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  1. Haemopoietic progenitor cells in human peripheral blood

    International Nuclear Information System (INIS)

    Zwaan, F.E.

    1980-01-01

    The purpose of the investigation reported is to purify haemopoietic progenitor cells from human peripheral blood using density gradient centrifugation in order to isolate a progenitor cell fraction without immunocompetent cells. The purification technique of peripheral blood flow colony forming unit culture (CFU-c) by means of density gradient centrifugation and a combined depletion of various rosettes is described. The results of several 'in vitro' characteristics of purified CFU-c suspensions and of the plasma clot diffusion chamber culture technique are presented. Irradiation studies revealed that for both human bone marrow and peripheral blood the CFU-c were less radioresistant than clusters. Elimination of monocytes (and granulocytes) from the test suspensions induced an alteration in radiosensitivity pararmeters. The results obtained with the different techniques are described by analysing peripheral progenitor cell activity in myeloproliferative disorders. (Auth.)

  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...... research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies....

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

    Science.gov (United States)

    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

    2010-11-01

    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. Invasin of Yersinia pseudotuberculosis activates human peripheral B cells.

    OpenAIRE

    Lundgren, E; Carballeira, N; Vazquez, R; Dubinina, E; Bränden, H; Persson, H; Wolf-Watz, H

    1996-01-01

    The Yersinia pseudotuberculosis cell surface-located protein invasin was found to promote binding between the pathogen and resting peripheral B cells via beta 1 integrin receptors (CD29). B cells responded by expressing several activation markers and by growing, In contrast, T cells did not react, although these cells express CD29. An isogenic invA mutant failed to activate B cells. The mutation could be complemented by providing the invA+ gene in trans. Purified invasin alone did not activat...

  6. 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.

  7. 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

  8. Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

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    Kai Fu

    Full Text Available Human serum albumin (HSA is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA before a First-in-human (FIH trial, we compared OsrHSA and plasma-derived HSA (pHSA, evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs. The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+ and CD8(+ T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α, interleukin (IL-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

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

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    Nan Yu

    Full Text Available 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.

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. [Human herpesvirus-6 pneumonitis following autologous peripheral blood stem cell transplantation].

    Science.gov (United States)

    Saitoh, Yuu; Gotoh, Moritaka; Yoshizawa, Seiichiro; Akahane, Daigo; Fujimoto, Hiroaki; Ito, Yoshikazu; Ohyashiki, Kazuma

    2018-01-01

    A-46-year-old man was diagnosed with peripheral T cell lymphoma, not otherwise specified. He achieved a complete remission after pirarubicin, cyclophosphamide, vincristine, and prednisolone (THP-COP) therapy and successful autologous peripheral blood stem-cell transplantation (AutoSCT). However, 6 months post AutoSCT, he complained of fever. Chest computed tomography of the patient displayed bilateral interstitial pneumonitis. Human herpesvirus-6 (HHV-6) DNA was detected in his bronchoalveolar lavage fluid. Therefore, the patient was confirmed for HHV-6 pneumonitis. The treatment with foscarnet was effective, and no relapse was noticed in the patient. Besides, we have experienced pneumonitis of unknown origin in some patients after autologous or allogeneic stem-cell transplantations. Moreover, most of the above patients were clinically diagnosed using serum or plasma markers. Therefore, examining respiratory symptoms after AutoSCT would enable a more accurate diagnosis as well as treatment of patients with HHV-6 pneumonitis.

  12. Peripheral Nerve Regeneration by Secretomes of Stem Cells from Human Exfoliated Deciduous Teeth.

    Science.gov (United States)

    Sugimura-Wakayama, Yukiko; Katagiri, Wataru; Osugi, Masashi; Kawai, Takamasa; Ogata, Kenichi; Sakaguchi, Kohei; Hibi, Hideharu

    2015-11-15

    Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbecco's modified Eagle's medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration.

  13. Research on effects of ionizing radiation of human peripheral blood white cell adhesive molecules

    International Nuclear Information System (INIS)

    Li Haijun; Cheng Ying; Le Chen; Min Rui

    2008-01-01

    Objective: To investigate the links between expression and function of adhesive molecule on the surface of irradiated peripheral blood white cells. Methods: Heparinized human peripheral blood was exposed to γ rays with different dose. At the different post-radiation time adhesive molecule expression on cellular surface was determined by double fluorescence labeling antibodies which were against adhesive molecule and special mark of granulocyte or mononuclear cell respectively with flow cytometry, and cellular adhesive ability to different matrixes mediated by adhesive molecule was estimated by commercializing enzyme-linked immunosorbent assay kit and crystalviolet dying. Results: A decline pattern of CD11b on surface of mononuclear cells and CD29 on surface of granulocyte with irradiation dose increase was found. The changes of adhesive ability of mononuclear cells to substance of β1-integrin and collagen-I was well related with irradiation dose. Conclusion: Good relationship shown by the changes of adhesive molecule expression and adhesive ability mediated by the molecules on the surface of peripheral blood white cells with radiation dose was primary base of further research on indicting exposure dose by biomarker. (authors)

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-11-01

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

  19. Donating Peripheral Blood Stem Cells

    Science.gov (United States)

    ... Print this page My Cart Donating peripheral blood stem cells Peripheral blood stem cell (PBSC) donation is a nonsurgical procedure to collect ... Donating bone marrow Donor experiences videos Peripheral blood stem cell (PBSC) donation is one of two methods of ...

  20. The DNA damage of high doses of X-ray on human peripheral blood nucleated cell's and sperm

    International Nuclear Information System (INIS)

    Wang Hui; Zoulian; Jiang Qisheng; Li Fengsheng; He Rui; Song Xiujun

    2011-01-01

    Objective: To detect the DNA damage of high doses of X-ray on human peripheral blood nucleated cell's and sperm by single cell gel electrophoresis (SCGE). Evaluation the level of DNA damage of human peripheral blood nucleated cell's and sperm after high doses of X-ray. Methods: Using human peripheral blood with normal blood routine and normal sperm,give the dose of 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy X-ray radiation with energy of 6MU. Detect the percentage of comet-like tail, tail length and content of DNA in tail of whole blood cell's DNA and sperm's DNA by SCGE technique in 1 hour. Results: The peripheral blood nucleated cell's and sperm's comet rate were 1.00±0.10%, 2.1±1.5%, respectively, have an evidently variance in 0 Gy group (υ=18, t=2.31>1.734, P 1.734, P 1.734, P<0.05). The peripheral blood nucleated cell's and sperm's comet rate were all 100%, 100%, have no-statistical significance in 8 Gy, 10 Gy group. Conclusion: The evidence is powerful enough. That the sperm's SCGE is more sensitive than peripheral blood nucleated cell's SCGE in reflect the X-ray damage in a certain extent (2-6 Gy). (authors)

  1. Dissecting interferon-induced transcriptional programs in human peripheral blood cells.

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    Simon J Waddell

    2010-03-01

    Full Text Available 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 alpha, beta, omega and gamma, IL12 and TNFalpha; and (2 characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, 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.

  2. Human adipose stromal cells expanded in human serum promote engraftment of human peripheral blood hematopoietic stem cells in NOD/SCID mice

    International Nuclear Information System (INIS)

    Kim, Su Jin; Cho, Hyun Hwa; Kim, Yeon Jeong; Seo, Su Yeong; Kim, Han Na; Lee, Jae Bong; Kim, Jae Ho; Chung, Joo Seop; Jung, Jin Sup

    2005-01-01

    Human mesenchymal stem cells (hMSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles, and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle, and neuron. Therefore, hMSC are attractive candidates for cell and gene therapy. The optimal conditions for hMSC expansion require medium supplemented with fetal bovine serum (FBS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FBS proteins. In this study, we cultured human adipose stromal cells (hADSC) and bone marrow stroma cells (HBMSC) in human serum (HS) during their isolation and expansion, and demonstrated that they maintain their proliferative capacity and ability for multilineage differentiation and promote engraftment of peripheral blood-derived CD34(+) cells mobilized from bone marrow in NOD/SCID mice. Our results indicate that hADSC and hBMSC cultured in HS can be used for clinical trials of cell and gene therapies, including promotion of engraftment after allogeneic HSC transplantation

  3. Grass immunotherapy induces inhibition of allergen-specific human peripheral blood mononuclear cell proliferation.

    Science.gov (United States)

    Baskar, S; Hamilton, R G; Norman, P S; Ansari, A A

    1997-02-01

    The peripheral blood mononuclear cells (PBMC) from humans allergic to grass pollens (GR+ subjects) show strong in vitro proliferative responses to purified allergens from Lolium perenne pollen Lol p 1, and to a lesser extent to Lol p 2 and Lol p 3. By contrast, PBMC from grass allergic patients undergoing immunotherapy (GR + IT subjects) exhibit a very poor Lol p-specific proliferative response, similar to that observed in nongrass allergic subjects (GR-subjects). Unlike GR-subjects, both GR+ and GR + IT subjects have high levels of antigen-specific serum IgG and IgE antibodies to Lol p 1, Lol p 2 and Lol p 3. While GR+ subjects exhibit a significant correlation between antigen-specific serum antibody and PBMC responses, GR + IT subjects do not show a correlation between the two responses. The possible mechanisms by which immunotherapy may modulate allergen-specific T cell proliferative response are discussed.

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

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    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.

  5. Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood.

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    Gu, Haihui; Huang, Xia; Xu, Jing; Song, Lili; Liu, Shuping; Zhang, Xiao-Bing; Yuan, Weiping; Li, Yanxin

    2018-06-15

    Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established.

  6. Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry.

    Science.gov (United States)

    Kunicki, Matthew A; Amaya Hernandez, Laura C; Davis, Kara L; Bacchetta, Rosa; Roncarolo, Maria-Grazia

    2018-01-01

    Human CD3 + CD4 + Th cells, FOXP3 + T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3 + CD4 + T cell compartment remains questionable. In this study, we examined CD3 + CD4 + T cell populations by single-cell mass cytometry. We characterize the CD3 + CD4 + Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3 + CD4 + Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4) + FOXP3 + Treg and CD183 (CXCR3) + T-bet + Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3 + CD4 + T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3 + CD4 + T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Sporothrix schenckii sensu stricto and Sporothrix brasiliensis Are Differentially Recognized by Human Peripheral Blood Mononuclear Cells

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    José A. Martínez-Álvarez

    2017-05-01

    Full Text Available Sporothrix schenckii sensu stricto and S. brasiliensis are usually associated to sporotrichosis, a subcutaneous mycosis worldwide distributed. Comparative analyses between these two species indicate they contain genetic and physiological differences that are likely to impact the interaction with host cells. Here, we study the composition of the cell wall from conidia, yeast-like cells and germlings of both species and found they contained the same sugar composition. The carbohydrate proportion in the S. schenckii sensu stricto wall was similar across the three cell morphologies, with exception in the chitin content, which was significantly different in the three morphologies. The cell wall from germlings showed lower rhamnose content and higher glucose levels than other cell morphologies. In S. brasiliensis, the wall sugars were constant in the three morphologies, but glucose was lower in yeast-like cells. In S. schenckii sensu stricto cells most of chitin and β1,3-glucan were underneath wall components, but in S. brasiliensis germlings, chitin was exposed at the cell surface, and β1,3-glucan was found in the outer part of the conidia wall. We also compared the ability of these cells to stimulate cytokine production by human peripheral blood mononuclear cells. The three S. schenckii sensu stricto morphologies stimulated increased levels of pro-inflammatory cytokines, when compared to S. brasiliensis cells; while the latter, with exception of conidia, stimulated higher IL-10 levels. Dectin-1 was a key receptor for cytokine production during stimulation with the three morphologies of S. schenckii sensu stricto, but dispensable for cytokine production stimulated by S. brasiliensis germlings. TLR2 and TLR4 were also involved in the sensing of Sporothrix cells, with a major role for the former during cytokine stimulation. Mannose receptor had a minor contribution during cytokine stimulation by S. schenckii sensu stricto yeast-like cells and

  8. Comparative Analysis of the Regulatory T Cells Dynamics in Peripheral Blood in Human and Porcine Polytrauma

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    Rafael Serve

    2018-03-01

    Full Text Available BackgroundSeverely injured patients experience substantial immunological stress in the aftermath of traumatic insult, which often results in systemic immune dysregulation. Regulatory T cells (Treg play a key role in the suppression of the immune response and in the maintenance of immunological homeostasis. Little is known about their presence and dynamics in blood after trauma, and nothing is known about Treg in the porcine polytrauma model. Here, we assessed different subsets of Treg in trauma patients (TP and compared those to either healthy volunteers (HV or data from porcine polytrauma.MethodsPeripheral blood was withdrawn from 20 TP with injury severity score (ISS ≥16 at the admittance to the emergency department (ED, and subsequently on day 1 and at day 3. Ten HV were included as controls (ctrl. The porcine polytrauma model consisted of a femur fracture, liver laceration, lung contusion, and hemorrhagic shock resulting in an ISS of 27. After polytrauma, the animals underwent resuscitation and surgical fracture fixation. Blood samples were withdrawn before and immediately after trauma, 24 and 72 h later. Different subsets of Treg, CD4+CD25+, CD4+CD25+FoxP3+, CD4+CD25+CD127−, and CD4+CD25+CD127−FoxP3+ were characterized by flow cytometry.ResultsAbsolute cell counts of leukocytes were significantly increasing after trauma, and again decreasing in the follow-up in human and porcine samples. The proportion of human Treg in the peripheral blood of TP admitted to the ED was lower when compared to HV. Their numbers did not recover until 72 h after trauma. Comparable data were found for all subsets. The situation in the porcine trauma model was comparable with the clinical data. In porcine peripheral blood before trauma, we could identify Treg with the typical immunophenotype (CD4+CD25+CD127−, which were virtually absent immediately after trauma. Similar to the human situation, most of these cells expressed FoxP3, as assessed by

  9. Differential induction from X-irradiated human peripheral blood monocytes to dendritic cells

    International Nuclear Information System (INIS)

    Yoshino, Hironori; Takahashi, Kenji; Monzen, Satoru; Kashiwakura, Ikuo

    2008-01-01

    Dendritic cells (DCs) are a type of antigen-presenting cell which plays an essential role in the immune system. To clarify the influences of ionizing radiation on the differentiation to DCs, we focused on human peripheral blood monocytes and investigated whether X-irradiated monocytes can differentiate into DCs. The non-irradiated monocytes and 5 Gy-irradiated monocytes were induced into immature DCs (iDCs) and mature DCs (mDCs) with appropriate cytokine stimulation, and the induced cells from each monocyte expressed each DC-expressing surface antigen such as CD40, CD86 and HLA-DR. However, the expression levels of CD40 and CD86 on the iDCs derived from the 5 Gy-irradiated monocytes were higher than those of iDCs derived from non-irradiated monocytes. Furthermore, the mDCs derived from 5 Gy-irradiated monocytes had significantly less ability to stimulate allogeneic T cells in comparison to the mDCs derived from non-irradiated monocytes. There were no significant differences in the phagocytotic activity of the iDCs and cytokines detected in the supernatants conditioned by the DCs from the non-irradiated and irradiated monocytes. These results suggest that human monocytes which are exposed to ionizing radiation can thus differentiate into DCs, but there is a tendency that X-irradiation leads to an impairment of the function of DCs. (author)

  10. 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...... 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......, cholesterol and glycosylated hemoglobin (HbA1c). In the group of men, there were significant positive associations between alcohol intake, HbA1c and FPG-sensitive sites in multivariate analysis. The levels of metabolic risk factors were positively associated with age, yet only few subjects fulfilled all...

  11. The production of collagenase by adherent mononuclear cells cultured from human peripheral blood.

    Science.gov (United States)

    Louie, J S; Weiss, J; Ryhänen, L; Nies, K M; Rantala-Ryhänen, S; Uitto, J

    1984-12-01

    Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque centrifugation, and the cells adherent to plastic substrata were cultured in serum-free media supplemented with lactalbumin hydrolysate. These cell cultures, which consisted predominantly of monocyte-macrophages as judged by nonspecific esterase staining, accumulated collagenase in the medium. This collagenase resembled other vertebrate collagenases in that it cleaved native triple-helical type I collagen at a locus 3/4-length away from the amino-terminal end of the molecule. The collagenase activity was inhibited by Na2EDTA, dithiothreitol, and fetal calf serum, while the addition of Ca++ or N-ethylmaleimide enhanced the enzyme activity. The accumulation of collagenase in the culture media was markedly enhanced by the incubation of cells with concanavalin A or phorbol myristic acetate. In the presence of cycloheximide, the levels of collagenase activity were markedly reduced, suggesting that active protein synthesis was required to express the enzyme activity. In additional experiments, monocytes were further purified by counterflow centrifugation-elutriation. The collagenase production was markedly increased in cultures enriched in monocyte-macrophages and devoid of polymorphonuclear leukocytes. The accumulation of collagenase in monocyte cultures incubated for 48 hours in the presence of concanavalin A or phorbol myristic acetate was of the same order of magnitude as in parallel cultures containing the same number of polymorphonuclear leukocytes purified by Ficoll-Hypaque centrifugation and Plasmagel sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Simulated night shift work induces circadian misalignment of the human peripheral blood mononuclear cell transcriptome.

    Science.gov (United States)

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

    2018-05-22

    Misalignment of the endogenous circadian timing system leads to disruption of physiological rhythms and may contribute to the development of the deleterious health effects associated with night shift work. However, the molecular underpinnings remain to be elucidated. Here, we investigated the effect of a 4-day simulated night shift work protocol on the circadian regulation of the human transcriptome. Repeated blood samples were collected over two 24-hour measurement periods from eight healthy subjects under highly controlled laboratory conditions before and 4 days after a 10-hour delay of their habitual sleep period. RNA was extracted from peripheral blood mononuclear cells to obtain transcriptomic data. Cosinor analysis revealed a marked reduction of significantly rhythmic transcripts in the night shift condition compared with baseline at group and individual levels. Subsequent analysis using a mixed-effects model selection approach indicated that this decrease is mainly due to dampened rhythms rather than to a complete loss of rhythmicity: 73% of transcripts rhythmically expressed at baseline remained rhythmic during the night shift condition with a similar phase relative to habitual bedtimes, but with lower amplitudes. Functional analysis revealed that key biological processes are affected by the night shift protocol, most notably the natural killer cell-mediated immune response and Jun/AP1 and STAT pathways. These results show that 4 days of simulated night shifts leads to a loss in temporal coordination between the human circadian transcriptome and the external environment and impacts biological processes related to the adverse health effects associated to night shift work.

  13. 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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Ming-Wei [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Yu, Sung-Liang [Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Lin, Wen-Chang [Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China); Tsai, Ching-Hui [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Chen, Po-Hua [School of Medicine, National Taiwan University, Taipei, Taiwan (China); Lee, Yungling Leo, E-mail: leolee@ntu.edu.tw [Institute of Epidemiology and Preventive Medicine, College of Public Health, National Taiwan University, Taipei, Taiwan (China); Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan (China)

    2016-08-15

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

  16. Direct Conversion of Human Fibroblasts into Schwann Cells that Facilitate Regeneration of Injured Peripheral Nerve In Vivo.

    Science.gov (United States)

    Sowa, Yoshihiro; Kishida, Tsunao; Tomita, Koichi; Yamamoto, Kenta; Numajiri, Toshiaki; Mazda, Osam

    2017-04-01

    Schwann cells (SCs) play pivotal roles in the maintenance and regeneration of the peripheral nervous system. Although transplantation of SCs enhances repair of experimentally damaged peripheral and central nerve tissues, it is difficult to prepare a sufficient number of functional SCs for transplantation therapy without causing adverse events for the donor. Here, we generated functional SCs by somatic cell reprogramming procedures and demonstrated their capability to promote peripheral nerve regeneration. Normal human fibroblasts were phenotypically converted into SCs by transducing SOX10 and Krox20 genes followed by culturing for 10 days resulting in approximately 43% directly converted Schwann cells (dSCs). The dSCs expressed SC-specific proteins, secreted neurotrophic factors, and induced neuronal cells to extend neurites. The dSCs also displayed myelin-forming capability both in vitro and in vivo. Moreover, transplantation of the dSCs into the transected sciatic nerve in mice resulted in significantly accelerated regeneration of the nerve and in improved motor function at a level comparable to that with transplantation of the SCs obtained from a peripheral nerve. The dSCs induced by our procedure may be applicable for novel regeneration therapy for not only peripheral nerve injury but also for central nerve damage and for neurodegenerative disorders related to SC dysfunction. Stem Cells Translational Medicine 2017;6:1207-1216. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  17. Combustible and non-combustible tobacco product preparations differentially regulate human peripheral blood mononuclear cell functions.

    Science.gov (United States)

    Arimilli, Subhashini; Damratoski, Brad E; Prasad, G L

    2013-09-01

    Natural killer (NK) cells and T cells play essential roles in innate and adaptive immune responses in protecting against microbial infections and in tumor surveillance. Although evidence suggests that smoking causes immunosuppression, there is limited information whether the use of smokeless tobacco (ST) products affects immune responses. In this study, we assessed the effects of two preparations of cigarette smoke, ST extract and nicotine on T cell and NK cell responses using Toll-like receptor-ligand stimulated human peripheral blood mononuclear cells (PBMCs). The tobacco product preparations (TPPs) tested included whole smoke conditioned media (WS-CM), total particulate matter (TPM) and a ST product preparation in complete artificial saliva (ST/CAS). The PBMCs were stimulated with polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS). A marked reduction of the expression of intracellular IFN-γ and TNF-α was evident in NK cells and T cells treated with WS-CM and TPM. Consistently, attenuation of ligand-induced secretion of cytokines (IL-1β, IL-10, IL-12 and TNF-α) from PBMCs treated with WS-CM and TPM were observed. While the treatment with TPPs did not alter the expression of the maturation marker CD69, WS-CM and TPM inhibited the cytolytic activity of human PBMCs. Suppression of perforin by WS-CM was also detected. Although interference from the vehicle confounded the interpretation of effects of ST/CAS, some effects were evident only at high concentrations. Nicotine treatment minimally impacted expression of cytokines and cytolytic activity. Data presented herein suggests that the function of NK cells and T cells is influenced by exposure to TPPs (based on equi-nicotine units) in the following order: WS-CM>TPM>ST/CAS. These findings are consistent with the hypothesis put forward by others that chronic smoking leads to immunosuppression, an effect that may contribute to increased microbial infections and cancer incidence among smokers

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

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    Xihe Tang

    2016-03-01

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

  19. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

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    Molenaar Douwe

    2010-11-01

    Full Text Available Abstract Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10 and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs. Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for

  20. Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells.

    Science.gov (United States)

    Chen, Yung-Ching; Wu, King-Chuen; Huang, Bu-Miin; So, Edmund Cheung; Wang, Yang-Kao

    2018-05-01

    Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high-density culture performed with TGF-β-driven chondrogenic induction medium. Treatment of the Midazolam dose-dependently inhibited chondrogenesis, examined using Alcian blue-stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor-β-induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam-induced congenital malformations of the musculoskeletal system through PBR. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  1. Screening and fractionation of plant extracts with antiproliferative activity on human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Souza-Fagundes Elaine M

    2002-01-01

    Full Text Available Three hundred and thirteen extracts from 136 Brazilian plant species belonging to 36 families were tested for their suppressive activity on phytohemaglutinin (PHA stimulated proliferation of human peripheral blood mononuclear cells (PBMC. The proliferation was evaluated by the amount of [³H]-thymidine incorporated by the cells. Twenty extracts inhibited or strongly reduced the proliferation in a dose-dependent manner at doses between 10 and 100 µg/ml. Three of these extracts appeared to be non-toxic to lymphocytes, according to the trypan blue permeability assay and visual inspection using optical microscopy. Bioassay-guided fractionation of Alomia myriadenia extract showed that myriadenolide, a labdane diterpene known to occur in this species, could account for the observed activity of the crude extract. Using a similar protocol, an active fraction of the extract from Gaylussacia brasiliensis was obtained. Analysis of the ¹H and13C NMR spectra of this fraction indicates the presence of an acetylated triterpene whose characterization is underway. The extract of Himatanthus obovatus is currently under investigation.

  2. Allium sativum L. regulates in vitro IL-17 gene expression in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Moutia, Mouna; Seghrouchni, Fouad; Abouelazz, Omar; Elouaddari, Anass; Al Jahid, Abdellah; Elhou, Abdelhalim; Nadifi, Sellama; Jamal Eddine, Jamal; Habti, Norddine; Badou, Abdallah

    2016-09-29

    Allium sativum L. (A.S.) "garlic", one of the most interesting medicinal plants, has been suggested to contain compounds that could be beneficial in numerous pathological situations including cancer. In this work, we aimed to assess the immunomodulatory effect of A.S. preparation on human peripheral blood mononuclear cells from healthy individuals. Nontoxic doses of A.S. were identified using MTT assay. Effects on CD4+ or CD8+ T lymphocyte proliferation were studied using flow cytometry. The effect of A.S. on cytokine gene expression was studied using qRT-PCR. Finally, qualitative analysis of A.S. was performed by HPLC approach. Data were analyzed statistically by one-way ANOVA test. The nontoxic doses of A.S. preparation did not affect neither spontaneous nor TCR-mediated CD4+ or CD8+ T lymphocyte proliferation. Interestingly, A.S. exhibited a statistically significant regulation of IL-17 gene expression, a cytokine involved in several inflammatory and autoimmune diseases. In contrast, the expression of IL-4, an anti-inflammatory cytokine, was unaffected. Qualitative analysis of A.S. ethanol preparation indicated the presence of three polyphenol bioactive compounds, which are catechin, vanillic acid and ferulic acid. The specific inhibition of the pro-inflammatory cytokine, IL-17 without affecting cell proliferation in human PBMCs by the Allium sativum L. preparation suggests a potential valuable effect of the compounds present in this plant for the treatment of inflammatory diseases and cancer, where IL-17 is highly expressed. The individual contribution of these three compounds to this global effect will be assessed.

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

    OpenAIRE

    Esteves Mabel B.; Marques-Santos Luis F.; Affonso-Mitidieri Ottília R.; Rumjanek Vivian M.

    2005-01-01

    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 b...

  4. Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

    OpenAIRE

    Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

    2001-01-01

    A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-...

  5. Cytokine secretion from human peripheral blood mononuclear cells cultured in vitro with metal particles.

    Science.gov (United States)

    Cachinho, Sandra C P; Pu, Fanrong; Hunt, John A

    2013-04-01

    The failure of implanted medical devices can be associated with changes in the production of cytokines by cells of the immune system. Cytokines released by peripheral blood mononuclear cells upon contact with metal particles were quantified to understand their role in implantation intergration and their importance as messengers in the recruitment of T-lymphocytes at the implantation site. Opsonization was utilised to understand the influence of serum proteins on particle-induced cytokine production and release. Different metal compositions were used in the particulate format, Titanium (Ti), Titanium alloy (Ti6Al4V), and Stainless Steel 316L (SS), and were cultured in vitro with a mixed population of monocytes/macrophages and lymphocytes. The cells were also exposed to an exogenous stimulant mixture of phytohemagglutinin-P and interferon-gamma (IFN-γ) and opsonized particles with human serum. Interleukins, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IFN-γ, and tumor necrosis factor-alpha (TNF-α) were investigated using enzyme-linked immunosorbent assay as they are an indicator of the inflammation evoked by particulate metals. It has been experimentally evidenced that metal particles induced higher amounts of IL-6 and IL-1 but very low amounts of TNF-α. T-lymphocyte activation was evaluated by the quantification of IL-2 and IFN-γ levels. The results showed that nonopsonized and opsonized metal particles did not induce the release of increased levels of IL-2 and IFN-γ. Copyright © 2013 Wiley Periodicals, Inc.

  6. Preliminary study on biological dosimetry using alkaline single cell gel electrophoresis of human peripheral lymphocytes

    International Nuclear Information System (INIS)

    Liu Qingjie; Lu Xue; Feng Jiangbing; Chen Deqing; Chen Xiaosui

    2006-01-01

    Objective: To explore the feasibility of alkaline single cell gel electrophoresis (SCGE) in biological dosimetry of ionizing radiation. Methods: Normal peripheral blood samples from two healthy males were exposed to different doses coblat-60 gamma-rays, ranged from 0 to 5 Gy, and the tail length (TL) and Oliver tail moment (TM) of the lymphocytes were analyzed with SCGE. The dose-effect curves of TL and TM were fitted respectively. The TL and TM of lymphocytes for eight radiation workers were analyzed with SCGE, cumulative doses were estimated using the fitted TL and TM equations, and then compared with the recorded monitoring doses. Results: The TLs or TMs of normal human lymphocytes were increased with the irradiation doses, and its relationship can be fitted with a linear-quadratic equations: Y=13.59 + 20.87X - 2.27 X 2 for TL, and Y = 8.50 + 15.04X - 1.43X 2 for TM, respectively (Y denotes TL or TM value, X is radiation dose). The doses estimated with TM equation were closer to the recorded monitoring doses than that with TL equation. Conclusions: The TM in lymphocytes analyzed with SCGE is a promising radiation biological dosimeter. (authors)

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

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    Claire Jones

    Full Text Available Opa proteins are major surface-expressed proteins located in the Neisseria meningitidis outer membrane, and are potential meningococcal vaccine candidates. Although Opa proteins elicit high levels of bactericidal antibodies following immunisation in mice, progress towards human clinical trials has been delayed due to previous findings that Opa inhibits T cell proliferation in some in vitro assays. However, results from previous studies are conflicting, with different Opa preparations and culture conditions being used. We investigated the effects of various Opa+ and Opa- antigens from N. meningitidis strain H44/76 in a range of in vitro conditions using peripheral blood mononuclear cells (PBMCs and purified CD4+ T cells, measuring T cell proliferation by CFSE dilution using flow cytometry. Wild type recombinant and liposomal Opa proteins inhibited CD4+ T cell proliferation after stimulation with IL-2, anti-CD3 and anti-CD28, and these effects were reduced by mutation of the CEACAM1-binding region of Opa. These effects were not observed in culture with ex vivo PBMCs. Opa+ and Opa- OMVs did not consistently exert a stimulatory or inhibitory effect across different culture conditions. These data do not support a hypothesis that Opa proteins would be inhibitory to T cells if given as a vaccine component, and T cell immune responses to OMV vaccines are unlikely to be significantly affected by the presence of Opa proteins.

  8. Killing effect of peripheral blood mononuclear cells irradiated by γ ray on human gastric cancer MKN-28 cell

    International Nuclear Information System (INIS)

    Wu Daocheng; Zhang Xianqing; Mu Shijie; Liu Zhongxiang; Xia Aijun; Huang Xiaofeng; An Qunxing

    2007-01-01

    Objective: To observe the killing effect of peripheral blood mononuclear cells (PBMCs) irradiated by γ ray on cultured human gastric cancer cell line MKN-28. Methods: The experiment were divided into MKN-28 tumor cell control group, PBMCs groups and MKN-28 cells with irradiated or non-irradiated PBMCs co-culture groups. Radidation dosage were from 0.5 to 3 Gy, acridine orange/ethidium bromide (AO/EB) staining were used to observe the kill effect of PBMCs on tumor cells in different period. Results: After culture for 144h, the dead cells of several dosage irradiated PBMCs are much more than those of non-irradiated PBMCs group. At 240 hours of culture, the alive PBMCs deareses in number in both irradiated and non-irradiared groups, but decreases in radiated groups are more obvious. After culture for 72 h in the co-cultured groups, the difference is not evident among all radiation dosage groups. After 96-240 h of co-culture, the killing effect of 0.5-2Gy irradiated PBMCs on tumor cells is very strong, especially in 1Gy group, but the killing effect of PBMCs irradiated by 2.5-3Gy on tumor cells were weaker than that of 0.5-2Gy irradiated groups. At 240 hours co-cultured groups irradiated by 2.5-3Gy, tumor cells still survive and proliferate. Conclusion: Gamma ray irradiation have killing effect to some PBMCs. The cytocidal effect of PBMCs irradiated by 0.5-2Gy on tumor cells were increased. Chemotaxis and cytocidal effect of tumor cells to postirradiated PBMCs were also found. The killing effect of PBMCs irradiated by 2.5 and 3 Gy on tumor cells were restrained. (authors)

  9. [Inhibitory effect and mechanism of tofacitinib on the secretion of cytokines by T cells in human peripheral blood].

    Science.gov (United States)

    Wu, Kunlun; Zhao, Jun; Wu, Qiongli; Wu, Changyou

    2017-11-01

    Objective To study the inhibitory effect of tofacitinib on the production of cytokines by T cells in human peripheral blood and its mechanism. Methods Peripheral blood mononuclear cells (PBMCs) and purified T cells were cultured and stimulated with anti-CD3 plus anti-CD28 antibodies in the presence or absence of tofacitinib (0.5 μmol/L). The levels of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) in the culture supernatants were detected by ELISA, and the expressions of activated molecules CD69 and CD25 on the surface of CD4 + and CD8 + T cells, the production of cytokines and the phosphorylation of signal transducers and transcriptional activators STAT1, STAT3, STAT4 in T cells were examined by flow cytometry. At the same time, the proliferation and apoptosis of T cells were observed by 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and the flow cy tometry with annexin V-FITC/PI, respectively. Results Tofacitinib inhibited the production of IFN-γ, TNF-α and the expression of CD25 on T cells from the peripheral blood. In addition, the proliferation and the phosphorylation of STAT1, STAT3, STAT4 by T cells were also depressed. However, tofacitinib had no effect on the secretion of IL-2, the expression of CD69 and the apoptosis of T cells. Conclusion Tofacitinib can inhibit the secretion of IFN-γ and TNF-α by T cells in the peripheral blood, and its mechanism might be related to the inhibitory effect of tofacitinib on the activation, proliferation and signal transduction in T cells.

  10. Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells.

    Science.gov (United States)

    Laranjeira, Paula; Pedrosa, Monia; Pedreiro, Susana; Gomes, Joana; Martinho, Antonio; Antunes, Brigida; Ribeiro, Tania; Santos, Francisco; Trindade, Helder; Paiva, Artur

    2015-01-05

    The different distribution of T cells among activation/differentiation stages in immune disorders may condition the outcome of mesenchymal stromal cell (MSC)-based therapies. Indeed, the effect of MSCs in the different functional compartments of T cells is not completely elucidated. We investigated the effect of human bone marrow MSCs on naturally occurring peripheral blood functional compartments of CD4(+) and CD8(+) T cells: naive, central memory, effector memory, and effector compartments. For that, mononuclear cells (MNCs) stimulated with phorbol myristate acetate (PMA) plus ionomycin were cultured in the absence/presence of MSCs. The percentage of cells expressing tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), and interleukin-2 (IL-2), IL-17, IL-9, and IL-6 and the amount of cytokine produced were assessed by flow cytometry. mRNA levels of IL-4, IL-10, transforming growth factor-beta (TGF-β), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in purified CD4(+) and CD8(+) T cells, and phenotypic and mRNA expression changes induced by PMA + ionomycin stimulation in MSCs, were also evaluated. MSCs induced the reduction of the percentage of CD4(+) and CD8(+) T cells producing TNF-α, IFNγ, and IL-2 in all functional compartments, except for naive IFNγ(+)CD4(+) T cells. This inhibitory effect differentially affected CD4(+) and CD8(+) T cells as well as the T-cell functional compartments; remarkably, different cytokines showed distinct patterns of inhibition regarding both the percentage of producing cells and the amount of cytokine produced. Likewise, the percentages of IL-17(+), IL-17(+)TNF-α(+), and IL-9(+) within CD4(+) and CD8(+) T cells and of IL-6(+)CD4(+) T cells were decreased in MNC-MSC co-cultures. MSCs decreased IL-10 and increased IL-4 mRNA expression in stimulated CD4(+) and CD8(+) T cells, whereas TGF-β was reduced in CD8(+) and augmented in CD4(+) T cells, with no changes for CTLA4. Finally, PMA

  11. Microculture assay for isolation of human immunodeficiency virus type 1 and for titration of infected peripheral blood mononuclear cells.

    Science.gov (United States)

    Dimitrov, D H; Melnick, J L; Hollinger, F B

    1990-04-01

    To define the optimal conditions for human immunodeficiency virus (HIV) detection in microcultures, experiments were conducted with different ratios of patient and donor peripheral blood mononuclear cells (PBMCs). Donor/patient PBMC ratios ranged from 1:1 to 1:125. Optimal results were obtained when 1,500,000 donor cells were cocultured with equal or smaller quantities of patient PBMCs. Thus, virologic endpoints could be achieved by diluting patient cells. Smaller numbers of donor cells, with or without larger numbers of patients cells, resulted in lower rates of HIV isolation. Similarly, the direct stimulation of patient PBMCs with phytohemagglutinin without the addition of normal donor cells lowered the sensitivity of the assay significantly. We suggest that a microculture procedure using a fixed quantity of donor cells with different dilutions of patient cells may be useful for monitoring changing HIV levels during antiviral therapy.

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

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    Yoshikazu Kishino

    Full Text Available 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.

  13. Association between age and repair of oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    damaged DNA in peripheral blood mononuclear cells (PBMCs). We isolated PBMCs from subjects aged 18-83 years, as part of a health survey of the Danish population that focussed on lifestyle factors. The level of DNA repair activity was measured as incisions on potassium bromate-damaged DNA by the comet...... assay. There was an inverse association between age and DNA repair activity with a 0.65% decline in activity per year from age 18 to 83 (95% confidence interval: 0.16-1.14% per year). Univariate regression analysis also indicated inverse associations between DNA repair activity and waist-hip ratio (P...

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

    Directory of Open Access Journals (Sweden)

    M Majumdar

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  18. 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. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. 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.

  20. Peripheral T-Cell Lymphoma

    Science.gov (United States)

    ... Non-Hodgkin Lymphoma Peripheral T-Cell Lymphoma Primary Central Nervous System Lymphoma T-Cell Lymphoma Transformed Mycosis Fungoides Waldenstrom Macroglobulinemia Young Adult Lymphoma Overview Treatment Options Relapsed/Refractory Long-term ...

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

    International Nuclear Information System (INIS)

    Andrade, Lidia Maria; Campos, Tarcisio Passos Ribeiro de; Leite, M.F.; Goes, A.M.

    2005-01-01

    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 60 Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min -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 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)

  2. 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)

  3. 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

  4. Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

    Science.gov (United States)

    Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

    2001-01-01

    A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-γ on either protein or mRNA levels. The anergic state of CD4+CD25+ T cells is not reversible by the addition of anti-CD28, anti–CTLA-4, anti–transforming growth factor β, or anti–IL-10 antibody. However, the refractory state of CD4+CD25+ T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties. PMID:11390435

  5. Generation of dendritic cells from human bone marrow mononuclear cells: advantages for clinical application in comparison to peripheral blood monocyte derived cells.

    Science.gov (United States)

    Bai, L; Feuerer, M; Beckhove, P; Umansky, V; Schirrmacher, V

    2002-02-01

    Dendritic cells (DCs) currently used for vaccination in clinical studies to induce immunity against malignant cells are normally generated from peripheral blood-derived monocytes. Here we studied conditions for the generation of DCs from unseparated human bone marrow (BM) mononuclear cells and compared them functionally with DCs from blood. The two types of DCs, from bone marrow (BM-DC) and peripheral blood (BL-DC), were generated in parallel from the same normal healthy donors by culturing in serum-free X-VIVO 20 medium containing GM-CSF and IL-4, and then the phenotypes and functions were compared. BM-DC generation occurred in 14 days and involved proliferative expansion from CD34 stem cells and differentiation while BL-DC generation occurred in 7 days from CD14 monocytes and involved only differentiation. A 7- to 25-fold higher number of DCs could be obtained from BM than from blood. BM-DC had similar phenotypes as BL-DC. The capacity to stimulate MLR reactivity in allogeneic T lymphocytes was higher with BM-DC than that with BL-DC. Also, the capacity to stimulate autologous memory T cell responses to tetanus toxoid (TT) or tuberculin (PPD) was higher with BM-DC than with BL-DC. These results suggest that BM-DC as produced here may be a very economic and useful source of professional antigen-presenting cells for anti-tumor immunotherapeutic protocols.

  6. Effects of Platelets on Platelet Concentrate Product on the Activation of Human Peripheral Blood Monocyte Cells

    Directory of Open Access Journals (Sweden)

    N Sadat Razavi Hoseini

    2016-02-01

    Full Text Available Introduction: Monocytes can interact with platelets due to their surface molecules such as P-selectin glycoprotein ligand-1 (PSGL-1, and form monocyte-platelet complex. In the present study, the effects of platelets interaction of platelet concentrates (PCs and peripheral blood monocytes were investigated in vitro as a model to predict the probable interactions of these cells and consequently activation of monocytes. Methods: In this experimental study, units of whole blood and PCs were prepared from Tehran Blood Transfusion Center. After isolation of monocytes from the whole blood, these cells were treated with PC- derived platelets. The activation of monocytes was assessed before and after treatment by the analysis of the respiratory burst of monocytes using dihydrorhodamine 123 (DHR-123. The study data were analyzed using the non-parametric test of Wilcoxon. Results: The purity of monocytes was determined as 86.1±2 using NycoPrep method. The respiratory burst of monocytes was increased after exposure with platelets. In fact, the difference was significant when platelets were used on the 5th day of storage (P=0.001. Conclusions: The study findings revealed that platelets have an efficient capacity to stimulate and activate monocytes. The possible involvement of molecules in the interaction of platelet-monocyte demand to be further studied in future.

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

    Science.gov (United States)

    Kazemi, E.; Mortazavi, S. M. J.; Ali-Ghanbari, A.; Sharifzadeh, S.; Ranjbaran, R.; Mostafavi-pour, Z.; Zal, F.; Haghani, M.

    2015-01-01

    Background Despite numerous studies over a decade, it still remains controversial about the biological effects of RF EMF emitted by mobile phone telephony. Objective Here we investigated the effect of 900 MHz GSM on the induction of oxidative stress and the level of intracellular reactive oxygen species (ROS) in human mononuclear cells, monocytes and lymphocytes as defence system cells. Method 6 ml Peripheral Blood samples were obtained from 13 healthy volunteers (21-30 year-old). Each sample was devided into 2 groups: one was exposed RF radiation emitted from a mobile phone simulator for 2 hour and the other used as control group which was not exposed to any fields. After that, mononuclear cells were isolated from peripheral blood by density gradient centrifugation in Ficoll-Paque. The intracellular ROS content in monocytes and lymphocytes was measured by the CM-H2DCFDA fluorescence probe using flowcytometry technique. Results Our results showed significant increase in  ROS production after exposure in population rich in monocytes. This effect was not significant in population rich in lymphocytes in comparison with non exposed cells. Conclusion The results obtained in this study clearly showed the oxidative stress induction capability of RF electromagnetic field in the portion of PBMCs mostly in monocytes, like the case of exposure to micro organisms, although the advantages or disadvantages of this effect should be evaluated. PMID:26396966

  8. 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.

  9. Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

    NARCIS (Netherlands)

    van Hemert, Saskia; Meijerink, Marjolein; Molenaar, Douwe; Bron, Peter A.; de Vos, Paul; Kleerebezem, Michiel; Wells, Jerry M.; Marco, Maria L.

    2010-01-01

    Background: Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic

  10. Increased ICAM-1 Expression in Transformed Human Oral Epithelial Cells: Molecular Mechanism and Functional Role in Peripheral Blood Mononuclear Cell Adhesion and Lymphokine-Activated-Killer Cell Cytotoxicity

    Science.gov (United States)

    Huang, George T.-J.; Zhang, Xinli; Park, No-Hee

    2012-01-01

    The intercellular adhesion molecule-1 (ICAM-1, CD54) serves as a counter-receptor for the β2-integrins, LFA-1 and Mac-1, which are expressed on leukocytes. Although expression of ICAM-1 on tumor cells has a role in tumor progression and development, information on ICAM-1 expression and its role in oral cancer has not been established. Normal human oral keratinocytes (NHOK), human papilloma virus (HPV)-immortalized human oral keratinocyte lines (HOK-16B, HOK-18A, and HOK-18C), and six human oral neoplastic cell lines (HOK-16B-BaP-T1, SCC-4, SCC-9, HEp-2, Tu-177 and 1483) were used to study ICAM-1 expression and its functional role in vitro. Our results demonstrated that NHOK express negligible levels of ICAM-1, whereas immortalized human oral keratinocytes and cancer cells express significantly higher levels of ICAM-1, except for HOK-16B-BaP-T1 and HEp-2. Altered mRNA half-lives did not fully account for the increased accumulation of ICAM-1 mRNA. Adhesion of peripheral blood mononuclear cells (PBMC) to epithelial cells correlated with cell surface ICAM-1 expression levels. This adhesion was inhibited by antibodies specific for either ICAM-1 or LFA-1/Mac-1, suggesting a role for these molecules in adhesion. In contrast, lymphokine-activated-killer (LAK) cell cytotoxic killing of epithelial cells did not correlate with ICAM-1 levels or with adhesion. Nonetheless, within each cell line, blocking of ICAM-1 or LFA-1/Mac-1 reduced LAK cells killing, suggesting that ICAM-1 is involved in mediating this killing. PMID:10938387

  11. Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry.

    Directory of Open Access Journals (Sweden)

    Bo Langhoff Hønge

    Full Text Available Live peripheral blood mononuclear cells (PBMCs can be frozen and thawed for later analyses by adding and removing a cryoprotectant, such as dimethyl sulfoxide (DMSO. Laboratories across the world use various procedures, but published evidence of optimal thawing procedures is scarce.PBMCs were separated from blood collected from healthy Danish blood donors, and stored at -80°C after adding of DMSO. The essential steps in the thawing procedure were modified and performance was evaluated by flow cytometry with respect to the percentage and total yield of viable PMBCs.The best-performing washing medium was Roswell Park Memorial Institute (RPMI 1640 at 37°C with 20% fetal bovine serum. When using 10 mL washing medium in a 15-mL Falcon tube, samples should be centrifuged for at least 10 minutes at 500 g. We failed to detect any differences between the tested methods of mixing PBMCs with washing medium. Likewise, neither the thawing duration nor centrifugation temperature (20°C and 37°C had any effect. PBMCs could be incubated (rested for up to eight hours in a 37°C 5% CO2 incubator without affecting cell counts, but incubating PBMCs for 16 hours significantly decreased viability and recovery. In general, high viability was not necessarily associated with high recovery.Changing the thawing procedure significantly impacted PBMC viability and live cell recovery. Evaluating both viability and live PBMC recovery are necessary to evaluate method performance. Investigation of differential loss of PBMC subtypes and phenotypic changes during thawing and incubation requires further evaluation.

  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. Fundamental studies on ADCC (antibody-dependent cell-mediated cytotoxicity) of human peripheral blood leukocytes using sheep red blood cells as target cells, and the effect of erythrophagocytosis

    International Nuclear Information System (INIS)

    Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

    1979-01-01

    We investigated antibody-dependent cell-mediated cytotoxicity (ADCC) of human peripheral blood leukocytes by using 51 Cr-labelled sheep red blood cells (SRBC) as target cells and anti-SRBC rabbit antibody. Lysis of SRBC was mediated by either human peripheral lymphoid cells or phagocytes (Monocytes and granulocytes). SRBC were useful as target cells in ADCC assay against human lymphoid cells, since decreased cytotoxic activity of phagocyte-contaminated crude lymphocyte fraction was recovered by elimination of contaminating phagocytes. The monocytes inhibited ADCC of lymphoid cells through phagocytosis of SRBC. This assay system may be useful for estimating not only Fc receptor-mediated cytotoxicity but also Fc receptor-mediated phagocytic activity of human peripheral blood leukocytes. (author)

  14. 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

  15. The Generation of Human γδT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture.

    Science.gov (United States)

    Watanabe, Daisuke; Koyanagi-Aoi, Michiyo; Taniguchi-Ikeda, Mariko; Yoshida, Yukiko; Azuma, Takeshi; Aoi, Takashi

    2018-01-01

    γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αβT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αβT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer. Stem Cells Translational Medicine 2018;7:34-44. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  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. Individual differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

    International Nuclear Information System (INIS)

    Oriya, Asami; Takahashi, Kenji; Kashiwakura, Ikuo; Inanami, Osamu; Kuwabara, Mikinori; Miura, Toshiaki; Abe, Yoshinao

    2008-01-01

    The aim of this study is to evaluate the individual differences in radiosensitivity of lineage-committed myeloid hematopoietic progenitors, colony-forming cells (CFC), detected in steady-state human peripheral blood (PB). Mononuclear cells were prepared from the buffy-coat of 30 individuals PB, and were assayed for CFC by semi-solid culture supplemented with cytokines. X irradiation was performed in the range of 0.5-4 Gy at a dose rate of about 80 cGy/min. The mean number of hematopoietic progenitor cells is 5866±3408 in 1 ml of buffy-coat, suggesting that the erythroid progenitor cells are the major population. The total CFC radiosensitivity parameter D 0 and n value are 1.18±0.24 and 1.89±0.98, respectively. Using a linear regression analysis, a statistically significant correlation is observed between the D 0 value and the surviving fraction at 4 Gy (r=0.611 p 0 parameter and the level of antioxidants, plasma uric acid, plasma bilirubin, and intracellular glutathione. The present study demonstrates that there are large individual differences in the radiosensitivity of hematopoietic progenitor cells as detected in steady-state human PB. These differences demonstrate almost no correlation with plasma or intracellular antioxidants. The prediction of individual differences in radiosensitivity of CFC can only be measured by 4 Gy irradiation. (author)

  18. 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...

  19. Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

    Science.gov (United States)

    Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

    2018-05-01

    Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

  20. Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease.

    Science.gov (United States)

    Jones, Simon P; Franco, Nunzio F; Varney, Bianca; Sundaram, Gayathri; Brown, David A; de Bie, Josien; Lim, Chai K; Guillemin, Gilles J; Brew, Bruce J

    2015-01-01

    The kynurenine pathway is a fundamental mechanism of immunosuppression and peripheral tolerance. It is increasingly recognized as playing a major role in the pathogenesis of a wide variety of inflammatory, neurodegenerative and malignant disorders. However, the temporal dynamics of kynurenine pathway activation and metabolite production in human immune cells is currently unknown. Here we report the novel use of flow cytometry, combined with ultra high-performance liquid chromatography and gas chromatography-mass spectrometry, to sensitively quantify the intracellular expression of three key kynurenine pathway enzymes and the main kynurenine pathway metabolites in a time-course study. This is the first study to show that up-regulation of indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monoxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) is lacking in lymphocytes treated with interferon gamma. In contrast, peripheral monocytes showed a significant elevation of kynurenine pathway enzymes and metabolites when treated with interferon gamma. Expression of IDO-1, KMO and QPRT correlated significantly with activation of the kynurenine pathway (kynurenine:tryptophan ratio), quinolinic acid concentration and production of the monocyte derived, pro-inflammatory immune response marker: neopterin. Our results also describe an original and sensitive methodological approach to quantify kynurenine pathway enzyme expression in cells. This has revealed further insights into the potential role of these enzymes in disease processes.

  1. Whole thorax irradiation of non-human primates induces persistent nuclear damage and gene expression changes in peripheral blood cells.

    Directory of Open Access Journals (Sweden)

    Shanaz A Ghandhi

    Full Text Available We investigated the cytogenetic and gene expression responses of peripheral blood cells of non-human primates (NHP, Macaca mulatta that were whole-thorax irradiated with a single dose of 10 Gy. In this model, partial irradiation of NHPs in the thoracic region (Whole Thorax Lung Irradiation, WTLI allows the study of late radiation-induced lung injury, while avoiding acute radiation syndromes related to hematopoietic and gastrointestinal injury. A transient drop in circulating lymphocytes and platelets was seen by 9 days, followed by elevations in respiratory rate, circulating neutrophils, lymphocytes, and monocytes at 60-100 days, corresponding to computed tomography (CT and histologic evidence of pneumonitis, and elective euthanasia of four animals. To evaluate long-term DNA damage in NHP peripheral blood lymphocytes after 10 Gy WTLI, we used the cytokinesis-block micronucleus (CBMN assay to measure chromosomal aberrations as post-mitotic micronuclei in blood samples collected up to 8 months after irradiation. Regression analysis showed significant induction of micronuclei in NHP blood cells that persisted with a gradual decline over the 8-month study period, suggesting long-term DNA damage in blood lymphocytes after WTLI. We also report transcriptomic changes in blood up to 30 days after WTLI. We isolated total RNA from peripheral blood at 3 days before and then at 2, 5 and 30 days after irradiation. We identified 1187 transcripts that were significantly changed across the 30-day time course. From changes in gene expression, we identified biological processes related to immune responses, which persisted across the 30-day study. Response to oxygen-containing compounds and bacteria were implicated by gene-expression changes at the earliest day 2 and latest, day 30 time-points. Gene expression changes suggest a persistent altered state of the immune system, specifically response to infection, for at least a month after WTLI.

  2. Whole thorax irradiation of non-human primates induces persistent nuclear damage and gene expression changes in peripheral blood cells.

    Science.gov (United States)

    Ghandhi, Shanaz A; Turner, Helen C; Shuryak, Igor; Dugan, Gregory O; Bourland, J Daniel; Olson, John D; Tooze, Janet A; Morton, Shad R; Batinic-Haberle, Ines; Cline, J Mark; Amundson, Sally A

    2018-01-01

    We investigated the cytogenetic and gene expression responses of peripheral blood cells of non-human primates (NHP, Macaca mulatta) that were whole-thorax irradiated with a single dose of 10 Gy. In this model, partial irradiation of NHPs in the thoracic region (Whole Thorax Lung Irradiation, WTLI) allows the study of late radiation-induced lung injury, while avoiding acute radiation syndromes related to hematopoietic and gastrointestinal injury. A transient drop in circulating lymphocytes and platelets was seen by 9 days, followed by elevations in respiratory rate, circulating neutrophils, lymphocytes, and monocytes at 60-100 days, corresponding to computed tomography (CT) and histologic evidence of pneumonitis, and elective euthanasia of four animals. To evaluate long-term DNA damage in NHP peripheral blood lymphocytes after 10 Gy WTLI, we used the cytokinesis-block micronucleus (CBMN) assay to measure chromosomal aberrations as post-mitotic micronuclei in blood samples collected up to 8 months after irradiation. Regression analysis showed significant induction of micronuclei in NHP blood cells that persisted with a gradual decline over the 8-month study period, suggesting long-term DNA damage in blood lymphocytes after WTLI. We also report transcriptomic changes in blood up to 30 days after WTLI. We isolated total RNA from peripheral blood at 3 days before and then at 2, 5 and 30 days after irradiation. We identified 1187 transcripts that were significantly changed across the 30-day time course. From changes in gene expression, we identified biological processes related to immune responses, which persisted across the 30-day study. Response to oxygen-containing compounds and bacteria were implicated by gene-expression changes at the earliest day 2 and latest, day 30 time-points. Gene expression changes suggest a persistent altered state of the immune system, specifically response to infection, for at least a month after WTLI.

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

    Directory of Open Access Journals (Sweden)

    Sonia Cabrera

    2015-09-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► Butachlor exhibited strong binding affinity with DNA and produced 8-oxodG adducts. ► Butachlor induced DNA strand breaks and micronuclei formation in PBMN cells. ► Butachlor induced ROS and dissipation of mitochondrial membrane potential in cells. ► Butachlor resulted in cell cycle arrest and eventually caused cellular necrosis. -- Abstract: 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 24 h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow

  5. Do protons and X-rays induce cell-killing in human peripheral blood lymphocytes by different mechanisms?

    Science.gov (United States)

    Miszczyk, J; Rawojć, K; Panek, A; Borkowska, A; Prasanna, P G S; Ahmed, M M; Swakoń, J; Gałaś, A

    2018-02-01

    Significant progress has been made in the technological and physical aspects of dose delivery and distribution in proton therapy. However, mode of cell killing induced by protons is less understood in comparison with X-rays. The purpose of this study is to see if there is any difference in the mode of cell-killing, induced by protons and X-rays in an ex vivo human peripheral blood lymphocyte (HPBL) model. HPBL were irradiated with 60 MeV proton beam or 250-kVp X-rays in the dose range of 0.3-4.0 Gy. Frequency of apoptotic and necrotic cells was determined by the Fluorescein (FITC)-Annexin V labelling procedure, 1 and 4 h after irradiation. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis and necrosis. Chip-based DNA Ladder Assay was used to confirm radiation-induced apoptosis. Ex vivo irradiation of HPBL with proton beams of 60 MeV or 250 kVp X-rays resulted in apoptotic as well as necrotic modes of cell-killing, which were evident at both 1 and 4 h after irradiation in the whole dose and time range. Generally, our results indicated that protons cause relatively higher yields of cell death that appears to be necrosis compared to X-rays. The analysis also demonstrates that radiation type and dose play a critical role in mode of cell-killing. Obtained results suggest that X-rays and protons induce cell-killing by different modes. Such differences in cell-killing modes may have implications on the potential of a given therapeutic modality to cause immune modulation via programmed cell death (X-rays) or necrotic cell death (proton therapy). These studies point towards exploring for gene expression biomarkers related necrosis or apoptosis to predict immune response after proton therapy.

  6. Effect of low-dose gamma radiation on HIV replication in human peripheral blood mononuclear cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada); Conway, B. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Dept. of Medicine; Montaner, J.S.G. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Dept. of Medicine]|[Canadian HIV Trials Network, Vancouver (Canada); O`Shaughnessy, M.V. [British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada)]|[British Columbia Centre for Excellence in HIV/AIDS, British Columbia (Canada). Faculty of Medicine]|[Canadian HIV Trials Network, Vancouver (Canada); Greenstock, C.L. [AECL Research, Chalk River, Ontario (Canada). Radiation Biology and Health Physics Branch

    1996-08-01

    Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of {gamma}-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. We have studied the effects of {gamma}-radiation on HIV replication in mono-nuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy {gamma}-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting. (author).

  7. Effect of low-dose gamma radiation on HIV replication in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Xu, Y.; Conway, B.; O'Shaughnessy, M.V.; Greenstock, C.L.

    1996-01-01

    Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of γ-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. We have studied the effects of γ-radiation on HIV replication in mono-nuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy γ-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting. (author)

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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

  9. 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.

  10. Evidence from Human and Animal Studies: Pathological Roles of CD8(+) T Cells in Autoimmune Peripheral Neuropathies.

    Science.gov (United States)

    Yang, Mu; Peyret, Corentin; Shi, Xiang Qun; Siron, Nicolas; Jang, Jeong Ho; Wu, Sonia; Fournier, Sylvie; Zhang, Ji

    2015-01-01

    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 indicates 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 neuropathies. 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.

  11. Evidence from Human and Animal Studies: Pathological Roles of CD8+ T Cells in Autoimmune Peripheral Neuropathies

    Science.gov (United States)

    Yang, Mu; Peyret, Corentin; Shi, Xiang Qun; Siron, Nicolas; Jang, Jeong Ho; Wu, Sonia; Fournier, Sylvie; Zhang, Ji

    2015-01-01

    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 indicates 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 neuropathies. 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. PMID:26528293

  12. Effects of Secondary Metabolites of Permafrost Bacillus sp. on Cytokine Synthesis by Human Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Kalenova, L F; Kolyvanova, S S; Bazhin, A S; Besedin, I M; Mel'nikov, V P

    2017-06-01

    We studied the effects of secondary metabolites of Bacillus sp. isolated from late Neogene permafrost on secretion of proinflammatory (TNF-α, IL-1β, IL-8, IL-2, and IFNγ) and antiinflammatory (IL-4 and IL-10) cytokines by human peripheral blood mononuclear cells. It was found that metabolites of Bacillus sp. produced more potent effect on cytokine secretion than mitogen phytohemagglutinin and metabolites of Bacillus cereus, medicinal strain IP5832. Activity of metabolites depended on the temperature of bacteria incubation. "Cold" metabolites of Bacillus sp. (isolated at -5°C) primarily induced Th1-mediated secretion of IFNγ, while "warm" metabolites (obtained at 37°C) induced Th2-mediated secretion of IL-4. The results suggest that Bacillus sp. metabolites are promising material for the development of immunomodulating drugs.

  13. 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.

  14. 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

    2010-06-01

    Full Text Available 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.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.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 implementation in human clinical trials.

  15. Regulation of tumor necrosis factor gene expression by ionizing radiation in human myeloid leukemia cells and peripheral blood monocytes

    International Nuclear Information System (INIS)

    Sherman, M.L.; Datta, R.; Hallahan, D.E.; Weichselbaum, R.R.; Kufe, D.W.

    1991-01-01

    Previous studies have demonstrated that ionizing radiation induces the expression of certain cytokines, such as TNF alpha/cachectin. However, there is presently no available information regarding the molecular mechanisms responsible for the regulation of cytokine gene expression by ionizing radiation. In this report, we describe the regulation of the TNF gene by ionizing radiation in human myeloid leukemia cells. The increase in TNF transcripts by x rays was both time- and dose-dependent as determined by Northern blot analysis. Similar findings were obtained in human peripheral blood monocytes. Transcriptional run-on analyses have demonstrated that ionizing radiation stimulates the rate of TNF gene transcription. Furthermore, induction of TNF mRNA was increased in the absence of protein synthesis. In contrast, ionizing radiation had little effect on the half-life of TNF transcripts. These findings indicate that the increase in TNF mRNA observed after irradiation is regulated by transcriptional mechanisms and suggest that production of this cytokine by myeloid cells may play a role in the pathophysiologic effects of ionizing radiation

  16. Microarray analysis of gene expression in peripheral blood mononuclear cells from dioxin-exposed human subjects

    International Nuclear Information System (INIS)

    McHale, Cliona M.; Zhang, Luoping; Hubbard, Alan E.; Zhao, Xin; Baccarelli, Andrea; Pesatori, Angela C.; Smith, Martyn T.; Landi, Maria Teresa

    2007-01-01

    Tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen and exerts toxic effects on the skin (chloracne). Effects on reproductive, immunological, and endocrine systems have also been observed in animal models. TCDD acts through the aryl hydrocarbon receptor (AhR) pathway influencing largely unknown gene networks. An industrial accident in Seveso, Italy in 1976 exposed thousands of people to substantial quantities of TCDD. Twenty years after the exposure, this study examines global gene expression in the mononuclear cells of 26 Seveso female never smokers, with similar age, alcohol consumption, use of medications, and background plasma levels of 22 dioxin congeners unrelated to the Seveso accident. Plasma dioxin levels were still elevated in the exposed subjects. We performed analyses in two different comparison groups. The first included high-exposed study subjects compared with individuals with background TCDD levels (average plasma levels 99.4 and 6.7 ppt, respectively); the second compared subjects who developed chloracne after the accident, and those who did not develop this disease. Overall, we observed a modest alteration of gene expression based on dioxin levels or on chloracne status. In the comparison between high levels and background levels of TCDD, four histone genes were up-regulated and modified expression of HIST1H3H was confirmed by real-time PCR. In the comparison between chloracne case-control subjects, five hemoglobin genes were up-regulated. Pathway analysis revealed two major networks for each comparison, involving cell proliferation, apoptosis, immunological and hematological disease, and other pathways. Further examination of the role of these genes in dioxin induced-toxicity is warranted

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

    Directory of Open Access Journals (Sweden)

    Lídia Maria Andrade

    2005-10-01

    Full Text Available 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 60 Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min-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 60Co irradiation were compared to damage caused by cell exposure to 10% methanol. The 50 Gy dose of irradiation did not stimulate nuclus 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.Radioterapia utilizando radiação gama é uma modalidade comum no tratamento do câncer de mama. A proposta deste estudo é investigar a resposta biológica in vitro da linhagem celular MDAMB-231 de câncer de mama humano e células do sangue periférico humano (PBMC expostas à irradiação pelo Co60 em frações simples de 10Gy, 25Gy e 50Gy e 136,4cGy min-1 rate. As células foram irradiadas a temperatura ambiente usando o equipamento de radioterapia Theratron 80 radiotherapy system. A resposta biológica, após irradiação gama, foi avaliada através do ensaio do MTT para viabilidade celular e o do ensaio com Iodeto de Propídio para visualização do dano nuclear, além da eletroforese em gel de agarose. Os danos nucleares induzidos pelo Co60 foram comparados aos danos causados pela exposição das células à solução de metanol a 10%. Nós observamos que a dose de 50Gy não estimulou a mesma quantidade de danos nucleares que a solução de metanol a 10% nas c

  18. Interaction of rotavirus with human peripheral blood mononuclear cells: plasmacytoid dendritic cells play a role in stimulating memory rotavirus specific T cells in vitro.

    Science.gov (United States)

    Mesa, Martha C; Rodríguez, Luz-Stella; Franco, Manuel A; Angel, Juana

    2007-09-15

    We studied the interaction of RV with human peripheral blood mononuclear cells (PBMC) from adult volunteers. After exposure of PBMC to rhesus RV (RRV), T and B lymphocytes, NK cells, monocytes, and myeloid and plasmacytoid dendritic cells expressed RV non-structural proteins, at variable levels. Expression of these RV proteins was abolished if infection was done in the presence of anti-VP7 neutralizing antibodies or 10% autologous serum. Supernatants of RRV exposed PBMC contained TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-2 and IL-10. Plasmacytoid DC were found to be the main source of IFN-alpha production, and in their absence the production of IFN-gamma and the frequency of RV specific T cells that secrete IFN-gamma diminished. Finally, we could not detect RV-antigen associated with the PBMC or expression of RV non-structural proteins in PBMC of acutely RV-infected children. Thus, although PBMC are susceptible to the initial steps of RV infection, most PBMC of children with RV-gastroenteritis are not infected.

  19. Time and dose dependent expression in the proteome of human peripheral blood mononuclear cells with γ-irradiation

    International Nuclear Information System (INIS)

    Nishad, S.; Ghosh, Anu

    2014-01-01

    The aim of present study is to investigate time and dose dependent differential protein expression pattern of human peripheral blood mononuclear cells (PBMCs) after acute gamma irradiation. For this purpose, PBMCs extracted from eight healthy individuals were irradiated using 60 Co gamma rays (0.3 Gy and 1 Gy) and compared with sham irradiated controls. Total proteins were extracted 1 and 4 hour post irradiation and analyzed using 2-D gel electrophoresis. A fold change of 1.5 in spot intensity was considered as 'biological significant'. Protein identification was performed by MALDI-TOF mass spectrometry. The MS/MS spectra were interrogated using Mascot 2.1 for searching against SWISS-PROT database. One-hour post irradiation, 18 proteins showed a significant difference between the sham (0 Gy) and 0.3 Gy irradiated group (6 proteins up-regulated and 12 proteins down-regulated) and 17 proteins between the sham (0 Gy) and 1 Gy irradiated group (9 proteins up-regulated and 8 down-regulated). Four hours after irradiation, 16 proteins were differentially expressed between the sham irradiated and 0.3 Gy treated group (5 proteins up-regulated and 11 proteins downregulated). Relatively high dose of 1 Gy showed modulation of 13 proteins (5 proteins upregulated and 8 proteins down regulated) after 4 hours. There were 15 proteins that were observed both at the early time point of 1-hour and the late time point of 4-hour. Important among these were, proteins involved in cytoskeletal organization like Actin, Plastin-2, Vinculin, PDZ and LIM domain protein, WD repeat containing protein and the chaperone proteins like HSP 90-alpha and Protein disulfide-isomerase A3. Proteins like thiol specific antioxidant peroxiredoxin-6 (indicating increased levels of ROS and oxidative stress) showed dose specific expression while proteins like Ras-related Rap-1b-like protein (involved in cell survival) were observed with both 0.3 Gy and 1 Gy. During the study, human peripheral blood

  20. Effect of paricalcitol and GcMAF on angiogenesis and human peripheral blood mononuclear cell proliferation and signaling.

    Science.gov (United States)

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco; Amato, Marcello; Aterini, Stefano

    2012-01-01

    In addition to its role in calcium homeostasis and bone mineralization, vitamin D is involved in immune defence, cardiovascular function, inflammation and angiogenesis, and these pleiotropic effects are of interested in the treatment of chronic kidney disease. Here we investigated the effects of paricalcitol, a nonhypercalcemic vitamin D analogue, on human peripheral blood mononuclear cell proliferation and signaling, and on angiogenesis. These effects were compared with those of a known inhibitor of angiogenesis pertaining to the vitamin D axis, the vitamin D-binding protein-derived Gc-macrophage activating factor (GcMAF). Since the effects of vitamin D receptor agonists are associated with polymorphisms of the gene coding for the receptor, we measured the effects of both compounds on mononuclear cells harvested from subjects harboring different BsmI polymorphisms. Paricalcitol inhibited mononuclear cell viability with the bb genotype showing the highest effect. GcMAF, on the contrary, stimulated cell proliferation, with the bb genotype showing the highest stimulatory effect. Both compounds stimulated 3'-5'-cyclic adenosine monophosphate formation in mononuclear cells with the highest effect on the bb genotype. Paricalcitol and GcMAF inhibited the angiogenesis induced by proinflammatory prostaglandin E1. Polymorphisms of the vitamin D receptor gene, known to be associated with the highest responses to vitamin D receptor agonists, are also associated with the highest responses to GcMAF. These results highlight the role of the vitamin D axis in chronic kidney disease, an axis which includes vitamin D, its receptor and vitamin D-binding protein-derived GcMAF.

  1. Two immunosuppressive compounds from the mushroom Rubinoboletus ballouii using human peripheral blood mononuclear cells by bioactivity-guided fractionation.

    Science.gov (United States)

    Li, Long-Fei; Chan, Ben Chung-Lap; Yue, Grace Gar-Lee; Lau, Clara Bik-San; Han, Quan-Bin; Leung, Ping-Chung; Liu, Ji-Kai; Fung, Kwok-Pui

    2013-10-15

    Rubinoboletus ballouii is an edible mushroom wildly grown in Yunnan province, China. Up till now, little was known about the chemical and biological properties of this mushroom. The aim of this study was to investigate the immunomodulatory effects of the ethanolic extract of Rubinoboletus ballouii and its fractions on human peripheral blood mononuclear cells (PBMCs) using bioactivity-guided fractionation. The crude extract of the fruiting bodies of RB was fractionated by high-speed counter current chromatography (HSCCC). Twelve fractions were obtained and the third fraction (Fraction C) exerted the most potent anti-inflammatory activities in mitogen-activated PBMCs. Further fractionation of fraction C led to the isolation of two single compounds which were elucidated as 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin, respectively. The results showed that both 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin exhibited significant immunosuppressive effects on phytohemagglutinin (PHA)-stimulated human PBMCs by inhibiting [methyl-(3)H]-thymidine uptake and inflammatory cytokines productions such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon (IFN)-γ and IL-1β. Besides, 1-ribofuranosyl-s-triazin-2(1H)-one was firstly found in natural resources, and pistillarin was also isolated from the family Boletaceae for the first time. They exhibited great potential in developing as anti-inflammatory reagents. Copyright © 2013 Elsevier GmbH. All rights reserved.

  2. The effects of high dose ionizing radiation on transcriptional regulation and paracrine signaling in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Beer, L.

    2015-01-01

    While it has long been accepted that direct cell-cell interactions and the replacement of injured tissues with injected cells exerts therapeutic effects, it is currently believed that, in addition, paracrine factors released from different cell types activate cytoprotective and regenerative processes. Cells are now seen as bioreactors that produce and release soluble factors which might be used as therapeutics. We have previously shown that peripheral blood mononuclear cells (PBMCs) release a plethora of paracrine factors that enhance wound healing, attenuate myocardial damage following acute myocardial infarction, abolish microvascular obstruction, improve neurological outcome after acute ischemic stroke and spinal cord injury and protect mice from experimental autoimmune myocarditis. These PBMC derived paracrine factors may exert their effects via the induction of cytoprotective pathways, augmentation of angiogenesis, induction of NO-depended vasodilation and inhibition of VASP dependent platelet aggregation, as well as driving auto-reactive CD4+ cells into apoptosis. To enhance the cellular secretory capacity, treatments which induce stress responses, such as hypoxic preconditioning or ionizing irradiation (IR), have been developed. Although these effects have been evaluated in several disease states there is little data available on the cellular effects of ionizing irradiation on human PBMCs and their secretome. In this study, we have thus undertaken to investigate the effects of IR on human PBMCs in terms of the induction of transcriptional changes and release of pleiotropic paracrine factors. There are three primary aims of this doctoral thesis: 1. To investigate cellular processes activated or repressed in human PBMCs following high dose ionizing radiation (60Gy) and high density cell cultivation (25*10"6 cells/ml) for up to 24 hours. 2. To identify paracrine factors released from these cells using a multi-methodical biochemical/bioinformatics approach. 3

  3. Changes in Transcriptional Output of Human Peripheral Blood Mononuclear Cells Following Resistance Exercise

    Science.gov (United States)

    2011-01-01

    immunodeficiency virus (HIV), or had been diagnosed with acquired immune deficiency syndrome (AIDS). Participants were also excluded if they were... Laron Z (1995) Mathematical formulae for the prediction of the residual beta cell function during the first two years of disease in children and

  4. Gene expression dose-response changes in microarrays after exposure of human peripheral lung epithelial cells to nickel(II).

    Science.gov (United States)

    Cheng, Robert Y S; Zhao, Ailian; Alvord, W Gregory; Powell, Douglas A; Bare, Robert M; Masuda, Akira; Takahashi, Takashi; Anderson, Lucy M; Kasprzak, Kazimierz S

    2003-08-15

    Occupational exposure to nickel compounds is associated with lung cancer risk; both genotoxic and epigenetic mechanisms have been proposed. For comprehensive examination of the acute effects of nickel(II) acetate on gene expression in cultured human peripheral lung epithelial HPL1D cells, microarray analyses were carried out with cDNA chips (approximately 8000 cDNAs). Cells were exposed for 24 h to nontoxic (50, 100, and 200 microM) or toxic (400, 800, and 1600 microM) nickel(II) concentrations. Cluster analysis was applied to the 868 genes with > or = 2-fold change at any concentration. Two main clusters showed marked up- or down-regulation at the highest, toxic concentrations. The data further subdivided into 10 highly cohesive clusters with high probability, and of these only 2 had the same response trend at low nontoxic as at high concentrations, an observation of clear relevance to the process of high- to low-dose extrapolation in risk assessment. There were 113 genes showing > or = 2-fold change at the three lower nontoxic concentrations, those most relevant to in vivo carcinogenesis. In addition to expected responses of metallothionein, ferritin, and heat-shock proteins, the results revealed for the first time changed expression of some potential cancer-related genes in response to low-dose Ni(II): RhoA, dyskerin, interferon regulatory factor 1, RAD21 homologue, and tumor protein, translationally controlled. Overall, most of the genes impacted by nontoxic concentrations of nickel(II) acetate related to gene transcription, protein synthesis and stability, cytoskeleton, signaling, metabolism, cell membrane, and extracellular matrix.

  5. Immunoglobulin production induced in vitro by glucocorticoid hormones: T cell-dependent stimulation of immunoglobulin production without B cell proliferation in cultures of human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    Grayson, J.; Dooley, N.J.; Koski, I.R.; Blaese, R.M.

    1981-01-01

    The direct effects of steroid hormones on the production of immunoglobulins and DNA synthesis by human T and B lymphocytes was evaluated in cultures of peripheral blood mononuclear cells. As detected by a reverse hemolytic plaque assay, the addition of 0.1 mM to 10 nM hydrocortisone to lymphocytes in culture in the absence of other stimulants or mitogens, resulted in the dramatic induction of immunoglobulin production with responses comparable to those seen in similar cultures stimulated with pokeweed mitogen. Steroid-stimulated immunoglobulin production was first seen after 48 h and peaked at 8-10 d of culture. The production of IgG, IgA, and IgM was induced following incubation with steroid. Glucocorticoids, but not estrogens or androgens, were capable of mediating this effect, and only compounds with affinity for the glucocorticoid receptor were active. The induction of immunoglobulin production was dependent on both T cells and monocytes; cultures depleted of either cell type did not produce immunoglobulin when stimulated with glucocorticoid hormones. Proliferation of B cells or T cells could not be detected by [/sup 3/H]thymidine incorporation or total cell recovery from steroid-stimulated cultures, even though such cultures demonstrated marked increases in immunoglobulin production. The mechanism responsible for this functional maturation of B cells to become high rate immunoglobulin producing cells is as yet undefined, although it appears to involve more than merely steroid mediated inactivation of suppressor T cells

  6. Effects of vitamin K3 and K5 on proliferation, cytokine production, and regulatory T cell-frequency in human peripheral-blood mononuclear cells.

    Science.gov (United States)

    Hatanaka, Hiroshige; Ishizawa, Hitomi; Nakamura, Yurie; Tadokoro, Hiroko; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

    2014-03-18

    The effects of vitamin K (VK) derivatives VK3 and VK5 on human immune cells have not been extensively investigated. We examined the effects of VK3 and VK5 on proliferation, apoptosis, cytokine production, and CD4+CD25+Foxp3+ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Anti-proliferative effects of VK3 and VK5 on T-cell mitogen activated PBMCs were assessed by WST assay procedures. Apoptotic cells were determined as Annexin V positive/propidium iodide (PI) negative cells. Cytokine concentrations in the supernatant of the culture medium were measured with bead-array procedures followed by analysis with flow cytometry. The CD4+CD25+Foxp3+Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. VK3 and VK5 suppressed the mitogen-activated proliferation of PBMCs significantly at 10-100μM (p<0.05). The data also suggest that VK3 and VK5 promote apoptosis in the mitogen-activated T cells. VK3 and VK5 significantly inhibited the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 10-100μM (p<0.05). In contrast, VK3 and VK5 significantly increased Treg cell-frequency in the activated PBMCs at concentrations more than 10μM (p<0.001). Our data suggest that VK3 and VK5 attenuate T cell mediated immunity by inhibiting the proliferative response and inducing apoptosis in activated T cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. 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....

  8. Analyses of 123 Peripheral Human Immune Cell Subsets: Defining Differences with Age and between Healthy Donors and Cancer Patients Not Detected in Analysis of Standard Immune Cell Types

    Directory of Open Access Journals (Sweden)

    Lauren M. Lepone

    2016-03-01

    Full Text Available Recent advances in human immunology have led to the identification of novel immune cell subsets and the biological function of many of these subsets has now been identified. The recent US Food and Drug Administration approval of several immunotherapeutics for the treatment of a variety of cancer types and the results of ongoing immunotherapy clinical studies requires a more thorough interrogation of the immune system. We report here the use of flow cytometry-based analyses to identify 123 immune cell subsets of peripheral blood mononuclear cells. The use of these panels defines multiple differences in younger (< 40 years vs. older (≥ 40 years individuals and between aged-matched apparently healthy individuals and metastatic cancer patients, aspects not seen in the analysis of the following standard immune cell types: CD8, CD4, natural killer, natural killer-T, regulatory T, myeloid derived suppressor cells, conventional dendritic cells (DCs, plasmacytoid DCs and B cells. The use of these panels identifying 123 immune cell subsets may aid in the identification of patients who may benefit from immunotherapy, either prior to therapy or early in the immunotherapeutic regimen, for the treatment of cancer or other chronic or infectious diseases.

  9. Cultured human peripheral blood mononuclear cells alter their gene expression when challenged with endocrine-disrupting chemicals

    International Nuclear Information System (INIS)

    Wens, B.; De Boever, P.; Verbeke, M.; Hollanders, K.; Schoeters, G.

    2013-01-01

    Endocrine disrupting chemicals (EDCs) have the potential to interfere with the hormonal system and may negatively influence human health. Microarray analysis was used in this study to investigate differential gene expression in human peripheral blood cells (PBMCs) after in vitro exposure to EDCs. PBMCs, isolated from blood samples of four male and four female healthy individuals, were exposed in vitro for 18 h to either a dioxin-like polychlorinated biphenyl (PCB126, 1 μM), a non-dioxin-like polychlorinated biphenyl (PCB153, 10 μM), a brominated flame retardant (BDE47, 10 μM), a perfluorinated alkyl acid (PFOA, 10 μM) or bisphenol (BPA, 10 μM). ANOVA analysis revealed a significant change in the expression of 862 genes as a result of EDC exposure. The gender of the donors did not affect gene expression. Hierarchical cluster analysis created three groups and clustered: (1) PCB126-exposed samples, (2) PCB153 and BDE47, (3) PFOA and BPA. The number of differentially expressed genes varied per compound and ranged from 60 to 192 when using fold change and multiplicity corrected p-value as filtering criteria. Exposure to PCB126 induced the AhR signaling pathway. BDE47 and PCB153 are known to disrupt thyroid metabolism and exposure influenced the expression of the nuclear receptors PPARγ and ESR2, respectively. BPA and PFOA did not induce significant changes in the expression of known nuclear receptors. Overall, each compound produced a unique gene expression signature affecting pathways and GO processes linked to metabolism and inflammation. Twenty-nine genes were significantly altered in expression under all experimental conditions. Six of these genes (HSD11B2, MMP11, ADIPOQ, CEL, DUSP9 and TUB) could be associated with obesity and metabolic syndrome. In conclusion, microarray analysis identified that PBMCs altered their gene expression response in vitro when challenged with EDCs. Our screening approach has identified a number of gene candidates that warrant

  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

    2018-02-01

    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. 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

    2018-01-01

    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.

  12. Anti-inflammatory compound resveratrol suppresses homocysteine formation in stimulated human peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Schroecksnadel, Katharina; Winkler, Christiana; Wirleitner, Barbara; Schennach, Harald; Weiss, Günter; Fuchs, Dietmar

    2005-01-01

    Inflammation, immune activation and oxidative stress play a major role in the pathogenesis of cardiovascular disorders. In addition to markers of inflammation, moderate hyperhomocysteinemia is an independent risk factor for cardiovascular disease, and there is a link between the activation of immunocompetent cells and the enhanced formation of homocysteine in vitro. Likewise, anti-inflammatory drugs and nutrients rich in antioxidant vitamins are able to reduce cardiovascular risk and to slow down the atherogenic process. Resveratrol, a phenolic antioxidant synthesized in grapes and vegetables and present in wine, has also been supposed to be beneficial for the prevention of cardiovascular events. Apart from its strong antioxidant properties, resveratrol has also been demonstrated to act as an anti-inflammatory agent. In this study the influence of resveratrol on the production of homocysteine by stimulated human peripheral blood mononuclear cells (PBMCs) was investigated. Results were compared to earlier described effects of the anti-inflammatory compounds aspirin and salicylic acid and of the lipid-lowering drug atorvastatin. Stimulation of PBMCs with the mitogens concanavalin A and phytohemagglutinin induced significantly higher homocysteine accumulation in supernatants compared with unstimulated cells. Treatment with 10-100 muM resveratrol suppressed homocysteine formation in a dose-dependent manner. Resveratrol did not influence the release of homocysteine from resting PBMCs. The data suggest that resveratrol may prevent homocysteine accumulation in the blood by suppressing immune activation cascades and the proliferation of mitogen-driven T-cells. The effect of resveratrol to down-regulate the release of homo-cysteine was comparable to the decline of neopterin concentrations in the same experiments. The suppressive effect of resveratrol was very similar to results obtained earlier with aspirin, salicylic acid and atorvastatin; however, it appeared that doses

  13. IL-7 Enhances Thymic Human T Cell Development in "Human Immune System" Rag2-/-IL-2R{gamma}c-/- Mice without Affecting Peripheral T Cell Homeostasis

    NARCIS (Netherlands)

    van Lent, Anja U.; Dontje, Wendy; Nagasawa, Maho; Siamari, Rachida; Bakker, Arjen Q.; Pouw, Stephan M.; Maijoor, Kelly A.; Weijer, Kees; Cornelissen, Jan J.; Blom, Bianca; Di Santo, James P.; Spits, Hergen; Legrand, Nicolas

    2009-01-01

    IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported. By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7 is approximately 100-fold less potent than human IL-7

  14. 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

  15. 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.

  16. Action of the poison of Apis mellifera bee and gamma radiation on bone marrow cells of Wistar rats and on lymphocytes of human peripheral blood

    International Nuclear Information System (INIS)

    Varanda, E.A.

    1987-01-01

    ''In vivo'' and ''in vitro'' experiments are performed to determine the radioprotective action of the poison of Apis mellifera bees. The frequency of chromosome aberrations, induced by gamma radiation, is studied in two assays: ''in vivo'' in bone marrow cells from Wistar rats and ''in vitro'' in human peripheral blood lymphocyte cultures. The sister chromatid exchanges (SCE) are studied in the ''in vitro'' assays. (M.A.C.) [pt

  17. Human Peripheral Blood Mononuclear Cells Exhibit Heterogeneous CD52 Expression Levels and Show Differential Sensitivity to Alemtuzumab Mediated Cytolysis

    Science.gov (United States)

    Rao, Sambasiva P.; Sancho, Jose; Campos-Rivera, Juanita; Boutin, Paula M.; Severy, Peter B.; Weeden, Timothy; Shankara, Srinivas; Roberts, Bruce L.; Kaplan, Johanne M.

    2012-01-01

    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. PMID:22761788

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Dose-effect of ionizing radiation-induced PIG3 gene expression alteration in human lymphoblastoid AHH-1 cells and human peripheral blood lymphocytes.

    Science.gov (United States)

    Liu, Qing-Jie; Zhang, De-Qin; Zhang, Qing-Zhao; Feng, Jiang-Bin; Lu, Xue; Wang, Xin-Ru; Li, Kun-Peng; Chen, De-Qing; Mu, Xiao-Feng; Li, Shuang; Gao, Ling

    2015-01-01

    To identify new ionizing radiation (IR)-sensitive genes and observe the dose-effect of gene expression alteration (GEA) induced by IR. Microarray was used to screen the differentially expressed genes in human lymphoblastoid cells (AHH-1) using three doses of (60)Co γ-rays (0.5-8 Gy at 1 Gy/min). Given that p53-inducible gene 3 (PIG3) was consistently upregulated, the GEA of PIG3 in AHH-1 cells and human peripheral blood lymphocytes (HPBL) induced by γ-rays (1 Gy/min) was measured at messenger RNA (mRNA) and protein levels. The GEA of PIG3 in AHH-1 cells exposed to neutron radiation (californium-252, 0.073 Gy/min) was also quantified. PIG3 was one of the seven differentially expressed genes found in the microarray analysis. The PIG3 mRNA and protein levels in AHH-1 cells were significantly increased from 1-10 Gy of γ-rays 8-72 h or 8-168 h after exposure, respectively. The enhancement was also observed in AHH-1 cells from 0.4-1.6 Gy of neutrons 48 h post-irradiation. The PIG3 mRNA levels (mRNA copy numbers) in HPBL were significantly increased from 1-8 Gy of γ-rays within 4-24 h post-irradiation, but the highest increase in signal-to-noise responsiveness is approximately two-fold, which was less than that of AHH-1 (approximately 20-fold). IR can upregulate the PIG3 gene expression in AHH-1 and HPBL in the early phase after exposure; however, the IR induced expression levels of PIG3 are greater in AHH-1 than HPBL.

  20. Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines

    International Nuclear Information System (INIS)

    Nelson, S.L.; Grosovsky, A.J.; Jones, I.M.; Burkhart-Schultz, K.; Fuscoe, J.C.

    1995-01-01

    We have examined the extent of of HPRT - total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT - deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (>1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT - total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT - large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions. 24 refs., 6 figs., 1 tab

  1. Activation of human B lymphocytes. 8. Differential radiosensitivity of subpopulations of lymphoid cells involved in the polyclonally-induced PFC responses of peripheral blood B lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Fauci, A S; Pratt, K R; Whalen, G [National Inst. of Allergy and Infectious Diseases, Bethesda, MD (USA)

    1978-11-01

    The differential effect of various doses of irradiation on subpopulations of human peripheral blood lymphoid cells involved in the pokeweed mitogen induced PFC response against sheep red blood cells was studied. The plaque forming B cells were quite sensitive to low doses of irradiation with complete suppression of responses at 300 to 500 rad. On the contrary, helper T-cell function was resistant to 2000 rad. Co-culture of irradiated T cells with autologous or allogeneic B cells resulted in marked enhancement of PFC responses consistent with the suppression of naturally occurring suppressor cells with a resulting pure helper effect. Irradiated T-cell-depleted suspensions failed to produce this effect as did heat killed T cells, whereas mitomycin C treated T cells gave effects similar to irradiated T cells. These findings are consistent with a lack of requirement of cell division for a T-cell helper effect and a requirement of mitosis or another irradiation sensitive, mitomycin C sensitive process for a T-suppressor cell effect. These studies have potential relevance in the evaluation of subpopulations of human lymphoid cells involved in antibody production in normal individuals and in disease states.

  2. Effect of 60Co γ-ray irradiation on cytoskeleton of human peripheral blood monocytes with whole mount cell electron microscopy in vitro

    International Nuclear Information System (INIS)

    Chen Xiaomei; Guo Yuhua; Yin Zhiwei

    1992-01-01

    Whole mount cell electron microscopy was used in combination with selective extraction to prepare cytoskeletal framework. Cytoskeleton prepared by Triton X-100 treatment of human peripheral blood monocytes appeared on electron microscopy as a highly organized and interconnected three-dimensional matrix of different fibrous elements. By three-dimensional visualization of Triton X-100 resistant cytoskeletons it was demonstrated that different doses of 60 Co γ-rays caused distinctive and reproducible alterations of the cytoskeleton of intact human peripheral blood monocytes in vitro. The alterations were similar to those caused by cytochalasin B and by colchicine. From these observations and other workers'studies, it is presumed that 60 Co γ-ray irradiation may inhibit cytoplasmic microtubule and microfilament assembling

  3. DNA damage response and role of shelterin complex in human peripheral blood mononuclear cells exposed to gamma radiation

    International Nuclear Information System (INIS)

    Saini, Divyalakshmi; Das, Birajalaxmi

    2013-01-01

    Telomeres are the DNA protein structures that cap the ends of linear DNA. It consists of short repetitive DNA sequences (TTAGGG)n and specialized telomere binding proteins. There are six telomeric proteins (TRF1, TRF2, TIN2, TERF2, PTOP and POT1) called as shelterin complex/telosome which maintains telomere integrity. The function of this 'telosome' is to protect the natural ends of the chromosomes from being recognized as artificial DNA breaks, thereby preventing chromosome end-to-end fusions. DNA Damage Response (DDR) induced by radiation and its interaction with telomeric protein complex is poorly understood in human PBMCs at G 0 stage. Alterations in either telomeric DNA or telomere binding proteins can impair the function of the telosome, which may lead to senescence or apoptosis. Ionizing radiation which induces a plethora of DNA lesions in human cell may also alter the expression of telomere associated proteins. In the present study, we have made an attempt to study the DNA damage response of telomere proteins in human peripheral blood mononuclear cells exposed to gamma radiation. Venous blood samples were collected from eight random healthy volunteers and PBMCs were separated. Dose response as well as time point kinetics study was carried out at transcription as well as protein level. PBMCs were irradiated at various doses between 10 cGy to 2.0 Gy at a dose rate of 1.0 Gy/min. Total RNA was isolated for gene expression analysis at 0 hour and 4 hours respectively. cDNA was prepared and transcriptional pattern as studied using real time q-PCR where Taqman probes were used. Time point kinetics of transcriptional pattern of TRF1, TRF2, TIN2, TERF2, PTOP and POT1 was carried out at 0 min, 15 min, 30 min, 60 min, and 120 min for two different doses (1.0 Gy and 2.0 Gy). Dose response and time point kinetics of TRF2 was studied at similar doses using confocal microscopy. Our results revealed that at 2.0 Gy there was a two fold increase at the level of transcription

  4. Winter to summer change in vitamin D status reduces systemic inflammation and bioenergetic activity of human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Emily K. Calton

    2017-08-01

    Full Text Available Background: Vitamin D status [25(OHD] has recently been reported to be associated with altered cellular bioenergetic profiles of peripheral blood mononuclear cells (PBMCs. No study has tracked the seasonal variation of 25(OHD and its putative influence on whole body energy metabolism, cellular bioenergetic profiles, inflammatory markers and clinical chemistry. Material and methods: Whole body energy metabolism and substrate utilisation were measured by indirect calorimetry. PBMCs obtained from the same subjects were isolated from whole blood, counted and freshly seeded. Bioenergetic analysis (mitochondrial stress test and glycolysis stress test was performed using the Seahorse XFe96 flux analyser. 25(OHD was assessed using the Architect immunoassay method. Results: 25(OHD increased by a median (IQR of 14.40 (20.13 nmol/L (p75 nmol/L. The absolute change in 25(OHD was not associated with altered bioenergetics. Conclusion: Seasonal improvements in 25(OHD was associated with reduced systemic inflammation, PBMC bioenergetic profiles and whole body energy metabolism. These observational changes in PBMC bioenergetics were most pronounced in those who had insufficient 25(OHD in winter. The data warrants confirmation through cause and effect study designs. Keywords: Peripheral blood mononuclear cells, Bioenergetics, Vitamin D, Season, Inflammation, Insulin sensitivity

  5. Consumption of selenium-enriched broccoli increases cytokine production in human peripheral blood mononuclear cells stimulated ex vivo, a preliminary human intervention study.

    Science.gov (United States)

    Bentley-Hewitt, Kerry L; Chen, Ronan K-Y; Lill, Ross E; Hedderley, Duncan I; Herath, Thanuja D; Matich, Adam J; McKenzie, Marian J

    2014-12-01

    Selenium (Se) is a micronutrient essential for human health, including immune function. Previous research indicates that Se supplementation may cause a shift from T helper (Th)1- to Th2-type immune responses. We aim to test the potential health promoting effects of Se-enriched broccoli. In a human trial, 18 participants consumed control broccoli daily for 3 days. After a 3-day wash-out period, the participants were provided with Se-enriched broccoli containing 200 μg of Se per serving for 3 days. Plasma and peripheral blood mononuclear cell (PBMC) samples were collected at the start and end of each broccoli feeding period for analysis of total Se and measurement of cytokine production from PBMC stimulated with antigens ex vivo. Plasma Se content remained consistent throughout the control broccoli feeding period and the baseline of the Se-enriched broccoli period (1.22 μmol/L) and then significantly increased following 3 days of Se-enriched broccoli feeding. Interleukin (IL-2, IL-4, IL-5, IL-13, and IL-22) production from PBMC significantly increased after 3 days of Se-enriched broccoli feeding compared with baseline. This study indicates that consumption of Se-enriched broccoli may increase immune responses toward a range of immune challenges. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Analysis of mtDNT 4977bp deletion induced by ionizing radiation in human peripheral blood nucleated cells using real-time PCR

    International Nuclear Information System (INIS)

    Fan Tianli; Wang Ping; Han Lin; Liu Yulong; Liu Yumin

    2010-01-01

    To detect mitochondrial DNA(mtDNA) 4977bp deletion(triangle open mtDNA 4977 ) in human peripheral blood nucleated cells exposed to ionizing radiation in vitro by using real-time PCR, and explore possibility of the index as biodosimetry for estimating biological dose in radiation accident,six healthy individuals' peripheral blood was collected,and the blood samples were irradiated with 0,1,2,3,4 and 5 Gy 60 Co gamma-ray. The triangle open mtDNA 4977 and total mtDNA copy number(mtDNA total ) in the mtDNA samples were detected, and then the deletion rates were calculated. The results showed that the mtDNA total and triangle open mtDNA 4977 copy number, and the deletion rates of mtDNA 4977bp in the mtDNA samples from 6 healthy individuals' blood exposed to 1-5 Gy radiation were higher than that with the samples exposed to 0 Gy radiation(p 0.05). The results indicated that ionizing radiation can induce accumulation of the triangle open mtDNA 4977 and increase of mtDNA total copy number in human peripheral blood nucleated cells,but both the mtDNA 4977bp deletion and exposure dose(0-5 Gy) were not obviously correlated. (authors)

  7. Molecular cloning of human T-cell lymphotrophic virus type I-like proviral genome from the peripheral lymphocyte DNA of a patient with chronic neurologic disorders

    International Nuclear Information System (INIS)

    Reddy, E.P.; Mettus, R.V.; DeFreitas, E.; Wroblewska, Z.; Cisco, M.; Koprowski, H.

    1988-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-I), the etiologic agent of human T-cell leukemia, has recently been shown to be associated with neurologic disorders such as tropical spastic paraparesis, HTLV-associated myelopathy, and possibly with multiple sclerosis. In this communication, the authors have examined one specific case of neurologic disorder that can be classified as multiple sclerosis or tropical spastic paraparesis. The patient suffering from chronic neurologic disorder was found to contain antibodies to HTLV-I envelope and gag proteins in his serum and cerebrospinal fluid. Lymphocytes from peripheral blood and cerebrospinal fluid of the patient were shown to express viral RNA sequences by in situ hybridization. Southern blot analysis of the patient lymphocyte DNA revealed the presence of HTLV-I-related sequences. Blot-hybridization analysis of the RNA from fresh peripheral lymphocytes stimulated with interleukin 2 revealed the presence of abundant amounts of genomic viral RNA with little or no subgenomic RNA. They have clones the proviral genome from the DNA of the peripheral lymphocytes and determined its restriction map. This analysis shows that this proviral genome is very similar if not identical to that of the prototype HTLV-I genome

  8. Human umbilical cord derived mesenchymal stem cells promote interleukin-17 production from human peripheral blood mononuclear cells of healthy donors and systemic lupus erythematosus patients.

    Science.gov (United States)

    Ren, S; Hu, J; Chen, Y; Yuan, T; Hu, H; Li, S

    2016-03-01

    Inflammation instigated by interleukin (IL)-17-producing cells is central to the development and pathogenesis of several human autoimmune diseases and animal models of autoimmunity. The expansion of IL-17-producing cells from healthy donors is reportedly promoted by mesenchymal stem cells derived from fetal bone marrow. In the present study, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were examined for their effects on lymphocytes from healthy donors and from patients with systemic lupus erythematosus (SLE). Significantly higher levels of IL-17 were produced when CD4(+) T cells from healthy donors were co-cultured with hUC-MSCs than those that were cultured alone. Blocking experiments identified that this effect might be mediated partially through prostaglandin E2 (PGE2 ) and IL-1β, without IL-23 involvement. We then co-cultured hUC-MSCs with human CD4(+) T cells from systemic lupus erythematosus patients. Ex-vivo inductions of IL-17 by hUC-MSCs in stimulated lymphocytes were significantly higher in SLE patients than in healthy donors. This effect was not observed for IL-23. Taken together, our results represent that hUC-MSCs can promote the IL-17 production from CD4(+) T cells in both healthy donor and SLE patients. PGE2 and IL-1β might also be partially involved in the promotive effect of hUC-MSCs. © 2015 British Society for Immunology.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. In vivo effects of myeloablative alkylator therapy on survival and differentiation of MGMTP140K-transduced human G-CSF-mobilized peripheral blood cells.

    Science.gov (United States)

    Cai, Shanbao; Hartwell, Jennifer R; Cooper, Ryan J; Juliar, Beth E; Kreklau, Emi; Abonour, Rafat; Goebel, W Scott; Pollok, Karen E

    2006-05-01

    High-intensity alkylator-based chemotherapy is required to eradicate tumors expressing high levels of O6-methylguanine DNA methyltransferase (MGMT). This treatment, however, can lead to life-threatening myelosuppression. We investigated a gene therapy strategy to protect human granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells (MPB) from a high-intensity alkylator-based regimen. We transduced MPB with an oncoretroviral vector that coexpresses MGMT(P140K) and the enhanced green fluorescent protein (EGFP) (n = 5 donors). At 4 weeks posttransplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, cohorts were not treated or were treated with low- or high-intensity alkylating chemotherapy. In the high-intensity-treated cohort, it was necessary to infuse NOD/SCID bone marrow (BM) to alleviate hematopoietic toxicity. At 8 weeks posttreatment, human CD45+ cells in the BM of mice treated with either regimen were EGFP+ and contained MGMT-specific DNA repair activity. In cohorts receiving low-intensity therapy, both primitive and mature hematopoietic cells were present in the BM. Although B-lymphoid and myeloid cells were resistant to in vivo drug treatment in cohorts that received high-intensity therapy, no human CD34+ cells or B-cell precursors were detected. These data suggest that improved strategies to optimize repair of DNA damage in primitive human hematopoietic cells are needed when using high-intensity anti-cancer therapy.

  12. 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.

  13. 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,

  14. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    Science.gov (United States)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  15. Human eosinophils modulate peripheral blood mononuclear cell response to Schistosoma mansoni adult worm antigen in vitro.

    Science.gov (United States)

    Tweyongyere, R; Namanya, H; Naniima, P; Cose, S; Tukahebwa, E M; Elliott, A M; Dunne, D W; Wilson, S

    2016-08-01

    High numbers of eosinophils are observed in parasitic infections and allergic diseases, where they are proposed to be terminally differentiated effector cells that play beneficial role in host defence, or cause harmful inflammatory response. Eosinophils have been associated with killing of schistosomulae in vitro, but there is growing evidence that eosinophils can play additional immuno-regulatory role. Here, we report results of a study that examines peripheral blood mononuclear cell (PBMC) cytokine responses to Schistosoma mansoni adult worm antigen (SWA) when stimulated alone or enriched with autologous eosinophils. Production of the Th-2 type cytokines interleukin (IL)-4, IL-5 and IL-13 was lower (P = 0·017, 0·018 and eosinophil cultures than in PBMC-only cultures stimulated with SWA. Substantial levels of IL-13, IL-10, interferon gamma and tumour necrosis factor alpha were recorded in cultures of eosinophils, but none of these cytokines showed significant association with the observed eosinophil-induced drop in cytokine responses of PBMC. Transwell experiments suggested that the observed effect is due to soluble mediators that downmodulate production of Th-2 type cytokines. This study shows that eosinophils may down-modulate schistosome-specific Th-2 type cytokine responses in S. mansoni-infected individuals. The mechanism of this immune modulation remains to be elucidated. © 2016 The Authors. Parasite Immunology Published by John Wiley & Sons Ltd.

  16. 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.

  17. Extremely low frequency electromagnetic field exposure does not modulate Toll-like receptor signaling in human peripheral blood mononuclear cells

    NARCIS (Netherlands)

    Kleijn, de S.; Bouwens, M.; Verburg-van Kemenade, B.M.L.; Cuppen, J.J.M.; Ferwerda, G.; Hermans, P.

    2011-01-01

    The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling

  18. An Integrated Workflow To Assess Technical and Biological Variability of Cell Population Frequencies in Human Peripheral Blood by Flow Cytometry.

    Science.gov (United States)

    Burel, Julie G; Qian, Yu; Lindestam Arlehamn, Cecilia; Weiskopf, Daniela; Zapardiel-Gonzalo, Jose; Taplitz, Randy; Gilman, Robert H; Saito, Mayuko; de Silva, Aruna D; Vijayanand, Pandurangan; Scheuermann, Richard H; Sette, Alessandro; Peters, Bjoern

    2017-02-15

    In the context of large-scale human system immunology studies, controlling for technical and biological variability is crucial to ensure that experimental data support research conclusions. In this study, we report on a universal workflow to evaluate both technical and biological variation in multiparameter flow cytometry, applied to the development of a 10-color panel to identify all major cell populations and T cell subsets in cryopreserved PBMC. Replicate runs from a control donation and comparison of different gating strategies assessed the technical variability associated with each cell population and permitted the calculation of a quality control score. Applying our panel to a large collection of PBMC samples, we found that most cell populations showed low intraindividual variability over time. In contrast, certain subpopulations such as CD56 T cells and Temra CD4 T cells were associated with high interindividual variability. Age but not gender had a significant effect on the frequency of several populations, with a drastic decrease in naive T cells observed in older donors. Ethnicity also influenced a significant proportion of immune cell population frequencies, emphasizing the need to account for these covariates in immune profiling studies. We also exemplify the usefulness of our workflow by identifying a novel cell-subset signature of latent tuberculosis infection. Thus, our study provides a universal workflow to establish and evaluate any flow cytometry panel in systems immunology studies. Copyright © 2017 by The American Association of Immunologists, Inc.

  19. 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.

  20. Naturally induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells.

    Science.gov (United States)

    Goverse, A; Rouppe van der Voort, J; Roppe van der Voort, C; Kavelaars, A; Smant, G; Schots, A; Bakker, J; Helder, J

    1999-10-01

    Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones alpha-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue--these nematodes normally invade the roots of potato plants--suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.

  1. The influence of gender- and age-related differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

    International Nuclear Information System (INIS)

    Kato, Kengo; Kashiwakura, Ikuo; Kuwabara, Mikinori

    2011-01-01

    To investigate the importance of gender and aging on the individual radiosensitivity of lineage-committed myeloid hematopoietic stem/progenitor cells (HSPCs) detected in mononuclear cells (MNCs) of steady-state human peripheral blood (PB), the clonogenic survival of HPCs, including colony-forming unit-granulocyte macrophage; burst-forming unit-erythroid; colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte cells derived from MNCs exposed to 0.5 Gy and 2 Gy X-irradiation were estimated. MNCs were prepared from the buffy-coats of 59 healthy individual blood donors. The results showed that large individual differences exist in the number of HSPCs, as well as in the surviving fraction of cells. Furthermore, the number of progenitor cells strongly correlated with their surviving fraction, suggesting that the radiosensitivity of hematopoietic progenitor cells decreases with the number of cells in the 10 5 cells population. A statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of an individual, however, none of these correlations were observed after 2 Gy irradiation. No statistically significant difference was observed in individual radiosensitivity between males and females at either radiation dose. The present results indicated a correlation between the individual responsiveness of HSPCs to ionizing irradiation, especially to low dose irradiation, and aging. (author)

  2. Allogeneic Peripheral Blood Stem Cell Harvest

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. Allogeneic Peripheral Blood Stem Cell Harvest. Mobilization protocol. G-CSF 10 mcg/Kg / day for 5 days. Pheresis. Cobe Spectra; Haemonetics mcs+. Enumeration. CD34 counts; Cfu-GM assays.

  3. Genotoxic damage in cultured human peripheral blood lymphocytes ...

    African Journals Online (AJOL)

    Falaq Naz

    2012-06-29

    Jun 29, 2012 ... Genotoxic damage in cultured human peripheral blood lymphocytes of oral ... catechol estrogens and quinines, via redox reactions causes oxidative damage to .... volume was prepared for each donor. About, 0.8 ml of cell sus .... duce the adverse effects of OCs, such as the reduction in the estrogen content.

  4. Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes

    International Nuclear Information System (INIS)

    Hakoda, Masayuki; Akiyama, Mitoshi; Kyoizumi, Seishi; Kobuke, Kyoko; Awa, A.A.

    1987-07-01

    Approximately 80 % of human peripheral blood T-lymphocytes could be cloned in the presence of crude Interleukin-2, phytohemagglutinin, and X-irradiated autologous lymphocytes and Raji B-cells. This modified cloning method was used to measure the in vivo frequency of HGPRT-deficient mutant T-lymphocytes. Repeated experiments using blood from the same individuals revealed that the frequency of mutant cells was almost constant for each individual even though the cloning efficiency of lymphocytes varied somewhat from experiment to experiment. Approximately 80 % of both wild-type unselected and 6-thioguanine-resistant colonies had helper/inducer and about 20 % had suppressor/cytotoxic T-lymphocyte markers. No difference was observed in the distribution of lymphocyte subsets between wild and mutant lymphocyte colonies. (author)

  5. 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

  6. 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.

    Directory of Open Access Journals (Sweden)

    Tetsuro Tamaki

    Full Text Available 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

  7. Electron microscopic study of the myelinated nerve fibres and the perineurial cell basement membrane in the diabetic human peripheral nerves

    International Nuclear Information System (INIS)

    ElBarrany, Wagih G.; Hamdy, Raid M.; AlHayani, Abdulmonem A.; Jalalah, Sawsan M.

    2009-01-01

    To study the quantitative and ultrastructural changes in myelinated nerve fibers and the basement membranes of the perineurial cells in diabetic nerves. The study was performed at the Department of Anatomy, Faculty of Medicine, King Abdul-Aziz University, Jeddah, Saudi Arabia from 2003 to 2005. Human sural nerves were obtained from 15 lower limbs and 5 diabetic nerve biopsies. The total mean and density of myelinated nerve fibers per fascicle were calculated, with density of microtubules and mitochondria in the axoplasm. The number of the perineurial cell basement membrane layers was counted, and thickness of the basement membrane was measured. Among the 15 diabetic and 5 normal human sural nerves, the average diameters, number and surface area of myelinated nerve fibers and axonal microtubules density were found to be less in diabetic nerves. Mitochondrial density was higher in diabetic axons. Thickness of the perineurial cell basement membrane had a greater mean, but the number of perineurial cell layers was less than that of the diabetic group. The inner cellular layer of the perineurium of the diabetic nerves contained large vacuoles containing electron-dense degenerated myelin. A few specimens showed degenerated myelinated nerve fibers, while others showed recovering ones. Retracted axoplasms were encountered with albumin extravasation. Diabetes caused an increase in perineurial permeability. The diabetic sural nerve showed marked decrease in the myelinated nerve fibres, increase degenerated mitochondria, and decreased microtubules. (author)

  8. The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation.

    Science.gov (United States)

    Weidele, Kathrin; Beneke, Sascha; Bürkle, Alexander

    2017-04-01

    NAD + is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD + consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD + levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NAD + levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD + concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD + levels may be limiting under conditions of genotoxic stress and that boosting the NAD + system with nicotinic acid can improve genomic stability. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. HIV-1 isolation from infected peripheral blood mononuclear cells

    NARCIS (Netherlands)

    Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A.; Schuitemaker, Hanneke; Scarlatti, Gabriella

    2014-01-01

    Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and

  10. Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ali, Al-Yousef Sulaiman; Musarrat, Javed

    2012-01-01

    The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (μg/g) were determined as Fe (34160.0 ± 1.38), Ni (150.8 ± 0.78), Cu (99.3 ± 0.56) and Cr (64.0 ± 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O 2 • − ) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: ► CFA consists of spherical crystalline nanoparticles in size range of 11–25 nm. ► Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. ► CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. ► Comet and CBMN assays revealed DNA and chromosomal breakage in PBMN cells. ► CFA-NPs resulted in mitochondrial membrane damage in PBMN cells.

  11. Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

    Energy Technology Data Exchange (ETDEWEB)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Ali, Al-Yousef Sulaiman [Department of Medical Laboratory Sciences, College of Applied Medical Science, University of Dammam, P.O. Box 1683, Hafr Al Batin-31991 (Saudi Arabia); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Department of Agricultural Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh202002 (India)

    2012-10-15

    The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles ({mu}g/g) were determined as Fe (34160.0 {+-} 1.38), Ni (150.8 {+-} 0.78), Cu (99.3 {+-} 0.56) and Cr (64.0 {+-} 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-a-vis -Aq extract. Generation of superoxide anions (O{sub 2} Bullet {sup -}) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: Black-Right-Pointing-Pointer CFA consists of spherical crystalline nanoparticles in size range of 11-25 nm. Black-Right-Pointing-Pointer Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. Black-Right-Pointing-Pointer CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. Black-Right-Pointing-Pointer Comet and CBMN assays revealed DNA and chromosomal

  12. T-cell receptor repertoire of human peripheral CD161hiTRAV1-2+ MAIT cells revealed by next generation sequencing and single cell analysis.

    Science.gov (United States)

    Held, Kathrin; Beltrán, Eduardo; Moser, Markus; Hohlfeld, Reinhard; Dornmair, Klaus

    2015-09-01

    Mucosal-associated invariant T (MAIT) cells are a T-cell subset that expresses a conserved TRAV1-2 (Vα7.2) T-cell receptor (TCR) chain and the surface marker CD161. They are involved in the defence against microbes as they recognise small organic molecules of microbial origin that are presented by the non-classical MHC molecule 1 (MR1). MAIT cells express a semi-restricted TCR α chain with TRAV1-2 preferentially linked to TRAJ33, TRAJ12, or TRAJ20 which pairs with a limited set of β chains. To investigate the TCR repertoire of human CD161(hi)TRAV1-2(+) T cells in depth we analysed the α and β chains of this T-cell subset by next generation sequencing. Concomitantly we analysed 132 paired α and β chains from single cells to assess the αβ pairing preferences. We found that the CD161(hi)TRAV1-2(+) TCR repertoire in addition to the typical MAIT TCRs further contains polyclonal elements reminiscent of classical αβ T cells. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  13. Absence of DNA double-strand breaks in human peripheral blood mononuclear cells after 3 Tesla magnetic resonance imaging assessed by γH2AX flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Fasshauer, Martin; Staab, Wieland; Sohns, Jan M.; Ritter, Christian; Lotz, Joachim [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Kruewel, Thomas; Stahnke, Vera C. [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); Zapf, Antonia [University Medical Center Goettingen, Department of Medical Statistics, Goettingen (Germany); Rave-Fraenk, Margret [University Medical Center Goettingen, Department of Radiotherapy and Radiooncology, Goettingen (Germany); Steinmetz, Michael [German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Pediatric Cardiology and Intensive Care Medicine, University Medical Center Goettingen (Germany); Unterberg-Buchwald, Christina [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Cardiology and Pneumology, University Medical Center Goettingen (Germany); Schuster, Andreas [German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Cardiology and Pneumology, University Medical Center Goettingen (Germany)

    2018-03-15

    Magnetic resonance imaging (MRI) is regarded as a non-harming and non-invasive imaging modality with high tissue contrast and almost no side effects. Compared to other cross-sectional imaging modalities, MRI does not use ionising radiation. Recently, however, strong magnetic fields as applied in clinical MRI scanners have been suspected to induce DNA double-strand breaks in human lymphocytes. In this study we investigated the impact of 3-T cardiac MRI examinations on the induction of DNA double-strand breaks in peripheral mononuclear cells by γH2AX staining and flow cytometry analysis. The study cohort consisted of 73 healthy non-smoking volunteers with 36 volunteers undergoing CMRI and 37 controls without intervention. Differences between the two cohorts were analysed by a mixed linear model with repeated measures. Both cohorts showed a significant increase in the γH2AX signal from baseline to post-procedure of 6.7 % (SD 7.18 %) and 7.8 % (SD 6.61 %), respectively. However, the difference between the two groups was not significant. Based on our study, γH2AX flow cytometry shows no evidence that 3-T MRI examinations as used in cardiac scans impair DNA integrity in peripheral mononuclear cells. (orig.)

  14. 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...

  15. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    International Nuclear Information System (INIS)

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-01-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with 125 I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture

  16. Transfer of unstable chromosomal aberrations in human peripheral lymphocytes at cell division and their significance for the aberration frequency

    International Nuclear Information System (INIS)

    Stephan, G.; Chang Tsangpi.

    1986-04-01

    In 48 h cultures, the fraction of human lymphocytes in 2nd mitosis was found to be between 0 and 42.5% (mean value 8.7%). The X-ray exposure from irradiating with 2 Gy resulted in a cell cycle delay which varied from donor to donor. A loss of nearly 50% of dicentric chromosomes and acentric fragments from unstable chromosomes occurred at cell division, while centric rings were not impeded. When dicentric chromosomes, or acentric fragments are found in 2nd mitosis, they show a characteristic differential staining, which means that chromatides at cell division fall free and are replicated in daughter cells. When plotting dose effect curves of dicentric chromosomes, up to 20% of 2nd mitosis fractions have little influence on the aberration rate. This may be additionally verified as part of the 'biological dosimetry' in a person with 24% of 2nd mitosis. When the rates of dicentric chromosomes exclusively evaluated from 1st mitosis after irradiation with 2.0 Gy were related to the donors age, no age-dependent sensitivity to radiation could be observed. Aberration rates which deviate from person to person are comparable to the results achieved by conventional staining methods. (orig./MG) [de

  17. Vaccine-Mediated Mechanisms Controlling Replication of Francisella tularensis in Human Peripheral Blood Mononuclear Cells Using a Co-culture System

    Directory of Open Access Journals (Sweden)

    Kjell Eneslätt

    2018-02-01

    Full Text Available Cell-mediated immunity (CMI is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to

  18. Winter to summer change in vitamin D status reduces systemic inflammation and bioenergetic activity of human peripheral blood mononuclear cells.

    Science.gov (United States)

    Calton, Emily K; Keane, Kevin N; Raizel, Raquel; Rowlands, Jordan; Soares, Mario J; Newsholme, Philip

    2017-08-01

    Vitamin D status [25(OH)D] has recently been reported to be associated with altered cellular bioenergetic profiles of peripheral blood mononuclear cells (PBMCs). No study has tracked the seasonal variation of 25(OH)D and its putative influence on whole body energy metabolism, cellular bioenergetic profiles, inflammatory markers and clinical chemistry. Whole body energy metabolism and substrate utilisation were measured by indirect calorimetry. PBMCs obtained from the same subjects were isolated from whole blood, counted and freshly seeded. Bioenergetic analysis (mitochondrial stress test and glycolysis stress test) was performed using the Seahorse XF e 96 flux analyser. 25(OH)D was assessed using the Architect immunoassay method. 25(OH)D increased by a median (IQR) of 14.40 (20.13)nmol/L (pwinter to summer and was accompanied by significant improvements in indices of insulin sensitivity, McAuley's index (p=0.019) and quantitative insulin sensitivity check index (p=0.028). PBMC mitochondrial parameters basal respiration, non-mitochondrial respiration, ATP production, proton leak, and maximal respiration decreased in summer compared to winter. Similarly, PBMC glycolytic parameters glycolytic activity, glucose response, and glycolytic capacity were all reduced in summer compared to winter. There was also a trend for absolute resting metabolic rate (RMR) to decrease (p=0.066). Markers of systemic inflammation MCP-1, IL-6, IL-8, IL-10, and IL-12p70 decreased significantly in summer compared to winter. Participants who entered winter with a low 25(OH)D (winter 25(OH)D concentrations of 50-75nmol/L or >75nmol/L. The absolute change in 25(OH)D was not associated with altered bioenergetics. Seasonal improvements in 25(OH)D was associated with reduced systemic inflammation, PBMC bioenergetic profiles and whole body energy metabolism. These observational changes in PBMC bioenergetics were most pronounced in those who had insufficient 25(OH)D in winter. The data warrants

  19. Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: an advantageous alternative to ES and iPS cells.

    Science.gov (United States)

    Amoh, Yasuyuki; Kanoh, Maho; Niiyama, Shiro; Hamada, Yuko; Kawahara, Katsumasa; Sato, Yuichi; Hoffman, Robert M; Katsuoka, Kensei

    2009-08-01

    The optimal source of stem cells for regenerative medicine is a major question. Embryonic stem (ES) cells have shown promise for pluripotency but have ethical issues and potential to form teratomas. Pluripotent stem cells have been produced from skin cells by either viral-, plasmid- or transposon-mediated gene transfer. These stem cells have been termed induced pluripotent stem cells or iPS cells. iPS cells may also have malignant potential and are inefficiently produced. Embryonic stem cells may not be suited for individualized therapy, since they can undergo immunologic rejection. To address these fundamental problems, our group is developing hair follicle pluripotent stem (hfPS) cells. Our previous studies have shown that mouse hfPS cells can differentiate to neurons, glial cells in vitro, and other cell types, and can promote nerve and spinal cord regeneration in vivo. hfPS cells are located above the hair follicle bulge in what we have termed the hfPS cell area (hfPSA) and are nestin positive and keratin 15 (K-15) negative. Human hfPS cells can also differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In the present study, human hfPS cells were transplanted in the severed sciatic nerve of the mouse where they differentiated into glial fibrillary-acidic-protein (GFAP)-positive Schwann cells and promoted the recovery of pre-existing axons, leading to nerve generation. The regenerated nerve recovered function and, upon electrical stimulation, contracted the gastrocnemius muscle. The hfPS cells can be readily isolated from the human scalp, thereby providing an accessible, autologous and safe source of stem cells for regenerative medicine that have important advantages over ES or iPS cells. (c) 2009 Wiley-Liss, Inc.

  20. 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.

  1. Determination of Zidovudine Triphosphate Intracellular Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Individuals by Tandem Mass Spectrometry

    Science.gov (United States)

    Font, Eva; Rosario, Osvaldo; Santana, Jorge; García, Hermes; Sommadossi, Jean-Pierre; Rodriguez, Jose F.

    1999-01-01

    Nucleoside reverse transcriptase inhibitors (NRTIs) used against the human immunodeficiency virus (HIV) need to be activated intracellularly to their triphosphate moiety to inhibit HIV replication. Intracellular concentrations of these NRTI triphosphates, especially zidovudine triphosphate (ZDV-TP), are relatively low (low numbers of femtomoles per 106 cells) in HIV-infected patient peripheral blood mononuclear cells. Recently, several methods have used either high-performance liquid chromatography (HPLC) or solid-phase extraction (SPE) coupled with radioimmunoassay to obtain in vivo measurements of ZDV-TP. The limit of detection (LOD) by these methods ranged from 20 to 200 fmol/106 cells. In this report, we describe the development of a method to determine intracellular ZDV-TP concentrations in HIV-infected patients using SPE and HPLC with tandem mass spectrometry for analysis. The LOD by this method is 4.0 fmol/106 cells with a linear concentration range of at least 4 orders of magnitude from 4.0 to 10,000 fmol/106 cells. In hispanic HIV-infected patients, ZDV-TP was detectable even when the sampling time after drug administration was 15 h. Intracellular ZDV-TP concentrations in these patients ranged from 41 to 193 fmol/106 cells. The low LOD obtained with this method will provide the opportunity for further in vivo pharmacokinetic studies of intracellular ZDV-TP in different HIV-infected populations. Furthermore, this methodology could be used to perform simultaneous detection of two or more NRTIs, such as ZDV-TP and lamivudine triphosphate. PMID:10582890

  2. Adenovirus-specific T-cell Subsets in Human Peripheral Blood and After IFN-γ Immunomagnetic Selection.

    Science.gov (United States)

    Qian, Chongsheng; Wang, Yingying; Cai, Huili; Laroye, Caroline; De Carvalho Bittencourt, Marcelo; Clement, Laurence; Stoltz, Jean-François; Decot, Véronique; Reppel, Loïc; Bensoussan, Danièle

    2016-01-01

    Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

  3. 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; Raaby, Ellen Margrethe Nedergaard

    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......-IgE stimulation. Here, the SR profile identified the CD133(+) PBdMC as the most active cells regarding secretion of IL-10, IL-13, GM-CSF, and TNF-α. Cells from all three culture protocols, however, produced IL-10 spontaneously at comparable levels. We recommend validating mast cell cultures by means of molecular...

  4. Protection of ionizing radiation-induced cytogenetic damage by hydroalcoholic extract of Cynodon dactylon in Chinese hamster lung fibroblast cells and human peripheral blood lymphocytes.

    Science.gov (United States)

    Rao, Bola Sadashiva Satish; Upadhya, Dinesh; Adiga, Satish Kumar

    2008-01-01

    The radiomodulatory potential of hydroalcoholic extract of a medicinal plant Cynodon dactylon (family: Poaceae) against radiation-induced cytogenetic damage was analyzed using Chinese hamster lung fibroblast (V79) cells and human peripheral blood lymphocytes (HPBLs) growing in vitro. Induction of micronuclei was used as an index of cytogenetic damage, evaluated in cytokinesis blocked binucleate cells. The hydroalcoholic Cynodon dactylon extract (CDE) rendered protection against the radiation-induced DNA damage, as evidenced by the significant (p<0.001) reduction in micronucleated binucleate cells (MNBNC%) after various doses of CDE treatment in V79 cells and HPBLs. The optimum dose of CDE (40 and 50 microg/ml in HPBLs and V79 cells, respectively) with the greatest reduction in micronuclei was further used in combination with various doses of gamma radiation (0.5, 1, 2, 3, and 4 Gy) exposed 1 h after CDE treatment. A linear dose-dependent MNBNC% increase in radiation alone group was observed, while 40/50 microg/ml CDE significantly resulted in the reduction of MNBNC%, compared to the respective radiation alone groups. CDE resulted in a dose-dependent increase in free radical scavenging ability against various free radicals, viz., 2, 2-diphenyl-2-picryl-hydrazyl (DPPH); 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS); superoxide anion (O2*-); hydroxyl radical (OH*) and nitric oxide radical (NO*) generated in vitro. Also, an excellent (70%) inhibition of lipid peroxidation in vitro was observed at a dose of 300 microg/ml CDE, attaining the saturation point at higher doses. The present findings demonstrated the radioprotective effect of CDE, also rendering protection against radiation-induced genomic instability and DNA damage. The observed radioprotective effect may be partly attributed to the free radical scavenging and antilipid peroxidative potential of CDE.

  5. Mast cell degranulation breaks peripheral tolerance.

    Science.gov (United States)

    de Vries, V C; Wasiuk, A; Bennett, K A; Benson, M J; Elgueta, R; Waldschmidt, T J; Noelle, R J

    2009-10-01

    Mast cells (MC) have been shown to mediate regulatory T-cell (T(reg))-dependent, peripheral allograft tolerance in both skin and cardiac transplants. Furthermore, T(reg) have been implicated in mitigating IgE-mediated MC degranulation, establishing a dynamic, reciprocal relationship between MC and T(reg) in controlling inflammation. In an allograft tolerance model, it is now shown that intragraft or systemic MC degranulation results in the transient loss of T(reg) suppressor activities with the acute, T-cell dependent rejection of established, tolerant allografts. Upon degranulation, MC mediators can be found in the skin, T(reg) rapidly leave the graft, MC accumulate in the regional lymph node and the T(reg) are impaired in the expression of suppressor molecules. Such a dramatic reversal of T(reg) function and tissue distribution by MC degranulation underscores how allergy may causes the transient breakdown of peripheral tolerance and episodes of acute T-cell inflammation.

  6. 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.

  7. 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

  8. 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.

  9. Expression of human immunodeficiency virus (HIV) in naturally infected peripheral blood mononuclear cells: comparison of a standard co-culture technique with a newly developed microculture method.

    Science.gov (United States)

    Eberlein, B; Baur, A; Neundorfer, M; Jahn, G

    1991-05-01

    Peripheral blood mononuclear cells (PBMCs) from 29 patients infected with human immunodeficiency virus (HIV) were cultured by two different methods. One was the standard co-culture technique, the other a newly developed microculture method. In this assay 10(6) PBMCs were cultivated in 250 microliters medium, no activating agents or allogeneic cells were present. P24 antigen production measured by this method was found in 7 out of 11 PBMC cultures of patients in the Walter Reed (WR) stage 1 or 2, whereas only 4 samples were positive by the co-culture procedure. Cultures from patients in the later stages of the disease (WR 5/6) showed a higher p24 production by the co-culture method than by the microculture assay. It is assumed that rapidly growing HIV strains can be better assessed by the co-culture method which may select for these strains. P24 expression can be more easily obtained by the microculture technique even in cases where slowly replicating strains may be present. In conclusion, results from the microculture procedure described may be a useful supplementation to findings observed by the co-culture method.

  10. Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from X-irradiated human peripheral blood hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

    2009-01-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34 + hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34 + cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34 + cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34 + cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34 + cells. (author)

  11. Characterization of glucocerebrosidase in peripheral blood cells and cultured blastoid cells

    NARCIS (Netherlands)

    Aerts, J. M.; Heikoop, J.; van Weely, S.; Donker-Koopman, W. E.; Barranger, J. A.; Tager, J. M.; Schram, A. W.

    1988-01-01

    We have characterized glucocerebrosidase in various cell types of peripheral blood of control subjects and in cultured human blastoid cells. The intracellular level of glucocerebrosidase in cultured blastoid cells (10-30 nmol substrate hydrolyzed/h.mg protein) resembles closely values observed for

  12. Multielemental fractionation in human peripheral blood mononuclear cells by size exclusion liquid chromatography coupled to UV and ICP-MS detection.

    Science.gov (United States)

    Alvarado, Gladys; Murillo, Miguel

    2010-10-01

    An analytical methodology is presented in this work to determine metal-biomolecule complexes size distribution patterns of several elements, among different compounds present in human peripheral blood mononuclear cells (PBMC). A hyphenated technique based on size exclusion chromatography (SEC) coupled online to UV and inductively coupled plasma mass spectrometry (ICP-MS) detection is used. Two different SEC columns with separation ranges between 1,500-1,000,000 relative molecular mass (M(r)) (Nanofilm SEC-250) and 5,000 and 100,000 relative molecular mass (M(r)) (TSK-Gel G2000 SW) are used with 10 mmol/L tris-HCl at pH 7.3 as mobile phase. Retention behavior (retention time and peak-area ratios) remained unchanged for several successive separations. Metal-containing compounds are found to a wide range of M(r). Copper-zinc superoxide dismutase, copper and zinc metallothionein, and copper and zinc transferrin are identified in PBMC samples. A high M(r) (147,000) metal-binding protein containing copper and zinc and a high M(r) (107,000) manganese-binding protein were also found; however, these remained unknown.

  13. 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.

  14. 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 trafficking, promote early response, mediating C-myc related proliferation, promote antiapoptotic activity and protects

  15. Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene

    Directory of Open Access Journals (Sweden)

    Viviana Meraviglia

    2018-03-01

    Full Text Available Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3, encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies.Resource tableImage 1Unique stem cell lines identifierEURACi001-AEURACi002-AEURACi003-AAlternative names of stem cell linesB2CAV3 (EURACi001-AL1CAV3 (EURACi002-AN1CAV3 (EURACi003-AInstitutionInstitute for Biomedicine, Eurac ResearchContact information of distributorAlessandra Rossini (alessandra.rossini@eurac.eduType of cell linesiPSCsOriginHumanCell sourcePeripheral blood mononuclear cells (PBMCsMethod of reprogrammingElectroporation of episomal vectors (pCXLE hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hULMultiline rationaleNon-isogenic cell lines obtained from patients with mutations in the same gene (CAV3Gene modificationNOType of modificationSpontaneous mutationsAssociated diseaseCaveolinopathiesGene/locusHeterozygous CAV3 c.Δ184–192 (EURACi001-AHeterozygous CAV3 c.303 TGG > TGC (EURACi002-AHeterozygous CAV3 c.233 ACG > AAG (EURACi003-AMethod of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock dateJanuary 2016 (EURACi001-ASeptember 2016 (EURACi002-AMay 2016 (EURACi003-ACell line repository/bankN/AEthical approvalPeripheral blood was collected from patients after signing the informed consent provided by Cell Line and DNA Biobank from Patients Affected by Genetic Diseases, member of the

  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.

    Science.gov (United States)

    Cifre, Margalida; Díaz-Rúa, Rubén; Varela-Calviño, Rubén; Reynés, Bàrbara; Pericás-Beltrán, Jordi; Palou, Andreu; Oliver, Paula

    2017-04-01

    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. 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. 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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Aberrant phenotypes in peripheral T cell lymphomas.

    Science.gov (United States)

    Hastrup, N; Ralfkiaer, E; Pallesen, G

    1989-01-01

    Seventy six peripheral T cell lymphomas were examined immunohistologically to test their reactivity with a panel of monoclonal antibodies against 11 T cell associated antigens (CD1-8, CD27, UCHL1, and the T cell antigen receptor). Sixty two (82%) lymphomas showed aberrant phenotypes, and four main categories were distinguished as follows: (i) lack of one or several pan-T cell antigens (49, 64% of the cases); (ii) loss of both the CD4 and CD8 antigens (11, 15% of the cases); (iii) coexpression of the CD4 and CD8 antigens (13, 17% of the cases); and (iv) expression of the CD1 antigen (eight, 11% of the cases). No correlation was seen between the occurrence of aberrant phenotypes and the histological subtype. It is concluded that the demonstration of an aberrant phenotype is a valuable supplement to histological assessment in the diagnosis of peripheral T cell lymphomas. It is recommended that the panel of monoclonal antibodies against T cell differentiation antigens should be fairly large, as apparently any antigen may be lost in the process of malignant transformation. Images Figure PMID:2469701

  18. Generation of human induced pluripotent stem cells (EURACi001-A, EURACi002-A, EURACi003-A) from peripheral blood mononuclear cells of three patients carrying mutations in the CAV3 gene.

    Science.gov (United States)

    Meraviglia, Viviana; Benzoni, Patrizia; Landi, Sara; Murano, Carmen; Langione, Marianna; Motta, Benedetta M; Baratto, Serena; Silipigni, Rosamaria; Di Segni, Marina; Pramstaller, Peter P; DiFrancesco, Dario; Gazzerro, Elisabetta; Barbuti, Andrea; Rossini, Alessandra

    2018-03-01

    Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Peripheral T cell lymphoma: Not otherwise specified

    Directory of Open Access Journals (Sweden)

    Anusha H Pai

    2015-01-01

    Full Text Available Peripheral T cell lymphoma (PTCL is a heterogeneous group of hematological tumors originating from mature T cells, which constitutes less than 15% of all non-Hodgkins lymphomas in adults. Primary cutaneous PTCL-not otherwise specified (NOS represent a subgroup of PTCLs with no consistent immunophenotypic, genetic or clinical features. PTCL-NOS frequently has an aggressive course with a tendency for systemic involvement, however, a well-defined therapeutic and prognostic approach has not been outlined yet. We report a case of PTCL-NOS with multiple cutaneous lesions in a young adult male with an emphasis on the treatment modality used.

  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

    Czech Academy of Sciences Publication Activity Database

    Himlová, L.; Kubies, Dana; Hulejová, H.; Bartová, J.; Riedel, Tomáš; Štikarová, J.; Suttnar, J.; Pešáková, V.

    2016-01-01

    Roč. 2016, č. 2016 (2016), 8769347_1-8769347_15 ISSN 0962-9351 R&D Projects: GA MZd(CZ) NT13297; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 Keywords : peripheral blood mononuclear cells * cytokine * osteoblasts Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.232, year: 2016

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

    Directory of Open Access Journals (Sweden)

    Leni Moldovan

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

  2. The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation.

    Science.gov (United States)

    Delpino, Andrea; Castelli, Mauro

    2002-01-01

    In human rabdomiosarcoma cells (TE671/RD) chronic exposure to 500 nM thapsigargin (a powerful inhibitor of the endoplasmic reticulum Ca2+-ATPases) resulted in the induction of the stress protein GRP78/BIP. Making use of the surface biotinylation method, followed by the isolation of the GRP78 using ATP-agarose affinity chromatography, it was found that a fraction of the thapsigargin-induced GRP78 is expressed on the cell surface. The presence of GRP78 on the membrane of thapsigargin-treated cells was confirmed by fractionation of cell lysates into a soluble and a membrane fraction, followed by Western blot analysis with an anti-GRP78 antibody. It was also found that conspicuous amounts of GRP78 are present in the culture medium collected from thapsigargin-treated cultures. This extracellular GRP78 originates mostly by an active release from intact cells and does not result solely from the leakage of proteins from dead cells. Moreover, small amounts of circulating, free GRP78 and naturally-occurring anti-GRP78 autoantibodies were detected in the peripheral circulation of healthy human individuals.

  3. Establishment of human iPSC line NCCSi003-A from CD34+cells of peripheral blood collected during apheresis of healthy donor from Indian ethnicity

    Directory of Open Access Journals (Sweden)

    Sophia Fernandes

    2018-03-01

    Full Text Available We present generation of iPSCs from CD34+ cells isolated from peripheral blood, collected during apheresis of a healthy female individual. We nucleofected the CD34+cells by episomal vectors containing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (dominant negative mutation in p53. The resultant colonies showed cobble-stone appearance and stained positive for alkaline phosphatase. The colonies demonstrated presence of pluripotency markers by immunofluorescence, flow-cytometry and PCR. The plasmids were lost from cells subsequently during passages as assessed by PCR. Karyotype analysis demonstrated a stable genome. The cells had capability to differentiate to cells from all three-germ lineages in vitro.

  4. HIV-1 isolation from infected peripheral blood mononuclear cells.

    Science.gov (United States)

    Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A; Schuitemaker, Hanneke; Scarlatti, Gabriella

    2014-01-01

    Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.

  5. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. 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.

  7. Analyses of 123 Peripheral Human Immune Cell Subsets: Defining Differences with Age and between Healthy Donors and Cancer Patients not Detected in Analysis of Standard Immune Cell Types

    Directory of Open Access Journals (Sweden)

    Lauren M. Lepone

    2016-03-01

    suppressor cells, conventional dendritic cells (DCs, plasmacytoid DCs and B cells. The use of these panels identifying 123 immune cell subsets may aid in the identi‐ fication of patients who may benefit from immunotherapy, either prior to therapy or early in the immunotherapeutic regimen, for the treatment of cancer or other chronic or infectious diseases.

  8. TXNIP regulates peripheral glucose metabolism in humans

    DEFF Research Database (Denmark)

    Parikh, Hemang; Carlsson, Emma; Chutkow, William A

    2007-01-01

    combined human insulin/glucose clamp physiological studies with genome-wide expression profiling to identify thioredoxin interacting protein (TXNIP) as a gene whose expression is powerfully suppressed by insulin yet stimulated by glucose. In healthy individuals, its expression was inversely correlated...... expression is consistently elevated in the muscle of prediabetics and diabetics, although in a panel of 4,450 Scandinavian individuals, we found no evidence for association between common genetic variation in the TXNIP gene and T2DM. CONCLUSIONS: TXNIP regulates both insulin-dependent and insulin......-independent pathways of glucose uptake in human skeletal muscle. Combined with recent studies that have implicated TXNIP in pancreatic beta-cell glucose toxicity, our data suggest that TXNIP might play a key role in defective glucose homeostasis preceding overt T2DM....

  9. A Modified Ficoll-Paque Gradient Method for Isolating Mononuclear Cells from the Peripheral and Umbilical Cord Blood of Humans for Biobanks and Clinical Laboratories.

    Science.gov (United States)

    Jia, Yanjuan; Xu, Hui; Li, Yonghong; Wei, Chaojun; Guo, Rui; Wang, Fang; Wu, Yu; Liu, Jing; Jia, Jing; Yan, Junwen; Qi, Xiaoming; Li, Yuanting; Gao, Xiaoling

    2018-04-01

    Although the Ficoll-Paque method is classically used to isolate peripheral blood mononuclear cells (PBMCs), modifications in this method are required for a more rapid and economic output for biobanks and clinical laboratories, particularly in developing countries. In this study, we addressed this issue by modifying the Ficoll-Paque method for the isolation of PBMCs or mononuclear cells from the peripheral and the umbilical cord blood of healthy and diseased (infected, anemic, and chronic obstructive pulmonary disease) adult individuals. In the modified method, we initiated the cell isolation process from the buffy coat layer, which appears in the interface between the plasma and sediments after centrifugation, instead of using the whole blood as described in the classic method. Although the PBMC yield by the modified method was about 12% less than in the classic method, the number of PBMCs isolated by the modified method was more than one million, which is enough for different research/diagnostic purposes, such as multi-omics detection. Assessment of cell viability and purity by hematology analyzer and trypan blue showed no significant difference between the viability and purity of the PBMCs isolated by these two methods in almost all groups, except samples from the infected and cord blood groups, where lower PBMC purity with higher granulocyte contamination were observed. In addition, at delayed processing time points, all parameters for the two methods were decreased in a time-dependent manner, especially at 8, 12, or 24 hours after the sample collection. In summary, the performance of PBMC isolation by the classic and modified methods mainly relies on the PBMC ratio in original samples. The modified method could be preferred for PBMC isolation because of its time and cost savings, especially for the biobanks and clinical laboratories in developing countries.

  10. Therapeutic options in peripheral T cell lymphoma

    Directory of Open Access Journals (Sweden)

    Yaping Zhang

    2016-04-01

    Full Text Available Abstract Peripheral T cell lymphoma (PTCL is a rare and heterogeneous group of non-Hodgkin lymphomas with a very poor prognosis. The standard first-line treatments have resulted in unsatisfactory patient outcomes. With the exception of low-risk anaplastic lymphoma kinase (ALK-positive anaplastic large cell lymphoma (ALCL, the majority of patients relapse rapidly; the current 5-year overall survival rates are only 10–30 %. Novel targeted therapies and combination chemotherapies are required for the treatment of patients with PTCL. In recent years, some retrospective and prospective studies have been performed concerning PTCL. Consequently, a number of novel agents and their relevant combination therapies have been identified, including histone deacetylase inhibitors, immunoconjugates, antifolates, monoclonal antibodies, immunomodulatory agents, nucleoside analogs, proteasome inhibitors, kinase inhibitors, bendamustine, l-asparaginase, and other targeted agents. It is hoped that these innovative approaches will finally improve outcomes in patients with PTCL. This review summarizes the currently available approaches for the treatment of PTCL with an emphasis on potential new agents, including the role of stem cell transplantation.

  11. Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Frémond, Marie-Louise; Uggenti, Carolina; Van Eyck, Lien; Melki, Isabelle; Bondet, Vincent; Kitabayashi, Naoki; Hertel, Christina; Hayday, Adrian; Neven, Bénédicte; Rose, Yoann; Duffy, Darragh; Crow, Yanick J; Rodero, Mathieu P

    2017-07-01

    Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNβ reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNβ promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies. © 2017, American College of Rheumatology.

  12. 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. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Giovanni Nardo

    Full Text Available 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. (paper)

  15. Rag Deletion in Peripheral T Cells Blocks TCR Revision

    Science.gov (United States)

    Hale, J. Scott; Ames, Kristina T.; Boursalian, Tamar E.; Fink, Pamela J.

    2010-01-01

    Mature CD4+Vβ5+ T cells that recognize a peripherally expressed endogenous superantigen are tolerized either by deletion or T cell receptor (TCR) revision. In Vβ5 transgenic mice, this latter tolerance pathway results in the appearance of CD4+Vβ5−TCRβ+ T cells, coinciding with Rag1, Rag2, and TdT expression and the accumulation of Vβ-DJβ recombination intermediates in peripheral CD4+ T cells. Because post-thymic RAG-dependent TCR rearrangement has remained controversial, we sought to definitively determine whether TCR revision is an extrathymic process that occurs in mature peripheral T cells. We now show that Rag deletion in post-positive selection T cells in Vβ5 transgenic mice blocks TCR revision in vivo, and that mature peripheral T cells sorted to remove cells bearing endogenous TCRβ chains can express newly generated TCRβ molecules in adoptive hosts. These findings unambiguously demonstrate post-thymic, RAG-dependent TCR rearrangement and define TCR revision as a tolerance pathway that targets mature peripheral CD4+ T cells. PMID:20435935

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-15

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

  17. 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.

  18. 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...... 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...... to exhaustion depend mostly on the relative intensity of exercise and are limb-specific....

  19. Mouse forward genetics in the study of the peripheral nervous system and human peripheral neuropathy

    Science.gov (United States)

    Douglas, Darlene S.; Popko, Brian

    2009-01-01

    Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics. Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as a human disease gene causing multiple forms of peripheral neuropathy, to the more recent identification of the claw paw and sprawling mutations, forward genetics has long been a tool for probing the physiology, pathogenesis, and genetics of the PNS. Even as spontaneous and mutagenized mice continue to enable the identification of novel genes, provide allelic series for detailed functional studies, and generate models useful for clinical research, new methods, such as the piggyBac transposon, are being developed to further harness the power of forward genetics. PMID:18481175

  20. 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.

  1. Peripheral T cell lymphoma: clinical utility of romidepsin

    Directory of Open Access Journals (Sweden)

    Sawey K

    2012-06-01

    Full Text Available Jasmine Zain, Kathryn SaweyNYU Langone Medical Center, New York, USAIntroduction: Direct therapeutic targets, such as aberrant tumor cell genes and tumor cell markers, have been the focus of cancer treatment for more than 50 years. The resulting damage to normal cells and emergence of drug-resistant tumor cells after exposure to conventional chemotherapy have led researchers to study indirect targets, like the tumor vasculature. A more recent indirect approach involves targeting the epigenetic modifiers, DNA methyltransferase and histone deacetylase. Histone deacetylase inhibitors have been shown to be active cytotoxic agents in T cell lymphoma. The current treatments approved by the US Food and Drug Administration for relapsed cutaneous T cell lymphoma are vorinostat and romidepsin. The diversity and rarity of peripheral T cell lymphomas present a challenge for effective treatment. With their poor overall survival rate, new targeted therapies need to be developed.Keywords: peripheral T cell lymphoma, treatment, romidepsin

  2. 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

  3. 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...

  4. 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 O 2 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.

  5. 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...... simultaneously. The dominant marker of these E- Fc- cells was surface Ig, and during 4 days of culture this population did not alter its surface markers. Subset 2 was obtained in two ways following rosette centrifugation with AET-treated SRBC and rabbit anti-human Ig-coated autologous RBC. This 'Null cell...

  6. 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.

  7. Explant culture of human peripheral lung. I. Metabolism of benzo[alpha]pyrene

    DEFF Research Database (Denmark)

    Stoner, G.D.; Harris, C.C.; Autrup, Herman

    1978-01-01

    the predominant alveolar epithelial cell type. Lamellar inclusion bodies were released from the type 2 cells and accumulated in the alveolar spaces. The metabolism of benzo[alpha]pyrene (BP) in human lung explants cultured for up to 7 days was investigated. Human lung explants had measurable aryl hydrocarbon......Human lung explants have been maintained in vitro for a period of 25 days. Autoradiographic studies indicated that the broncholar epithelial cells, type 2 alveolar epithelial cells, and stromal fibroblasts incorporated 3H-thymidine during the culture. After 7 to 10 days, type 2 cells were...... hydroxylase activity and could metabolize BP into forms that were bound to cellular DNA and protein. Peripheral lung had significantly lower aryl hydrocarbon hydroxylase activity than cultured bronchus but both tissues had similar binding levels of BP to DNA. Radioautographic studies indicated that all cell...

  8. Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

    Science.gov (United States)

    Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe

    2011-04-01

    Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  9. 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.

  10. Peripheral Glial Cells in the Development of Diabetic Neuropathy

    Science.gov (United States)

    Gonçalves, Nádia Pereira; Vægter, Christian Bjerggaard; Pallesen, Lone Tjener

    2018-01-01

    The global prevalence of diabetes is rapidly increasing, affecting more than half a billion individuals within the next few years. As diabetes negatively affects several physiological systems, this dramatic increase represents not only impaired quality of life on the individual level but also a huge socioeconomic challenge. One of the physiological consequences affecting up to half of diabetic patients is the progressive deterioration of the peripheral nervous system, resulting in spontaneous pain and eventually loss of sensory function, motor weakness, and organ dysfunctions. Despite intense research on the consequences of hyperglycemia on nerve functions, the biological mechanisms underlying diabetic neuropathy are still largely unknown, and treatment options lacking. Research has mainly focused directly on the neuronal component, presumably from the perspective that this is the functional signal-transmitting unit of the nerve. However, it is noteworthy that each single peripheral sensory neuron is intimately associated with numerous glial cells; the neuronal soma is completely enclosed by satellite glial cells and the length of the longest axons covered by at least 1,000 Schwann cells. The glial cells are vital for the neuron, but very little is still known about these cells in general and especially how they respond to diabetes in terms of altered neuronal support. We will discuss current knowledge of peripheral glial cells and argue that increased research in these cells is imperative for a better understanding of the mechanisms underlying diabetic neuropathy. PMID:29770116

  11. 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.

  12. A simple method for human peripheral blood monocyte Isolation

    Directory of Open Access Journals (Sweden)

    Marcos C de Almeida

    2000-04-01

    Full Text Available We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

  13. The Radiation Effect on Peripheral Blood Cell

    International Nuclear Information System (INIS)

    Lee, Tae June; Kwon, Hyoung Cheol; Kim, Jung Soo; Im, Sun Kyun; Choi, Ki Chul

    1988-01-01

    To evaluate radiation effect on the hematopoietic system, we analyzed 44 patients who were treated with conventionally fractionated radiation therapy (RT) at Chonbuk National University Hospital. According to the treatment sites, we classified them into three groups: group I as head and neck, group II as thorax, and group III as pelvis. White blood cell, lymphocyte, platelet and hemoglobin were checked before and during RT The results were as follow; 1. White blood cell (WBC) and lymphocyte count were declined from the first week of RT to the third week, and then slightly recovered after the third or fourth week. There was prominent decrease in lymphocyte counts than WBC. 2. Platelet counts were declined until the second week of the RT, showed slight recovery at fourth week in all groups. Hemoglobin values were slightly decreased in the first week and then recovered the level of pretreatment value, gradually. 3. Lymphocyte count were declined significantly on group III(p<0.01), WBC and platelet counts were decreased on group II but statistically not significant

  14. 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

  15. 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. © 2015 Wiley Periodicals, Inc.

  16. Endurance exercise training increases peripheral vascular response in human fingers.

    Science.gov (United States)

    Katayama, K; Shimoda, M; Maeda, J; Takemiya, T

    1998-10-01

    The purpose of this study was to clarify whether peripheral vascular response to alteration of transmural pressure is changed by endurance exercise training. The healthy male subjects (training group; n = 6) performed endurance exercise training that consisted of cycle ergometer exercise 5 d.week-1 and 30 min.d-1 for a period of 8 weeks. Changes in the peripheral vascular response to alteration of transmural pressure in the human finger were measured by a differential digital photoplethysmogram (DeltaDPG) and blood pressure during passive movement of the arm to different vertical hand positions relative to heart level. Following 8 weeks of endurance training, percent changes in DeltaDPG from heart level in the training group increased significantly (mean +/- SD, -48.1 +/- 7. 3 to -58.7 +/- 9.3% at the lowered position, 46.1 +/- 13.4 to 84.6 +/- 8.8% at the elevated position, ppressure, also significantly changed in the training group over the 8 weeks (5.6 +/- 1.3 to 2.7 +/- 1.6 mV. V-1.s-1.mmHg-1 at the lowered position, 30.0 +/- 12.4 to 54.4 +/- 18. 9 mV.V-1.s-1.mmHg-1 at the elevated position ). Maximal oxygen uptake (V.O2 max) was significantly increased in the training group. On the other hand, the control group (n = 6) showed no significant changes in all parameters for 8 weeks. Therefore these results suggest that endurance exercise training induces an increase in peripheral vascular response to alteration of transmural pressure in the human finger.

  17. Isolation and partial characterization of peripheral blood CD4+ T cell clones expressing γδT cell receptors

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; Akiyama, Mitoshi; Hirai, Yuko; Kusunoki, Yoichiro.

    1990-06-01

    Rare T cell clones bearing both CD4 and T cell receptors (TCRγ and TCRδ) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCRδ1 antibodies. All the clones established were reactive with anti-TCRγδ1 antibody, whereas only about 20 % of the clones showed reactivity with anti-δTCS1 antibody. Unlike CD4 + T cells bearing TCRαβ, all the clones tested were lectin-dependent and showed CD3 antibody-redirected cytolytic activity. About 60 % exhibited natural killer cell-like activity. Immunoprecipitation analysis of TCRγδ showed that each clone expressed either a disulfide-linked or nondisulfide-linked heterodimer consisting of 37-44 kilodalton TCRγ and TCRδ chains. Southern blot analyses of TCRγ and TCRδ genes revealed some identical rearrangement patterns, suggesting the limited heterogeneity of CD4 + TCRγδ + T cells in peripheral blood. (author)

  18. 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...... skeletal muscle, cutaneous and subcutaneous tissues of the limbs indicate that the situation is more complex. Measurements have been carried out during acute as well as chronic sympathetic denervation. Spinal sympathetic reflex mechanisms have been evaluated in tetraplegic patients, where supraspinal...

  19. Synthesis of Multivalent Glycoconjugates Containing the Immunoactive LELTE Peptide: Effect of Glycosylation on Cellular Activation and Natural Killing by Human Peripheral Blood Mononuclear Cells

    Czech Academy of Sciences Publication Activity Database

    Renaudet, O.; Křenek, Karel; Bossu, I.; Dumy, P.; Kádek, A.; Adámek, David; Vaněk, O.; Kavan, Daniel; Gažák, Radek; Šulc, Miroslav; Bezouška, K.; Křen, Vladimír

    2010-01-01

    Roč. 132, č. 19 (2010), s. 6800-6808 ISSN 0002-7863 R&D Projects: GA MŠk(CZ) LC06010; GA MŠk 1M0505; GA AV ČR IAA400200503; GA ČR GA303/09/0477; GA ČR GD305/09/H008 Institutional research plan: CEZ:AV0Z50200510 Keywords : KILLER-CELLS * TN ANTIGEN * RECEPTOR Subject RIV: EE - Microbiology, Virology Impact factor: 9.019, year: 2010

  20. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Junk, Michael; Salzwedel, Judy; Sindlinger, Thilo; Bürkle, Alexander; Moreno-Villanueva, Maria

    2014-01-01

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  1. Amyotrophic Lateral Sclerosis Multiprotein Biomarkers in Peripheral Blood Mononuclear Cells

    OpenAIRE

    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

    2011-01-01

    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...

  2. 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.

  3. 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.

  4. Effect of Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L. on Th1- and Th2-cytokine responses and human leukocyte antigen-DR expression in peripheral blood mononuclear cells of septic patients.

    Science.gov (United States)

    Wu, Huang-Pin; Lin, Yin-Ku

    2018-05-10

    Many traditional Chinese medicines (TCM), such as Eucommia ulmoides Oliv., Gynostemma pentaphyllum (Thunb.) Makino, and Curcuma longa L., have been reported to have various immune-modulatory effects. To determine the effects of extracts from these three TCM on type 1 T help (Th1)- and Th2-cytokine responses and human leukocyte antigen (HLA)-DR expression in peripheral blood mononuclear cells (PBMCs) obtained from septic patients. Lipopolysaccharide (LPS)-stimulated PBMCs of healthy controls and septic patients were cultured for 48 hs with or without 0.05/0.1 mg/ml of TCM extract. HLA-DR expression in monocytes was detected using flow cytofluorimetry. The interferon [IFN]-γ, tumor necrosis factor [TNF]-α, interleukin (IL)- 2, IL-5, IL-10, and IL-13 levels in supernatants were measured with a human enzyme-linked immunosorbent assay. Treatment with either 0.05 or 0.1 mg/ml of C. longa L. extract significantly restored the percentage of HLA-DR-positive monocytes, which was decreased by LPS in control and patient groups. Treatment with 0.05 or 0.1 mg/ml E. ulmoides Oliv. and C.longa L. extract decreased IL-10 production from LPS-stimulated PBMCs of controls and patients. In patients with sepsis, C. longa L. extract decreased IL-10 production to a greater degree than did E. ulmoides Oliv extract. Although IFN-γ, TNF-α, or IL-13 productions from LPS-stimulated PBMCs were influenced by E. ulmoides Oliv., G. pentaphyllum (Thunb.) Makino, or C. longa L. in control or sepsis groups in this study, only the influence of IL-10 was consistent in both control and sepsis groups. By enhancing monocyte HLA-DR expression and decreasing IL-10 production, C. longa L. might help restore inflammatory responses in septic patients to eradicate pathogens. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Pegylated interferons Lambda-1a and alfa-2a display different gene induction and cytokine and chemokine release profiles in whole blood, human hepatocytes and peripheral blood mononuclear cells.

    Science.gov (United States)

    Freeman, J; Baglino, S; Friborg, J; Kraft, Z; Gray, T; Hill, M; McPhee, F; Hillson, J; Lopez-Talavera, J C; Wind-Rotolo, M

    2014-06-01

    Pegylated interferon-lambda-1a (Lambda), a type III interferon (IFN) in clinical development for the treatment of chronic HCV infection, has shown comparable efficacy and an improved safety profile to a regimen based on pegylated IFN alfa-2a (alfa). To establish a mechanistic context for this improved profile, we investigated the ex vivo effects of Lambda and alfa on cytokine and chemokine release, and on expression of IFN-stimulated genes (ISGs) in primary human hepatocytes and peripheral blood mononuclear cells (PBMCs) from healthy subjects. Our findings were further compared with changes observed in blood analysed from HCV-infected patients treated with Lambda or alfa in clinical studies. mRNA transcript and protein expression of the IFN-λ-limiting receptor subunit was lower compared with IFN-α receptor subunits in all cell types. Upon stimulation, alfa and Lambda induced ISG expression in hepatocytes and PBMCs, although in PBMCs Lambda-induced ISG expression was modest. Furthermore, alfa and Lambda induced release of cytokines and chemokines from hepatocytes and PBMCs, although differences in their kinetics of induction were observed. In HCV-infected patients, alfa treatment induced ISG expression in whole blood after single and repeat dosing. Lambda treatment induced modest ISG expression after single dosing and showed no induction after repeat dosing. Alfa and Lambda treatment increased IP-10, iTAC, IL-6, MCP-1 and MIP-1β levels in serum, with alfa inducing higher levels of all mediators compared with Lambda. Overall, ex vivo and in vivo induction profiles reported in this analysis strongly correlate with clinical observations of fewer related adverse events for Lambda vs those typically associated with alfa. © 2014 John Wiley & Sons Ltd.

  6. Biodistribution of Liver-Derived Mesenchymal Stem Cells After Peripheral Injection in a Hemophilia A Patient.

    Science.gov (United States)

    Sokal, Etienne M; Lombard, Catherine Anne; Roelants, Véronique; Najimi, Mustapha; Varma, Sharat; Sargiacomo, Camillo; Ravau, Joachim; Mazza, Giuseppe; Jamar, François; Versavau, Julia; Jacobs, Vanessa; Jacquemin, Marc; Eeckhoudt, Stéphane; Lambert, Catherine; Stéphenne, Xavier; Smets, Françoise; Hermans, Cédric

    2017-08-01

    With the exception of liver transplantation, there is no cure for hemophilia, which is currently managed by preemptive replacement therapy. Liver-derived stem cells are in clinical development for inborn and acquired liver diseases and could represent a curative treatment for hemophilia A. The liver is a major factor VIII (FVIII) synthesis site, and mesenchymal stem cells have been shown to control joint bleeding in animal models of hemophilia. Adult-derived human liver stem cells (ADHLSCs) have mesenchymal characteristics and have been shown able to engraft in and repopulate both animal and human livers. Thus, the objectives were to evaluate the potency of ADHLSCs to control bleeding in a hemophilia A patient and assess the biodistribution of the cells after intravenous injection. A patient suffering from hemophilia A was injected with repeated doses of ADHLSCs via a peripheral vein (35 million In-oxine-labeled cells, followed by 125 million cells the next day, and 3 infusions of 250 million cells every 2 weeks thereafter; total infusion period, 50 days). After cell therapy, we found a temporary (15 weeks) decrease in the patient's FVIII requirements and severe bleeding complications, despite a lack of increase in circulating FVIII. The cells were safely administered to the patient via a peripheral vein. Biodistribution analysis revealed an initial temporary entrapment of the cells in the lungs, followed by homing to the liver and to a joint afflicted with hemarthrosis. These results suggest the potential use of ADHLSCs in the treatment of hemophilia A.

  7. An Assessment of the Effects of Different Dose Levels of Gamma Rays on HPRT Gene of T-Cells from Human Peripheral Blood

    International Nuclear Information System (INIS)

    Bahreyni, M. T.; Rezaee, M.

    2004-01-01

    Ionizing radiation has been shown to produce a broad range of genetic aberrations in human and other species. Most of the genetic aberrations are deletions. To study genetic alterations, an assessment of somatic ell gene mutations induced by ionizing radiation is proper method. In this study, the intragenic and total gene deletions of 18 HPRT-mutants derived from T-lymphocytes and induced by gamma rays were analyzed. PCR amplification of individual HPRT exons and multiplex PCR. HPRT-mutants were isolated by treatment of irradiated samples with 6-thioguanine. MPCR and PCR of individual exons of HPRT demonstrated that the intragenic and total gene deletions were not significantly different. The samples including more than one deletion had non-random significantly higher frequency. Mapping of all intragenic deleltion exhibited a nonrandom distribution. Middle part of HPRT gene was more sensitive to gamma rays. The sensitivity was increased with radiation dose. This study showed that the size of deletions are dose dependent. Our results suggest that alterations in T- lymphocytes mutant genes, induced deletions, size of deletions and distribution of DNA breakpoints appeared to be dependent on low LET radiation dose. (Author) 11 refs

  8. Antigenicity of Leishmania-Activated C-Kinase Antigen (LACK in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters

    Directory of Open Access Journals (Sweden)

    Laura Fernández

    2018-04-01

    Full Text Available Leishmania-activated C-kinase antigen (LACK is a highly conserved protein among Leishmania species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65 expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL. Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to Leishmania infantum (L. infantum or induce protection in L. infantum-infected hamsters [visceral leishmaniasis (VL model], has not yet been analyzed. The present work examines the ex vivo immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an L. infantum area. It also evaluates the vaccine potential of LACK against L. infantum infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response. Further, 78% of PBMC samples that responded to soluble Leishmania antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of Leishmania DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with L. infantum, and the parasite protective effect of prime/boost vaccination in hamsters with DNA

  9. Effect of drinking water disinfection by-products in human peripheral blood lymphocytes and sperm.

    Science.gov (United States)

    Ali, Aftab; Kurzawa-Zegota, Malgorzata; Najafzadeh, Mojgan; Gopalan, Rajendran C; Plewa, Michael J; Anderson, Diana

    2014-12-01

    Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses. Copyright © 2014. Published by Elsevier B.V.

  10. Peripheral vasodilatation determines cardiac output in exercising humans

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...... arterial ATP infusion at rest. Exercise and ATP infusion increased cardiac output, leg blood flow and vascular conductance (P heart rate by up to 54 beats min(−1), cardiac output did not change in any of the three...... demonstrate that the elevated cardiac output during steady-state exercise is regulated by the increase in skeletal muscle blood flow and venous return to the heart, whereas the increase in heart rate appears to be secondary to the regulation of cardiac output....

  11. Rapid resetting of human peripheral clocks by phototherapy during simulated night shift work.

    Science.gov (United States)

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

    2017-11-24

    A majority of night shift workers have their circadian rhythms misaligned to their atypical schedule. While bright light exposure at night is known to reset the human central circadian clock, the behavior of peripheral clocks under conditions of shift work is more elusive. The aim of the present study was to quantify the resetting effects of bright light exposure on both central (plasma cortisol and melatonin) and peripheral clocks markers (clock gene expression in peripheral blood mononuclear cells, PBMCs) in subjects living at night. Eighteen healthy subjects were enrolled to either a control (dim light) or a bright light group. Blood was sampled at baseline and on the 4 th day of simulated night shift. In response to a night-oriented schedule, the phase of PER1 and BMAL1 rhythms in PBMCs was delayed by ~2.5-3 h (P shift was observed for the other clock genes and the central markers. Three cycles of 8-h bright light induced significant phase delays (P night-oriented schedule and a rapid resetting effect of nocturnal bright light exposure on peripheral clocks.

  12. Generation of “Off-the-Shelf” Natural Killer Cells from Peripheral Blood Cell-Derived Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Jieming Zeng

    2017-12-01

    Full Text Available Summary: Current donor cell-dependent strategies can only produce limited “made-to-order” therapeutic natural killer (NK cells for limited patients. To provide unlimited “off-the-shelf” NK cells that serve many recipients, we designed and demonstrated a holistic manufacturing scheme to mass-produce NK cells from induced pluripotent stem cells (iPSCs. Starting with a highly accessible human cell source, peripheral blood cells (PBCs, we derived a good manufacturing practice-compatible iPSC source, PBC-derived iPSCs (PBC-iPSCs for this purpose. Through our original protocol that excludes CD34+ cell enrichment and spin embryoid body formation, high-purity functional and expandable NK cells were generated from PBC-iPSCs. Above all, most of these NK cells expressed no killer cell immunoglobulin-like receptors (KIRs, which renders them unrestricted by recipients' human leukocyte antigen genotypes. Hence, we have established a practical “from blood cell to stem cells and back with less (less KIRs” strategy to generate abundant “universal” NK cells from PBC-iPSCs for a wide range of patients. : To provide unlimited “off-the-shelf” NK cells that serve many recipients, Zeng and colleagues demonstrate a manufacturing scheme to mass-produce NK cells from peripheral blood cell-derived iPSCs (PBC-iPSCs. Through their original protocol, high-purity functional NK cells are generated from PBC-iPSCs. Most of these NK cells express no killer cell immunoglobulin-like receptors, which renders them unrestricted by recipients' HLA genotypes. Keywords: induced pluripotent stem cells, peripheral blood cells, natural killer cells, killer cell immunoglobulin-like receptors, cell therapy, immunotherapy, cancer, cytotoxicity

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

    International Nuclear Information System (INIS)

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

    1985-01-01

    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

  14. 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.

  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. Does the preference of peripheral versus central venous access in peripheral blood stem cell collection/yield change stem cell kinetics in autologous stem cell transplantation?

    Science.gov (United States)

    Dogu, Mehmet Hilmi; Kaya, Ali Hakan; Berber, Ilhami; Sari, İsmail; Tekgündüz, Emre; Erkurt, Mehmet Ali; Iskender, Dicle; Kayıkçı, Ömur; Kuku, Irfan; Kaya, Emin; Keskin, Ali; Altuntaş, Fevzi

    2016-02-01

    Central venous access is often used during apheresis procedure in stem cell collection. The aim of the present study was to evaluate whether central or peripheral venous access has an effect on stem cell yield and the kinetics of the procedure and the product in patients undergoing ASCT after high dose therapy. A total of 327 patients were retrospectively reviewed. The use of peripheral venous access for stem cell yield was significantly more frequent in males compared to females (p = 0.005). Total volume of the product was significantly lower in central venous access group (p = 0.046). As being a less invasive procedure, peripheral venous access can be used for stem cell yield in eligible selected patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. The comparison of CT findings between peripheral pulmonary squamous cell carcinoma and pulmonary adenocarcinoma

    International Nuclear Information System (INIS)

    Tan Guosheng; Yang Xufeng; Zhou Xuhui; Li Ziping; Fan Miao; Chen Jindi

    2007-01-01

    Objective: To compare the principal HRCT features of peripheral pulmonary squamous cell carcinoma and pulmonary adenocarcinoma and to explore their pathological mechanism, in order to improve the recognition of the CT signs of peripheral pulmonary carcinoma. Methods: The principal HRCT signs of thirty-five cases with pathologically proved peripheral pulmonary squamous cell carcinoma and forty cases with pathologically proved peripheral pulmonary adenocarcinoma were analyzed retrospectively to explore the relationship between CT features and pathological findings. Results: The main features of peripheral pulmonary squamous cell carcinoma included larger masses, clear boundary, superficial sublobes and intra-tumor necrosis. While peripheral pulmonary adenocarcinoma mostly demonstrated as smaller nodules, deep sublobes, spiculations, spiculate protuberance, pleural indentation, vessel converging signs, and vacuole signs. The different of these above findings of peripheral pulmonary squamous cell carcinoma and adenocarcinoma were significant (P<0.05). Peripheral pulmonary squamous cell carcinoma may depict bronchial casts and polygonal nodules; and peripheral pulmonary adenocarcinoma may demonstrate ground glass-like nodules. Conclusion: The difference of the CT findings between peripheral pulmonary squamous cell carcinoma and peripheral adenocarcinoma is based on their different histological features and biological behaviors. It is possible to differentiate them before operation in combination with clinical information. (authors)

  18. Transplantation of olfactory ensheathing cells as adjunct cell therapy for peripheral nerve injury.

    Science.gov (United States)

    Radtke, Christine; Wewetzer, Konstantin; Reimers, Kerstin; Vogt, Peter M

    2011-01-01

    Traumatic events, such as work place trauma or motor vehicle accident violence, result in a significant number of severe peripheral nerve lesions, including nerve crush and nerve disruption defects. Transplantation of myelin-forming cells, such as Schwann cells (SCs) or olfactory ensheathing cells (OECs), may be beneficial to the regenerative process because the applied cells could mediate neurotrophic and neuroprotective effects by secretion of chemokines. Moreover, myelin-forming cells are capable of bridging the repair site by establishing an environment permissive to axonal regeneration. The cell types that are subject to intense investigation include SCs and OECs either derived from the olfactory bulb or the olfactory mucosa, stromal cells from bone marrow (mesenchymal stem cells, MSCs), and adipose tissue-derived cells. OECs reside in the peripheral and central nervous system and have been suggested to display unique regenerative properties. However, so far OECs were mainly used in experimental studies to foster central regeneration and it was not until recently that their regeneration-promoting activity for the peripheral nervous system was recognized. In the present review, we summarize recent experimental evidence regarding the regenerative effects of OECs applied to the peripheral nervous system that may be relevant to design novel autologous cell transplantation therapies. © 2011 Cognizant Comm. Corp.

  19. Peripheral benzodiazepine receptors are decreased during cocaine withdrawal in humans.

    Science.gov (United States)

    Javaid, J I; Notorangelo, M P; Pandey, S C; Reddy, P L; Pandey, G N; Davis, J M

    1994-07-01

    In the present study, homovanillic acid in plasma (pHVA) and benzodiazepine receptors (3H-PK11195 binding) in neutrophil membranes were determined in blood obtained from cocaine-dependent (DSM-III-R) adult male inpatients at baseline-(within 72 hr of last cocaine use) and after 3 weeks of cocaine abstinence, and normal controls. The mean (+/- SEM) pHVA at baseline (10.3 ng/ml +/- 1.1) was similar to normals and did not change after 3 weeks of cocaine abstinence. Similarly, the binding indices of benzodiazepine receptors in cocaine-dependent subjects as a group were not significantly different than in normal controls. In 10 cocaine-dependent subjects, however, where both blood samples were available, the number of 3H-PK11195 binding sites was significantly (p < 0.05) decreased after 3 weeks of cocaine abstinence (mean +/- sem: Bmax = 6371 +/- 657 fmol/mg protein) compared with baseline (Bmax = 7553 +/- 925 fmol/mg protein), although there were no differences in the binding affinity (mean +/- sem: KD = 8.6 +/- 1.2 nmol/L after 3 weeks of abstinence compared with 8.1 +/- 1.0 nmol/L at baseline). These preliminary results suggest that peripheral benzodiazepine receptors may play an important role in the pathophysiology of cocaine withdrawal in cocaine-dependent human subjects.

  20. Cytogenetic Low-Dose Hyperradiosensitivity Is Observed in Human Peripheral Blood Lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Seth, Isheeta [Department of Biological Sciences, Wayne State University, Detroit, Michigan (United States); Joiner, Michael C. [Department of Radiation Oncology, Wayne State University, Detroit, Michigan (United States); Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, Detroit, Michigan (United States)

    2015-01-01

    Purpose: The shape of the ionizing radiation response curve at very low doses has been the subject of considerable debate. Linear-no-threshold (LNT) models are widely used to estimate risks associated with low-dose exposures. However, the low-dose hyperradiosensitivity (HRS) phenomenon, in which cells are especially sensitive at low doses but then show increased radioresistance at higher doses, provides evidence of nonlinearity in the low-dose region. HRS is more prominent in the G2 phase of the cell cycle than in the G0/G1 or S phases. Here we provide the first cytogenetic mechanistic evidence of low-dose HRS in human peripheral blood lymphocytes using structural chromosomal aberrations. Methods and Materials: Human peripheral blood lymphocytes from 2 normal healthy female donors were acutely exposed to cobalt 60 γ rays in either G0 or G2 using closely spaced doses ranging from 0 to 1.5 Gy. Structural chromosomal aberrations were enumerated, and the slopes of the regression lines at low doses (0-0.4 Gy) were compared with doses of 0.5 Gy and above. Results: HRS was clearly evident in both donors for cells irradiated in G2. No HRS was observed in cells irradiated in G0. The radiation effect per unit dose was 2.5- to 3.5-fold higher for doses ≤0.4 Gy than for doses >0.5 Gy. Conclusions: These data provide the first cytogenetic evidence for the existence of HRS in human cells irradiated in G2 and suggest that LNT models may not always be optimal for making radiation risk assessments at low doses.

  1. 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.

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

    Directory of Open Access Journals (Sweden)

    Tanja Sofrenovic

    Full Text Available 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.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.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.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.

  3. 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 Hansen

    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...... in the presence of 30% autologous serum. Blocking the CR2 ligand-binding site with monoclonal antibody (mAb) FE8 resulted in significant reduction (37.9+/-11.9%) in C3-fragment deposition, whereas MAC formation was only marginally affected (12.1+/-22.2% reduction). Blocking the CR1 binding-site resulted...

  4. 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

    PROBLEM: The T-cell immunoglobulin and mucin domain (TIM) family is a relatively newly described group of molecules with a conserved structure and important immunological functions. Identification of Galectin-9 as a ligand for TIM-3 has established the Galectin-9/TIM-3 pathway as an important...... 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...... 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...

  5. Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Sabrina Krause

    2018-02-01

    Full Text Available Background: Child maltreatment (CM and attachment experiences are closely linked to alterations in the human oxytocin (OXT system. However, human data about oxytocin receptor (OXTR protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation.Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC and plasma OXT levels were determined by radioimmunoassay (RIA in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ to assess adverse childhood experiences. Attachment representations (secure vs. insecure were classified using the Adult Attachment Projective Picture System (AAP and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D.Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women.Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system.

  6. Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells.

    Science.gov (United States)

    Krause, Sabrina; Boeck, Christina; Gumpp, Anja M; Rottler, Edit; Schury, Katharina; Karabatsiakis, Alexander; Buchheim, Anna; Gündel, Harald; Kolassa, Iris-Tatjana; Waller, Christiane

    2018-01-01

    Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system.

  7. Evaluation of an Immunomodulator Drug as a Radioprotectant on Human Peripheral Blood Lymphocytes In Vitro

    Directory of Open Access Journals (Sweden)

    Zahra Sattarpour

    2018-01-01

    Full Text Available Background: IMOD™, a selenium enriched extract of the plants Tanacetum vulgare, Urtica dioica, and Rosa canina, has an excellent effect on oxidative stress. In this study, we investigated the radioprotective effects of this immunomodulatory drug on human peripheral blood lymphocytes. Methods: Peripheral blood samples obtained from venipuncture of the brachial vein were treated with IMOD™ (5, 10, 15, 20 μl for 30 min and Cobalt 60 γ-rays (0.25, 0.5, 1, 2 Gy as the test groups and cultured with the control. We used the micronuclei assay, cell death detection, and cell toxicity assay to analyze the treatment effects. Results: The frequency of micronuclei were 1.66 (0 Gy, 5.33 (0.25 Gy, 9.67 (0.5 Gy, 17.67 (1 Gy, and 23.67 (2 Gy in the irradiated lymphocytes (P<0.001. The percentage of micronuclei frequency reduced to 20%, 26.83%, 37.68%, 16%, and 20.47% with IMOD™. Apoptosis and necrosis decreased significantly in the IMOD™ treated groups (P<0.05. Conclusion: IMOD™ may protect these cells against ionizing radiation.

  8. The Human Cell Atlas.

    Science.gov (United States)

    Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

    2017-12-05

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

  9. 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

  10. Survival and PHA-stimulation of #betta#-irradiated human peripheral blood T lymphocyte subpopulations

    International Nuclear Information System (INIS)

    Schwartz, J.L.; Darr, D.C.; Daulden, M.E.

    1983-01-01

    Human peripheral blood T lymphocyte subpopulations were identified and isolated on the basis of their ability to bind IgG (T-G), IgM (T-M), or neither immunoglobulin class (T-null). Lymphocytes were exposed to 0, 0.5, 1.0, 2.5 or 5.0 Gy of 60 Co #betta#-rays either as a T-cell suspension or as separated T cell subsets. Survival curves, determined 5 days after irradiation, revealed that each subset has radiosensitive and radioresistant portions, and that the T-G cell is the most sensitive subset. Mitotic indices of 48-h cultures showed that the response of unirradiated T lymphocytes to PHA varied greatly among the subsets, the highest indices being obtained for the T-M and the lowest for the T-G cells. With the possible exception of the T-G cells, the subsets are realtively resistant to mitotic effects of #betta#-rays. T-G cells suppress the PHA-induced mitotic response of the other T lymphocyte subsets, and this suppressor effect is radiosensitive, being abolished by 1.0 Gy. It is concluded that lymphocytes exposed to >= 1 Gy of #betta#-rays will have very few dividing B lymphocytes or T-G cells. This together with radiation-induced loss of T-G suppressor action means that the predominant lymphocyte types in mitosis after >=1 Gy are the radioresistant T-M and T-null cells. (orig.)

  11. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    Science.gov (United States)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  12. 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.

  13. Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-gamma production of T cells.

    Science.gov (United States)

    Pierkes, M; Bellinghausen, I; Hultsch, T; Metz, G; Knop, J; Saloga, J

    1999-02-01

    Recent studies provide evidence that venom immunotherapy (VIT) alters the pattern of cytokine production by inducing an allergen-specific T-cell shift in cytokine expression from TH2 (IL-4, IL-5) to TH1 (IFN-gamma) cytokines and also inducing the production of IL-10. This study was carried out to analyze whether these changes in cytokine production of T cells already observed 1 week after the initiation of VIT in subjects with wasp venom allergy also influence the reactivity of effector cells, such as mast cells and basophils. All subjects included in this study had a history of severe systemic allergic reactions to wasp stings and positive skin test responses with venom and venom-specific IgE in the sera. Peripheral blood leukocytes were isolated before and after the initiation of VIT (rush therapy reaching a maintenance dose of 100 microg venom injected subcutaneously within 1 week) and preincubated with or without addition of IL-10, IFN-gamma, IL-10 + IFN-gamma, anti-IL-10, or anti-IFN-gamma. After stimulation with wasp venom, histamine and sulfidoleukotriene release were assessed by ELISA and compared with spontaneous release and total histamine content. After the induction of VIT, venom-induced absolute and relative histamine and sulfidoleukotriene release were reduced. This was at least partially due to the induction of IFN-gamma and IL-10 production, because (1) neutralization of IL-10 and IFN-gamma by mAbs partially restored the release after the initiation of VIT and (2) the addition of exogenous IFN-gamma and IL-10 caused a statistically significant diminution of the venom-induced histamine and sulfidoleukotriene release before VIT. Depletion of CD2(+) T cells also restored the releasability after VIT. These data indicate that T cells (producing IL-10 and IFN-gamma after VIT) play a key role for the inhibition of histamine and sulfidoleukotriene release of effector cells.

  14. CNS infiltration of peripheral immune cells: D-Day for neurodegenerative disease?

    Science.gov (United States)

    Rezai-Zadeh, Kavon; Gate, David; Town, Terrence

    2009-12-01

    While the central nervous system (CNS) was once thought to be excluded from surveillance by immune cells, a concept known as "immune privilege," it is now clear that immune responses do occur in the CNS-giving rise to the field of neuroimmunology. These CNS immune responses can be driven by endogenous (glial) and/or exogenous (peripheral leukocyte) sources and can serve either productive or pathological roles. Recent evidence from mouse models supports the notion that infiltration of peripheral monocytes/macrophages limits progression of Alzheimer's disease pathology and militates against West Nile virus encephalitis. In addition, infiltrating T lymphocytes may help spare neuronal loss in models of amyotrophic lateral sclerosis. On the other hand, CNS leukocyte penetration drives experimental autoimmune encephalomyelitis (a mouse model for the human demyelinating disease multiple sclerosis) and may also be pathological in both Parkinson's disease and human immunodeficiency virus encephalitis. A critical understanding of the cellular and molecular mechanisms responsible for trafficking of immune cells from the periphery into the diseased CNS will be key to target these cells for therapeutic intervention in neurodegenerative diseases, thereby allowing neuroregenerative processes to ensue.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Gene expression in human peripheral blood 48 hours after exposure to ionizing radiation

    Data.gov (United States)

    National Aeronautics and Space Administration — Analysis of human peripheral blood 48 hours after irradiation ex vivo with graded doses of gamma rays. Results have been used in building and testing classifiers to...

  17. microRNA expression profiles in human peripheral blood lymphocytes cultured in modeled microgravity

    Data.gov (United States)

    National Aeronautics and Space Administration — In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a...

  18. Gene expression profiling of human peripheral blood lymphocytes cultured in modeled microgravity

    Data.gov (United States)

    National Aeronautics and Space Administration — In the present study we analyzed miRNA and mRNA expression profiles in human peripheral blood lymphocytes (PBLs) incubated in microgravity condition simulated by a...

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

    Directory of Open Access Journals (Sweden)

    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

  20. The human cell atlas

    DEFF Research Database (Denmark)

    Regev, Aviv; Teichmann, Sarah A.; Lander, Eric S.

    2017-01-01

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international...... collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells...... in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early...

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

    OpenAIRE

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

    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 hiPS...

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

    Directory of Open Access Journals (Sweden)

    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

  3. Effects of mercury on the proliferation of human peripheral lymphocytes in vitro

    International Nuclear Information System (INIS)

    Piwecka, K.; Poniedzialek, B.; Rzymski, P.; Karczewski, J.; Zurawski, J.; Wiktorowicz, K.

    2011-01-01

    Our project aimed to investigate the effects of mercury on the proliferation of human peripheral lymphocytes in vitro. The lymphocytes were isolated from the blood collected from healthy donors at Regionalne Centrum Krwiodawstwa i Krwiolecznictwa in Poznan, Poland. For the purpose of cell culture, the lymphocyte suspension (25 · 10 4 cells/ml) in Eagle's medium supplemented with 10% fetal calf serum was prepared. Phytohaemagglutinin-L (PHA-L) was used in a concentration of 2.5 mg/ml to stimulate cell proliferation. Mercuric chloride (HgCl 2 ) in four different concentrations (1 μM, 10 μM, 50 μM, 100 μM) and [3H]-thymidine were added after 48 hours of incubation and the cell culture was continued for the next 24 hours. The rate of lymphocyte proliferation was measured by [3H]-thymidine incorporation method with a liquid scintillation counter. Results indicate that higher concentrations of mercury (50 μM, 100 μM) inhibit the [3H]-thymidine incorporation of human peripheral lymphocytes in vitro. The incorporation was lower than the control sample by 65% at a concentration of 50 μM, while at a concentration of 100 μM it fell to virtually zero. Moreover, the phase of lymphocyte proliferation cycle affected by mercuric chloride was also investigated. For this purpose HgCl 2 in 2 concentrations (10 μM, 50 μM) was added to the cell culture in 4 different time points: at the start of the cell culture and after 4, 24, and 48 hours of incubation. After 48 hours, [3H]-thymidine was added and the cell culture was continued for an additional 24 hours. The rate of cell proliferation was estimated by [3H]-thymidine incorporation using a liquid scintillation counter. The inhibition effect was observed in samples with metal added at the start of the cell culture and after 4 h of incubation, i.e. at the initial phase of the lymphocyte proliferation cycle. (authors)

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Transcription factor fos-related antigen-2 induces progressive peripheral vasculopathy in mice closely resembling human systemic sclerosis.

    Science.gov (United States)

    Maurer, Britta; Busch, Nicole; Jüngel, Astrid; Pileckyte, Margarita; Gay, Renate E; Michel, Beat A; Schett, Georg; Gay, Steffen; Distler, Jörg; Distler, Oliver

    2009-12-08

    Microvascular damage is one of the first pathological changes in systemic sclerosis. In this study, we investigated the role of Fos-related antigen-2 (Fra-2), a transcription factor of the activator protein-1 family, in the peripheral vasculopathy of systemic sclerosis and examined the underlying mechanisms. Expression of Fra-2 protein was significantly increased in skin biopsies of systemic sclerosis patients compared with healthy controls, especially in endothelial and vascular smooth muscle cells. Fra-2 transgenic mice developed a severe loss of small blood vessels in the skin that was paralleled by progressive skin fibrosis at 12 weeks of age. The reduction in capillary density was preceded by a significant increase in apoptosis in endothelial cells at week 9 as detected by immunohistochemistry. Similarly, suppression of Fra-2 by small interfering RNA prevented human microvascular endothelial cells from staurosporine-induced apoptosis and improved both the number of tubes and the cumulative tube lengths in the tube formation assay. In addition, cell migration in the scratch assay and vascular endothelial growth factor-dependent chemotaxis in a modified Boyden chamber assay were increased after transfection of human microvascular endothelial cells with Fra-2 small interfering RNA, whereas proliferation was not affected. Fra-2 is present in human systemic sclerosis and may contribute to the development of microvasculopathy by inducing endothelial cell apoptosis and by reducing endothelial cell migration and chemotaxis. Fra-2 transgenic mice are a promising preclinical model to study the mechanisms and therapeutic approaches of the peripheral vasculopathy in systemic sclerosis.

  8. Decline in peripheral blood NKG2D+CD3+CD56+ NKT cells in metastatic colorectal cancer patients.

    Science.gov (United States)

    Gharagozloo, M; Rezaei, A; Kalantari, H; Bahador, A; Hassannejad, N; Maracy, M; Nouri, N; Sedghi, M; Ghazanfari, H; Bayat, B

    2018-01-01

    Colorectal cancer (CRC) is one of the main causes of cancer deaths in the world. This cancer can be divided into non-metastatic and metastatic CRC stages. CD3+CD56+ NKT cell subsets are a minor T cell subset in peripheral blood and conduct the killing of tumor cells in direct manner. Little is obvious about levels and surface markers of these cells such as NKG2D in different cancers, especially in CRC. We included 15 non-metastatic (low-grade), 11 non-metastatic (high-grade), 10 metastatic colorectal cancer patients and 18 healthy controls. The percentages of CD3+CD56+ NKT cells and NKG2D+CD56+ NKT cells from samples were analyzed by flow cytometry in peripheral blood mononuclear cells (PBMCs) of samples. We found that there was a significantly lower number of NKG2D+CD3+CD56+ cells in peripheral blood of patients with metastatic colorectal cancer compared with normal controls (77.53 ± 5.79 % vs 90.74 ± 9.84 %; pNKT cells was significantly lower in patients with metastatic colorectal cancer compared to healthy controls strengthens the hypothesis that NKT cells can play a substantial role in the protection against human colorectal cancer, and this opens up avenues for novel studies about elucidating the other aspects of tumor surveillance in CRC progression and immunotherapy (Tab. 2, Fig. 2, Ref. 46).

  9. 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

    Directory of Open Access Journals (Sweden)

    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

  10. In vitro X-ray irradiation of human peripheral blood T lymphocytes enhances suppressor function

    International Nuclear Information System (INIS)

    Ogawa, H.; Tsunematsu, T.

    1983-01-01

    The effect of in vitro X-ray irradiation on human peripheral blood T lymphocytes was studied with regard to their suppressor activity related to the concanavalin A (Con A)-induced suppressor system. To generate suppressor T lymphocytes, purified human T lymphocytes were incubated for 3 days in the first culture, with or without Con A. These lymphocytes were irradiated with various doses of X-ray before, mid or after the culture. After doing a second culture for 6 days, the suppressive influence of these cells on T lymphocyte proliferation rates stimulated with allogeneic mononuclear cells, and B lymphocyte proliferation rates stimulated with pokeweed mitogen was measured. Irradiation of cultures to which Con A had not been added induced much the same level of suppressor activity as seen in the cultures with Con A. The suppressor activity gradually increased with time from the irradiation to the suppressor cell assay. Suppressor T lymphocytes were resistant to X-ray irradiation and independent of DNA synthesis. However, irradiation-induced enhancement was minimal in cultures incubated with con A, regardless of the irradiation time. (author)

  11. Induction of mitotic micronuclei by X-ray contrast media in human peripheral lymphocytes

    International Nuclear Information System (INIS)

    Parvez, Z.; Moncada, R.; Kormano, M.; Satokari, K.; Eklund, R.

    1987-01-01

    In vitro and in vivo cytogenetic effects of X-ray contrast media (CM) were determined by scoring micronuclei (MN) in 72-h cultures of human peripheral lymphocytes. Both ionic (sodium meglumine diatrizoate, methylglucamine diatrizoate, and sodium meglumine ioxaglate and nonionic CM (iosimide, iopromide, iohexol and iotrolan) were able to induce MN in lymphocytes. Based upon their calculated percent probabilities for MN induction, these agents could be ranked in their decreasing order of probability, as iosimide > sodium meglumine ioxaglate > iohexol > sodium meglumine diatrizoate > iopromide > methylglucamine diatrizoate > iotrolan. Stepwise logistic regression analysis of the data indicated that the frequency of MN in CM-exposed lymphocyte cultures was significantly higher than the frequency of MN in control cultures (P < 0.001). In clinical studies where 14 patients were injected with an ionic CM methylglucamine diatrizoate, lymphocyte cultures from 10 patients showed higher frequencies of MN. The differences between pre- and post-CM counts of MN were significant in a Mann-Whitney U test (P < 0.05). The effect of X-irradiation on MN formation in lymphocytes was separately determined and was found to be insignificant. These results indicate that irrespective of ionic and osmolality differences, X-ray contrast agents are capable of producing chromosomal damage in peripheral lymphocytes. Further studies are required to establish molecular mechanisms in the observed cytogenetic effects of CM in cell cultures. (Auth.)

  12. Investigation of micronuclei induction in human peripheral blood lymphocytes exposed in vitro to EMF RF

    International Nuclear Information System (INIS)

    Kolomiets, Irina A.; Triapitsina, Galina A.; Polevik, Nikolai D.; Pryakhin, Evgeny A.

    2008-01-01

    Full text: The widespread application of cellular phones is of great concern in view possible consequences for human health. The aim of this study is to assess the capability of electromagnetic fields (EMF) RF with frequency 925 MHz and modulation 217 Hz to induce genotoxic effects as evaluated by the in vitro micronucleus assay on peripheral blood lymphocytes. The flasks of peripheral blood samples collected from healthy volunteers (5 men and 5 women) were placed just on the oscillator of emitting antenna. The signals were produced by the laboratory research plant and were evaluated at four specific absorption rates (SARs) - 0; 0.29; 1.2; 8.1 W/kg. SARs were determined by the calorimetric method. Phytohaemagglutinin stimulated lymphocytes were exposed three times for 10 minutes in the G o (the first 30 minutes after the beginning of cultivation), S (24 hours later), G 2 -M (after 48 hours from the beginning of cultivation) stages of the cell cycle. 72-hours cultures of lymphocytes were examined to determine the extent of micronuclei. The Mann-Whitney U-test was used to evaluate the significance for comparison. The data indicated a significant increase of micronuclei in human lymphocytes exposed to EMF RF (6.5 ± 0.51 0/00; 7.1 ± 0.66 0/00; 7.0 ± 0.50 0/00) in comparison with sham-exposed lymphocytes (3.0 ± 0.60 0/00). There was not revealed a dose-dependent increase of micronuclei in human lymphocytes. It was suggested that the increase of micronuclei in lymphocytes is explicated by a particularity of EMF RF just near the oscillator of emitting antenna. (author)

  13. Effect of peripheral morphine in a human model of acute inflammatory pain

    DEFF Research Database (Denmark)

    Lillesø, J; Hammer, N A; Pedersen, J L

    2000-01-01

    Several studies have demonstrated the presence of opioid inducible receptors on peripheral nerves and peripheral antinociceptive effects of opioids. However, the effects of peripheral opioid administration in man are controversial. Our study used a randomized, double-blind, placebo-controlled, th......Several studies have demonstrated the presence of opioid inducible receptors on peripheral nerves and peripheral antinociceptive effects of opioids. However, the effects of peripheral opioid administration in man are controversial. Our study used a randomized, double-blind, placebo......-controlled, three-way crossover design in a human model of acute inflammatory pain (heat injury). We studied 18 healthy volunteers who each received morphine locally (2 mg), morphine systemically (2 mg), or placebo on three separate study days. The subjects received morphine infiltration subcutaneously (s.c.). 1 h......, but local morphine infiltration neither reduced pain during the burn, nor primary or secondary hyperalgesia to mechanical and heat stimuli after the burn. In conclusion, peripherally applied morphine had no acute antinociceptive effects in this human model of acute inflammatory pain....

  14. Paracetamol (acetaminophen) attenuates in vitro mast cell and peripheral blood mononucleocyte cell histamine release induced by N-acetylcysteine.

    Science.gov (United States)

    Coulson, James; Thompson, John Paul

    2010-02-01

    The treatment of acute paracetamol (acetaminophen) poisoning with N-acetylcysteine (NAC) is frequently complicated by an anaphylactoid reaction to the antidote. The mechanism that underlies this reaction is unclear. We used the human mast cell line 1 (HMC-1) and human peripheral blood mononucleocytes (PBMCs) to investigate the effects of NAC and paracetamol on histamine secretion in vitro. HMC-1 and human PBMCs were incubated in the presence of increasing concentrations of NAC +/- paracetamol. Cell viability was determined by the Trypan Blue Assay, and histamine secretion was measured by ELISA. NAC was toxic to HMC-1 cells at 100 mg/mL and to PBMCs at 67 mg/mL. NAC increased HMC-1 and PBMC histamine secretion at concentrations of NAC from 20 to 50 mg/mL and 2.5 to 100 mg/mL, respectively. NAC-induced histamine secretion by both cell types was reduced by co-incubation with 2.5 mg/mL of paracetamol. Paracetamol (acetaminophen) is capable of modifying histamine secretion in vitro. This may explain the clinical observation of a lower incidence of adverse reactions to NAC in vivo when higher concentrations of paracetamol are present than when paracetamol concentrations are low. Paracetamol (acetaminophen) attenuates in vitro mast cell and PBMC cell histamine release induced by NAC.

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  2. Quantification of BCR-ABL transcripts in peripheral blood cells and ...

    African Journals Online (AJOL)

    Purpose: To investigate the feasibility of using peripheral blood plasma samples as surrogates for blood cell sampling for quantification of breakpoint cluster region-Abelson oncogene (BCR-ABL) transcript levels to monitor treatment responses in chronic myeloid leukemia (CML) patients. Methods: Peripheral blood samples ...

  3. Chromosome aberrations in human peripheral lymphocytes induced by single or fractionated X-irradiation

    International Nuclear Information System (INIS)

    Ivanov, B.; Leonard, A.; Deknyudt, G.

    1980-01-01

    Investigated is the effect of single (125 and 250 R) and fractionated (2x125 R) irradiation on the output of chromosome aberrations in lymphocytes of human peripheral blood kept between irradiations at the temperature of 5 deg C. The single irradiation is carried out immediately after vein-puncture. In the case of fractionated irradiation the first dose of 125R is given after vein-puncture, the second, in the interval of 2, 8 and 24 hours. Blood is cultivated immediately after two irradiations in order to prepare metaphase plates for cytogenic analysis. Repair processes in cell heritage structures are not realised in blood irradiated by fractions which is kept at 5 deg C between irradiations. On the contrary, chromosome fragments, interstitial deletions, aberrant cells and cell breaks are found in a large amount in blood irradiated by fractions. They have appeared with the authentically high statistic difference as compared with the cells irradiated one time with the same dose. This effect is probably attained due to blood preservation

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

    Science.gov (United States)

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

    2014-12-01

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

  5. 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.

  6. Comparison of immunological characteristics of peripheral, splenic and tonsilar naïve B cells by differential gene expression meta-analyses.

    Science.gov (United States)

    Chokeshai-u-saha, Kaj; Lepoivre, Cyrille; Grieco, Luca; Nguyen, Catherine; Ruxrungtham, Kiat

    2012-12-01

    Naïve B cells isolated from peripheral blood, spleen and tonsil are commonly used in human B cell studies. However, little has been written about their possible variations in immunological properties. This study compared differential gene expression in human naive B subsets by meta-analysis using expression data available in Gene Expression Onimbus (GEO). Gene expression files of the Affymetrix Human Genome U133A Array (Affymetrix) were downloaded to collect 21 total array data samples of peripheral naïve B cells (n=10), splenic naïve B cells (n=2), tonsilar naïve B cells (n=3), peripheral memory B cells (n=4) and splenic memory B cells (n=2). Prior to differential gene expression analyses, data were normalized in order to reduce non-biological variation among the datasets. Comparisons of peripheral naive B cells with their splenic and tonsilar counterparts showed remarkable differences in terms of gene expression (29 and 202 genes, respectively). However, only minor differences were detected between splenic and tonsilar naive B cells (10 genes), consistent with the clustering results classifying both of them as lymphoid naive B cells. Differential gene expression results also implied higher stimulating states of lymphoid naive B cells when compared with peripheral blood naive B cells. These included enhanced expressions of CD27, CR2, EGR1, GADD45B, ICAM1, ICOSLG, IGHA, IL6, MMP9, SAMSN1, SMAD7, TNFAIP3, but reduced HLA-DOB expression. Our findings suggest that results generated from peripheral naive B cells may not always be applicable to the biological activities of other lymphoid naïve B cells. Nonetheless, further biological study is warranted.

  7. 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.

  8. HLA-C is necessary for optimal human immunodeficiency virus type 1 infection of human peripheral blood CD4 lymphocytes.

    Science.gov (United States)

    Baroni, Miriam; Matucci, Andrea; Scarlatti, Gabriella; Soprana, Elisa; Rossolillo, Paola; Lopalco, Lucia; Zipeto, Donato; Siccardi, Antonio G; De Santis, Claudio

    2010-01-01

    The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors. Infectability by HIV (strains IIIB and Bal and primary isolates) was significantly reduced (P=0.016) in silenced cells compared with cells that maintained HLA-C expression in 10 of the 11 PBL donors. Normal infectability was resumed, together with HLA-C expression, when the effect of siRNA interference waned after several days in culture. Additional confirmation of the HLA-C effect was obtained in several assays employing HLA-C-positive and -negative cell lines, a number of HIV strains and also pseudoviruses. In particular, viruses pseudotyped with env genes from HIV strains AC10 and QH0692.42 were assayed on siRNA-silenced lymphocytes from three healthy donors: the differences in infection with pseudoviruses were even higher than those observed in infections with normal viruses.

  9. Potential antioxidant and cytoprotective effects of essential oil extracted from Cymbopogon citratus on OxLDL and H2O2 LDL induced Human Peripheral Blood Mononuclear Cells (PBMC

    Directory of Open Access Journals (Sweden)

    Jamuna S.

    2017-06-01

    Full Text Available Cymbopogon citratus (lemon grass is commonly used in traditional folk medicine. The essential oil extracted from C. citratus has been reported as a potential anti-oxidant and anti-inflammatory agent. This study has been designed to explore the protective effect of C. citratus (lemon grass against modified LDL (OxLDL and H2O2 LDL induced cytotoxicity in Peripheral Blood Mononuclear Cells (PBMC. The essential oil extracted from C. citratus (EOC was subjected to FT-IR spectroscopic analysis for the identification of functional groups. In vitro antioxidant assays were carried out to assess the electron donating capability of EOC as compared with a known standard L-ascorbic acid. The cytoprotective effects of EOC were determined in PBMC induced with modified LDL. Spectra obtained from FT-IR analysis showed the presence of functional groups in EOC such as H-bonded, OH stretching, NH stretching, aldehydeCH stretching, aldehyde/ketoneCO stretching, CC-stretching, CH3 bending, CH in plane bending. EOC has greater antioxidant property when compared with the standard L-ascorbic acid. EOC at all test concentrations demonstrated free radical scavenging activity and cytoprotective effect when challenged against modified LDL in PBMC. The above results show EOC as a promising antioxidant and cytoprotective agent.

  10. The uptake kinetics and immunotoxic effects of microcystin-LR in human and chicken peripheral blood lymphocytes in vitro

    International Nuclear Information System (INIS)

    Lankoff, Anna; Carmichael, Wayne W.; Grasman, Keith A.; Yuan, Moucun

    2004-01-01

    Microcystin-LR is a cyanobacterial heptapeptide that presents acute and chronic hazards to animal and human health. We investigated the influence of this toxin on human and chicken immune system modulation in vitro. Peripheral blood lymphocytes were treated with microcystin-LR at environmentally relevant doses of 1, 10 and 25 μg/ml for 12, 24, 48, 72 h (for proliferation assay cells were treated for 72 h). T-cell and B-cell proliferation as well as apoptosis and necrosis were determined in human and chicken samples. IL-2 and IL-6 production by human lymphocytes also was measured. In addition, uptake kinetics of microcystin-LR into human and chicken peripheral blood lymphocytes were calculated by Liquid Chromatography (LS) /Mass Spectrometry (MS) analysis. At the highest dose microcystin-LR decreased T-cell proliferation and all doses of microcystin-LR inhibited B-cell proliferation. The frequency of apoptotic and necrotic cells increased in a dose and time-dependent manner. Human lymphocytes responded to stimulation with microcystin-LR by increased production of IL-6 and decreased production of IL-2. Human lymphocytes were able to uptake from 0.014 to 1.663 μg/ml and chicken lymphocytes from 0.035 to 1.733 μg/ml of the microcystin-LR added to the cultures, depending on the treatment time and dose. In conclusion, microcystin-LR acted as an immunomodulator in cytokine production and down-regulated lymphocyte functions by induction of apoptosis and necrosis. However, further studies dealing with the influence of microcystin-LR on expression cytokine genes and transcription factors are necessary to confirm these hypotheses

  11. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    International Nuclear Information System (INIS)

    Doi, Keiko; Fujimoto, Takahiro; Okamura, Tadashi; Ogawa, Masahiro; Tanaka, Yoko; Mototani, Yasumasa; Goto, Motohito; Ota, Takeharu; Matsuzaki, Hiroshi; Kuroki, Masahide; Tsunoda, Toshiyuki; Sasazuki, Takehiko; Shirasawa, Senji

    2012-01-01

    Highlights: ► We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. ► Zfat-deficiency leads to reduction in the number of the peripheral T cells. ► Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. ► Decreased expression of IL-7Rα, IL-2Rα and IL-2 in Zfat-deficient peripheral T cells. ► Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  12. Gene expression patterns in CD4+ peripheral blood cells in healthy subjects and stage IV melanoma patients.

    Science.gov (United States)

    Felts, Sara J; Van Keulen, Virginia P; Scheid, Adam D; Allen, Kathleen S; Bradshaw, Renee K; Jen, Jin; Peikert, Tobias; Middha, Sumit; Zhang, Yuji; Block, Matthew S; Markovic, Svetomir N; Pease, Larry R

    2015-11-01

    Melanoma patients exhibit changes in immune responsiveness in the local tumor environment, draining lymph nodes, and peripheral blood. Immune-targeting therapies are revolutionizing melanoma patient care increasingly, and studies show that patients derive clinical benefit from these newer agents. Nonetheless, predicting which patients will benefit from these costly therapies remains a challenge. In an effort to capture individual differences in immune responsiveness, we are analyzing patterns of gene expression in human peripheral blood cells using RNAseq. Focusing on CD4+ peripheral blood cells, we describe multiple categories of immune regulating genes, which are expressed in highly ordered patterns shared by cohorts of healthy subjects and stage IV melanoma patients. Despite displaying conservation in overall transcriptome structure, CD4+ peripheral blood cells from melanoma patients differ quantitatively from healthy subjects in the expression of more than 2000 genes. Moreover, 1300 differentially expressed genes are found in transcript response patterns following activation of CD4+ cells ex vivo, suggesting that widespread functional discrepancies differentiate the immune systems of healthy subjects and melanoma patients. While our analysis reveals that the transcriptome architecture characteristic of healthy subjects is maintained in cancer patients, the genes expressed differentially among individuals and across cohorts provide opportunities for understanding variable immune states as well as response potentials, thus establishing a foundation for predicting individual responses to stimuli such as immunotherapeutic agents.

  13. Analysis of Peripheral B Cell Subsets in Patients With Allergic Rhinitis.

    Science.gov (United States)

    Luo, Jing; Guo, Huanhuan; Liu, Zhuofu; Peng, Tao; Hu, Xianting; Han, Miaomiao; Yang, Xiangping; Zhou, Xuhong; Li, Huabin

    2018-05-01

    Recent evidence suggests that B cells can both promote and inhibit the development and progression of allergic disease. However, the characteristics of B cell subsets in patients with allergic rhinitis (AR) have not been well documented. This study aimed to analyze the characteristics of B cell subsets in the peripheral blood of AR patients. Forty-seven AR patients and 54 healthy controls were enrolled in this study, and the B cell subsets in peripheral blood of all subjects were analyzed by flow cytometry. Moreover, the serum total immunoglobulin E (IgE) and IgE concentrations secreted into the cultured peripheral blood mononuclear cells (PBMCs) were measured by using enzyme-linked immunosorbent assay. We found the peripheral blood of AR patients contained higher percentages of memory B cells, plasma cells, and CD19⁺CD24(hi)CD27⁺ regulatory B cells (Bregs) than those of age-matched healthy controls (PB cells and CD19⁺CD24(hi)CD38(hi) Bregs were significantly lower in AR patients than in healthy individuals (PB cells or plasma cells and decreases in CD19⁺CD24(hi)CD38(hi) Breg cells in the peripheral blood. Copyright © 2018 The Korean Academy of Asthma, Allergy and Clinical Immunology · The Korean Academy of Pediatric Allergy and Respiratory Disease.

  14. 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...

  15. 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....

  16. Primary nodal peripheral T-cell lymphomas: diagnosis and therapeutic considerations

    Directory of Open Access Journals (Sweden)

    Luis Alberto de Pádua Covas Lage

    2015-08-01

    Full Text Available Nodal peripheral T-cell lymphomas are a rare group of neoplasms derived from post-thymic and activated T lymphocytes. A review of scientific articles listed in PubMed, Lilacs, and the Cochrane Library databases was performed using the term "peripheral T-cell lymphomas". According to the World Health Organization classification of hematopoietic tissue tumors, this group of neoplasms consists of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS, angioimmunoblastic T-cell lymphoma (AITL, anaplastic large cell lymphoma-anaplastic lymphoma kinase positive (ALCL-ALK+, and a provisional entity called anaplastic large cell lymphoma-anaplastic lymphoma kinase negative (ALCL-ALK-. Because the treatment and prognoses of these neoplasms involve different principles, it is essential to distinguish each one by its clinical, immunophenotypic, genetic, and molecular features. Except for anaplastic large cell lymphoma-anaplastic lymphoma kinase positive, which has no adverse international prognostic index, the prognosis of nodal peripheral T-cell lymphomas is worse than that of aggressive B-cell lymphomas. Chemotherapy based on anthracyclines provides poor outcomes because these neoplasms frequently have multidrug-resistant phenotypes. Based on this, the current tendency is to use intensified cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP regimens with the addition of new drugs, and autologous hematopoietic stem cell transplantation. This paper describes the clinical features and diagnostic methods, and proposes a therapeutic algorithm for nodal peripheral T-cell lymphoma patients.

  17. Isolation, propagation, and titration of human immunodeficiency virus type 1 from peripheral blood of infected individuals

    NARCIS (Netherlands)

    Schuitemaker, Hanneke; Kootstra, Neeltje A.

    2005-01-01

    HIV-1 can be isolated from peripheral blood mononuclear cells and is easily propagated on primary cells in vitro. Here we describe the method for bulk isolation of the HIV-1 quasispecies and a limiting dilution virus isolation protocol by which single coexisting clones can be obtained. In addition,

  18. Conformally integrated stent cell resonators for wireless monitoring of peripheral artery disease

    KAUST Repository

    Viswanath, Anupam; Green, Scott Ryan; Kosel, Jü rgen; Gianchandani, Yogesh B.

    2013-01-01

    This paper presents the design and in vitro evaluation of magnetoelastic sensors intended for wireless monitoring of tissue accumulation in peripheral artery stents. The sensors, shaped like stent cells, are fabricated from 28-μm thick foils

  19. Biological radiation dose estimation by chromosomal aberrations analysis in human peripheral blood (dose-effect curve)

    International Nuclear Information System (INIS)

    Al-Achkar, W.

    2001-09-01

    In order to draw a dose-effect curve, experimentally gamma ray induced chromosomal aberrations in human peripheral lymphocytes from eight healthy people were studied. Samples from 4 males and 4 females were irradiated in tubes with 0.15, 0.25, 0.5, 1, 1.5, 2, 2.5 gray of gamma ray (Co 60 at dose rate 0.3 Gy/min). Irradiated and control samples were incubated in 37 centigrade for 48 hours cell cultures. Cell cultures then were stopped and metaphases spread, Giemsa stained to score the induced chromosomal aberrations. Chromosomal aberrations from 67888 metaphases were scored. Curves from the total number of dicentrics, dicentrics + rings and total numbers of breaks in cell for each individual or for all people were drawn. An increase of all chromosomal aberrations types with the elevation of the doses was observed. The yield of chromosome aberrations is related to the dose used. These curves give a quick useful estimation of the accidentally radiation exposure. (author)

  20. Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    Science.gov (United States)

    Kuligina, Elena V.; Bariakin, Dmitry N.; Kozlov, Vadim V.; 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. PMID:28127559

  1. The effect of peripheral lymphoid cells on the incidence of lethal graft versus host disease following allogeneic mouse bone marrow transplantation

    International Nuclear Information System (INIS)

    Almaraz, R.; Ballinger, W.; Sachs, D.H.; Rosenberg, S.A.

    1983-01-01

    Experiments were performed to study the role of circulating lymphoid cells in the incidence of lethal graft versus host disease (GVHD) in radiation-induced fully allogeneic mouse chimeras. The incidence of GVHD was reduced significantly in BALB/c leads to C57BL/6 radiation chimeras if bone marrow donors were exsanguinated immediately prior to marrow harvest. Chimeras resulting from the injection of bone marrow from bled donors exhibited only donor cells in spleen, bone marrow and peripheral blood and normal levels of Thy 1+ and Ia+ cells were found in each of these lymphoid compartments. The addition of as few as 3 X 10(4) peripheral mononuclear cells to the marrow from exsanguinated donors uniformly led to lethal GVHD. 51 Cr-labeled cell traffic studies revealed that prior exsanguination of marrow donors led to about a 70% reduction in the number of circulating mononuclear cells contaminating the bone marrow at the time of marrow harvest. This decrease in contaminating peripheral cells was calculated to be in the appropriate range to account for the decreased GVHD seen when marrow from exsanguinated donors was used. It thus appears that peripheral cells contaminating marrow can be an important factor in causing lethal GVHD in allogeneic radiation chimeras. These results raise the possibility that the fulminant GVHD seen in human marrow transplantation is in part due to the major contamination of bone marrow with peripheral blood that results from the techniques currently used for human bone marrow harvest

  2. Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry.

    Science.gov (United States)

    Liegler, T J; Hyun, W; Yen, T S; Stites, D P

    1995-05-01

    Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a

  3. Roles of neural stem cells in the repair of peripheral nerve injury.

    Science.gov (United States)

    Wang, Chong; Lu, Chang-Feng; Peng, Jiang; Hu, Cheng-Dong; Wang, Yu

    2017-12-01

    Currently, researchers are using neural stem cell transplantation to promote regeneration after peripheral nerve injury, as neural stem cells play an important role in peripheral nerve injury repair. This article reviews recent research progress of the role of neural stem cells in the repair of peripheral nerve injury. Neural stem cells can not only differentiate into neurons, astrocytes and oligodendrocytes, but can also differentiate into Schwann-like cells, which promote neurite outgrowth around the injury. Transplanted neural stem cells can differentiate into motor neurons that innervate muscles and promote the recovery of neurological function. To promote the repair of peripheral nerve injury, neural stem cells secrete various neurotrophic factors, including brain-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor and hepatocyte growth factor. In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways.

  4. Isolation of Human Innate Lymphoid Cells.

    Science.gov (United States)

    Krabbendam, Lisette; Nagasawa, Maho; Spits, Hergen; Bal, Suzanne M

    2018-06-29

    Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8 + T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce. As the frequency of ILCs is low, their isolation requires extensive depletion of other cell types. The lack of unique cell surface antigens further complicates the identification of these cells. Here, methods for ILC isolation and characterization from human peripheral blood and different tissues are described. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

  5. Histochemical, light and electron microscopic study of polonium-210 induced peripheral tumours in hamster lungs -evidence implicating the Clara Cell as the cell of origin

    International Nuclear Information System (INIS)

    Kennedy, A.R.; McGandy, R.B.; Little, J.B.

    1977-01-01

    Peripheral lung tumors induced in Syrian golden hamsters by intratracheally administered polonium-210 ( 210 Po) are similar to the peripheral lung tumours induced in many species by a variety of carcinogens. In addition, they show many of the histopathological features observed in human bronchiolar-alveolar carcinomas. Serial sacrifice studies of hamsters exposed to multiple instillations of 210 Po have been carried our to identify the cell of origin of these tumors. By means of thin, plastic (glycol methacrylate) sections, electron microscopy, and histochemistry, it is concluded that the bronchiolar Clara cell is the probable cell of origin, and that this view is generally compatible with many of the reported cytological characteristics of the human tumor. (author)

  6. 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.

  7. Treatment of Arsenite Intoxication-Induced Peripheral Vasculopathy with Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Yi-Hung Chiang

    2018-03-01

    Full Text Available Arsenite (As, a notorious toxic metal, is ubiquitously distributed in the earth and poses a serious threat to human health. Histopathological lesions of As intoxication are known as thromboangiitis obliterans, which are resistant to current treatment and often lead to lower limb amputation. In this study, we attempt to find that treatment with mesenchymal stem cells (MSCs may be effective for As-induced vasculopathy. We first conducted an in vitro study with a co-culture system containing human MSCs and human umbilical vein endothelial cells (HUVECs and treated individual and co-cultured cells with various concentrations of arsenite. We also designed an in vivo study in which Sprague Dawley (SD rats received periodic intraperitoneal (IP injections of 16 ppm arsenite for 12 weeks. MSCs were harvested from BALB/c mice that were transplanted via tail vein injection. We found that there was significantly higher cellular viability in human mesenchymal stem cells (hMSCs than in HUVECs under concentrations of arsenite between 15 and 25 μM. The Annexin V apoptosis assay further confirmed this finding. Cytokine array assay for As-conditioned media revealed an elevated vascular endothelial growth factor (VEGF level secreted by MSCs, which is crucial for HUVEC survival and was evaluated by an siRNA VEGF knockdown test. In the in vivo study, we demonstrated early apoptotic changes in the anterior tibial vessels of As-injected SD rats with a Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assay, but these apoptotic changes were less frequently observed upon MSCs transplantation, indicating that the cytoprotective effect of MSCs successfully protected against As-induced peripheral vasculopathy. The feasibility of MSCs to treat and /or prevent the progression of As-induced vasculopathy is justified. Further clinical studies are required to demonstrate the therapeutic efficacy of MSCs in patients suffering from As intoxication with

  8. Peripheral blood CD34+ cell count as a predictor of adequacy of hematopoietic stem cell collection for autologous transplantation

    Directory of Open Access Journals (Sweden)

    Combariza, Juan F.

    2016-10-01

    Full Text Available Introduction: In order to carry out an autologous transplantation, hematopoietic stem cells should be mobilized to peripheral blood and later collected by apheresis. The CD34+ cell count is a tool to establish the optimal time to begin the apheresis procedure. Objective: To evaluate the association between peripheral blood CD34+ cell count and the successful collection of hematopoietic stem cells. Materials and methods: A predictive test evaluation study was carried out to establish the usefulness of peripheral blood CD34+ cell count as a predictor of successful stem cell collection in patients that will receive an autologous transplantation. Results: 77 patients were included (median age: 49 years; range: 5-66. The predominant baseline diagnosis was lymphoma (53.2 %. The percentage of patients with successful harvest of hematopoietic stem cells was proportional to the number of CD34+cells in peripheral blood at the end of the mobilization procedure. We propose that more than 15 CD34+cells/μL must be present in order to achieve an adequate collection of hematopoietic stem cells. Conclusion: Peripheral blood CD34+ cell count is a useful tool to predict the successful collection of hematopoietic stem cells.

  9. Nipah virus infects specific subsets of porcine peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Beata Stachowiak

    Full Text Available Nipah virus (NiV, a zoonotic paramyxovirus, is highly contagious in swine, and can cause fatal infections in humans following transmission from the swine host. The main viral targets in both species are the respiratory and central nervous systems, with viremia implicated as a mode of dissemination of NiV throughout the host. The presented work focused on the role of peripheral blood mononuclear cells (PBMC in the viremic spread of the virus in the swine host. B lymphocytes, CD4-CD8-, as well as CD4+CD8- T lymphocytes were not permissive to NiV, and expansion of the CD4+CD8- cells early post infection was consistent with functional humoral response to NiV infection observed in swine. In contrast, significant drop in the CD4+CD8- T cell frequency was observed in piglets which succumbed to the experimental infection, supporting the hypothesis that antibody development is the critical component of the protective immune response. Productive viral replication was detected in monocytes, CD6+CD8+ T lymphocytes and NK cells by recovery of infectious virus in the cell supernatants. Virus replication was supported by detection of the structural N and the non-structural C proteins or by detection of genomic RNA increase in the infected cells. Infection of T cells carrying CD6 marker, a strong ligand for the activated leukocyte cell adhesion molecule ALCAM (CD166 highly expressed on the microvascular endothelial cell of the blood-air and the blood-brain barrier may explain NiV preferential tropism for small blood vessels of the lung and brain.

  10. Effects of exogenous and endogenous IL-2 on irradiated human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    Zhang Lansheng; Wang Ninghai; Luan Meiling

    1993-08-01

    Human peripheral blood lymphocytes were irradiated with 1 to 40 Gy of γ-ray, and then cultured with PHA to prepare supernatant containing IL-2 for observation of kinetics of endogenous IL-2 production and reversion of lymphocyte proliferation after adding a highly purified IL-2. IL-2 activity was determined by the ability to sustain IL-2 dependent cell line (CTLL), lymphocyte proliferation was determined by 3 H-TdR incorporation and T lymphocyte subsets by monoclonal antibodies. The experimental results showed that lymphocytes exposed to 60 Co synthesized less DNA than nonirradiated lymphocytes. The inhibitory effect can partially reversed by purified IL-2 at the γ-ray dose range of 1 to 10 Gy, while irradiation with 2.5 Gy resulted in a reduction of T cells and T subsets, and increase in CD + 4 /CD + 8 ratio. The ratio of subsets recovered after adding IL-2. The kinetics of IL-2 production showed that the endogenous IL-2 production rose markedly with increasing dose of irradiation at the range of 1 to 10 Gy, and the peak of IL-2 production was at the γ-ray dose of 10 Gy

  11. Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

    Science.gov (United States)

    Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

    1989-04-01

    Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

  12. Natural killer (NK)-cell activity in sorted subsets of peripheral blood mononuclear cells from patients with severe combined immunodeficiency

    NARCIS (Netherlands)

    ten Berge, R. J.; Schellekens, P. T.; Budding-Koppenol, A.; Dooren, L. J.; Vossen, J. M.

    1987-01-01

    Natural killer-cell activity for K562 target cells was measured in 13 patients with severe combined immunodeficiency before bone marrow transplantation. Both unseparated peripheral blood mononuclear cells and sorted cell subsets (B73.1 positive, B73.1 negative, OKT3 positive, OKT3 negative) were

  13. Generation of integration-free induced pluripotent stem cells (GZHMUi001-A by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient

    Directory of Open Access Journals (Sweden)

    Yuchang Chen

    2017-08-01

    Full Text Available 47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease.

  14. Distribution of Peripheral Memory T Follicular Helper Cells in Patients with Schistosomiasis Japonica.

    Directory of Open Access Journals (Sweden)

    Xiaojun Chen

    Full Text Available Schistosomiasis is a helminthic disease that affects more than 200 million people. An effective vaccine would be a major step towards eliminating the disease. Studies suggest that T follicular helper (Tfh cells provide help to B cells to generate the long-term humoral immunity, which would be a crucial component of successful vaccines. Thus, understanding the biological characteristics of Tfh cells in patients with schistosomiasis, which has never been explored, is essential for vaccine design.In this study, we investigated the biological characteristics of peripheral memory Tfh cells in schistosomiasis patients by flow cytometry. Our data showed that the frequencies of total and activated peripheral memory Tfh cells in patients were significantly increased during Schistosoma japonicum infection. Moreover, Tfh2 cells, which were reported to be a specific subpopulation to facilitate the generation of protective antibodies, were increased more greatly than other subpopulations of total peripheral memory Tfh cells in patients with schistosomiasis japonica. More importantly, our result showed significant correlations of the percentage of Tfh2 cells with both the frequency of plasma cells and the level of IgG antibody. In addition, our results showed that the percentage of T follicular regulatory (Tfr cells was also increased in patients with schistosomiasis.Our report is the first characterization of peripheral memory Tfh cells in schistosomasis patients, which not only provides potential targets to improve immune response to vaccination, but also is important for the development of vaccination strategies to control schistosomiasis.

  15. Assessment of in vitro radiosensitivity of human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    Knox, S.J.; Shifrine, M.; Rosenblatt, L.S.

    1980-01-01

    The proliferative capacity of sensitive lymphocyte progenitor cells, from thirty-one clinically normal adults, was evaluated following in vitro x-irradiation (0-400R). Radiation effects were studied using both whole blood and lymphocyte-enriched mononuclear cell fractions in the lymphocyte stimulation test and colony formation assay with 6 different mitogens and antigens. Radiation dose-response survival curves were determined for the different test groups. The sensitivity of the different assay systems is compared and normative values are presented that may be used for comparison purposes to determine the relative radiosensitivity of atypical individuals and groups of individuals

  16. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  17. Alterations on peripheral B cell subsets following an acute uncomplicated clinical malaria infection in children

    Directory of Open Access Journals (Sweden)

    Ng'ang'a Zipporah W

    2008-11-01

    Full Text Available Abstract Background The effects of Plasmodium falciparum on B-cell homeostasis have not been well characterized. This study investigated whether an episode of acute malaria in young children results in changes in the peripheral B cell phenotype. Methods Using flow-cytofluorimetric analysis, the B cell phenotypes found in the peripheral blood of children aged 2–5 years were characterized during an episode of acute uncomplicated clinical malaria and four weeks post-recovery and in healthy age-matched controls. Results There was a significant decrease in CD19+ B lymphocytes during acute malaria. Characterization of the CD19+ B cell subsets in the peripheral blood based on expression of IgD and CD38 revealed a significant decrease in the numbers of naive 1 CD38-IgD+ B cells while there was an increase in CD38+IgD- memory 3 B cells during acute malaria. Further analysis of the peripheral B cell phenotype also identified an expansion of transitional CD10+CD19+ B cells in children following an episode of acute malaria with up to 25% of total CD19+ B cell pool residing in this subset. Conclusion Children experiencing an episode of acute uncomplicated clinical malaria experienced profound disturbances in B cell homeostasis.

  18. Effector/memory CD8+ T cells synergize with co-stimulation competent macrophages to trigger autoimmune peripheral neuropathy.

    Science.gov (United States)

    Yang, Mu; Shi, Xiang Qun; Peyret, Corentin; Oladiran, Oladayo; Wu, Sonia; Chambon, Julien; Fournier, Sylvie; Zhang, Ji

    2018-04-05

    Autoimmune peripheral neuropathy (APN) such as Guillain Barre Syndrome (GBS) is a debilitating illness and sometimes life threatening. The molecular and cellular mechanisms remain elusive but exposure to environmental factors including viral/bacterial infection and injury is highly associated with disease incidence. We demonstrated previously that both male and female B7.2 (CD86) transgenic L31 and L31/CD4KO mice develop spontaneous APN. Here we further reveal that CD8 + T cells in these mice exhibit an effector/memory phenotype, which bears a resemblance to the CD8 + T cell response following persistent cytomegalovirus (CMV) infection in humans and mice, whilst CMV has been considered as one of the most relevant pathogens in APN development. These activated, peripheral myelin Ag specific CD8 + T cells are required for the disease initiation. While an injury to a peripheral nerve results in Wallerian degeneration in control littermates, the same injury accelerates the development of APN in other non-injured nerves of L31 mice which have a predisposed inflammatory background consisting of effector/memory CD8 + T (CD8 + T EM ) cells. However, CD8 + T EM cells alone are not sufficient. A certain threshold of B7.2 expression on nerve macrophages is an additional requisite. Our findings reveal that indeed, the synergism between CD8 + T EM cells and co-stimulation competent macrophages is crucial in inducing autoimmune-mediated peripheral neuropathy. The identification of decisive molecular/cellular players connecting environmental triggers and the occurrence of APN provides opportunities to prevent disease onset, reduce relapses and develop new therapeutic strategies. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

  19. Human innate lymphoid cells.

    Science.gov (United States)

    Mjösberg, Jenny; Spits, Hergen

    2016-11-01

    Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune responses. As such, ILCs make up interesting therapeutic targets for several diseases. In patients with allergy and asthma, group 2 innate lymphoid cells produce high amounts of IL-5 and IL-13, thereby contributing to type 2-mediated inflammation. Group 3 innate lymphoid cells are implicated in intestinal homeostasis and psoriasis pathology through abundant IL-22 production, whereas group 1 innate lymphoid cells are accumulated in chronic inflammation of the gut (inflammatory bowel disease) and lung (chronic obstructive pulmonary disease), where they contribute to IFN-γ-mediated inflammation. Although the ontogeny of mouse ILCs is slowly unraveling, the development of human ILCs is far from understood. In addition, the growing complexity of the human ILC family in terms of previously unrecognized functional heterogeneity and plasticity has generated confusion within the field. Here we provide an updated view on the function and plasticity of human ILCs in tissue homeostasis and disease. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  20. Adult Stem Cell Based Enhancement of Nerve Conduit for Peripheral Nerve Repair

    Science.gov (United States)

    2016-10-01

    accompanied by injuries to peripheral nerves; if not repaired, the trauma can lead to significant dysfunction and disability . While nerves have the ability to...recovery, minimized disability , and increased quality of life for our wounded warriors. 2. KEYWORDS: Stem Cell, Nerve Conduit, Peripheral Nerve...would be a paradigm shift away from ordering X-rays at 10-12 weeks and only ordering a CT scan. It has the potential to change the standard of care

  1. Isolation of Human Skin Dendritic Cell Subsets.

    Science.gov (United States)

    Gunawan, Merry; Jardine, Laura; Haniffa, Muzlifah

    2016-01-01

    Dendritic cells (DCs) are specialized leukocytes with antigen-processing and antigen-presenting functions. DCs can be divided into distinct subsets by anatomical location, phenotype and function. In human, the two most accessible tissues to study leukocytes are peripheral blood and skin. DCs are rare in human peripheral blood (skin covering an average total surface area of 1.8 m(2) has approximately tenfold more DCs than the average 5 L of total blood volume (Wang et al., J Invest Dermatol 134:965-974, 2014). DCs migrate spontaneously from skin explants cultured ex vivo, which provide an easy method of cell isolation (Larsen et al., J Exp Med 172:1483-1493, 1990; Lenz et al., J Clin Invest 92:2587-2596, 1993; Nestle et al., J Immunol 151:6535-6545, 1993). These factors led to the extensive use of skin DCs as the "prototype" migratory DCs in human studies. In this chapter, we detail the protocols to isolate DCs and resident macrophages from human skin. We also provide a multiparameter flow cytometry gating strategy to identify human skin DCs and to distinguish them from macrophages.

  2. Melanin-concentrating hormone in peripheral circulation in the human.

    Science.gov (United States)

    Naufahu, J; Alzaid, F; Fiuza Brito, M; Doslikova, B; Valencia, T; Cunliffe, A; Murray, J F

    2017-03-01

    Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide with a well-characterised role in energy homeostasis and emergent roles in diverse physiologic functions such as arousal, mood and reproduction. Work to date has predominantly focused on its hypothalamic functions using animal models; however, little attention has been paid to its role in circulation in humans. The aims of this study were to (a) develop a radioimmunoassay for the detection of MCH in human plasma; (b) establish reference ranges for circulating MCH and (c) characterise the pattern of expression of circulating MCH in humans. A sensitive and specific RIA was developed and cross-validated by RP-HPLC and MS. The effective range was 19.5-1248 pg MCH/mL. Blood samples from 231 subjects were taken to establish a reference range of 19.5-55.4 pg/mL for fasting MCH concentrations. There were no significant differences between male and female fasting MCH concentrations; however, there were correlations between MCH concentrations and BMI in males and females with excess fat (P < 0.001 and P = 0.020) and between MCH concentrations and fat mass in females with excess fat (P = 0.038). Plasma MCH concentrations rose significantly after feeding in a group of older individuals (n = 50, males P = 0.006, females P = 0.023). There were no robust significant correlations between fasting or post-prandial MCH and resting metabolic rate, plasma glucose, insulin or leptin concentrations although there were correlations between circulating MCH and leptin concentrations in older individuals (P = 0.029). These results indicate that the role of circulating MCH may not be reflective of its regulatory hypothalamic role. © 2017 Society for Endocrinology.

  3. Peripheral blood stem cell harvest in patients with limited stage small-cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Katakami, Nobuyuki; Takakura, Shunji; Fujii, Hiroshi; Nishimura, Takashi; Umeda, Bunichi [Kobe City General Hospital (Japan)

    2000-06-01

    Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) induced mobilization of peripheral blood stem cells (PBSC) was performed in patients with limited stage small-cell lung cancer. Chemotherapy consisted of cisplatin/etoposide or cisplatin/adriamycin/etoposide. The amounts of CD34 positive cells and granulocyte-macrophage colony forming units (CFU-GM) collected during 2-3 courses of apheresis were 3.1{+-}2.9 x 10{sup 6}/kg (n=10) and 3.1{+-}1.5 x 10{sup 5}/kg (n=8) , respectively. Adequate amounts of PBSC were also harvested even in patients treated with concurrent chemoradiotherapy. Eight patients were successfully treated with high-dose chemotherapy consisting of ifosfamide, carboplatin and etoposide with PBSC transfusion. The patients'-bone marrow reconstruction was rapid and no treatment-related death was observed. (author)

  4. Florid cemento-osseous dysplasia and peripheral giant cell granuloma in a patient with neurofibromatosis 1.

    Science.gov (United States)

    Sarmento, Dmitry José de Santana; Carvalho, Sérgio Henrique Gonçalves de; Araújo, José Cadmo Wanderley Peregrino de; Carvalho, Marianne de Vasconcelos; Silveira, Éricka Janine Dantas da

    2017-01-01

    We report a 35-year-old mulatto female patient with neurofibromatosis Type 1 who presented with facial asymmetry. The patient had two lesions: florid cemento-osseous dysplasia associated with peripheral giant cell granuloma. She was referred for surgical treatment of the peripheral giant cell granuloma and the florid cemento-osseous dysplasia was treated conservatively by a multidisciplinary team. So far, no changes have been observed in the patient's clinical status. We observed no recurrence of peripheral giant cell granuloma. To the best of our knowledge, the present case is the first report of a patient with neurofibromatosis Type 1 associated with a giant cell lesion and florid cemento-osseous dysplasia.

  5. Florid cemento-osseous dysplasia and peripheral giant cell granuloma in a patient with neurofibromatosis 1*

    Science.gov (United States)

    Sarmento, Dmitry José de Santana; de Carvalho, Sérgio Henrique Gonçalves; de Araújo Filho, José Cadmo Wanderley Peregrino; Carvalho, Marianne de Vasconcelos; da Silveira, Éricka Janine Dantas

    2017-01-01

    We report a 35-year-old mulatto female patient with neurofibromatosis Type 1 who presented with facial asymmetry. The patient had two lesions: florid cemento-osseous dysplasia associated with peripheral giant cell granuloma. She was referred for surgical treatment of the peripheral giant cell granuloma and the florid cemento-osseous dysplasia was treated conservatively by a multidisciplinary team. So far, no changes have been observed in the patient's clinical status. We observed no recurrence of peripheral giant cell granuloma. To the best of our knowledge, the present case is the first report of a patient with neurofibromatosis Type 1 associated with a giant cell lesion and florid cemento-osseous dysplasia. PMID:28538890

  6. Trophic Effects of Dental Pulp Stem Cells on Schwann Cells in Peripheral Nerve Regeneration.

    Science.gov (United States)

    Yamamoto, Tsubasa; Osako, Yohei; Ito, Masataka; Murakami, Masashi; Hayashi, Yuki; Horibe, Hiroshi; Iohara, Koichiro; Takeuchi, Norio; Okui, Nobuyuki; Hirata, Hitoshi; Nakayama, Hidenori; Kurita, Kenichi; Nakashima, Misako

    2016-01-01

    Recently, mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient, which has high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSC transplantation on peripheral nerve regeneration. Effects of MDPSC transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSC transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labeling demonstrated reduced apoptosis and increased proliferation in resident Schwann cells in the MDPSC transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration, and antiapoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.

  7. Activation of peripheral blood mononuclear cells by dengue virus infection depotentiates balapiravir.

    Science.gov (United States)

    Chen, Yen-Liang; Abdul Ghafar, Nahdiyah; Karuna, Ratna; Fu, Yilong; Lim, Siew Pheng; Schul, Wouter; Gu, Feng; Herve, Maxime; Yokohama, Fumiaki; Wang, Gang; Cerny, Daniela; Fink, Katja; Blasco, Francesca; Shi, Pei-Yong

    2014-02-01

    In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC(50)). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC(50) was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.

  8. The thin-section CT, pathological and clinical findings of peripheral small squamous cell lung carcinomas

    International Nuclear Information System (INIS)

    Yamamoto, Takahito; Saito, Haruhiro; Kondo, Tetsuro

    2010-01-01

    We analyzed thin-section CT, pathological, and clinical findings of peripheral lung squamous cell carcinomas, with diameters of less than 20 mm and compared these findings with solid type adenocarcinomas. CT findings of polygonal shapes, notches, pleural thickness, and cavities are more frequently found in squamous cell carcinomas than in adenocarcinomas. The pathological types can be classified in two groups: Solid types, Scirrhous types. The 5 year survival rate after resection is 64.5%, which is poorer than survival rate for solid type adenocarcinomas. It is vital to diagnose and treat peripheral squamous cell carcinomas as early as possible. (author)

  9. Frequency and genetic characterization of V(DD)J recombinants in the human peripheral blood antibody repertoire.

    Science.gov (United States)

    Briney, Bryan S; Willis, Jordan R; Hicar, Mark D; Thomas, James W; Crowe, James E

    2012-09-01

    Antibody heavy-chain recombination that results in the incorporation of multiple diversity (D) genes, although uncommon, contributes substantially to the diversity of the human antibody repertoire. Such recombination allows the generation of heavy chain complementarity determining region 3 (HCDR3) regions of extreme length and enables junctional regions that, because of the nucleotide bias of N-addition regions, are difficult to produce through normal V(D)J recombination. Although this non-classical recombination process has been observed infrequently, comprehensive analysis of the frequency and genetic characteristics of such events in the human peripheral blood antibody repertoire has not been possible because of the rarity of such recombinants and the limitations of traditional sequencing technologies. Here, through the use of high-throughput sequencing of the normal human peripheral blood antibody repertoire, we analysed the frequency and genetic characteristics of V(DD)J recombinants. We found that these recombinations were present in approximately 1 in 800 circulating B cells, and that the frequency was severely reduced in memory cell subsets. We also found that V(DD)J recombination can occur across the spectrum of diversity genes, indicating that virtually all recombination signal sequences that flank diversity genes are amenable to V(DD)J recombination. Finally, we observed a repertoire bias in the diversity gene repertoire at the upstream (5') position, and discovered that this bias was primarily attributable to the order of diversity genes in the genomic locus. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

  10. The peripheral NK cell repertoire after kidney transplantation is modulated by different immunosuppressive drugs

    Directory of Open Access Journals (Sweden)

    Christine eNeudoerfl

    2013-02-01

    Full Text Available In the context of kidney transplantation, little is known about the involvement of NK cells in the immune reaction leading to either rejection or immunological tolerance under immunosuppression. Therefore, the peripheral NK cell repertoire of patients after kidney transplantation was investigated in order to identify NK cell subsets that may be associated with the individual immune status at the time of their protocol biopsies for histopathological evaluation of the graft. Alterations in the peripheral NK cell repertoire could be correlated to the type of immunosuppression, i.e. calcineurin-inhibitors like CyclosporinA vs. Tacrolimus with or without addition of mTOR inhibitors. Here, we could demonstrate that the NK cell repertoire in peripheral blood of kidney transplant patients differs significantly from healthy individuals. The presence of donor-specific antibodies was associated with reduced numbers of CD56dim NK cells. Moreover, in patients, down-modulation of CD16 and CD6 on CD56dim NK cells was observed with significant differences between CyclosporinA- and Tac-treated patients. Tac-treatment was associated with decreased CD69, HLA-DR and increased CD94/NKG2A expression in CD56dim NK cells indicating that the quality of the immunosuppressive treatment impinges on the peripheral NK cell repertoire. In vitro studies with PBMC of healthy donors showed that this modulation of CD16, CD6, CD69, and HLA-DR could also be induced experimentally. The presence of calcineurin or mTOR inhibitors had also functional consequences regarding degranulation and IFN--production against K562 target cells, respectively. In summary, we postulate that the NK cell composition in peripheral blood of kidney transplanted patients represents an important hallmark of the efficacy of immunosuppression and may be even informative for the immune status after transplantation in terms of rejection vs. drug-induced allograft tolerance. Thus,NK cells can serve as sensors

  11. ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Keiko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute of Life Sciences for the Next Generation of Women Scientists, Fukuoka University, Fukuoka (Japan); Fujimoto, Takahiro [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Okamura, Tadashi [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ogawa, Masahiro [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tanaka, Yoko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Mototani, Yasumasa; Goto, Motohito [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ota, Takeharu; Matsuzaki, Hiroshi [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Kuroki, Masahide [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tsunoda, Toshiyuki [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Sasazuki, Takehiko [Institute for Advanced Study, Kyushu University, Fukuoka (Japan); Shirasawa, Senji, E-mail: sshirasa@fukuoka-u.ac.jp [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

  12. Peripheral mononuclear cell resistin mRNA expression is increased in type 2 diabetic women.

    Science.gov (United States)

    Tsiotra, Panayoula C; Tsigos, Constantine; Anastasiou, Eleni; Yfanti, Eleni; Boutati, Eleni; Souvatzoglou, Emmanouil; Kyrou, Ioannis; Raptis, Sotirios A

    2008-01-01

    Resistin has been shown to cause insulin resistance and to impair glucose tolerance in rodents, but in humans its physiological role still remains elusive. The aim of this study was to examine whether resistin mRNA expression in human peripheral mononuclear cells (PBMCs) and its corresponding plasma levels are altered in type 2 diabetes. Resistin mRNA levels were easily detectable in human PBMC, and found to be higher in DM2 compared to healthy women (P = .05). Similarly, mononuclear mRNA levels of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 were all significantly higher in DM2 compared to control women (P DM2 women (P = .051), and overall, they correlated significantly with BMI (r = 0.406, P = .010) and waist circumference (r = 0.516, P = .003), but not with fasting insulin levels or HOMA-IR. Resistin mRNA expression is increased in PBMC from DM2 women, together with increased expression of the inflammatory cytokines IL-1beta, TNF-alpha, and IL-6, independent of obesity. These results suggest that resistin and cytokines might contribute to the low-grade inflammation and the increased atherogenic risk observed in these patients.

  13. Peripheral Mononuclear Cell Resistin mRNA Expression Is Increased in Type 2 Diabetic Women

    Directory of Open Access Journals (Sweden)

    Panayoula C. Tsiotra

    2008-01-01

    Full Text Available Resistin has been shown to cause insulin resistance and to impair glucose tolerance in rodents, but in humans its physiological role still remains elusive. The aim of this study was to examine whether resistin mRNA expression in human peripheral mononuclear cells (PBMCs and its corresponding plasma levels are altered in type 2 diabetes. Resistin mRNA levels were easily detectable in human PBMC, and found to be higher in DM2 compared to healthy women (P=.05. Similarly, mononuclear mRNA levels of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 were all significantly higher in DM2 compared to control women (P<.001. The corresponding plasma resistin levels were slightly, but not significantly, increased in DM2 women (P=.051, and overall, they correlated significantly with BMI (r=0.406, P=.010 and waist circumference (r=0.516, P=.003, but not with fasting insulin levels or HOMA-IR. Resistin mRNA expression is increased in PBMC from DM2 women, together with increased expression of the inflammatory cytokines IL-1β, TNF-α, and IL-6, independent of obesity. These results suggest that resistin and cytokines might contribute to the low-grade inflammation and the increased atherogenic risk observed in these patients.

  14. Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair

    NARCIS (Netherlands)

    Shakhbazau, A.; Kawasoe, J.; Hoyng, S.A.; Kumar, R.; van Minnen, J.; Verhaagen, J.; Midha, R.

    2012-01-01

    Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a

  15. Peripheral blood cells among community residents living near nuclear power plants

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yuan-Teh; Hsu, Hsiu-Ching; Chien, Kuo-Liong; Yang, Chi-Yu; Chen, Wen Jone [Department of Internal Medicine, National Taiwan University College of Medicine, No. 7 Chungshan South Road, 10020 Taipei (Taiwan, Province of China); Sung, Fung C. [Institute of Environmental Health, National Taiwan University College of Public Health, Taipei (Taiwan, Province of China); Lin, Ruey S. [Institute of Epidemiology, National Taiwan University College of Public Health, Taipei (Taiwan, Province of China)

    2001-12-03

    Information about hematopoieses as a result of exposure to very low levels of radiation is scarce. To investigate the human hematopoietic effect of very low level radiation exposure, measurements of peripheral blood components were performed among 3602 men and women, aged 35 and above, living in a community near two nuclear power installations in Chinshan, Taiwan. The radiation level that each individual was exposed to was represented by a surrogate level, '+', a transformed distance from each individual's residence to the two power plants D{sub 1} and D{sub 2}. In addition to comparing average hematology measurements, multiple regression analyses were done to include age, gender, smoking, drinking status and the surrogate radiation exposure level as independent variables. Univariate and bivariate analyses showed that the hematology measurements had significant associations with age, gender, smoking or drinking. The multiple regression analyses revealed that significant positive associations with '+' were found for hemoglobin, hematocrit, platelet, white blood cell and red blood cell. The platelet count might increase for 208.7x10{sup 3}/{mu}l if the exposure from the nuclear plants increased by one exposure unit. This type of association implies that those who lived closer to the nuclear power installation had a higher blood cell count; we suspect that this could be a type of radiation hormesis.

  16. Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage.

    Science.gov (United States)

    Chen, Liming; Liu, Yinghui; Dong, Liangliang; Chu, Xiaoxia

    2015-03-01

    Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p edaravone offers protection from radiation-induced cytogenetic alterations.

  17. Catabolism of exogenously supplied thymidine to thymine and dihydrothymine by platelets in human peripheral blood

    International Nuclear Information System (INIS)

    Pero, R.W.; Johnson, D.; Olsson, A.

    1984-01-01

    The interference of platelets with the estimation of unscheduled DNA synthesis in human peripheral mononuclear leukocytes following genotoxic exposure was studied. A 96% reduction in the unscheduled DNA synthesis value was achieved by incubating [ 3 H]thymidine with platelet-rich plasma for 5 hr at 37 degrees. Using radioactive thymine-containing compounds, together with quantitative analyses based on thin-layer and ion-exchange chromatographies, we have shown that thymidine was converted to thymine which, in turn, was converted to dihydrothymine in platelet-rich plasma. The enzymes responsible were separated from platelet lysates by gel filtration and were identified as thymidine phosphorylase and dihydrothymine dehydrogenase. The phosphorylase reversibly catalyzed the formation of thymine from thymidine and converted bromodeoxyuridine to bromouracil. The dehydrogenase reversibly catalyzed the interconversion of thymine and dihydrothymine in a reaction dependent on NADP(H), and it was inhibited by diazouracil and by thymine. Nearly all the thymidine-catabolizing activity found in whole blood samples supplied exogenously with thymidine was accounted for by the platelets. Since most genetic toxicological tests that use blood samples do not involve removing platelets from the blood cell cultures, then it is concluded that precautions should be taken in the future to determine the influence of platelets on these test systems. This is particularly true for methods dependent on thymidine pulses such as unscheduled DNA synthesis, or those dependent on bromodeoxyuridine, such as sister chromatid exchanges, since this nucleoside is also a substrate for thymidine phosphorylase

  18. Distinct kinetics of memory B-cell and plasma-cell responses in peripheral blood following a blood-stage Plasmodium chabaudi infection in mice.

    Directory of Open Access Journals (Sweden)

    Eunice W Nduati

    2010-11-01

    Full Text Available B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC. To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(- IgM(- CD19(+ B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+ plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25 or secondary (day 10 infection. CD138(+ plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood.

  19. Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.

    Science.gov (United States)

    Zhu, Peixuan; Stanton, Melissa L; Castle, Erik P; Joseph, Richard W; Adams, Daniel L; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei; Ho, Thai H

    2016-07-02

    Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.

  20. C-Reactive Protein Impairs Dendritic Cell Development, Maturation, and Function: Implications for Peripheral Tolerance

    Directory of Open Access Journals (Sweden)

    Rachel V. Jimenez

    2018-03-01

    Full Text Available C-reactive protein (CRP is the prototypical acute phase reactant, increasing in blood concentration rapidly and several-fold in response to inflammation. Recent evidence indicates that CRP has an important physiological role even at low, baseline levels, or in the absence of overt inflammation. For example, we have shown that human CRP inhibits the progression of experimental autoimmune encephalomyelitis (EAE in CRP transgenic mice by shifting CD4+ T cells away from the TH1 and toward the TH2 subset. Notably, this action required the inhibitory Fcγ receptor IIB (FcγRIIB, but did not require high levels of human CRP. Herein, we sought to determine if CRP’s influence in EAE might be explained by CRP acting on dendritic cells (DC; antigen presenting cells known to express FcγRIIB. We found that CRP (50 µg/ml reduced the yield of CD11c+ bone marrow-derived DCs (BMDCs and CRP (≥5 μg/ml prevented their full expression of major histocompatibility complex class II and the co-stimulatory molecules CD86 and CD40. CRP also decreased the ability of BMDCs to stimulate antigen-driven proliferation of T cells in vitro. Importantly, if the BMDCs were genetically deficient in mouse FcγRIIB then (i the ability of CRP to alter BMDC surface phenotype and impair T cell proliferation was ablated and (ii CD11c-driven expression of a human FCGR2B transgene rescued the CRP effect. Lastly, the protective influence of CRP in EAE was fully restored in mice with CD11c-driven human FcγRIIB expression. These findings add to the growing evidence that CRP has important biological effects even in the absence of an acute phase response, i.e., CRP acts as a tonic suppressor of the adaptive immune system. The ability of CRP to suppress development, maturation, and function of DCs implicates CRP in the maintenance of peripheral T cell tolerance.

  1. Cytokine release from human peripheral blood leucocytes incubated with endotoxin with and without prior infection with influenza virus

    DEFF Research Database (Denmark)

    Banner, Jytte; Smith, H; Sweet, C

    1993-01-01

    Previous work with a neonatal ferret model for human SIDS had indicated that inflammation caused by a combination of influenza virus and bacterial endotoxin may be a cause of human SIDS. To determine whether cytokines may be involved in this inflammatory response, levels of interleukin (IL)-1 beta......, IL-6 and tumour necrosis factor (TNF)-alpha were examined, using ELISA assays, in culture supernatants of human peripheral blood leucocytes infected with influenza virus and subsequently incubated with endotoxin. Levels of TNF-alpha were increased compared to cells incubated with virus or endotoxin...... alone. Levels of IL-1 beta were also increased whereas levels of IL-6 were generally not enhanced. Cytokines appeared within 1-2 h of stimulation with virus or endotoxin and increased subsequently to reach maximum titres between 16 and 20 h post treatment. While levels of cytokine were much lower when...

  2. Sensitivity of human peripheral lymphocyte chromosomes to various X-ray doses and subsequent storage in Plexiglass or glass containers

    International Nuclear Information System (INIS)

    Ivanov, B.; Bulanova, M.; Geogieva, I.

    1979-01-01

    A study was performed to determine whether chromosomal aberrations produced in vitro by various X-ray doses in human lymphocytes were affected by post-irradiation storage of the blood in plastic or glass containers. Following X-ray doses of up to 400 R, the yields of cells with aberrations and the incidence of dicentrics, rings, interstitial deletions, symmetrical changes and chromosome fragments increased with dose. After storage of the irradiated lymphocytes in either Plexiglass or glass, the values for exchange aberrations, deletions and aberrant cells were compared. The only statistically significant difference was a slight increase in the percentage of aberrant cells stored in the plastic containers at the 400 R dose level. It was concluded that plastics appear to have a sensitizing effect on the genetic structure of the peripheral lymphocyte and thus the use of this material to store blood in biological dosimetry studies should be discouraged. (U.K.)

  3. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin

    2011-01-01

    To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

  4. Mantle Cell Hyperplasia of Peripheral Lymph Nodes as Initial Manifestation of Sickle Cell Disease.

    Science.gov (United States)

    Monabbati, Ahmad; Noori, Sadat; Safaei, Akbar; Ramzi, Mani; Eghbali, Seyedsajjad; Adib, Ali

    2016-01-01

    Sickle cell disease (SCD) is a well known hemoglobinopathy with usual manifestations including anemia, hyperbilirubinemia, and vasoocclusive complications. Despite presence of mild splenomegaly in early phase of the disease, lymphadenopathy is not an often finding of SCD. We introduce an undiagnosed case of SCD who presented in third decade of his life with multiple cervical lymphadenopathies and mild splenomegaly persistent for about five years. Histopathologic examination of the resected lymph nodes showed expansion of the mantle cell layers of secondary follicles as well as several monomorphic mantle cell nodules. To rule out possibility of a malignant process involving lymph nodes, an immunohistochemical panel was ordered which was in favor of benign mantle cell hyperplasia. Immunoglobulin gene rearrangement study showed no clonal bands and confirmed benign nature of the process. Respecting mild abnormalities on Complete Blood Count, peripheral blood smear was reviewed revealing some typical sickle red blood cells as well as rare nucleated red blood cells. Solubility test for hemoglobin (HB) S was positive. Hemoglobin electrophoresis confirmed diagnosis of homozygous HbS disease.

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

    Directory of Open Access Journals (Sweden)

    Hayato Fukusumi

    2016-01-01

    Full Text Available 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.

  6. The development of human mast cells. An historical reappraisal

    International Nuclear Information System (INIS)

    Ribatti, Domenico

    2016-01-01

    The understanding of mast cell (MC) differentiation is derived mainly from in vitro studies of different stages of stem and progenitor cells. The hematopoietic lineage development of human MCs is unique compared to other myeloid-derived cells. Human MCs originate from CD34"+/CD117"+/CD13"+multipotent hematopoietic progenitors, which undergo transendothelial recruitment into peripheral tissues, where they complete differentiation. Stem cell factor (SCF) is a major chemotactic factor for MCs and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of MCs. Because MC maturation is influenced by local microenvironmental factors, different MC phenotypes can develop in different tissues and organs. - Highlights: • Human mast cells originate from CD34/CD117/CD13 positive multipotent hematopoietic progenitors. • Stem cell factor is a major chemotactic factor for mast cells and their progenitors. • Different mast cell phenotypes can develop in different tissues and organs.

  7. Human leukaemic cells

    International Nuclear Information System (INIS)

    Andronikashvili, E.L.; Mosulishvili, L.M.; Belokobil'skiy, A.I.; Kharabadze, N.E.; Shonia, N.I.; Desai, L.S.; Foley, G.E.

    1976-01-01

    The results of the determination of trace elements in nucleic acids and histones in human leukaemic cells by activation analysis are reported. The Cr 2+ , Fe 2+ , Zn 2+ , Co 2+ and Sb 2+ content of DNA and RNA of leukaemic cells compared to that of lymphocytes from a patient with infectious mononucleosis or a normal donor are shown tabulated. Similar comparisons are shown for the same trace metal content of histones isolated from the same type of cells. It is felt that the results afford further interesting speculation that trace metals may be involved in the interactions between histones and DNA (especially at the binding sites of histones to DNA), which affect transcription characteristics. (U.K.)

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

    NARCIS (Netherlands)

    Diaz-Rua, R.; Keijer, J.; Caimari, A.; Schothorst, van E.M.; Oliver, P.; Palou, A.

    2015-01-01

    Peripheral blood mononuclear cells (PBMC) are accessible in humans and their gene expression pattern was shown to reflect overall physiological response of the body to a specific stimulus, such as diet. We aimed to study the impact of sustained intake (4 months) of diets with an unbalanced

  9. Paradoxical expression of IL-28B mRNA in peripheral blood in human T-cell leukemia virus Type-1 mono-infection and co-infection with hepatitis C Virus

    Directory of Open Access Journals (Sweden)

    Kamihira Shimeru

    2012-02-01

    Full Text Available Abstract Background Human T-cell leukemia virus type-1 (HTLV-1 carriers co-infected with and hepatitis C virus (HCV have been known to be at higher risk of their related diseases than mono-infected individuals. The recent studies clarified that IL-28B polymorphism rs8099917 is associated with not only the HCV therapeutic response by IFN, but also innate immunity and antiviral activity. The aim of our research was to clarify study whether IL-28B gene polymorphism (rs8099917 is associated with HTLV-1/HCV co-infection. Results The genotyping and viral-serological analysis for 340 individuals showed that IL-28B genotype distribution of rs8099917 SNP did not differ significantly by respective viral infection status. However, the IL-28B mRNA expression level was 3.8 fold higher in HTLV-1 mono-infection than HTLV-1/HCV co-infection. The high expression level was associated with TT (OR, 6.25, whiles the low expression was associated with co-infection of the two viruses (OR, 9.5. However, there was no association between down-regulation and ATL development (OR, 0.8. Conclusion HTLV-1 mono-infection up-regulates the expression of IL-28B transcripts in genotype-dependent manner, whiles HTLV-1/HCV co-infection down-regulates regardless of ATL development.

  10. Equine peripheral blood mononuclear cells proliferate in response to tetanus toxoid antigen.

    Science.gov (United States)

    McKelvie, J; Little, S; Foster, A P; Cunningham, F M; Hamblin, A

    1998-01-01

    It has been reported that equine peripheral blood mononuclear cells (PBMNs) do not proliferate in response to tetanus toxoid (TT) (Frayne and Stokes 1995, Research in Veterinary Science 59, 79-81). Here we demonstrate that lymphocyte proliferation responses to TT, which are characteristic of a recall antigen, may be achieved under certain culture conditions. Given that TT vaccination is routinely applied to many horses, TT is a suitable antigen for the investigation of cellular immune responses by peripheral blood mononuclear cells in the horse.

  11. Human enterovirus in the gastrocnemius of patients with peripheral arterial disease.

    Science.gov (United States)

    Kim, Julian K S; Zhu, Zhen; Casale, George; Koutakis, Panagiotis; McComb, Rodney D; Swanson, Stanley; Thompson, Jonathan; Miserlis, Dimitrios; Johanning, Jason M; Haynatzki, Gleb; Pipinos, Iraklis I

    2013-08-06

    Peripheral arterial disease (PAD) is characterized by myofiber degeneration and loss of function in muscles of the lower limbs. Human enterovirus (HEV) infection has been implicated in the pathogenesis of a number of muscle diseases. However, its association with PAD has not been studied. In this study, we tested the hypothesis that infectious HEV is present in skeletal muscle of patients with PAD and is associated with severity of disease. Gastrocnemius biopsies from 37 patients with PAD and 14 controls were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity. HEV RNA was detected in 54% of the biopsies from patients with PAD but was not detected in muscle biopsies from control patients. This difference in prevalence among PAD and control patients was significant at P<0.001. Viral RNA copy numbers were increased significantly at the later stages of disease; Fontaine Stage IV (10(5.50) copies/mg muscle wet weight, at P<0.005) and Stage III (10(4.87) copies/mg, at P<0.010) compared to Stage II (10(2.50) copies/mg). Viral replication was confirmed by the presence of the negative-strand of viral RNA in all specimens positive for HEV RNA. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein. Our data identified infectious HEV in the gastrocnemius of PAD patients but not in controls. Viral copy number and prevalence of infection were higher in the later stages of disease. Our data point to the need for further studies to determine the contribution of HEV infection to the pathophysiology of PAD.

  12. Electrical Stimulation to Enhance Axon Regeneration After Peripheral Nerve Injuries in Animal Models and Humans.

    Science.gov (United States)

    Gordon, Tessa

    2016-04-01

    Injured peripheral nerves regenerate their lost axons but functional recovery in humans is frequently disappointing. This is so particularly when injuries require regeneration over long distances and/or over long time periods. Fat replacement of chronically denervated muscles, a commonly accepted explanation, does not account for poor functional recovery. Rather, the basis for the poor nerve regeneration is the transient expression of growth-associated genes that accounts for declining regenerative capacity of neurons and the regenerative support of Schwann cells over time. Brief low-frequency electrical stimulation accelerates motor and sensory axon outgrowth across injury sites that, even after delayed surgical repair of injured nerves in animal models and patients, enhances nerve regeneration and target reinnervation. The stimulation elevates neuronal cyclic adenosine monophosphate and, in turn, the expression of neurotrophic factors and other growth-associated genes, including cytoskeletal proteins. Electrical stimulation of denervated muscles immediately after nerve transection and surgical repair also accelerates muscle reinnervation but, at this time, how the daily requirement of long-duration electrical pulses can be delivered to muscles remains a practical issue prior to translation to patients. Finally, the technique of inserting autologous nerve grafts that bridge between a donor nerve and an adjacent recipient denervated nerve stump significantly improves nerve regeneration after delayed nerve repair, the donor nerves sustaining the capacity of the denervated Schwann cells to support nerve regeneration. These reviewed methods to promote nerve regeneration and, in turn, to enhance functional recovery after nerve injury and surgical repair are sufficiently promising for early translation to the clinic.

  13. Leptomeningeal Cells Transduce Peripheral Macrophages Inflammatory Signal to Microglia in Reponse to Porphyromonas gingivalis LPS

    Directory of Open Access Journals (Sweden)

    Yicong Liu

    2013-01-01

    Full Text Available We report here that the leptomeningeal cells transduce inflammatory signals from peripheral macrophages to brain-resident microglia in response to Porphyromonas gingivalis (P.g. LPS. The expression of Toll-like receptor 2 (TLR2, TLR4, TNF-α, and inducible NO synthase was mainly detected in the gingival macrophages of chronic periodontitis patients. In in vitro studies, P.g. LPS induced the secretion of TNF-α and IL-1β from THP-1 human monocyte-like cell line and RAW264.7 mouse macrophages. Surprisingly, the mean mRNA levels of TNF-α and IL-1β in leptomeningeal cells after treatment with the conditioned medium from P.g. LPS-stimulated RAW264.7 macrophages were significantly higher than those after treatment with P.g. LPS alone. Furthermore, the mean mRNA levels of TNF-α and IL-1β in microglia after treatment with the conditioned medium from P.g. LPS-stimulated leptomeningeal cells were significantly higher than those after P.g. LPS alone. These observations suggest that leptomeninges serve as an important route for transducing inflammatory signals from macrophages to microglia by secretion of proinflammatory mediators during chronic periodontitis. Moreover, propolis significantly reduced the P.g. LPS-induced TNF-α and IL-1 β production by leptomeningeal cells through inhibiting the nuclear factor-κB signaling pathway. Together with the inhibitory effect on microglial activation, propolis may be beneficial in preventing neuroinflammation during chronic periodontitis.

  14. Lower percentage of CD8+ T cells in peripheral blood of patients with sporotrichosis.

    Science.gov (United States)

    Zhu, Mingji; Xu, Yaqin; An, Lin; Jiang, Jinlan; Zhang, Xu; Jiang, Rihua

    2016-07-01

    To characterize the peripheral immunity and immunity response of patients with sporotrichosis, in this study we determined the lymphocyte subsets in the peripheral blood of Chinese patients with sporotrichosis. In this retrospective study, peripheral blood was collected from 69 sporotrichosis patients (37, fixed cutaneous form; 32 lymphocutaneous) and 66 healthy controls. Lymphocyte subsets were analyzed using flow cytometry. Compared to controls, the percentage of CD8+ T cells was lower in sporotrichosis patients. The percentage of CD8+ T cells in peripheral blood tended to become lower with disease duration and disease severity, although the difference was not statistically significant for either acute, subacute and chronic patients or fixed cutaneous and lymphocutaneous patients. Our data indicate that the decrease of CD8+ T cells in peripheral blood of patients with sporotrichosis is associated with disease severity, although the difference was not statistically significant for either duration or clinical forms of the disease. Combining antifungal agents and immunomodulators in patients with long disease duration and lymphocutaneous may be more beneficial than antifungal monotherapy. Copyright © 2016. Published by Elsevier Inc.

  15. Phytohemagglutinin (PHA) stimulation of peripheral-blood lymphocytes and stem cell take

    Energy Technology Data Exchange (ETDEWEB)

    Astaldi, G [Blood Research Foundation Center, Tortona, Italy; Karanovic, D; Vettori, P P; Karanovic, J; Piletic, O

    1974-01-01

    The effect of PHA-stimulation of peripheral-blood lymphocytes on the spleen-colony formation in irradiated rats was examined. 25-day old Wistar rats underwent total-body irradiation (600 R), and they were used as recipients. On the other hand, 2 and /sup 1///sub 2/ month old untreated Wistar rats were used as donors of peripheral-blood lymphocytes, which were obtained by sedimentation with Dextraven from defibrinated blood. Four rat lots were used. The 1st one did not receive irradiation, and was kept as ''blank control.'' The 2nd one was just irradiated and kept as ''radiated control.'' The 3rd and the 4th rat lots of the series were irradiated, but the former lot was injected i.v. with 5 x 10/sup 7/ peripheral-blood untreated lymphocytes, whereas the fourth lot was injected i.v. with the same amount of lymphocytes, which were previously incubated in vitro for 24 hrs with PHA-M (Difco). The results showed that the PHA-incubation of transplanted peripheral-blood lymphocytes significantly increases the number and size of the macroscopic spleen colonies, in relationship to the colonies which occurs after transplantation of untreated lymphocytes. Histo-cytological observation clearly showed that the colonies formed after injection of mitogen-pretreated peripheral-blood lymphocytes were predominantly of erythroid type and, then, of non-differentiated cells. Only a few of them were of a mixed type, consisting of both undifferentiated cells and erythroid cells.

  16. Intravenous Transplantation of Mesenchymal Stromal Cells to Enhance Peripheral Nerve Regeneration

    Directory of Open Access Journals (Sweden)

    Stella M. Matthes

    2013-01-01

    Full Text Available Peripheral nerve injury is a common and devastating complication after trauma and can cause irreversible impairment or even complete functional loss of the affected limb. While peripheral nerve repair results in some axonal regeneration and functional recovery, the clinical outcome is not optimal and research continues to optimize functional recovery after nerve repair. Cell transplantation approaches are being used experimentally to enhance regeneration. Intravenous infusion of mesenchymal stromal cells (MSCs into spinal cord injury and stroke was shown to improve functional outcome. However, the repair potential of intravenously transplanted MSCs in peripheral nerve injury has not been addressed yet. Here we describe the impact of intravenously infused MSCs on functional outcome in a peripheral nerve injury model. Rat sciatic nerves were transected followed, by intravenous MSCs transplantation. Footprint analysis was carried out and 21 days after transplantation, the nerves were removed for histology. Labelled MSCs were found in the sciatic nerve lesion site after intravenous injection and regeneration was improved. Intravenously infused MSCs after acute peripheral nerve target the lesion site and survive within the nerve and the MSC treated group showed greater functional improvement. The results of study suggest that nerve repair with cell transplantation could lead to greater functional outcome.

  17. Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

    Science.gov (United States)

    Denys, A; Allain, F; Foxwell, B; Spik, G

    1997-08-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

  18. Impact of Autologous and Allogeneic Stem Cell Transplantation in Peripheral T-Cell Lymphomas

    Directory of Open Access Journals (Sweden)

    Peter Reimer

    2010-01-01

    Full Text Available Peripheral T/NK-cell lymphomas (PTCLs are rare malignancies characterized by poor prognosis. So far, no standard therapy has been established, due to the lack of randomised studies. High-dose therapy and autologous stem cell transplantation (HDT-autoSCT have shown good feasibility with low toxicity in retrospective studies. In relapsing and refractory PTCL several comparison analyses suggest similar efficacy for PTCL when compared with aggressive B-cell lymphoma. In the upfront setting, prospective data show promising results with a long-lasting overall survival in a relevant subset of patients. Achieving a complete remission at transplantation seems to be the most important prognostic factor. Allogeneic stem cell transplantation (alloSCT has been investigated only as salvage treatment. Especially when using reduced intensity conditioning regimen, eligible patients seem to benefit from this approach. To define the role for upfront stem cell transplantation a randomised trial by the German High-Grade Non-Hodgkin Lymphoma Study Group comparing HDT-autoSCT and alloSCT will be initiated this year.

  19. Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay.

    Science.gov (United States)

    Al-Amrah, Hadba Jar-Allah; Aboznada, Osama Abdullah; Alam, Mohammad Zubair; ElAssouli, M-Zaki Mustafa; Mujallid, Mohammad Ibrahim; ElAssouli, Sufian Mohamad

    2014-12-01

    Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 µl of cell suspension was mixed with 85 µl of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 µl of smoke condensate and subjected for comet assay. The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.

  20. Effects of chronic morphine and morphine withdrawal on gene expression in rat peripheral blood mononuclear cells.

    OpenAIRE

    Desjardins , Stephane; Belkai , Emilie; Crete , Dominique; Cordonnier , Laurie; Scherrmann , Jean-Michel; Noble , Florence; Marie-Claire , Cynthia

    2008-01-01

    International audience; Chronic morphine treatment alters gene expression in brain structures. There are increasing evidences showing a correlation, in gene expression modulation, between blood cells and brain in psychological troubles. To test whether gene expression regulation in blood cells could be found in drug addiction, we investigated gene expression profiles in peripheral blood mononuclear (PBMC) cells of saline and morphine-treated rats. In rats chronically treated with morphine, th...

  1. Mesenchymal stem cells isolated from peripheral blood and umbilical cord Wharton’s jelly

    Directory of Open Access Journals (Sweden)

    Trivanović Drenka

    2013-01-01

    Full Text Available Introduction. Mesenchymal stem cells (MSCs are a promising tool for regenerative medicine, but due to the heterogeneity of their populations, different sources and isolation techniques, the characteristics defining MSCs are inconsistent. Objective. The aim of this study was to compare the characteristics of MSCs derived from two different human tissues: peripheral blood (PB-MSCs and umbilical cord Wharton’s Jelly (UC-MSCs. Methods. The PB-MSC and UC-MSC were isolated by adherence to plastic after gradient-density separation or an explant culture method, respectively, and compared regarding their morphology, clonogenic efficiency, proliferating rates, immunophenotype and differentiation potential. Results. MSCs derived from both sources exhibit similar morphology, proliferation capacity and multilineage (osteogenic, chondrogenic, adipogenic and myogenic differentiation potential. Differences were observed in the clonogenic capacity and the immunophenotype, since UC-MSCs showed higher CFU-F (colony-forming units-fibroblastic cloning efficiency, as well as higher embryonic markers (Nanog, Sox2, SSEA4 expression. When additional surface antigens were analyzed by flow cytometry (CD44, CD90, CD105, CD33, CD34, CD45, CD11b, CD235a or immunofluorescent labeling (vimentin, STRO-1 and α-smooth muscle actin, most appeared to have similar epitope profiles irrespective of MSC source. Conclusion. The results obtained demonstrated that both MSCs represent good alternative sources of adult MSCs that could be used in cell therapy applications. [Projekat Ministarstva nauke Republike Srbije, br. 175062

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Titanium biomaterials with complex surfaces induced aberrant peripheral circadian rhythms in bone marrow mesenchymal stromal cells.

    Science.gov (United States)

    Hassan, Nathaniel; McCarville, Kirstin; Morinaga, Kenzo; Mengatto, Cristiane M; Langfelder, Peter; Hokugo, Akishige; Tahara, Yu; Colwell, Christopher S; Nishimura, Ichiro

    2017-01-01

    Circadian rhythms maintain a high level of homeostasis through internal feed-forward and -backward regulation by core molecules. In this study, we report the highly unusual peripheral circadian rhythm of bone marrow mesenchymal stromal cells (BMSCs) induced by titanium-based biomaterials with complex surface modifications (Ti biomaterial) commonly used for dental and orthopedic implants. When cultured on Ti biomaterials, human BMSCs suppressed circadian PER1 expression patterns, while NPAS2 was uniquely upregulated. The Ti biomaterials, which reduced Per1 expression and upregulated Npas2, were further examined with BMSCs harvested from Per1::luc transgenic rats. Next, we addressed the regulatory relationship between Per1 and Npas2 using BMSCs from Npas2 knockout mice. The Npas2 knockout mutation did not rescue the Ti biomaterial-induced Per1 suppression and did not affect Per2, Per3, Bmal1 and Clock expression, suggesting that the Ti biomaterial-induced Npas2 overexpression was likely an independent phenomenon. Previously, vitamin D deficiency was reported to interfere with Ti biomaterial osseointegration. The present study demonstrated that vitamin D supplementation significantly increased Per1::luc expression in BMSCs, though the presence of Ti biomaterials only moderately affected the suppressed Per1::luc expression. Available in vivo microarray data from femurs exposed to Ti biomaterials in vitamin D-deficient rats were evaluated by weighted gene co-expression network analysis. A large co-expression network containing Npas2, Bmal1, and Vdr was observed to form with the Ti biomaterials, which was disintegrated by vitamin D deficiency. Thus, the aberrant BMSC peripheral circadian rhythm may be essential for the integration of Ti biomaterials into bone.

  4. Electrical Stimulation to Enhance Axon Regeneration After Peripheral Nerve Injuries in Animal Models and Humans

    OpenAIRE

    Gordon, Tessa

    2016-01-01

    Injured peripheral nerves regenerate their lost axons but functional recovery in humans is frequently disappointing. This is so particularly when injuries require regeneration over long distances and/or over long time periods. Fat replacement of chronically denervated muscles, a commonly accepted explanation, does not account for poor functional recovery. Rather, the basis for the poor nerve regeneration is the transient expression of growth-associated genes that accounts for declining regene...

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

    Science.gov (United States)

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

    2013-12-01

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

  6. PHENYTOIN-ASSOCIATED LYMPHOADENOPATHY MIMICKING A PERIPHERAL T-CELL LYMPHOMA

    Directory of Open Access Journals (Sweden)

    Mark E. Johns

    2010-09-01

    Full Text Available We report a case of phenytoin-induced pseudolymphoma in a 28-year-old male with a history of autism and seizure disorder.  The patient presented with bilateral cervical lymphadenopathy that was shown to be moderately to markedly FDG-avid on a whole body PET/CT scan.  Flow cytometry analysis of peripheral blood and bone marrow mononuclear cells detected identical T cell population with aberrant immunophenotype.  Additionally, a TCR beta gene was found to be clonally rearranged in both peripheral blood and bone marrow supporting a clonal origin of atypical T cells. However, no such clonal population of T-cells could be detected in a pathologic specimen obtained from an excisional biopsy of one of the patient’s cervical lymph nodes. After discontinuing the patient’s phenytoin, his lymphadenopathy has nearly completely resolved and circulation clonal T cell population disappeared with 12 months of follow-up.

  7. Human leukaemic cells

    International Nuclear Information System (INIS)

    Andronikashvili, E.L.; Mosulishvili, L.M.; Belokobil'skiy, A.I.; Kharabadze, N.E.; Shonia, N.I.; Desai, L.S.; Foley, G.E.

    1976-01-01

    Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis, and from normal donors. DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high content of Cr 2+ , Sb 2+ , Fe 2+ , Zn 2+ , whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co 2+ content was 20-fold higher than in DNA of normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn 2+ , which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr 2+ , Sb 2+ and Co 2+ , whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones. (author)

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

    DEFF Research Database (Denmark)

    Ibitokou, Samad; Oesterholt, Mayke; Brutus, Laurent

    2012-01-01

    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 ...

  9. In vitro radiation protection of peripheral blood mononuclear cells by Tetragona Clavipes Propolis

    Energy Technology Data Exchange (ETDEWEB)

    Nogueira, Naira de Souza Gomes [Universidade Federal do Amazonas (ICET/UFAM), Manaus (Brazil). Instituto de Ciências Exatas e Tecnologia; Campos, Tarcísio Passos Ribeiro; Montaño Valencia, Carlos Julio; Falcão, Patrícia Lima [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte (Brazil). Departamento de Técnicas Nucleares

    2017-07-01

    Background: Research in breast cancer has shown notable relevance in recent years, bringing serious concern on the public health policies due to its high incidence and mortality, especially in Brazil. Limitations due to the toxic effects in the normal tissues and the depletion in the immune system have often been present in the breast cancer radiotherapy and chemotherapy. It is a promise a drug development of vegetal origin that induce immune system protection in patients submitted to radiotherapy and chemotherapy, if such compounds inhibit the depletion of the circulating cell number. Objectives: To evaluate the in vitro effect of the extract of Tetragona Clavipes Propolis (TCP) in the viability of the human Peripheral Blood Mononuclear Cells (PBMC). Methods: The non-irradiated control, the irradiated control and the radiated and exposed to TCP (n=3) (GIE) groups were established. The cells were irradiated with predetermined doses of 2 and 5Gy, supplemented with TCP-extracts in aqueous solution at concentrations of 0.5, 1, 5 and 10 %. Post-irradiated sample was collected and viability assay was carried out at the time of 24, 48 and 72h. Results: A significant decrease of PBMC viability was observed after 2 and 5Gy. However, GIE showed an increase of cell viability, especially in the 5 and 10% concentrations of the extract incubated in culture, even after 5Gy. Conclusions: The findings showed that the aqueous extract of TCP is an exogenous protective agent to irradiated PBMC. This study opens a relevant perspective on the role of Propolis use as an adjuvant agent in protecting the immune system of patients undergoing breast radiotherapy. (author)

  10. In vitro radiation protection of peripheral blood mononuclear cells by Tetragona Clavipes Propolis

    International Nuclear Information System (INIS)

    Nogueira, Naira de Souza Gomes; Campos, Tarcísio Passos Ribeiro; Montaño Valencia, Carlos Julio; Falcão, Patrícia Lima

    2017-01-01

    Background: Research in breast cancer has shown notable relevance in recent years, bringing serious concern on the public health policies due to its high incidence and mortality, especially in Brazil. Limitations due to the toxic effects in the normal tissues and the depletion in the immune system have often been present in the breast cancer radiotherapy and chemotherapy. It is a promise a drug development of vegetal origin that induce immune system protection in patients submitted to radiotherapy and chemotherapy, if such compounds inhibit the depletion of the circulating cell number. Objectives: To evaluate the in vitro effect of the extract of Tetragona Clavipes Propolis (TCP) in the viability of the human Peripheral Blood Mononuclear Cells (PBMC). Methods: The non-irradiated control, the irradiated control and the radiated and exposed to TCP (n=3) (GIE) groups were established. The cells were irradiated with predetermined doses of 2 and 5Gy, supplemented with TCP-extracts in aqueous solution at concentrations of 0.5, 1, 5 and 10 %. Post-irradiated sample was collected and viability assay was carried out at the time of 24, 48 and 72h. Results: A significant decrease of PBMC viability was observed after 2 and 5Gy. However, GIE showed an increase of cell viability, especially in the 5 and 10% concentrations of the extract incubated in culture, even after 5Gy. Conclusions: The findings showed that the aqueous extract of TCP is an exogenous protective agent to irradiated PBMC. This study opens a relevant perspective on the role of Propolis use as an adjuvant agent in protecting the immune system of patients undergoing breast radiotherapy. (author)

  11. Epigenetics of peripheral B cell differentiation and the antibody response

    Directory of Open Access Journals (Sweden)

    Hong eZan

    2015-12-01

    Full Text Available Epigenetic modifications, such as histone post-translational modifications, DNA methylation, and alteration of gene expression by non-coding RNAs, including microRNAs (miRNAs and long non-coding RNAs (lncRNAs, are heritable changes that are independent from the genomic DNA sequence. These regulate gene activities and, therefore, cellular functions. Epigenetic modifications act in concert with transcription factors and play critical roles in B cell development and differentiation, thereby modulating antibody responses to foreign- and self-antigens. Upon antigen encounter by mature B cells in the periphery, alterations of these lymphocytes epigenetic landscape are induced by the same stimuli that drive the antibody response. Such alterations instruct B cells to undergo immunoglobulin class switch DNA recombination (CSR and somatic hypermutation (SHM, as well as differentiation to memory B cells or long-lived plasma cells for the immune memory. Inducible histone modifications, together with DNA methylation and miRNAs modulate the transcriptome, particularly the expression of activation-induced cytidine deaminase (AID, which is essential for CSR and SHM, and factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1. These inducible B cell-intrinsic epigenetic marks guide the maturation of antibody responses. Combinatorial histone modifications also function as histone codes to target CSR and, possibly, SHM machinery to the Ig loci by recruiting specific adaptors that can stabilize CSR/SHM factors. In addition, lncRNAs, such as recently reported lncRNA-CSR and an lncRNA generated through transcription of the S region that form G-quadruplex structures, are also important for CSR targeting. Epigenetic dysregulation in B cells, including the aberrant expression of non-coding RNAs and alterations of histone modifications and DNA methylation, can result in aberrant antibody responses to foreign antigens

  12. Toxicity to sensory neurons and Schwann cells in experimental linezolid-induced peripheral neuropathy.

    Science.gov (United States)

    Bobylev, Ilja; Maru, Helina; Joshi, Abhijeet R; Lehmann, Helmar C

    2016-03-01

    Peripheral neuropathy is a common side effect of prolonged treatment with linezolid. This study aimed to explore injurious effects of linezolid on cells of the peripheral nervous system and to establish in vivo and in vitro models of linezolid-induced peripheral neuropathy. C57BL/6 mice were treated with linezolid or vehicle over a total period of 4 weeks. Animals were monitored by weight, nerve conduction studies and behavioural tests. Neuropathic changes were assessed by morphometry on sciatic nerves and epidermal nerve fibre density in skin sections. Rodent sensory neuron and Schwann cell cultures were exposed to linezolid in vitro and assessed for mitochondrial dysfunction. Prolonged treatment with linezolid induced a mild, predominantly small sensory fibre neuropathy in vivo. Exposure of Schwann cells and sensory neurons to linezolid in vitro caused mitochondrial dysfunction primarily in neurons (and less prominently in Schwann cells). Sensory axonopathy could be partially prevented by co-administration of the Na(+)/Ca(2+) exchanger blocker KB-R7943. Clinical and pathological features of linezolid-induced peripheral neuropathy can be replicated in in vivo and in vitro models. Mitochondrial dysfunction may contribute to the axonal damage to sensory neurons that occurs after linezolid exposure. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Detection of free gastric cancer cell in peripheral and portal blood

    Energy Technology Data Exchange (ETDEWEB)

    Bang, Ho Yoon; Lee, Jong Inn [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1998-01-01

    In fact, there is no definite treatment modality after liver or hematogenous metastasis in the gastric cancer. So it is important to develop a new method to predict the high risk patients for systemic recurrence. If we can detect metastatic cell in circulation, it may be beneficial in assessing tumor progression, metastatic potential and prognosis. To establish the RT-PCR methodology for detection of CEA expressing cancer cells in peripheral and portal blood and to define the relationship between peripheral and portal blood detection rate of gastric cancer patients, we performed RT-PCR analysis with peripheral and portal blood samples from 24 patients with gastric cancer (stage Ia,b, n=3; stage II, n=2; stage IIIa, n=9; stage IIIb, n=7; stage IV, n=3) and checked serum CEA level preoperatively. Mean age was 49.2 years old and male : female was 1.2 : 2 (13:11 patients). The mean serum CEA level was 10.4 ng/ml and that was higher than normal in only 2 cases. There was no positive case of tumor cell in portal and peripheral blood using RT-PCR and CEA gene specific primer. Our results indicate that the incidence of circulating cancer cells is unexpectedly very low even in advanced gastric cancer patients. (author). 20 refs.

  14. Detection of free gastric cancer cell in peripheral and portal blood

    International Nuclear Information System (INIS)

    Bang, Ho Yoon; Lee, Jong Inn

    1998-01-01

    In fact, there is no definite treatment modality after liver or hematogenous metastasis in the gastric cancer. So it is important to develop a new method to predict the high risk patients for systemic recurrence. If we can detect metastatic cell in circulation, it may be beneficial in assessing tumor progression, metastatic potential and prognosis. To establish the RT-PCR methodology for detection of CEA expressing cancer cells in peripheral and portal blood and to define the relationship between peripheral and portal blood detection rate of gastric cancer patients, we performed RT-PCR analysis with peripheral and portal blood samples from 24 patients with gastric cancer (stage Ia,b, n=3; stage II, n=2; stage IIIa, n=9; stage IIIb, n=7; stage IV, n=3) and checked serum CEA level preoperatively. Mean age was 49.2 years old and male : female was 1.2 : 2 (13:11 patients). The mean serum CEA level was 10.4 ng/ml and that was higher than normal in only 2 cases. There was no positive case of tumor cell in portal and peripheral blood using RT-PCR and CEA gene specific primer. Our results indicate that the incidence of circulating cancer cells is unexpectedly very low even in advanced gastric cancer patients. (author). 20 refs

  15. Isolating peripheral lymphocytes by density gradient centrifugation and magnetic cell sorting.

    Science.gov (United States)

    Brosseron, Frederic; Marcus, Katrin; May, Caroline

    2015-01-01

    Combining density gradient centrifugation with magnetic cell sorting provides a powerful tool to isolate blood cells with high reproducibility, yield, and purity. It also allows for subsequent separation of multiple cell types, resulting in the possibility to analyze different purified fractions from one donor's sample. The centrifugation step divides whole blood into peripheral blood mononuclear cells (PBMC), erythrocytes, and platelet-rich plasma. In the following, lymphocyte subtypes can be consecutively isolated from the PBMC fraction. This chapter describes enrichment of erythrocytes, CD14-positive monocytes and CD3-positive T lymphocytes. Alternatively, other cell types can be targeted by using magnetic beads specific for the desired subpopulation.

  16. The importance of Notch signaling in peripheral T-cell lymphomas

    DEFF Research Database (Denmark)

    Kamstrup, Maria Rørbæk; Biskup, Edyta; Gjerdrum, Lise Mette Rahbek

    2014-01-01

    Peripheral T-cell lymphomas (PTLs) represent an area of high medical need. Previously, we demonstrated high expression of Notch, a known oncogene, in primary cutaneous anaplastic large cell lymphoma (ALCL). In this study, we performed immunohistochemical staining for Notch1 in lymph nodes from PTL...... cases) (p > 0.05). In the ALK+ ALCL cell line, Karpas-299, pharmacological inhibition of Notch with γ-secretase inhibitor (GSI) I was far more potent than with GSI IX, XX and XXI with regard to cell viability and apoptosis. In conclusion, PTL tumor cells have prominent Notch1 expression and treatment...... with Notch inhibitors has cytotoxic effects....

  17. [Mobilization of peripheral blood stem cells with plerixafor in poor mobilizer patients].

    Science.gov (United States)

    Sancho, Juan-Manuel; Duarte, Rafael; Medina, Laura; Querol, Sergi; Marín, Pedro; Sureda, Anna

    2016-09-02

    Poor mobilization of peripheral blood stem cells (CD34(+) cells) from bone marrow is a frequent reason for not reaching the autologous stem cell trasplantation (SCT) procedure in patients diagnosed with lymphoma or myeloma. Plerixafor, a reversible inhibitor of the binding of stromal cell-derived factor 1 to its cognate receptor CXCR4, has demonstrated a higher capacity for the mobilization of peripheral blood stem cells in combination with granulocyte colony stimulating factor (G-CSF) compared with G-CSF alone. For this reason, plerixafor is now indicated for poor mobilizer myeloma or lymphoma patients. Some studies have recently indicated that a pre-emptive strategy of plerixafor use during first mobilization, according to the number of CD34(+) mobilized cells in peripheral blood or to the harvested CD34(+) cells after first apheresis, could avoid mobilization failures and re-mobilizations, as well as the delay of autologous SCT. The aim of this consensus was to perform a review of published studies on pre-emptive strategy and to establish common recommendations for hospitals in Catalonia and Balearics on the use of pre-emptive plerixafor. For the Consensus, physicians from participant hospitals met to review previous studies as well as previous own data about plerixafor use. The GRADE system was used to qualify the available evidence and to establish recommendations on the use of pre-emptive plerixafor. After a review of the literature, the expert consensus recommended the administration of pre-emptive plerixafor for multiple myeloma or lymphoma patients with a CD34+ cell count lower than 10 cells/μL in peripheral blood (measured in the morning of day 4 of mobilization with G-CSF or after haematopietic recovery in the case of mobilization with chemotherapy plus G-CSF). Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

  18. Investigation of cell adhesion in chitosan membranes for peripheral nerve regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Carvalho, Cristiana R.; López-Cebral, Rita; Silva-Correia, Joana; Silva, Joana M.; Mano, João F.; Silva, Tiago H. [3B' s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark – Parque de Ciência e Tecnologia, 4805-017, Barco, Guimarães (Portugal); ICVS/3B' s - PT Government Associate Laboratory, Braga, Guimarães (Portugal); Freier, Thomas [MEDOVENT GmbH, Friedrich-Koenig-Str. 3, D-55129 Mainz (Germany); Reis, Rui L. [3B' s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark – Parque de Ciência e Tecnologia, 4805-017, Barco, Guimarães (Portugal); ICVS/3B' s - PT Government Associate Laboratory, Braga, Guimarães (Portugal); Oliveira, Joaquim M., E-mail: miguel.oliveira@dep.uminho.pt [3B' s Research Group - Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark – Parque de Ciência e Tecnologia, 4805-017, Barco, Guimarães (Portugal); ICVS/3B' s - PT Government Associate Laboratory, Braga, Guimarães (Portugal)

    2017-02-01

    Peripheral nerve injuries have produced major concerns in regenerative medicine for several years, as the recovery of normal nerve function continues to be a significant clinical challenge. Chitosan (CHT), because of its good biocompatibility, biodegradability and physicochemical properties, has been widely used as a biomaterial in tissue engineering scaffolding. In this study, CHT membranes were produced with three different Degrees of Acetylation (DA), envisioning its application in peripheral nerve regeneration. The three CHT membranes (DA I: 1%, DA II: 2%, DA III: 5%) were extensively characterized and were found to have a smooth and flat surface, with DA III membrane having slightly higher roughness and surface energy. All the membranes presented suitable mechanical properties and did not show any signs of calcification after SBF test. Biodegradability was similar for all samples, and adequate to physically support neurite outgrowth. The in vitro cell culture results indicate selective cell adhesion. The CHT membranes favoured Schwann cells invasion and proliferation, with a display of appropriate cytoskeletal morphology. At the same time they presented low fibroblast infiltration. This fact may be greatly beneficial for the prevention of fibrotic tissue formation, a common phenomenon impairing peripheral nerve regeneration. The great deal of results obtained during this work permitted to select the formulation with the greatest potential for further biological tests. - Highlights: • Three chitosan membranes were produced with very specific degrees of acetylation (DA I: 1%, DA II: 2%, DA III: 5%). • Physicochemical characterization of the membranes showed their suitability for peripheral nerve regeneration purposes. • In vitro cellular tests confirmed the potential of the membranes as peripheral nerve regeneration systems. • The results indicated that DA III membrane should be the one considered for further peripheral nerve regeneration studies.

  19. Investigation of cell adhesion in chitosan membranes for peripheral nerve regeneration

    International Nuclear Information System (INIS)

    Carvalho, Cristiana R.; López-Cebral, Rita; Silva-Correia, Joana; Silva, Joana M.; Mano, João F.; Silva, Tiago H.; Freier, Thomas; Reis, Rui L.; Oliveira, Joaquim M.

    2017-01-01

    Peripheral nerve injuries have produced major concerns in regenerative medicine for several years, as the recovery of normal nerve function continues to be a significant clinical challenge. Chitosan (CHT), because of its good biocompatibility, biodegradability and physicochemical properties, has been widely used as a biomaterial in tissue engineering scaffolding. In this study, CHT membranes were produced with three different Degrees of Acetylation (DA), envisioning its application in peripheral nerve regeneration. The three CHT membranes (DA I: 1%, DA II: 2%, DA III: 5%) were extensively characterized and were found to have a smooth and flat surface, with DA III membrane having slightly higher roughness and surface energy. All the membranes presented suitable mechanical properties and did not show any signs of calcification after SBF test. Biodegradability was similar for all samples, and adequate to physically support neurite outgrowth. The in vitro cell culture results indicate selective cell adhesion. The CHT membranes favoured Schwann cells invasion and proliferation, with a display of appropriate cytoskeletal morphology. At the same time they presented low fibroblast infiltration. This fact may be greatly beneficial for the prevention of fibrotic tissue formation, a common phenomenon impairing peripheral nerve regeneration. The great deal of results obtained during this work permitted to select the formulation with the greatest potential for further biological tests. - Highlights: • Three chitosan membranes were produced with very specific degrees of acetylation (DA I: 1%, DA II: 2%, DA III: 5%). • Physicochemical characterization of the membranes showed their suitability for peripheral nerve regeneration purposes. • In vitro cellular tests confirmed the potential of the membranes as peripheral nerve regeneration systems. • The results indicated that DA III membrane should be the one considered for further peripheral nerve regeneration studies.

  20. Peripheral tissue homing receptor control of naïve, effector, and memory CD8 T cell localization in lymphoid and non-lymphoid tissues.

    Science.gov (United States)

    Brinkman, C Colin; Peske, J David; Engelhard, Victor Henry

    2013-01-01

    T cell activation induces homing receptors that bind ligands on peripheral tissue vasculature, programing movement to sites of infection and injury. There are three major types of CD8 effector T cells based on homing receptor expression, which arise in distinct lymphoid organs. Recent publications indicate that naïve, effector, and memory T cell migration is more complex than once thought; while many effectors enter peripheral tissues, some re-enter lymph nodes (LN), and contain central memory precursors. LN re-entry can depend on CD62L or peripheral tissue homing receptors. Memory T cells in LN tend to express the same homing receptors as their forebears, but often are CD62Lneg. Homing receptors also control CD8 T cell tumor entry. Tumor vasculature has low levels of many peripheral tissue homing receptor ligands, but portions of it resemble high endothelial venules (HEV), enabling naïve T cell entry, activation, and subsequent effector activity. This vasculature is associated with positive prognoses in humans, suggesting it may sustain ongoing anti-tumor responses. These findings reveal new roles for homing receptors expressed by naïve, effector, and memory CD8 T cells in controlling entry into lymphoid and non-lymphoid tissues.

  1. The intensity of the 1602 cm-1 band in human cells is related to mitochondrial activity

    NARCIS (Netherlands)

    Pully, V.V.; Otto, Cornelis

    2009-01-01

    We report a Raman band at 1602 cm−1 in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal

  2. Identification of early B cell precursors (stage 1 and 2 hematogones) in the peripheral blood.

    Science.gov (United States)

    Kurzer, Jason H; Weinberg, Olga K

    2018-05-25

    Differentiating malignant B-lymphoblasts from early benign B cell precursors (hematogones) is a vital component of the diagnosis of B-lymphoblastic leukaemia. It has been previously reported that only late-stage B cell precursors circulate in the peripheral blood. Consequently, flow cytometric detection of cells with immunophenotypic findings similar to earlier stage precursors in the peripheral blood justifiably raises concern for involvement by B-lymphoblastic leukaemia. We report here, however, that benign early B cell precursors can indeed be detected in the peripheral blood, thus complicating the interpretation of flow cytometric findings derived from these sample types. A retrospective search of our collective databases identified 13 cases containing circulating early stage B cell precursors. The patients ranged in age from 15 days to 85 years old. All positive cases demonstrated that the earlier B cell precursors were associated with later stage precursors, a finding that could help differentiate these cells from B-lymphoblastic leukaemia. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  3. The determination of lymphoid cell chimerism using peripheral blood lymphocytes from murine bone marrow chimeras

    International Nuclear Information System (INIS)

    Skidmore, B.J.; Miller, L.S.

    1978-01-01

    A simple, rapid and accurate method was devised for determining lymphoid cell chimerism in bone marrow-reconstituted mice. Chimeras were produced by reconstituting lethally irradiated mice with semi-allogeneic bone marrow cells. Lymphocytes from the peripheral blood of individual chimeric mice were purified by sedimentation in dextran solution and differential flotation in Ficoll-Hypaque gradients. From 250-500 μl of blood, 1-7 x 10 5 cells were routinely obtained. The extent of chimerism was determined serologically by using peripheral blood lymphocytes as target cells in a dye exclusion microcytotoxicity assay. Using this new technique, approximately 80% of the reconstituted mice were found to be repopulated with lymphocytes of the donor type. (Auth.)

  4. Assessment of Red Blood Cell Parameters and Peripheral Smear at ...

    African Journals Online (AJOL)

    Cold agglutination disease (CAD) is characterized by an auto‑antibody which is able to agglutinate red blood cells (RBCs) at temperatures lower than that of the body, and subsequently to activate the complement system responsible for lysis of RBCs. Patients show hemolytic anemia of varying degrees of severity, which ...

  5. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kuzmina, Nina S., E-mail: nin-kuzmin@youndex.ru; Lapteva, Nellya Sh.; Rubanovich, Alexander V.

    2016-04-15

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24–77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19–77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2–51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62–11.76, p=3.9×10{sup −7}). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10{sup −5}). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10{sup −7}). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging

  6. Hypermethylation of gene promoters in peripheral blood leukocytes in humans long term after radiation exposure

    International Nuclear Information System (INIS)

    Kuzmina, Nina S.; Lapteva, Nellya Sh.; Rubanovich, Alexander V.

    2016-01-01

    Some human genes known to undergo age-related promoter hypermethylation. These epigenetic modifications are similar to those occurring in the course of certain diseases, e.g. some types of cancer, which in turn may also associate with age. Given external genotoxic factors may additionally contribute to hypermethylation, this study was designed to analyzes, using methylation-sensitive polymerase chain reaction (PCR), the CpG island hypermethylation in RASSF1A, CDKN2A (including p16/INK4A and p14/ARF) and GSTP1 promoters in peripheral blood leukocytes of individuals exposed to ionizing radiation long time ago. One hundred and twenty-four irradiated subjects (24–77 years old at sampling: 83 Chernobyl Nuclear Power Plant clean-up workers, 21 nuclear workers, 20 residents of territories with radioactive contamination) and 208 unirradiated volunteers (19–77 years old at sampling) were enrolled. In addition, 74 non-exposed offspring (2–51 years old at sampling) born to irradiated parents were examined. The frequency of individuals displaying promoter methylation of at least one gene in exposed group was significantly higher as compared to the control group (OR=5.44, 95% CI=2.62–11.76, p=3.9×10 −7 ). No significant difference was found between the frequency of subjects with the revealed promoter methylation in the group of offspring born to irradiated parents and in the control group. The increase in the number of methylated loci of RASSF1A and p14/ARF was associated with age (β=0.242; p=1.7×10 −5 ). In contrast, hypermethylation of p16/INK4A and GSTP1 genes correlated with the fact of radiation exposure only (β=0.290; p=1.7×10 −7 ). The latter finding demonstrates that methylation changes in blood leukocytes of healthy subjects exposed to radiation resemble those reported in human malignancies. Additional studies are required to identify the dose-response of epigenetic markers specifically associating with radiation-induced premature aging and/or with

  7. Induction and Maintenance of CX3CR1-Intermediate Peripheral Memory CD8+ T Cells by Persistent Viruses and Vaccines

    Directory of Open Access Journals (Sweden)

    Claire Louse Gordon

    2018-04-01

    Full Text Available Summary: The induction and maintenance of T cell memory is critical to the success of vaccines. A recently described subset of memory CD8+ T cells defined by intermediate expression of the chemokine receptor CX3CR1 was shown to have self-renewal, proliferative, and tissue-surveillance properties relevant to vaccine-induced memory. We tracked these cells when memory is sustained at high levels: memory inflation induced by cytomegalovirus (CMV and adenovirus-vectored vaccines. In mice, both CMV and vaccine-induced inflationary T cells showed sustained high levels of CX3R1int cells exhibiting an effector-memory phenotype, characteristic of inflationary pools, in early memory. In humans, CX3CR1int CD8+ T cells were strongly induced following adenovirus-vectored vaccination for hepatitis C virus (HCV (ChAd3-NSmut and during natural CMV infection and were associated with a memory phenotype similar to that in mice. These data indicate that CX3CR1int cells form an important component of the memory pool in response to persistent viruses and vaccines in both mice and humans. : Gordon et al. demonstrate that CX3CR1int peripheral memory T cells are a substantial component of memory inflation induced by persistent CMVs and adenoviral vaccination. They are characterized by sustained proliferation and an effector-memory phenotype linked to these expanded CD8+ T cell memory responses. Core phenotypic features are shared by humans and mice. Keywords: cytomegalovirus, T cells, memory, adenovirus, vaccination, CX3CR1, memory inflation, mouse, human

  8. Interstitial mononuclear cell infiltrates in chronic rejection of the kidney and correlation with peripheral blood.

    OpenAIRE

    Jeong, H. J.; Hong, S. W.; Kim, Y. S.; Kim, M. S.; Choi, I. H.; Park, K.; Choi, I. J.

    1996-01-01

    To investigate the characteristics of interstitial inflammatory cells and possible involvement of nudelta T cells, 16 renal allograft biopsies showing chronic rejection were stained by immunohistochemical method and correlated with the data of peripheral blood evaluated by flow cytometry. For immunophenotyping, fresh frozen sections were stained with monoclonal antibodies against CD3, CD4, CD8, CD68, CD56, TCRdelta1 and HLA DR. Paraffin embedded tissue was stained with CD45RO, CD20-Cy and CD6...

  9. Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

    Energy Technology Data Exchange (ETDEWEB)

    Ruskamo, Salla [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Yadav, Ravi P. [Banaras Hindu University, Varanasi (India); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Sharma, Satyan; Lehtimäki, Mari [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Laulumaa, Saara [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Aggarwal, Shweta; Simons, Mikael [Max Planck Institute for Experimental Medicine, Göttingen (Germany); Bürck, Jochen; Ulrich, Anne S. [Karlsruhe Institute for Technology (KIT), Karlsruhe (Germany); Juffer, André H. [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Kursula, Inari [University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); Kursula, Petri, E-mail: petri.kursula@oulu.fi [University of Oulu, Oulu (Finland); University of Oulu, Oulu (Finland); Helmholtz Centre for Infection Research (CSSB-HZI), German Electron Synchrotron (DESY), Hamburg (Germany); University of Hamburg, Hamburg (Germany)

    2014-01-01

    The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structural analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.

  10. Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus

    Directory of Open Access Journals (Sweden)

    Tchitchek Nicolas

    2012-06-01

    Full Text Available Abstract Background African Green Monkeys (AGM are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution. Results We have exhaustively sequenced an Expression Sequence Tag (EST library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes. Conclusions The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

  11. Novel Adult Stem Cells for Peripheral Nerve Regeneration

    Science.gov (United States)

    2013-09-01

    presentations related to this project o Days of Molecular Medicine-Regenerative Medicine, Hong Kong . 11/2011 Figure 4. Differentiation of MVSCs into...School, Boston. 10/2012 o Distinguished Seminar Series, Mechanical and Industrial Engineering, University of Toronto, Canada. 11/2012 o International...10% of the cells in the carotid arterial tunica media of SM-MHC-Cre/LoxP- EGFP mice were not labelled with EGFP, indicating the existence of a small

  12. Multispectral Imaging Analysis of Circulating Tumor Cells in Negatively Enriched Peripheral Blood Samples.

    Science.gov (United States)

    Miller, Brandon; Lustberg, Maryam; Summers, Thomas A; Chalmers, Jeffrey J

    2017-01-01

    A variety of biomarkers are present on cells in peripheral blood of patients with a variety of disorders, including solid tumor malignancies. While rare, characterization of these cells for specific protein levels with the advanced technology proposed, will lead to future validation studies of blood samples as "liquid biopsies" for the evaluation of disease status and therapeutic response. While circulating tumor cells (CTCs) have been isolated in the blood samples of patients with solid tumors, the exact role of CTCs as clinically useful predictive markers is still debated. Current commercial technology has significant bias in that a positive selection technology is used that preassumes specific cell surface markers (such as EpCAM) are present on CTCs. However, CTCs with low EpCAM expression have been experimentally demonstrated to be more likely to be missed by this method. In contrast, this application uses a previously developed, technology that performs a purely negative enrichment methodology on peripheral blood, yielding highly enriched blood samples that contain CTCs as well as other, undefined cell types. The focus of this contribution is the use of multispectral imaging of epifluorescent, microscopic images of these enriched cells in order to help develop clinically relevant liquid biopsies from peripheral blood samples.

  13. Monoclonal antibody to a subset of human monocytes found only in the peripheral blood and inflammatory tissues

    Energy Technology Data Exchange (ETDEWEB)

    Zwadlo, G.; Schlegel, R.; Sorg, C.

    1986-07-15

    A monoclonal antibody is described that was generated by immunizing mice with cultured human blood monocytes. The antibody (27E10) belongs to the IgG1 subclass and detects a surface antigen at M/sub r/ 17,000 that is found on 20% of peripheral blood monocytes. The antigen is increasingly expressed upon culture of monocytes, reaching a maximum between days 2 and 3. Stimulation of monocytes with interferon-..gamma.. (IFN-..gamma..), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and lipopolysaccharide (LPS) Ylalanine (fMLP) increased the 27E10 antigen density. The amount of 27E10-positive cells is not or is only weakly affected. The antigen is absent from platelets, lymphotyces, and all tested human cell lines, yet it cross-reacts with 15% of freshly isolated granulocytes. By using the indirect immunoperoxidase technique, the antibody is found to be negative on cryostat sections of normal human tissue (skin, lung, and colon) and positive on only a few monocyte-like cells in liver and on part of the cells of the splenic red pulp. In inflammatory tissue, however, the antibody is positive on monocytes/macrophages and sometimes on endothelial cells and epidermal cells, depending on the stage and type of inflammation, e.g., BCG ranulomas are negative, whereas psoriasis vulgaris, atopic dermatitis, erythrodermia, pressure urticaria, and periodontitis contain positively staining cells. In contact eczemas at different times after elicitation (6 hr, 24 hr, and 72 hr), the 27E10 antigen is seen first after 24 hr on a few infiltrating monocytes/macrophages, which increase in numbers after 72 hr.

  14. Characterization of γδ regulatory T cells from peripheral blood in patients with multiple myeloma

    International Nuclear Information System (INIS)

    Ma, Yongyong; Lei, Huyi; Tan, Jie; Xuan, Li; Wu, Xiuli; Liu, Qifa

    2016-01-01

    γδ regulatory T cells are able to inhibit the activation and function of T cells involved in antigen-specific immune responses. This study aimed to investigate the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in patients diagnosed as multiple myeloma (MM). We measured the levels of γδ T cells, the distribution and clonally amplified TCR Vγ and VδT cells in peripheral blood of healthy donors, patients recently diagnosed with MM, and MM patients in remission cohorts. In addition, we evaluated the ability of γδ regulatory T cells to inhibit the proliferation of CD4+CD25- T cells and detected the expression of immunoregulatory-associated molecules. We found that the levels of γδ regulatory T cells from the peripheral blood in patients of MM were significantly higher than those in healthy donors. Comparison of γδT regulatory cells function in MM and healthy donors showed similarly inhibitory effects on the proliferation of T cells. Additionally, TLR8 expression level increased significantly in MM patients compared to healthy donors, while the expression levels of Foxp3, CD25, CTLA4, GITR, GATA3 and Tbet in MM patients and healthy donors showed no significant difference. Taken together, our study reveals the potential role of γδ regulatory T cells in inhibiting anti-tumor immune responses in MM patients.

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

    Science.gov (United States)

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

    2014-12-01

    The n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to enhance the effect of chemotherapeutic drugs in clinical studies in cancer patients and to induce apoptotic tumor cell death in vitro. Until now, EPA and DHA have never been investigated in multiple myeloma (MM). Human myeloma cells (L363, OPM-1, OPM-2 and U266) and normal peripheral blood mononuclear cells were exposed to EPA and DHA, and effects on mitochondrial function and apoptosis, caspase-3 activation, gene expression and drug toxicity were measured. Exposure to EPA and DHA induced apoptosis and increased sensitivity to bortezomib in MM cells. Importantly, they did not affect viability of normal human peripheral mononuclear cells. Messenger RNA expression arrays showed that EPA and DHA modulated genes involved in multiple signaling pathways including nuclear factor (NF) κB, Notch, Hedgehog, oxidative stress and Wnt. EPA and DHA inhibited NFκB activity and induced apoptosis through mitochondrial perturbation and caspase-3 activation. Our study suggests that EPA and DHA induce selective cytotoxic effects in MM and increase sensitivity to bortezomib and calls for further exploration into a potential application of these n-3 polyunsaturated fatty acids in the therapy of MM. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Impact of a Low CD34+ Cell Dose on Allogeneic Peripheral Blood Stem Cell Transplantation.

    Science.gov (United States)

    Yamamoto, Chihiro; Ogawa, Hiroyasu; Fukuda, Takahiro; Igarashi, Aiko; Okumura, Hirokazu; Uchida, Naoyuki; Hidaka, Michihiro; Nakamae, Hirohisa; Matsuoka, Ken-Ichi; Eto, Tetsuya; Ichinohe, Tatsuo; Atsuta, Yoshiko; Kanda, Yoshinobu

    2018-04-01

    Although the CD34 + cell dose in allogeneic peripheral blood stem cell transplantation (PBSCT) is considered to be associated with transplantation outcomes, a lower acceptable threshold has not been defined. We retrospectively analyzed 2919 adult patients with hematologic malignancies who underwent related PBSCT in Japan between 2001 and 2014. According to the number of CD34 + cells in the graft, we categorized 2494 patients in the standard group (2 to 5 × 10 6 cells/kg), 377 patient in the low group (1 to 2 × 10 6 cells/kg), and 48 patients in the very low group (<1 × 10 6 cells/kg). Compared with the standard group, the low and very low groups showed delayed neutrophil recovery (93.8%, 89.5%, and 78.3%, respectively at day +28; P < .001) and platelet recovery (69.3%, 53.0%, and 45.5%, respectively at day +28; P < .001). The 2-year overall survival (OS) in the 3 groups was 45.5%, 45.3%, and 29.8%, respectively, with inferior survival in the very low group. However, a higher percentage of high-risk patients may account for the inferior survival in the very low group, and no significant difference in OS was found in a multivariate analysis. There were no differences in relapse, nonrelapse mortality, or the development of graft-versus-host disease among the 3 groups. In conclusion, allogeneic PBSCT with low CD34 + cell doses of 1 to 2 × 10 6 cells/kg gives acceptable results, whereas further investigations are needed to evaluate the effects of lower doses of <1 × 10 6 cells/kg owing to the smaller number and the higher percentage of patients with adverse prognostic factors in this cohort. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  17. Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases.

    Science.gov (United States)

    Massart, C; Caroff, G; Maugendre, D; Genetet, N; Gibassier, J

    1999-01-01

    For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.

  18. Genetic variation and epigenetic modification of the prodynorphin gene in peripheral blood cells in alcoholism.

    Science.gov (United States)

    D'Addario, Claudio; Shchetynsky, Klementy; Pucci, Mariangela; Cifani, Carlo; Gunnar, Agneta; Vukojević, Vladana; Padyukov, Leonid; Terenius, Lars

    2017-06-02

    Dynorphins are critically involved in the development, maintenance and relapse of alcoholism. Alcohol-induced changes in the prodynorphin gene expression may be influenced by both gene polymorphisms and epigenetic modifications. The present study of human alcoholics aims to evaluate DNA methylation patterns in the prodynorphin gene (PDYN) promoter and to identify single nucleotide polymorphisms (SNPs) associated with alcohol dependence and with altered DNA methylation. Genomic DNA was isolated from peripheral blood cells of alcoholics and healthy controls, and DNA methylation was studied in the PDYN promoter by bisulfite pyrosequencing. In alcoholics, DNA methylation increased in three of the seven CpG sites investigated, as well as in the average of the seven CpG sites. Data stratification showed lower increase in DNA methylation levels in individuals reporting craving and with higher levels of alcohol consumption. Association with alcoholism was observed for rs2235751 and the presence of the minor allele G was associated with reduced DNA methylation at PDYN promoter in females and younger subjects. Genetic and epigenetic factors within PDYN are related to risk for alcoholism, providing further evidence of its involvement on ethanol effects. These results might be of relevance for developing new biomarkers to predict disease trajectories and therapeutic outcome. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Expression of candidate genes associated with obesity in peripheral white blood cells of Mexican children

    Science.gov (United States)

    Ulloa-Martínez, Marcela; Burguete-García, Ana I.; Murugesan, Selvasankar; Hoyo-Vadillo, Carlos; Cruz-Lopez, Miguel

    2016-01-01

    Introduction Obesity is a chronic, complex, and multifactorial disease, characterized by excess body fat. Diverse studies of the human genome have led to the identification of susceptibility genes that contribute to obesity. However, relatively few studies have addressed specifically the association between the level of expression of these genes and obesity. Material and methods We studied 160 healthy and obese unrelated Mexican children aged 6 to 14 years. We measured the transcriptional expression of 20 genes associated with obesity, in addition to the biochemical parameters, in peripheral white blood cells. The detection of mRNA levels was performed using the OpenArray Real-Time PCR System (Applied Biosystems). Results Obese children exhibited higher values of fasting glucose (p = 0.034), fasting insulin (p = 0.004), low-density lipoprotein (p = 0.006), triglycerides (p GHRL (p = 0.0060) and FTO (p = 0.0348) genes. Conclusions Our results suggest that changes in the expression level of the studied genes are involved in biological processes implicated in the development of childhood obesity. Our study contributes new perspectives for a better understanding of biological processes involved in obesity. The protocol was approved by the National Committee and Ethical Committee Board from the Mexican Social Security Institute (IMSS) (IMSS FIS/IMSS/PRIO/10/011). PMID:27695486

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

    Directory of Open Access Journals (Sweden)

    Nisha Jain Garg

    2016-02-01

    Full Text Available Trypanosoma cruzi (Tc infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs of normal healthy controls (N/H, n = 30 and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25 or clinically symptomatic (C/S, n = 28 with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol and resolved by two-dimensional gel electrophoresis (2D-GE. After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.

  1. Neutron distribution in the central cell and a peripheral cell of the Fontenay-aux-Roses pile

    International Nuclear Information System (INIS)

    Roullier, F.

    1958-01-01

    The distribution of the neutron density has been determined in the central cell and a peripheral cell of the pile at Fontenay-aux-Roses. This measurement was carried out by the autoradiographic method with manganese detectors. The neutron density distribution in the uranium rod has already been studied. The measurement was completed by the study of the neutron density in the complete cell by means of detectors placed in the uranium and in the heavy water. (author) [fr

  2. Interleukin-4 and interferon-¿ production by Leishmania stimulated peripheral blood mononuclear cells from nonexposed individuals

    DEFF Research Database (Denmark)

    Kurtzhals, J A; Kemp, M; Poulsen, L K

    1995-01-01

    of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-gamma production was abrogated by depletion of CD2+ or CD4+ but not CD8+ cells. CD2+ or CD4+ but not CD8+ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating...... call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.......Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-gamma was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated...

  3. Low-frequency electrical stimulation induces the proliferation and differentiation of peripheral blood stem cells into Schwann cells.

    Science.gov (United States)

    Gu, Xudong; Fu, Jianming; Bai, Jing; Zhang, Chengwen; Wang, Jing; Pan, Wenping

    2015-02-01

    Functional recovery after peripheral nerve injury remains a tough problem at present. Specifically, a type of glial cell exists in peripheral nerves that promotes axonal growth and myelin formation and secretes various active substances, such as neurotrophic factors, extracellular matrix and adherence factors. These substances have important significance for the survival, growth and regeneration of nerve fibers. Numerous recent studies have shown that electrical stimulation can increase the number of myelinated nerve fibers. However, whether electrical stimulation acts on neurons or Schwann cells has not been verified in vivo. This study investigates low-frequency electrical stimulation-induced proliferation and differentiation of peripheral blood stem cells into Schwann cells and explores possible mechanisms. Peripheral blood stem cells from Sprague-Dawley rats were primarily cultured. Cells in passage 3 were divided into 4 groups: a low-frequency electrical stimulation group (20 Hz, 100 μs, 3 V), a low-frequency electrical stimulation+PD98059 (blocking the extracellular signal-regulated kinase [ERK] signaling pathway) group, a PD98059 group and a control group (no treatment). After induction, the cells were characterized. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide assay was employed to measure the absorbance values at 570 nm in the 4 groups. A Western blot assay was used to detect the expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) in each group. No significant difference in cell viability was detected before induction. Peripheral blood stem cells from the 4 groups differentiated into Schwann cells. Phosphorylated ERK 1/2, cyclin D1 and CDK4 protein levels were highest in the low-frequency electrical stimulation group and lowest in the ERK blockage group. Phosphorylated ERK 1/2, cyclin D1 and CDK4 protein levels in the low-frequency electrical stimulation+ERK blockage group were lower than those in the low-frequency electrical

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

    Directory of Open Access Journals (Sweden)

    SE Hosseini

    2014-02-01

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

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

    DEFF Research Database (Denmark)

    Glue, C; Millner, A; Bødtger, Uffe

    2002-01-01

    It has recently been shown that plasticizers are present in indoor air dust, which may lead to human exposure via the inhalation route. Moreover, studies have indicated that plasticizers may possess adjuvant effects increasing the health damaging potential of allergens. The aim of this study...... was to investigate the in vitro effect of metabolites of phthalate plastisizers, such as whether an adjuvant effect is paralleled by changes of the cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells (PBMCs) from allergics and non-allergics. The toxicity monitored by cell...... viability was determined by incubating THP-1 cells with a 10-fold dilution series of monophthalates for 24 h. At different points in time cytokine expression (IL-1beta, IL-6, IL-12alpha (p35)) in THP-1 cells incubated with non-toxic concentrations of monophthalate (2-20 microg/ml)+/-LPS (1 microg/ml) were...

  6. Recipient dendritic cells, but not B cells, are required antigen-presenting cells for peripheral alloreactive CD8+ T-cell tolerance.

    Science.gov (United States)

    Mollov, J L; Lucas, C L; Haspot, F; Gaspar, J Kurtz C; Guzman, A; Sykes, M

    2010-03-01

    Induction of mixed allogeneic chimerism is a promising approach for achieving donor-specific tolerance, thereby obviating the need for life-long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti-CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T-cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient-type B cells from chimeras that were tolerant to donor still promoted CD8 T-cell tolerance, but their role could not be replaced by donor-type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL-10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.

  7. Peripheral Lymphoid Volume Expansion and Maintenance Are Controlled by Gut Microbiota via RALDH+ Dendritic Cells.

    Science.gov (United States)

    Zhang, Zongde; Li, Jianjian; Zheng, Wencheng; Zhao, Guang; Zhang, Hong; Wang, Xiaofei; Guo, Yaqian; Qin, Chuan; Shi, Yan

    2016-02-16

    Lymphocyte homing to draining lymph nodes is critical for the initiation of immune responses. Secondary lymphoid organs of germ-free mice are underdeveloped. How gut commensal microbes remotely regulate cellularity and volume of secondary lymphoid organs remains unknown. We report here that, driven by commensal fungi, a wave of CD45(+)CD103(+)RALDH(+) cells migrates to the peripheral lymph nodes after birth. The arrival of these cells introduces high amounts of retinoic acid, mediates the neonatal to adult addressin switch on endothelial cells, and directs the homing of lymphocytes to both gut-associated lymphoid tissues and peripheral lymph nodes. In adult mice, a small number of these RALDH(+) cells might serve to maintain the volume of secondary lymphoid organs. Homing deficiency of these cells was associated with lymph node attrition in vitamin-A-deficient mice, suggesting a perpetual dependence on retinoic acid signaling for structural and functional maintenance of peripheral immune organs. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Interleukin-6, C-reactive protein, and expression of human leukocyte antigen-DR on peripheral blood mononuclear cells in patients after laparoscopic vs. conventional bowel resection - A randomized study

    NARCIS (Netherlands)

    Dunker, M. S.; ten Hove, T.; Bemelman, W. A.; Slors, J. F. M.; Gouma, D. J.; van Deventer, S. J. H.

    2003-01-01

    PURPOSE: The aim of the study was to investigate the effect of surgical trauma in terms of approach (laparoscopic vs. conventional surgery) and extent of bowel resection (ileocolic resection vs. colectomy) on interleukin-6 level, C-reactive protein level, and expression of human leukocyte antigen-DR

  9. Reciprocal patterns of allergen-induced GATA-3 expression in peripheral blood mononuclear cells from atopics vs. non-atopics.

    Science.gov (United States)

    Macaubas, C; Lee, P T; Smallacombe, T B; Holt, B J; Wee, C; Sly, P D; Holt, P G

    2002-01-01

    T helper (Th)2 cytokines are considered to play a central role in the induction and expression of allergic disease. However, the relative importance of individual cytokines is unclear, and overall disease pathogenesis appears to involve the coordinate activities of a range of Th2 cytokines acting in sequence or in parallel. The present study examines an alternative approach to the study of cytokine gene function in atopy, focusing instead upon T cell transcription factors (TFs) which play a role in the regulation of multiple cytokine genes. To investigate the allergen-induced expression of the TF GATA-3 and c-Maf in peripheral blood mononuclear cells (PBMCs) and in cytokine-driven Th polarization. PBMC from house dust mite (HDM)-atopic and non-atopics were stimulated in vitro with allergen or anti-CD3/IL-2. TF expression was analysed by semiquantitative RT-PCR and major findings were validated by real-time PCR. Cell separations were performed to analyse the contribution of CD45RO+ cells. CD4+ cord blood cells were Th1 or Th2 polarized in vitro by exogenous cytokines and TF expression analysed by Northern blot and real-time PCR. Results We demonstrate for the first time that during differentiation of CD4+ CD45RA+ naïve human T cells towards Th2 commitment, and during allergen-specific reactivation of peripheral CD4+ CD45RO+ Th2 memory cells in established atopics, expression of the Th2-associated TF GATA-3 is rapidly up-regulated, whereas T cells from non-atopics display equally rapid GATA-3 down-regulation under identical conditions of allergen stimulation. These findings identify Th2-associated TFs as key determinants of the atopic phenotype, suggesting their unique potential as therapeutic targets for disease control.

  10. Detection of tetanus toxoid-specific memory T cells in equine lymph nodes but not in peripheral blood.

    Science.gov (United States)

    Frayne, J; Stokes, C R

    1995-07-01

    The use of tetanus toxoid as a recall antigen to investigate equine immune responses would be, in theory, a useful and cost-effective model in vitro. However, by using various regimens for culturing peripheral blood mononuclear cells from horses previously immunised with toxoid no proliferative response to the antigen was obtained in vitro, whereas lymph node mononuclear cells from the same animals proliferated significantly in response to it. The lack of response by the peripheral blood mononuclear cells was not due to the presence of a suppressive factor but to a lack of recognition of the antigen by the T cells of the peripheral blood.

  11. Lipid Accumulation in Peripheral Blood Dendritic Cells and Anticancer Immunity in Patients with Lung Cancer

    Directory of Open Access Journals (Sweden)

    Ryo Arai

    2018-01-01

    Full Text Available We studied the subsets of peripheral blood dendritic cells (DCs and lipid accumulation in DCs to investigate the involvement of DCs in the decreased anticancer immunity of advanced lung cancer patients. We analyzed the population of DC subsets in peripheral blood using flow cytometry. We then determined lipid accumulation in the DCs using BODIPY 650/665, a fluorophore with an affinity for lipids. Compared with healthy controls, the number of DCs in the peripheral blood of treatment-naive cancer patients was significantly reduced. In patients with stage III + IV disease, the numbers of myeloid DCs (mDCs and plasmacytoid DCs were also significantly reduced. Lipid accumulation in DCs evaluated based on the fluorescence intensity of BODIPY 650/665 was significantly higher in stage III + IV lung cancer patients than in the controls. In the subset analysis, the fluorescence was highest for mDCs. The intracellularly accumulated lipids were identified as triglycerides. A decreased mixed leukocyte reaction was observed in the mDCs from lung cancer patients compared with those from controls. Taken together, the results show that lung cancer patients have a notably decreased number of peripheral blood DCs and their function as antigen-presenting cells is decreased due to their high intracellular lipid accumulation. Thereby, anticancer immunity is suppressed.

  12. Phytohemagglutinin (PHA) stimulation of peripheral-blood lymphocytes and stem cell take

    Energy Technology Data Exchange (ETDEWEB)

    Astaldi, G. (Blood Research Foundation Center, Tortona, Italy); Karanovic, D.; Vettori, P.P.; Karanovic, J.; Piletic, O.

    1974-01-01

    The effect of PHA-stimulation of peripheral-blood lymphocytes on the spleen-colony formation in irradiated rats was examined. 25-day old Wistar rats underwent total-body irradiation (600 R), and they were used as recipients. On the other hand, 2 and /sup 1///sub 2/ month old untreated Wistar rats were used as donors of peripheral-blood lymphocytes, which were obtained by sedimentation with Dextraven from defibrinated blood. Four rat lots were used. The 1st one did not receive irradiation, and was kept as ''blank control.'' The 2nd one was just irradiated and kept as ''radiated control.'' The 3rd and the 4th rat lots of the series were irradiated, but the former lot was injected i.v. with 5 x 10/sup 7/ peripheral-blood untreated lymphocytes, whereas the fourth lot was injected i.v. with the same amount of lymphocytes, which were previously incubated in vitro for 24 hrs with PHA-M (Difco). The results showed that the PHA-incubation of transplanted peripheral-blood lymphocytes significantly increases the number and size of the macroscopic spleen colonies, in relationship to the colonies which occurs after transplantation of untreated lymphocytes. Histo-cytological observation clearly showed that the colonies formed after injection of mitogen-pretreated peripheral-blood lymphocytes were predominantly of erythroid type and, then, of non-differentiated cells. Only a few of them were of a mixed type, consisting of both undifferentiated cells and erythroid cells.

  13. Sleep Deprivation Impairs the Human Central and Peripheral Nervous System Discrimination of Social Threat.

    Science.gov (United States)

    Goldstein-Piekarski, Andrea N; Greer, Stephanie M; Saletin, Jared M; Walker, Matthew P

    2015-07-15

    Facial expressions represent one of the most salient cues in our environment. They communicate the affective state and intent of an individual and, if interpreted correctly, adaptively influence the behavior of others in return. Processing of such affective stimuli is known to require reciprocal signaling between central viscerosensory brain regions and peripheral-autonomic body systems, culminating in accurate emotion discrimination. Despite emerging links between sleep and affective regulation, the impact of sleep loss on the discrimination of complex social emotions within and between the CNS and PNS remains unknown. Here, we demonstrate in humans that sleep deprivation impairs both viscerosensory brain (anterior insula, anterior cingulate cortex, amygdala) and autonomic-cardiac discrimination of threatening from affiliative facial cues. Moreover, sleep deprivation significantly degrades the normally reciprocal associations between these central and peripheral emotion-signaling systems, most prominent at the level of cardiac-amygdala coupling. In addition, REM sleep physiology across the sleep-rested night significantly predicts the next-day success of emotional discrimination within this viscerosensory network across individuals, suggesting a role for REM sleep in affective brain recalibration. Together, these findings establish that sleep deprivation compromises the faithful signaling of, and the "embodied" reciprocity between, viscerosensory brain and peripheral autonomic body processing of complex social signals. Such impairments hold ecological relevance in professional contexts in which the need for accurate interpretation of social cues is paramount yet insufficient sleep is pervasive. Copyright © 2015 the authors 0270-6474/15/3510135-11$15.00/0.

  14. Factors affecting autologous peripheral blood hematopoietic stem cell collections by large-volume leukapheresis: a single center experience

    Directory of Open Access Journals (Sweden)

    Araci Massami Sakashita

    2011-06-01

    Full Text Available Objective: To evaluate factors affecting peripheral bloodhematopoietic stem cell yield in patients undergoing large-volumeleukapheresis for autologous peripheral blood stem cell collection.Methods: Data from 304 consecutive autologous peripheral bloodstem cell donors mobilized with hematopoietic growth factor (usually G-CSF, associated or not with chemotherapy, at Hospital Israelita Albert Einstein between February 1999 and June 2010 were retrospectively analyzed. The objective was to obtain at least 2 x 106CD34+ cells/kg of body weight. Pre-mobilization factors analyzedincluded patient’s age, gender and diagnosis. Post mobilizationparameters evaluated were pre-apheresis peripheral white bloodcell count, immature circulating cell count, mononuclear cell count,peripheral blood CD34+ cell count, platelet count, and hemoglobinlevel. The effect of pre and post-mobilization factors on hematopoietic stem cell collection yield was investigated using logistic regression analysis (univariate and multivariate approaches. Results: Premobilization factors correlating to poor CD34+ cell yield in univariate analysis were acute myeloid leukemia (p = 0.017 and other hematological diseases (p = 0.023. Significant post-mobilization factors included peripheral blood immature circulating cells (p = 0.001, granulocytes (p = 0.002, hemoglobin level (p = 0.016, and CD34+ cell concentration (p < 0.001 in the first harvesting day. However, according to multivariate analysis, peripheral blood CD34+ cell content (p < 0.001 was the only independent factor that significantly correlated to poor hematopoietic stem cell yield. Conclusion: In this study, peripheral blood CD34+ cell concentration was the only factor significantly correlated to yield in patients submitted to for autologous collection.

  15. Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Armando de M. Carvalho

    2013-09-01

    Full Text Available The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs. Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

  16. [Prostatic granulomas revealing a peripheral T-cell lymphoma].

    Science.gov (United States)

    Foguem, C; Curlier, E; Rouamba, M-M; Regent, A; Philippe, P

    2009-02-01

    The presence of granulomas on tissue biopsie has been reported in a wide range of disorders. The clinical presentation and the diagnostic work-up of granulomatosis can be difficult as it is illustrated in the following report. A 59-year-old patient was referred in 2002 for a granulomatous prostatitis. Physical examination was normal. Except for the increase of prostate-specific antigen (which motivated a biopsy), the laboratory results were normal. Thoracic CT-scan disclosed mediastinal lymph nodes. A minor salivary gland biopsy was consistent with the diagnosis of sarcoidosis. In 2004, the patient presented an epidermal necrolysis, and in 2005 the deterioration of general status raised suspicion of a lymphoproliferative disorder. Liver and bone marrow biopsies revealed a granulomatous process. Despite steroid therapy, the patient died. Autopsy discloses a anaplasic T cell lymphoma. This report illustrates the relationship between sarcoidosis and lymphoma as a mode of presentation, a complication, or an accidental but misleading association? The association between anaplastic lymphoma and sarcoidosis is exceptional.

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

    Directory of Open Access Journals (Sweden)

    Longo Dan L

    2011-03-01

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

  18. Polycystic ovary syndrome and the peripheral blood white cell count.

    LENUS (Irish Health Repository)

    Herlihy, A C

    2012-02-01

    This retrospective cross-sectional study examined if the white cell count (WCC) is increased in women with polycystic ovary syndrome (PCOS) and if so, is it due to PCOS or to the associated obesity? Body mass index (BMI) was calculated and body composition was measured using bioelectrical impedance analysis. Of the 113 women studied, 36 had PCOS and 77 did not. The mean WCC was higher in the PCOS group compared with the non-PCOS group (8.9 x 10(9)\\/l vs 7.4 x 10(9)\\/l p = 0.002). This increase was due to a higher neutrophil count (5.6 x 10(9)\\/l vs 4.3 x 10(9)\\/l; p = 0.003). There was a leucocytosis (WCC >11 x 10(9)\\/l) present in 19% of the PCOS group compared with 1% in the non-PCOS group (p < 0.001). The neutrophil count was abnormally high (>7.7 x 10(9)\\/l) in 14% of the PCOS group compared with 4% in the non-PCOS group (p < 0.001). On regression analysis, however, the only independent variable which explained both the increased WCC and the increased neutrophil count was PCOS. We found that PCOS is associated with an increased WCC due to increased neutrophils, which supports the evidence that PCOS is associated with low-grade inflammation. The increase appears to be due to the underlying PCOS, and not to the increased adiposity associated with PCOS.

  19. Effects of folic acid deficiency and MTHFRC677T polymorphisms on cytotoxicity in human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    Wu Xiayu; Liang Ziqing; Zou Tianning; Wang Xu

    2009-01-01

    Apoptosis (APO) and necrosis (NEC) are two different types of cell death occurring in response to cellular stress factors. Cells with DNA damage may undergo APO or NEC. Folate is an essential micronutrient associated with DNA synthesis, repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) regulates intracellular folate metabolism. Folate deficiency and MTHFR C677T polymorphisms have been shown to be related to DNA damage. To verify the cytotoxic effects of folate deficiency on cells with different MTHFR C677T genotypes, 15 human peripheral lymphocyte cases with different MTHFR C677T genotypes were cultured in folic acid (FA)-deficient and -sufficient media for 9 days. Cytotoxicity was quantified using the frequencies of APO and NEC as endpoints, the nuclear division index (NDI), and the number of viable cells (NVC). These results showed that FA is an important factor in reducing cytotoxicity and increasing cell proliferation. Lymphocytes with the TT genotype proliferated easily under stress and exhibited different responses to FA deficiency than lymphocytes with the CC and CT genotypes. A TT individual may accumulate more cytotoxicity under cytotoxic stress, suggesting that the effects of FA deficiency on cytotoxicity are greater than the effects in individuals with the other MTHFR C677T variants.

  20. The effect of stress on core and peripheral body temperature in humans.

    Science.gov (United States)

    Vinkers, Christiaan H; Penning, Renske; Hellhammer, Juliane; Verster, Joris C; Klaessens, John H G M; Olivier, Berend; Kalkman, Cor J

    2013-09-01

    Even though there are indications that stress influences body temperature in humans, no study has systematically investigated the effects of stress on core and peripheral body temperature. The present study therefore aimed to investigate the effects of acute psychosocial stress on body temperature using different readout measurements. In two independent studies, male and female participants were exposed to a standardized laboratory stress task (the Trier Social Stress Test, TSST) or a non-stressful control task. Core temperature (intestinal and temporal artery) and peripheral temperature (facial and body skin temperature) were measured. Compared to the control condition, stress exposure decreased intestinal temperature but did not affect temporal artery temperature. Stress exposure resulted in changes in skin temperature that followed a gradient-like pattern, with decreases at distal skin locations such as the fingertip and finger base and unchanged skin temperature at proximal regions such as the infra-clavicular area. Stress-induced effects on facial temperature displayed a sex-specific pattern, with decreased nasal skin temperature in females and increased cheek temperature in males. In conclusion, the amplitude and direction of stress-induced temperature changes depend on the site of temperature measurement in humans. This precludes a direct translation of the preclinical stress-induced hyperthermia paradigm, in which core temperature uniformly rises in response to stress to the human situation. Nevertheless, the effects of stress result in consistent temperature changes. Therefore, the present study supports the inclusion of body temperature as a physiological readout parameter of stress in future studies.

  1. Genome engineering in human cells.

    Science.gov (United States)

    Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

    2014-01-01

    Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

  2. Influence of age on the proliferation and peripheralization of thymic T cells

    International Nuclear Information System (INIS)

    Hirokawa, K.; Utsuyama, M.; Katsura, Y.; Sado, T.

    1988-01-01

    Bone marrow cells obtained from B10.Thy-1.1 mice (H-2b, Thy-1.1) were injected directly into the thymus of C57BL/6 mice (H-2b,Thy 1.2) of various ages. Thymocyte precursors in the injected donor-bone marrow cells could proliferate in the thymic microenvironment in the following manner: first, preferentially proliferating into the subcapsular cortex; and second, spreading to the whole layer of the cortex, a portion of them gradually moving into the medulla. The proliferation of donor-type thymocytes was most pronounced when intrathymic injection of bone marrow cells (ITB) was performed in newborn mice and especially prominent in week-old mice; it took approximately ten weeks for donor-type thymocytes to finish the whole course of proliferation, differentiation, and emigration to the periphery. When ITB was performed in mice 4 weeks of age and older, the proliferation of donor-type thymocytes was retarded at onset, less pronounced in magnitude, and disappeared earlier. Emigration of donor-type T cells from the thymus to the peripheral lymphoid tissues occurred most rapidly when ITB was performed in newborn mice, and these T cells continued to reside thereafter in the peripheral lymphoid tissues. However, when ITB was performed in mice 4 weeks of age and older, the number of emigrated T cells in the spleen decreased (about a tenth of that in newborn mice) and, moreover, these T cells resided only transiently in the spleen. It was suggested that T cells emigrating from the thymus of mice from newborn to 2 weeks of age are long-lived, whereas those from the thymus in mice 4 weeks of age and older are short-lived. However, when 4-week-old young adult mice were treated by irradiation or hydrocortisone, the thymic capacity was enhanced in terms of proliferation and peripheralization of thymocytes, and emigrated T cells became long-lived

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    -out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production...... of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A 3-wk diet with high BX content enhanced IL-1β responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 h of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation...

  4. Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: implications for an anticancer mechanism.

    Science.gov (United States)

    Shamim, Uzma; Hanif, Sarmad; Ullah, M F; Azmi, Asfar S; Bhat, Showket H; Hadi, S M

    2008-08-01

    It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.

  5. Gene Transfer Properties and Structural Modeling of Human Stem Cell-derived AAV

    OpenAIRE

    Smith, Laura J; Ul-Hasan, Taihra; Carvaines, Sarah K; Van Vliet, Kim; Yang, Ethel; Wong, Kamehameha K; Agbandje-McKenna, Mavis; Chatterjee, Saswati

    2014-01-01

    Adeno-associated virus (AAV) vectors are proving to be remarkably successful for in vivo gene delivery. Based upon reports of abundant AAV in the human marrow, we tested CD34+ hematopoietic stem cells for the presence of natural AAV. Here, we report for the first time, the presence of novel AAV variants in healthy CD34+ human peripheral blood stem cells. The majority of healthy peripheral blood stem cell donors were found to harbor AAV in their CD34+ cells. Every AAV isolated from CD34+ cells...

  6. Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

    Science.gov (United States)

    Chen, Yuchang; Ou, Zhanhui; Song, Bing; Xian, Yexing; Ouyang, Shuming; Xie, Yuhuan; Xue, Yanting; Sun, Xiaofang

    2017-08-01

    47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease. Copyright © 2017. Published by Elsevier B.V.

  7. Peripheral Hole Acceptor Moieties on an Organic Dye Improve Dye‐Sensitized Solar Cell Performance

    Science.gov (United States)

    Hao, Yan; Gabrielsson, Erik; Lohse, Peter William; Yang, Wenxing; Johansson, Erik M. J.; Hagfeldt, Anders

    2015-01-01

    Investigation of charge transfer dynamics in dye‐sensitized solar cells is of fundamental interest and the control of these dynamics is a key factor for developing more efficient solar cell devices. One possibility for attenuating losses through recombination between injected electrons and oxidized dye molecules is to move the positive charge further away from the metal oxide surface. For this purpose, a metal‐free dye named E6 is developed, in which the chromophore core is tethered to two external triphenylamine (TPA) units. After photoinduced electron injection into TiO2, the remaining hole is rapidly transferred to a peripheral TPA unit. Electron–hole recombination is slowed down by 30% compared to a reference dye without peripheral TPA units. Furthermore, it is found that the added TPA moieties improve the electron blocking effect of the dye, retarding recombination of electrons from TiO2 to the cobalt‐based electrolyte. PMID:27722076

  8. Counter-flow elutriation of clinical peripheral blood mononuclear cell concentrates for the production of dendritic and T cell therapies

    OpenAIRE

    Stroncek, David F; Fellowes, Vicki; Pham, Chauha; Khuu, Hanh; Fowler, Daniel H; Wood, Lauren V; Sabatino, Marianna

    2014-01-01

    Introduction Peripheral blood mononuclear cells (PBMC) concentrates collected by apheresis are frequently used as starting material for cellular therapies, but the cell of interest must often be isolated prior to initiating manufacturing. Study design and methods The results of enriching 59 clinical PBMC concentrates for monocytes or lymphocytes from patients with solid tumors or multiple myeloma using a commercial closed system semi-automated counter-flow elutriation instrument (Elutra, Teru...

  9. Multivariate analysis using high definition flow cytometry reveals distinct T cell repertoires between the foetal-maternal interface and the peripheral blood

    Directory of Open Access Journals (Sweden)

    Michelle eNeller

    2014-02-01

    Full Text Available The human T-cell compartment is a complex system and while some information is known on repertoire composition and dynamics in the peripheral blood, little is known on repertoire composition at different anatomical sites. Here, we determine the T-cell receptor β-variable (TRBV repertoire at the decidua and compare it with the peripheral blood during normal pregnancy and preclampsia. We found total T-cell subset disparity of up to 58% between sites, including large signature TRBV expansions unique to the foetal-maternal interface. Defining the functional nature and specificity of compartment-specific T-cells will be necessary if we are to understand localised immunity, tolerance and pathogenesis.

  10. Adipose-Derived Stem Cells Promote Peripheral Nerve Regeneration In Vivo without Differentiation into Schwann-Like Lineage.

    Science.gov (United States)

    Sowa, Yoshihiro; Kishida, Tsunao; Imura, Tetsuya; Numajiri, Toshiaki; Nishino, Kenichi; Tabata, Yasuhiko; Mazda, Osam

    2016-02-01

    During recent decades, multipotent stem cells were found to reside in the adipose tissue, and these adipose-derived stem cells were shown to play beneficial roles, like those of Schwann cells, in peripheral nerve regeneration. However, it has not been well established whether adipose-derived stem cells offer beneficial effects to peripheral nerve injuries in vivo as Schwann cells do. Furthermore, the in situ survival and differentiation of adipose-derived stem cells after transplantation at the injured peripheral nerve tissue remain to be fully elucidated. Adipose-derived stem cells and Schwann cells were transplanted with gelatin hydrogel tubes at the artificially blunted sciatic nerve lesion in mice. Neuroregenerative abilities of them were comparably estimated. Cre-loxP-mediated fate tracking was performed to visualize survival in vivo of transplanted adipose-derived stem cells and to investigate whether they differentiated into Schwann linage cells at the peripheral nerve injury site. The transplantation of adipose-derived stem cells promoted regeneration of axons, formation of myelin, and restoration of denervation muscle atrophy to levels comparable to those achieved by Schwann cell transplantation. The adipose-derived stem cells survived for at least 4 weeks after transplantation without differentiating into Schwann cells. Transplanted adipose-derived stem cells did not differentiate into Schwann cells but promoted peripheral nerve regeneration at the injured site. The neuroregenerative ability was comparable to that of Schwann cells. Adipose-derived stem cells at an undifferentiated stage may be used as an alternative cell source for autologous cell therapy for patients with peripheral nerve injury.

  11. T cell-mediated increased osteoclast formation from peripheral blood as a mechanism for crohn's disease-associated bone loss

    NARCIS (Netherlands)

    Oostlander, A.E.; Everts, V.; Schoenmaker, T.; Bravenboer, N.; van Vliet, S.J.; van Bodegraven, A.A.; Lips, P.; de Vries, T.J.

    2012-01-01

    The pathophysiology of osteoporosis in patients with Crohn's disease (CD) is still not completely elucidated. In this study, we evaluated osteoclastogenesis from peripheral blood cells of CD patients and studied the role of lymphocytes and inflammatory cytokines in this process. Peripheral blood

  12. PIM kinases as potential therapeutic targets in a subset of peripheral T cell lymphoma cases.

    Directory of Open Access Journals (Sweden)

    Esperanza Martín-Sánchez

    Full Text Available Currently, there is no efficient therapy for patients with peripheral T cell lymphoma (PTCL. The Proviral Integration site of Moloney murine leukemia virus (PIM kinases are important mediators of cell survival. We aimed to determine the therapeutic value of PIM kinases because they are overexpressed in PTCL patients, T cell lines and primary tumoral T cells. PIM kinases were inhibited genetically (using small interfering and short hairpin RNAs and pharmacologically (mainly with the pan-PIM inhibitor (PIMi ETP-39010 in a panel of 8 PTCL cell lines. Effects on cell viability, apoptosis, cell cycle, key proteins and gene expression were evaluated. Individual inhibition of each of the PIM genes did not affect PTCL cell survival, partially because of a compensatory mechanism among the three PIM genes. In contrast, pharmacological inhibition of all PIM kinases strongly induced apoptosis in all PTCL cell lines, without cell cycle arrest, in part through the induction of DNA damage. Therefore, pan-PIMi synergized with Cisplatin. Importantly, pharmacological inhibition of PIM reduced primary tumoral T cell viability without affecting normal T cells ex vivo. Since anaplastic large cell lymphoma (ALK+ ALCL cell lines were the most sensitive to the pan-PIMi, we tested the simultaneous inhibition of ALK and PIM kinases and found a strong synergistic effect in ALK+ ALCL cell lines. Our findings suggest that PIM kinase inhibition could be of therapeutic value in a subset of PTCL, especially when combined with ALK inhibitors, and might be clinically beneficial in ALK+ ALCL.

  13. A