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  1. Artikel ; Online: Efficient isolation of highly purified tonsil B lymphocytes using RosetteSep with allogeneic human red blood cells

    Deans Julie P / Unruh Tammy L / Zuccolo Jonathan

    BMC Immunology, Vol 10, Iss 1, p

    2009  Band 30

    Abstract: Abstract Background Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to ... ...

    Abstract Abstract Background Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step, using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes, whereas single cell suspensions from tonsils contain relatively few red blood cells. Results B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however, the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells, but not sheep red blood cells, reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells. Conclusion RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods.
    Schlagwörter Immunologic diseases. Allergy ; RC581-607 ; Specialties of internal medicine ; RC581-951 ; Internal medicine ; RC31-1245 ; Medicine ; R ; DOAJ:Allergy and Immunology ; DOAJ:Medicine (General) ; DOAJ:Health Sciences
    Thema/Rubrik (Code) 610
    Sprache Englisch
    Erscheinungsdatum 2009-05-01T00:00:00Z
    Verlag BioMed Central
    Dokumenttyp Artikel ; Online
    Datenquelle BASE - Bielefeld Academic Search Engine (Lebenswissenschaftliche Auswahl)

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  2. Artikel ; Online: Efficient isolation of highly purified tonsil B lymphocytes using RosetteSep with allogeneic human red blood cells.

    Zuccolo, Jonathan / Unruh, Tammy L / Deans, Julie P

    BMC immunology

    2009  Band 10, Seite(n) 30

    Abstract: Background: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate ... ...

    Abstract Background: Human tonsils are a rich source of B lymphocytes exhibiting a variety of phenotypes and activation states. Existing methods of purification are time consuming or costly. The aim of the present study was to optimize conditions to isolate large numbers of highly purified primary B lymphocytes from tonsils in a short and cost-effective single step, using a commercially available reagent designed for purifying cells from whole blood (RosetteSep). This technique relies on the presence of the large excess of red blood cells in whole blood for the formation of immunorosettes, whereas single cell suspensions from tonsils contain relatively few red blood cells.
    Results: B cell enrichment from tonsils was achieved using RosetteSep with no modification to the whole blood procedure; however, the degree of purity depended on the extent of red blood cell contamination of the starting tonsil cell suspension. Addition of a 50-fold excess of allogeneic human red blood cells, but not sheep red blood cells, reproducibly resulted in high levels of purity. Depletion of mononuclear cells from the donor red blood cells eliminated potential contamination with allogeneic B cells.
    Conclusion: RosetteSep reagent can be used in combination with allogeneic human red blood cells to reproducibly isolate tonsil B lymphocytes to high levels of purity with no change in phenotype or loss of cells. This method provides considerable time and cost savings compared to other methods.
    Mesh-Begriff(e) Animals ; B-Lymphocytes/cytology ; B-Lymphocytes/immunology ; Cell Separation ; Cost-Benefit Analysis ; Erythrocytes/immunology ; Erythrocytes/metabolism ; HLA Antigens ; Humans ; Immunologic Techniques/economics ; Palatine Tonsil/cytology ; Rosette Formation ; Sheep ; Time Factors
    Chemische Substanzen HLA Antigens
    Sprache Englisch
    Erscheinungsdatum 2009-05-27
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1471-2172
    ISSN (online) 1471-2172
    DOI 10.1186/1471-2172-10-30
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  3. Artikel ; Online: Therapeutic (high) doses of rituximab activate calcium mobilization and inhibit B-cell growth via an unusual mechanism triggered independently of both CD20 and Fcgamma receptors.

    Unruh, Tammy L / Zuccolo, Jonathan / Beers, Stephen A / Kanevets, Uliana / Shi, Yan / Deans, Julie P

    Journal of immunotherapy (Hagerstown, Md. : 1997)

    2010  Band 33, Heft 1, Seite(n) 30–39

    Abstract: Rituximab is a CD20-specific monoclonal antibody that effectively targets and depletes B lymphocytes in vivo, primarily via indirect cytotoxic mechanisms. Direct effects on B cells may also contribute to B-cell depletion but are less clearly defined. In ... ...

    Abstract Rituximab is a CD20-specific monoclonal antibody that effectively targets and depletes B lymphocytes in vivo, primarily via indirect cytotoxic mechanisms. Direct effects on B cells may also contribute to B-cell depletion but are less clearly defined. In this report, we demonstrate that monomeric rituximab, at the high concentrations found in plasma following infusion of therapeutic doses, induces prolonged low-amplitude release of calcium from thapsigargin-sensitive intracellular stores and reduces the growth of Ramos B cells in culture. Intracellular calcium release was triggered via a signaling pathway distinct from the lipid raft-dependent and src family kinase-dependent pathway that is activated by CD20 hypercrosslinking or B-cell receptor association. The response was independent of both CD20 and Fc receptor binding, and was also triggered by some, but not all, irrelevant monoclonal IgG1 antibodies. The data indicate that unique regions within IgG may contribute to direct effects of therapeutic monoclonal antibodies delivered at suprasaturating concentrations.
    Mesh-Begriff(e) Antibodies, Monoclonal/administration & dosage ; Antibodies, Monoclonal, Murine-Derived ; Antigens, CD20/metabolism ; B-Lymphocytes/drug effects ; Calcium Signaling/drug effects ; Cell Line ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Chromatography, High Pressure Liquid ; Humans ; Immunologic Factors/administration & dosage ; Receptors, Fc/metabolism ; Rituximab
    Chemische Substanzen Antibodies, Monoclonal ; Antibodies, Monoclonal, Murine-Derived ; Antigens, CD20 ; Immunologic Factors ; Receptors, Fc ; Rituximab (4F4X42SYQ6)
    Sprache Englisch
    Erscheinungsdatum 2010-01
    Erscheinungsland United States
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1064067-8
    ISSN 1537-4513 ; 1053-8550 ; 1524-9557
    ISSN (online) 1537-4513
    ISSN 1053-8550 ; 1524-9557
    DOI 10.1097/CJI.0b013e3181b290f1
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  4. Artikel ; Online: Quantification of CD20 mRNA and protein levels in chronic lymphocytic leukemia suggests a post-transcriptional defect.

    Sarro, Shannon M / Unruh, Tammy L / Zuccolo, Jonathan / Sanyal, Ratna / Luider, Joanne M / Auer-Grzesiak, Iwona A / Mansoor, Adnan / Deans, Julie P

    Leukemia research

    2010  Band 34, Heft 12, Seite(n) 1670–1673

    Abstract: Chronic lymphocytic leukemia is less effectively treated than other B cell malignancies with the anti-CD20 agent, rituximab, presumably due, at least in part, to low CD20 expression. CD20 expression is typically measured by flow cytometry, which may not ... ...

    Abstract Chronic lymphocytic leukemia is less effectively treated than other B cell malignancies with the anti-CD20 agent, rituximab, presumably due, at least in part, to low CD20 expression. CD20 expression is typically measured by flow cytometry, which may not be quantitative. This study was undertaken to measure total CD20 protein in CLL B cells using quantitative immunoblot analysis. The results demonstrated that total CD20 protein levels were consistently decreased by ∼60% in CLL B cells with low CD20 fluorescence staining. Surprisingly, real-time polymerase chain reaction analysis showed that CD20 mRNA levels were normal or close to normal, depending on the comparative B cell population, and did not correlate well with protein expression. We conclude that CD20 protein is substantially decreased in CLL due to a post-transcriptional defect.
    Mesh-Begriff(e) Antigens, CD20/biosynthesis ; B-Lymphocytes/metabolism ; B-Lymphocytes/pathology ; Female ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/metabolism ; Leukemia, Lymphocytic, Chronic, B-Cell/pathology ; Male ; Neoplasm Proteins/biosynthesis ; RNA, Messenger/biosynthesis ; RNA, Neoplasm/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Transcription, Genetic
    Chemische Substanzen Antigens, CD20 ; Neoplasm Proteins ; RNA, Messenger ; RNA, Neoplasm
    Sprache Englisch
    Erscheinungsdatum 2010-12
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 752396-8
    ISSN 1873-5835 ; 0145-2126
    ISSN (online) 1873-5835
    ISSN 0145-2126
    DOI 10.1016/j.leukres.2010.06.031
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  5. Artikel: Expression of MS4A and TMEM176 Genes in Human B Lymphocytes.

    Zuccolo, Jonathan / Deng, Lili / Unruh, Tammy L / Sanyal, Ratna / Bau, Jeremy A / Storek, Jan / Demetrick, Douglas J / Luider, Joanne M / Auer-Grzesiak, Iwona A / Mansoor, Adnan / Deans, Julie P

    Frontiers in immunology

    2013  Band 4, Seite(n) 195

    Abstract: The MS4A gene family in humans includes CD20 and at least 15 other genes. CD20 exists as homo-oligomers in the plasma membrane, however different MS4A proteins expressed in the same cell may hetero-oligomerize. Given the importance of CD20 in B-cell ... ...

    Abstract The MS4A gene family in humans includes CD20 and at least 15 other genes. CD20 exists as homo-oligomers in the plasma membrane, however different MS4A proteins expressed in the same cell may hetero-oligomerize. Given the importance of CD20 in B-cell function and as a therapeutic target, we sought to explore the potential for CD20 hetero-oligomerization with other MS4A proteins. We investigated expression in primary human B-cells of the four MS4A genes previously shown to be expressed in human B-cell lines (MS4A4A, MS4A6A, MS4A7, MS4A8B), as well as two genes comprising the closely related TMEM176 gene family, with a view to identifying candidates for future investigation at the protein level. TMEM176A and TMEM176B transcripts were either not detected, or were detected at relatively low levels in a minority of donor B-cell samples. MS4A4A and MS4A8B transcripts were not detected in any normal B-cell sample. MS4A6A and MS4A7 transcripts were detected at low levels in most samples, however the corresponding proteins were not at the plasma membrane when expressed as GFP conjugates in BJAB cells. We also examined expression of these genes in chronic lymphocytic leukemia (CLL), and found that it was similar to normal B-cells with two exceptions. First, whereas MS4A4A expression was undetected in normal B-cells, it was expressed in 1/14 CLL samples. Second, compared to expression levels in normal B-cells, MS4A6A transcripts were elevated in 4/14 CLL samples. In summary, none of the MS4A/TMEM176 genes tested was expressed at high levels in normal or in most CLL B-cells. MS4A6A and MS4A7 were expressed at low levels in most B-cell samples, however the corresponding proteins may not be positioned at the plasma membrane. Altogether, these data suggest that CD20 normally does not form hetero-oligomers with other MS4A proteins and that there are unlikely to be other MS4A proteins in CLL that might provide useful alternate therapeutic targets.
    Sprache Englisch
    Erscheinungsdatum 2013-07-15
    Erscheinungsland Switzerland
    Dokumenttyp Journal Article
    ZDB-ID 2606827-8
    ISSN 1664-3224
    ISSN 1664-3224
    DOI 10.3389/fimmu.2013.00195
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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  6. Artikel: Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor.

    Mutch, Cathlin M / Sanyal, Ratna / Unruh, Tammy L / Grigoriou, Lana / Zhu, Minghua / Zhang, Weiguo / Deans, Julie P

    International immunology

    2007  Band 19, Heft 1, Seite(n) 19–30

    Abstract: Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct ... ...

    Abstract Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation. Both adaptors were distributed predominantly at the plasma membrane in resting cells and co-clustered with other raft-associated proteins; however, they distributed differently in buoyant membranes isolated by either detergent resistance or non-detergent methods, indicating that they might localize to distinct rafts. After activation, LAB/NTAL was internalized and co-localized with the BCR while PAG/Cbp remained on the cell surface. BCR internalization was reduced in LAB/NTAL-deficient murine B cells, suggesting a regulatory role for LAB/NTAL in activation-induced internalization of the BCR. The cytoplasmic domain of LAB/NTAL, and not the transmembrane/juxtamembrane region, was found to be essential for its internalization.
    Mesh-Begriff(e) Adaptor Proteins, Vesicular Transport/genetics ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; B-Lymphocytes/immunology ; Cell Membrane/chemistry ; Cell Membrane/physiology ; Cells, Cultured ; Endocytosis ; Green Fluorescent Proteins/genetics ; Lymphocyte Activation ; Mice ; Receptors, Antigen, B-Cell/physiology
    Chemische Substanzen Adaptor Proteins, Vesicular Transport ; LAB protein, mouse ; Receptors, Antigen, B-Cell ; Green Fluorescent Proteins (147336-22-9)
    Sprache Englisch
    Erscheinungsdatum 2007-01
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1013745-2
    ISSN 1460-2377 ; 0953-8178
    ISSN (online) 1460-2377
    ISSN 0953-8178
    DOI 10.1093/intimm/dxl118
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  7. Artikel: Cholesterol depletion inhibits src family kinase-dependent calcium mobilization and apoptosis induced by rituximab crosslinking.

    Unruh, Tammy L / Li, Haidong / Mutch, Cathlin M / Shariat, Neda / Grigoriou, Lana / Sanyal, Ratna / Brown, Christopher B / Deans, Julie P

    Immunology

    2005  Band 116, Heft 2, Seite(n) 223–232

    Abstract: The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B-cell non-Hodgkin's lymphoma and antibody-based autoimmune diseases. Mechanisms mediating B-cell depletion by rituximab are not completely understood and may ... ...

    Abstract The monoclonal antibody (mAb) rituximab produces objective clinical responses in patients with B-cell non-Hodgkin's lymphoma and antibody-based autoimmune diseases. Mechanisms mediating B-cell depletion by rituximab are not completely understood and may include direct effects of signalling via the target antigen CD20. Like most but not all CD20 mAbs, rituximab induces a sharp change in the solubility of the CD20 protein in the non-ionic detergent Triton-X-100, reflecting a dramatic increase in the innate affinity of CD20 for membrane raft signalling domains. Apoptosis induced by rituximab hypercrosslinking has been shown to require src family kinases (SFK), which are enriched in rafts. In this report we provide experimental evidence that SFK-dependent apoptotic signals induced by rituximab are raft dependent. Cholesterol depletion prevented the association of hypercrosslinked CD20 with detergent-insoluble rafts, and attenuated both calcium mobilization and apoptosis induced with rituximab. CD20 cocapped with the raft-associated transmembrane adaptor LAB/NTAL after hypercrosslinking with CD20 mAbs, regardless of their ability to induce a change in the affinity of CD20 for rafts. Taken together, the data demonstrate that CD20 hypercrosslinking via rituximab activates SFKs and downstream signalling events by clustering membrane rafts in which antibody-bound CD20 is localized in a high-affinity configuration.
    Mesh-Begriff(e) Antibodies, Monoclonal/pharmacology ; Antibodies, Monoclonal, Murine-Derived ; Antigens, CD20/metabolism ; Antineoplastic Agents/pharmacology ; Apoptosis/drug effects ; Burkitt Lymphoma/immunology ; Burkitt Lymphoma/metabolism ; Burkitt Lymphoma/pathology ; Calcium/metabolism ; Cholesterol/physiology ; Humans ; Rituximab ; Signal Transduction/drug effects ; Signal Transduction/immunology ; Tumor Cells, Cultured ; src-Family Kinases/physiology
    Chemische Substanzen Antibodies, Monoclonal ; Antibodies, Monoclonal, Murine-Derived ; Antigens, CD20 ; Antineoplastic Agents ; Rituximab (4F4X42SYQ6) ; Cholesterol (97C5T2UQ7J) ; src-Family Kinases (EC 2.7.10.2) ; Calcium (SY7Q814VUP)
    Sprache Englisch
    Erscheinungsdatum 2005-10
    Erscheinungsland England
    Dokumenttyp Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 80124-0
    ISSN 1365-2567 ; 0019-2805 ; 0953-4954
    ISSN (online) 1365-2567
    ISSN 0019-2805 ; 0953-4954
    DOI 10.1111/j.1365-2567.2005.02213.x
    Datenquelle MEDical Literature Analysis and Retrieval System OnLINE

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