G/G genotype PBMC, but had no effect on the level of TNF-α from non-G/G genotype PBMC. It was concluded that TNF-α gene polymorphism was related to the TNF-α production from triptolide-inhibited PBMC culture in healthy humans.

  14. Cooperative Effects of Corticosteroids and Catecholamines upon Immune Deviation of the Type-1/Type-2 Cytokine Balance in Favor of Type-2 Expression in Human Peripheral Blood Mononuclear Cells

    Science.gov (United States)

    Salicru, A. N.; Sams, Clarence F.; Marshall, G. D.

    2007-01-01

    A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high levels of catecholamines (CT) and corticosteroids (CS). Although both CS and CT individually can inhibit the production of T-helper 1 (TH1, type-1 like) cytokines and simultaneously promote the production of T-helper 2 (TH2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination CT and CS in immune responses that may be more physiologically relevant. We therefore investigated the combined effects of in vitro CT and CS upon the type-1/type-2 cytokine balance of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of superimposed acute and chronic stress. Results demonstrated a significant decrease in type-1 cytokine production (IFN-gamma) and a significant increase in type-2 cytokine production (IL-4, IL-10) in our CS+CT incubated cultures when compared to either CT or CS agents alone. Furthermore, variable enhancement of type-1/type-2 immune deviation occurred depending upon when the CT was added. The data suggest that CS can increase the sensitivity of PBMC to the immunomodulatory effects of CT and establishes an in vitro model to study the combined effects of in vivo type-1/type-2 cytokine alterations observed in acute and chronic stress.

  15. Reconstitution of the subclass-specific expression of CD4 in thymocytes and peripheral T cells of transgenic mice: identification of a human CD4 enhancer

    OpenAIRE

    1993-01-01

    During thymic maturation, CD4-CD8-TCR- immature thymocytes differentiate through a CD4+CD8+TCRlo intermediate into two functionally distinct mature T cell subsets: helper T cells expressing CD4 and a major histocompatibility complex (MHC) class II-restricted T cell receptor (TCR), and cytotoxic T cells expressing CD8 and and MHC class I-restricted TCR. The mutually exclusive expression of CD4 and CD8 is maintained in the periphery during expansion of these mature T cell subsets. To elucidate ...

  16. 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

  17. Schwann cells express erythropoietin receptor and represent a major target for Epo in peripheral nerve injury.

    Science.gov (United States)

    Li, Xiaoqing; Gonias, Steven L; Campana, W Marie

    2005-09-01

    Erythropoietin (Epo) expresses potent neuroprotective activity in the peripheral nervous system; however, the underlying mechanism remains incompletely understood. In this study, we demonstrate that Epo is upregulated in sciatic nerve after chronic constriction injury (CCI) and crush injury in rats, largely due to local Schwann cell production. In uninjured and injured nerves, Schwann cells also express Epo receptor (EpoR), and its expression is increased during Wallerian degeneration. CCI increased the number of Schwann cells at the injury site and the number was further increased by exogenously administered recombinant human Epo (rhEpo). To explore the activity of Epo in Schwann cells, primary cultures were established. These cells expressed cell-surface Epo receptors, with masses of 71 and 62 kDa, as determined by surface protein biotinylation and affinity precipitation. The 71-kDa species was rapidly but transiently tyrosine-phosphorylated in response to rhEpo. ERK/MAP kinase was also activated in rhEpo-treated Schwann cells; this response was blocked by pharmacologic antagonism of JAK-2. RhEpo promoted Schwann cell proliferation, as determined by BrdU incorporation. Cell proliferation was ERK/MAP kinase-dependent. These results support a model in which Schwann cells are a major target for Epo in injured peripheral nerves, perhaps within the context of an autocrine signaling pathway. EpoR-induced cell signaling and Schwann cell proliferation may protect injured peripheral nerves and promote regeneration.

  18. Thyroxine differentially modulates the peripheral clock: lessons from the human hair follicle.

    Directory of Open Access Journals (Sweden)

    Jonathan A Hardman

    Full Text Available The human hair follicle (HF exhibits peripheral clock activity, with knock-down of clock genes (BMAL1 and PER1 prolonging active hair growth (anagen and increasing pigmentation. Similarly, thyroid hormones prolong anagen and stimulate pigmentation in cultured human HFs. In addition they are recognized as key regulators of the central clock that controls circadian rhythmicity. Therefore, we asked whether thyroxine (T4 also influences peripheral clock activity in the human HF. Over 24 hours we found a significant reduction in protein levels of BMAL1 and PER1, with their transcript levels also decreasing significantly. Furthermore, while all clock genes maintained their rhythmicity in both the control and T4 treated HFs, there was a significant reduction in the amplitude of BMAL1 and PER1 in T4 (100 nM treated HFs. Accompanying this, cell-cycle progression marker Cyclin D1 was also assessed appearing to show an induced circadian rhythmicity by T4 however, this was not significant. Contrary to short term cultures, after 6 days, transcript and/or protein levels of all core clock genes (BMAL1, PER1, clock, CRY1, CRY2 were up-regulated in T4 treated HFs. BMAL1 and PER1 mRNA was also up-regulated in the HF bulge, the location of HF epithelial stem cells. Together this provides the first direct evidence that T4 modulates the expression of the peripheral molecular clock. Thus, patients with thyroid dysfunction may also show a disordered peripheral clock, which raises the possibility that short term, pulsatile treatment with T4 might permit one to modulate circadian activity in peripheral tissues as a target to treat clock-related disease.

  19. Effect of cell-surface components and metabolites of lactic acid bacteria and probiotic organisms on cytokine production and induction of CD25 expression in human peripheral mononuclear cells.

    Science.gov (United States)

    Ashraf, R; Vasiljevic, T; Smith, S C; Donkor, O N

    2014-05-01

    In the current study, the relative contribution of cell-surface components (CSC) and cell-free supernatants (CFS) in the immuno-modulatory properties of 17 strains of probiotic and lactic acid bacteria (LAB) was assessed. The production of pro- and antiinflammatory cytokines including IL-2, IL-4, IL-10, IL-12 p70, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β was measured at different time points after stimulation of buffy coat derived-peripheral blood mononuclear cells (PBMC) from healthy donors with CSC and CFS of probiotic and LAB. Results showed that CSC of probiotic and LAB strains induced production of T helper 1 and 2 type cytokines. Transforming growth factor-β was stimulated at highest concentrations, followed by IL-10 and TNF-α. The CFS of all tested bacterial strains induced PBMC for significantly high levels of IL-10 secretion compared with unstimulated cells, but the values were less than lipopolysaccharide-stimulated cells. Cytokines due to CFS stimulation showed declined concentration for IL-2, TNF-α, and IL-4, and complete disappearance of IL-12, IFN-γ, and transforming growth factor-β in the cultured medium at 96 h of incubation. Results of cytokine data demonstrate proinflammatory TNF-α immune responses are mainly directed through cell-surface structures of probiotic and LAB, but antiinflammatory immune responses are mediated both by metabolites and cell-surfaces of these bacteria. The induction of CD4(+)CD25(+) regulatory T cells after stimulation of PBMC with CSC and CFS of probiotic and LAB showed regulatory T cell activity appeared to be influenced both by the CSC and metabolites, but was principally triggered by cell surfaces of probiotic and LAB strains.

  20. Critical influence of the thymus on peripheral T cell homeostasis

    Science.gov (United States)

    Vianna, Pedro Henrique Oliveira; Canto, Fábio B.; Nogueira, Jeane S.; Nunes, Caroline Fraga Cabral Gomes; Bonomo, Adriana César

    2016-01-01

    Abstract Introduction A tight balance between regulatory CD4+Foxp3+ (Treg) and conventional CD4+Foxp3− (Tconv) T cell subsets in the peripheral compartment, maintained stable throughout most of lifetime, is essential for preserving self‐tolerance along with efficient immune responses. An excess of Treg cells, described for aged individuals, may critically contribute to their reported immunodeficiency. In this work, we investigated if quantitative changes in thymus emigration may alter the Treg/Tconv homeostasis regardless of the aging status of the peripheral compartment. Methods We used two different protocols to modify the rate of thymus emigration: thymectomy of adult young (4–6 weeks old) mice and grafting of young thymus onto aged (18 months old) hosts. Additionally, lymphoid cells from young and aged B6 mice were intravenously transferred to B6.RAG2−/− mice. Alterations in Treg and Tconv peripheral frequencies following these protocols were investigated after 30 days by flow cytometry. Results Thymectomized young mice presented a progressive increase in the Treg cell frequency, while the grafting of a functional thymus in aged mice restored the young‐like physiological Treg/Tconv proportion. Strikingly, T cells derived from young or aged splenocytes colonized the lymphopenic periphery of RAG−/− hosts to the same extent, giving rise to similarly elevated Treg cell levels irrespective of the age of the donor population. In the absence of thymus output, the Treg subset seems to survive longer, as confirmed by their lower proportion of Annexin‐V+ cells. Conclusions Our data suggest that the thymus‐emigrating population, harboring an adequate proportion of Treg/Tconv lymphocytes, may be essential to keep the Treg cell balance, independently of age‐related shifts intrinsic to the peripheral environment or to the T cell biology. PMID:27980781

  1. [The Analysis for Probable Reasons of Cd4+ T-Cell Activation Non-Linear Dependence on Extra Cellular Calcium Ion Concentration in Human Peripheral Blood in vitro].

    Science.gov (United States)

    Litvinov, I S

    2015-01-01

    The analysis for probable reasons of CD4+ T-cell activation non-linear dependence on [Ca2+]o in HPB in vitro is the general aim of current work. At the beginning we pursued the analysis of receptor-dependent (the mixture of monoclonal antibodies (mAbs) to CD3 and CD28 molecules) and receptor-independent (phorbol-myristate-acetate and ionomycin mixture) means to activate T cells in vitro with different [Ca2+]o in HPB. The key role of intracellular T-cell signaling systems in activated T cells in their non-similar sensitivity to calcium ions in the blood was shown. The analysis of differentiation next stages of CD4+ T-cell activation in vitro relatively [Ca2+]o in PHB demonstrates the key role of the earliest induction stages in non-similar sensitivity to calcium ions in CD4+ T-cell activation in vitro. According to the pursued analysis; the non-similar sensitivity of CD4+ T-cell in vitro to activation is in no-way connected with pace differences on the primary stages of activation process. The comparison of CD4+ memory T cells with their naive T-cell precursors in the cell activation process in hypocalcemia conditions was made in the separate experimental series. The 1st maximum consists in average of 85% CD4+CD45R0high CD69+ memory T cells. Naive CD4+CD45RAlowCD69+ T cells constitute the remainder 15%. The 2nd maximum almost completely consists of CD4+CD45R0+CD69+ memory T cells. The ratio between CD4+CD69+ T cell maximums depends on donor ages and represents linear dependence with R = -0.981. The most probable candidate on the role of CD4+ T cell, being capable of activation in hypocalcemia conditions, are memory T lymphocytes, being resistant to ionomycin action (I R) subset. To check this assumption the mononuclear cells and their IR-fraction were prepared from donor PB. Then the mononuclear cells and their IR-fraction were activated by mAbs mixture at different [EGTA] values. For IR-fraction, enriched with CD4+CD45RA-CD45R0+ memory T cells, slightly seen 1st

  2. Convergences and divergences of thymus- and peripherally-derived regulatory T cells in cancer

    OpenAIRE

    ALESSIA eBUROCCHI; MARIO PAOLO COLOMBO; Silvia ePICONESE

    2013-01-01

    The expansion of regulatory T cells (Treg) is a common event characterizing the vast majority of human and experimental tumors and it is now well established that Treg represent a crucial hurdle for a successful immunotherapy. Treg are currently classified, according to their origin, into thymus-derived (tTreg) or peripherally induced (pTreg) cells. Controversy exists over the prevalent mechanism accounting for Treg expansion in tumors, since both tTreg proliferation and de novo pTreg differe...

  3. Dentinogenic Ghost Cell Tumor of the Peripheral Variant Mimicking Epulis

    Directory of Open Access Journals (Sweden)

    Uddipan Kumar

    2010-01-01

    Full Text Available Dentinogenic ghost cell tumor (DGCT is an uncommon locally invasive odontogenic tumor regarded by many as a variant of calcifying odontogenic cyst. The peripheral variant of this clinical rarity appears as a well-circumscribed mass mimicking a nonspecific gingival enlargement. Microscopic appearance of odontogenic epithelium admixed with focal areas of dentinoid formation and sheets of ghost cells giving the definitive diagnosis of dentinogenic ghost cell tumor imply that microscopic examination is compulsory for any gingival mass. Van Gieson histochemical stain further confirmed the nature of dentinoid-like material. A complete workup of a case of peripheral dentinogenic ghost cell tumor is presented in this paper and the current concept as well as the appraisal of literature is presented.

  4. Regulation of cell adhesion strength by peripheral focal adhesion distribution.

    Science.gov (United States)

    Elineni, Kranthi Kumar; Gallant, Nathan D

    2011-12-21

    Cell adhesion to extracellular matrices is a tightly regulated process that involves the complex interplay between biochemical and mechanical events at the cell-adhesive interface. Previous work established the spatiotemporal contributions of adhesive components to adhesion strength and identified a nonlinear dependence on cell spreading. This study was designed to investigate the regulation of cell-adhesion strength by the size and position of focal adhesions (FA). The cell-adhesive interface was engineered to direct FA assembly to the periphery of the cell-spreading area to delineate the cell-adhesive area from the cell-spreading area. It was observed that redistributing the same adhesive area over a larger cell-spreading area significantly enhanced cell-adhesion strength, but only up to a threshold area. Moreover, the size of the peripheral FAs, which was interpreted as an adhesive patch, did not directly govern the adhesion strength. Interestingly, this is in contrast to the previously reported functional role of FAs in regulating cellular traction where sizes of the peripheral FAs play a critical role. These findings demonstrate, to our knowledge for the first time, that two spatial regimes in cell-spreading area exist that uniquely govern the structure-function role of FAs in regulating cell-adhesion strength.

  5. Primary cutaneous peripheral T-cell lymphoma, unspecified with an indolent clinical course: a distinct peripheral T-cell lymphoma?

    LENUS (Irish Health Repository)

    Ryan, A J A

    2012-02-01

    Primary cutaneous peripheral T-cell lymphomas (PTL), unspecified, are rare lymphomas, with a poor prognosis. They grow and disseminate rapidly, leading to widespread disease. We report a case of PTL, unspecified occurring on the nose. Despite its aggressive histology, this tumour behaved indolently. It is remarkably similar, clinically and histologically, to four recently described cases that occurred on the ear.

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

    NARCIS (Netherlands)

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

    2017-01-01

    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

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

    NARCIS (Netherlands)

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

    2017-01-01

    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 outbr

  8. Evaluation of genotoxic activity of tenofovir disoproxil fumarate in human peripheral lymphocytes

    Directory of Open Access Journals (Sweden)

    Kubra Kurt

    2016-06-01

    Full Text Available Purpose: Antiretroviral drugs used in the treatment of HIV (Human Immunodeficiency Virus iinfection, treat by preventing the proliferation of HIV in human body. People with HIV have to use this drugs for lifelong because of inability of the drugs to eradicate the viruses. In this study, we investigated the in vitro genotoxic activity of tenofovir disoproxil fumarate one of the antiretroviral drugs, in human peripheral lymphocytes. Material and Methods: The cells were treated with four different concentrations of tenofovir disoproxil fumarate for 24 and 48 hours. The levels of sister chromatid exchanges, chromosomal aberrations, and micronucleus in the cells were examined for the genotoxic activity of tenofovir disoproxil fumarate. Mitotic index, proliferation index, and nucleer division index of treated cells were also determined for the cytotoxic effect of tenofovir disoproxil fumarate. Results: There was no significant differences in the level of sister chromatid exchanges, chromosomal aberrations, and micronucleus in human lyphocytes treated with all concetrations of tenofovir disoproxil fumarate for all treatment period as compared to control group. Similarly, it was observed that treatment of tenofovir disoproxil fumarate did not affect the mitotic index, proliferation index, and nucleer division index values. Conclusion: As a result, in this study, it is demonstrated that tenofovir disoproxil fumarate did not have genotoxic or cytotoxic effect in the human peripheral lymphocytes. [Cukurova Med J 2016; 41(2.000: 229-235

  9. Sensory Adaptation in Naive Peripheral CD4 T Cells

    OpenAIRE

    Smith, Katy; Seddon, Benedict; Purbhoo, Marco A.; Zamoyska, Rose; Fisher, Amanda G.; Merkenschlager, Matthias

    2001-01-01

    T cell receptor interactions with peptide/major histocompatibility complex (pMHC) ligands control the selection of T cells in the thymus as well as their homeostasis in peripheral lymphoid organs. Here we show that pMHC contact modulates the expression of CD5 by naive CD4 T cells in a process that requires the continued expression of p56lck. Reduced CD5 levels in T cells deprived of pMHC contact are predictive of elevated Ca2+ responses to subsequent TCR engagement by anti-CD3 or nominal anti...

  10. 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.

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

    OpenAIRE

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

    2017-01-01

    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...

  12. Lenalidomide Therapy for Patients With Relapsed and/or Refractory, Peripheral T-Cell Lymphomas

    Science.gov (United States)

    2012-04-18

    Peripheral T-cell Lymphomas; Adult T-cell Leukemia; Adult T-cell Lymphoma; Peripheral T-cell Lymphoma Unspecified; Angioimmunoblastic T-cell Lymphoma; Anaplastic Large Cell Lymphoma; T/Null Cell Systemic Type; Cutaneous t-Cell Lymphoma With Nodal/Visceral Disease

  13. Hydrogen sulfide is essential for Schwann cell responses to peripheral nerve injury.

    Science.gov (United States)

    Park, Byung Sun; Kim, Hyun-Wook; Rhyu, Im Joo; Park, Chan; Yeo, Seung Geun; Huh, Youngbuhm; Jeong, Na Young; Jung, Junyang

    2015-01-01

    Hydrogen sulfide (H2 S) functions as a physiological gas transmitter in both normal and pathophysiological cellular events. H2 S is produced from substances by three enzymes: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST). In human tissues, these enzymes are involved in tissue-specific biochemical pathways for H2 S production. For example, CBS and cysteine aminotransferase/MST are present in the brain, but CSE is not. Thus, we examined the expression of H2 S production-related enzymes in peripheral nerves. Here, we found that CSE and MST/cysteine aminotransferase, but not CBS, were present in normal peripheral nerves. In addition, injured sciatic nerves in vivo up-regulated CSE in Schwann cells during Wallerian degeneration (WD); however, CSE was not up-regulated in peripheral axons. Using an ex vivo sciatic nerve explant culture, we found that the inhibition of H2 S production broadly prevented the process of nerve degeneration, including myelin fragmentation, axonal degradation, Schwann cell dedifferentiation, and Schwann cell proliferation in vitro and in vivo. Thus, these results indicate that H2 S signaling is essential for Schwann cell responses to peripheral nerve injury. Hydrogen sulfide (H2 S) functions as a physiological gas transmitter in both normal and pathophysiological cellular events. H2 S is produced from cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (MST). Here, we found that CSE and MST/CAT were present in normal peripheral nerves. Injured static nerves in vivo up-regulated CSE in Schwann cells during Wallerian degeneration, but CSE was not up-regulated in peripheral axons.

  14. Inhibitory activity of the white wine compounds, tyrosol and caffeic acid, on lipopolysaccharide-induced tumor necrosis factor-alpha release in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Giovannini, L; Migliori, M; Filippi, C; Origlia, N; Panichi, V; Falchi, M; Bertelli, A A E; Bertelli, A

    2002-01-01

    The objective of this study was to assess whether tyrosol and caffeic acid are able to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha release. TNF is one of the most important cytokines involved in inflammatory reactions. The results show that both tyrosol and caffeic acid are able to inhibit LPS-induced TNF-alpha release from human monocytes, even at low doses. Their mechanisms of action are discussed and we conclude that high doses of the two compounds are not required to achieve effective inhibition of inflammatory reactions due to TNF-alpha release.

  15. 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.

  16. Immunomodulatory activity of a novel, synthetic beta-glucan (β-glu6 in murine macrophages and human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Xiaofei Li

    Full Text Available Natural β-glucans extracted from plants and fungi have been used in clinical therapies since the late 20th century. However, the heterogeneity of natural β-glucans limits their clinical applicability. We have synthesized β-glu6, which is an analog of the lentinan basic unit, β-(1→6-branched β-(1→3 glucohexaose, that contains an α-(1→3-linked bond. We have demonstrated the stimulatory effect of this molecule on the immune response, but the mechanisms by which β-glu6 activates innate immunity have not been elucidated. In this study, murine macrophages and human PBMCs were used to evaluate the immunomodulatory effects of β-glu6. We showed that β-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway in murine macrophages. Additionally, β-glu6 enhanced the secretion of large levels of cytokines and chemokines, including CD54, IL-1α, IL-1β, IL-16, IL-17, IL-23, IFN-γ, CCL1, CCL3, CCL4, CCL12, CXCL10, tissue inhibitor of metalloproteinase-1 (TIMP-1 and G-CSF in murine macrophages as well as IL-6, CCL2, CCL3, CCL5, CXCL1 and macrophage migration inhibitory factor (MIF in human PBMCs. In summary, it demonstrates the immunomodulatory activity of β-glu6 in innate immunity.

  17. Peripheral blood TIM-3 positive NK and CD8+ T cells throughout pregnancy

    DEFF Research Database (Denmark)

    Meggyes, Matyas; Miko, Eva; Polgar, Beata;

    2014-01-01

    of TIM-3+ peripheral blood mononuclear cells during healthy human pregnancy. METHODS OF STUDY: 57 healthy pregnant women [first trimester (n = 16); second trimester (n = 19); third trimester (n = 22)] and 30 non-pregnant controls were enrolled in the study. We measured the surface expression of TIM-3...... negative regulator of Th1 immunity and tolerance induction. Data about the TIM-3/Gal-9 pathway in the pathogenesis of human diseases is emerging, but their possible role during human pregnancy is not precisely known. The aim of our study was to investigate the number, phenotype and functional activity...... by cytotoxic T cells, NK cells and NK cell subsets as well as Galectin-9 expression by regulatory T cells by flow cytometry. We analyzed the cytokine production and cytotoxicity of TIM3+ and TIM3- CD8 T and NK cells obtained from non-pregnant and healthy pregnant women at different stages of pregnancy by flow...

  18. Synthesis of multivalent glycoconjugates containing the immunoactive LELTE peptide: effect of glycosylation on cellular activation and natural killing by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Renaudet, Olivier; Krenek, Karel; Bossu, Isabelle; Dumy, Pascal; Kádek, Alan; Adámek, David; Vanek, Ondrej; Kavan, Daniel; Gazák, Radek; Sulc, Miroslav; Bezouska, Karel; Kren, Vladimír

    2010-05-19

    Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

  19. Role of Schwann cells in the regeneration of penile and peripheral nerves

    Directory of Open Access Journals (Sweden)

    Lin Wang

    2015-01-01

    Full Text Available Schwann cells (SCs are the principal glia of the peripheral nervous system. The end point of SC development is the formation of myelinating and nonmyelinating cells which ensheath large and small diameter axons, respectively. They play an important role in axon regeneration after injury, including cavernous nerve injury that leads to erectile dysfunction (ED. Despite improvement in radical prostatectomy surgical techniques, many patients still suffer from ED postoperatively as surgical trauma causes traction injuries and local inflammatory changes in the neuronal microenvironment of the autonomic fibers innervating the penis resulting in pathophysiological alterations in the end organ. The aim of this review is to summarize contemporary evidence regarding: (1 the origin and development of SCs in the peripheral and penile nerve system; (2 Wallerian degeneration and SC plastic change following peripheral and penile nerve injury; (3 how SCs promote peripheral and penile nerve regeneration by secreting neurotrophic factors; (4 and strategies targeting SCs to accelerate peripheral nerve regeneration. We searched PubMed for articles related to these topics in both animal models and human research and found numerous studies suggesting that SCs could be a novel target for treatment of nerve injury-induced ED.

  20. Dynamic regulation of Schwann cell enhancers after peripheral nerve injury.

    Science.gov (United States)

    Hung, Holly A; Sun, Guannan; Keles, Sunduz; Svaren, John

    2015-03-13

    Myelination of the peripheral nervous system is required for axonal function and long term stability. After peripheral nerve injury, Schwann cells transition from axon myelination to a demyelinated state that supports neuronal survival and ultimately remyelination of axons. Reprogramming of gene expression patterns during development and injury responses is shaped by the actions of distal regulatory elements that integrate the actions of multiple transcription factors. We used ChIP-seq to measure changes in histone H3K27 acetylation, a mark of active enhancers, to identify enhancers in myelinating rat peripheral nerve and their dynamics after demyelinating nerve injury. Analysis of injury-induced enhancers identified enriched motifs for c-Jun, a transcription factor required for Schwann cells to support nerve regeneration. We identify a c-Jun-bound enhancer in the gene for Runx2, a transcription factor induced after nerve injury, and we show that Runx2 is required for activation of other induced genes. In contrast, enhancers that lose H3K27ac after nerve injury are enriched for binding sites of the Sox10 and early growth response 2 (Egr2/Krox20) transcription factors, which are critical determinants of Schwann cell differentiation. Egr2 expression is lost after nerve injury, and many Egr2-binding sites lose H3K27ac after nerve injury. However, the majority of Egr2-bound enhancers retain H3K27ac, indicating that other transcription factors maintain active enhancer status after nerve injury. The global epigenomic changes in H3K27ac deposition pinpoint dynamic changes in enhancers that mediate the effects of transcription factors that control Schwann cell myelination and peripheral nervous system responses to nerve injury.

  1. Central nervous system mast cells in peripheral inflammatory nociception

    Directory of Open Access Journals (Sweden)

    Ellmeier Wilfried

    2011-06-01

    Full Text Available Abstract Background Functional aspects of mast cell-neuronal interactions remain poorly understood. Mast cell activation and degranulation can result in the release of powerful pro-inflammatory mediators such as histamine and cytokines. Cerebral dural mast cells have been proposed to modulate meningeal nociceptor activity and be involved in migraine pathophysiology. Little is known about the functional role of spinal cord dural mast cells. In this study, we examine their potential involvement in nociception and synaptic plasticity in superficial spinal dorsal horn. Changes of lower spinal cord dura mast cells and their contribution to hyperalgesia are examined in animal models of peripheral neurogenic and non-neurogenic inflammation. Results Spinal application of supernatant from activated cultured mast cells induces significant mechanical hyperalgesia and long-term potentiation (LTP at spinal synapses of C-fibers. Lumbar, thoracic and thalamic preparations are then examined for mast cell number and degranulation status after intraplantar capsaicin and carrageenan. Intradermal capsaicin induces a significant percent increase of lumbar dural mast cells at 3 hours post-administration. Peripheral carrageenan in female rats significantly increases mast cell density in the lumbar dura, but not in thoracic dura or thalamus. Intrathecal administration of the mast cell stabilizer sodium cromoglycate or the spleen tyrosine kinase (Syk inhibitor BAY-613606 reduce the increased percent degranulation and degranulated cell density of lumbar dural mast cells after capsaicin and carrageenan respectively, without affecting hyperalgesia. Conclusion The results suggest that lumbar dural mast cells may be sufficient but are not necessary for capsaicin or carrageenan-induced hyperalgesia.

  2. Chlorobenzenes, lindane and dieldrin induce apoptotic alterations in human peripheral blood lymphocytes (in vitro study).

    Science.gov (United States)

    Michałowicz, Jaromir; Mokra, Katarzyna; Rosiak, Karolina; Sicińska, Paulina; Bukowska, Bożena

    2013-11-01

    In this study, we have assessed apoptotic effect of 1,2,4-trichlorobenzene, hexachlorobenzene, lindane and dieldrin on human peripheral blood lymphocytes. We observed an increase in ROS formation and a decrease in mitochondrial transmembrane potential in the cells incubated with low concentrations of all compounds studied, in particular lindane and dieldrin. ROS formation and changes in mitochondrial transmembrane potential may have influenced caspase-3 activation, a crucial enzyme in the apoptotic process. Moreover, chlorobenzenes, and in particular lindane and dieldrin changed cells' membrane permeability and induced phosphatidylserine translocation, which confirmed that they are capable of inducing apoptosis in human lymphocytes. Apoptotic changes in human lymphocytes provoked by biologically relevant concentrations of these substances suggest that they may disturb function of immunological system especially among people occupationally exposed to their action. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. 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.

  4. Human severe combined immunodeficiency disease: phenotypic and functional characteristics of peripheral B lymphocytes.

    Science.gov (United States)

    Gougeon, M L; Drean, G; Le Deist, F; Dousseau, M; Fevrier, M; Diu, A; Theze, J; Griscelli, C; Fischer, A

    1990-11-01

    Human severe combined immunodeficiency disease (SCID) includes an X-chromosome-linked type characterized by a complete absence of mature T cells, hypogammaglobulinemia but normal or elevated number of B cells, suggesting that the disease results from a block in early T cell differentiation. It has been shown that B cells from obligate carrier women of this disorder exhibit the preferential use of the nonmutant X chromosome as the active X (as shown for T cells), suggesting that the SCID gene product has a direct effect on B cells as well as on T cells. To examine this question, we analyzed the phenotypic and functional characteristics of peripheral B cells from nine infants with SCID. We found a constant absence of spontaneously expressed activation Ag on B cell membrane from all SCID patients tested which contrasts with the phenotypic pattern exhibited by age-matched infants whom all cells bearing surface Ig express the 4F2 Ag and to a lesser extent the transferrin receptor. Concurrently, B cells from SCID patients have a profound impairment in their responses to stimuli that induce in vitro B cell proliferation and differentiation. Although rIL-2 and low-Mr B cell growth factor are potent inducers of proliferation on age-matched infants' B cells, they are poorly efficient in inducing proliferation of anti-mu-activated SCID B cells. This impairment is not related to the resting B cell phenotype of SCID B cells as shown by comparison with normal resting B cells. Furthermore, we observed an apparent block in B cell differentiation inasmuch as neither rIL-2 nor rIL-6 could support SAC-activated SCID B cell differentiation, both lymphokines being very efficient in inducing SAC-activated age-matched infants' B cell or purified resting B cell differentiation. These results suggest that the SCID gene defect has a direct effect on B cells and is required during B cell maturation.

  5. Prokineticins in central and peripheral control of human reproduction.

    Science.gov (United States)

    Traboulsi, Wael; Brouillet, Sophie; Sergent, Frederic; Boufettal, Houssine; Samouh, Naima; Aboussaouira, Touria; Hoffmann, Pascale; Feige, Jean Jacques; Benharouga, Mohamed; Alfaidy, Nadia

    2015-11-01

    Prokineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases.

  6. 人外周血间充质干细胞向肝样细胞诱导分化的实验研究%The study of ex vivo differentiation of hepatocyte-like cells induced from mesenchymal stem cells of human peripheral blood

    Institute of Scientific and Technical Information of China (English)

    王骥; 周海军; 董红燕; 李向农

    2012-01-01

    Objective To culture and amplify mesenchymal stem cells from human peripheral blood, and study the possibility of differentiating them into hepatocyte - like cells in vitro and observe the function of hepatocyte - like cells. Methods Human peripheral blood was obtained sterilely by ulnar vein from 13 donors. MSCs were isolated by ficoll or human mesenchymal stem cell enrichment cocktail. Mature cells were removed by the difference of average adhesion time. Purified cells induced by recombinant HGF, FGF -4 and OSM, were identified by the specific biological functions with biochemical assays in vitro. Results We could isolate and culture MSCs from peripheral blood by cocktail procedures rather than ficoll procedures. The expression of cell surface antigens resulted in the MSCs phenotype. In differentiated MSCs, gene expression of ALB and AFP were found by RT - PCR analysis, ALB and AFP protein were confirmed to express by immunofluorescence and Western blot. The ultrastruture of the cells in transmission electron microscope showed the induced cells were hepatocyte - like cells. Conclusion The existence of MSCs in human peripheral blood is confirmed by our study. These cells can be isolated by non - invasive method and differentiate into heppatocyte - like cells. This method provides us a potential seed cells source for liver tissue engineering.%目的 探讨人外周血来源间充质干细胞(mesenchymal stem cells,MSCs)的体外分离、培养及诱导分化成有功能肝样细胞的可行性.方法 静脉采集13例成人外周血,分别应用单纯密度梯度离心法及人间充质干细胞富集试剂+密度梯度离心两步法分离MSCs;将得到的MSCs经贴壁纯化扩增,以流式细胞仪检测细胞表面抗原后,经肝细胞生长因子(HGF)、成纤维细胞生长因子-4(FGF-4)及抑瘤素M(OSM)诱导分化成肝样细胞;以RT-PCR、免疫荧光染色及Western blot检测所得细胞中白蛋白(ALB)、甲胎蛋白(AFP)在基因及蛋白水

  7. The human BDNF gene: peripheral gene expression and protein levels as biomarkers for psychiatric disorders

    Science.gov (United States)

    Cattaneo, A; Cattane, N; Begni, V; Pariante, C M; Riva, M A

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) regulates the survival and growth of neurons, and influences synaptic efficiency and plasticity. The human BDNF gene consists of 11 exons, and distinct BDNF transcripts are produced through the use of alternative promoters and splicing events. The majority of the BDNF transcripts can be detected not only in the brain but also in the blood cells, although no study has yet investigated the differential expression of BDNF transcripts at the peripheral level. This review provides a description of the human BDNF gene structure as well as a summary of clinical and preclinical evidence supporting the role of BDNF in the pathogenesis of psychiatric disorders. We will discuss several mechanisms as possibly underlying BDNF modulation, including epigenetic mechanisms. We will also discuss the potential use of peripheral BDNF as a biomarker for psychiatric disorders, focusing on the factors that can influence BDNF gene expression and protein levels. Within this context, we have also characterized, for we believe the first time, the expression of BDNF transcripts in the blood, with the aim to provide novel insights into the molecular mechanisms and signaling that may regulate peripheral BDNF gene expression levels. PMID:27874848

  8. Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Holers, V.M.; Kotzin, B.L.

    1985-09-01

    The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.

  9. Ex vivo expansion of human peripheral blood progenitors.

    Science.gov (United States)

    Chabannon, C; Herrera-Rodriguez, D; Bardin, F; Mouren, M; Novakovitch, G; Blaise, D; Maraninchi, D; Mannoni, P

    1995-01-01

    Culture of human hematopoietic progenitors on a large scale could lead to several clinical applications within the near future, including the production of differentiated and functional cells, the increase in the number of early progenitors, especially stem cells, with such use as gene transfer, or the improvement of grafts used to limit the hematological toxicity associated with high-dose chemotherapy. In this case, one can still distinguish different objectives: improvement of grafts that contain low numbers of progenitors because of prior chemotherapies or because of marrow involvement for example, and qualitative changes in the graft content that would allow to envision the disappearance, or the further reduction, in the duration of absolute neutropenia that follows delivery of high dose chemotherapy ("nadir rescue"), despite substitution of mobilized blood cells to marrow cells and the in vivo use of hematopoietic growth factors. Additional advantages may be related to tumor purging in autologous expanded cells, and to the change in the ratio between hematopoietic progenitors and immunocompetent cells in allogeneic expanded populations. Therefore it appears that in vitro expansion currently raises two types of questions: the first ones are related to the definition of clinical or biological endpoints to be achieved, the second ones are related to "bioengineering", and deal with the efficiency and safety of progenitor cell cultures to be used for clinical applications. We here present preliminary results preparing future pilot clinical studies with ex vivo cultured human hematopoietic cells.

  10. 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.

  11. Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood†

    Science.gov (United States)

    Dickinson, Alexandra J.; Meyer, Megan; Pawlak, Erica A.; Gomez, Shawn; Jaspers, Ilona; Allbritton, Nancy L.

    2015-01-01

    Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56+CD45+CD3−CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine–fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting a

  12. Analysis of sphingosine kinase activity in single natural killer cells from peripheral blood.

    Science.gov (United States)

    Dickinson, Alexandra J; Meyer, Megan; Pawlak, Erica A; Gomez, Shawn; Jaspers, Ilona; Allbritton, Nancy L

    2015-04-01

    Sphingosine-1-phosphate (S1P), a lipid second messenger formed upon phosphorylation of sphingosine by sphingosine kinase (SK), plays a crucial role in natural killer (NK) cell proliferation, migration, and cytotoxicity. Dysregulation of the S1P pathway has been linked to a number of immune system disorders and therapeutic manipulation of the pathway has been proposed as a method of disease intervention. However, peripheral blood NK cells, as identified by surface markers (CD56(+)CD45(+)CD3(-)CD16) consist of a highly diverse population with distinct phenotypes and functions and it is unknown whether the S1P pathway is similarly diverse across peripheral blood NK cells. In this work, we measured the phosphorylation of sphingosine-fluorescein (SF) and subsequent metabolism of S1P fluorescein (S1PF) to form hexadecanoic acid fluorescein (HAF) in 111 single NK cells obtained from the peripheral blood of four healthy human subjects. The percentage of SF converted to S1PF or HAF was highly variable amongst the cells ranging from 0% to 100% (S1PF) and 0% to 97% (HAF). Subpopulations of cells with varying levels of S1PF formation and metabolism were readily identified. Across all subjects, the average percentage of SF converted to S1PF or HAF was 37 ± 36% and 12 ± 19%, respectively. NK cell metabolism of SF by the different subjects was also distinct with hierarchical clustering suggesting two possible phenotypes: low (50%) producers of S1PF. The heterogeneity of SK and downstream enzyme activity in NK cells may enable NK cells to respond effectively to a diverse array of pathogens as well as incipient tumor cells. NK cells from two subjects were also loaded with S1PF to assess the activity of S1P phosphatase (S1PP), which converts S1P to sphingosine. No NK cells (n = 41) formed sphingosine, suggesting that S1PP was minimally active in peripheral blood NK cells. In contrast to the SK activity, S1PP activity was homogeneous across the peripheral blood NK cells, suggesting

  13. Geno- and cytotoxicity of salinomycin in human nasal mucosa and peripheral blood lymphocytes.

    Science.gov (United States)

    Scherzad, Agmal; Hackenberg, Stephan; Schramm, Carolin; Froelich, Katrin; Ginzkey, Christian; Hagen, Rudolf; Kleinsasser, Norbert

    2015-06-01

    Salinomycin is usually applied in stock breading but has also been described as a promising agent against cancer stem cells (CSC). However, knowledge about the toxicity of this ionophor substance is incomplete. The aim of this study was to investigate cyto- and genotoxic effects of salinomycin in human non-malignant cells. Primary human nasal mucosa cells (monolayer and mini organ cultures) and peripheral blood lymphocytes from 10 individuals were used to study the cytotoxic effects of salinomycin (0.1-175 μM) by annexin-propidiumiodide- and MTT-test. The comet assay was performed to evaluate DNA damage. Additionally, the secretion of interleukin-8 was analyzed by ELISA. Flow cytometry and MTT assay revealed significant cytotoxic effects in nasal mucosa cells and lymphocytes at low salinomycin concentrations of 10-20 μM. No genotoxic effects could be observed. IL-8 secretion was elevated at 5 μM. Salinomycin-induced cytotoxic and pro-inflammatory effects were seen at concentrations relevant for anti-cancer treatment. Concurrent to the evaluation of salinomycin application in experimental oncology, adverse effects in non-malignant cells need to be monitored and reduced as much as possible. Further studies are also warranted to evaluate the toxic effects in a variety of human cell systems, e.g., liver, kidney and muscle cells.

  14. Effect of Human Periodontal Ligament Fibroblasts on the Differentiation of Peripheral Blood Mononuclear Cells.%牙周膜成纤维细胞对外周血单个核细胞分化的影响

    Institute of Scientific and Technical Information of China (English)

    陈建明; 兰泽栋; 刘嵘

    2011-01-01

    目的:探讨牙周膜成纤维细胞在破骨细胞形成过程中作用;观察破骨样细胞的生长过程.方法:本实验以含有1α,25(OH)2D3和地塞米松的培养基将牙周膜成纤维细胞、单个核细胞分别进行单独或直接共培养,每3d对TRAP阳性多核破骨细胞的数量及牙本质磨片的吸收陷窝数目和面积分别进行记录、计算.结果:不同时间段间的TRAP阳性单个核细胞与TRAP阳性多核细胞数目相比较,差异具有统计学意义(P<0.05);同时,不同组间的吸收陷窝数目和面积比较,差异具有显著性(P<0.001).牙周膜成纤维细胞明显增加了共培养组TRAP阳性多核细胞数量、吸收陷窝数目和面积.然而牙周膜成纤维细胞组与单个核细胞组之间的吸收陷窝数目与吸收陷窝面积差异无统计学意义.结论:末梢血单个核细胞需在牙周膜成纤维细胞存在的条件下,才能形成多核的破骨样细胞.%Objective: To investigate the effect of human periodontal ligament fibroblasts (PDLF) on the formation of osteoclast like cells(OLC) in vitro, and to probe the process of their growth. Methods: Human PDLF and peripheral blood mononuclear cells (PBMC) were co-cultured in the presence of 1α.25(OH)2D3 and dexamethasone for 30 days. Then the numbers of tartrate-resistant acidic phosphatase (TRAP) positive multi-nucleate cells and resorptive pits and the area of resorptive pits were recorded and accumulated every 3 days. Results: There was statistical significant difference in the numbers of TRAP positive multi-nucleate cells (P<0. 05) at different stages. So were the number of resorptive pits and the area of resorptive pits (P<0. 001). And PDLF also significantly increased the number of OLC and resorptive pits and the area of resorptive pits in PDLF-PBMC co-culture (P<0. 001). But no statistical significance had been found between PDLF culture and PBMC culture on the number of resorptive pits and the area of resorptive pits

  15. Synergistic effect of DHT and IGF-1 hyperstimulation in human peripheral blood lymphocytes.

    Science.gov (United States)

    Imperlini, Esther; Spaziani, Sara; Mancini, Annamaria; Caterino, Marianna; Buono, Pasqualina; Orrù, Stefania

    2015-06-01

    The abuse of mixed or combined performance-enhancing drugs is widespread among athletes and amateurs, adults and adolescents. Clinical studies demonstrated that misuse of these doping agents is associated with serious adverse effects to many organs in human. Previously, we demonstrated in human peripheral blood lymphocytes that high doses of anabolic androgenic steroids, such as dihydrotestosterone (DHT) and growth factors, such as insulin-like growth factor-1 (IGF-1), have effects at gene and protein levels. Supraphysiological treatments of DHT and IGF-1 affected the expression of genes involved in skeletal muscle disorders as well as in cell-mediated immunological response. At protein level, DHT hyperdosage affects cell motility and apoptosis; IGF-1 hyperstimulation triggers an active cytoskeletal reorganization and an overproduction of immune response- and inflammation-related cytokines. In this study, we investigate the combined effects of DHT and IGF-1 hyperdosage in peripheral blood lymphocytes using a differential proteomic approach. DHT and IGF-1 combined treatment affects cell adhesion, migration, and survival through modulation of expression levels of cytokines and paxillin-signaling-related proteins, and activation of several pathways downstream focal adhesion kinase. Our results indicate a synergistic effect of DHT and IGF-1 which has potential implications for health risk factors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Isolation of labile Fcgamma-receptors from human peripheral blood lymphocytes and production of an antiserum.

    Science.gov (United States)

    Sandilands, G P; Peel, M G; Froebel, K S; Belch, J J; MacSween, R N

    1985-05-01

    In this study, we have isolated membranelabile Fcgamma-receptors (i.e. FcgammaR I) from normal human peripheral blood lymphocytes and have produced a rabbit antiserum to this protein. Using this antiserum, we have shown that membrane-labile and membrane-stable (i.e. FcgammaR II) Fcgamma-receptors are antigenically distinct and that these two forms of the receptors probably coexist on the same lymphocyte subpopulation. Moreover, it was apparent that lymphocyte FcgammaR Is are distinct from FcgammaRs expressed on other cell types (e.g. monocytes, polymorphs and spermatozoa). Preliminary evidence does suggest, however, that human platelets express an FcgammaR which is antigenically similar to human lymphocyte FcgammaR I.

  17. Long-term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor

    Science.gov (United States)

    Kuzin, Igor; Sun, Hongliang; Moshkani, Safiekhatoon; Feng, Changyong; Mantalaris, Athanasios; Wu, JH David; Bottaro, Andrea

    2011-01-01

    Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g. T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three-dimensional bioreactor system for long-term, functional culture of human bone marrow cells on macroporous microspheres in a packed-bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell-enriched areas. Strikingly, these cultures generated a significant number of antibody-producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three-dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo. PMID:21309085

  18. Transplantation? Peripheral Stem Cell/Bone Marrow/Cord Blood

    Directory of Open Access Journals (Sweden)

    Itır Sirinoglu Demiriz

    2012-01-01

    Full Text Available The introduction of peripheral stem cell (PSC and cord blood (CB as an alternative to bone marrow (BM recently has caused important changes on hematopoietic stem cell transplantation (HSCT practice. According to the CIBMTR data, there has been a significant decrease in the use of bone marrow and increase in the use of PSC and CB as the stem cell source for HSCT performed during 1997–2006 period for patients under the age of 20. On the other hand, the stem cell source in 70% of the HSCT procedures performed for patients over the age of 20 was PSC and the second most preferred stem cell source was bone marrow. CB usage is very limited for the adult population. Primary disease, stage, age, time and urgency of transplantation, HLA match between the patient and the donor, stem cell quantity, and the experience of the transplantation center are some of the associated factors for the selection of the appropriate stem cell source. Unfortunately, there is no prospective randomized study aimed to facilitate the selection of the correct source between CB, PSC, and BM. In this paper, we would like to emphasize the data on stem cell selection in light of the current knowledge for patient populations according to their age and primary disease.

  19. Peripheral injection of human umbilical cord blood stimulates neurogenesis in the aged rat brain

    Directory of Open Access Journals (Sweden)

    Sanberg Paul R

    2008-02-01

    Full Text Available Abstract Background Neurogenesis continues to occur throughout life but dramatically decreases with increasing age. This decrease is mostly related to a decline in proliferative activity as a result of an impoverishment of the microenvironment of the aged brain, including a reduction in trophic factors and increased inflammation. Results We determined that human umbilical cord blood mononuclear cells (UCBMC given peripherally, by an intravenous injection, could rejuvenate the proliferative activity of the aged neural stem/progenitor cells. This increase in proliferation lasted for at least 15 days after the delivery of the UCBMC. Along with the increase in proliferation following UCBMC treatment, an increase in neurogenesis was also found in the aged animals. The increase in neurogenesis as a result of UCBMC treatment seemed to be due to a decrease in inflammation, as a decrease in the number of activated microglia was found and this decrease correlated with the increase in neurogenesis. Conclusion The results demonstrate that a single intravenous injection of UCBMC in aged rats can significantly improve the microenvironment of the aged hippocampus and rejuvenate the aged neural stem/progenitor cells. Our results raise the possibility of a peripherally administered cell therapy as an effective approach to improve the microenvironment of the aged brain.

  20. Peripheral injection of human umbilical cord blood stimulates neurogenesis in the aged rat brain

    Science.gov (United States)

    Bachstetter, Adam D; Pabon, Mibel M; Cole, Michael J; Hudson, Charles E; Sanberg, Paul R; Willing, Alison E; Bickford, Paula C; Gemma, Carmelina

    2008-01-01

    Background Neurogenesis continues to occur throughout life but dramatically decreases with increasing age. This decrease is mostly related to a decline in proliferative activity as a result of an impoverishment of the microenvironment of the aged brain, including a reduction in trophic factors and increased inflammation. Results We determined that human umbilical cord blood mononuclear cells (UCBMC) given peripherally, by an intravenous injection, could rejuvenate the proliferative activity of the aged neural stem/progenitor cells. This increase in proliferation lasted for at least 15 days after the delivery of the UCBMC. Along with the increase in proliferation following UCBMC treatment, an increase in neurogenesis was also found in the aged animals. The increase in neurogenesis as a result of UCBMC treatment seemed to be due to a decrease in inflammation, as a decrease in the number of activated microglia was found and this decrease correlated with the increase in neurogenesis. Conclusion The results demonstrate that a single intravenous injection of UCBMC in aged rats can significantly improve the microenvironment of the aged hippocampus and rejuvenate the aged neural stem/progenitor cells. Our results raise the possibility of a peripherally administered cell therapy as an effective approach to improve the microenvironment of the aged brain. PMID:18275610

  1. Pulmonary Involvement of Peripheral T-cell Lymphoma, Unspecified: A Case Report

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Jin; Shim, Hyo Sub; Ham, Seok Jin; Kim, Tae Hoon; Kim, Sang Jin [Gangnam Severance Hospital, Seoul (Korea, Republic of)

    2010-01-15

    Peripheral T-cell lymphoma is a rare type of lymphoma that's derived from postthymic lymphoid T cells. Pulmonary involvement of peripheral T-cell lymphoma of the unspecified type is very rare and the imaging findings of this illness have rarely been reported. We present here a case of peripheral T-cell lymphoma of the unspecified type with a cavitary lesion in the lung parenchyma, and we pathologically confirmed this illness by performing video-assisted thoracoscopic surgery.

  2. Convergences and Divergences of Thymus- and Peripherally Derived Regulatory T Cells in Cancer

    OpenAIRE

    Burocchi, Alessia; Colombo, Mario P.; Piconese, Silvia

    2013-01-01

    The expansion of regulatory T cells (Treg) is a common event characterizing the vast majority of human and experimental tumors and it is now well established that Treg represent a crucial hurdle for a successful immunotherapy. Treg are currently classified, according to their origin, into thymus-derived Treg (tTreg) or peripherally induced Treg (pTreg) cells. Controversy exists over the prevalent mechanism accounting for Treg expansion in tumors, since both tTreg proliferation and de novo pTr...

  3. 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...

  4. Post-prandial rise of microvesicles in peripheral blood of healthy human donors.

    Science.gov (United States)

    Suštar, Vid; Bedina-Zavec, Apolonija; Stukelj, Roman; Frank, Mojca; Ogorevc, Eva; Janša, Rado; Mam, Keriya; Veranič, Peter; Kralj-Iglič, Veronika

    2011-03-21

    Microvesicles isolated from body fluids are membrane - enclosed fragments of cell interior which carry information on the status of the organism. It is yet unclear how metabolism affects the number and composition of microvesicles in isolates from the peripheral blood. To study the post - prandial effect on microvesicles in isolates from the peripheral blood of 21 healthy donors, in relation to blood cholesterol and blood glucose concentrations. The average number of microvesicles in the isolates increased 5 hours post - prandially by 52%; the increase was statistically significant (p = 0.01) with the power P = 0.68, while the average total blood cholesterol concentration, average low density lipoprotein cholesterol concentration (LDL-C) and average high density lipoprotein cholesterol concentration (HDL-C) all remained within 2% of their fasting values. We found an 11% increase in triglycerides (p = 0.12) and a 6% decrease in blood glucose (p microvesicles negatively correlated with the post - fasting total cholesterol concentration (r = - 0.46, p = 0.035) while the difference in the number of microvesicles in the isolates between post - prandial and post - fasting states negatively correlated with the respective difference in blood glucose concentration (r = - 0.39, p = 0.05). In a population of healthy human subjects the number of microvesicles in isolates from peripheral blood increased in the post - prandial state. The increase in the number of microvesicles was affected by the fasting concentration of cholesterol and correlated with the decrease in blood glucose.

  5. Peripheral T cell cytokine responses for diagnosis of active tuberculosis.

    Directory of Open Access Journals (Sweden)

    Johannes Nemeth

    Full Text Available BACKGROUND: A test for diagnosis of active Tuberculosis (TB from peripheral blood could tremendously improve clinical management of patients. METHODS: Of 178 prospectively enrolled patients with possible TB, 60 patients were diagnosed with pulmonary and 27 patients with extrapulmonary TB. The frequencies of Mycobacterium tuberculosis (MTB specific CD4(+ T cells and CD8(+ T cells producing cytokines were assessed using overnight stimulation with purified protein derivate (PPD or early secretory antigenic target (ESAT-6, respectively. RESULTS: Among patients with active TB, an increased type 1 cytokine profile consisting of mainly CD4(+ T cell derived interferon (IFN-γ was detectable. Despite contributing to the cytokine profile as a whole, the independent diagnostic performance of one cytokine producing T cells as well as polyfunctional T cells was poor. IFN-γ/Interleukin(IL-2 cytokine ratios discriminated best between active TB and other diseases. CONCLUSION: T cells producing one cytokine and polyfunctional T cells have a limited role in diagnosis of active TB. The significant shift from a "memory type" to an "effector type" cytokine profile may be useful for further development of a rapid immune-diagnostic tool for active TB.

  6. A Method for Functional Quantification of the Reflex Effect of Human Peripheral Nerve Stimulation

    OpenAIRE

    Zehr, E. P.; Komiyama, Tomoyushi; Stein, R B

    2000-01-01

    E.P. Zehr, KOMIYAMA, T. and R.B. Stein. A Method for Functional Quantification of the Reflex Effect of Human Peripheral Nerve Stimulation. Adv. Exerc. Sports Physiol., Vol.6, No.1 pp25-32, 2000. We have developed simple method that accounts for the overrall function of reflex effects occurring in the surface electrimyogran (EMG) after human nerve stimulation. This method involves the subtraction of pre-stimulus EMG levels from EMG modulation curves obtained after human peripheral nerve stimul...

  7. 不同年龄段前列腺外周带基质细胞超微结构差异%Ultrastructural Difference of Stromal Cells from Normal Human Prostate Peripheral Zone of Varying Ages

    Institute of Scientific and Technical Information of China (English)

    王永传; 韩邦旻; 朱平; 杨洁; 夏术阶

    2011-01-01

    目的:研究不同年龄段前列腺外周带基质细胞超微结构并探讨其意义.方法:透射电镜观察3例年轻组(23-、26-、32-岁)和3例老年组(56-、64-、71-岁)正常前列腺外周带和3例前列腺癌组织的超微结构.Masson'sTrichrome染色法观察前列腺外周带基质胶原的分布情况.结果:年轻组、老年组前列腺外周带间质均以平滑肌细胞分布为主,间杂成纤维细胞,但老年组平滑肌细胞和成纤维细胞体积较大、形态不规则.与正常年轻组相比,老年组前列腺外周带成纤维细胞存在明显老化和肌化改变,部分细胞内与蛋白合成相关的细胞器粗面内质网发达,呈现肌成纤维细胞特征.与年轻组和老年组外周带相比,前列腺癌基质中功能更加活跃的肌成纤维细胞和胶原蛋白数量增加明显.Masson's Trichrome 染色支持超微观察到的基质胶原分布差异:年轻组<老年组<前列腺癌组.结论:不同年龄段前列腺外周带基质细胞超微结构间存在差异,老年组前列腺外周带和前列腺癌基质组织中具有活性的肌成纤维细胞的增加可能是造成老年前列腺癌高发和恶性进展的重要病理基础之一.%Objective: To characterize the phenotype of stromal cells from the human prostate peripheral zone (PZ) of varying ages.Methods: The ultrastrictural features ofstromal cells from normal prostate PZ of 3 young donors (23, 26 and 32 years old), 3 old donors (56, 64 and 71 years old) and 3 cases of prostate cancer tissue specimen were compared by transmission electron microscopy, also the distribution of collagen were determined by Masson's Trichrome staining.Results: Smooth muscle cells in the stroma of PZ-old and PCa groups were larger in size and number than those in the PZ-young group.Fibroblasts in the stroma of PZ-old and PCa groups showed phenotype of myodifferentiation and more active cellular function, characterized by prominent protein synthesis associated

  8. The role of complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) in promoting C3 fragment deposition and membrane attack complex formation on normal peripheral human B cells

    DEFF Research Database (Denmark)

    Nielsen, Claus Henrik; Pedersen, Morten Løbner; Marquart, Hanne Vibeke

    2002-01-01

    Normal human B lymphocytes are known to activate the alternative pathway (AP) of complement, leading to C3-fragment deposition and membrane attack complex (MAC) formation. The process is mediated via complement receptor type 2 (CR2, CD21), with complement receptor type 1 (CR1, CD35) playing...... a subsidiary role. In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B lymphocytes under circumstances where all complement pathways are operational. C3-fragment deposition and MAC formation were assessed on human peripheral B lymphocytes......) bearing CR1, however, markedly reduced both C3-fragment deposition and MAC formation. Our data suggest that C3-fragment deposition and MAC formation on B lymphocytes in vivo may involve both AP and classical pathway activation, with CR1 contributing significantly to the latter. On the other hand...

  9. Apheresis techniques for collection of peripheral blood progenitor cells.

    Science.gov (United States)

    Moog, Rainer

    2004-12-01

    The combination of effective mobilisation protocols and efficient use of apheresis machines has caused peripheral blood progenitor cells (PBPC) transplantation to grow rapidly. The development of apheresis technology has improved over the years. Today PBSC procedures have changed towards systems to minimise operator interaction and to reduce the collection of undesired cells such as polymorphonuclear cells and platelets using functionally closed, sterile environments for PBSC collection in keeping with Good Manufacturing Practice guidelines. Blood cell separators with continuous flow technique allow the processing of more blood than intermittent flow devices resulting in higher PBSC yields. Large volume leukapheresis with the processing of 3-4-fold donor's/patient's blood volume can increase the number of collected progenitor cells. Therefore, intermittent flow cell separators are indicated if only single vein access is available. Anticoagulant induced hypocalcaemia is an often observed side effect in long lasting PBPC harvesting and monitoring of electrolytes should be performed especially at the end of the apheresis procedure to supplement low levels of potassium, calcium or magnesium. Refinement and improvement of collection techniques continue to add to the armamentarium of current approaches for cancer and non-malignant conditions and will enable future strategies.

  10. Autologous bone marrow cell therapy for peripheral arterial disease

    Directory of Open Access Journals (Sweden)

    Botti C

    2012-09-01

    Full Text Available C Botti, C Maione, A Coppola, V Sica, G CobellisDepartment of General Pathology, Second University of Naples, Naples, ItalyAbstract: Inadequate blood supply to tissues caused by obstruction of arterioles and/or capillaries results in ischemic injuries – these injuries can range from mild (eg, leg ischemia to severe conditions (eg, myocardial infarction, stroke. Surgical and/or endovascular procedures provide cutting-edge treatment for patients with vascular disorders; however, a high percentage of patients are currently not treatable, owing to high operative risk or unfavorable vascular involvement. Therapeutic angiogenesis has recently emerged as a promising new therapy, promoting the formation of new blood vessels by the introduction of bone marrow–derived stem and progenitor cells. These cells participate in the development of new blood vessels, the enlargement of existing blood vessels, and sprouting new capillaries from existing blood vessels, providing evidence of the therapeutic utility of these cells in ischemic tissues. In this review, the authors describe peripheral arterial disease, an ischemic condition affecting the lower extremities, summarizing different aspects of vascular regeneration and discussing which and how stem cells restore the blood flow. The authors also present an overview of encouraging results from early-phase clinical trials using stem cells to treat peripheral arterial disease. The authors believe that additional research initiatives should be undertaken to better identify the nature of stem cells and that an intensive cooperation between laboratory and clinical investigators is needed to optimize the design of cell therapy trials and to maximize their scientific rigor. Only this will allow the results of these investigations to develop best clinical practices. Additionally, although a number of stem cell therapies exist, many treatments are performed outside international and national regulations and many

  11. Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen

    OpenAIRE

    2003-01-01

    Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC) directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plas...

  12. Peripheral blood cell signatures of Plasmodium falciparum infection during pregnancy.

    Directory of Open Access Journals (Sweden)

    Samad Ibitokou

    Full Text Available Sequestration of Plasmodium falciparum-infected erythrocytes in placental intervillous spaces causes inflammation and pathology. Knowledge of the profiles of immune cells associated with the physiopathology of pregnancy-associated malaria (PAM is scarce. We conducted a longitudinal, prospective study, both in Benin and Tanzania, including ∼1000 pregnant women in each site with systematic follow-up at scheduled antenatal visits until delivery. We used ex vivo flow cytometry to identify peripheral blood mononuclear cell (PBMC profiles that are associated with PAM and anaemia, determining the phenotypic composition and activation status of PBMC in selected sub-groups with and without PAM both at inclusion and at delivery in a total of 302 women. Both at inclusion and at delivery PAM was associated with significantly increased frequencies both of B cells overall and of activated B cells. Infection-related profiles were otherwise quite distinct at the two different time-points. At inclusion, PAM was associated with anaemia, with an increased frequency of immature monocytes and with a decreased frequency of regulatory T cells (Treg. At delivery, infected women presented with significantly fewer plasmacytoid dendritic cells (DC, more myeloid DC expressing low levels of HLA-DR, and more effector T cells (Teff compared to uninfected women. Independent associations with an increased risk of anaemia were found for altered antigen-presenting cell frequencies at inclusion, but for an increased frequency of Teff at delivery. Our findings emphasize the prominent role played by B cells during PAM whenever it arises during pregnancy, whilst also revealing signature changes in other circulating cell types that, we conclude, primarily reflect the relative duration of the infections. Thus, the acute, recently-acquired infections present at delivery were marked by changes in DC and Teff frequencies, contrasting with infections at inclusion, considered chronic in

  13. Schwann cell mitochondria as key regulators in the development and maintenance of peripheral nerve axons.

    Science.gov (United States)

    Ino, Daisuke; Iino, Masamitsu

    2017-03-01

    Formation of myelin sheaths by Schwann cells (SCs) enables rapid and efficient transmission of action potentials in peripheral axons, and disruption of myelination results in disorders that involve decreased sensory and motor functions. Given that construction of SC myelin requires high levels of lipid and protein synthesis, mitochondria, which are pivotal in cellular metabolism, may be potential regulators of the formation and maintenance of SC myelin. Supporting this notion, abnormal mitochondria are found in SCs of neuropathic peripheral nerves in both human patients and the relevant animal models. However, evidence for the importance of SC mitochondria in myelination has been limited, until recently. Several studies have recently used genetic approaches that allow SC-specific ablation of mitochondrial metabolic activity in living animals to show the critical roles of SC mitochondria in the development and maintenance of peripheral nerve axons. Here, we review current knowledge about the involvement of SC mitochondria in the formation and dysfunction of myelinated axons in the peripheral nervous system.

  14. Sulforaphane mitigates cadmium-induced toxicity pattern in human peripheral blood lymphocytes and monocytes.

    Science.gov (United States)

    Alkharashi, Nouf Abdulkareem Omer; Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2017-10-01

    Cadmium (Cd) is a highly toxic and widely distributed heavy metal that induces various diseases in humans through environmental exposure. Therefore, alleviation of Cd-induced toxicity in living organisms is necessary. In this study, we investigated the protective role of sulforaphane on Cd-induced toxicity in human peripheral blood lymphocytes and monocytes. Sulforaphane did not show any major reduction in the viability of lymphocytes and monocytes. However, Cd treatment at a concentration of 50μM induced around 69% cell death. Treatment of IC10-Cd and 100μM sulforaphane combination for 24 and 48h increased viability by 2 and 9% in cells subjected to Cd toxicity, respectively. In addition, IC25 of Cd and 100μM sulforaphane combination recovered 17-20% of cell viability. Cd induced apoptotic and necrotic cell death. Sulforaphane treatment reduced Cd-induced cell death in lymphocytes and monocytes. Our results clearly indicate that when the cells were treated with Cd+sulforaphane combination, sulforaphane decreased the Cd-induced cytotoxic effect in lymphocytes and monocytes. In addition, sulforaphane concentration plays a major role in the alleviation of Cd-induced toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. [SEX HORMONE INFLUENCE ON PERIPHERAL NATURAL KILLER CELLS COUNT].

    Science.gov (United States)

    Ivanov, P; Konova, E; Blajeva, Sv; Lukanov, Tsv; Angelova, P; Georgieva, V; Totev, V; Komsa-Penkova, R

    2015-01-01

    Proper evaluation of immunological factors connected with pregnancy establishment increased the possibility for exact treatment in high risk gestation cases. Hormonal changes during an ovarian cycle may affect immune response, which is crucial for the embryonic implantation. Peripheral Natural killer (pNK) cells are key components of immune systems and their activities could be regulated by sex hormones. In the present study we investigated the effects of estrogen fluctuation on the number of NK cells in vivo during the early follicular and middle luteal phase of menstrual cycle. In 63 healthy women with at least one full term pregnancy and regular menstrual cycle with duration between 24 and 32 days, blood samples have been collected twice for investigation of CD3/CD16/CD56 positive lymphocytes. The mean pNK count in follicular phase was 11.6% with 4.7% variation. The median was 10.6%. The mean pNK count in luteal phase was 12.1% with 5.1% variation, respectively median for cell number 11.8%. The two-tailed t-test comparison did not find any statistical difference despite the slight elevation of pNK cells count in luteal phase. The insignificant variation in pNK cells count objected the suggestion to evaluate immunological status in women with adverse pregnancy outcome in specific phase of menstrual cycle.

  16. 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.

  17. Stem Cell Transplant (Peripheral Blood, Bone Marrow, and Cord Blood Transplants)

    Science.gov (United States)

    ... cells , they are not the same as the embryos’ stem cells that are studied in cloning and ... March 16, 2016. National Cancer Institute. Bone Marrow Transplantation and Peripheral Blood Stem Cell Transplantation . August 12, ...

  18. Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Benedetta Romi

    Full Text Available Helicobacter pylori is a gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ and chemokines (such as MIP-1β, RANTES in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other gram-negative bacteria. The cytotoxin-associated antigen-A (CagA, was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.

  19. Up-front autologous stem-cell transplantation in peripheral T-cell lymphoma

    DEFF Research Database (Denmark)

    d'Amore, Francesco; Relander, Thomas; Lauritzsen, Grete F;

    2012-01-01

    Systemic peripheral T-cell lymphomas (PTCLs) respond poorly to conventional therapy. To evaluate the efficacy of a dose-dense approach consolidated by up-front high-dose chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) in PTCL, the Nordic Lymphoma Group (NLG) conducted a large p...

  20. Lymphoid Cell-Glioma Cell Interaction Enhances Cell Coat Production by Human Gliomas: Novel Suppressor Mechanism

    Science.gov (United States)

    Dick, Steven J.; Macchi, Beatrice; Papazoglou, Savvas; Oldfield, Edward H.; Kornblith, Paul L.; Smith, Barry H.; Gately, Maurice K.

    1983-05-01

    Certain human glioma lines produce mucopolysaccharide coats that impair the generation of cytolytic lymphocytes in response to these lines in vitro. Coat production is substantially enhanced by the interaction of glioma cells with a macromolecular factor released by human peripheral blood mononuclear cells in culture. This interaction thus constitutes an unusual mechanism by which inflammatory cells may nonspecifically suppress the cellular immune response to at least one class of solid tumors in humans.

  1. 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...

  2. 低浓度饮水型砷暴露对人群外周血血细胞参数的影响%Influence of low level arsenic exposure through drinking water on human peripheral blood cells

    Institute of Scientific and Technical Information of China (English)

    马彩凤; 云奋; 安全; 李云云; 苗艳玲; 田凤洁; 吕懿; 秦秀军; 刘力

    2015-01-01

    Objective To analyze the impact of low concentrations of arsenic exposure on peripheral blood cells in human being,and to provide a basis for screening early damage indicators of arsenic exposure.Methods In Datong City Shanxi Province,two neighboring districts with and without endemic arsenism were selected,in which economic development was balanced.The arsenic contents in drinking water of the two districts were 14.41-90.34 and 0.00-0.87 μg/L,respectively.One natural village in each district was selected.Aborigines (drinking local water for 15 or more years consecutively) were selected as research subjects (85 people as exposed group and 71 people as control group).All of the candidates had neither infectious nor genetic diseases nor contacted radiation and physicochemical factors which may cause the disease.Venous blood was collected and automatic blood cell analyzer was used to analyze the changes of blood parameters.Results Compared with the control group [lymphocyte (LYM):(1.58 ± 0.57) × 109/L,lymphocyte percentage (LYM%):(29.72 ± 8.32)%,mononuclear cell (MON):(0.47 ± 0.15) × 109/L,mononuclear cell percentage (MON%):(8.64 ± 1.97)%; red blood cell(RBC):(4.10 ± 0.58) × 109/L,hemoglobin (HGB):(111.11 ± 16.49)g/L,mean corpuscular hemoglobin (MCH):(27.68 ± 2.99)pg,mean corpuscular hemoglobin concentration (MCHC):(295.20 ± 36.82)g/L,red distribution width (RDW):(11.06 ± 1.08)%,mean cell volume (MCV):(92.69 ± 7.50)fl; platelet(PLT):(259.30 ± 74.97) × 109/L,mean platelet volume (MPV):(11.27 ± 1.31)fl,platelet deposited (PCT):(0.28 ± 0.08)L/L,platelet volume width (PDW):(9.23 ± 2.29)%],some blood parameters had been changed in arsenic poisoning group:① LYM [(2.00 ± 0.90) × 109/L] and LYM% [(33.92 ± 9.70)%] were increased (t =-3.348,-2.873,all P < 0.05); MON[(0.15 ± 0.07) × 109/L],MON%[(2.53 ± 0.77)%] were decreased (t =16.309,24.599,all P < 0.05); ②RBC [(4.44 ± 0.46) × 109/L],HGB [136.59 ± 13.84)g/L],MCH [(30.85

  3. Chidamide in the treatment of peripheral T-cell lymphoma

    Science.gov (United States)

    Chan, Thomas S; Tse, Eric; Kwong, Yok-Lam

    2017-01-01

    Mature T-cell lymphomas are aggressive malignancies. Treatment outcome is poor with conventional chemotherapy. They are about twice as common in Asia as compared with other non-Asian countries. Histone proteins form the basic structure of chromatin, and their acetylation at lysine residues relaxes chromatin structure, facilitating gene transcription. Conversely, histone deacetylation, catalyzed by histone deacetylases, compacts chromatin and represses gene transcription. Histone deacetylase inhibitors are an important class of antineoplastic agents. Chidamide is a novel orally active benzamide-type histone deacetylase inhibitor that has shown in vitro activities against a wide array of neoplasms. In Phase I trials, chidamide showed preferential efficacy in mature T-cell lymphomas. In a pivotal Phase II trial of chidamide in 79 patients with relapsed or refractory mature T-cell lymphomas, an overall response rate of 28% (complete remission/complete remission unconfirmed: 14%) was achieved, with most responses occurring within the first 6 weeks of treatment. The median duration of response (DOR) was 9.9 (1.1–40.8) months. Of 22 responders, 19 patients (86%) had a DOR of ≥3 months and eight patients (36%) had a DOR of >12 months. Angioimmunoblastic T-cell lymphoma and anaplastic large cell lymphoma (anaplastic lymphoma kinase-negative) showed better response rates, with the most durable responses observed in angioimmunoblastic T-cell lymphoma patients. Safety profile was favorable, with very few cases of grade 3/4 toxicities observed. Chidamide is approved by the China Food and Drug Administration for the treatment of relapsed and refractory peripheral T-cell lymphomas. PMID:28138258

  4. MAJOR AND LYMPHOCYTE POPULATIONS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES AND THEIR REFERENCE VALUES, AS ASSAYED BY MULTI-COLOUR CYTOMETRY

    Directory of Open Access Journals (Sweden)

    S. V. Khaidukov

    2009-01-01

    Full Text Available Abstract. Determination of lymphocyte subpopulations and their phenotypes is an important diagnostic feature, in order to elucidate some disturbances connected with immune system functioning. However, insufficient data are obtained when analyzing only major populations of peripheral lymphocytes. In order to perform clinical diagnostics, the data about minor lymphocytic populations and activated cellular pools seem to be more pertinent.Studies of peripheral blood cell subpopulations of healthy donors performed in different Russian regions allowed to assess quantitative distribution intervals for both major and minor immune cell subpopulations in humans. The results obtained, as compared with data from literature, provide an evidence for similar reference intervals for main immune cell subpopulations in healthy donors, independent on their habitation area.Present work has resulted into development of algorithms for cytometric studies and generation of certain panels of monoclonal antibodies enabling evaluation of all main lymphocyte subpopulations, as well as their minor subsets participating in emerging immune response. The distribution intervals have been estimated for such minor subpopulations, as B1- and B2-lymphocytes, memory B-cells, γδ- and αβT-cells, regulatory and naїve T-cells, cytotoxic and secretory NK-cell polupations.The results of present study, while been performed with peripheral blood of healthy donors, may provide a basis of reference values when studying subpopulation profile of immune cells.

  5. Effects of peripheral-type benzodiazepine receptor ligands on Ehrlich tumor cell proliferation.

    Science.gov (United States)

    Sakai, Mônica; Fonseca, Evelise Souza Monteiro; Oloris, Silvia Catarina Salgado; Matsuzaki, Patrícia; Otake, Andréia Hanada; Leite, Kátia Ramos Moura; Massoco, Cristina Oliveira; Dagli, Maria Lúcia Zaidan; Palermo-Neto, João

    2006-11-21

    Peripheral-type benzodiazepine receptors have been found throughout the body, and particularly, in high numbers, in neoplastic tissues such as the ovary, liver, colon, breast, prostate and brain cancer. Peripheral-type benzodiazepine receptor expression has been associated with tumor malignity, and its subcellular localization is important to define its function in tumor cells. We investigated the presence of peripheral-type benzodiazepine receptors in Ehrlich tumor cells, and the in vitro effects of peripheral-type benzodiazepine receptors ligands on tumor cell proliferation. Our results demonstrate the presence of peripheral-type benzodiazepine receptor in the nucleus of Ehrlich tumor cells (85.53+/-12.60%). They also show that diazepam and Ro5-4864 (peripheral-type benzodiazepine receptor agonists) but not clonazepam (a molecule with low affinity for the peripheral-type benzodiazepine receptor) decreased the percentage of tumor cells in G0-G1 phases and increased that of cells in S-G2-M phases. The effects of those agonists were prevented by PK11195 (a peripheral-type benzodiazepine receptor antagonist) that did not produce effects by itself. Altogether, these data suggest that the presence of peripheral-type benzodiazepine receptor within the nucleus of Ehrlich tumor cells is associated with tumor malignity and proliferation capacity.

  6. The Role of Human Adult Peripheral and Umbilical Cord Blood Platelet-Rich Plasma on Proliferation and Migration of Human Skin Fibroblasts.

    Science.gov (United States)

    Hashemi, Seyedeh-Sara; Mahmoodi, Mahdokht; Rafati, Ali Reza; Manafi, Farzad; Mehrabani, Davood

    2017-05-01

    Wound healing is a complex and dynamic process following damage in tissue structures. Due to extensive skin damage caused by burn injuries, this study determined the role of human adult peripheral and umbilical cord blood platelet-rich plasma on proliferation and migration in human skin fibroblasts. Platelet-rich plasma (5, 10, 15, 20 and 50% PRP) from human umbilical cord blood and adult peripheral blood were provided and added to fibroblasts cultured from a human skin sample. Migration and proliferation of fibroblasts were assessed in comparison to 10% FBS and by the fibroblast responses to a concentration gradient. All components of the umbilical cord blood PRP significantly stimulated the growth of fibroblasts when compared to the negative control. Fibroblast growth was enhanced in a dose dependent manner. All fibroblast cultures retained normal morphology. No significant difference was noted between umbilical cord blood and adult peripheral blood PRP preparations regarding cell proliferation and migration, but the difference to 10% FBS was significant. 1% and 50% PRP reduced cellular proliferation. The 20% umbilical cord blood PRP and 10% adult peripheral blood PRP had a significant stimulatory effect on the migration of the skin fibroblast cells in comparison with 10% FBS. As PRP could promote the migration and proliferation of dermal fibroblasts, it can be safely added in cultures when treatment of chronic wounds without triggering the immune response is needed.

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

    Directory of Open Access Journals (Sweden)

    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

  8. 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

  9. Detection of cancer cells in peripheral blood with nested RT-PCR and itssignificance in patients with gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Jia Zeng Xia; Hao Ran Yin; Zheng Gang Zhu; Min yan

    2000-01-01

    AIM To study the detection of micrometastasis in peripheral blood of patients with gastric carcinomas andits clinical significance.METHODS A cytokeratin 19 (CK19)-specific nested reverse transcriptase-polimerase chain reaction (RT-PCR) assay was developed to detect CK19 expressing cancer cells, the sensitivity was determined by serialdilution method using CK19 expressing gastric cancer cells, the specificity was assessed by examining 12negative controls and 12 positive controls. Then pre-operative peripheral blood from 42 patients with gastriccancer was detected and the relationship between positive results and biological behavior was studied.RESULTS CK19mRNA was expressed in all the 12 gastric cancer tissues but not in peripheral blood from12 healthy individuals;sensitivity of nested RT-PCR amplification for CK19mRNA was confirmed to be 1/106 by serial dilution method using human gastric cancer line SGC-7901; micrometastases in pre-operativeperipheral blood were detected in 13 (30,9%) patients with gastric carcinomas, the frequency ofmicrometastasis in peripheral blood was significantly correlated with tumor size,depth of invasion and TNMstage (x2 test, P<0.05).CONCLUSION Nested RT-PCR amplification for CK19mRNA is a sensitive and specific method for thedetection of micrometastases in peripheral blood in gastric cancer patients; pre-operative detection ofmicrometastasis in peripheral blood may be helpful in the prediction of tumor progression.

  10. Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation.

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    Jacek M Witkowski

    2008-01-01

    Full Text Available Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+ cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.

  11. Peripheral endothelial cell damage after trephination of donor tissue.

    Science.gov (United States)

    Terry, Mark A; Saad, Hisham A; Shamie, Neda; Shah, Anand K

    2009-12-01

    To evaluate and quantify the degree and pattern of donor endothelial cell damage, which occurs with mechanical trephination of donor corneal tissue. Twenty donor corneal-scleral tissues were used for these paired experiments. The tissues were randomized for trephination with 10 tissues trephinated by an 8.0-mm-diameter Barron trephine (Katena, Denville, NJ), and 10 tissues trephinated with an 8.0-mm-diameter UltraFit Coronet trephine (distributed by Angiotech, British Columbia, Canada) by the same investigator. Trephinated corneal buttons were then stained with vital dye stain, and the endothelial layer image captured with digital photography. The images were then analyzed by digital planimetry, and the pattern and quantity of endothelial damage was determined by an investigator who was masked to the specific trephine used for the individual tissue. Trephination created a pattern of circular damage at the edge of the donor button in every case with no break in continuity of the circle, but some portions of the circle were wider than others. Occasional, scattered, peripheral small areas also displayed damage, but no significant striae, stretch, or other central damage was noted in any donor. The mean percent damage in the series was 6.35% +/- 0.90% (range: 4.33%-7.78%). The UltraFit Coronet trephinations averaged damage of 5.64% +/- 0.85% (range: 4.33%-6.69%), and the Barron trephinations averaged damage of 6.50% +/- 0.95% (range: 4.92%-7.78%). Although 8 of 10 experimental pairs of trephinations demonstrated less peripheral endothelial damage with the UltraFit Coronet trephine, the mean damage between each group did not reach statistical significance in this small series. (P = 0.08) Donor mechanical trephination of full-thickness corneal tissue creates relatively consistent amounts of peripheral edge damage and likely no central endothelial damage. There may exist differences in edge damage between different mechanical trephination systems, and a direct comparison

  12. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

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    Joo, Hyung Joon; Seo, Ha-Rim [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Jeong, Hyo Eun [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Chung, Seok [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Lim, Do-Sun, E-mail: dslmd@kumc.or.kr [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of)

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

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

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    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.

  14. Identification of myeloid derived suppressor cells in the peripheral blood of tumor bearing dogs

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    Sherger Matthew

    2012-10-01

    Full Text Available Abstract Background Myeloid derived suppressor cells (MDSCs are a recently described population of immune cells that significantly contribute to the immunosuppression seen in cancer patients. MDSCs are one of the most important factors that limit the efficacy of cancer immunotherapy (e.g. cancer vaccines and MDSC levels are increased in cancer in multiple species. Identifying and targeting MDSCs is actively being investigated in the field of human oncology and is increasingly being investigated in veterinary oncology. The treatment of canine cancer not only benefits dogs, but is being used for translational studies evaluating and modifcying candidate therapies for use in humans. Thus, it is necessary to understand the immune alterations seen in canine cancer patients which, to date, have been relatively limited. This study investigates the use of commercially available canine antibodies to detect an immunosuppressive (CD11blow/CADO48low cell population that is increased in the peripheral blood of tumor-bearing dogs. Results Commercially available canine antibodies CD11b and CADO48A were used to evaluate white blood cells from the peripheral blood cells of forty healthy control dogs and forty untreated, tumor-bearing dogs. Tumor-bearing dogs had a statistically significant increase in CD11blow/CADO48Alow cells (7.9% as compared to the control dogs (3.6%. Additionally, sorted CD11blow/CADO48Alow generated in vitro suppressed the proliferation of canine lymphocytes. Conclusions The purpose of this study was aimed at identifying potential canine specific markers for identifying MDSCs in the peripheral blood circulation of dogs. This study demonstrates an increase in a unique CD11blow/CADO48Alow cell population in tumor-bearing dogs. This immunophenotype is consistent with described phenotypes of MDSCs in other species (i.e. mice and utilizes commercially available canine-specific antibodies. Importantly, CD11blow/CADO48Alow from a tumor environment

  15. Peripheral T-cell count in sixty cases of cutaneous vasculitis

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    Mittal RR

    1996-01-01

    Full Text Available ABSTRACT: Clinically diagnosed 60 cases of cutaneous vasculitis (CV were collected from Dermato-venereology Department. T-cell count in the peripheral blood, a marker of cell-mediated immunity, was done in all the cases by Thomson Method 1977. Statistically significant increase in peripheral T-lymphocytes was seen in 15/60 (25 percent cases of CV.

  16. Phenotypic, ultra-structural and functional characterization of bovine peripheral blood dendritic cell subsets

    Science.gov (United States)

    Dendritic cells (DC) are multifunctional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets...

  17. Tumour necrosis factor production and natural killer cell activity in peripheral blood during treatment with recombinant tumour necrosis factor

    OpenAIRE

    Männel, Daniela N.; Kist, A.; Ho, A D; Räth, U.; Reichardt, P; Wiedenmann, B; Schlick, E.; Kirchner, H.

    1989-01-01

    Tumour necrosis factor (TNF) has been found to be an important immunomodulator. Among other functions TNF activates natural killer (NK) cells and stimulates monocytes/macrophages in an autocrine fashion. TNF production and NK activity in peripheral blood mononuclear cells were determined in a clinical phase I study in which recombinant human (rh) TNF was administered as a continuous infusion weekly for a period of 8 weeks. Even though TNF production and NK activity were significantly reduced ...

  18. The correlation between T regulatory cells and autologous peripheral blood stem cell transplantation in multiple myeloma

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    Ayşe Pınar Erçetin

    2011-06-01

    Full Text Available Objective: Multiple myeloma (MM is characterized by malignant proliferation of plasmocytes and their precursors. T regulatory cells (Tregs have a role in immunosuppression and control of autoimmunity, and are currently an important topic in the study of immune response to tumor cells. The correlation between Tregs and autologous peripheral blood stem cell transplantation (APBSCT in MM has not been studied. The aim of this study was to compare CD4+CD25+FOXP3+ Treg, CD200, and PD-1 levels in MM patients that did and did not undergo APBSCT. Materials and Methods: Peripheral blood samples were collected from 28 MM patients ranging in age from 41 to 78 years for analysis of CD4CD25+ FOXP3+ Tregs, PD-1 (CD279, and CD200. Peripheral blood mononuclear cells were isolated via density gradient centrifugation. Four-color flow cytometry was performed. Using a sequential gating strategy, Tregs were identified as CD4+CD25+FOXP3+ T-cells. Results were analyzed using the Mann Whitney U non-parametric test and a compare means test. p values 0.05. Conclusion: Treg levels were higher in the patients that underwent APBSCT. Tregs are crucial for the induction and maintenance of peripheral tolerance to self-antigens. In addition, Tregs can suppress immune responses to tumor antigens; however, APBSCT and Treg levels were not correlated with CD200 or PD-1 expression. Relationship of Tregs with prognosis needs to be determined by studies that include larger cohorts.

  19. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins

    Science.gov (United States)

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes. PMID:27212953

  20. Establishment of Human Neural Progenitor Cells from Human Induced Pluripotent Stem Cells with Diverse Tissue Origins.

    Science.gov (United States)

    Fukusumi, Hayato; Shofuda, Tomoko; Bamba, Yohei; Yamamoto, Atsuyo; Kanematsu, Daisuke; Handa, Yukako; Okita, Keisuke; Nakamura, Masaya; Yamanaka, Shinya; Okano, Hideyuki; Kanemura, Yonehiro

    2016-01-01

    Human neural progenitor cells (hNPCs) have previously been generated from limited numbers of human induced pluripotent stem cell (hiPSC) clones. Here, 21 hiPSC clones derived from human dermal fibroblasts, cord blood cells, and peripheral blood mononuclear cells were differentiated using two neural induction methods, an embryoid body (EB) formation-based method and an EB formation method using dual SMAD inhibitors (dSMADi). Our results showed that expandable hNPCs could be generated from hiPSC clones with diverse somatic tissue origins. The established hNPCs exhibited a mid/hindbrain-type neural identity and uniform expression of neural progenitor genes.

  1. Peripheral stem cell transplantation in non-Hodgkin's lymphoma patients.

    Science.gov (United States)

    Kessinger, A; Vose, J M; Bierman, P J; Bishop, M; Armitage, J O

    1993-01-01

    Transplantation of circulating progenitor/stem cells collected before and stored during administration of marrow-ablative antitumor therapy has restored sustained hematopoiesis for patients with a variety of malignancies. One of the most common diseases so treated is refractory or relapsed non-Hodgkin's lymphoma (NHL). Autologous peripheral stem cell transplantation (PSCT) often has been used rather than autologous bone marrow transplantation (ABMT) because NHL commonly involves the bone marrow, and because, in some situations, PSCT provides earlier engraftment than ABMT. Between July 1986 and September 1992, 170 adult patients with refractory or relapsed NHL were treated with high-dose therapy and PSCT at the University of Nebraska Medical Center (UNMC). With a median follow-up of 469 days for the evaluable survivors, the actuarial progression-free survival for 167 patients at 6 years after PSCT was 30%. High-dose therapy and PSCT for NHL patients has resulted in long-term progression-free survival and probably cure for some patients. The role of PSCT in this disease continues to evolve.

  2. 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.

  3. Auraptene from Ferula szowitsiana protects human peripheral lymphocytes against oxidative stress.

    Science.gov (United States)

    Soltani, Fatemeh; Mosaffa, Fatemeh; Iranshahi, Mehrdad; Karimi, Gholamreza; Malekaneh, Mohammad; Haghighi, Fatemeh; Behravan, Javad

    2010-01-01

    The antigenotoxicity effects of auraptene on DNA damage in human peripheral lymphocytes were studied using alkaline single cell gel electrophoresis. Auraptene at concentrations of 5, 10, 25, 50, 100, 200 and 400 microM was tested under simultaneous treatment with 25 microM H(2)O(2). The data are expressed as % tail DNA and compared with ascorbic acid at concentrations of 25, 50, 100, 200 and 400 microM. Auraptene significantly reduced the genotoxicity of H(2)O(2 )at concentrations higher than 25 microM (p 0.05). It seems that the significant antigenotoxicity effects of auraptene may be due to the prenyl moiety and also the suppression of superoxide anion (O(2) (-)) generation. This study suggests that the antigenotoxic property of auraptene is of great pharmacological importance and might be beneficial for cancer prevention.

  4. Stem Cell Heterogeneity of Mononucleated Cells from Murine Peripheral Blood: Molecular Analysis

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    Muhammad Dain Yazid

    2011-01-01

    Full Text Available The main purpose of this paper was to determine the heterogeneity of primary isolated mononucleated cells that originated from the peripheral blood system by observing molecular markers. The isolated cells were cultured in complete medium for 4 to 7 days prior to the separation of different cell types, that is, adherent and suspension. Following a total culture time of 14 days, adherent cells activated the Cd105 gene while suspension cells activated the Sca-1 gene. Both progenitor markers, Cbfa-1 and Ostf-1, were inactivated in both suspension and adherent cells after 14-day culture compared to cells cultured 3 days in designated differentiation medium. In conclusion, molecular analyses showed that primary mononucleated cells are heterogeneous, consisting of hematopoietic stem cells (suspension and mesenchymal stem cells (adherent while both cells contained no progenitor cells.

  5. Association of the frequency of peripheral natural killer T cells with nonalcoholic fatty liver disease

    Institute of Scientific and Technical Information of China (English)

    Cheng-Fu Xu; Chao-Hui Yu; You-Ming Li; Lei Xu; Juan Du; Zhe Shen

    2007-01-01

    AIM: To investigate whether changes in the frequency of peripheral natural killer T (NKT) cells were correlated with liver disease in patients who had metabolic predispositions to nonalcoholic fatty liver disease (NAFLD).METHODS: Peripheral blood samples were obtained from 60 Chinese NAFLD patients and 60 age and gender matched healthy controls. The frequency of peripheral NKT cells was detected by flow cytometry. Clinical and laboratory data were collected for further analysis.RESULTS: NAFLD patients had a lower frequency of peripheral NKT cells than healthy controls (1.21% ± 0.06% vs 1.62% ± 0.07%, P < 0.001). Further analysis revealed that the frequency of peripheral NKT cells was negatively correlated with body mass index, waist circumference and serum levels of alanine aminotransferase. Logistic regression analysis revealed that elevated body mass index [hazard ratio (HR):2.991], aspartate aminotransferase levels (HR: 1.148)and fasting blood sugar (HR: 3.133) increased the risk of NAFLD, whereas an elevated frequency of peripheral NKT cells (HR: 0.107) decreased the risk.CONCLUSION: Changes in the frequency of peripheral NKT cells were correlated with NAFLD and a decreased frequency of peripheral NKT cells was a risk factor for NAFLD.

  6. Harvesting, processing and inventory management of peripheral blood stem cells

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    Mijovic Aleksandar

    2007-01-01

    Full Text Available By 2003, 97% autologous transplants and 65% of allogeneic transplants in Europe used mobilised peripheral blood stem cells (PBSC. Soon after their introduction in the early 1990′s, PBSC were associated with faster haemopoietic recovery, fewer transfusions and antibiotic usage, and a shorter hospital stay. Furthermore, ease and convenience of PBSC collection made them more appealing than BM harvests. Improved survival has hitherto been demonstrated in patients with high risk AML and CML. However, the advantages of PBSC come at a price of a higher incidence of extensive chronic GVHD. In order to be present in the blood, stem cells undergo the process of "mobilisation" from their bone marrow habitat. Mobilisation, and its reciprocal process - homing - are regulated by a complex network of molecules on the surface of stem cells and stromal cells, and enzymes and cytokines released from granulocytes and osteoclasts. Knowledge of these mechanisms is beginning to be exploited for clinical purposes. In current practice, stem cell are mobilised by use of chemotherapy in conjunction with haemopoietic growth factors (HGF, or with HGF alone. Granulocyte colony stimulating factor has emerged as the single most important mobilising agent, due to its efficacy and a relative paucity of serious side effects. Over a decade of use in healthy donors has resulted in vast experience of optimal dosing and administration, and safety matters. PBSC harvesting can be performed on a variety of cell separators. Apheresis procedures are nowadays routine, but it is important to be well versed in the possible complications in order to avoid harm to the patient or donor. To ensure efficient collection, harvesting must begin when sufficient stem cells have been mobilised. A rapid, reliable, standardized blood test is essential to decide when to begin harvesting; currently, blood CD34+ cell counting by flow cytometry fulfils these criteria. Blood CD34+ cell counts strongly

  7. A SYSTEM AND A DEVICE FOR ISOLATING CIRCULATING TUMOR CELLS FROM THE PERIPHERAL BLOOD IN VIVO

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    Michal Mego

    2015-08-01

    Full Text Available Circulating tumor cells (CTC play a crucial role in disseminating tumors and in the metastatic cascade. CTCs are found only in small numbers, and the limited amount of isolated CTCs makes it impossible to characterize them closely. This paper presents a proposal for a new system for isolating CTCs from the peripheral blood in vivo. The system enables CTCs to be isolated from the whole blood volume for further research and applications. The proposed system consists of magnetic nanoparticles covered by monoclonal antibodies against a common epithelial antigen, large supermagnets, which are used to control the position of the nanoparticles within the human body, and a special wire made of a magnetic core wrapped in a non-magnetic shell. The system could be used not only for isolating CTCs, but also for in vivo isolation of other rare cells from the peripheral blood, including hematopoietic and/or mesenchymal stem cells, with applications in regenerative medicine and/or in stem cell transplantation.

  8. Stimulation with Concanavalin-A Induces IL-17 Production by Canine Peripheral T Cells

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    Michelle G. Ritt

    2015-04-01

    Full Text Available The characteristics of canine IL-17-producing cells are incompletely understood. Expression of mRNA encoding orthologs of IL-17 and the IL-17 receptor has been documented in tissues from dogs with arthritis, inflammatory bowel disease, and lymphoma; however, no associations have been found between IL-17 gene expression and disease phenotype in these conditions. Robust assessment of the role of IL-17-producing cells in dogs will require measuring the frequency of these cells in health and disease in balance with other lymphocyte subsets. The aim of this study was to confirm that the T-cell IL-17 response in dogs is evolutionarily conserved. Canine peripheral blood mononuclear cells were stimulated with Concanavalin A with or without polarizing cytokines. We used a canine specific IL-17 ELISA and flow cytometry to identify IL-17-producing T cells. Accumulation of intracellular IL-17 was observed in stimulated CD4 and CD8 T cells. The addition of pro-inflammatory cytokines appeared to enhance polarization of canine CD4 T cells to the Th17 phenotype. Conversely, the addition of IL-2 in the presence of TGF-β resulted in expansion of Treg cells. We conclude that canine IL-17-producing cells behave similarly to those from humans and mice when stimulated with mitogens and polarized with pro-inflammatory or immune regulatory cytokines.

  9. Measurement of the Phagocytic Activity of Human Peripheral Blood Using a Highly Sensitive Fluorometric Detection Device Without Hemolysis.

    Science.gov (United States)

    Zhang, Ran; Inagawa, Hiroyuki; Takahashi, Masaru; Kawanishi, Hisami; Kazumura, Kimiko; Tsuchiya, Hiroshi; Morishita, Naokazu; Kobayashi, Yutaro; Masaki, Tsutomu; Kobara, Hideki; Soma, Gen-Ichiro

    2017-07-01

    Phagocytes recognize pathogens that enter the body as well as other abnormal and foreign materials that may exist within an organism (such as dead cells, oxidized lipids, and denatured proteins), and phagocytose and eliminate them to maintain a healthy state. In a previous study a simple prototype device was used, under development by Hamamatsu Photonics (Prototype), that detects fluorescence to determine the phagocytic activity of the murine macrophage cell line J774.1. The present study aimed to determine whether it was possible to detect phagocytic activity in a slight amount of human peripheral blood without using hemolysis. Three microliters of human peripheral blood was drawn from the fingertip and mixed with 30 μg of pH-sensitive fluorescent particles. The fluorescence intensity of the human peripheral blood sample was then measured using the Prototype in development, cultured for 2 h at 37°C, and then re-measured. The phagocytes were observed under fluorescence microscopy and the phagocytosis rate of CD11b-positive cells was verified with a flow cytometer. The phagocytic activity of non-hemolyzed human peripheral blood was measured using the Prototype under development; fluorescence after phagocytosis was detected. Furthermore, this was confirmed by both fluorescence microscopy and flow cytometry. The precision of the measurements of human peripheral blood phagocytic activity was verified with the Prototype using samples from three healthy individuals. The relationship between blood sugar levels and phagocytic activity before and after meal times was determined. Concerning exercise, phagocytic activity tended to decrease, although salivary amylase level increased in the healthy individual examined after exercise. The simple Prototype can measure phagocytic activity in a small amount of peripheral blood without hemolysis. The device allows for rapid and minimally-invasive detection of changes in phagocytic activity, which has conventionally been difficult

  10. Aire-Overexpressing Dendritic Cells Induce Peripheral CD4+ T Cell Tolerance

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    Dongbei Li

    2015-12-01

    Full Text Available Autoimmune regulator (Aire can promote the ectopic expression of peripheral tissue-restricted antigens (TRAs in thymic medullary epithelial cells (mTECs, which leads to the deletion of autoreactive T cells and consequently prevents autoimmune diseases. However, the functions of Aire in the periphery, such as in dendritic cells (DCs, remain unclear. This study’s aim was to investigate the effect of Aire-overexpressing DCs (Aire cells on the functions of CD4+ T cells and the treatment of type 1 diabetes (T1D. We demonstrated that Aire cells upregulated the mRNA levels of the tolerance-related molecules CD73, Lag3, and FR4 and the apoptosis of CD4+ T cells in STZ-T1D mouse-derived splenocytes. Furthermore, following insulin stimulation, Aire cells decreased the number of CD4+ IFN-γ+ T cells in both STZ-T1D and WT mouse-derived splenocytes and reduced the expression levels of TCR signaling molecules (Ca2+ and p-ERK in CD4+ T cells. We observed that Aire cells-induced CD4+ T cells could delay the development of T1D. In summary, Aire-expressing DCs inhibited TCR signaling pathways and decreased the quantity of CD4+IFN-γ+ autoreactive T cells. These data suggest a mechanism for Aire in the maintenance of peripheral immune tolerance and provide a potential method to control autoimmunity by targeting Aire.

  11. Aire-Overexpressing Dendritic Cells Induce Peripheral CD4+ T Cell Tolerance

    Science.gov (United States)

    Li, Dongbei; Li, Haijun; Fu, Haiying; Niu, Kunwei; Guo, Yantong; Guo, Chuan; Sun, Jitong; Li, Yi; Yang, Wei

    2015-01-01

    Autoimmune regulator (Aire) can promote the ectopic expression of peripheral tissue-restricted antigens (TRAs) in thymic medullary epithelial cells (mTECs), which leads to the deletion of autoreactive T cells and consequently prevents autoimmune diseases. However, the functions of Aire in the periphery, such as in dendritic cells (DCs), remain unclear. This study’s aim was to investigate the effect of Aire-overexpressing DCs (Aire cells) on the functions of CD4+ T cells and the treatment of type 1 diabetes (T1D). We demonstrated that Aire cells upregulated the mRNA levels of the tolerance-related molecules CD73, Lag3, and FR4 and the apoptosis of CD4+ T cells in STZ-T1D mouse-derived splenocytes. Furthermore, following insulin stimulation, Aire cells decreased the number of CD4+ IFN-γ+ T cells in both STZ-T1D and WT mouse-derived splenocytes and reduced the expression levels of TCR signaling molecules (Ca2+ and p-ERK) in CD4+ T cells. We observed that Aire cells-induced CD4+ T cells could delay the development of T1D. In summary, Aire-expressing DCs inhibited TCR signaling pathways and decreased the quantity of CD4+IFN-γ+ autoreactive T cells. These data suggest a mechanism for Aire in the maintenance of peripheral immune tolerance and provide a potential method to control autoimmunity by targeting Aire. PMID:26729097

  12. Cytotoxicity of Betel leaf (Piper betel L. against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by human peripheral blood mononuclear cells

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    Suprapto Ma’at

    2012-06-01

    Full Text Available Background: Betel leaf (Piper betel L. has been used in modern and traditional medicine as antiseptic, antibacterial, and also prevention of plaque accumulation, but it still can stimulate cancer in lime-piper betel quid. Betel leaf also has anti-inflammatory properties. Purpose: The purpose of this study was examine the cytotoxicity of Betel leaf extract (BLE against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMC stimulated with LPS. Methods: MTT assay was used to investigate the survival rate of the culture with the survival rate result of the given culture extract 4%, 2% and 1% about 82%, 83.4% and 85%. There was no significant difference between treatment with various concentrations of the extract and the control (p>0.05. To evaluate the effect of Betel leaf extracts on the production of cytokines, proinflammatory was conducted by incubating the extracts of betel leaf with peripheral blood mononuclear cells stimulated with lipopolysaccharide. Peripheral blood mononuclear cells were obtained from healthy volunteers isolated by density centrifugation method using Ficoll-Hypaque. Once coupled with various concentrations of betel leaf extract and lipopolysaccharide, and then incubated for 24 hours, the culture supernatant was used to determine the level of IFN-γ and TNF-α by ELISA method. Results: It is known that the survival rates of BLE 4%, 2% and 1% were 82%, 83.4% and 85%. There was no significant of difference between several concentrations of BLE and those in the control group (p>0.05. The production of IFN-γ and TNF-α stimulated with LPS was no significant difference between BLE 4%, 2% and 1% and that in the control group (p>0.05. Conclusion: It can be concluded that BLE is not toxic against primary culture of chicken embryo fibroblast, and the production of IFN-γ and TNF-α by PBMC was not affected by BLE.Latar belakang: Daun

  13. CYTOMEGALOVIRUS INTERSTITIAL PNEUMONITIS FOLLOWING ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION

    Institute of Scientific and Technical Information of China (English)

    XU Xiao-hua; HUANG Lian-sheng; ZHANG Xiao-hong; ZHU Kang-er; XU Yang; WU Dong; ZHAO Xiao-ying

    2005-01-01

    Objective: To explore the risk factors and prophylaxis and treatment of cytomegalovirus interstitial pneumonitis(CMV-IP) after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Methods: 43 patients who received allo-PBSCT were allocated to either a Gancyclovir(GCV)-prophylaxis group (n=19) or a non-GCV prophylaxis group (n=24).A comparison was made of the incidence of CMV-IP in patients given or not given prophylactic gancyclovir. Results: 9patients in non-GCV prophylaxis group developed late CMV-IP (P<0.05). Graft-versus-host-disease (GVHD) may be associated with a high risk of CMV-IP. 5 cases of CMV-IP were successfully treated with GCV, but 3 cases died of CMV-IP.The most common adverse event of GCV was neutropenia, but was reversible. Conclusion: CMV infection was a major cause of interstitial pneumonitis after allo-PBSCT, which correlated strongly with the severity of GVHD. Gancyclovir was shown to be effective in both prophylaxis and treatment of CMV-IP.

  14. The modulating effect of royal jelly consumption against radiation-induced apoptosis in human peripheral blood leukocytes

    Directory of Open Access Journals (Sweden)

    Navid Rafat

    2016-01-01

    Full Text Available The present work was designed to assess the radioprotective effect of royal jelly (RJ against radiation-induced apoptosis in human peripheral blood leukocytes. In this study, peripheral blood samples were obtained on days 0, 4, 7, and 14 of the study from six healthy male volunteers taking a 1000 mg RJ capsule orally per day for 14 consecutive days. On each sampling day, all collected whole blood samples were divided into control and irradiated groups which were then exposed to the selected dose of 4 Gy X-ray. Percentage of apoptotic cells (Ap % was evaluated for all samples immediately after irradiation (Ap0 and also after a 24 h postirradiation incubation at 37°C in 5% CO2 (Ap24 by the use of neutral comet assay. Concerning Ap0, collected data demonstrated that the percentage of apoptotic cells in both control and irradiated groups did not significantly change during the study period. However, with respect to Ap24, the percentage of apoptotic cells in irradiated groups gradually reduced during the experiment, according to which a significant decrease was found after 14 days RJ consumption (P = 0.002. In conclusion, the present study revealed the protective role of 14 days RJ consumption against radiation-induced apoptosis in human peripheral blood leukocytes.

  15. Modelled temperature-dependent excitability behaviour of a generalised human peripheral sensory nerve fibre

    CSIR Research Space (South Africa)

    Smit, Jacoba E

    2009-09-01

    Full Text Available The objective of this study was to determine if a recently developed human Ranvier node model, which is based on a modified version of the Hodgkin-Huxley model, could predict the excitability behaviour in human peripheral sensory nerve fibres...

  16. Proteomic Analysis Identifies Outcome-Predictive Clusters in Patients with Peripheral T-Cell Lymphoma, Not otherwise specified

    DEFF Research Database (Denmark)

    Ludvigsen, Maja; Pedersen, Martin Bjerregård; Poulsen, T.S.

    2014-01-01

    Proteomic Analysis Identifies Outcome-Predictive Clusters in Patients with Peripheral T-Cell Lymphoma, Not otherwise specified......Proteomic Analysis Identifies Outcome-Predictive Clusters in Patients with Peripheral T-Cell Lymphoma, Not otherwise specified...

  17. Natural Killer Cell Assessment in Peripheral Circulation and Bronchoalveolar Lavage Fluid of Patients with Severe Sepsis: A Case Control Study

    Science.gov (United States)

    Souza-Fonseca-Guimaraes, Paulo; Guimaraes, Fernando; Natânia De Souza-Araujo, Caroline; Maria Boldrini Leite, Lidiane; Cristina Senegaglia, Alexandra; Nishiyama, Anita; Souza-Fonseca-Guimaraes, Fernando

    2017-01-01

    Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-γ). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56bright or dim) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis. PMID:28287491

  18. Natural Killer Cell Assessment in Peripheral Circulation and Bronchoalveolar Lavage Fluid of Patients with Severe Sepsis: A Case Control Study.

    Science.gov (United States)

    Souza-Fonseca-Guimaraes, Paulo; Guimaraes, Fernando; Natânia De Souza-Araujo, Caroline; Maria Boldrini Leite, Lidiane; Cristina Senegaglia, Alexandra; Nishiyama, Anita; Souza-Fonseca-Guimaraes, Fernando

    2017-03-12

    Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-γ). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56(bright or dim)) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis.

  19. Novel agents inhibit human leukemic cells

    Institute of Scientific and Technical Information of China (English)

    Wei-ping YU; Juan LI

    2012-01-01

    Ouabain (OUA) and pyrithione zinc (PZ) have been proved as the potential drugs for treating acute myeloid leukemia (AML).Selected from a screening among 1040 Food and Drug Administration-approved pharmacological agents,both drugs showability to induce apoptosis of the culturing AML cells,exhibiting the poisoning effect on the cells.Studies also reveal the efficiency of the drugs in inhibiting the growth of human AML cells injected into the mice lacking of immunity and killing primary AML cells from the peripheral blood of AML patients[1].

  20. Tissue engineering with peripheral blood-derived mesenchymal stem cells promotes the regeneration of injured peripheral nerves.

    Science.gov (United States)

    Pan, Mengjie; Wang, Xianghai; Chen, Yijing; Cao, Shangtao; Wen, Jinkun; Wu, Guofeng; Li, Yuanyuan; Li, Lixia; Qian, Changhui; Qin, Zhenqi; Li, Zhenlin; Tan, Dandan; Fan, Zhihao; Wu, Wutian; Guo, Jiasong

    2017-06-01

    Peripheral nerve injury repair can be enhanced by Schwann cell (SC) transplantation, but clinical applications are limited by the lack of a cell source. Thus, alternative systems for generating SCs are desired. Herein, we found the peripheral blood-derived mesenchymal stem cells (PBMSCs) could be induced into SC like cells with expressing SC-specific markers (S100, P75NTR and CNPase) and functional factors (NGF, NT-3, c-Fos, and Krox20). When the induced PBMSCs (iPBMSCs) were transplanted into crushed rat sciatic nerves, they functioned as SCs by wrapping the injured axons and expressing myelin specific marker of MBP. Furthermore, iPBMSCs seeded in an artificial nerve conduit to bridge a 10-mm defect in a sciatic nerve achieved significant nerve regeneration outcomes, including axonal regeneration and remyelination, nerve conduction recovery, and restoration of motor function, and attenuated myoatrophy and neuromuscular junction degeneration in the target muscle. Overall, the data from this study indicated that PBMSCs can transdifferentiate towards SC-like cells and have potential as grafting cells for nerve tissue engineering. Copyright © 2017. Published by Elsevier Inc.

  1. 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

  2. [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.

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

    Directory of Open Access Journals (Sweden)

    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.

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  11. A Splenic Marginal Zone-Like Peripheral Blood CD27+B220− B Cell Population Is Preferentially Depleted in HIV Type 1-Infected Individuals

    OpenAIRE

    Morrow, Matthew; Valentin, Antonio; Little, Richard; Yarchoan, Robert; Pavlakis, George N.

    2008-01-01

    Peripheral blood CD27+ B cells are reduced in HIV-1-infected individuals. In healthy individuals, the human peripheral blood CD27+ B cell pool consists of two subsets defined by the expression, or lack thereof, of the CD45 isoform B220. We investigated the presence of circulating B220+ and B220− memory B cells in HIV+ individuals and found that the reduction in CD27+ memory B cells occurs primarily among CD27+B220− B cells. Studies conducted using healthy controls indicate that CD27+B220− B c...

  12. Bridging long gap peripheral nerve injury using skeletal muscle-derived multipotent stem cells

    Institute of Scientific and Technical Information of China (English)

    Tetsuro Tamaki

    2014-01-01

    Long gap peripheral nerve injuries usually reulting in life-changing problems for patients. Skeletal muscle derived-multipotent stem cells (Sk-MSCs) can differentiate into Schwann and perineurial/endoneurial cells, vascular relating pericytes, and endothelial and smooth muscle cells in the damaged peripheral nerve niche. Application of the Sk-MSCs in the bridging conduit for repairing long nerve gap injury resulted favorable axonal regeneration, which showing supe-rior effects than gold standard therapy--healthy nerve autograft. This means that it does not need to sacriifce of healthy nerves or loss of related functions for repairing peripheral nerve injury.

  13. Increased ability of peripheral blood B cells from patients with rheumatoid arthritis to produce interleukin 1 in vitro.

    Directory of Open Access Journals (Sweden)

    Yamamura,Masahiro

    1990-12-01

    Full Text Available Twenty-four patients with rheumatoid arthritis (RA and 20 normal controls were examined for the ability of their peripheral blood B cells to produce interleukin 1 (IL-1 with or without lipopolysaccharide (LPS. B cells were purified from peripheral blood by negative selection methods (i.e., removal of adherent cells and sheep red blood cell rosette-forming cells, followed by treatment with monoclonal antibodies (OKT3 and OKM1 and complement. The amount of IL-1 in B cell culture supernatants (SN was measured by thymocyte and fibroblast proliferation assays and an enzyme-linked immunosorbent assay for IL-1 alpha and beta. As a group, cultured B cells from patients with RA, both spontaneously and when stimulated with LPS, produced higher levels of IL-1 than those from normal controls. IL-1 production by RA B cells with LPS had a weak but positive correlation with disease activity. Moreover, RA B cell culture SN with elevated levels of IL-1 had a synergistic effect on the growth of anti-human IgM (anti-mu stimulated B cells. In separate experiments, the growth of RA B cells was significantly promoted by IL-1 beta both with and without anti-mu stimulation. These results suggest that B cell-derived IL-1 may be involved in the B cell clonal expansion of RA through its own activity as a B cell stimulatory factor.

  14. 线粒体DNA含量与HIV相关脂肪营养不良的关系%Association between the change regularity of peripheral blood mononuclear cell mitochondrial deoxyribonucleic acid content and human immunodeficiency virus-related lipodystrophy

    Institute of Scientific and Technical Information of China (English)

    吴鹏; 顾卫斌; 张璐; 李雁凌; 邱志峰; 韩扬; 谢静; 李太生

    2011-01-01

    Objective To investigate the change regularity of peripheral blood mononuclear cell ( PBMC) mtDNA ( mitochondrial deoxyribonucleic acid) content and its association with HIV-LD ( human immunodeficiency virus-related lipodystrophy ) in HAART ( highly active antiretroviral therapy). Methods At baseline, Months 6 and 24 of therapy, the cryopreserved PBMC were collected from 33 patients on a regular follow-up at our clinic. Among them, 17 had HIV-LD. Then total DNA was extracted and mtDNA content quantified by real-time PCR (polymerase chain reaction). Results The HIV/AIDS patients had a lower content of PBMC mtDNA (2-△△Ct) than the healthy controls at baseline (9.578 vs 17. 195, P <0.01). The mtDNA content was lower in the HIV-LD group than that in the no LD (NLD) group at each timepoint of therapy (13.619 vs5.775, 6.360 vs 1.387, 7. 170 vs 1.266, all P<0.05). In the HIV-LD group, the half- and 2-year PBMC mtDNA content was markedly lower than those at baseline (both P <0. 05 ). And the change of mtDNA content (within half a year) was earlier than the onset of clinical HIV-LD at one year later. In the NLD group, the PBMC mtDNA content have an insignificant change after therapy.The mtDNA content decreased significantly in stavudine (d4T)-containing regimen group after treatment (P <0. 01) , but showed no significant change in zidovudine ( AZT) -containing regimen group after therapy.Conclusion The decreased content of PBMC mtDNA after HIV infection and during HAART therapy is associated with HIV-LD. Nucleoside reverse transcriptase inhibitor, especially d4T, plays an important role in the progression of HIV-LD.%目的 研究高效联合抗反转录病毒治疗(HAART)过程中外周血单核淋巴细胞(PBMC)内线粒体DNA(mtDNA)含量变化规律及其与人类免疫缺陷病毒(HIV)感染相关脂肪营养不良(HIV-LD)的相关性.方法 通过实时定量PCR(real-time PCR),对2002年5月至2008年5月北京协和医院感染内

  15. Peripheral sensory cells in the cephalic sensory organs of Lymnaea stagnalis.

    Science.gov (United States)

    Wyeth, Russell C; Croll, Roger P

    2011-07-01

    The peripheral nervous system in gastropods plays a key role in the neural control of behaviors, but is poorly studied in comparison with the central nervous system. Peripheral sensory neurons, although known to be widespread, have been studied in a patchwork fashion across several species, with no comprehensive treatment in any one species. We attempted to remedy this limitation by cataloging peripheral sensory cells in the cephalic sensory organs of Lymnaea stagnalis employing backfills, vital stains, histochemistry, and immunohistochemistry. By using at least two independent methods to corroborate observations, we mapped four different cell types. We have found two different populations of bipolar sensory cells that appear to contain catecholamines(s) and histamine, respectively. Each cell had a peripheral soma, an epithelial process bearing cilia, and a second process projecting to the central nervous system. We also found evidence for two populations of nitric oxide-producing sensory cells, one bipolar, probably projecting centrally, and the second unipolar, with only a single epithelial process and no axon. The various cell types are presumably either mechanosensory or chemosensory, but the complexity of their distributions does not allow formation of hypotheses regarding modality. In addition, our observations indicate that yet more peripheral sensory cell types are present in the cephalic sensory organs of L. stagnalis. These results are an important step toward linking sensory cell morphology to modality. Moreover, our observations emphasize the size of the peripheral nervous system in gastropods, and we suggest that greater emphasis be placed on understanding its role in gastropod neuroethology.

  16. Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicits TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

    Institute of Scientific and Technical Information of China (English)

    HongZhou; GuofuDing; WeiLiu; LiangxiWang; YonglingLu; HongweiCao; JiangZheng

    2004-01-01

    Lipopolysaccharide (LPS [endotoxin]), the principal component of the outer membrane of gram-negative bacteria, stimulate various cell types to release numerous proinflammatory mediators such as TNF-α, IL-6 and IL-12, which may damage cells and lead to organ injury, even sepsis and septic shock. Toll-like receptor 4(TLR4) has been identified as the receptor involved in the recognition of LPS, but the role of LPS uptake inactivating signal transduction remains controversial. In the present study, TNF-α was used as a marker of macrophages/monocytes activated by LPS, and CQ was used as an inhibitor of endosome mature in order to definitude what stage the signal transduction elicited by LPS was interrupted. We found that there indeed existed internalization of LPS and internalization partially participated in LPS signaling since CQ inhibitedcytokine release, and decreased accumulation of FITC-LPS in hPBMC. In contrast, anti-hTLR4 antibodycould decrease cytokines' release, but no inhibition on accumulation of FITC-LPS. This result revealed thatinhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells. But TLR4 on thecell surface didn't possibly participated in internalization of LPS. Thus, LPS signaling and internalizationcannot be viewed as mutually independent processes. Cellular & Molecular Immunology. 2004;1(5):373-377.

  17. Methylene blue modulates transendothelial migration of peripheral blood cells.

    Directory of Open Access Journals (Sweden)

    Isabella Werner

    Full Text Available Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1 were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial

  18. Methylene blue modulates transendothelial migration of peripheral blood cells.

    Science.gov (United States)

    Werner, Isabella; Guo, Fengwei; Bogert, Nicolai V; Stock, Ulrich A; Meybohm, Patrick; Moritz, Anton; Beiras-Fernandez, Andres

    2013-01-01

    Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose

  19. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    Directory of Open Access Journals (Sweden)

    Anna V. Savelyeva

    2017-01-01

    Full Text Available Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs’ fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches.

  20. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions.

    Science.gov (United States)

    Savelyeva, Anna V; Kuligina, Elena V; Bariakin, Dmitry N; Kozlov, Vadim V; Ryabchikova, Elena I; Richter, Vladimir A; Semenov, Dmitry V

    2017-01-01

    Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches.

  1. Assay of Peripheral Regulatory Vδ1 T Cells in Ankylosing Spondylitis and its Significance

    Science.gov (United States)

    Wang, Hongliang; Sun, Na; Li, Ka; Tian, Jiguang; Li, Jianmin

    2016-01-01

    Background Ankylosing spondylitis (AS) involves inflammation at the sacroiliac joint and spine attachment site. This study aimed to observe the ratio and function of peripheral regulatory Vδ1 T cells in AS patients to investigate their roles in AS pathogenesis. Material/Methods Peripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation from AS patients and healthy controls. Flow cytometry was used to determine the ratio between Vδ1 and CD4 T cells of PBMC in AS patients and controls. Flow cytometry sorting (FCS) was used to obtain Vδ1 and naïve CD4 T cells with purity higher than 90%. CFSE staining method was used to detect the effect of Vδ1 T cells on proliferation of naïve CD4 T cells. The effect of Vδ1 T cells on secretion of IFN-γ from naïve CD4 T cells and the ability to secrete IL-10 from Vδ1 T cells were determined by flow cytometry. Results AS patients had significantly lower Vδ1 T cell ratio in PBMC compared to controls (p<0.05), but their CD4 T cell ratio was significantly elevated (p<0.05). Functional assay showed suppression of naïve CD4 T cell proliferation and IFN-γ secretion by peripheral Vδ1 T cells in AS patients (p<0.01). AS patients also had lower IL-10 secreting level from peripheral derived Vδ1 T cells (p<0.01). Conclusions The immune suppression of peripheral Vδ1 T cell in AS patient increases the ratio of peripheral CD4 T cells and IFN-γ level, leading to AS pathogenesis. This immune suppression is mainly due to suppressed IL-10 secretion. PMID:27598263

  2. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

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    Restu Syamsul Hadi

    2015-12-01

    Full Text Available BACKGROUND Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. METHODS In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. RESULTS Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1- fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity CONCLUSIONS Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  3. Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

    Directory of Open Access Journals (Sweden)

    Restu Syamsul Hadi

    2014-08-01

    Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

  4. Human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Abdallah, Basem; Kassem, Moustapha

    2008-01-01

    Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

  5. Aire-Overexpressing Dendritic Cells Induce Peripheral CD4⁺ T Cell Tolerance.

    Science.gov (United States)

    Li, Dongbei; Li, Haijun; Fu, Haiying; Niu, Kunwei; Guo, Yantong; Guo, Chuan; Sun, Jitong; Li, Yi; Yang, Wei

    2015-12-29

    Autoimmune regulator (Aire) can promote the ectopic expression of peripheral tissue-restricted antigens (TRAs) in thymic medullary epithelial cells (mTECs), which leads to the deletion of autoreactive T cells and consequently prevents autoimmune diseases. However, the functions of Aire in the periphery, such as in dendritic cells (DCs), remain unclear. This study's aim was to investigate the effect of Aire-overexpressing DCs (Aire cells) on the functions of CD4⁺ T cells and the treatment of type 1 diabetes (T1D). We demonstrated that Aire cells upregulated the mRNA levels of the tolerance-related molecules CD73, Lag3, and FR4 and the apoptosis of CD4⁺ T cells in STZ-T1D mouse-derived splenocytes. Furthermore, following insulin stimulation, Aire cells decreased the number of CD4⁺ IFN-γ⁺ T cells in both STZ-T1D and WT mouse-derived splenocytes and reduced the expression levels of TCR signaling molecules (Ca(2+) and p-ERK) in CD4⁺ T cells. We observed that Aire cells-induced CD4⁺ T cells could delay the development of T1D. In summary, Aire-expressing DCs inhibited TCR signaling pathways and decreased the quantity of CD4⁺IFN-γ⁺ autoreactive T cells. These data suggest a mechanism for Aire in the maintenance of peripheral immune tolerance and provide a potential method to control autoimmunity by targeting Aire.

  6. Molybdate modulates mitogen and cyclosporin responses of human peripheral blood lymphocytes.

    Science.gov (United States)

    Michelis, Fotios V; Delitheos, Andreas; Tiligada, Ekaterini

    2011-07-01

    The trace element molybdenum (Mo) is an essential component of key physiological systems in animals, plants and microorganisms. The molybdate oxoanion MoO(4)(2-) has been demonstrated to cause diverse yet poorly understood biochemical and pharmacological effects, such as non-specific inhibition of phosphatases and stabilization of steroid receptors. This study aimed to investigate the effects of molybdate on the activation of human peripheral blood lymphocytes (hPBLs) ex vivo and its potential interaction with the widely used immunosuppressant drug cyclosporin A (CsA). Lymphocyte activation was evaluated by performing multiple experiments determining blastogenesis in cultured peripheral blood lymphocytes obtained from 5 healthy volunteers, following stimulation induced by phytohemagglutinin (PHA), in the absence or presence of 0.05-10 mM sodium molybdate or/and 2.5-30 μg/mL CsA. Blastogenesis was assessed by a morphometric assay based on the relative proportions of unactivated lymphocytes, activated lymphoblasts and cells with aberrant morphology after PHA-induced activation. Molybdate concentrations up to 1 mM showed no effect on lymphocyte blastogenesis, while higher concentrations exerted immunosuppressive actions on cultured hPBLs. Co-administration of 0.1 mM sodium molybdate with CsA, at doses up to 20 μg/mL, induced no alteration in the response of cultured hPBLs to CsA. However, molybdate potentiated the immunosuppressive action of higher CsA concentrations, implying a likely dose-related synergistic interaction of the two agents in PHA-stimulated blood lymphocytes. These observations are indicative of the possible biological importance of molybdate oxoanions in the modulation of hPBL activation that may have pharmacological consequences during the therapeutic application of immunomodulatory drugs.

  7. Phenotypic Changes and Impaired Function of Peripheral γδ T Cells in Patients With Sepsis.

    Science.gov (United States)

    Liao, Xue-Lian; Feng, Ting; Zhang, Jiang-Qian; Cao, Xing; Wu, Qi-Hong; Xie, Zhi-Chao; Kang, Yan; Li, Hong

    2017-09-01

    Recent studies demonstrated the significant loss of gamma delta T (γδ T) cells in patients with sepsis. Given the distinct functions of γδ T cells in human anti-infection immunity, we are interested in evaluating the phenotype and function of peripheral γδ T cells in septic patients and determining their prognostic implication. This prospective study has been conducted in three intensive care units of a university hospital. During the period from October 2014 to June 2015, we enrolled 107 patients who were consecutively admitted and diagnosed with severe sepsis or septic shock (excluding previous immunosuppression) and 45 healthy controls. Using flow cytometry, we analyzed the in vivo percentage of γδ T cells in cluster of differentiation (CD)3 cells from peripheral blood mononuclear cells as well as their expression of surface markers (CD69, natural-killer group 2 member D [NKG2D], programmed death receptor 1 [PD-1]) and intracellular cytokines (interferon-γ [IFN-γ], interleukin [IL]-17, IL-10, transforming growth factor-β [TGF-β]). Then we further evaluated the different responses of γδ T cells after the antigen stimulation ex vivo by measuring CD69 and IFN-γ expression. Lastly, we conducted the multiple logistic regressions to analyze the risk factor for prognosis. Compared with control group, γδ T cells in septic patients displayed a decrease in percentage, increase in CD69, decrease in NKG2D, and increase in cytokine expression (pro-inflammatory IFN-γ, IL-17, anti-inflammatory IL-10, TGF-β) in vivo. After the antigen stimulation ex vivo, both CD69 and IFN-γ expression in γδ T cells were significantly lower in septic patients than control group. Importantly, the decrease in CD69 and IFN-γ expression was more pronounced in non-survivors than survivors. Multiple logistic regression analysis revealed that lower expression of IFN-γ after stimulation is a dependent risk factor that associated with patient 28-day death in septic patients (OR

  8. Changes of Number and Function of Late Endothelial Progenitor Cells in Peripheral Blood of COPD Patients Combined with Pulmonary Hypertension.