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Sample records for mutant b-cell lines

  1. Cooperativity between the J and S elements of class II major histocompatibility complex genes as enhancers in normal and class II- negative patient and mutant B cell lines

    PubMed Central

    1995-01-01

    The class II major histocompatibility complex genes all contain in their proximal promoters three cis-elements called S, X, and Y that are conserved in both sequence and position, and a fourth element, J, conserved in sequence but not in position. J, X, and Y and, to some extent, S, have been shown to be functionally important in regulation of expression of these genes. In the present study, a protein factor that binds cooperatively to the S plus J elements of the promoter of the class II major histocompatibility complex gene DPA has been detected. Moreover, functional cooperativity between S and J in activation of the enhancerless -40 interferon-beta (-40 IFN-beta) promoter has been demonstrated. Finally, the latter assay appears to subdivide complementation group A of class II negative human B cell lines that includes both mutants generated in vitro and cells from patients with the bare lymphocyte syndrome (type II). In three of these cell lines, the enhancerless -40 IFN-beta promoter containing the S plus J elements was functionally active, while in the others it was inactive. PMID:7790817

  2. Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic defects defined by complementation analysis.

    PubMed Central

    Bénichou, B; Strominger, J L

    1991-01-01

    Expression of class II major histocompatibility complex antigens in defective B-lymphoblastoid cell lines from patients with class II antigen deficiency and from in vitro mutants generated with the same phenotype was studied. By heterogenetic fusion experiments, at least three, and probably four, complementation groups were defined. Furthermore, clone 13 (a DR-, DP-, but DQ+ cell line) appeared to belong to the RJ2.2.5 complementation group, for which all other members are DR-, DP-, and also DQ-. Thus, it is hypothesized that the cell lines of this group lack the activity of a gene that can differentially regulate the DR/DP and the DQ promoters. Images PMID:1852002

  3. Reversion of a transcriptionally defective MHC class II-negative human B-cell mutant.

    PubMed Central

    Ombra, M N; Perfetto, C; Autiero, M; Anzisi, A M; Pasquinelli, R; Maffei, A; Del Pozzo, G; Guardiola, J

    1993-01-01

    RJ2.2.5, a mutant derived from the human B-lymphoma cell, Raji, is unable to express the MHC class II genes because of a recessive transcriptional defect attributed to the lack of an activator function. We report the isolation of a RJ2.2.5 revertant, namely AR, in which the expression of the mRNAs encoded by these genes is restored. Comparison of the binding of nuclear extracts or of partially purified nuclear preparations from the wild-type, the mutant and the revertant cells to a conserved MHC class II promoter element, the X-box, showed no alteration in the mobility of the complexes thus formed. However, in extracts from RJ2.2.5, and other MHC class II negative cell lines, such as HeLa, the amount of complex observed was significantly higher than in wild-type Raji cells. Furthermore, the binding activity exhibited by the AR revertant was lower than that of the RJ2.2.5 and higher than that of Raji. The use of specific monoclonal antibodies indicated that in all cases c-Jun and c-Fos or antigenically related proteins were required for binding. An inverse correlation between the level of DNA-protein complex formed and the level of MHC class II gene mRNA expressed in the three cell lines was apparent, suggesting that overexpression of a DNA binding factor forming complexes with class II promoter elements may cause repression of MHC class II transcription. A model which reconciles the previously ascertained recessivity of the phenotype of the mutation carried by RJ2.2.5 with the findings reported here is discussed. Images PMID:8441650

  4. A human follicular lymphoma B cell line hypermutates its functional immunoglobulin genes in vitro.

    PubMed

    Wu, H; Pelkonen, E; Knuutila, S; Kaartinen, M

    1995-12-01

    The functional immunoglobulin (Ig) genes of B lymphocytes undergo somatic mutations during immune responses. These mutations modify the antigen binding site of the immunoglobulins, thereby enhancing the average affinity of the antibodies produced. The molecular mechanism underlying these B cell hypermutations remains unresolved, partly because it is difficult to grow normal B cells in long-term cell cultures and because there is no suitable transformed or malignant B cell line which generates mutations in its immunoglobulin genes in vitro. Here, we show that the recently established follicular lymphoma line HF-1.3.4 generates somatic hypermutations in vitro at a high frequency of 0.7 x 10(-6) mutations per base pair per generation in standard cell cultures (RPMI 1640 + 5% fetal calf serum). This shows for the first time that B cell hypermutation can occur without T cells or T cell factors. The mutation frequency increased approximately tenfold to 1 x 10(-5) mutations/base pair/generation with B cell-specific growth factors (interleukins-2 and -4 and three antibodies stimulatory to HF-1.3.4 cells). This HF-1.3.4 lymphoma line may help to elucidate the molecular mechanism of Ig gene hypermutation.

  5. Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire

    SciTech Connect

    Magari, Masaki; Kanehiro, Yuichi; Todo, Kagefumi; Ikeda, Mika; Kanayama, Naoki Ohmori, Hitoshi

    2010-05-28

    Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SW{Delta}C, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SW{Delta}C cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SW{Delta}C cells, although the protein might be highly susceptible to degradation. In DT40-SW{Delta}C cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SW{Delta}C cells may be useful for constructing Ab libraries for efficient screening of mAbs in vitro.

  6. Protein kinase C inhibitor sotrastaurin selectively inhibits the growth of CD79 mutant diffuse large B-cell lymphomas.

    PubMed

    Naylor, Tara L; Tang, Huaping; Ratsch, Boris A; Enns, Andreas; Loo, Alice; Chen, Liqing; Lenz, Peter; Waters, Nigel J; Schuler, Walter; Dörken, Bernd; Yao, Yung-Mae; Warmuth, Markus; Lenz, Georg; Stegmeier, Frank

    2011-04-01

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.

  7. Modulation of a human lymphoblastoid B cell line by cyclic AMP. Ig secretion and phosphatidylcholine metabolism

    SciTech Connect

    Shearer, W.T.; Patke, C.L.; Gilliam, E.B.; Rosenblatt, H.M.; Barron, K.S.; Orson, F.M.

    1988-09-01

    A transformed human B cell line, LA350, was found to be sensitive to cAMP-elevating agents by responding with rapid (0 to 2 h) severalfold elevations of intracellular cAMP to treatment with cholera toxin, isobutylmethylxanthine (IBMX), forskolin, and dibutyryl cAMP (all p less than 0.001). These cAMP-elevating agents also produced significant inhibitions of subsequent (48 to 72 h) Ig secretion by the same B cells as measured by a reverse hemolytic plaque assay and an enzyme-linked immunoadsorbent assay for IgM (both p less than 0.001). PMA- and IBMX-treated cells were particularly responsive to the effects of cholera toxin, showing a doubling of cAMP content and profound decrease in Ig production (p less than 0.001). Because our previous studies had correlated activation of the metabolic turnover of the phosphatidylcholine (PC) fraction of membrane phospholipids with enhanced Ig secretion, we examined the sensitivity of PC metabolism to cAMP in control and PMA-stimulated cells. Formation of PC was found to be inhibited by forskolin and IBMX (both p less than 0.002) but breakdown of PC was stimulated (p less than 0.001). These findings imply that as the enzymatic products of PC, choline phosphate and diacylglycerol, are depleted due to the combined effects of cAMP upon synthesis and turnover of PC, there is a decrease in Ig secretion. Since diacylglycerol activates protein kinase C, it appears reasonable that Ig secretion is at least partially regulated by cAMP-responsive alterations in PC metabolism produced by protein kinase C-induced phosphorylation. We conclude that the early cAMP-sensitive changes in PC metabolism in this activated B cell line may signal for subsequent alterations in Ig secretion.

  8. Efficient Infection of a Human B Cell Line with Cell-Free Kaposi's Sarcoma-Associated Herpesvirus

    PubMed Central

    Dollery, Stephen J.; Santiago-Crespo, Rey J.; Kardava, Lela; Moir, Susan

    2014-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is causatively linked to two B cell lymphoproliferative disorders, multicentric Castleman's disease and primary effusion lymphoma. Latently infected B cells are a major KSHV reservoir, and virus activation from tonsillar B cells can result in salivary shedding and virus transmission. Paradoxically, human B cells (primary and continuous) are notoriously refractory to infection, thus posing a major obstacle to the study of KSHV in this cell type. By performing a strategic search of human B cell lymphoma lines, we found that MC116 cells were efficiently infected by cell-free KSHV. Upon exposure to recombinant KSHV.219, enhanced green fluorescent protein reporter expression was detected in 17 to 20% of MC116 cells. Latent-phase transcription and protein synthesis were detected by reverse transcription-PCR and detection of latency-associated nuclear antigen expression, respectively, in cell lysates and individual cells. Selection based on the puromycin resistance gene in KSHV.219 yielded cultures with all cells infected. After repeated passaging of the selected KSHV-infected cells without puromycin, latent KSHV was maintained in a small fraction of cells. Infected MC116 cells could be induced into lytic phase with histone deacetylase inhibitors, as is known for latently infected non-B cell lines, and also selectively by the B cell-specific pathway involving B cell receptor cross-linking. Lytic-phase transition was documented by red fluorescent protein reporter expression, late structural glycoprotein (K8.1A, gH) detection, and infectious KSHV production. MC116 cells were CD27−/CD10+, characteristic of transitional B cells. These findings represent an important step in the establishment of an efficient continuous B cell line model to study the biologically relevant steps of KSHV infection. IMPORTANCE PMID:24257608

  9. [Molecular analysis of space mutant line of kidney bean].

    PubMed

    Zhang, J; Li, J G; Wang, P S; Wang, X Q; Jiang, X C

    2000-12-01

    Objective. To identify the occurrence of gene mutant in mutant lines in the offspring of Kidney bean seeds under space flight condition. Method. Kidney bean seeds were carried onboard a recoverable satellite for 15 days in space and were planted on the ground after recovery. Five mutant lines showing variation in the form of leaf blade and their parents were analyzed with RAPD technique. Result. 50 random 10-mer primers were used in this study, among which 20 primers generated 180 polymorphic DNA bands, their size ranged from 200 bp to 2000 bp. 3 primers amplified obviously different bands in the DNA of mutant lines in comparison with that of the control. Conclusion. This is the first molecular analysis of the mutant lines of Kidney bean generated by space mutagenesis at DNA level. The result of RAPD analysis indicated that distinct variations were demonstrated in the DNA of mutant lines as compared with that of the original control.

  10. Comparative proteomic analysis of drug sodium iron chlorophyllin addition to Hep 3B cell line.

    PubMed

    Zhang, Jun; Wang, Wenhai; Yang, Fengying; Zhou, Xinwen; Jin, Hong; Yang, Peng-yuan

    2012-09-21

    The human hepatoma 3B cell line was chosen as an experimental model for in vitro test of drug screening. The drugs included chlorophyllin and its derivatives such as fluo-chlorophyllin, sodium copper chlorophyllin, and sodium iron chlorophyllin. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method was used in this study to obtain the primary screening results. The results showed that sodium iron chlorophyllin had the best LC(50) value. Proteomic analysis was then performed for further investigation of the effect of sodium iron chlorophyllin addition to the Hep 3B cell line. The proteins identified from a total protein extract of Hep 3B before and after the drug addition were compared by two-dimensional-gel-electrophoresis. Then 32 three-fold differentially expressed proteins were successfully identified by MALDI-TOF-TOF-MS. There are 29 unique proteins among those identified proteins. These proteins include proliferating cell nuclear antigen (PCNA), T-complex protein, heterogeneous nuclear protein, nucleophosmin, heat shock protein A5 (HspA5) and peroxiredoxin. HspA5 is one of the proteins which are involved in protecting cancer cells against stress-induced apoptosis in cultured cells, protecting them against apoptosis through various mechanisms. Peroxiredoxin has anti-oxidant function and is related to cell proliferation, and signal transduction. It can protect the oxidation of other proteins. Peroxiredoxin has a close relationship with cancer and can eventually become a disease biomarker. This might help to develop a novel treatment method for carcinoma cancer.

  11. Surface IgM mediated regulation of RAG gene expression in E mu-N-myc B cell lines.

    PubMed Central

    Ma, A; Fisher, P; Dildrop, R; Oltz, E; Rathbun, G; Achacoso, P; Stall, A; Alt, F W

    1992-01-01

    Transgenic mice carrying either the c-myc or N-myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre-B and B cell lymphomas (E mu-c-myc and E mu-N-myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v-myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre-B stage of development-including the recombinase activating genes RAG-1 and RAG-2. Continued RAG-1 and RAG-2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross-linking of the surface immunoglobulin on these lines with an anti-mu antibody leads to rapid, specific and reversible down-regulation of RAG-1 and RAG-2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG-1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre-B to B cell transition. Images PMID:1628630

  12. T cell binding to B lymphoid cell lines in humans: a marker for T-B cell interaction?

    PubMed

    Goust, J M; Fudenberg, H H

    1983-04-15

    Binding of human circulating T cells to established normal and malignant B cell lines results in rosette formation. The percentage of B cells, circulating T cells, and thymocytes able to bind to the B-LCL Raji were 0%, 59 +/- 4% and 61 +/- 6%, respectively. The percentage of rosettes formed between Raji cells and circulating mononuclear cells from 92 normal individuals was 27.8 +/- 5.3%, and remained stable over several months. This phenomenon seems to involve relatively mature B cells, and a T cell marker which appears early in T cell ontogeny. In the peripheral blood, most of the B-LCL binding T cells exhibit a 'helper-inducer' phenotype, as determined with the monoclonal antibodies Leu 3a and OKT4. However, a significant percentage of T cells with so-called 'cytotoxic-suppressor' markers (Leu 2a and OKT8) also bind to B-LCL. The T cells involved in this morphological interactive reaction with B cells might conceivably be specifically involved in regulating B cell functions. Enumeration of this particular subset may be useful in conditions where abnormal T-B cell interactions are suspected. PMID:6601166

  13. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

    PubMed

    Dai, Haiping; Ehrentraut, Stefan; Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G; MacLeod, Roderick A F

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings

  14. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines.

    PubMed

    Dai, Haiping; Ehrentraut, Stefan; Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G; MacLeod, Roderick A F

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2-10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings

  15. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines

    PubMed Central

    Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G.; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G.; MacLeod, Roderick A. F.

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings

  16. The transcriptional program of a human B cell line in response to Myc

    PubMed Central

    Schuhmacher, Marino; Kohlhuber, Franz; Hölzel, Michael; Kaiser, Carmen; Burtscher, Helmut; Jarsch, Michael; Bornkamm, Georg W.; Laux, Gerhard; Polack, Axel; Weidle, Ulrich H.; Eick, Dirk

    2001-01-01

    The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes. PMID:11139609

  17. B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

    PubMed

    Zwollo, Patty; Ray, Jocelyn C; Sestito, Michael; Kiernan, Elizabeth; Wiens, Gregory D; Kaattari, Steve; StJacques, Brittany; Epp, Lidia

    2015-01-01

    Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative

  18. Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines

    PubMed Central

    1986-01-01

    Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell- stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes. PMID:3086481

  19. Malignant hematopoietic cell lines: in vitro models for the study of primary mediastinal B-cell lymphomas.

    PubMed

    Drexler, Hans G; Ehrentraut, Stefan; Nagel, Stefan; Eberth, Sonja; MacLeod, Roderick A F

    2015-01-01

    Primary mediastinal B-cell lymphoma (PMBL) is a highly aggressive disease with a unique set of biological, clinical, morphological, immunological and in particular genetic features that in the molecular era of defining lymphomas clearly distinguishes it as a separate entity from other diffuse large B-cell lymphomas (DLBCL). A precise molecular diagnosis of PMBL can be achieved by gene expression profiling. The signature gene expression profile of PMBL is more closely related to classic Hodgkin lymphoma (cHL) than to other DLBCL subgroups. A number of common genetic aberrations in PMBL and cHL further underscore their close relationship. To investigate the pathobiology of lymphomas in depth, many groups have turned to cell lines that are suitable models facilitating molecular studies and providing unique insights. For the purposes of the current perspective, we focus on four bona fide PMBL-derived cell lines (FARAGE, KARPAS-1106, MEDB-1, U-2940) that we identified and validated as such through hierarchical cluster analysis among a large collection of leukemia-lymphoma cell lines. These gene expression profiles showed that the four PMBL cell lines represent a distinct entity and are most similar to cHL cell lines, confirming derivation from a related cell type. A validated cell line resource for PMBL should assist those seeking druggable targets in this entity. This review aims to provide a comprehensive overview of the currently available cellular models for the study of PMBL. PMID:25480038

  20. Functional differences between immunoglobulins M and D expressed on the surface of an immature B-cell line.

    PubMed Central

    Tisch, R; Roifman, C M; Hozumi, N

    1988-01-01

    Crosslinked IgM molecules expressed on the surface of immature B cells mediate responses that inhibit further development, in contrast to the activational and proliferative events that follow crosslinking of the mu heavy chain in mature B cells. Concomitant with this change in IgM signaling capacity is the appearance of surface IgD, which has been proposed to modulate the response elicited by the mu heavy chain. In an attempt to gain insight into the mechanism(s) by which surface IgM is able to generate such disparate responses, delta heavy chain gene transfectants of the murine B-cell lymphoma line WEHI-231 were established. WEHI-231 cells resemble phenotypically immature B cells, in addition to being highly susceptible to the growth-inhibitory effect of surface IgM cross-linking. Endogenous mu and exogenous delta heavy chains expressed on the surface of the transfectants were compared for their role in cell proliferation and on gene expression. Our results indicate that the growth-inhibitory response is associated only with the mu heavy chain and that surface IgD does not mediate such a response. Furthermore, in contrast to IgM, IgD molecules appear to have an inductive effect on the expression of Myc and the endogenous mu and exogenous delta Ig heavy chain genes but not on the expression of the housekeeping gene encoding beta 2-microglobulin. These findings suggest that IgM and IgD are functionally distinct when expressed on the surface of an immature B cell. Images PMID:3137579

  1. Two distinct genetic loci regulating class II gene expression are defective in human mutant and patient cell lines.

    PubMed Central

    Yang, Z; Accolla, R S; Pious, D; Zegers, B J; Strominger, J L

    1988-01-01

    Heterokaryons were prepared and analyzed shortly after cell fusion using two mutant class-II-negative human B cell lines (RJ 2.2.5 and 6.1.6) and a cell line (TF) from a patient with a class-II-negative Bare Lymphocyte Syndrome. The resulting transient heterokaryons were analyzed by using an anti-HLA-DR monoclonal antibody to assess the cell surface expression of HLA-DR (the major subtype of class II antigens) by immunofluorescence microscopy and by using uniformly 32P-labeled SP6 RNA probes in Northern blots and RNase protection assays to assess mRNA synthesis. We find that class II gene expression in a B cell line from a Bare Lymphocyte Syndrome patient (TF) is rescued by a B cell line which expresses class II antigens indicating that this disease, at least in part, is caused by a defect(s) in a genetic locus encoding a factor(s) necessary for class II gene expression. Secondly, reciprocal genetic complementation was demonstrated in the heterokaryons 6.1.6 x RJ 2.2.5 and TF x RJ 2.2.5 (but not in TF x 6.1.6) by detection of cell surface DR by immunofluorescence microscopy and by a novel class II mRNA typing technique which allows characterization of distinct class II alleles. Thus, the two mutants generated in vitro have defects at two different genetic loci encoding specific regulatory factors necessary for human class II gene expression. One of these mutant cell lines, but not the other, complements the defect in the patient cell line, TF. Images PMID:2458252

  2. Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line.

    PubMed

    Sturm, E; Zimmerman, T L; Crawford, A R; Svetlov, S I; Sundaram, P; Ferrara, J L; Karpen, S J; Crawford, J M

    2000-01-01

    Endotoxemia leads to cytokine-mediated alterations of the hepatocellular sodium-taurocholate-cotransporting polypeptide (ntcp). We hypothesized that stimulated macrophages are essential transducers for down-regulating hepatocellular bile salt uptake in response to endotoxin (lipopolysaccharide [LPS]) exposure. Using an in vitro model, we exposed mouse macrophages (IC-21 cell line) to LPS for 24 hours. Concentrations of cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 increased 10.6-fold, 12.5-fold, and 444-fold, respectively, in LPS-conditioned IC-21 medium (CM) versus unconditioned IC-21 medium (UM). WIF-B rat hepatoma hybrid cells were incubated with either CM or UM or treated directly with medium containing recombinant TNF-alpha, IL-1beta, and IL-6. [(3)H]Taurocholate ([(3)H]TC) uptake decreased in WIF-B cells exposed to either TNF-alpha (54% of control), IL-1beta (78%), IL-6 (55%) as single additives, or in triple combination (TCC) (43%). A virtually identical decrease was observed after exposing WIF-B cells to CM (52%, P <.001). LPS had no direct effect on [(3)H]TC uptake. CM treatment did not decrease L-alanine transport in WIF-B cells. Blocking antibodies against TNF-alpha, IL-1beta, and IL-6 restored the diminished [(3)H]TC uptake in cells exposed to TCC and CM to 87% and 107% of controls, respectively. Northern blotting revealed that ntcp messenger RNA (mRNA) expression was significantly reduced in WIF-B cells after exposure to CM, and in primary rat hepatocytes exposed to CM or TNF-alpha (68%, 14%, and 29% of control, respectively). We conclude that macrophages and their ability to secrete the cytokines TNF-alpha, IL-1beta, and IL-6 may be essential in mediating the endotoxin-induced cholestatic effect of decreased hepatocellular bile salt uptake. PMID:10613737

  3. Apoptotic induction in B-cell acute lymphoblastic leukemia cell lines treated with a protein kinase Cβ inhibitor.

    PubMed

    Saba, Nakhle S; Levy, Laura S

    2011-05-01

    B-cell acute lymphoblastic leukemia (B-ALL) in adults exhibits a 5-year disease-free survival rate of only 25-40% after currently available treatment. Protein kinase Cβ (PKCβ) is under active consideration as a rational therapeutic target in several B-cell malignancies, but studies of its possible utility in B-ALL are lacking. Expression of PKCβ1 and PKCβ2 isoforms was demonstrated in five B-ALL cell lines characterized by distinctive chromosomal translocations, and sensitivity to PKCβ-selective inhibition was examined. Inhibitor treatment resulted in a dose-dependent reduction in viability in all cell lines, although pro-B ALL with t(4;11)(q21;q23) was most sensitive. Apoptotic induction was evident after 24-48 h of treatment, and an inhibition of cell cycle progression was detected in one cell line. Treatment resulted in a rapid induction of poly(ADP-ribose) polymerase (PARP) cleavage, indicating caspase-3-mediated apoptosis, and a rapid reduction in phosphorylation of AKT and its downstream target glycogen synthase kinase 3β (GSK3β). These results indicate that PKCβ targeting should be considered as a potential treatment option in B-ALL.

  4. Assessment of carbonic anhydrase IX expression and extracellular pH in B-cell lymphoma cell line models

    PubMed Central

    Chen, Liu Qi; Howison, Christine M.; Spier, Catherine; Stopeck, Alison T.; Malm, Scott W.; Pagel, Mark D.; Baker, Amanda F.

    2015-01-01

    The expression of carbonic anhydrase (CA IX) and it’s relation to acidosis in lymphomas has not been widely studied. We investigated the protein expression of CA IX in a human B-cell lymphoma tissue microarray, and in Raji, Ramos, and Granta 519 lymphoma cell lines and tumor models, while also investigating the relation with hypoxia. An imaging method, acidoCEST MRI, was used to estimate lymphoma xenograft extracellular pH (pHe). Our results showed that clinical lymphoma tissues and cell line models in vitro and in vivo had moderate CA IX expression. Although in vitro studies showed that CA IX expression was induced by hypoxia, in vivo studies did not show this correlation. Untreated lymphoma xenograft tumor pHe had acidic fractions, and an Acidity Score was qualitatively correlated with CA IX expression. Therefore, CA IX is expressed in B-cell lymphomas and is qualitatively correlated with extracellular acidosis in xenograft tumor models. PMID:25130478

  5. Piperlongumine inhibits the proliferation and survival of B-cell acute lymphoblastic leukemia cell lines irrespective of glucocorticoid resistance

    SciTech Connect

    Han, Seong-Su; Han, Sangwoo; Kamberos, Natalie L.

    2014-09-26

    Highlights: • PL inhibits the proliferation of B-ALL cell lines irrespective of GC-resistance. • PL selectively kills B-ALL cells by increasing ROS, but not normal counterpart. • PL does not sensitize majority of B-ALL cells to DEX. • PL represses the network of constitutively activated TFs and modulates their target genes. • PL may serve as a new therapeutic molecule for GC-resistant B-ALL. - Abstract: Piperlongumine (PL), a pepper plant alkaloid from Piper longum, has anti-inflammatory and anti-cancer properties. PL selectively kills both solid and hematologic cancer cells, but not normal counterparts. Here we evaluated the effect of PL on the proliferation and survival of B-cell acute lymphoblastic leukemia (B-ALL), including glucocorticoid (GC)-resistant B-ALL. Regardless of GC-resistance, PL inhibited the proliferation of all B-ALL cell lines, but not normal B cells, in a dose- and time-dependent manner and induced apoptosis via elevation of ROS. Interestingly, PL did not sensitize most of B-ALL cell lines to dexamethasone (DEX). Only UoC-B1 exhibited a weak synergistic effect between PL and DEX. All B-ALL cell lines tested exhibited constitutive activation of multiple transcription factors (TFs), including AP-1, MYC, NF-κB, SP1, STAT1, STAT3, STAT6 and YY1. Treatment of the B-ALL cells with PL significantly downregulated these TFs and modulated their target genes. While activation of AURKB, BIRC5, E2F1, and MYB mRNA levels were significantly downregulated by PL, but SOX4 and XBP levels were increased by PL. Intriguingly, PL also increased the expression of p21 in B-ALL cells through a p53-independent mechanism. Given that these TFs and their target genes play critical roles in a variety of hematological malignancies, our findings provide a strong preclinical rationale for considering PL as a new therapeutic agent for the treatment of B-cell malignancies, including B-ALL and GC-resistant B-ALL.

  6. Cloning and Expression of CD19, a Human B-Cell Marker in NIH-3T3 Cell Line

    PubMed Central

    Abbasi-Kenarsari, Hajar; Shafaghat, Farzaneh; Baradaran, Behzad; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

    2015-01-01

    Background CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Methods Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5α competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using KpnI and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Results Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells (29 and 93%, respectively). Conclusion Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera. PMID:25926951

  7. Enhancement of anti-tumor activity of natural killer cells by BALL-1, a B cell lymphoma line.

    PubMed

    Hirashima, M; Yoshida, N; Seki, M; Okada, H; Takamura, S; Mihara, Y

    1998-04-01

    The anti-tumor activity of human peripheral blood mononuclear cells (PBMC) against various tumor cell line cells (K562, Daudi, KMG-2, and KATOIII) was enhanced by coculture with irradiated BALL-1, but not with other irradiated B cell line cells (NALM-1, Namalwa, and Daudi). PBMC cocultured with BALL-1, however, failed to exhibit evident cytotoxicity against autologous concanavalin A-induced lymphoblasts. The enhancement of the anti-tumor activity seemed not to be correlated with EBNA and HLA-DR expression on B cell line cells. Monoclonal antibodies (mAbs) against interleukin (IL)-2, interferon-gamma, IL-12, IL-15, tumor necrosis factor-alpha and lymphotoxin showed little or no suppression of the anti-tumor activity of PBMC treated with irradiated BALL-1. Furthermore, the culture supernatants of BALL-1 failed to enhance the anti-tumor activity of PBMC, suggesting no involvement of soluble factors in the induction of the anti-tumor activity. The anti-tumor activity of PBMC treated with BALL-1 was synergistically enhanced by an additional IL-2 stimulation. Periodate-lysine-paraformaldehyde-fixed, but not ethanol- or acetone-fixed, BALL-1 could significantly enhance the anti-tumor activity. Furthermore, BALL-1-derived membrane fraction, but not that of Daudi, enhances the anti-tumor activity. It was thus suggested that some membrane glycoproteins on the cell surface of BALL-1 play a crucial role in the induction of the anti-tumor activity. By analysis using mAbs against human leukocytes, we found that depletion of CD11b, CD16, and CD56-positive cells resulted in decreased anti-tumor activity, suggesting that the main effector cells in the BALL-1-induced anti-tumor activity were natural killer (NK) cells. The present results thus raise the possibility that BALL-1, probably via membrane glycoproteins, modulates NK cell-mediated anti-tumor activity. PMID:9617349

  8. Development of a robust method for establishing B cell lines using Epstein-Barr Virus.

    PubMed

    Danjoh, Inaho; Sone, Hiyori; Shirota, Ryoko; Hiroyama, Takashi; Nakamura, Yukio

    2012-08-01

    B lymphoblastoid cell lines (B-LCLs) are generally established from B lymphocytes by infection with Epstein-Barr virus (EBV). As their genomic structure is stable in culture, B-LCLs are a valuable resource for many types of analysis. The efficiency of establishing B-LCLs from freshly obtained blood samples from healthy individuals is almost 100 %; however, for blood samples stored inappropriately after collection or held in long-term storage as peripheral blood mononuclear cells (PBMCs) in liquid nitrogen, the efficiency of B-LCL establishment can be considerably lower. To date, we have established more than 550 B-LCLs from 685 PBMC samples that have been stored in liquid nitrogen for over 20 yr. The PBMCs were prepared from blood samples donated by individuals belonging to native minority ethnic groups in outlying regions of South America and elsewhere. The establishment of B-LCLs from this material is difficult, and failure results in the waste of valuable and rare samples. We sought to improve our success rate for establishing B-LCLs from these difficult and irreplaceable samples by a detailed examination of each step of the process. The analysis showed that two parameters were particularly critical to the success rate: the density of the PBMCs plated after EBV infection and the EBV titer. These observations shed light on cases where establishment of B-LCLs was hard due to the small number of PBMCs or damage to the cells.

  9. Human B Cell-Derived Lymphoblastoid Cell Lines Constitutively Produce Fas Ligand and Secrete MHCII+FasL+ Killer Exosomes

    PubMed Central

    Klinker, Matthew W.; Lizzio, Vincent; Reed, Tamra J.; Fox, David A.; Lundy, Steven K.

    2013-01-01

    Immune suppression mediated by exosomes is an emerging concept with potentially immense utility for immunotherapy in a variety of inflammatory contexts, including allogeneic transplantation. Exosomes containing the apoptosis-inducing molecule Fas ligand (FasL) have demonstrated efficacy in inhibiting antigen-specific immune responses upon adoptive transfer in animal models. We report here that a very high frequency of human B cell-derived lymphoblastoid cell lines (LCL) constitutively produce MHCII+FasL+ exosomes that can induce apoptosis in CD4+ T cells. All LCL tested for this study (>20 independent cell lines) showed robust expression of FasL, but had no detectable FasL on the cell surface. Given this intracellular sequestration, we hypothesized that FasL in LCL was retained in the secretory lysosome and secreted via exosomes. Indeed, we found both MHCII and FasL proteins present in LCL-derived exosomes, and using a bead-based exosome capture assay demonstrated the presence of MHCII+FasL+ exosomes among those secreted by LCL. Using two independent experimental approaches, we demonstrated that LCL-derived exosomes were capable of inducing antigen-specific apoptosis in autologous CD4+ T cells. These results suggest that LCL-derived exosomes may present a realistic source of immunosuppressive exosomes that could reduce or eliminate T cell-mediated responses against donor-derived antigens in transplant recipients. PMID:24765093

  10. VH mutant rabbits lacking the VH1a2 gene develop a2+ B cells in the appendix by gene conversion-like alteration of a rearranged VH4 gene.

    PubMed

    Sehgal, D; Mage, R G; Schiaffella, E

    1998-02-01

    We investigated the molecular basis for the appearance of V(H)a2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of V(H) genes because of a small deletion encompassing V(H)1a2, the 3'-most gene in the V(H) locus. The V(H)1 gene is the major source of V(H)a allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits. We separated appendix cells from a 6-wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM+ a2+ and IgM+ a2- populations by reverse transcriptase-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that, in a2+ B cells, the first D proximal functional gene in Alicia rabbits, V(H)4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream V(H) genes were identified as potential gene sequence donors; V(H)9 was found to be the most frequently used gene donor. Among the a2- B cells, y33 was the most frequently rearranged gene.

  11. Ras is not required for the interleukin 3-induced proliferation of a mouse pro-B cell line, BaF3.

    PubMed

    Terada, K; Kaziro, Y; Satoh, T

    1995-11-17

    It has been demonstrated that Ras is involved in interleukin 3 (IL-3)-stimulated signal transduction in various hematopoietic cultured cells (Satoh, T., Nakafuku, M., Miyajima, A., and Kaziro, Y. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3314-3318; Duronio, V., Welham, M. J., Abraham, S., Dryden, P., and Schrader, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1587-1591). However, it has not been fully understood which of IL-3-promoted cellular responses, i.e. proliferation, survival, and differentiation, requires Ras function. We employed a system of inducible expression of the dominant-negative (S17N) or dominant-active (G12V) mutant of Ras in BaF3 mouse pro-B cell line to analyze the role of Ras in IL-3-stimulated signal transduction. Induction of the dominant-negative Ras(S17N) effectively inhibited the IL-3-induced activation of c-Raf-1 and mitogen-activated protein kinase (MAPK). Furthermore, the activation of fos gene promoter following IL-3 stimulation was almost completely abolished when Ras(S17N) was induced. Under these conditions, Ras(S17N) exhibited no inhibitory effect on IL-3-dependent proliferation assessed by the increase of cell numbers and a mitochondrial enzyme activity. The results indicate that Ras-dependent pathways, including the Raf/MAPK/Fos pathway, are dispensable for IL-3-induced growth stimulation. When BaF3 cells were treated with a tyrosine kinase inhibitor, herbimycin A, IL-3-dependent proliferation of the cells was impaired, suggesting that tyrosine kinase-mediated pathways are critical for growth promotion. On the other hand, apoptotic cell death caused by deprivation of IL-3 was prevented by the induction of the activated mutant Ras(G12V), although the rate of cell number increase was markedly reduced. Thus, it is likely that Ras-independent pathways play important roles to facilitate the proliferation although they may not be essential for IL-3-stimulated antiapoptotic signal transduction.

  12. Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

    PubMed Central

    Barth, C F; Humphries, E H

    1988-01-01

    The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte. Images PMID:2854197

  13. The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eμ-MYC driven B-cell lymphoma

    PubMed Central

    Walton, Mike I.; Eve, Paul D.; Hayes, Angela; Henley, Alan T.; Valenti, Melanie R.; De Haven Brandon, Alexis K.; Box, Gary; Boxall, Kathy J.; Tall, Matthew; Swales, Karen; Matthews, Thomas P.; McHardy, Tatiana; Lainchbury, Michael; Osborne, James; Hunter, Jill E.; Perkins, Neil D.; Aherne, G. Wynne; Reader, John C.; Raynaud, Florence I.; Eccles, Suzanne A.; Collins, Ian; Garrett, Michelle D.

    2016-01-01

    CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition. PMID:26295308

  14. The clinical development candidate CCT245737 is an orally active CHK1 inhibitor with preclinical activity in RAS mutant NSCLC and Eµ-MYC driven B-cell lymphoma.

    PubMed

    Walton, Mike I; Eve, Paul D; Hayes, Angela; Henley, Alan T; Valenti, Melanie R; De Haven Brandon, Alexis K; Box, Gary; Boxall, Kathy J; Tall, Matthew; Swales, Karen; Matthews, Thomas P; McHardy, Tatiana; Lainchbury, Michael; Osborne, James; Hunter, Jill E; Perkins, Neil D; Aherne, G Wynne; Reader, John C; Raynaud, Florence I; Eccles, Suzanne A; Collins, Ian; Garrett, Michelle D

    2016-01-19

    CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4 nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220 nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition. PMID:26295308

  15. PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line.

    PubMed

    Kumar, Vipul; Alt, Frederick W; Frock, Richard L

    2016-09-20

    Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B-cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro-B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.

  16. Enhanced insulin-receptor tyrosine kinase activity associated with chromosomal translocation (1;19) in a pre-B-cell leukemia line.

    PubMed

    Newman, J D; Harrison, L C; Eckardt, G S; Jack, I

    1992-02-01

    The gene for the insulin receptor has been assigned to chromosome 19 near the breakpoint of the translocation t(1;19) which occurs in 25% of pre-B-cell leukemias. Insulin receptors in a pre-B-cell leukemia cell line (ACV) with t(1;19) were found to have 2-fold higher affinity for insulin, 5-fold higher basal and insulin-stimulated beta sub-unit autophosphorylation, and 2-fold higher basal and 4-fold higher insulin-stimulated beta sub-unit kinase activity on the synthetic peptide poly(Glu,Tyr), compared to receptors in a B-cell line (ADD) with normal karyotype from the same patient. ACV cells had a novel 13-kb receptor mRNA species and expressed a DNA polymorphism localized to the tyrosine kinase domain of the receptor gene. These findings suggest that t(1;19) in the ACV cell may result in rearrangement of the insulin receptor gene and translation of a receptor with enhanced tyrosine kinase activity. PMID:1310491

  17. Downregulation of internal enhancer activity contributes to abnormally low immunoglobulin expression in the MedB-1 mediastinal B-cell lymphoma cell line.

    PubMed

    Ritz, Olga; Leithäuser, Frank; Hasel, Cornelia; Brüderlein, Silke; Ushmorov, Alexey; Möller, Peter; Wirth, Thomas

    2005-02-01

    Primary mediastinal B-cell lymphoma (PMBL) is a highly aggressive tumour with a unique pattern of clinical, morphological, immunological and genetic features distinct from other diffuse large B-cell lymphomas. PMBLs are characterized by a mature B-cell phenotype, but they typically lack immunoglobulin (Ig) gene expression. The PMBL cell line MedB-1 shares many characteristic properties of the primary tumour, including low-level Ig production despite a functionally rearranged IgVH gene and absence of 'crippling' mutations. In this study, a search was undertaken for reasons for downregulated Ig expression. Similar levels of the B-cell-specific transcription factors BOB.1/OBF.1 and PU.1 were found in MedB-1 cells to those in the Ig-producing UM-1 lymphoblastoid cell line. However, MedB-1 lacked the Oct2 transcription factor. Reporter assays showed that Ig-type promoters were active in MedB-1 cells. In contrast, activity of the intronic heavy chain enhancer was dramatically reduced. Ectopic expression of Oct2 was able partially to restore enhancer activity but transcription from the endogenous IgVH gene could not be rescued. Therefore, the role of epigenetic factors in the downregulation of Ig was investigated. Methylated histone 3 lysine 9, a reliable marker of chromatin silencing, was not detected in MedB-1 promoter and enhancer regions. Inhibition of DNA methyltransferase and of histone deacetylases also did not reactivate Ig production. These data suggest the existence of alternative mechanisms of Ig inhibition in MedB-1 cells, different from chromatin silencing and the lack of Oct2. PMID:15682441

  18. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines.

    PubMed

    Horsch, Marion; Aguilar-Pimentel, Juan Antonio; Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T; Lund, Anders H; Lee, Icksoo; Grossman, Lawrence I; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; de Angelis, Martin Hrabĕ; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype-envirotype interactions for other diseases.

  19. Cox4i2, Ifit2, and Prdm11 Mutant Mice: Effective Selection of Genes Predisposing to an Altered Airway Inflammatory Response from a Large Compendium of Mutant Mouse Lines

    PubMed Central

    Bönisch, Clemens; Côme, Christophe; Kolster-Fog, Cathrine; Jensen, Klaus T.; Lund, Anders H.; Lee, Icksoo; Grossman, Lawrence I.; Sinkler, Christopher; Hüttemann, Maik; Bohn, Erwin; Fuchs, Helmut; Ollert, Markus; Gailus-Durner, Valérie; Hrabĕ de Angelis, Martin; Beckers, Johannes

    2015-01-01

    We established a selection strategy to identify new models for an altered airway inflammatory response from a large compendium of mutant mouse lines that were systemically phenotyped in the German Mouse Clinic (GMC). As selection criteria we included published gene functional data, as well as immunological and transcriptome data from GMC phenotyping screens under standard conditions. Applying these criteria we identified a few from several hundred mutant mouse lines and further characterized the Cox4i2tm1Hutt, Ifit2tm1.1Ebsb, and Prdm11tm1.1ahl lines following ovalbumin (OVA) sensitization and repeated OVA airway challenge. Challenged Prdm11tm1.1ahl mice exhibited changes in B cell counts, CD4+ T cell counts, and in the number of neutrophils in bronchoalveolar lavages, whereas challenged Ifit2tm1.1Ebsb mice displayed alterations in plasma IgE, IgG1, IgG3, and IgM levels compared to the challenged wild type littermates. In contrast, challenged Cox4i2tm1Hutt mutant mice did not show alterations in the humoral or cellular immune response compared to challenged wild type mice. Transcriptome analyses from lungs of the challenged mutant mouse lines showed extensive changes in gene expression in Prdm11tm1.1ahl mice. Functional annotations of regulated genes of all three mutant mouse lines were primarily related to inflammation and airway smooth muscle (ASM) remodeling. We were thus able to define an effective selection strategy to identify new candidate genes for the predisposition to an altered airway inflammatory response under OVA challenge conditions. Similar selection strategies may be used for the analysis of additional genotype – envirotype interactions for other diseases. PMID:26263558

  20. Isolation and characterization of mutant CHO cell lines with compartment-specific resistance to brefeldin A

    PubMed Central

    1994-01-01

    22 CHOBFY (BFY) cell lines were isolated at a frequency 2-30 x 10(-7) from mutagenized populations on the basis of their ability to grow in the presence of 1 microgram/ml brefeldin A (BFA). Four of the five mutant lines tested are genetically stable and none of the mutant lines characterized degrade this drug. Immunofluorescence studies reveal that whereas early endosomes and the Golgi complex have nearly identical BFA sensitivities in the parent CHO line, the relative sensitivities of these two organelles were dramatically altered in all six mutant lines tested. Four cell lines maintain normal Golgi appearance at a BFA concentration as high as 10 micrograms/ml. Mutant lines show wide variation in the level of resistance to growth inhibition by BFA, but none of the mutant lines characterized grow above 2 micrograms/ml BFA. This specific growth inhibition is observed under conditions where Golgi morphology and function remain unaffected, suggesting that some factor(s) unrelated to Golgi function remains sensitive to BFA in BFY mutant lines. These observations provide strong evidence for the presence of multiple, organelle-specific targets for BFA. Cell-free measurements with membrane extracts establish that resistance to BFA in BFY-1 cells involves a membrane-associated factor distinct from ARFs and coatomers. This collection of mutant lines may prove valuable for the identification of intracellular target(s) for BFA and/or of effectors that interact upstream or downstream with these targets, thereby uncovering the cascade which regulates assembly of organelle- specific coats. PMID:8027187

  1. Expression of the B cell repertoire in lpr mice; abnormal expansion of a few VHJ558 germ-line genes.

    PubMed Central

    Alarcón-Riquelme, M E; Fernández, C

    1995-01-01

    Analysis of the VH gene repertoire of the J558 family was done in lipopolysaccharide (LPS)-stimulated resting cells and in vivo activated cells derived from C57Bl/6-lpr mice (IghCb). Using a restriction fragment length polymorphism (RFLP) based on digestion with the restriction enzyme Pstl, the expression of the subfamilies of the J558 family of VH genes could be determined. The J558 subfamily repertoire of resting B cells of the lpr mice was similar to that of the normal mice, while the J558 repertoire of the in vivo-activated cells was altered: analysis and sequencing of the IgM-expressed J558 repertoire of a sick female mouse showed that 50% of the J558 genes were represented by a single VH gene rearrangement, showing that its expansion was monoclonal. Furthermore, this same rearrangement made up to 90% of the J558 repertoire in the IgG2a+ population, showing that it had been preferentially selected, expanded and switched. However, compared with its IgM counterpart, it showed no evidence of somatic hypermutation. PMID:7851020

  2. Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project.

    PubMed

    Díez, Paula; Droste, Conrad; Dégano, Rosa M; González-Muñoz, María; Ibarrola, Nieves; Pérez-Andrés, Martín; Garin-Muga, Alba; Segura, Víctor; Marko-Varga, Gyorgy; LaBaer, Joshua; Orfao, Alberto; Corrales, Fernando J; De Las Rivas, Javier; Fuentes, Manuel

    2015-09-01

    A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of

  3. Isolation of a heterogeneous population of temperature-sensitive mutants of measles virus from persistently infected human lymphoblastoid cell lines.

    PubMed

    Ju, G; Udem, S; Rager-Zisman, B; Bloom, B R

    1978-06-01

    Two human lymphoblastoid B-cell lines, WI-L2 and 8866, were infected with the Edmonston strain of measles virus at a multiplicity of infection of 10(-6), and stable persistent infections were established. By immunofluorescence and electron microscopy, the vast majority of cells from both cell lines were expressing viral antigens and releasing virion-like particles. However, very little infectious virus could be detected at 37 degrees C, either by an infectious centers assay or by titration of supernates from persistently infected cultures. When cultures were shifted to 31 degrees C, the cells released a population of virus that was temperature-sensitive. Clonal analysis of supernatant virus at 31 degrees C revealed a highly heterogeneous population of temperature-sensitive mutants, differing in plating efficiency ratios, thermolability, and antigen production at the nonpermissive temperature. Factors such as interferon, defective interfering particles, and extracellular virus do not appear to be important in maintaining the persistent carrier state. These studies have important implications for persistent infections of lymphoid cells in vivo, and the slow neurological diseases associated with measles, subacute sclerosing panencephalitis, and multiple sclerosis.

  4. A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR(-) B cells.

    PubMed

    Boisson, Bertrand; Wang, Yong-Dong; Bosompem, Amma; Ma, Cindy S; Lim, Annick; Kochetkov, Tatiana; Tangye, Stuart G; Casanova, Jean-Laurent; Conley, Mary Ellen

    2013-11-01

    Approximately 90% of patients with isolated agammaglobulinemia and failure of B cell development have mutations in genes required for signaling through the pre–B cell and B cell receptors. The nature of the gene defect in the majority of remaining patients is unknown. We recently identified 4 patients with agammaglobulinemia and markedly decreased numbers of peripheral B cells. The B cells that could be detected had an unusual phenotype characterized by the increased expression of CD19 but the absence of a B cell receptor. Genetic studies demonstrated that all 4 patients had the exact same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. E555K in the transfected cells localized normally to the nucleus and resulted in a dominant negative effect when bound to DNA as a homodimer with wild-type E47. Mutant E47 did permit DNA binding by a tissue-specific heterodimeric DNA-binding partner, myogenic differentiation 1 (MYOD). These findings document a mutational hot-spot in E47 and represent an autosomal dominant form of agammaglobulinemia. Further, they indicate that E47 plays a critical role in enforcing the block in development of B cell precursors that lack functional antigen receptors. PMID:24216514

  5. A new non-Hodgkin's B-cell line (DoHH2) with a chromosomal translocation t(14;18)(q32;q21).

    PubMed

    Kluin-Nelemans, H C; Limpens, J; Meerabux, J; Beverstock, G C; Jansen, J H; de Jong, D; Kluin, P M

    1991-03-01

    A spontaneously growing EBV-negative B-cell line (DoHH2) was established from the pleural fluid cells of a 60-year-old man with centroblastic/centrocytic non-Hodgkin's lymphoma, that had transformed into an immunoblastic lymphoma. The pleural fluid cells and the DoHH2 cells expressed IgG lambda, were reactive with CD10 and CD19 monoclonal antibodies, and showed by cytogenetic analysis 48,XY, +7, +del(12)(q24), t(14;18)(q32;q21). Southern blot analysis of mini-satellite DNA patterns, and of rearrangements of the immunoglobulin genes and bcl-2, confirmed that the cell line was derived from the patient's clonal lymphoma cells. Direct nucleotide sequence analysis on polymerase chain reaction (PCR) products of the t(14;18) junction revealed an identical sequence for the JH-bcl-2 junction at JH6 and in the major breakpoint region of bcl-2 in both the original tumor cells and the DoHH2 cell line. The cell line was valuable as a standard quantification control for PCR analysis of the t(14;18) breakpoint. Titration experiments demonstrated the detection of up to one tumor cell in 10(5) normal blood lymphocytes.

  6. The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines.

    PubMed

    Strauss, Sandra J; Higginbottom, Karen; Jüliger, Simone; Maharaj, Lenushka; Allen, Paul; Schenkein, David; Lister, T Andrew; Joel, Simon P

    2007-03-15

    Bortezomib is a proteasome inhibitor with proven efficacy in multiple myeloma and non-Hodgkin's lymphoma. This study reports the effects of bortezomib in B-cell lymphoma cell lines with differing sensitivity to bortezomib to investigate factors that influence sensitivity. Bortezomib induced a time- and concentration-dependent reduction in cell viability in five lymphoma cell lines, with EC(50) values ranging from 6 nmol/L (DHL-7 cells) to 25 nmol/L (DHL-4 cells) after 72 h. Bortezomib cytotoxicity was independent of p53 function, as all cell lines exhibited mutations by sequence analysis. The difference in sensitivity was not explained by proteasome or nuclear factor-kappaB (NF-kappaB) inhibition as these were similar in the most and least sensitive cells. NF-kappaB inhibition was less marked than that of a specific NF-kappaB inhibitor, Bay 11-7082. Cell cycle analysis showed a marked G(2)-arrested population in the least sensitive DHL-4 line only, an effect that was not present with Bay 11-7082 treatment. Conversely, in DHL-7 cells, bortezomib treatment resulted in cells moving into an aberrant mitosis, indicative of mitotic catastrophe that may contribute to increased sensitivity to bortezomib. These studies show that although bortezomib treatment had similar effects on apoptotic and NF-kappaB signaling pathways in these cell lines, different cell cycle effects were observed and induction of a further mechanism of cell death, mitotic catastrophe, was observed in the more sensitive cell line, which may provide some pointers to the difference in sensitivity between cell lines. An improved understanding of how DHL-7 cells abrogate the G(2)-M cell cycle checkpoint may help identify targets to increase the efficacy of bortezomib.

  7. Complement receptor type 2 mediates infection of the human CD4-negative Raji B-cell line with opsonized HIV.

    PubMed

    Boyer, V; Delibrias, C; Noraz, N; Fischer, E; Kazatchkine, M D; Desgranges, C

    1992-12-01

    Opsonization of the HTLV-RF and HTLV-IIIB strains of HIV-1 with normal human HIV seronegative serum under conditions that allow complement activation resulted in the productive infection of cells of the Raji B lymphoblastoid cell line. Under the same experimental conditions, no infection of Raji cells was observed with unopsonized virus. Infection of Raji cells with complement-opsonized HIV-1 was totally suppressed by preblocking the function of CR2 (the C3dg receptor, CD21) on the cells with a monoclonal anti-CR2 antibody cross-linked with rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Infection of Raji cells occurred independently of CD4 since the cells lacked the expression of CD4 antigen and of CD4 transcripts. Thus, Raji cells may be infected with complement-opsonized HIV independently of CD4 and in the absence of antibodies. By mediating and/or enhancing HIV infection, complement and complement receptors contribute to extend the range of target cells to the virus and may increase infection in patients with a low viral load. PMID:1281336

  8. The CD40 ligand expressed by human B cells costimulates B cell responses.

    PubMed

    Grammer, A C; Bergman, M C; Miura, Y; Fujita, K; Davis, L S; Lipsky, P E

    1995-05-15

    The possibility that activated B cells might express a ligand for CD40 that was of functional importance for B cell responses was examined by using highly purified human peripheral blood B cells, as well as a variety of B lymphoblastoid cell lines and hybridomas. Following stimulation with the combination of a calcium ionophore and a phorbol ester, human B cells bound a soluble fusion protein containing the extracellular portion of CD40 and the Fc region of IgG1 (CD40.Ig). A variety of B cell lines and hybridomas also bound CD40.Ig, either constitutively or after activation. In addition, CD40.Ig specifically immunoprecipitated a 33-kDa glycoprotein from surface 125I-labeled activated B cells. The nucleotide sequence of the coding region of the CD40 ligand mRNA amplified by RT-PCR from activated T cells and B cell lines was identical. The CD40 ligand expressed on human B cells was important functionally because homotypic aggregation of CD40 ligand-expressing B cells was inhibited by the CD40.Ig construct. Additionally, RNA and DNA synthesis as well as Ig production by polyclonally activated, highly purified peripheral B cells and a variety of B cell lines were inhibited significantly by the CD40.Ig construct. Finally, B cell lines expressing the CD40 ligand induced Ig production from resting normal B cells in a CD40-dependent manner. These results indicate that human B cells express a ligand for CD40 that is identical with that expressed by activated T cells and that the B cell-expressed CD40 ligand plays an important role in facilitating responses of activated B cells.

  9. Copper phytoextraction in tandem with oilseed production using commercial cultivars and mutant lines of sunflower.

    PubMed

    Kolbas, A; Mench, M; Herzig, R; Nehnevajova, E; Bes, C M

    2011-01-01

    Use of sunflower (Helianthus annuus L.) for Cu phytoextraction and oilseed production on Cu-contaminated topsoils was investigated in afield trial at a former wood preservation site. Six commercial cultivars and two mutant lines were cultivated in plots with and without the addition of compost (5% w/w) and dolomitic limestone (0.2% w/w). Total soil Cu ranged from 163 to 1170 mg kg(-1). In soil solutions, Cu concentration varied between 0.16-0.93 mg L(-1). The amendment increased soil pH, reduced Cu exposure and promoted sunflower growth. Stem length, shoot and capitulum biomasses, seed yield, and shoot and leaf Cu concentrations were measured. At low total soil Cu, shoot Cu mineralomass was higher in commercial cultivars, Le., Salut, Energic, and Countri, whereas competition and shading affected morphological traits of mutants. Based on shoot yield (7 Mg DW ha(-1)) and Cu concentration, the highest removal was 59 g Cu ha(-1). At high total soil Cu, shoot Cu mineralomass peaked for mutants (e.g., 52 g Cu ha(-1) for Mutant 1 line) and cultivars Energic and Countri. Energic seed yield (3.9 Mg air-DW ha(-1)) would be sufficient to produce oil Phenotype traits and shoot Cu removal depended on sunflower types and Cu exposure.

  10. Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

    PubMed Central

    Long, E O; Gross, N; Wake, C T; Mach, J P; Carrel, S; Accolla, R; Mach, B

    1982-01-01

    HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6821356

  11. Peripheral blood lymphocyte/monocyte ratio predicts outcome for patients with diffuse large B cell lymphoma after standard first-line regimens.

    PubMed

    Li, Yan-Li; Pan, Yue-Yin; Jiao, Yang; Ning, Jie; Fan, Yin-Guang; Zhai, Zhi-Min

    2014-04-01

    To determine whether peripheral blood absolute lymphocyte/absolute monocyte counts ratio (ALC/AMC ratio) at diagnosis predicts survival of diffuse large B cell lymphoma (DLBCL) patients treated with standard first-line regimens, we retrospectively analyzed 244 patients with DLBCL who were treated with standard cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone, or rituximab-cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone. Progression-free survival and overall survival (PFS and OS) were estimated using the Kaplan-Meier method and two-tailed log-rank; The Cox proportional hazards model was used to evaluate ALC/AMC ratio as prognostic factors when adjusting for the International Prognostic Index (IPI). On univariate and multivariate analyses performed with factors included in the IPI, the ALC/AMC ratio at diagnosis remained an independent predictor of OS and PFS (OS: P < 0.001; PFS: P < 0.001). Patients with lower ALC/AMC ratio (<3.8) seemed to have lower complete remission rate, 2-year PFS and 3-year OS when compared to patients with ALC/AMC ratio ≥3.8, respectively (26 versus 90 %, P < 0.001; 18 versus 82 %, P < 0.001; 24 versus 86 %; P < 0.001, respectively). Moreover, the ALC/AMC ratio was able to further risk-stratify IPI 0-2 and three-five risk patient groups, respectively. The ALC/AMC ratio at the time of diagnosis may provide additional prognostic information beyond that of the IPI for patients with DLBCL who receive standard first-line regimens.

  12. Selection and molecular characterization of a high tocopherol accumulation rice mutant line induced by gamma irradiation.

    PubMed

    Hwang, Jung Eun; Ahn, Joon-Woo; Kwon, Soon-Jae; Kim, Jin-Baek; Kim, Sang Hoon; Kang, Si-Yong; Kim, Dong Sub

    2014-11-01

    Tocopherols are micronutrients with antioxidant properties. They are synthesized by photosynthetic bacteria and plants, and play important roles in animal and human nutrition. In this study, we isolated a new rice mutant line with elevated tocopherol content (MRXII) from an in vitro mutagenized population induced by gamma irradiation. The mutant exhibited greater seed longevity than the control, indicating a crucial role for tocopherols in maintaining viability during quiescence, and displayed faster seedling growth during the early growth stage. To study the molecular mechanism underlying vitamin E biosynthesis, we examined the expression patterns of seven rice genes encoding vitamin E biosynthetic enzymes. Accumulation levels of the OsVTE2 transcript and OsVTE2 protein in the MRXII mutant were significantly higher than in the control. Sequence analysis revealed that the MRXII mutant harbored a point mutation in the OsVTE2 promoter region, which resulted in the generation of MYB transcription factor-binding cis-element. These results help identify the promoter regions that regulate OsVTE2 transcription, and offer insights into the regulation of tocopherol content.

  13. Physiological Basis and Transcriptional Profiling of Three Salt-Tolerant Mutant Lines of Rice

    PubMed Central

    Domingo, Concha; Lalanne, Eric; Catalá, María M.; Pla, Eva; Reig-Valiente, Juan L.; Talón, Manuel

    2016-01-01

    Salinity is a complex trait that affects growth and productivity in many crops, including rice. Mutation induction, a useful tool to generate salt tolerant plants, enables the analysis of plants with similar genetic background, facilitating the understanding of the salt tolerance mechanisms. In this work, we generated three salt tolerant mutant lines by irradiation of a salt-sensitive cultivar plants and screened M2 plants at seedling stage in the presence of high salinity. These three lines, SaT20, SaS62, and SaT58, showed different responses to salinity, but exhibited similar phenotype to wild type plants, except SaT20 that displayed shorter height when grown in the absence of salt. Under salt conditions, all three mutants and the parental line showed similar reduction in yield, although relevant differences in other physiological parameters, such as Na+ accumulation in healthy leaves of SaT20, were registered. Microarray analyses of gene expression profiles in roots revealed the occurrence of common and specific responses in the mutants. The three mutants showed up-regulation of responsive genes, the activation of oxido-reduction process and the inhibition of ion transport. The participation of jasmonate in the plant response to salt was evident by down-regulation of a gene coding for a jasmonate O-methyltransferase. Genes dealing with lipid transport and metabolism were, in general, up-regulated except in SaS62, that also exhibited down-regulation of genes involved in ion transport and Ca2+ signal transduction. The two most tolerant varieties, SaS62 and SaT20, displayed lower levels of transcripts involved in K+ uptake. The physiological study and the description of the expression analysis evidenced that the three lines showed different responses to salt: SaT20 showed a high Na+ content in leaves, SaS62 presented an inhibition of lipid metabolism and ion transport and SaT58 differs in both features in the response to salinity. The analysis of these salt

  14. B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial Cold Water Disease (BCWD) is a chronic disease of rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp...

  15. Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran propolis in a B-cell lymphoma cell line.

    PubMed

    Alday, Efrain; Valencia, Dora; Carreño, Ana Laura; Picerno, Patrizia; Piccinelli, Anna Lisa; Rastrelli, Luca; Robles-Zepeda, Ramon; Hernandez, Javier; Velazquez, Carlos

    2015-12-01

    Propolis is a resinous substance produced by honeybees (Apis mellifera) from the selective collection of exudates and bud secretions from several plants. In previous works, we reported the antiproliferative activity of Sonoran propolis (SP) on cancer cells; in addition we suggested the induction of apoptosis after treatment with SP due to the presence of morphological changes and a characteristic DNA fragmentation pattern. Herein, in this study we demonstrated that the antiproliferative effect of SP is induced through apoptosis in a B-cell lymphoma cancer cell line, M12.C3.F6, by an annexin V-FITC/Propidium iodide double labeling. This apoptotic effect of SP resulted to be mediated by modulations in the loss of mitochondrial membrane potential (ΔΨm) and through activation of caspases signaling pathway (3, 8 and 9). Afterward, in order to characterize the chemical constituents of SP that induce apoptosis in cancer cells, an HPLC-PDA-ESI-MS/MS method followed by a preparative isolation procedure and NMR spectroscopy analysis have been used. Eighteen flavonoids, commonly described in propolis from temperate regions, were characterized. Chrysin, pinocembrin, pinobanksin and its ester derivatives are the main constituents of SP and some of them have never been reported in SP. In addition, two esters of pinobanksin (8 and 13) are described by first time in propolis samples in general. The antiproliferative activity on M12.C3.F6 cells through apoptosis induction was exhibited by pinobanksin (4), pinobanksin-3-O-propanoate (14), pinobanksin-3-O-butyrate (16), pinobanksin-3-O-pentanoate (17), and the already reported galangin (11), chrysin (9) and CAPE. To our knowledge this is the first report of bioactivity of pinobanksin and some of its ester derivatives as apoptosis inducers. Further studies are needed to advance in the understanding of the molecular basis of apoptosis induction by SP and its constituents, as well as the structure-activity relationship of them.

  16. CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines.

    PubMed

    Kijimoto-Ochiai, S; Uede, T

    1995-06-01

    Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.

  17. HDAC1,2 inhibition impairs EZH2- and BBAP- mediated DNA repair to overcome chemoresistance in EZH2 gain-of-function mutant diffuse large B-cell lymphoma

    PubMed Central

    Tharkar, Shweta; Quayle, Steven N.; Shearstone, Jeffrey R.; Jones, Simon; McDowell, Maria E.; Wellman, Hannah; Tyler, Jessica K.; Cairns, Bradley R.; Chandrasekharan, Mahesh B.; Bhaskara, Srividya

    2015-01-01

    Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2GOF) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2GOF DLBCL cells overexpress another chemotherapy resistance factor − B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2GOF DLBCL cells. PMID:25605023

  18. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines

    PubMed Central

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate. PMID:26519470

  19. Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines.

    PubMed

    Wilson, Robert; McGuire, Christina; Mohun, Timothy

    2016-01-01

    The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate.

  20. B cells in transplantation

    PubMed Central

    Dijke, Esme I.; Platt, Jeffrey L.; Blair, Paul; Clatworthy, Menna R.; Patel, Jignesh K.; Kfoury, A.G.; Cascalho, Marilia

    2016-01-01

    B cell responses underlie the most vexing immunological barriers to organ transplantation. Much has been learned about the molecular mechanisms of B cell responses to antigen and new therapeutic agents that specifically target B cells or suppress their functions are available. Yet, despite recent advances, there remains an incomplete understanding about how B cell functions determine the fate of organ transplants and how, whether or when potent new therapeutics should optimally be used. This gap in understanding reflects in part the realization that besides producing antibodies, B cells can also regulate cellular immunity, contribute to the genesis of tolerance and induce accommodation. Whether non-specific depletion of B cells, their progeny or suppression of their functions would undermine these non-cognate functions and whether graft outcome would suffer as a result is unknown. These questions were discussed at a symposium on “B cells in transplantation” at the 2015 ISHLT annual meeting. Those discussions are summarized here and a new perspective is offered. PMID:26996930

  1. Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

    PubMed

    Muthusamy, Annamalai; Jayabalan, Narayanasamy

    2013-01-01

    The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

  2. Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

    PubMed Central

    Chandhok, Gursimran; Horvath, Judit; Aggarwal, Annu; Bhatt, Mohit; Zibert, Andree; Schmidt, Hartmut HJ

    2016-01-01

    AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines. PMID:27122662

  3. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    PubMed

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

  4. Clinical significance of co-expression of MYC and BCL2 protein in aggressive B-cell lymphomas treated with a second line immunochemotherapy.

    PubMed

    Miura, Katsuhiro; Takahashi, Hiromichi; Nakagawa, Masaru; Izu, Asami; Sugitani, Masahiko; Kurita, Daisuke; Sakagami, Masashi; Ohtake, Shimon; Uchino, Yoshihito; Hojo, Atsuko; Kodaira, Hitomi; Yagi, Mai; Kobayashi, Yujin; Iriyama, Noriyoshi; Kobayashi, Sumiko; Kiso, Satomi; Hirabayashi, Yukio; Hatta, Yoshihiro; Takei, Masami

    2016-01-01

    The clinical significance of concurrent expression of MYC and BCL2 protein, known as "double-expressor lymphoma" (DEL), among patients with relapsed or refractory aggressive B-cell lymphomas, remains unclear. A retrospective analysis was performed of 38 patients treated with a salvage treatment consisting of rituximab, ifosfamide, etoposide, cytarabine and dexamethasone followed by consolidative high-dose chemotherapies. A total of 17 cases (45%) were categorized as DEL using immunohistochemical assay with a cut-off value of positivity of 40% for MYC and 50% for BCL2, respectively. DEL was associated with a lower overall response rate (35% vs 71%, p = 0.0481), worse 2-year progression-free survival (9% vs 67%, p = 0.001) and overall survival (35% vs 71%, p = 0.037). This analysis suggests that DEL is common among patients with relapsed/refractory aggressive B-cell lymphomas and that such patients require novel treatment strategies.

  5. A lacZ reporter gene expression atlas for 313 adult KOMP mutant mouse lines

    PubMed Central

    Pasumarthi, Ravi K.; Baridon, Brian; Djan, Esi; Trainor, Amanda; Griffey, Stephen M.; Engelhard, Eric K.; Rapp, Jared; Li, Bowen; de Jong, Pieter J.; Lloyd, K.C. Kent

    2015-01-01

    Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼80% of mutants showed specific staining in one or more tissues, while ∼20% showed no specific staining, ∼13% had staining in only one tissue, and ∼25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known. PMID:25591789

  6. Genetic Variability and Selection Criteria in Rice Mutant Lines as Revealed by Quantitative Traits

    PubMed Central

    Oladosu, Yusuff; Rafii, M. Y.; Abdullah, Norhani; Abdul Malek, Mohammad; Rahim, H. A.; Hussin, Ghazali; Abdul Latif, Mohammad; Kareem, Isiaka

    2014-01-01

    Genetic based knowledge of different vegetative and yield traits play a major role in varietal improvement of rice. Genetic variation gives room for recombinants which are essential for the development of a new variety in any crop. Based on this background, this work was carried out to evaluate genetic diversity of derived mutant lines and establish relationships between their yield and yield components using multivariate analysis. To achieve this objective, two field trials were carried out on 45 mutant rice genotypes to evaluate their growth and yield traits. Data were taken on vegetative traits and yield and its components, while genotypic and phenotypic coefficients, variance components, expected genetic advance, and heritability were calculated. All the genotypes showed variations for vegetative traits and yield and its components. Also, there was positive relationship between the quantitative traits and the final yield with the exception of number of tillers. Finally, the evaluated genotypes were grouped into five major clusters based on the assessed traits with the aid of UPGMA dendrogram. So hybridization of group I with group V or group VI could be used to attain higher heterosis or vigour among the genotypes. Also, this evaluation could be useful in developing reliable selection indices for important agronomic traits in rice. PMID:25431777

  7. The membrane IgM-associated proteins MB-1 and Ig-beta are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line.

    PubMed

    Matsuuchi, L; Gold, M R; Travis, A; Grosschedl, R; DeFranco, A L; Kelly, R B

    1992-04-15

    The B-cell antigen receptors consist of membrane immunoglobulins (mIgs) noncovalently associated with two accessory proteins, MB-1 and Ig-beta. We used transfection into a nonlymphoid cell line to test whether MB-1 and Ig-beta were sufficient to promote cell surface expression of mIgM capable of signal transduction. Expression of MB-1 and Ig-beta, but not MB-1 alone, allowed high-level surface expression of mIgM in the AtT20 endocrine cell line, which presumably lacks other B-cell-specific components. The reconstituted antigen receptor was capable of mediating some of the signaling reactions characteristic of mIgM in B lymphocytes. Crosslinking mIgM on transfected AtT20 cells stimulated tyrosine phosphorylation of MB-1 and Ig-beta and also increased the amount of phosphatidylinositol 3-kinase activity that could be precipitated with anti-phosphotyrosine antibodies. When total cell lysates were analyzed by anti-phosphotyrosine immunoblotting, however, no induced phosphorylation of more abundant proteins was detected. Moreover, crosslinking of the receptor in AtT20 cells did not stimulate inositol phospholipid breakdown. Thus, the transfected B-cell antigen receptor could initiate some signal transduction events but AtT20 cells may lack components required for other signaling events associated with mIgM.

  8. Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

    PubMed

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

  9. Identification of circulating maternal T and B lymphocytes in uncomplicated severe combined immunodeficiency by HLA typing of subpopulations of T cells separated by the fluorescence-activated cell sorter and of Epstein Barr virus-derived B cell lines.

    PubMed

    Geha, R S; Reinherz, E

    1983-06-01

    Circulating maternal T cells were sought in a child with severe combined immunodeficiency (SCID) and no evidence of acute graft-vs-host disease, but who had small numbers (9 to 11%) of circulating T3-positive cells. HLA typing of unfractionated peripheral blood lymphocytes (PBL) and of isolated E rosette-forming cells (37 to 44% of PBL) failed to reveal the presence of maternal lymphocytes. T3-positive cells isolated by the fluorescence-activated cell sorter, however, expressed exclusively maternal HLA antigens. A lymphoblastoid B cell line established by infecting the patient's PBL with Epstein Barr virus then expressed exclusively maternal HLA antigens. The presence of maternal T and B cells in uncomplicated SCID may be more common than thought previously and calls for a careful assessment of the origin of any mature T cells that are present in affected infants. In addition, the presence of maternal cells in SCID may complicate the infant's therapy.

  10. UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts.

    PubMed

    Collins, A R; Mitchell, D L; Zunino, A; de Wit, J; Busch, D

    1997-01-01

    Rodent UV-sensitive mutant cell lines of complementation groups 6 and 8 are the genetic counterparts of human Cockayne syndrome CS-B and CS-A, respectively. The original mutant in this group, UV61, was described as defective in cyclobutane pyrimidine dimer removal after high doses of UV. We have examined the responses of several cell lines from group 6 to low doses of UV irradiation, and find that these mutants have wild-type capacity for DNA repair as indicated by incision, cyclobutane pyrimidine dimer, and (6-4) photoproduct removal. ERCC6, the product of the gene defective in CS-B and group 6 mutants, is implicated in the regulation of repair of actively transcribed genes in Cockayne syndrome; however, this protein clearly is not required for the processing of low levels of damage in CHO cells, which occurs remarkably efficiently, 40-50% of dimers being removed in both wild-type and group 6 mutants in 5 hours following 0.1 Jm(-2) of UV. The group 8 mutant cell line US31, on the other hand, is very deficient in repair of UV damage, showing a more extreme phenotype than is seen in the corresponding human syndrome CS-A. In both complementation groups, expression of mutations in a gene involved in regulation of DNA repair takes very different forms in human and rodent cells. PMID:9118967

  11. Expression of an anthranilate synthase from maize mutant bf-1 in maize line HiII

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize mutant bf-1 was one of a series of maize mutants generated by radiation from the Bikini Atoll atomic bomb test in 1946. It is characterized by blue fluorescence in seedlings and anthers under ultraviolet illumination and by mutant plants giving off a characteristic grape-like odor due to the ...

  12. Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

    PubMed

    Schafer, I A; Jamieson, A M; Petrelli, M; Price, B J; Salzman, G C

    1979-01-01

    Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

  13. Recurrent mutations of the STAT6 DNA binding domain in primary mediastinal B-cell lymphoma.

    PubMed

    Ritz, Olga; Guiter, Chrystelle; Castellano, Flavia; Dorsch, Karola; Melzner, Julia; Jais, Jean-Philippe; Dubois, Gwendoline; Gaulard, Philippe; Möller, Peter; Leroy, Karen

    2009-08-01

    Primary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma, characterized by a constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma. Although many cancers exhibit constitutive JAK-STAT pathway activation, mutations of STAT genes have not been reported in neoplasms. Here, we show that MedB-1 PMBL-derived and L1236 Hodgkin-derived cell lines and 20 of 55 (36%) PMBL cases harbor heterozygous missense mutations in STAT6 DNA binding domain, whereas no mutation was found in 25 diffuse large B-cell lymphoma samples. In 3 cases, somatic origin was indicated by the absence of the mutations in the nontumoral tissue. The pattern of STAT6 mutations was different from the classical features of somatic hypermutations. The mutant STAT6 proteins showed a decreased DNA binding ability in transfected HEK cells, but no decrease in expression of STAT6 canonical target genes was observed in PMBL cases with a mutated STAT6 gene. Although the oncogenic properties of STAT6 mutant proteins remain to be determined, their recurrent selection in PMBL strongly argues for their involvement in the pathogenesis of this aggressive B-cell lymphoma. PMID:19423726

  14. The B-Cell-Specific src-Family Kinase Blk Is Dispensable for B-Cell Development and Activation

    PubMed Central

    Texido, Gemma; Su, I-hsin; Mecklenbräuker, Ingrid; Saijo, Kaoru; Malek, Sami N.; Desiderio, Stephen; Rajewsky, Klaus; Tarakhovsky, Alexander

    2000-01-01

    The B-cell lymphocyte kinase (Blk) is a src-family protein tyrosine kinase specifically expressed in B-lineage cells of mice. The early onset of Blk expression during B-cell development in the bone marrow and the high expression levels of Blk in mature B cells suggest a possible important role of Blk in B-cell physiology. To study the in vivo function of Blk, mice homozygous for the targeted disruption of the blk gene were generated. In homozygous mutant mice, neither blk mRNA nor Blk protein is expressed. Despite the absence of Blk, the development, in vitro activation, and humoral immune responses of B cells to T-cell-dependent and -independent antigens are unaltered. These data are consistent with functional redundancy of Blk in B-cell development and immune responses. PMID:10648608

  15. Analysis of mutational signatures in exomes from B-cell lymphoma cell lines suggest APOBEC3 family members to be involved in the pathogenesis of primary effusion lymphoma

    SciTech Connect

    Wagener, R.; Alexandrov, L. B.; Montesinos-Rongen, M.; Schlesner, M.; Haake, A.; Drexler, H. G.; Richter, J.; Bignell, G. R.; McDermott, U.; Siebert, R.

    2015-02-04

    Here, primary effusion lymphoma (PEL) is a rare large B-cell neoplasm particularly affecting immunodeficient hosts with an increased incidence in young or middle-aged males infected with the HIV.1 The clinical outcome of patients with PEL is unfavorable with a median survival of <6 months.1 PEL has been closely associated with human herpes virus 8 (HHV8, previously called Kaposi sarcoma herpesvirus) infection.1 In some cases a coinfection of HHV8 with the Epstein–Barr Virus (EBV) has been described.1 HHV8 encodes various genes homologous to cellular genes that have proliferative and anti-apoptotic functions.2 Although HHV8 is supposed to be a major driver of PEL, it alone is not sufficient for a full-blown lymphomagenesis.2 PEL usually shows complex karyotypes with many chromosomal aberrations.3 This chromosomal complexity might be driven by the viral infection and lead to genetic alterations cooperating with HHV8 in PEL lymphomagenesis.4

  16. Analysis of mutational signatures in exomes from B-cell lymphoma cell lines suggest APOBEC3 family members to be involved in the pathogenesis of primary effusion lymphoma

    DOE PAGESBeta

    Wagener, R.; Alexandrov, L. B.; Montesinos-Rongen, M.; Schlesner, M.; Haake, A.; Drexler, H. G.; Richter, J.; Bignell, G. R.; McDermott, U.; Siebert, R.

    2015-02-04

    Here, primary effusion lymphoma (PEL) is a rare large B-cell neoplasm particularly affecting immunodeficient hosts with an increased incidence in young or middle-aged males infected with the HIV.1 The clinical outcome of patients with PEL is unfavorable with a median survival of <6 months.1 PEL has been closely associated with human herpes virus 8 (HHV8, previously called Kaposi sarcoma herpesvirus) infection.1 In some cases a coinfection of HHV8 with the Epstein–Barr Virus (EBV) has been described.1 HHV8 encodes various genes homologous to cellular genes that have proliferative and anti-apoptotic functions.2 Although HHV8 is supposed to be a major driver ofmore » PEL, it alone is not sufficient for a full-blown lymphomagenesis.2 PEL usually shows complex karyotypes with many chromosomal aberrations.3 This chromosomal complexity might be driven by the viral infection and lead to genetic alterations cooperating with HHV8 in PEL lymphomagenesis.4« less

  17. Regulation of interleukin-6 and interleukin-6R alpha (gp80) expression by murine immunoglobulin-secreting B-cell hybridomas.

    PubMed Central

    Iwasaki, T; Hamano, T; Fujimoto, J; Kakishita, E

    1998-01-01

    We have examined the contribution of endogenous interleukin-6 (IL-6) to the differentiation of murine B-cell hybridomas. AT73 was established by somatic hybridization between BALB/c mice B cells and 2.52M, a hypoxanthine-aminopterine-thymidine (HAT) medium-sensitive B-cell line mutant. It spontaneously secreted IgM, and addition of exogenous IL-6 augmented IgM secretion. Triggering of CD40 led to an augmentation of IL-6 expression and IgM secretion. Blocking the binding of IL-6 to its cellular receptor through the use of inhibitory monoclonal antibodies inhibited CD40-induced IgM secretion, suggesting a possible autocrine role of IL-6 for the differentiation of a CD40-activated B-cell hybridoma. Co-triggering with CD40 and B-cell receptor or activation through CD40 and IL-4 led to a synergistic augmentation of IL-6 expression as well as additive IgM secretion; this was followed by a marked decrease in the expression of B-cell surface markers on the cell membrane. Furthermore, under conditions where IL-6 expression was augmented, gp80 expression was down-regulated, suggesting a negative feedback mechanism in this B-cell hybridoma. These findings provide a role by which T-cell-dependent activation through CD40 regulates an IL-6 autocrine loop, controlling B-cell differentiation. Images Figure 4 PMID:9659221

  18. Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

    PubMed Central

    Zieg, J; Clayton, C E; Ardeshir, F; Giulotto, E; Swyryd, E A; Stark, G R

    1983-01-01

    Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines. Images PMID:6656764

  19. Synergistic effects of eIF4A and MEK inhibitors on proliferation of NRAS-mutant melanoma cell lines.

    PubMed

    Malka-Mahieu, Hélène; Girault, Isabelle; Rubington, Margot; Leriche, Melissa; Welsch, Caroline; Kamsu-Kom, Nyam; Zhao, Qian; Desaubry, Laurent; Vagner, Stéphan; Robert, Caroline

    2016-09-16

    Activating mutations of the NRAS (neuroblastoma rat sarcoma viral oncogene) protein kinase, present in many cancers, induce a constitutive activation of both the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway and the PI(3)K-AKT-mTOR, pathway. This in turn regulates the formation of the eIF4F eukaryotic translation initiation complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, which binds to the 7-methylguanylate cap (m(7)G) at the 5' end of messenger RNAs. Small molecules targeting MEK (MEKi: MEK inhibitors) have demonstrated activity in NRAS-mutant cell lines and tumors, but resistance sets in most cases within months of treatment. Using proximity ligation assays, that allows visualization of the binding of eIF4E to the scaffold protein eIF4G, generating the active eIF4F complex, we have found that resistance to MEKi is associated with the persistent formation of the eIF4F complex in MEKi-treated NRAS-mutant cell lines. Furthermore, inhibiting the eIF4A component of the eIF4F complex, with a small molecule of the flavagline/rocaglate family, synergizes with inhibiting MEK to kill NRAS-mutant cancer cell lines.

  20. Interaction of Staphylococci with Human B cells

    PubMed Central

    Nygaard, Tyler K.; Kobayashi, Scott D.; Freedman, Brett; Porter, Adeline R.; Voyich, Jovanka M.; Otto, Michael; Schneewind, Olaf; DeLeo, Frank R.

    2016-01-01

    Staphylococcus aureus is a leading cause of human infections worldwide. The pathogen produces numerous molecules that can interfere with recognition and binding by host innate immune cells, an initial step required for the ingestion and subsequent destruction of microbes by phagocytes. To better understand the interaction of this pathogen with human immune cells, we compared the association of S. aureus and S. epidermidis with leukocytes in human blood. We found that a significantly greater proportion of B cells associated with S. epidermidis relative to S. aureus. Complement components and complement receptors were important for the binding of B cells with S. epidermidis. Experiments using staphylococci inactivated by ultraviolet radiation and S. aureus isogenic deletion mutants indicated that S. aureus secretes molecules regulated by the SaeR/S two-component system that interfere with the ability of human B cells to bind this bacterium. We hypothesize that the relative inability of B cells to bind S. aureus contributes to the microbe’s success as a human pathogen. PMID:27711145

  1. The effect of interferon-alpha on the ecto 5'-nucleotidase of human lymphoblastoid B-cell lines depends on the class of immunoglobulin secreted.

    PubMed Central

    Johnson, S M

    1991-01-01

    Thirteen immunoglobulin-secreting mycoplasma-free human cell lines were treated with increasing concentrations of lymphoblastoid interferon-alpha (IFN-alpha) in order to study the activity of their CD73 ecto-5'-nucleotidase (5'N), their rate of growth and their immunoglobulin (Ig) production. Although IFN-alpha did not immediately affect the activity of the 5'N on the cell plasma membranes, the class of Ig secreted by the cell lines determined whether culturing the cells in the presence of IFN-alpha gave a small increase in the 5'N enzyme activity (IgM), or had no effect (IgE), or showed a marked decrease in activity (IgG). The 5'N activity of two IgG4-secreting clones was more suppressed by IFN-alpha than that of the four IgG1-secreting clones. The clone with the highest 5'N was killed by IFN-alpha. A high 5'N activity inhibited the growth rate of the cells, since the rate of growth of the three IgG-producing lines with high 5'N was increased when their 5'N was inhibited by increasing concentrations of IFN-alpha. The growth rate of three other Ig-producing lines was uninhibited by up to 10(5) U/ml IFN-alpha, whereas the rest were partially or strongly inhibited. Excluding the clone which died, 10/11 lines or cultures increased their Ig/cell by a mean of 25% at 100 U/ml IFN-alpha; their total Ig production also increased despite any growth inhibition. One IgG-secreting clone decreased its Ig production at 100 U/ml IFN-alpha by 11%. The Ig/cell of 4/5 of the cell lines increased with IFN-alpha concentrations up to at least 10(4) U/ml. The increase in Ig/cell was not related to the class of Ig, the growth rate of the cells or the amount of 5'N. PMID:1937572

  2. Study of radiosensitive Drosophila lines. XI. Induction of recessive sex-linked lethal mutations in females of the mutant line rad(2)201/sup G1/

    SciTech Connect

    Varentsova, E.R.

    1986-05-01

    The authors have studied the frequency of occurrence of recessive sex-linked lethal mutations (RSLLM) after treatment of the females with ..gamma..-rays as a function of the dose (from 5 to 20 Gy) and oogenesis stage. They have shown that within the dose range used the oocytes of the 14th and 7th development stage are more sensitive in females of the mutant line than in those of the control. They detected significant differences in the frequency of occurrence of RSLLM between the 14th and 7th stages of development of oocytes for both Drosophila lines investigated.

  3. Delay of treatment change following objective progression on first-line erlotinib in EGFR-mutant lung cancer

    PubMed Central

    Lo, Peter C.; Dahlberg, Suzanne E.; Nishino, Mizuki; Johnson, Bruce E.; Sequist, Lecia V.; Jackman, David M.; Jänne, Pasi A; Oxnard, Geoffrey R.

    2015-01-01

    Background Erlotinib is a highly active EGFR kinase inhibitor approved for first-line use in lung cancers harboring EGFR mutations. Anecdotal experience suggests this drug may provide continued disease control following objective progression of disease (PD), however this has not been systematically studied. Methods Patients with RECIST-defined PD on three prospective trials of first-line erlotinib in advanced lung cancer were studied retrospectively, comparing progression characteristics of cases with and without EGFR sensitizing mutations. Factors influencing time to treatment change (TTC), defined as the time from PD until start of a new systemic therapy or death, were studied. Rate of tumor progression was assessed by comparing tumor measurements between the PD scan and the preceding scan. Results 92 eligible patients were studied: 42 with an EGFR sensitizing mutation and 50 without. The EGFR-mutant cohort had a slower rate of progression (p = 0.003) and a longer TTC (p < 0.001). Among EGFR-mutant cancers, 28 (66%) continued single-agent erlotinib following PD and 21 (50%) were able to delay change in systemic therapy for >3 months; only 2 received local debulking therapy during that period. Multivariate analysis of EGFR-mutant cases demonstrated that longer time to progression, slower rate of progression, and lack of new extrathoracic metastases were associated with a longer TTC. Conclusions A change in systemic therapy can commonly be delayed in patients with EGFR-mutant lung cancer objectively progressing on first-line erlotinib, particularly in those with a longer time to progression, a slow rate of progression, and lack of new extrathoracic metastases. PMID:25876525

  4. Characterization of cis-regulatory elements of the c-myc promoter responding to human GM-CSF or mouse interleukin 3 in mouse proB cell line BA/F3 cells expressing the human GM-CSF receptor.

    PubMed Central

    Watanabe, S; Ishida, S; Koike, K; Arai, K

    1995-01-01

    Interleukin 3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) activates c-fos, c-jun, and c-myc genes and proliferation in both hematopoietic and nonhematopoietic cells. Using a series of deletion mutants of the beta subunit of human GM-CSF receptor (hGMR) and inhibitors of tyrosine kinase, two distinct signaling pathways, one for activation of c-fos and c-jun genes, and the other for cell proliferation and activation of c-myc gene have been elucidated. In contrast to wealth of information on the pathway leading to activation of c-fos/c-jun genes, knowledge of the latter is scanty. To clarify the mechanisms of activation of c-myc gene by cytokines, we established a transient transfection assay in mouse proB cell line BA/F3 cells expressing hGMR. Analyses of hGMR beta subunit mutants revealed two cytoplasmic regions involved in activation of the c-myc promoter, one is essential and the other is dispensable but enhances the activity. These regions are located at the membrane proximal and the distal regions covering amino acid positions 455-544 and 544-589, respectively. Characterization of cis-acting regulatory elements of the c-myc gene showed that the region containing the P2 promoter initiation site is sufficient to mediate the response to mIL-3 or hGM-CSF. Electrophoretic mobility shift assay using an oligonucleotide corresponding to the distal putative E2F binding site revealed that p107/E2F complex, the negative regulator of E2F, decreased, and free E2F increased after mIL-3 stimulation. These results support the thesis that mIL-3 or hGM-CSF regulates the c-myc promoter by altering composition of the E2F complexes at E2F binding site. Images PMID:7579683

  5. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    PubMed

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

  6. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    PubMed

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

  7. B Cells, Antibodies, and More

    PubMed Central

    Hoffman, William; Lakkis, Fadi G.

    2016-01-01

    B cells play a central role in the immunopathogenesis of glomerulonephritides and transplant rejection. B cells secrete antibodies that contribute to tissue injury via multiple mechanisms. In addition, B cells contribute to disease pathogenesis in autoimmunity and alloimmunity by presenting antigens as well as providing costimulation and cytokines to T cells. B cells also play an immunomodulatory role in regulating the immune response by secreting cytokines that inhibit disease onset and/or progression. B cell–targeted approaches for treating immune diseases of the kidney and other organs have gained significant momentum. However, much remains to be understood about B-cell biology in order to determine the timing, duration, and context of optimal therapeutic response to B cell–targeted approaches. In this review, we discuss the multifaceted roles of B cells as enhancers and regulators of immunity with relevance to kidney disease and transplantation. PMID:26700440

  8. EBV latent membrane proteins (LMPs) 1 and 2 as immunotherapeutic targets: LMP-specific CD4+ cytotoxic T cell recognition of EBV-transformed B cell lines.

    PubMed

    Haigh, Tracey A; Lin, Xiaorong; Jia, Hui; Hui, Edwin P; Chan, Anthony T C; Rickinson, Alan B; Taylor, Graham S

    2008-02-01

    The EBV-latent membrane proteins (LMPs) 1 and 2 are among only three viral proteins expressed in EBV-associated Hodgkin's lymphoma and nasopharyngeal carcinoma. Since these tumors are HLA class I and class II-positive, the LMPs could serve as both CD8+ and CD4+ T cell targets. In contrast to CD8 responses, very little is known about CD4 responses to LMPs. In this study, we describe CD4+ T cell clones defining four LMP1- and three LMP2-derived peptide epitopes and their restricting alleles. All clones produced Th1-like cytokines in response to peptide and most killed peptide-loaded target cells by perforin-mediated lysis. Although clones to different epitopes showed different functional avidities in peptide titration assays, avidity per se was a poor predictor of the ability to recognize naturally infected B lymphoblastoid cell lines (LCLs) expressing LMPs at physiologic levels. Some epitopes, particularly within LMP1, consistently mediated strong LCL recognition detectable in cytokine release, cytotoxicity, and outgrowth inhibition assays. Using cyclosporin A to selectively block cytokine release, we found that CD4+ T cell cytotoxicity is the key effector of LCL outgrowth control. We therefore infer that cytotoxic CD4+ T cells to a subset of LMP epitopes could have therapeutic potential against LMP-expressing tumors.

  9. Analysis of spontaneous chromosomal rearrangements in neuroblasts of genetically unstable mutant lines of Drosophila melanogaster

    SciTech Connect

    Derzhavets, E.M.; Kim, A.I.; Aslanyan, M.M.

    1988-11-01

    The spectrum and frequency of chromosomal aberrations in the somatic cells of III instar larvae of Drosophila melanogaster mutator line were studied using three of its derivatives (sbt, if, and w/sup a/) and line w as control. It has been demonstrated that the frequency of anaphases with bridges and acentric fragments increases in the neuroblast of flies of the mutator line as well as in the neuroblasts of the larvae of the lines sbt, if, and w/sup a/. The metaphase analysis revealed that the mutator line and its derivatives are characterized by higher frequencies of chromosomal aberrations as compared to the control. Chromatid breaks are predominant type of rearrangements. These results, suggest probably presence of the specific mutator factor or factors in the line studied, affecting chromosomal structure and, possibly, activating migration of the mobile genetic elements in the mutator line.

  10. Biallelic deletion within 16p13.13 including SOCS-1 in Karpas1106P mediastinal B-cell lymphoma line is associated with delayed degradation of JAK2 protein.

    PubMed

    Melzner, Ingo; Weniger, Marc A; Bucur, Alexandra J; Brüderlein, Silke; Dorsch, Karola; Hasel, Cornelia; Leithäuser, Frank; Ritz, Olga; Dyer, Martin J S; Barth, Thomas F E; Möller, Peter

    2006-04-15

    Activity of Janus kinase 2 (JAK2) in the JAK2/STAT5 signaling pathway is critically controlled by suppressor of cytokine signaling-1 (SOCS-1). We have previously shown that SOCS-1 is biallelically mutated in the primary mediastinal B-cell lymphoma (PMBL) cell line MedB-1, resulting in impaired JAK2 degradation and sustained phospho-JAK2 action. SOCS-1 is frequently mutated in PMBL tumor primaries. Here, we report that the PMBL cell line Karpas1106P has a biallelic deletion of the SOCS-1 region on chromosome 16p13.13. By fluorescence in situ hybridization and microsatellite analysis, this deletion was narrowed down to a range of 650 kb to 1.48 Mb. Like MedB-1, Karpas1106P harbors gains of the JAK2 gene on chromosomal region 9p24 and elevated levels of JAK2 mRNA. Nevertheless, JAK2 protein was not increased but constitutively phosphorylated in Karpas1106P cells. In analogy to MedB-1 cells, Karpas1106P cells exhibited a retarded degradation of de novo synthesized JAK2 protein revealed by pulse/chase experiments. Therefore, we conclude that loss of SOCS-1 function either by mutation or by the complete deletion of the gene plays an important role in the dysregulation of JAK/STAT signaling in Karpas1106P and PMBL. PMID:16287070

  11. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors

    PubMed Central

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D.; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L.; Gapin, Laurent; Marrack, Philippa; Kappler, John W.

    2016-01-01

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

  12. Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines, in Yeast, and in Vivo*

    PubMed Central

    Mbefo, Martial Kamdem; Fares, Mohamed-Bilal; Paleologou, Katerina; Oueslati, Abid; Yin, Guowei; Tenreiro, Sandra; Pinto, Madalena; Outeiro, Tiago; Zweckstetter, Markus; Masliah, Eliezer; Lashuel, Hilal A.

    2015-01-01

    Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms. PMID:25657004

  13. Reduced Levels of Hspa9 Attenuates Stat5 Activation in Mouse B-cells

    PubMed Central

    Krysiak, Kilannin; Tibbitts, Justin F.; Shao, Jin; Liu, Tuoen; Ndonwi, Matthew; Walter, Matthew J.

    2014-01-01

    HSPA9 is located on chromosome 5q31.2 in humans, a region that is commonly deleted in patients with myeloid malignancies [del(5q)], including myelodysplastic syndromes (MDS). HSPA9 expression is reduced by 50% in patients with del(5q)-associated MDS, consistent with haploinsufficient levels. Zebrafish mutants and knockdown studies in human and mouse cells have implicated a role for HSPA9 in hematopoiesis. To comprehensively evaluate the effects of Hspa9 haploinsufficiency on hematopoiesis, we generated an Hspa9 knockout mouse model. While homozygous knockout of Hspa9 is embryonic lethal, mice with heterozygous deletion of Hspa9 (Hspa9+/−) are viable and have a 50% reduction in Hspa9 expression. Hspa9+/− mice have normal basal hematopoiesis and do not develop MDS. However, Hspa9+/− mice have a cell- intrinsic reduction in bone marrow CFU-PreB colony formation without alterations in the number of B-cell progenitors in vivo, consistent with a functional defect in Hspa9+/− B-cell progenitors. We further reduced Hspa9 expression (<50%) using RNAi and observe reduced B-cell progenitors in vivo, indicating that appropriate levels (≥50%) of Hspa9 are required for normal B- lymphopoiesis in vivo. Knockdown of Hspa9 in an IL-7 dependent mouse B-cell line reduced Stat5 phosphorylation following IL-7 receptor stimulation, supporting a role for Hspa9 in Stat5 signaling in B-cells. Collectively, these data implicate a role for Hspa9 in B-lymphopoiesis and Stat5 activation downstream of IL-7 signaling. PMID:25550197

  14. Inhibition of Cell Proliferation in an NRAS Mutant Melanoma Cell Line by Combining Sorafenib and α-Mangostin

    PubMed Central

    Xia, Yun; Li, Ying; Westover, Kenneth D.; Sun, Jiaming; Chen, Hongxiang; Zhang, Jianming; Fisher, David E.

    2016-01-01

    α-Mangostin is a natural product commonly used in Asia for cosmetic and medicinal applications including topical treatment of acne and skin cancer. Towards finding new pharmacological strategies that overcome NRAS mutant melanoma, we performed a cell proliferation-based combination screen using a collection of well-characterized small molecule kinase inhibitors and α-Mangostin. We found that α-Mangostin significantly enhances Sorafenib pharmacological efficacy against an NRAS mutant melanoma cell line. The synergistic effects of α-Mangostin and Sorafenib were associated with enhanced inhibition of activated AKT and ERK, induced ER stress, and reduced autophagy, eventually leading to apoptosis. The structure of α-Mangostin resembles several inhibitors of the Retinoid X receptor (RXR). MITF expression, which is regulated by RXR, was modulated by α-Mangostin. Molecular docking revealed that α-Mangostin can be accommodated by the ligand binding pocket of RXR and may thereby compete with RXR-mediated control of MITF expression. In summary, these data demonstrate an unanticipated synergy between α-Mangostin and sorafenib, with mechanistic actions that convert a known safe natural product to a candidate combinatorial therapeutic agent. PMID:27152946

  15. Altered Glycine Decarboxylation Inhibition in Isonicotinic Acid Hydrazide-Resistant Mutant Callus Lines and in Regenerated Plants and Seed Progeny

    PubMed Central

    Zelitch, Israel; Berlyn, Mary B.

    1982-01-01

    Isonicotinic acid hydrazide (INH), an inhibitor of the photorespiratory pathway blocking the conversion of glycine to serine and CO2, has been used as a selective agent to obtain INH-resistant tobacco (Nicotiana tabacum) callus cells. Of 22 cell lines that were INH-resistant, none were different from wild-type cells in their ability to take up [3H]INH or to oxidize INH to isonicotinic acid. In 7 of the 22 cell lines, INH resistance was associated with decreased inhibition of NAD-dependent glycine decarboxylation activity in isolated mitochondrial preparations. In the cell line that was most extensively investigated (I 24), this biochemical phenotype (exhibiting a 3-fold higher Ki with INH) was observed in leaf mitochondria of regenerated plants and of plants produced from them by self-fertilization. After crosses between resistant and sensitive plants, the decreased inhibition of glycine decarboxylation was observed among F2 and backcross progeny only in those plants previously identified as INH-resistant by callus growth tests. In contrast, in siblings identified as INH-sensitive, glycine decarboxylation was inhibited by INH at the wild-type level. This demonstration of the transfer of an altered enzyme property from callus to regenerated plants and through seed progeny fulfills an important requirement for the use of somatic cell genetics to produce biochemical mutants of higher plants. PMID:16662158

  16. Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells.

    PubMed Central

    Mermer, B; Felber, B K; Campbell, M; Pavlakis, G N

    1990-01-01

    A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev. Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans. Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus. HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function. Images PMID:2186373

  17. The lower peripheral blood lymphocyte/monocyte ratio assessed during routine follow-up after standard first-line chemotherapy is a risk factor for predicting relapse in patients with diffuse large B-cell lymphoma.

    PubMed

    Yan-Li, Li; Kang-Sheng, Gu; Yue-Yin, Pan; Yang, Jiao; Zhi-Min, Zhai

    2014-03-01

    A specific predictor during routine follow-up to ascertain risk for relapse after standard first-line chemotherapy in non-Hodgkin's lymphoma (NHL) has not been identified, although blood counts, lactate dehydrogenase (LDH) and imaging studies, such as computed tomography (CT) scans or positron emission tomography, have been recommended. Therefore, we studied the absolute lymphocyte count/absolute monocyte count ratio (ALC/AMC ratio) as a marker of poststandard first-line chemotherapy for predicting relapse in patients with diffuse large B-cell lymphoma (DLBCL). 220 consecutive DLBCL patients, originally diagnosed, treated with CHOP or R-CHOP and followed up at two institutions. ALC/AMC ratio was obtained at the time of confirmed relapse or last follow-up. Patients at the time of confirmed relapse (n = 163) had a lower ALC/AMC ratio compared with those at last follow-up (n = 57) (P < 0.001). ALC/AMC ratio at the time of confirmed relapse was a strong predictor for relapse with an area under the curve = 0.813 (P < 0.001). The sensitivity and specificity for ALC/AMC ratio at the time of confirmed relapse or at last follow-up were 68.1% and 87.7%, respectively, and the relative risk of relapse with an ALC/AMC ratio < 2.8 at the time of confirmed relapse or at last follow-up was 1.845 with an odds ratio of 15.247 (95% cumulative incidence: 6.473-35.916) after CHOP or R-CHOP in DLBCL. Patients with an ALC/AMC ratio (< 2.8) had a higher cumulative hazard rate of relapse compared with an ALC/AMC ratio (≥2.8) (P < 0.001). This study suggests that the lower ALC/AMC ratio can be used as a marker to assess risk of DLBCL relapse during routine follow-up after standard first-line chemotherapy.

  18. Multiple pathways of DNA double-strand break processing in a mutant Indian muntjac cell line

    SciTech Connect

    Bouffler, S.D.; Jha, B.; Johnson, R.T. )

    1990-09-01

    DNA break processing is compared in the Indian muntjac cell lines, SVM and DM. The initial frequencies and resealing of X-ray generated single- and double-strand breaks are similar in the two cell lines. Inhibiting the repair of UV damage leads to greater double-strand breakage in SVM than in DM, and some of these breaks are not repaired; however, repair-associated single-strand breakage and resealing are normal. Dimethylsulfate also induces excess double-strand breakage in SVM, and these breaks are irreparable. Restricted plasmids are reconstituted correctly in SVM at approximately 30% of the frequency observed in DM. Thus SVM has a reduced capacity to repair certain types of double-strand break. This defect is not due to a DNA ligase deficiency. We conclude that DNA double-strand breaks are repaired by a variety of pathways within mammalian cells and that the structure of the break or its mode of formation determines its subsequent fate.

  19. Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: analysis of phosphoprotein dynamics in a B cell lymphoma cell line.

    PubMed

    Cutillas, Pedro R; Geering, Barbara; Waterfield, Mike D; Vanhaesebroeck, Bart

    2005-08-01

    Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a non-isotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase Cgamma2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospholipase Cgamma2 phosphorylation sites. Our results illustrate the capacity of this conceptually

  20. Impact of high-dose chemotherapy and autologous transplantation as first-line therapy on the survival of high-risk diffuse large B cell lymphoma patients: a single-center study in Japan.

    PubMed

    Inano, Shojiro; Iwasaki, Makoto; Iwamoto, Yoshihiro; Sueki, Yuki; Fukunaga, Akiko; Yanagita, Soshi; Arima, Nobuyoshi

    2014-02-01

    High-dose chemotherapy (HDT), together with autologous stem cell transplantation (ASCT), plays an important role in the treatment of diffuse large B cell lymphoma (DLBCL), especially as second-line therapy. However, its significance in up-front settings remains to be elucidated. In our institute, patients with DLBCL in both the high-intermediate and high international prognostic index (IPI) groups initially underwent CHOP/R-CHOP treatment followed by HDT/ASCT at upfront settings between 2002 and 2011. We retrospectively analyzed 25 patients who were all treated with upfront HDT/ASCT. We excluded one patient who failed to undergo transplantation because of primary refractory disease from the analysis. The median follow-up was 77 months (range 17-110 months). Five-year overall survival (OS) and progression-free survival (PFS) were 91.7 and 79.2 %, respectively, which were higher than the equivalents in previous studies. The OS and PFS in the high-risk group were lower than those in the high-intermediate group. Treatment-related mortalities or fatal complication were not observed. Our results confirm that HDT/ASCT for high-risk aggressive lymphoma is a feasible and promising therapy, but patients with high IPI continued to have poor prognoses; improvements in treatment strategy are clearly needed. Since HDT/ASCT is an aggressive treatment option associated with long-term complications, we need to identify patient groups that will gain the maximum benefit from HDT/ASCT in the upfront setting. PMID:24338743

  1. Activated mast cells promote differentiation of B cells into effector cells

    PubMed Central

    Palm, Anna-Karin E.; Garcia-Faroldi, Gianni; Lundberg, Marcus; Pejler, Gunnar; Kleinau, Sandra

    2016-01-01

    Based on the known accumulation of mast cells (MCs) in B cell-dependent inflammatory diseases, including rheumatoid arthritis, we hypothesized that MCs directly modulate B cells. We show here that degranulated, and to a lesser extent naïve or IgE-sensitized, MCs activate both naïve and B cell receptor-activated B cells. This was shown by increased proliferation, blast formation, and expression of CD19, MHC class II and CD86 in the B cells. Further, MCs stimulated the secretion of IgM and IgG in IgM+ B cells, indicating that MCs can induce class-switch recombination in B cells. We also show that coculture of MCs with B cells promotes surface expression of L-selectin, a homing receptor, on the B cells. The effects of MCs on B cells were partly dependent on cell-cell contact and both follicular and marginal zone B cells could be activated by MCs. Our findings suggest that degranulated MCs support optimal activation of B cells, a finding that is in line with in vivo studies showing that MCs frequently degranulate in the context of B-cell driven pathologies such as arthritis. Together, our findings show that MCs have the capacity to differentiate B cells to effector cells. PMID:26847186

  2. Isolation and characterization of liver epithelial cell lines from wild-type and mutant TgN737Rpw mice.

    PubMed Central

    Richards, W. G.; Yoder, B. K.; Isfort, R. J.; Detilleux, P. G.; Foster, C.; Neilsen, N.; Woychik, R. P.; Wilkinson, J. E.

    1997-01-01

    The Tg737 gene encodes a tetratricopeptide repeat containing protein that, when disrupted in TgN737Rpw mutant mice, results in pleiotropic phenotypes that include the proliferation of epithelial cells. In the kidney and liver, this causes a phenotype that resembles autosomal recessive polycystic kidney disease. In the liver, the affected epithelial cells morphologically and immunologically resemble oval cells. Here we describe the isolation, culture, and characterization of epithelial cell lines derived from the livers of wild-type, heterozygous, and homozygous TgN737Rpw mice. Essentially homogeneous cell cultures were established and the expression of liver markers was examined by reverse transcriptase polymerase chain reaction and by immunohistochemistry. All of the cell lines reacted to the A6 antibody that was raised against mouse oval cells and expressed markers seen in oval cells. Cells transplanted into the interscapular fat pads of isogenic mice formed well defined ductular structures. Furthermore, in transfection experiments, we have demonstrated the involvement of Tg737 in cellular proliferation. Images Figure 1 Figure 2 Figure 3 PMID:9094975

  3. Glucocorticoid-regulated glycoprotein maturation in wild-type and mutant rat cell lines

    PubMed Central

    1986-01-01

    Glucocorticoid hormones can regulate the posttranslational maturation of mouse mammary tumor virus (MTV) precursor polyproteins in M1.54, a stably infected rat hepatoma cell line. We have used complement- mediated cytolysis to recover variants of M1.54 that fail to express MTV cell surface glycoproteins in a hormone-regulated manner (Firestone, G.L., and K.R. Yamamoto, 1983, Mol. Cell. Biol., 3:149- 160). One such clonal isolate, CR4, is similar to wild-type with respect to synthesis of MTV mRNAs, production of the MTV glycoprotein precursor (gPr74env) and a glycosylated maturation product (gp51), and hormone-induced processing of two MTV phosphoproteins. In contrast, three viral cell surface glycoproteins (gp78, gp70, and gp32) and one extracellular species (gp70s), which derive from gPr74env in glucocorticoid-treated wild-type cells, fail to appear in CR4. CR4 showed no apparent alterations in proliferation rate, cell shape, or expression of total functional mRNA and bulk glycoproteins. We conclude that the genetic lesion in CR4 defines a highly selective hormone- regulated glycoprotein maturation pathway that alters the fate of a restricted subset of precursor species. PMID:3023398

  4. Characterization of cottonseed nutrients composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines for fuzzless seed trait under well-watered and water stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton mutant near isogenic lines (NILs) for fuzzless seed trait has been used to investigate cell biology, genetic, and molecular processes of fiber initiation, development, fiber yield and quality. However, there is no information available on the effect of fuzzless seed trait on cottonseed nutrie...

  5. Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines.

    PubMed

    Riberdy, J M; Avva, R R; Geuze, H J; Cresswell, P

    1994-06-01

    We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II-associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild-type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.

  6. HLA-A2 as a target for cell-mediated lympholysis: evidence from immunoselected HLA-A2 negative mutant cell lines.

    PubMed

    Andreotti, P E; Apgar, J R; Cresswell, P

    1980-07-01

    Cloned mutants of the human B lymphoblastoid cell line SB have been isolated using mutagenesis with ethyl methanesulfonate followed by negative selection with an anti-HLA-A2 serum and complement. Absorption analysis with 125I Staphylococcus aureus protein A binding to antibody sensitized cells. HLA typing, and immune precipitation analysis showed the mutants to be serologically identical to the SB parent except for the loss of HLA-A2. When tested as target cells for cell-mediated lympholysis by cytotoxic T lymphocytes generated in the mixed lymphocyte response, the SB and mutant cell lines demonstrated comparable susceptibility when the putative targets were HLA antigens other than HLA-A2. However, when compared for susceptibility to lysis by cytotoxic T lymphocytes considered to be HLA-A2 specific, the SB parent was effectively killed whereas little or no killing of the HLA-A2 mutants was observed. The results provide a new line of evidence that HLA antigens recognized by antibody can also be the true molecular targets for cytotoxic T lymphocytes. PMID:7263311

  7. Introduction of mutant p53 into a wild-type p53-expressing glioma cell line confers sensitivity to Ad-p53-induced apoptosis.

    PubMed Central

    Cerrato, J. A.; Yung, W. K.; Liu, T. J.

    2001-01-01

    Transient expression of the tumor suppressor gene p53 via adenoviral-mediated gene transfer induces apoptosis in glioma cells expressing mutant p53, while causing cell cycle arrest in cells with wild-type p53. To determine whether a change in p53 status of a wild-type p53-expressing cell line such as U-87 MG would alter its apoptotic resistant phenotype in response to Ad-p53 infection, we generated cell lines U-87-175.4 and U-87-175.13 via retroviral-mediated gene transfer of the p53 (175H) mutant into the U-87 MG parental line. Control cell lines U-87-Lux.6 and U-87-Lux.8 were also generated and express the reporter gene luciferase. Both U-87-175.4 and U-87-175.13, but not control cell lines, exhibited morphology characteristic of apoptosis after Ad-p53 infection. Furthermore, expression of other p53 mutants (248W, 273H) in U-87 MG also sensitized cells to Ad-p53-induced apoptosis. Apoptosis was confirmed by TUNEL and cell cycle analysis. Several p53 response genes were examined in cells infected with Ad-p53, and among these, BCL2, p21WAF1/CIP1, CPP32/caspase 3, and PARP showed differences in expression between U87-175 and U87-Lux cell lines. Taken together, our data demonstrate that the introduction of p53 mutants in U-87 MG promotes an apoptotic response in association with adenoviral-mediated wild-type p53 gene transfer. These results underscore the importance of glioma p53 genotype for predicting tumor response to p53-based gene therapy. PMID:11296482

  8. Identification of the human mature B cell miRNome

    PubMed Central

    Basso, Katia; Sumazin, Pavel; Morozov, Pavel; Schneider, Christof; Maute, Roy L.; Kitagawa, Yukiko; Mandelbaum, Jonathan; Haddad, Joseph; Chen, Chang-Zheng; Califano, Andrea; Dalla-Favera, Riccardo

    2009-01-01

    The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B-cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naïve, germinal-center, memory) and from a Burkitt lymphoma cell-line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B-cell libraries. Most notably, the B-cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B-cell development, immune function, and lymphomagenesis. PMID:19446474

  9. [Regulatory B cells in human autoimmune diseases].

    PubMed

    Miyagaki, Tomomitsu

    2015-01-01

    B cells have been generally considered to be positive regulators of immune responses because of their ability to produce antigen-specific antibodies and to activate T cells through antigen presentation. Impairment of B cell development and function may cause autoimmune diseases. Recently, specific B cell subsets that can negatively regulate immune responses have been described in mouse models of a wide variety of autoimmune diseases. The concept of those B cells, termed regulatory B cells, is now recognized as important in the murine immune system. Among several regulatory B cell subsets, IL-10-producing regulatory B cells are the most widely investigated. On the basis of discoveries from studies of such mice, human regulatory B cells that produce IL-10 in most cases are becoming an active area of research. There have been emerging data suggesting the importance of human regulatory B cells in various diseases. Revealing the immune regulation mechanisms of human regulatory B cells in human autoimmune diseases could lead to the development of novel B cell targeted therapies. This review highlights the current knowledge on regulatory B cells, mainly IL-10-producing regulatory B cells, in clinical research using human samples. PMID:26725860

  10. Temperature-Sensitive Mutants of a Chinese Hamster Cell Line. I. Selection of Clones with Defective Macromolecular Biosynthesis

    PubMed Central

    Roufa, Donald J.; Reed, Susan J.

    1975-01-01

    Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxy-uridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39° and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39° to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115–47 and 115–53) also stop incorporating radioactive amino acids and uridine within six hours at 39°. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33° after 8 hours at 39°, both resumed DNA synthesis immediately (< 1 hour). Reversal of defective DNA synthesis in both mutants was sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication. PMID:1232024

  11. Multiple Curricula for B Cell Developmental Programming.

    PubMed

    Rothenberg, Ellen V

    2016-09-20

    B-1 B cells differ from conventional B-2 B cells functionally, but how these differences relate to the ontogeny of these lineages has been unclear. Two recent Immunity articles, Kristiansen et al. (2016) and Montecino-Rodriguez et al. (2016), now provide insight into the origins of B-1 and B-2 B cells, revealing a multi-layered developmental program and successive waves of B cell precursors.

  12. Multiple Curricula for B Cell Developmental Programming.

    PubMed

    Rothenberg, Ellen V

    2016-09-20

    B-1 B cells differ from conventional B-2 B cells functionally, but how these differences relate to the ontogeny of these lineages has been unclear. Two recent Immunity articles, Kristiansen et al. (2016) and Montecino-Rodriguez et al. (2016), now provide insight into the origins of B-1 and B-2 B cells, revealing a multi-layered developmental program and successive waves of B cell precursors. PMID:27653594

  13. Dissociation of two signals required for activation of resting B cells.

    PubMed Central

    Julius, M H; von Boehmer, H; Sidman, C L

    1982-01-01

    Cellular interactions involved in the T cell-dependent activation of B cells were analyzed by using lines and clones of helper T cells specific for determinants expressed on the B cell surface. Activation of male antigen-, M locus-, and H-2-specific T cells was shown to support polyclonal Ig production by a population of B cells that did not require T-cell-B-cell interaction for induction/amplification. However, these T cells alone did not activate gradient-purified small (resting) B cells. The activation of small B cells was shown to require not only a signal derived through an antigen-specific T-helper cell-B cell interaction but in addition a second signal that could be provided by anti-Ig antibodies. PMID:6979046

  14. Identification of bovine B cell reactive and B cell specific monoclonal antibodies.

    PubMed

    Mukwedeya, D T; Takamatsu, H; Parkhouse, R M

    1993-11-01

    All monoclonal antibodies (mAbs) submitted to the workshop panel were screened for reactivity with bovine surface immunoglobulin (sIg)+ cells (gated small dense lymphocytes from peripheral blood) by fluorescence activated cell sorter (FACS) analysis. Eighteen temporary clusters--TCs 1-12, 15, 16, 18, 19, 25 and 26--contained mAbs reactive with sIg+ cells. mAb BAS21A (unclustered) and CC92 (TC25) were also reactive with sIg+ cells. Further FACS analysis with B cells from peripheral blood lymphocytes and mesenteric lymph nodes, and B and T lymphoma cell lines, indicated that the majority of mAbs within TCs 2, 4, 15, 18 and 26 reacted specifically with bovine B cells. Bovine B cell specific mAbs within these clusters were TH14B, IL-A55, CACT101A, MUC76A from TC4, VPM30, GC65A, CACT65A from TC15, IL-A58, CC56, CC70, IL-A65 from TC18, and CC57 and 26A9 from TC26. Three mAbs--IL-A65, CC70, and BAQ15A--within TC18 defined WC3; mAbs TD9 and CC56 may also be related to WC3.

  15. Determination of essential fatty acid composition among mutant lines of Canola (Brassica napus), through high pressure liquid chromatography.

    PubMed

    Raza, Ghulam; Siddique, Aquil; Khan, Imtiaz Ahmad; Ashraf, Muhammed Yasin; Khatri, Abdullah

    2009-12-01

    The present study aimed to quantify the methyl esters of lenoleic acid (LA), gamma-lenolenic acid (LNA) and oleic acid (OL) in the oil of Brassica napus mutants. Five stable mutants (ROO-75/1, ROO-100/6, ROO-125/12, ROO-125/14, and ROO-125/17) of B. napus cv. 'Rainbow' (P) and three mutants (W97-95/16, W97-0.75/11 and W97-.075/13) of B. napus cv. 'Westar' (P) at M6 stage, exhibiting better yield and yield components, were analyzed for essential fatty acids. The highest seed yield was observed in the mutant (ROO-100/6) followed by ROO-125/14 of Rainbow, that is, 34% and 32% higher than their parent plants, respectively. Westar mutant W97-75/11 also showed 30% higher seed yield than its parent plant. High performance liquid chromatography analysis of the composition of fatty acids indicated that OL was the most dominant fatty acid, ranging from 39.1 to 66.3%; LA was second (15.3-41.6%) and LNA was third (18.1-28.9%). Mutant ROO-125/14 showed higher OL contents than parent (Rainbow). These results are expected to support the approval of ROO-125/14 in the National Uniform Varietal Yield Trials (NUVYT) as a new variety based on high oil quality.

  16. Early B-cell Factor 1 Regulates the Expansion of B-cell Progenitors in a Dose-dependent Manner*

    PubMed Central

    Åhsberg, Josefine; Ungerbäck, Jonas; Strid, Tobias; Welinder, Eva; Stjernberg, Jenny; Larsson, Malin; Qian, Hong; Sigvardsson, Mikael

    2013-01-01

    Transcription factor doses are of importance for normal and malignant B-lymphocyte development; however, the understanding of underlying mechanisms and functional consequences of reduced transcription factor levels is limited. We have analyzed progenitor and B-lineage compartments in mice carrying heterozygote mutations in the E2a, Ebf1, or Pax5 gene. Although lymphoid progenitors from Ebf1 or Pax5 heterozygote mice were specified and lineage-restricted in a manner comparable with Wt progenitors, this process was severely impaired in E2a heterozygote mutant mice. This defect was not significantly enhanced upon combined deletion of E2a with Ebf1 or Pax5. Analysis of the pre-B-cell compartment in Ebf1 heterozygote mice revealed a reduction in cell numbers. These cells expressed Pax5 and other B-lineage-associated genes, and global gene expression analysis suggested that the reduction of the pre-B-cell compartment was a result of impaired pre-B-cell expansion. This idea was supported by a reduction in IL2Rα-expressing late pre-B-cells as well as by cell cycle analysis and by the finding that the complexity of the VDJ rearrangement patterns was comparable in Wt and Ebf1+/− pre-B-cells, although the number of progenitors was reduced. Heterozygote deletion of Ebf1 resulted in impaired response to IL7 in vitro and reduced expression levels of pre-BCR on the cell surface, providing possible explanations for the observed stage-specific reduction in cellular expansion. Thus, transcription factor doses are critical for specification as well as expansion of B-lymphoid progenitors, providing increased insight into the molecular regulation of B-cell development. PMID:24078629

  17. Memory B cells in mouse models.

    PubMed

    Bergmann, B; Grimsholm, O; Thorarinsdottir, K; Ren, W; Jirholt, P; Gjertsson, I; Mårtensson, I-L

    2013-08-01

    One of the principles behind vaccination, as shown by Edward Jenner in 1796, and host protection is immunological memory, and one of the cells central to this is the antigen-experienced memory B cell that responds rapidly upon re-exposure to the initiating antigen. Classically, memory B cells have been defined as progenies of germinal centre (GC) B cells expressing isotype-switched and substantially mutated B cell receptors (BCRs), that is, membrane-bound antibodies. However, it has become apparent over the last decade that this is not the only pathway to B cell memory. Here, we will discuss memory B cells in mice, as defined by (1) cell surface markers; (2) multiple layers; (3) formation in a T cell-dependent and either GC-dependent or GC-independent manner; (4) formation in a T cell-independent fashion. Lastly, we will touch upon memory B cells in; (5) mouse models of autoimmune diseases. PMID:23679222

  18. B Cell Autonomous TLR Signaling and Autoimmunity

    PubMed Central

    Meyer-Bahlburg, Almut; Rawlings, David J

    2009-01-01

    B cells play a central role in the pathogenesis of multiple autoimmune diseases and the recognition of importance of B cells in these disorders has grown dramatically in association with the remarkable success of B-cell depletion as a treatment for autoimmunity. The precise mechanisms that promote alterations in B cell tolerance remain incompletely defined. There is increasing evidence, however, that TLRs play a major role in these events. Stimulation of B cells via the TLR pathway not only leads to an increase in antibody production but also promotes additional changes including cytokine production and upregulation of activation markers increasing the effectiveness of B cells as APCs. Understanding the role of TLRs in systemic autoimmunity will not only provide insight into the disease pathogenesis but may also lead to the development of novel therapies. This article gives an overview of TLR signaling in B cells and the possible involvement of such signals in autoimmune diseases. PMID:18295736

  19. Low PSI content limits the photoprotection of PSI and PSII in early growth stages of chlorophyll b-deficient wheat mutant lines.

    PubMed

    Brestic, Marian; Zivcak, Marek; Kunderlikova, Kristyna; Sytar, Oksana; Shao, Hongbo; Kalaji, Hazem M; Allakhverdiev, Suleyman I

    2015-08-01

    In vivo analyses of electron and proton transport-related processes as well as photoprotective responses were carried out at different stages of growth in chlorophyll b (Chl b)-deficient mutant lines (ANK-32A and ANK-32B) and wild type (WT) of wheat (Triticum aestivum L.). In addition to a high Chl a-b ratio, ANK mutants had a lower content of photo-oxidizable photosystem I (PSI, P m), and several parameters indicated a low PSI/PSII ratio. Moreover, simultaneous measurements of Chl fluorescence and P700 indicated a shift of balance between redox poise of the PSII acceptor side and the PSII donor side, with preferential reduction of the plastoquinone pool, resulting in an over reduced PSI acceptor side (high Φ NA values). This was the probable reason for PSI inactivation observed in the ANK mutants, but not in WT. In later growth phases, we observed partial relief of "chlorina symptoms," toward WT. Measurements of ΔA 520 decay confirmed that, in early growth stages, the ANK mutants with low PSI content had a limited capacity to build up the transthylakoid proton gradient (ΔpH) needed to trigger non-photochemical quenching (NPQ) and to regulate the electron transport by cytochrome b 6/f. Later, the increase in the PSI/PSII ratio enabled ANK mutants to reach full NPQ, but neither over reduction of the PSI acceptor side nor PSI photoinactivation due to imbalance between the activity of PSII and PSI was mitigated. Thus, our results support the crucial role of proper regulation of linear electron transport in the protection of PSI against photoinhibition. Moreover, the ANK mutants of wheat showing the dynamic developmental changes in the PSI/PSII ratio are presented here as very useful models for further studies.

  20. Microbes and B cell development.

    PubMed

    Wesemann, Duane R

    2015-01-01

    Animals and many of their chronic microbial inhabitants form relationships of symbiotic mutualism, which occurs when coexisting life-forms derive mutual benefit from stable associations. While microorganisms receive a secure habitat and constant food source from vertebrate hosts, they are required for optimal immune system development and occupy niches otherwise abused by pathogens. Microbes have also been shown to provide vertebrate hosts with metabolic capabilities that enhance energy and nutrient uptake from the diet. The immune system plays a central role in the establishment and maintenance of host-microbe homeostasis, and B lineage cells play a key role in this regulation. Here, I reviewed the structure and function of the microbiota and the known mechanisms of how nonpathogenic microbes influence B cell biology and immunoglobulin repertoire development early in life. I also discuss what is known about how B lineage cells contribute to the process of shaping the composition of commensal/mutualistic microbe membership.

  1. Studies on radiosensitive lines of Drosophila. IX. Analysis of fertility and frequency of dominant lethal mutations in the gamma-irradiated females of the mutant line rad(2)201/sup G1/

    SciTech Connect

    Varentsova, E.R.; Sharygin, V.I.; Khromykh, Yu.M.

    1986-03-01

    Fertility and frequency of dominant lethal mutations (DLM) induced by gamma rays in females at the age of 0-5 h and 5-7 days were studied in the radiosensitive mutant rad(2)201/sup G1/ of Drosophila. It has been found that the oocytes of mutant lines are more radiosensitive as compared to those of the wild type flies when compared on the basis of DLM frequency obtained through the entire maturation period. The early oocytes of stages 2-7, i.e., at the stages corresponding to the recombination-defective properties of mutation rad(2)201/sup g1/ are the most sensitive. It has also been demonstrated that the gamma-ray doses exceeding 10 Gy cause a strong sterilizing effect in the mutant females as a result of destruction and resorption of the egg chamber, irradiated at the stages of previtellogenic growth of oocytes. In the radiosensitive mutant females, the sensitivity of the oocytes for DLM induction does not correlate with the sensitivity of the ovarian follicles toward the resorbing effect of gamma rays. The possible involvement of the mutant locus in the genetic processes in different specialized cells of the sexual pathway in Drosophila is discussed.

  2. Suppression of experimental autoimmune myasthenia gravis in IL-10 gene-disrupted mice is associated with reduced B cells and serum cytotoxicity on mouse cell line expressing AChR.

    PubMed

    Poussin, M A; Goluszko, E; Hughes, T K; Duchicella, S I; Christadoss, P

    2000-11-01

    To analyze the role of interleukin-10 (IL-10) in experimental autoimmune myasthenia gravis (EAMG) pathogenesis, we induced clinical EAMG in C57BL/6 and IL-10 gene-knockout (KO) mice. IL-10 KO mice had a lower incidence and severity of EAMG, with less muscle acetylcholine receptor (AChR) loss. AChR-immunized IL-10 KO mice showed a significantly higher AChR-specific proliferative response, altered cytokine response, lower number of class II-positive cells and B-cells, but a greater CD5(+)CD19(+) population than C57BL/6 mice. The lower clinical incidence in IL-10 KO could be explained not by a reduction of the quantity, but by a possible difference in the pathogenicity of anti-AChR antibodies.

  3. T-24.B-cell differentiation factor induces immunoglobulin secretion in human B cells without prior cell replication.

    PubMed

    Gallagher, G; Christie, J F; Stimson, W H; Guy, K; Dewar, A E

    1987-04-01

    Stimulation of B lymphocytes from B-cell chronic lymphocytic leukaemia (B-CLL) with 12-0-tetradecanoylphorbol-13-acetate (TPA) has shown that these cells are capable of differentiation (Totterman, Nilsson & Sundstrom, 1980). Increases in the expression of different class II MHC antigens (Guy et al., 1983, 1986) and responsiveness to growth factors (Kabelitz et al., 1985; Suzuki, Butler & Cooper, 1985) have been studied. Supernatant from the human bladder carcinoma line T-24 contains a B-cell differentiation factor (BCDF) able to induce immunoglobulin secretion from CESS cells. We investigated the induction of proliferation and immunoglobulin secretion in human B cells by studying the effects of this factor on B-CLL cells, in both the presence and absence of TPA. We report here that this material (termed T-24.BCDF) causes immunoglobulin secretion to be initiated in these cells, and that this is not accompanied by detectable DNA synthesis. These observations were extended to normal human B cells and demonstrate that human B cells can secrete immunoglobulin in the absence of clonal expansion. PMID:3495482

  4. Adhesion of Human B Cells to Germinal Centers in Vitro Involves VLA-4 and INCAM-110

    NASA Astrophysics Data System (ADS)

    Freedman, Arnold S.; Munro, J. Michael; Rice, G. Edgar; Bevilacqua, Michael P.; Morimoto, Chikao; McIntyre, Bradley W.; Rhynhart, Kurt; Pober, Jordan S.; Nadler, Lee M.

    1990-08-01

    Human B lymphocytes localize and differentiate within the microenvironment of lymphoid germinal centers. A frozen section binding assay was developed for the identification of those molecules involved in the adhesive interactions between B cells and lymphoid follicles. Activated human B cells and B cell lines were found to selectively adhere to germinal centers. The VLA-4 molecule on the lymphocyte and the adhesion molecule INCAM-110, expressed on follicular dendritic cells, supported this interaction. This cellular interaction model can be used for the study of how B cells differentiate.

  5. B-Cell Hematologic Malignancy Vaccination Registry

    ClinicalTrials.gov

    2015-09-15

    Monoclonal Gammopathy of Undetermined Significance; Multiple Myeloma; Waldenstrom Macroglobulinemia; Lymphocytosis; Lymphoma, Non-Hodgkin; B-Cell Chronic Lymphocytic Leukemia; Hematological Malignancies

  6. Oncogenic CARMA1 couples NF-κB and β-catenin signaling in diffuse large B-cell lymphomas

    PubMed Central

    Bognar, M K; Vincendeau, M; Erdmann, T; Seeholzer, T; Grau, M; Linnemann, J R; Ruland, J; Scheel, C H; Lenz, P; Ott, G; Lenz, G; Hauck, S M; Krappmann, D

    2016-01-01

    Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis. PMID:26776161

  7. Production of RANKL by Memory B Cells

    PubMed Central

    Meednu, Nida; Zhang, Hengwei; Owen, Teresa; Sun, Wen; Wang, Victor; Cistrone, Christopher; Rangel-Moreno, Javier; Xing, Lianping; Anolik, Jennifer H.

    2016-01-01

    Objective Rheumatoid arthritis (RA) is a systemic autoimmune disease that often leads to joint damage. The mechanisms of bone damage in RA are complex, involving activation of bone-resorbing osteoclasts (OCs) by synoviocytes and Th17 cells. This study was undertaken to investigate whether B cells play a direct role in osteoclastogenesis through the production of RANKL, the essential cytokine for OC development. Methods RANKL production by total B cells or sorted B cell subpopulations in the peripheral blood and synovial tissue from healthy donors or anti–cyclic citrullinated peptide–positive patients with RA was examined by flow cytometry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. To define direct effects on osteoclastogenesis, B cells were cocultured with CD14+ monocytes, and OCs were enumerated by tartrate-resistant acid phosphatase staining. Results Healthy donor peripheral blood B cells were capable of expressing RANKL upon stimulation, with switched memory B cells (CD27+IgD−) having the highest propensity for RANKL production. Notably, switched memory B cells in the peripheral blood from RA patients expressed significantly more RANKL compared to healthy controls. In RA synovial fluid and tissue, memory B cells were enriched and spontaneously expressed RANKL, with some of these cells visualized adjacent to RANK+ OC precursors. Critically, B cells supported OC differentiation in vitro in a RANKL-dependent manner, and the number of OCs was higher in cultures with RA B cells than in those derived from healthy controls. Conclusion These findings reveal the critical importance of B cells in bone homeostasis and their likely contribution to joint destruction in RA. PMID:26554541

  8. DNase I hypersensitive sites flank the mouse class II major histocompatibility complex during B cell development.

    PubMed Central

    Carson, S

    1991-01-01

    The mouse class II major histocompatibility complex (MHC) encodes a polymorphic, multigene family important in the immune response, and is expressed mainly on mature B cells, on certain types of dendritic cells and is also inducible by gamma-interferon on antigen presenting cells. To study the regulatory elements which control this expression pattern, we have examined the chromatin structure flanking the class II MHC region, in particular during B cell differentiation. Using a panel of well-characterised mouse cell lines specific for different stages of B cell development (pre-B, B, plasma cell) as well as non-B cell lines, we have mapped the DNase I hypersensitive (DHS) sites adjacent to the mouse MHC class II region. The results presented show, for the first time that there are specific hypersensitive sites flanking the class II MHC locus during pre B cell, B cell and plasma cell stages of B cell differentiation, irrespective of the status of class II MHC expression. These hypersensitive sites are not found in T cell, fibroblast or uninduced myelomonocytic cell lines. This suggests that these DHS sites define a developmentally stable, chromatin structure, which can be used as a marker of B cell lineage commitment and may indicate that a combination of these hypersensitive sites reflect regulatory proteins involved in the immediate expression of a particular class II MHC gene or possibly control of the entire locus. Images PMID:1923768

  9. Ginsenoside Production and Morphological Characterization of Wild Ginseng (Panax ginseng Meyer) Mutant Lines Induced by γ-irradiation (60Co) of Adventitious Roots

    PubMed Central

    Zhang, Jun-Ying; Bae, Tae-Woong; Boo, Kyung-Hwan; Sun, Hyeon-Jin; Song, In-Ja; Pham, Chi-Hoa; Ganesan, Markkandan; Yang, Dae-Hwa; Kang, Hong-Gyu; Ko, Suk-Min; Riu, Key-Zung; Lim, Pyung-Ok; Lee, Hyo-Yeon

    2011-01-01

    With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture. PMID:23717071

  10. Expression of sprouty2 inhibits B-cell proliferation and is epigenetically silenced in mouse and human B-cell lymphomas

    PubMed Central

    Frank, Matthew J.; Dawson, David W.; Bensinger, Steven J.; Hong, Jason S.; Knosp, Wendy M.; Xu, Lizhong; Balatoni, Cynthia E.; Allen, Eric L.; Shen, Rhine R.; Bar-Sagi, Dafna; Martin, Gail R.

    2009-01-01

    B-cell lymphoma is the most common immune system malignancy. TCL1 transgenic mice (TCL1-tg), in which TCL1 is ectopically expressed in mature lymphocytes, develop multiple B- and T-cell leukemia and lymphoma subtypes, supporting an oncogenic role for TCL1 that probably involves AKT and MAPK-ERK signaling pathway augmentation. Additional, largely unknown genetic and epigenetic alterations cooperate with TCL1 during lymphoma progression. We examined DNA methylation patterns in TCL1-tg B-cell tumors to discover tumor-associated epigenetic changes, and identified hypermethylation of sprouty2 (Spry2). Sprouty proteins are context-dependent negative or positive regulators of MAPK-ERK pathway signaling, but their role(s) in B-cell physiology or pathology are unknown. Here we show that repression of Spry2 expression in TCL1-tg mouse and human B-cell lymphomas and cell lines is associated with dense DNA hypermethylation and was reversed by inhibition of DNA methylation. Spry2 expression was induced in normal splenic B cells by CD40/B-cell receptor costimulation and regulated a negative feedback loop that repressed MAPK-ERK signaling and decreased B-cell viability. Conversely, loss of Spry2 function hyperactivated MAPK-ERK signaling and caused increased B-cell proliferation. Combined, these results implicate epigenetic silencing of Spry2 expression in B lymphoma progression and suggest it as a companion lesion to ectopic TCL1 expression in enhancing MAPK-ERK pathway signaling. PMID:19147787

  11. Molecular underpinning of B-cell anergy

    PubMed Central

    Yarkoni, Yuval; Getahun, Andrew; Cambier, John C.

    2010-01-01

    Summary A byproduct of the largely stochastic generation of a diverse B-cell specificity repertoire is production of cells that recognize autoantigens. Indeed, recent studies indicate that more than half of the primary repertoire consists of autoreactive B cells that must be silenced to prevent autoimmunity. While this silencing can occur by multiple mechanisms, it appears that most autoreactive B cells are silenced by anergy, wherein they populate peripheral lymphoid organs and continue to express unoccupied antigen receptors yet are unresponsive to antigen stimulation. Here we review molecular mechanisms that appear operative in maintaining the antigen unresponsiveness of anergic B cells. In addition, we present new data indicating that the failure of anergic B cells to mobilize calcium in response to antigen stimulation is not mediated by inactivation of stromal interacting molecule 1, a critical intermediary in intracellular store depletion-induced calcium influx. PMID:20727040

  12. CEACAM1 mediates B cell aggregation in central nervous system autoimmunity.

    PubMed

    Rovituso, Damiano M; Scheffler, Laura; Wunsch, Marie; Kleinschnitz, Christoph; Dörck, Sebastian; Ulzheimer, Jochen; Bayas, Antonios; Steinman, Lawrence; Ergün, Süleyman; Kuerten, Stefanie

    2016-01-01

    B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1(+) B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines, application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain -3 (TIM-3) on B cells, a novel molecule that has recently been described to induce anergy in T cells. Interestingly, elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall, these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. PMID:27435215

  13. Nek2 Is a Novel Regulator of B Cell Development and Immunological Response

    PubMed Central

    Zhou, Wen; Huang, Junwei; Yang, Ye; Wendlandt, Erik; Xu, Hongwei; Zhan, Fenghuang

    2014-01-01

    The serine/threonine kinase Nek2 is commonly found upregulated in a wide variety of neoplasms including diffuse large B cell lymphoma and multiple myeloma. High expression of Nek2 is implicated in the induction of chromosomal instability, promotion of cell proliferation, and drug resistance in tumor cells as well as a marker for poor clinical outcomes. Despite its well recorded involvement in chromosomal instability and neoplastic growth, little is known about the involvement of Nek2 in B cell development. Here we report the development of a transgenic mouse line with conditional expression of Nek2 in the B cell lineage and the effects it has on the development of B cells. Interestingly, we found that the overexpression of Nek2 does not induce spontaneous tumor formation within the transgenic mice up to 24 months after induction. Instead, overexpression of Nek2 in the B cell lineage affects the development of B cells by increasing the proportion of immature B cells in the bone marrow and decreasing B-1 B cells in peritoneal cavity. Furthermore, Nek2 transgenic mice develop spontaneous germinal centers and exhibit an enhanced T cell dependent immune response. Altogether, our data demonstrates a novel role for Nek2 in regulating B cell development and the immune response. PMID:25485281

  14. CEACAM1 mediates B cell aggregation in central nervous system autoimmunity

    PubMed Central

    Rovituso, Damiano M.; Scheffler, Laura; Wunsch, Marie; Dörck, Sebastian; Ulzheimer, Jochen; Bayas, Antonios; Steinman, Lawrence; Ergün, Süleyman; Kuerten, Stefanie

    2016-01-01

    B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1+ B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines, application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain −3 (TIM-3) on B cells, a novel molecule that has recently been described to induce anergy in T cells. Interestingly, elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall, these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. PMID:27435215

  15. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.

    PubMed

    Chan, O T; Hannum, L G; Haberman, A M; Madaio, M P; Shlomchik, M J

    1999-05-17

    The precise role of B cells in systemic autoimmunity is incompletely understood. Although B cells are necessary for expression of disease (Chan, O., and M.J. Shlomchik. 1998. J. Immunol. 160:51-59, and Shlomchik, M.J., M.P. Madaio, D. Ni, M. Trounstine, and D. Huszar. 1994. J. Exp. Med. 180:1295-1306), it is unclear whether autoantibody production, antigen presentation, and/or other B cell functions are required for the complete pathologic phenotype. To address this issue, two experimental approaches were used. In the first, the individual contributions of circulating antibodies and B cells were analyzed using MRL/MpJ-Faslpr (MRL/lpr) mice that expressed a mutant transgene encoding surface immunoglobulin (Ig), but which did not permit the secretion of circulating Ig. These mice developed nephritis, characterized by cellular infiltration within the kidney, indicating that B cells themselves, without soluble autoantibody production, exert a pathogenic role. The results indicate that, independent of serum autoantibody, functional B cells expressing surface Ig are essential for disease expression, either by serving as antigen-presenting cells for antigen-specific, autoreactive T cells, or by contributing directly to local inflammation.

  16. CD27-CD70 interactions regulate B-cell activation by T cells.

    PubMed Central

    Kobata, T; Jacquot, S; Kozlowski, S; Agematsu, K; Schlossman, S F; Morimoto, C

    1995-01-01

    CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis. PMID:7479974

  17. Disodium cromoglycate enhances ongoing immunoglobulin production in vitro in human B cells.

    PubMed Central

    Kimata, H; Yoshida, A; Ishioka, C; Mikawa, H

    1991-01-01

    The effect of disodium cromoglycate (DSCG) upon human immunoglobulin (Ig) isotypes and IgG subclasses production by purified B cells was studied. DSCG enhanced IgM, IgG1, IgG2, IgG3, IgG4 and IgA production in a dose-dependent fashion, while DSCG failed to induce IgE production at any concentrations tested by purified B cells. When B cells were separated into small resting and large activated B cells, DSCG failed to induce Ig production from small resting B cells in the presence or absence of Staphylococcus aureus Cowan strain I (SAC). In contrast, in large activated B cells DSCG significantly enhanced all types of Ig production (two-to threefold), especially IgG4 production (seven-to 11-fold), except IgE, which large B cells did not produce. The enhancement of IgG subclass production was not subclass switching, since DSCG failed to enhance IgG1 production in B cells depleted of surface IgG1+ cells (sIgG1+ cells). Similarly, DSCG did not enhance IgG2, IgG3 or IgG4 production from sIgG2-, sIgG3- or sIgG4- B cells, respectively, Interleukin-4 (IL-4) or interleukin-6 (IL-6) also enhanced Ig production except IgG4 from large activated B cells. The enhancing effect of DSCG was not mediated by IL-4 or IL-6 since anti-IL-4 or anti-IL-6 antibody failed to block the DSCG-induced enhancement. DSCG also enhanced IgG2 and IgM production from human B-cell lines GM-1500 and CBL, respectively. These results suggest that DSCG directly and preferentially stimulates activated B cells which are producing Ig and, in addition, enhances their Ig production. PMID:1904400

  18. Two maturation-associated mouse erythrocyte receptors of human B cells. I. Identification of four human B-cell subsets.

    PubMed Central

    Forbes, I J; Zalewski, P D; Valente, L; Gee, D

    1982-01-01

    Using rosetting tests with untreated mouse erythrocytes (M) and pronase-treated M (pro M), four human B cell subsets can be identified. Three of these, possessing the phenotypes BM+ pro M+, BM- pro M+ or BM- pro M-, constitute 17%, 61% and 22% of normal blood B cells respectively. The fourth subset, BM+ pro M-, does not occur in normal tissues but was found in the pre-B-cell line of Raji cells, indicating that this phenotype may be a marker for early B cells. Some differences in the proportion of each subset were found in cord blood, lymph nodes and tonsils. Surface-immunoglobulin-positive (SIg+) and -negative (SIg-) non-T cells were present in each subset. M and pro-M rosetting tests were applied to cells from blood of 27 cases of chronic lymphocytic leukaemia (CLL) and to cells from involved nodes, spleen or marrow in five cases of non-Hodgkin's lymphoma (NHL). In 15 cases of CLL, there was considerable increase in the BM+ pro M+ subset (BM+ pro M+ type CLL); in seven cases, there was a predominance of BM- pro M+ cells and in another four cases, BM- pro M- cells predominated. All five cases of NHL were greatly enriched in BM- pro M- cells. There was no obvious correlation between rosetting and other surface markers but BM- pro M- clones in CLL or NHL always stained brightly with FITC-anti-Ig. This was not found in BM+ pro M+ or BM- pro M+ clones. Rosette formation of neuraminidase-treated B cells with M identifies the same subset as B-pro-M rosetting in normals and CLL. Evidence is presented that two types of receptors are involved in M and pro-M rosetting, designated R1 and R2, binding to corresponding M ligands L1 and L2. M rosetting is due to R1-L1 binding while R2-L2 binding mediates B-pro-M rosetting. Shifts between subsets within the same clone in some cases of CLL suggest that the subsets are distinct maturational stage of B-cell development rather than families of B cells of different lineage. The following B-cell maturation sequence is proposed: R1+ R2

  19. B-cell markers in malignant B-cell lymphoma with scleroderma-like manifestation.

    PubMed

    Van Joost, T; Stolz, E; Blog, F B; Van der Kwast, T H; Vuzevski, V D; Van Dongen, J M

    1984-12-01

    A case is described of malignant B-cell lymphoma with scleroderma-like manifestation. Using different monoclonals as B-cell markers the tumor appeared to be positive for surface immunoglobulins (SmIg) and for B2-antigen, but negative for intracytoplasmic immunoglobulin (CIg), BA2- and FMC7-antigens. Therefore, the tumor could be determined as a highly differentiated Sm-positive early B-cell type of B-cell lymphoma. In this clinically rare manifestation of cutaneous B-cell lymphoma aspects of the cell morphology and of cellular mediated immunity are briefly discussed.

  20. Expression cloning of human B cell immunoglobulins.

    PubMed

    Wardemann, Hedda; Kofer, Juliane

    2013-01-01

    The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

  1. Primary cutaneous B-cell lymphoma.

    PubMed

    Bogle, Melissa A; Riddle, Christy C; Triana, Emily M; Jones, Dan; Duvic, Madeleine

    2005-09-01

    Primary cutaneous B-cell lymphomas include extranodal marginal zone B-cell lymphoma, follicular lymphoma, large B-cell lymphoma, and, rarely, mantle cell lymphoma. Our purpose in conducting this review was to determine the clinical and behavioral characteristics of primary cutaneous B-cell lymphomas, their relationship to infectious triggers, and therapeutic response. We conducted a retrospective chart review of 23 adult patients presenting to the dermatology clinic at M. D. Anderson Cancer Center with primary cutaneous B-cell lymphoma between January 1999 and May 2003. Primary cutaneous B-cell lymphomas generally present on the head and neck, with the trunk and extremities afflicted to a lesser extent. Patients were found to have serologic evidence of prior infection with Borrelia burgdorferi (n = 10), Helicobacter pylori (n = 5), and Epstein-Barr virus (n = 6). Overall, treatment of primary cutaneous B-cell lymphoma should involve multiple modalities; however, specific treatment aimed at concurrent or suspected infection, particularly B burgdorferi, is a helpful adjunct and may achieve complete remission in a small subset of patients.

  2. Auxin Transport and Ribosome Biogenesis Mutant/Reporter Lines to Study Plant Cell Growth and Proliferation under Altered Gravity

    NASA Astrophysics Data System (ADS)

    Valbuena, Miguel A.; Manzano, Ana I.; van Loon, Jack JWA.; Saez-Vasquez, Julio; Carnero-Diaz, Eugenie; Herranz, Raul; Medina, F. J.

    2013-02-01

    We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (μg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (μgRPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity.

  3. B Cells Are Multifunctional Players in Multiple Sclerosis Pathogenesis: Insights from Therapeutic Interventions

    PubMed Central

    Claes, Nele; Fraussen, Judith; Stinissen, Piet; Hupperts, Raymond; Somers, Veerle

    2015-01-01

    Multiple sclerosis (MS) is a severe disease of the central nervous system (CNS) characterized by autoimmune inflammation and neurodegeneration. Historically, damage to the CNS was thought to be mediated predominantly by activated pro-inflammatory T cells. B cell involvement in the pathogenesis of MS was solely attributed to autoantibody production. The first clues for the involvement of antibody-independent B cell functions in MS pathology came from positive results in clinical trials of the B cell-depleting treatment rituximab in patients with relapsing-remitting (RR) MS. The survival of antibody-secreting plasma cells and decrease in T cell numbers indicated the importance of other B cell functions in MS such as antigen presentation, costimulation, and cytokine production. Rituximab provided us with an example of how clinical trials can lead to new research opportunities concerning B cell biology. Moreover, analysis of the antibody-independent B cell functions in MS has gained interest since these trials. Limited information is present on the effects of current immunomodulatory therapies on B cell functions, although effects of both first-line (interferon, glatiramer acetate, dimethyl fumarate, and teriflunomide), second-line (fingolimod, natalizumab), and even third-line (monoclonal antibody therapies) treatments on B cell subtype distribution, expression of functional surface markers, and secretion of different cytokines by B cells have been studied to some extent. In this review, we summarize the effects of different MS-related treatments on B cell functions that have been described up to now in order to find new research opportunities and contribute to the understanding of the pathogenesis of MS. PMID:26734009

  4. B Cells Are Multifunctional Players in Multiple Sclerosis Pathogenesis: Insights from Therapeutic Interventions.

    PubMed

    Claes, Nele; Fraussen, Judith; Stinissen, Piet; Hupperts, Raymond; Somers, Veerle

    2015-01-01

    Multiple sclerosis (MS) is a severe disease of the central nervous system (CNS) characterized by autoimmune inflammation and neurodegeneration. Historically, damage to the CNS was thought to be mediated predominantly by activated pro-inflammatory T cells. B cell involvement in the pathogenesis of MS was solely attributed to autoantibody production. The first clues for the involvement of antibody-independent B cell functions in MS pathology came from positive results in clinical trials of the B cell-depleting treatment rituximab in patients with relapsing-remitting (RR) MS. The survival of antibody-secreting plasma cells and decrease in T cell numbers indicated the importance of other B cell functions in MS such as antigen presentation, costimulation, and cytokine production. Rituximab provided us with an example of how clinical trials can lead to new research opportunities concerning B cell biology. Moreover, analysis of the antibody-independent B cell functions in MS has gained interest since these trials. Limited information is present on the effects of current immunomodulatory therapies on B cell functions, although effects of both first-line (interferon, glatiramer acetate, dimethyl fumarate, and teriflunomide), second-line (fingolimod, natalizumab), and even third-line (monoclonal antibody therapies) treatments on B cell subtype distribution, expression of functional surface markers, and secretion of different cytokines by B cells have been studied to some extent. In this review, we summarize the effects of different MS-related treatments on B cell functions that have been described up to now in order to find new research opportunities and contribute to the understanding of the pathogenesis of MS. PMID:26734009

  5. Adhesive interactions regulate transcriptional diversity in malignant B cells.

    PubMed

    Nadav-Dagan, Liat; Shay, Tal; Dezorella, Nili; Naparstek, Elizabeth; Domany, Eytan; Katz, Ben-Zion; Geiger, Benjamin

    2010-04-01

    The genetic profiling of B-cell malignancies is rapidly expanding, providing important information on the tumorigenic potential, response to treatment, and clinical outcome of these diseases. However, the relative contributions of inherent gene expression versus microenvironmental effects are poorly understood. The regulation of gene expression programs by means of adhesive interactions was studied here in ARH-77 human malignant B-cell variants, derived from the same cell line by selective adhesion to a fibronectin matrix. The populations included cells that adhere to fibronectin and are highly tumorigenic (designated "type A" cells) and cells that fail to adhere to fibronectin and fail to develop tumors in vivo ("type F" cells). To identify genes directly affected by cell adhesion to fibronectin, type A cells deprived of an adhesive substrate (designated "AF cells") were also examined. Bioinformatic analyses revealed a remarkable correlation between cell adhesion and both B-cell differentiation state and the expression of multiple myeloma (MM)-associated genes. The highly adherent type A cells expressed higher levels of NFkappaB-regulated genes, many of them associated with MM. Moreover, we found that the transcription of several MM-related proto-oncogenes is stimulated by adhesion to fibronectin. In contrast, type F cells, which display poor adhesive and tumorigenic properties, expressed genes associated with higher levels of B-cell differentiation. Our findings indicate that B-cell differentiation, as manifested by gene expression profiles, is attenuated by cell adhesion to fibronectin, leading to upregulation of specific genes known to be associated with the pathogenesis of MM.

  6. Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line

    PubMed Central

    2014-01-01

    Background Mutations in NBN, the gene for Nijmegen Breakage Syndrome (NBS), are thought to predispose women to developing breast cancer, but a breast cancer cell line containing mutations in NBN has not yet been described. The p.R215W missense mutation occurs at sub-polymorphic frequencies in several populations. We aimed to investigate its functional impact in breast cancer cells from a carrier of this NBN mutation. Methods Breast cancer cell lines were screened by immunoblotting for NBN protein levels, and the NBN coding region was sequenced for mutation analysis. Radiosensitivity assays and functional studies were performed through immunocytochemistry and immunoblotting, and flow cytometry was employed to assess cell cycle progression. Impedance measurements were used to study the consequences of PARP1 inhibition. Statistical comparisons between cell lines were performed using t-tests. Results HCC1395 breast cancer cells exhibited reduced NBN protein levels. Direct sequencing identified the NBN p.R215W mutation in the hemizygous state, in addition to a truncation in BRCA1. Mutations in both genes were already present in the heterozygous state in the patient’s germline. HCC1395 cells were highly radiosensitive, susceptible to apoptosis and were deficient in the formation of NBN foci. There was also evidence for some impairment in the formation of γH2AX, MDC1, and 53BP1 foci after irradiation; these foci appeared smaller and irregular compared with repair foci in wild-type cells, although ATM signalling was largely unaffected. In line with their deficiency in NBN and BRCA1, HCC1395 cells were particularly sensitive to PARP1 inhibition. Conclusion Our results indicate that the p.R215W mutation in the HCC1395 breast cancer cell line impairs NBN function, making this cell line a potentially useful cellular model for studying defective NBN protein within a mutant BRCA1 background. PMID:24928521

  7. Enhanced Cultivation Of Stimulated Murine B Cells

    NASA Technical Reports Server (NTRS)

    Sammons, David W.

    1994-01-01

    Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

  8. Functional characterization of a new p53 mutant generated by homozygous deletion in a neuroblastoma cell line

    SciTech Connect

    Nakamura, Yohko; Ozaki, Toshinori; Niizuma, Hidetaka; Ohira, Miki; Kamijo, Takehiko; Nakagawara, Akira . E-mail: akiranak@chiba-cc.jp

    2007-03-23

    p53 is a key modulator of a variety of cellular stresses. In human neuroblastomas, p53 is rarely mutated and aberrantly expressed in cytoplasm. In this study, we have identified a novel p53 mutant lacking its COOH-terminal region in neuroblastoma SK-N-AS cells. p53 accumulated in response to cisplatin (CDDP) and thereby promoting apoptosis in neuroblastoma SH-SY5Y cells bearing wild-type p53, whereas SK-N-AS cells did not undergo apoptosis. We found another p53 (p53{delta}C) lacking a part of oligomerization domain and nuclear localization signals in SK-N-AS cells. p53{delta}C was expressed largely in cytoplasm and lost the transactivation function. Furthermore, a 3'-part of the p53 locus was homozygously deleted in SK-N-AS cells. Thus, our present findings suggest that p53 plays an important role in the DNA-damage response in certain neuroblastoma cells and it seems to be important to search for p53 mutations outside DNA-binding domain.

  9. Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line.

    PubMed

    Ware, C F; Krangel, M S; Pious, D; Burakoff, S J; Strominger, J L

    1983-09-01

    HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule. PMID:6193184

  10. Role of insulin-like growth factors and myogenin in the altered program of proliferation and differentiation in the NFB4 mutant muscle cell line.

    PubMed

    Sarbassov, D D; Stefanova, R; Grigoriev, V G; Peterson, C A

    1995-11-21

    In the present study we used the mutant muscle cell line NFB4 to study the balance between proliferation and myogenic differentiation. We show that removal of serum, which induced the parental C2C12 cells to withdraw from the cell cycle and differentiate, had little effect on NFB4 cells. Gene products characteristic of the proliferation state, such as c-Jun, continued to accumulate in the mutant cells in low serum, whereas those involved in differentiation, like myogenin, insulin-like growth factor II (IGF-II), and IGF-binding protein 5 (IGFBP-5) were undetectable. Moreover, NFB4 cells displayed a unique pattern of tyrosine phosphorylated proteins, especially in low serum, suggesting that the signal transduction pathway(s) that controls differentiation is not properly regulated in these cells. Treatment of NFB4 cells with exogenous IGF-I or IGF-II at concentrations shown to promote myogenic differentiation in wild-type cells resulted in activation of myogenin but not MyoD gene expression, secretion of IG-FBP-5, changes in tyrosine phosphorylation, and enhanced myogenic differentiation. Similarly, transfection of myogenin expression constructs also enhanced differentiation and resulted in activation of IGF-II expression, showing that myogenin and IGF-II cross-activate each other's expression. However, in both cases, the expression of Jun mRNA remained elevated, suggesting that IGFs and myogenin cannot overcome all aspects of the block to differentiation in NFB4 cells.

  11. Early B-cell factor 1 (EBF1) is critical for transcriptional control of SLAMF1 gene in human B cells.

    PubMed

    Schwartz, Anton M; Putlyaeva, Lidia V; Covich, Milica; Klepikova, Anna V; Akulich, Kseniya A; Vorontsov, Ilya E; Korneev, Kirill V; Dmitriev, Sergey E; Polanovsky, Oleg L; Sidorenko, Svetlana P; Kulakovskiy, Ivan V; Kuprash, Dmitry V

    2016-10-01

    Signaling lymphocytic activation molecule family member 1 (SLAMF1)/CD150 is a co-stimulatory receptor expressed on a variety of hematopoietic cells, in particular on mature lymphocytes activated by specific antigen, costimulation and cytokines. Changes in CD150 expression level have been reported in association with autoimmunity and with B-cell chronic lymphocytic leukemia. We characterized the core promoter for SLAMF1 gene in human B-cell lines and explored binding sites for a number of transcription factors involved in B cell differentiation and activation. Mutations of SP1, STAT6, IRF4, NF-kB, ELF1, TCF3, and SPI1/PU.1 sites resulted in significantly decreased promoter activity of varying magnitude, depending on the cell line tested. The most profound effect on the promoter strength was observed upon mutation of the binding site for Early B-cell factor 1 (EBF1). This mutation produced a 10-20 fold drop in promoter activity and pinpointed EBF1 as the master regulator of human SLAMF1 gene in B cells. We also identified three potent transcriptional enhancers in human SLAMF1 locus, each containing functional EBF1 binding sites. Thus, EBF1 interacts with specific binding sites located both in the promoter and in the enhancer regions of the SLAMF1 gene and is critical for its expression in human B cells.

  12. Smad-binding defective mutant of transforming growth factor beta type I receptor enhances tumorigenesis but suppresses metastasis of breast cancer cell lines.

    PubMed

    Tian, Fang; Byfield, Stacey DaCosta; Parks, W Tony; Stuelten, Christina H; Nemani, Deepa; Zhang, Ying E; Roberts, Anita B

    2004-07-01

    The role of transforming growth factor beta (TGF-beta) in carcinogenesis is complex, with tumor suppressor and pro-oncogenic activities depending on the particular tumor cell and its stage in malignant progression. We previously have demonstrated in breast cancer cell lines that Smad2/3 signaling played a dominant role in mediating tumor suppressor effects on well-differentiated breast cancer cell lines grown as xenografts and prometastatic effects on a more invasive, metastatic cell line. Our present data based on selective interference with activation of endogenous Smad2 and Smad3 by stable expression of a mutant form of the TGF-beta type I receptor (RImL45) unable to bind Smad2/3 but with a functional kinase again show that reduction in Smad2/3 signaling by expression of RImL45 enhanced the malignancy of xenografted tumors of the well-differentiated MCF10A-derived tumor cell line MCF10CA1h, resulting in formation of larger tumors with a higher proliferative index and more malignant histologic features. In contrast, expression of RImL45 in the more aggressive MCF10CA1a cell line strongly suppressed formation of lung metastases following tail vein injection. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in these cells. Using an in vitro assay, we further show that non-Smad signaling pathways, including p38 and c-Jun NH(2)-terminal kinase, cooperate with TGF-beta/Smads in enhancing migration of metastatic MCF10CA1a cells, but that, although necessary for migration, these other pathways are not sufficient for metastasis.

  13. Novel pyrazolo[3,4-d]pyrimidines as dual Src-Abl inhibitors active against mutant form of Abl and the leukemia K-562 cell line.

    PubMed

    El-Moghazy, Samir M; George, Riham F; Osman, Essam Eldin A; Elbatrawy, Ahmed A; Kissova, Miroslava; Colombo, Ambra; Crespan, Emmanuele; Maga, Giovanni

    2016-11-10

    Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity.

  14. The unexpected evolution of a case of diffuse large B-cell non-Hodgkin lymphoma.

    PubMed

    Găman, Amelia; Bold, Adriana; Găman, G

    2011-01-01

    The diffuse large B-cell lymphoma (DLBCL) represents the most common type of aggressive non-Hodgkin's lymphoma with a heterogeneous morphology, biology and clinical presentation. Gene expression profiling studies identified three distinct molecular subtypes of DLCBL arisen from B-cells at different stages of differentiation: germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, primary mediastinal B-cell lymphoma (PMBL). The most relevant oncogenic pathways in diffuse large B-cell lymphoma are: deregulated B-cell receptor/proliferation signaling, BCL6 and NF-kB constitutive expression, defects in apoptosis and neoangiogenesis. The treatment of DLBCL has been completely modified in the last ten years by combination of anti-CD20 monoclonal antibody (rituximab) and CHOP chemotherapy, which is now the first line therapy. In the last years, there have been reported several cases of progressive multifocal leukoencephalopathy (PML) at patients with rheumatoid arthritis treated with rituximab. Progressive multifocal leukoencephalopathy is possible as an adverse reaction to rituximab at patients treated with R-CHOP for diffuse large B-cell lymphoma. PMID:21655667

  15. Proinflammatory GM-CSF-producing B cells in multiple sclerosis and B cell depletion therapy.

    PubMed

    Li, Rui; Rezk, Ayman; Miyazaki, Yusei; Hilgenberg, Ellen; Touil, Hanane; Shen, Ping; Moore, Craig S; Michel, Laure; Althekair, Faisal; Rajasekharan, Sathy; Gommerman, Jennifer L; Prat, Alexandre; Fillatreau, Simon; Bar-Or, Amit

    2015-10-21

    B cells are not limited to producing protective antibodies; they also perform additional functions relevant to both health and disease. However, the relative contribution of functionally distinct B cell subsets in human disease, the signals that regulate the balance between such subsets, and which of these subsets underlie the benefits of B cell depletion therapy (BCDT) are only partially elucidated. We describe a proinflammatory, granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing human memory B cell subset that is increased in frequency and more readily induced in multiple sclerosis (MS) patients compared to healthy controls. In vitro, GM-CSF-expressing B cells efficiently activated myeloid cells in a GM-CSF-dependent manner, and in vivo, BCDT resulted in a GM-CSF-dependent decrease in proinflammatory myeloid responses of MS patients. A signal transducer and activator of transcription 5 (STAT5)- and STAT6-dependent mechanism was required for B cell GM-CSF production and reciprocally regulated the generation of regulatory IL-10-expressing B cells. STAT5/6 signaling was enhanced in B cells of untreated MS patients compared with healthy controls, and B cells reemerging in patients after BCDT normalized their STAT5/6 signaling as well as their GM-CSF/IL-10 cytokine secretion ratios. The diminished proinflammatory myeloid cell responses observed after BCDT persisted even as new B cells reconstituted. These data implicate a proinflammatory B cell/myeloid cell axis in disease and underscore the rationale for selective targeting of distinct B cell populations in MS and other human autoimmune diseases. PMID:26491076

  16. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations

    PubMed Central

    Xiao, Wenming; Stinson, Jeffrey R.; Lu, Wei; Chaigne-Delalande, Benjamin; Zheng, Lixin; Pittaluga, Stefania; Matthews, Helen F.; Schmitz, Roland; Jhavar, Sameer; Kuchen, Stefan; Kardava, Lela; Wang, Wei; Lamborn, Ian T.; Jing, Huie; Raffeld, Mark; Moir, Susan; Fleisher, Thomas A.; Staudt, Louis M.; Su, Helen C.

    2012-01-01

    Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)–induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis. PMID:23129749

  17. Congenital B cell lymphocytosis explained by novel germline CARD11 mutations.

    PubMed

    Snow, Andrew L; Xiao, Wenming; Stinson, Jeffrey R; Lu, Wei; Chaigne-Delalande, Benjamin; Zheng, Lixin; Pittaluga, Stefania; Matthews, Helen F; Schmitz, Roland; Jhavar, Sameer; Kuchen, Stefan; Kardava, Lela; Wang, Wei; Lamborn, Ian T; Jing, Huie; Raffeld, Mark; Moir, Susan; Fleisher, Thomas A; Staudt, Louis M; Su, Helen C; Lenardo, Michael J

    2012-11-19

    Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.

  18. The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

    PubMed Central

    2014-01-01

    Background The Medicago truncatula 2HA seed line is highly embryogenic while the parental line Jemalong rarely produces embryos. The 2HA line was developed from one of the rare Jemalong regenerates and this method for obtaining a highly regenerable genotype in M. truncatula is readily reproducible suggesting an epigenetic mechanism. Microarray transcriptomic analysis showed down regulation of an ETHYLENE INSENSITIVE 3-like gene in 2HA callus which provided an approach to investigating epigenetic regulation of genes related to ethylene signalling and the 2HA phenotype. Ethylene is involved in many developmental processes including somatic embryogenesis (SE) and is associated with stress responses. Results Microarray transcriptomic analysis showed a significant number of up-regulated transcripts in 2HA tissue culture, including nodule and embryo specific genes and transposon-like genes, while only a few genes were down-regulated, including an EIN3-like gene we called MtEIL1. This reduced expression was associated with ethylene insensitivity of 2HA plants that was further investigated. The weak ethylene insensitivity affected root and nodule development. Sequencing of MtEIL1 found no difference between 2HA and wild-type plants. DNA methylation analysis of MtEIL1 revealed significant difference between 2HA and wild-type plants. Tiling arrays demonstrated an elevated level of miRNA in 2HA plants that hybridised to the antisense strand of the MtEIL1 gene. AFLP-like methylation profiling revealed more differences in DNA methylation between 2HA and wild-type. Segregation analysis demonstrated the recessive nature of the eil1 phenotype and the dominant nature of the SE trait. Conclusions We have demonstrated that EIL1 of Medicago truncatula (MtEIL1) is epigenetically silenced in the 2HA seed line. The possible cause is an elevated level of miRNA that targets its 3’UTR and is also associated with DNA methylation of MtEIL1. Down regulation of MtEIL1 makes it possible to

  19. VPX mutants of HIV-2 are infectious in established cell lines but display a severe defect in peripheral blood lymphocytes.

    PubMed Central

    Guyader, M; Emerman, M; Montagnier, L; Peden, K

    1989-01-01

    Nucleotide sequence comparison between HIV-1, HIV-2 and SIV has revealed the presence of an open reading frame (ORF) in the central region of the genomes of HIV-2 and SIV that has no counterpart in HIV-1. This new ORF, called vpx, is highly conserved between HIV-2ROD and SIVmac. Using anti-peptide sera to the predicted protein and site-directed mutagenesis, we show that mutations in the vpx ORF eliminate the synthesis of a 16 kd protein in HIV-2 infected cells, confirming that this protein is the product of this gene. Full-length clones of HIV-2 containing these mutations are infectious in two permanent T lymphocytic cell lines and two monocytic cell lines. In contrast, we show that loss of VPX function results in a severe defect in the productive infection of human peripheral blood lymphocytes both in the amount of reverse transcriptase activity produced and in core protein expression. These findings suggest that the VPX protein plays an important role in the in vivo life cycle of the HIV-2/SIV viruses. Images PMID:2743977

  20. Human norovirus culture in B cells.

    PubMed

    Jones, Melissa K; Grau, Katrina R; Costantini, Veronica; Kolawole, Abimbola O; de Graaf, Miranda; Freiden, Pamela; Graves, Christina L; Koopmans, Marion; Wallet, Shannon M; Tibbetts, Scott A; Schultz-Cherry, Stacey; Wobus, Christiane E; Vinjé, Jan; Karst, Stephanie M

    2015-12-01

    Human noroviruses (HuNoVs) are a leading cause of foodborne disease and severe childhood diarrhea, and they cause a majority of the gastroenteritis outbreaks worldwide. However, the development of effective and long-lasting HuNoV vaccines and therapeutics has been greatly hindered by their uncultivability. We recently demonstrated that a HuNoV replicates in human B cells, and that commensal bacteria serve as a cofactor for this infection. In this protocol, we provide detailed methods for culturing the GII.4-Sydney HuNoV strain directly in human B cells, and in a coculture system in which the virus must cross a confluent epithelial barrier to access underlying B cells. We also describe methods for bacterial stimulation of HuNoV B cell infection and for measuring viral attachment to the surface of B cells. Finally, we highlight variables that contribute to the efficiency of viral replication in this system. Infection assays require 3 d and attachment assays require 3 h. Analysis of infection or attachment samples, including RNA extraction and RT-qPCR, requires ∼6 h.

  1. Ligand stimulation of ErbB4 and a constitutively-active ErbB4 mutant result in different biological responses in human pancreatic tumor cell lines

    SciTech Connect

    Mill, Christopher P.; Gettinger, Kathleen L.; Riese, David J.

    2011-02-15

    Pancreatic cancer is the fourth leading cause of cancer death in the United States. Indeed, it has been estimated that 37,000 Americans will die from this disease in 2010. Late diagnosis, chemoresistance, and radioresistance of these tumors are major reasons for poor patient outcome, spurring the search for pancreatic cancer early diagnostic and therapeutic targets. ErbB4 (HER4) is a member of the ErbB family of receptor tyrosine kinases (RTKs), a family that also includes the Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1), Neu/ErbB2/HER2, and ErbB3/HER3. These RTKs play central roles in many human malignancies by regulating cell proliferation, survival, differentiation, invasiveness, motility, and apoptosis. In this report we demonstrate that human pancreatic tumor cell lines exhibit minimal ErbB4 expression; in contrast, these cell lines exhibit varied and in some cases abundant expression and basal tyrosine phosphorylation of EGFR, ErbB2, and ErbB3. Expression of a constitutively-dimerized and -active ErbB4 mutant inhibits clonogenic proliferation of CaPan-1, HPAC, MIA PaCa-2, and PANC-1 pancreatic tumor cell lines. In contrast, expression of wild-type ErbB4 in pancreatic tumor cell lines potentiates stimulation of anchorage-independent colony formation by the ErbB4 ligand Neuregulin 1{beta}. These results illustrate the multiple roles that ErbB4 may be playing in pancreatic tumorigenesis and tumor progression.

  2. Receptor editing in a transgenic mouse model: site, efficiency, and role in B cell tolerance and antibody diversification.

    PubMed

    Pelanda, R; Schwers, S; Sonoda, E; Torres, R M; Nemazee, D; Rajewsky, K

    1997-12-01

    Mice carrying transgenic rearranged V region genes in their IgH and Igkappa loci to encode an autoreactive specificity direct the emerging autoreactive progenitors into a pre-B cell compartment, in which their receptors are edited by secondary Vkappa-Jkappa rearrangements and RS recombination. Editing is an efficient process, because the mutant mice generate normal numbers of B cells. In a similar nonautoreactive transgenic strain, neither a pre-B cell compartment nor receptor editing was seen. Thus, the pre-B cell compartment may have evolved to edit the receptors of autoreactive cells and later been generally exploited for efficient antibody diversification through the invention of the pre-B cell receptor, mimicking an autoreactive antibody to direct the bulk of the progenitors into that compartment.

  3. Igλ+ B cell development but not Igκ editing depends on NF-κB signals

    PubMed Central

    Derudder, Emmanuel; Cadera, Emily J; Vahl, J Christoph; Wang, Jing; Fox, Casey J.; Zha, Shan; van Loo, Geert; Pasparakis, Manolis; Schlissel, Mark S; Schmidt-Supprian, Marc; Rajewsky, Klaus

    2009-01-01

    By genetically ablating IκB kinase (IKK)-mediated NF-κB activation in the B cell lineage, and by analyzing a mouse mutant in which Igλ+ B cells are generated in the absence of rearrangements in Igk, we define two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-κB signaling. During the first phase, in which NF-κB signaling is dispensable, predominantly Igκ+ B cells are generated and undergo efficient receptor editing. In the second phase, predominantly Igλ+ B cells are generated, whose development is ontogenetically timed to occur after Igk rearrangements. This second phase of development is dependent on NF-κB signals, which can be substituted by transgenic expression of the pro-survival factor Bcl2. PMID:19412180

  4. Heat shock protein 60 activates B cells via the TLR4-MyD88 pathway.

    PubMed

    Cohen-Sfady, Michal; Nussbaum, Gabriel; Pevsner-Fischer, Meirav; Mor, Felix; Carmi, Pnina; Zanin-Zhorov, Alexandra; Lider, Ofer; Cohen, Irun R

    2005-09-15

    We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-gamma by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses. PMID:16148103

  5. Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

    PubMed

    Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

    2006-08-15

    The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

  6. Isolation and characterization of a novel B cell activation gene

    SciTech Connect

    Hong, J.X.; Wilson, G.L.; Fox, C.H.; Kehrl, J.H. )

    1993-05-01

    Using subtractive cDNA cloning, the authors have isolated a series of cDNA clones that are differentially expressed between B and T lymphocytes. Whereas some of the isolated cDNA are from known B cell-specific genes, many of them represent previously uncharacterized genes. One of these unknown genes was denoted as BL34. Northern blot analysis performed with the BL34 cDNA revealed a 1.6-kb mRNA transcript that was present at low levels in RNA extracted from resting B lymphocytes, but whose expression was markedly increased in RNA prepared from mitogen-activated B cells. Similarly, RNA prepared from several B cell lines treated with phorbol myristate acetate (PMA) contained high levels of BL34 mRNA. In contrast, RNA from purified T cells treated with phytohemagglutinin and PMA had undetectable amounts of BL34 mRNA. In addition, high levels of BL34 mRNA were detected in RNA purified from PBMC of a patient with B cell acute lymphocytic leukemia. Southern blot analysis of human DNA from various tissues and cells lines demonstrated that BL34 is a single-copy gene without evidence of rearrangement. Two full length BL34 cDNA were sequenced, and an open reading frame of 588 bp was identified that was predicted to encode for a 196 amino acid protein. Searches of several protein data bases failed to find any homologous proteins. To directly analyze the expression of BL34 mRNA in lymphoid tissues in situ, hybridization studies with human tonsil tissue sections were performed. BL34 mRNA was detected in a portion of the cells in the germinal center region and adjacent to the mantle region. Further characterization of the BL34 gene and its protein should lead to insights to its role in B cell function and the consequences of its over-expression in acute lymphocytic leukemia. 26 refs., 6 figs., 1 tab.

  7. Selective autoantibody production by Yaa+ B cells in autoimmune Yaa(+)- Yaa- bone marrow chimeric mice

    PubMed Central

    1991-01-01

    The accelerated autoimmune syndrome observed in BXSB/MpJ male mice is associated with the presence on the Y chromosome of an as yet unidentified mutant gene, designated Y chromosome-linked autoimmune acceleration (Yaa). To study the mechanisms by which the Yaa gene accelerates and/or induces the production of autoantibodies, we have developed double-congenic bone marrow chimeras containing B cells from autoimmune males carrying the Yaa gene, and from nonautoimmune male or female mice lacking it and differing by the Igh allotype. The analysis of the allotype of total immunoglobulins and anti-DNA antibodies in Yaa+ male-normal female (Yaa-) chimeric mice revealed that the selective activation of B cells from autoimmune Yaa+ male mice was responsible for the hypergammaglobulinemia and autoantibody production. This phenomenon was not due to an anti-HY interaction between female T helper cells and male B cells, because first, Yaa+ B cells were selectively stimulated to produce autoantibodies in Yaa+ male-Yaa- male chimeric mice; and second, normal male and female chimeras failed to develop an autoimmune syndrome. In addition, the fact that both B cell populations in Yaa(+)-Yaa- chimeras similarly responded to a foreign antigen, human IgG, argues against the possibility that the selective activation of Yaa+ B cells may be due to their hyper-responsiveness to T helper signals. We propose that a cognate interaction of T helper cells with Yaa+ B cells, because of possible T cell recognition of a Yaa-related molecule expressed on Yaa+ B cells, may be responsible for the acceleration and/or induction of autoantibodies in BXSB/MpJ mice. PMID:1834759

  8. Isogenic pairs of wild type and mutant induced pluripotent stem cell (iPSC) lines from Rett syndrome patients as in vitro disease model.

    PubMed

    Ananiev, Gene; Williams, Emily Cunningham; Li, Hongda; Chang, Qiang

    2011-01-01

    Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC), we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5-6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology. PMID:21966470

  9. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    PubMed Central

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher. PMID:8529116

  10. Effects of Lu-Do-Huang Extract (LDHE) on Apoptosis Induction in Human Hep3B Cells.

    PubMed

    Huang, Hui-Yu; Chen, Li-Han; Liu, Chen-Wei; Chien, Ting-Yi; Yu, Yu-Ping; Kao, Yu-Yu; Yang, Jo-Hsuan; Tsai, Ying-Chieh

    2015-06-30

    Lu-Do-Huang (Pracparatum mungo) is a fermented mung bean [corrected] (Vigna radiata) and has long been used as a traditional and functional food in Traditional Chinese Medicine, especially for treating a variety of liver disorders. The present study aimed to evaluate the apoptotic effects of Lu-Do-Huang ethanol extract (LDHE) on Hep3B cells, a human hepatoma cell line. A variety of cellular assays, flow cytometry and immunoblotting were used. Our results showed that LDHE significantly inhibited Hep3B cells growth. Additionally, the cell cycle assay showed that LDHE prevented Hep3B cell entry into S phase and led to an arrest of Hep3B cells in the G₀/G₁ phase. LDHE induced Hep3B cells to undergo apoptosis as determined through Hep3B cell morphology changes, increase of apoptotic bodies, apoptotic cells, DNA fragmentations and caspase activity. We further examined the protein expression of TRADD, FADD, and Bax to verify the possible apoptotic pathways. The results indicated that LDHE-induced apoptosis in Hep3B cells might be mediated [corrected] by an extrinsic signaling pathway leading to an induction of apoptosis in Hep3B cells. In conclusion, LDHE induced apoptosis and cell cycle arrest in Hep3B cells. Our data provide the evidences regarding the anti-hepatoma potential of LDHE in Hep3B cells.

  11. Human B cells have an active phagocytic capability and undergo immune activation upon phagocytosis of Mycobacterium tuberculosis.

    PubMed

    Zhu, Qi; Zhang, Min; Shi, Ming; Liu, Yang; Zhao, Qing; Wang, Wenjing; Zhang, Guangyun; Yang, Longxiu; Zhi, Jin; Zhang, Lin; Hu, Gengyao; Chen, Pin; Yang, Yining; Dai, Wen; Liu, Tingting; He, Ying; Feng, Guodong; Zhao, Gang

    2016-04-01

    The paradigm that B cells are nonphagocytic was taken for granted for a long time until phagocytic B cells were found in early vertebrate animals. Thereafter, limited evidence has shown that human B cells may also internalize bacteria. However, whether human B cells can actively phagocytose bacteria has been less extensively investigated; in particular, the mechanisms and significance of the phagocytosis require clarification. Here, we show that the human Raji B cell line can phagocytose both live and dead Mycobacterium tuberculosis (Mtb), and the phagocytosed Mtb in turn affects the immune functions of the B cells. After incubation of Raji cells with Mtb, our confocal microscopy, electron microscopy and flow cytometry data showed that Raji cells effectively engulfed Mtb as well as latex beads. The phagocytic rate was proportional to the incubation time and the amount of Mtb or beads added. Additionally, we found that normal human serum could enhance the ability of Raji cells to phagocytose Mtb, while heat-inactivated serum reversed this promoting effect. The phagocytic process of B cells could partially be inhibited by cytochalasin B, an actin inhibitor. Importantly, the phagocytosed Mtb could regulate B cell immune functions, such as stimulating IgM production and upregulating the expression of the antigen-presenting costimulatory molecules CD80 and CD86. Therefore, our results provide the first evidence that human B cells can phagocytose Mtb in an active manner that is independent of bacterial viability, and phagocytosed Mtb can in turn regulate the immune activation of B cells.

  12. B cell abnormalities in systemic lupus erythematosus

    PubMed Central

    2003-01-01

    Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154–CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater. PMID:15180894

  13. B cell abnormalities in systemic lupus erythematosus.

    PubMed

    Grammer, Amrie C; Lipsky, Peter E

    2003-01-01

    Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.

  14. Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques.

    PubMed

    Sadreddini, Sanam; Jadidi-Niaragh, Farhad; Younesi, Vahid; Pourlak, Tala; Afkham, Amir; Shokri, Fazel; Yousefi, Mehdi

    2016-07-01

    Several studies have been performed to develop effective neutralizing monoclonal antibodies. The Epstein-Barr virus (EBV) can efficiently immortalize B-cells to establish lymphoblastoid cell lines (LCL) and so it has been used extensively for transformation of B-cells to produce and secrete immunoglobulin. The present study addressed the effect of TLR7/8 agonist (R848), feeder cells layer and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) cell separation methods on the transformation efficiency of antibody-producing memory B-cells. For these studies, the antigen used for analyses of antibody formation was the tetanus neurotoxin (TeNT) derived from Clostridium tetani. The results here showed that employing an HFFF.PI6 feeder cell layer, R848 agonist and FACS-mediated purification of memory B-cells led to increased transformation efficiency. Altogether, the effects of the R848 and the feeder cells provided an efficient method for EBV transformation of human B-cells. Moreover, there was an advantage in using FACS sorting of B-cells over the MACS method in the context of EBV transformation and immortalization of precursors of antigen-specific B-cells.

  15. Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques.

    PubMed

    Sadreddini, Sanam; Jadidi-Niaragh, Farhad; Younesi, Vahid; Pourlak, Tala; Afkham, Amir; Shokri, Fazel; Yousefi, Mehdi

    2016-07-01

    Several studies have been performed to develop effective neutralizing monoclonal antibodies. The Epstein-Barr virus (EBV) can efficiently immortalize B-cells to establish lymphoblastoid cell lines (LCL) and so it has been used extensively for transformation of B-cells to produce and secrete immunoglobulin. The present study addressed the effect of TLR7/8 agonist (R848), feeder cells layer and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) cell separation methods on the transformation efficiency of antibody-producing memory B-cells. For these studies, the antigen used for analyses of antibody formation was the tetanus neurotoxin (TeNT) derived from Clostridium tetani. The results here showed that employing an HFFF.PI6 feeder cell layer, R848 agonist and FACS-mediated purification of memory B-cells led to increased transformation efficiency. Altogether, the effects of the R848 and the feeder cells provided an efficient method for EBV transformation of human B-cells. Moreover, there was an advantage in using FACS sorting of B-cells over the MACS method in the context of EBV transformation and immortalization of precursors of antigen-specific B-cells. PMID:27043044

  16. Shutoff of BZLF1 gene expression is necessary for immortalization of primary B cells by Epstein-Barr virus.

    PubMed

    Yu, Xianming; McCarthy, Patrick J; Wang, Zhenxun; Gorlen, Daniel A; Mertz, Janet E

    2012-08-01

    The BZLF1 gene controls the switch between latent and lytic infection by Epstein-Barr virus (EBV). We previously reported that both the ZV and ZIIR elements within the BZLF1 promoter, Zp, are potent transcription silencers within the context of an intact EBV genome. We report here identification of another sequence element, ZV', which synergized with ZV in repressing Zp via binding ZEB1 or ZEB2. We then determined the phenotype of a variant of EBV strain B95.8 in which the ZV, ZV', and ZIIR elements were concurrently mutated. HEK293 cell lines infected with this triple mutant (tmt) virus spontaneously synthesized 6- to 10-fold more viral BZLF1, BRLF1, BMRF1, and BLLF1 RNAs, 3- to 6-fold more viral Zta, Rta, and EAD proteins, 3- to 5-fold more viral DNA, and 7- to 9-fold more infectious virus than did 293 cell lines latently infected with either the ZV ZV' double mutant (dmt) or ZIIR mutant (mt) virus. While ZV ZV' ZIIR tmt EBV efficiently infected human primary blood B cells in vitro, it was highly defective in immortalizing them. Instead of the nearly complete silencing of BZLF1 gene expression that occurs within 4 days after primary infection with wild-type EBV, the ZV ZV' ZIIR tmt-infected cells continued to synthesize BZLF1 RNA, with 90% of them dying within 9 days postinfection. BL41 cells infected with this "superlytic" virus also exhibited increased synthesis of BZLF1 and BMRF1 RNAs. Thus, we conclude that the ZV, ZV', and ZIIR silencing elements act synergistically to repress transcription from Zp, thereby tightly controlling BZLF1 gene expression, which is crucial for establishing and maintaining EBV latency.

  17. Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma

    PubMed Central

    Sarkar, Aloke; Balakrishnan, Kumudha; Chen, Jefferson; Patel, Viralkumar; Neelapu, Sattva S.; McMurray, John S.; Gandhi, Varsha

    2016-01-01

    The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3–7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27–43%), but not in DKO MEFs or MEFs expressing respective Casp3–7 catalytic mutants (12–13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30–73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18–36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation. PMID:26658105

  18. Antigen-affinity controls pre-germinal centser B cell selection by promoting Mcl-1 induction through BAFF receptor signaling

    PubMed Central

    Wensveen, Felix M.; Slinger, Erik; van Attekum, Martijn HA; Brink, Robert; Eldering, Eric

    2016-01-01

    Upon antigen encounter, the responsive B cell pool undergoes stringent selection which eliminates cells with low B cell receptor (BCR) affinity. Already before formation of the germinal center, activated B cells of low-affinity are negatively selected in a process that is molecularly not well understood. In this study, we investigated the mechanism behind pre-GC affinity-mediated B cell selection. We applied affinity mutants of HEL antigen and found that rapidly after activation B cells become highly dependent on the cytokine BAFF. Moreover, expression of BAFF receptor CD268 is regulated in a BCR-affinity dependent fashion. High affinity responses via BAFF correlated with PI3K activation, which controlled expression of the pro-survival protein Mcl-1, and thereby increased survival. In the presence of excess BAFF, or in absence of the Mcl-1 antagonist Noxa, more low-affinity B cells survived the first two days after antigen encounter. This resulted in increased numbers of antigen-specific B cells of low affinity upon immunization and reduced the overall affinity of cells that contributed to the germinal center reaction. Our findings elucidate a crucial molecular pathway of B cell selection in the earliest phases of activation by identifying a novel link between BCR affinity and BAFF-R signaling towards Mcl-1. PMID:27762293

  19. Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma.

    PubMed

    Jardin, Fabrice; Pujals, Anais; Pelletier, Laura; Bohers, Elodie; Camus, Vincent; Mareschal, Sylvain; Dubois, Sydney; Sola, Brigitte; Ochmann, Marlène; Lemonnier, François; Viailly, Pierre-Julien; Bertrand, Philippe; Maingonnat, Catherine; Traverse-Glehen, Alexandra; Gaulard, Philippe; Damotte, Diane; Delarue, Richard; Haioun, Corinne; Argueta, Christian; Landesman, Yosef; Salles, Gilles; Jais, Jean-Philippe; Figeac, Martin; Copie-Bergman, Christiane; Molina, Thierry Jo; Picquenot, Jean Michel; Cornic, Marie; Fest, Thierry; Milpied, Noel; Lemasle, Emilie; Stamatoullas, Aspasia; Moeller, Peter; Dyer, Martin J S; Sundstrom, Christer; Bastard, Christian; Tilly, Hervé; Leroy, Karen

    2016-09-01

    Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein. Am. J. Hematol. 91:923-930, 2016. © 2016 Wiley Periodicals, Inc. PMID:27312795

  20. Oxidative damage-induced PCNA complex formation is efficient in xeroderma pigmentosum group A but reduced in Cockayne syndrome group B cells.

    PubMed

    Balajee, A S; Dianova, I; Bohr, V A

    1999-11-15

    Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases delta and epsilon, is essential for both DNA replication and repair. PCNA is required in the resynthesis step of nucleotide excision repair (NER). After UV irradiation, PCNA translocates into an insoluble protein complex, most likely associated with the nuclear matrix. It has not previously been investigated in vivo whether PCNA complex formation also takes place after oxidative stress. In this study, we have examined the involvement of PCNA in the repair of oxidative DNA damage. PCNA complex formation was studied in normal human cells after treatment with hydrogen peroxide, which generates a variety of oxidative DNA lesions. PCNA was detected by two assays, immunofluorescence and western blot analyses. We observed that PCNA redistributes from a soluble to a DNA-bound form during the repair of oxidative DNA damage. PCNA complex formation was analyzed in two human natural mutant cell lines defective in DNA repair: xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). XP-A cells are defective in overall genome NER while CS-B cells are defective only in the preferential repair of active genes. Immunofluorescent detection of PCNA complex formation was similar in normal and XP-A cells, but was reduced in CS-B cells. Consistent with this observation, western blot analysis in CS-B cells showed a reduction in the ratio of PCNA relocated as compared to normal and XP-A cells. The efficient PCNA complex formation observed in XP-A cells following oxidative damage suggests that formation of PCNA-dependent repair foci may not require the XPA gene product. The reduced PCNA complex formation observed in CS-B cells suggests that these cells are defective in the processing of oxidative DNA damage. PMID:10536158

  1. Identification of Highly Methylated Genes across Various Types of B-Cell Non-Hodgkin Lymphoma

    PubMed Central

    Bethge, Nicole; Honne, Hilde; Hilden, Vera; Trøen, Gunhild; Eknæs, Mette; Liestøl, Knut; Holte, Harald; Delabie, Jan; Smeland, Erlend B.; Lind, Guro E.

    2013-01-01

    Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients. PMID:24260260

  2. Rituximab does not reset defective early B cell tolerance checkpoints.

    PubMed

    Chamberlain, Nicolas; Massad, Christopher; Oe, Tyler; Cantaert, Tineke; Herold, Kevan C; Meffre, Eric

    2016-01-01

    Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve β cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti-B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti-B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti-B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti-B cell therapy. PMID:26642366

  3. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    PubMed Central

    Liao, Wei; Sharma, Sanjai

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells. PMID:27458535

  4. B Cell Adaptor Containing Src Homology 2 Domain (Bash) Links B Cell Receptor Signaling to the Activation of Hematopoietic Progenitor Kinase 1

    PubMed Central

    Tsuji, Sachiyo; Okamoto, Mariko; Yamada, Koichi; Okamoto, Noriaki; Goitsuka, Ryo; Arnold, Rudiger; Kiefer, Friedemann; Kitamura, Daisuke

    2001-01-01

    The B cell adaptor containing src homology 2 domain (BASH; also termed BLNK or SLP-65), is crucial for B cell antigen receptor (BCR)-mediated activation, proliferation, and differentiation of B cells. BCR-mediated tyrosine-phosphorylation of BASH creates binding sites for signaling effectors such as phospholipase Cγ (PLCγ)2 and Vav, while the function of its COOH-terminal src homology 2 domain is unknown. We have now identified hematopoietic progenitor kinase (HPK)1, a STE20-related serine/threonine kinase, as a protein that inducibly interacts with the BASH SH2 domain. BCR ligation induced rapid tyrosine-phosphorylation of HPK1 mainly by Syk and Lyn, resulting in its association with BASH and catalytic activation. BCR-mediated activation of HPK1 was impaired in Syk- or BASH-deficient B cells. The functional SH2 domain of BASH and Tyr-379 within HPK1 which we identified as a Syk-phosphorylation site were both necessary for interaction of both proteins and efficient HPK1 activation after BCR stimulation. Furthermore, HPK1 augmented, whereas its kinase-dead mutant inhibited IκB kinase β (IKKβ) activation by BCR engagement. These results reveal a novel BCR signaling pathway leading to the activation of HPK1 and subsequently IKKβ, in which BASH recruits tyrosine-phosphorylated HPK1 into the BCR signaling complex. PMID:11514608

  5. IRF8 regulates B-cell lineage specification, commitment, and differentiation

    PubMed Central

    Lee, Chang Hoon; Qi, Chenfeng; Tailor, Prafullakumar; Feng, Jianxun; Abbasi, Sadia; Atsumi, Toru

    2008-01-01

    PU.1, IKAROS, E2A, EBF, and PAX5 comprise a transcriptional network that orchestrates B-cell lineage specification, commitment, and differentiation. Here we identify interferon regulatory factor 8 (IRF8) as another component of this complex, and show that it also modulates lineage choice by hematopoietic stem cells (HSCs). IRF8 binds directly to an IRF8/Ets consensus sequence located in promoter regions of Sfpi1 and Ebf1, which encode PU.1 and EBF, respectively, and is associated with transcriptional repression of Sfpi1 and transcriptional activation of Ebf1. Bone marrows of IRF8 knockout mice (IRF8−/−) had significantly reduced numbers of pre-pro-B cells and increased numbers of myeloid cells. Although HSCs of IRF8−/− mice failed to differentiate to B220+ B-lineage cells in vitro, the defect could be rescued by transfecting HSCs with wild-type but not with a signaling-deficient IRF8 mutant. In contrast, overexpression of IRF8 in HSC-differentiated progenitor cells resulted in growth inhibition and apoptosis. We also found that IRF8 was expressed at higher levels in pre-pro-B cells than more mature B cells in wild-type mice. Together, these results indicate that IRF8 modulates lineage choice by HSCs and is part of the transcriptional network governing B-cell lineage specification, commitment, and differentiation. PMID:18799728

  6. Advances in Human B Cell Phenotypic Profiling

    PubMed Central

    Kaminski, Denise A.; Wei, Chungwen; Qian, Yu; Rosenberg, Alexander F.; Sanz, Ignacio

    2012-01-01

    To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (“Big Biology”), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort. PMID:23087687

  7. B Cell Lymphoma mimicking Rheumatoid Arthritis.

    PubMed

    Cosatti, M A; Pisoni, C N; Altuve, J L; Lorente, C

    2016-01-01

    Non Hodking´s lymphoma (NHL) may involve bones but synovial involvement is uncommon. We describe a patient who presented with polyarthritis, sicca symptoms and rash suggestive of rheumatoid arthritis. An atypical skin rash prompted skin and synovial biopsies. A diagnosis of synovial and skin malignant large B-cell lymphoma anaplastic subtype was performed. Chemotherapy with dexamethasone, vincristine and rituximab was started. Following treatment the patient had complete resolution of cutaneous and articular lymphoma manifestations. PMID:27419896

  8. Meningeal Infiltration of the Spinal Cord by Non-Classically Activated B Cells is Associated with Chronic Disease Course in a Spontaneous B Cell-Dependent Model of CNS Autoimmune Disease

    PubMed Central

    Dang, Amy K.; Tesfagiorgis, Yodit; Jain, Rajiv W.; Craig, Heather C.; Kerfoot, Steven M.

    2015-01-01

    We characterized B cell infiltration of the spinal cord in a B cell-dependent spontaneous model of central nervous system (CNS) autoimmunity that develops in a proportion of mice with mutant T and B cell receptors specific for myelin oligodendrocyte glycoprotein. We found that, while males are more likely to develop disease, females are more likely to have a chronic rather than monophasic disease course. B cell infiltration of the spinal cord was investigated by histology and FACs. CD4+ T cell infiltration was pervasive throughout the white and in some cases gray matter. B cells were almost exclusively restricted to the meninges, often in clusters reminiscent of those described in human multiple sclerosis. These clusters were typically found adjacent to white matter lesions and their presence was associated with a chronic disease course. Extensive investigation of these clusters by histology did not identify features of lymphoid follicles, including organization of T and B cells into separate zones, CD35+ follicular dendritic cells, or germinal centers. The majority of cluster B cells were IgD+ with little evidence of class switch. Consistent with this, B cells isolated from the spinal cord were of the naïve/memory CD38hi CD95lo phenotype. Nevertheless, they were CD62Llo and CD80hi compared to lymph node B cells suggesting that they were at least partly activated and primed to present antigen. Therefore, if meningeal B cells contribute to CNS pathology in autoimmunity, follicular differentiation is not necessary for the pathogenic mechanism. PMID:26441975

  9. Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma

    PubMed Central

    Hailfinger, Stephan; Lenz, Georg; Ngo, Vu; Posvitz-Fejfar, Anita; Rebeaud, Fabien; Guzzardi, Montserrat; Penas, Eva-Maria Murga; Dierlamm, Judith; Chan, Wing C.; Staudt, Louis M.; Thome, Margot

    2009-01-01

    A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy. PMID:19897720

  10. New insights in the regulation of human B cell differentiation

    PubMed Central

    Schmidlin, Heike; Diehl, Sean A.; Blom, Bianca

    2009-01-01

    B lymphocytes provide the cellular basis of the humoral immune response. All stages of this process, from B cell activation to formation of germinal centers and differentiation into memory B cells or plasma cells, are influenced by extrinsic signals and controlled by transcriptional regulation. Compared to naïve B cells, memory B cells display a distinct expression profile, which allows for their rapid secondary responses. Indisputably, many B cell malignancies result from aberrations in the circuitry controlling B cell function, particularly during the GC reaction. Here we review new insights into memory B cell subtypes, recent literature on transcription factors regulating human B cell differentiation, and further evidence for B cell lymphomagenesis emanating from errors during the GC cell reactions. PMID:19447676

  11. Germinal center B cells and mixed leukocyte reactions

    SciTech Connect

    Monfalcone, A.P.; Kosco, M.H.; Szakal, A.K.; Tew, J.G. )

    1989-09-01

    The present study was undertaken to determine if germinal center (GC) B cells are sufficiently activated to stimulate mixed leukocyte reactions (MLR). Percoll density fractionation and a panning technique with peanut agglutinin (PNA) were used to isolate GC B cells from the lymph nodes of immune mice. The GC B cells were treated with mitomycin C or irradiation and used to stimulate allogeneic or syngeneic splenic T cells in the MLR. Controls included high-density (HD) B cells prepared from spleens of the same mice and HD B cells activated with lipopolysaccharide (LPS) and dextran sulfate. GC B cells bound high amount sof PNA (i.e., PNAhi). Similarly, the LPS-dextran sulfate-activated B cells were PNAhi. Treatment with neuraminidase rendered the PNAlo HD B cells PNAhi. GC B cells and the LPS-dextran sulfate-activated HD B cells stimulated a potent MLR, while the untreated HD B cells did not. However, following neuraminidase treatment, the resulting PNAhi HD B cell population was able to induce an MLR. The PNA marker appeared to be an indicator of stimulatory activity, but incubating the cells with PNA to bind the cell surface ligand did not interfere with the MLR. GC B cells were also capable of stimulating a syngeneic MLR in most experiments although this was not consistently obtained. It appears that germinal centers represent a unique in vivo microenvironment that provides the necessary signals for B cells to become highly effective antigen-presenting cells.

  12. Epigenetics and B-cell Lymphoma

    PubMed Central

    Shaknovich, Rita; Melnick, Ari

    2011-01-01

    STRUCTURED ABSTRACT Purpose of review It has only recently become apparent that mutations in epigenetic mechanisms and perturbation of epigenomic patterning are frequent events in B-cell lymphomas. The purpose of this review is to highlight these new findings and provide a conceptual framework for understanding how epigenetic modifications might contribute to lymphomagenesis. Recent findings Somatic mutations affecting histone methyltransferases such as EZH2 and MLL2, histone demethylases including UTX and JMJD2C and histone acetyltransferases including CBP and p300 are recurrent and common in lymphomas. These mutations result in disruption of chromatin structure and functions of other proteins, ultimately causing aberrant transcriptional programming affecting multiple gene networks. Widespread perturbation of cytosine methylation patterning now appears to be a hallmark of B-cell lymphomas and occurs in specific patterns that can distinguish disease subtypes. Therapeutic targeting strategies can overcome abnormal epigenetic mechanisms and potently kill lymphoma cells. Summary Newly discovered epigenetic lesions may provide critical insights into the genesis of B-cell lymphomas but further studies are required to understand how they affect biological mechanism. Epigenetic lesions offer tremendous opportunities for the development of improved biomarkers and treatments. PMID:21577103

  13. CD4 T cell activation by B cells in human Leishmania (Viannia) infection

    PubMed Central

    2014-01-01

    Background An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated. Methods CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC. Results Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells. Conclusions The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to

  14. Loss of SIRT3 Provides Growth Advantage for B Cell Malignancies.

    PubMed

    Yu, Wei; Denu, Ryan A; Krautkramer, Kimberly A; Grindle, Kreg M; Yang, David T; Asimakopoulos, Fotis; Hematti, Peiman; Denu, John M

    2016-02-12

    B cell malignancies comprise a diverse group of cancers that proliferate in lymph nodes, bone marrow, and peripheral blood. SIRT3 (sirtuin 3) is the major deacetylase within the mitochondrial matrix that promotes aerobic metabolism and controls reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). There is controversy as to whether SIRT3 acts as an oncogene or a tumor suppressor, and here we investigated its role in B cell malignancies. In mantle cell lymphoma patient samples, we found that lower SIRT3 protein expression was associated with worse overall survival. Further, SIRT3 protein expression was reduced in chronic lymphocytic leukemia primary samples and malignant B cell lines compared to primary B cells from healthy donors. This lower level of expression correlated with hyperacetylation of IDH2 and SOD2 mitochondrial proteins, lowered enzymatic activities, and higher ROS levels. Overexpression of SIRT3 decreased proliferation and diminished the Warburg-like phenotype in SIRT3-deficient cell lines, and this effect is largely dependent on deacetylation of IDH2 and SOD2. Lastly, depletion of SIRT3 from malignant B cell lines resulted in greater susceptibility to treatment with an ROS scavenger but did not result in greater sensitivity to inhibition of the hypoxia-inducible factor-1α pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but hypoxia-inducible factor-1α-independent mechanisms. Our study suggests that SIRT3 acts as a tumor suppressor in B cell malignancies, and activating the SIRT3 pathway might represent a novel therapeutic approach for treating B cell malignancies. PMID:26631723

  15. Class-switched B cells display response to therapeutic B-cell depletion in rheumatoid arthritis

    PubMed Central

    Möller, Burkhard; Aeberli, Daniel; Eggli, Stefan; Fuhrer, Martin; Vajtai, Istvan; Vögelin, Esther; Ziswiler, Hans-Rudolf; Dahinden, Clemens A; Villiger, Peter M

    2009-01-01

    Introduction Reconstitution of peripheral blood (PB) B cells after therapeutic depletion with the chimeric anti-CD20 antibody rituximab (RTX) mimics lymphatic ontogeny. In this situation, the repletion kinetics and migratory properties of distinct developmental B-cell stages and their correlation to disease activity might facilitate our understanding of innate and adaptive B-cell functions in rheumatoid arthritis (RA). Methods Thirty-five 'RTX-naïve' RA patients with active arthritis were treated after failure of tumour necrosis factor blockade in an open-label study with two infusions of 1,000 mg RTX. Prednisone dose was tapered according to clinical improvement from a median of 10 mg at baseline to 5 mg at 9 and 12 months. Conventional disease-modifying antirheumatic drugs were kept stable. Subsets of CD19+ B cells were assessed by flow cytometry according to their IgD and CD27 surface expression. Their absolute number and relative frequency in PB were followed every 3 months and were determined in parallel in synovial tissue (n = 3) or synovial fluid (n = 3) in the case of florid arthritis. Results Six of 35 patients fulfilled the European League Against Rheumatism criteria for moderate clinical response, and 19 others for good clinical response. All PB B-cell fractions decreased significantly in number (P < 0.001) after the first infusion. Disease activity developed independently of the total B-cell number. B-cell repopulation was dominated in quantity by CD27-IgD+ 'naïve' B cells. The low number of CD27+IgD- class-switched memory B cells (MemB) in the blood, together with sustained reduction of rheumatoid factor serum concentrations, correlated with good clinical response. Class-switched MemB were found accumulated in flaring joints. Conclusions The present data support the hypothesis that control of adaptive immune processes involving germinal centre-derived, antigen, and T-cell-dependently matured B cells is essential for successful RTX treatment. PMID

  16. Regulation of B cell linker protein transcription by PU.1 and Spi-B in murine B cell acute lymphoblastic leukemia.

    PubMed

    Xu, Li S; Sokalski, Kristen M; Hotke, Kathryn; Christie, Darah A; Zarnett, Oren; Piskorz, Jan; Thillainadesan, Gobi; Torchia, Joseph; DeKoter, Rodney P

    2012-10-01

    B cell acute lymphoblastic leukemia (B-ALL) is frequently associated with mutations or chromosomal translocations of genes encoding transcription factors. Conditional deletion of genes encoding the E26-transformation-specific transcription factors, PU.1 and Spi-B, in B cells (ΔPB mice) leads to B-ALL in mice at 100% incidence rate and with a median survival of 21 wk. We hypothesized that PU.1 and Spi-B may redundantly activate transcription of genes encoding tumor suppressors in the B cell lineage. Characterization of aging ΔPB mice showed that leukemia cells expressing IL-7R were found in enlarged thymuses. IL-7R-expressing B-ALL cells grew in culture in response to IL-7 and could be maintained as cell lines. Cultured ΔPB cells expressed reduced levels of B cell linker protein (BLNK), a known tumor suppressor gene, compared with controls. The Blnk promoter contained a predicted PU.1 and/or Spi-B binding site that was required for promoter activity and occupied by PU.1 and/or Spi-B as determined by chromatin immunoprecipitation. Restoration of BLNK expression in cultured ΔPB cells opposed IL-7-dependent proliferation and induced early apoptosis. We conclude that the tumor suppressor BLNK is a target of transcriptional activation by PU.1 and Spi-B in the B cell lineage.

  17. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2

    PubMed Central

    1996-01-01

    Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions. PMID:8691147

  18. EAF2 mediates germinal centre B-cell apoptosis to suppress excessive immune responses and prevent autoimmunity

    PubMed Central

    Li, Yingqian; Takahashi, Yoshimasa; Fujii, Shin-ichiro; Zhou, Yang; Hong, Rongjian; Suzuki, Akari; Tsubata, Takeshi; Hase, Koji; Wang, Ji-Yang

    2016-01-01

    Regulated apoptosis of germinal centre (GC) B cells is critical for normal humoral immune responses. ELL-associated factor 2 (EAF2) regulates transcription elongation and has been shown to be an androgen-responsive potential tumour suppressor in prostate by inducing apoptosis. Here we show that EAF2 is selectively upregulated in GC B cells among various immune cell types and promotes apoptosis of GC B cells both in vitro and in vivo. EAF2 deficiency results in enlarged GCs and elevated antibody production during a T-dependent immune response. After immunization with type II collagen, mice lacking EAF2 produce high levels of collagen-specific autoantibodies and rapidly develop severe arthritis. Moreover, the mutant mice spontaneously produce anti-dsDNA, rheumatoid factor and anti-nuclear antibodies as they age. These results demonstrate that EAF2-mediated apoptosis in GC B cells limits excessive humoral immune responses and is important for maintaining self-tolerance. PMID:26935903

  19. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells

    PubMed Central

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  20. A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

    PubMed

    Lorenzo, Daniela; Duarte, Alejandra; Mundiñano, Juliana; Berguer, Paula; Nepomnaschy, Irene; Piazzon, Isabel

    2016-01-01

    B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed. PMID:27603942

  1. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

    PubMed Central

    Villar, Rina F.; Patel, Jinal; Weaver, Grant C.; Kanekiyo, Masaru; Wheatley, Adam K.; Yassine, Hadi M.; Costello, Catherine E.; Chandler, Kevin B.; McTamney, Patrick. M.; Nabel, Gary J.; McDermott, Adrian B.; Mascola, John R.; Carr, Steven A.; Lingwood, Daniel

    2016-01-01

    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire could be triggered by both complementarity to influenza HA and a separate mode of signaling that relied on multivalent ligation of BCR sialyl-oligosaccharide. The latter suggested a new mechanism for priming naïve B cell responses and manifested as the induction of SA-dependent pan-activation by peripheral blood B cells. BCR crosslinking in the absence of complementarity is a superantigen effect induced by some microbial products to subvert production of antigen-specific immune responses. B cell superantigen activity through affinity for BCR carbohydrate is discussed. PMID:27796362

  2. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling*♦

    PubMed Central

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu.; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I.; Nienhaus, G. Ulrich; Gierschik, Peter

    2015-01-01

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca2+. The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca2+ and regulation of Ca2+-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca2+ release from intracellular stores; (iii) Ca2+ entry from the extracellular compartment; and (iv) nuclear translocation of the Ca2+-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca2+ signaling. PMID:25903139

  3. Interleukin-induced increase in Ia expression by normal mouse B cells

    PubMed Central

    1984-01-01

    The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen- presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology. PMID:6432933

  4. Diffuse Large B-Cell Lymphoma Classification System That Associates Normal B-Cell Subset Phenotypes With Prognosis

    PubMed Central

    Dybkær, Karen; Bøgsted, Martin; Falgreen, Steffen; Bødker, Julie S.; Kjeldsen, Malene K.; Schmitz, Alexander; Bilgrau, Anders E.; Xu-Monette, Zijun Y.; Li, Ling; Bergkvist, Kim S.; Laursen, Maria B.; Rodrigo-Domingo, Maria; Marques, Sara C.; Rasmussen, Sophie B.; Nyegaard, Mette; Gaihede, Michael; Møller, Michael B.; Samworth, Richard J.; Shah, Rajen D.; Johansen, Preben; El-Galaly, Tarec C.; Young, Ken H.; Johnsen, Hans E.

    2015-01-01

    Purpose Current diagnostic tests for diffuse large B-cell lymphoma use the updated WHO criteria based on biologic, morphologic, and clinical heterogeneity. We propose a refined classification system based on subset-specific B-cell–associated gene signatures (BAGS) in the normal B-cell hierarchy, hypothesizing that it can provide new biologic insight and diagnostic and prognostic value. Patients and Methods We combined fluorescence-activated cell sorting, gene expression profiling, and statistical modeling to generate BAGS for naive, centrocyte, centroblast, memory, and plasmablast B cells from normal human tonsils. The impact of BAGS-assigned subtyping was analyzed using five clinical cohorts (treated with cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP], n = 270; treated with rituximab plus CHOP [R-CHOP], n = 869) gathered across geographic regions, time eras, and sampling methods. The analysis estimated subtype frequencies and drug-specific resistance and included a prognostic meta-analysis of patients treated with first-line R-CHOP therapy. Results Similar BAGS subtype frequencies were assigned across 1,139 samples from five different cohorts. Among R-CHOP–treated patients, BAGS assignment was significantly associated with overall survival and progression-free survival within the germinal center B-cell–like subclass; the centrocyte subtype had a superior prognosis compared with the centroblast subtype. In agreement with the observed therapeutic outcome, centrocyte subtypes were estimated as being less resistant than the centroblast subtype to doxorubicin and vincristine. The centroblast subtype had a complex genotype, whereas the centrocyte subtype had high TP53 mutation and insertion/deletion frequencies and expressed LMO2, CD58, and stromal-1–signature and major histocompatibility complex class II–signature genes, which are known to have a positive impact on prognosis. Conclusion Further development of a diagnostic platform using

  5. B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

    PubMed

    Knittel, Gero; Liedgens, Paul; Korovkina, Darya; Seeger, Jens M; Al-Baldawi, Yussor; Al-Maarri, Mona; Fritz, Christian; Vlantis, Katerina; Bezhanova, Svetlana; Scheel, Andreas H; Wolz, Olaf-Oliver; Reimann, Maurice; Möller, Peter; López, Cristina; Schlesner, Matthias; Lohneis, Philipp; Weber, Alexander N R; Trümper, Lorenz; Staudt, Louis M; Ortmann, Monika; Pasparakis, Manolis; Siebert, Reiner; Schmitt, Clemens A; Klatt, Andreas R; Wunderlich, F Thomas; Schäfer, Stephan C; Persigehl, Thorsten; Montesinos-Rongen, Manuel; Odenthal, Margarete; Büttner, Reinhard; Frenzel, Lukas P; Kashkar, Hamid; Reinhardt, H Christian

    2016-06-01

    The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL. PMID:27048211

  6. Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells

    PubMed Central

    Korniotis, Sarantis; Gras, Christophe; Letscher, Hélène; Montandon, Ruddy; Mégret, Jérôme; Siegert, Stefanie; Ezine, Sophie; Fallon, Padraic G.; Luther, Sanjiv A.; Fillatreau, Simon; Zavala, Flora

    2016-01-01

    The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. PMID:27396388

  7. Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase.

    PubMed

    Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

    2016-01-01

    The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT. PMID:26496927

  8. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

    PubMed

    Frenkel, Deborah; Zhang, Fengqiu; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Radwanska, Magdalena; Magez, Stefan; Black, Samuel J

    2016-07-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  9. Somatostatin Improved B Cells Mature in Macaques during Intestinal Ischemia-Reperfusion

    PubMed Central

    Liu, Ling; Tan, Qinghua; Hu, Bin; Wu, Hao; Wang, Chunhui; Liu, Rui; Tang, Chengwei

    2015-01-01

    Aims Intestinal ischemia-reperfusion has been taken as an important pathophysiological process for multiple organ dysfunctions in critical patients. Recent studies reported that dual expression programs of the B cells receptors and Toll-like receptors on B-lymphocytes permit these ubiquitous cells to integrate both adaptive and innate immune functions. Our previous studies found that somatostatin inhibited the intestinal inflammatory injury after ischemia-reperfusion in macaques. However, the changes of B cells and the effects of somatostatin on B cells after intestinal ischemia-reperfusion were unclear. Methods 15 macaques were divided into control, intestinal ischemia-reperfusion and somatostatin pretreatment groups. Immunohistochemistry was performed to identify the distributions of adaptive and innate immunity markers in the iliac mucosa. Hmy2.cir B lymphoblastoid cell line was cultured in vitro study. Enzyme-linked immunosorbent assay was used to measure IgM, IL-6 and SIgA, and the expressions of B cells transcription factors, PAX-5 and BLIMP-1, were detected by Western blotting. Results B2 lymphocytes in normal Peyer’s patches were presented the phenotype of PAX-5+CD20+CD5-. Ischemia-reperfusion increased the numbers and sizes of Peyer’s patches but with PAX-5+CD20-CD5- B cells, an unmatured set of B cells. Somatostatin partly kept the phenotype of mature B cells during ischemia-reperfusion. The innate immunity of B cells was inhibited whereas the adaptive immunity was increased in the intestinal mucosa in the somatostatin group, compared to the ischemia-reperfusion group. In vitro, somatostatin significantly inhibited IL-6 and promoted IgM by increasing the expression of both PAX-5 and BLIMP-1 in the proinflammatory condition. Conclusion Intestinal ischemia-reperfusion resulted in the proliferation of unmatured B cells which were involved in the augmentation of innate immunity. Somatostatin, with a bi-directional regulation function on innate as well as

  10. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells

    PubMed Central

    Frenkel, Deborah; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Magez, Stefan; Black, Samuel J.

    2016-01-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  11. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

    PubMed

    Frenkel, Deborah; Zhang, Fengqiu; Guirnalda, Patrick; Haynes, Carole; Bockstal, Viki; Radwanska, Magdalena; Magez, Stefan; Black, Samuel J

    2016-07-01

    After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice

  12. Analysis of FOXO1 mutations in diffuse large B-cell lymphoma | Office of Cancer Genomics

    Cancer.gov

    Abstract: Diffuse large B-cell lymphoma (DLBCL) accounts for 30% to 40% of newly diagnosed lymphomas and has an overall cure rate of approximately 60%. Previously, we observed FOXO1 mutations in non-Hodgkin lymphoma patient samples. To explore the effects of FOXO1 mutations, we assessed FOXO1 status in 279 DLBCL patient samples and 22 DLBCL-derived cell lines.

  13. Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

    PubMed

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M; Ng, Yen-Shing; Massad, Christopher; Oe, Tyler; Wu, Renee; Lavoie, Aubert; Walter, Jolan E; Notarangelo, Luigi D; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2015-11-17

    Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

  14. Primary Mediastinal B-Cell Lymphoma

    PubMed Central

    Pileri, Stefano A.; Gaidano, Gianluca; Zinzani, Pier Luigi; Falini, Brunangelo; Gaulard, Philippe; Zucca, Emanuele; Pieri, Federica; Berra, Eva; Sabattini, Elena; Ascani, Stefano; Piccioli, Milena; Johnson, Peter W. M.; Giardini, Roberto; Pescarmona, Edoardo; Novero, Domenico; Piccaluga, Pier Paolo; Marafioti, Teresa; Alonso, Miguel A.; Cavalli, Franco

    2003-01-01

    Although primary mediastinal (thymic) large B-cell lymphoma has been primarily studied, its precise phenotype, molecular characteristics, and histogenesis are still a matter of debate. The International Extranodal Lymphoma Study Group collected 137 such cases for extensive pathological review. Histologically, the lymphomatous growth was predominantly diffuse with fibrosis that induced compartmentalized cell aggregation. It consisted of large cells with varying degrees of nuclear polymorphism and clear to basophilic cytoplasm. On immunohistochemistry, the following phenotype was observed: CD45+, CD20+, CD79a+, PAX5/BSAP+, BOB.1+, Oct-2+, PU.1+, Bcl-2+, CD30+, HLA-DR+, MAL protein+/−, Bcl-6+/−, MUM1/IRF4+/−, CD10−/+, CD21−, CD15−, CD138−, CD68−, and CD3−. Immunoglobulins were negative both at immunohistochemistry and in situ hybridization. Molecular analysis, performed in 45 cases, showed novel findings. More than half of the cases displayed BCL-6 gene mutations, which usually occurred along with functioning somatic IgVH gene mutations and Bcl-6 and/or MUM1/IRF4 expression. The present study supports the concept that a sizable fraction of cases of this lymphoma are from activated germinal center or postgerminal center cells. However, it differs from other aggressive B-cell lymphomas in that it shows defective immunoglobulin production despite the expression of OCT-2, BOB.1, and PU.1 transcription factors and the lack of IgVH gene crippling mutations. PMID:12507907

  15. B cell biology: implications for treatment of systemic lupus erythematosus.

    PubMed

    Anolik, J H

    2013-04-01

    B cells are critical players in the orchestration of properly regulated immune responses, normally providing protective immunity without autoimmunity. Balance in the B cell compartment is achieved through the finely regulated participation of multiple B cell populations with different antibody-dependent and independent functions. Both types of functions allow B cells to modulate other components of the innate and adaptive immune system. Autoantibody-independent B cell functions include antigen presentation, T cell activation and polarization, and dendritic cell modulation. Several of these functions are mediated by the ability of B cells to produce immunoregulatory cytokines and chemokines and by their critical contribution to lymphoid tissue development and organization including the development of ectopic tertiary lymphoid tissue. Additionally, the functional versatility of B cells enables them to play either protective or pathogenic roles in autoimmunity. In turn, B cell dysfunction has been critically implicated in the pathophysiology of systemic lupus erythematosus (SLE), a complex disease characterized by the production of autoantibodies and heterogeneous clinical involvement. Thus, the breakdown of B cell tolerance is a defining and early event in the disease process and may occur by multiple pathways, including alterations in factors that affect B cell activation thresholds, B cell longevity, and apoptotic cell processing. Once tolerance is broken, autoantibodies contribute to autoimmunity by multiple mechanisms including immune-complex mediated Type III hypersensitivity reactions, type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines including IFNα, TNF and IL-1. The complexity of B cell functions has been highlighted by the variable success of B cell-targeted therapies in multiple autoimmune diseases, including those conventionally viewed as T cell-mediated conditions. Given the widespread

  16. Proteomics Based Identification of Proteins with Deregulated Expression in B Cell Lymphomas.

    PubMed

    Wu, Rui; Nijland, Marcel; Rutgers, Bea; Veenstra, Rianne; Langendonk, Myra; van der Meeren, Lotte E; Kluin, Philip M; Li, Guanwu; Diepstra, Arjan; Chiu, Jen-Fu; van den Berg, Anke; Visser, Lydia

    2016-01-01

    Follicular lymphoma and diffuse large B cell lymphomas comprise the main entities of adult B cell malignancies. Although multiple disease driving gene aberrations have been identified by gene expression and genomic studies, only a few studies focused at the protein level. We applied 2 dimensional gel electrophoresis to compare seven GC B cell non Hodgkin lymphoma (NHL) cell lines with a lymphoblastoid cell line (LCL). An average of 130 spots were at least two folds different in intensity between NHL cell lines and the LCL. We selected approximately 38 protein spots per NHL cell line and linked them to 145 unique spots based on the location in the gel. 34 spots that were found altered in at least three NHL cell lines when compared to LCL, were submitted for LC-MS/MS. This resulted in 28 unique proteins, a substantial proportion of these proteins were involved in cell motility and cell metabolism. Loss of expression of B2M, and gain of expression of PRDX1 and PPIA was confirmed in the cell lines and primary lymphoma tissue. Moreover, inhibition of PPIA with cyclosporine A blocked cell growth of the cell lines, the effect size was associated with the PPIA expression levels. In conclusion, we identified multiple differentially expressed proteins by 2-D proteomics, and showed that some of these proteins might play a role in the pathogenesis of NHL. PMID:26752561

  17. Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.

    PubMed

    Johnsen, Hans Erik; Bergkvist, Kim Steve; Schmitz, Alexander; Kjeldsen, Malene Krag; Hansen, Steen Møller; Gaihede, Michael; Nørgaard, Martin Agge; Bæch, John; Grønholdt, Marie-Louise; Jensen, Frank Svendsen; Johansen, Preben; Bødker, Julie Støve; Bøgsted, Martin; Dybkær, Karen

    2014-06-01

    Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy. PMID:23998255

  18. Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells

    PubMed Central

    Rakhmanov, Mirzokhid; Keller, Baerbel; Gutenberger, Sylvia; Foerster, Christian; Hoenig, Manfred; Driessen, Gertjan; van der Burg, Mirjam; van Dongen, Jacques J.; Wiech, Elisabeth; Visentini, Marcella; Quinti, Isabella; Prasse, Antje; Voelxen, Nadine; Salzer, Ulrich; Goldacker, Sigune; Fisch, Paul; Eibel, Hermann; Schwarz, Klaus; Peter, Hans-Hartmut; Warnatz, Klaus

    2009-01-01

    The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21low B cells are polyclonal, unmutated IgM+IgD+ B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21low B cells represent a human innate-like B cell population. PMID:19666505

  19. The life and death of a B cell.

    PubMed

    Defrance, Thierry; Casamayor-Pallejà, Montserrat; Krammer, Peter H

    2002-01-01

    Regulation of apoptosis in the B cell lineage has implications for homeostasis, quality control of the antibody response, and tolerance. In this chapter we examine the different checkpoints that control life and death decisions of B cells during the antigen-independent and antigen-dependent phases of their development. We discuss the cell death mechanism involved in elimination of unwanted B cells at different stages of their development as well as the signals that trigger or repress the apoptotic process. At the steady state, before or after development of an immune response, B cell apoptosis ensures that the antigen receptor (BCR) on newly produced B cells is functional and does not recognize self-antigens with high avidity. It also ensures that the size of the peripheral B cell compartment remains constant in spite of the continuous input of B cells from the bone marrow. All these processes are controlled by the mitochondrial death pathway and are thus perturbed by overexpression of the antiapoptotic members of the bcl-2 gene family. By contrast, the death receptor pathway plays a prominent role during the antigen-dependent phase of B cell development. Three sets of membrane molecules stand as crucial regulators of B cell survival. First, the BCR which plays a central but ambiguous role. On the one hand, it triggers death of B cells that recognize self-antigens or have been exposed to repeated antigenic stimulations. On the other hand, it promotes survival of the peripheral mature B cell pool and protects activated B cells from CD95-induced killing. Second, the death receptor Fas/CD95 which is instrumental in censoring B cells activated in a bystander fashion at the initiation of the response to T-dependent antigens. It also drives elimination of low-affinity and self-reactive B cell clones that arise through the process of somatic mutations during the germinal center reaction. As such, it contributes to the affinity maturation of the antibody response. Finally

  20. TIM-1 signaling is required for maintenance and induction of regulatory B cells.

    PubMed

    Yeung, M Y; Ding, Q; Brooks, C R; Xiao, S; Workman, C J; Vignali, D A A; Ueno, T; Padera, R F; Kuchroo, V K; Najafian, N; Rothstein, D M

    2015-04-01

    Apart from their role in humoral immunity, B cells can exhibit IL-10-dependent regulatory activity (Bregs). These regulatory subpopulations have been shown to inhibit inflammation and allograft rejection. However, our understanding of Bregs has been hampered by their rarity, lack of a specific marker, and poor insight into their induction and maintenance. We previously demonstrated that T cell immunoglobulin mucin domain-1 (TIM-1) identifies over 70% of IL-10-producing B cells, irrespective of other markers. We now show that TIM-1 is the primary receptor responsible for Breg induction by apoptotic cells (ACs). However, B cells that express a mutant form of TIM-1 lacking the mucin domain (TIM-1(Δmucin) ) exhibit decreased phosphatidylserine binding and are unable to produce IL-10 in response to ACs or by specific ligation with anti-TIM-1. TIM-1(Δmucin) mice also exhibit accelerated allograft rejection, which appears to be due in part to their defect in both baseline and induced IL-10(+) Bregs, since a single transfer of WT TIM-1(+) B cells can restore long-term graft survival. These data suggest that TIM-1 signaling plays a direct role in Breg maintenance and induction both under physiological conditions (in response to ACs) and in response to therapy through TIM-1 ligation. Moreover, they directly demonstrate that the mucin domain regulates TIM-1 signaling.

  1. Single molecule analysis of B cell receptor motion during signaling activation

    NASA Astrophysics Data System (ADS)

    Rey Suarez, Ivan; Koo, Peter; Mochrie, Simon; Song, Wenxia; Upadhyaya, Arpita

    B cells are an essential part of the adaptive immune system. They patrol the body looking for signs of infection in the form of antigen on the surface of antigen presenting cells. The binding of the B cell receptor (BCR) to antigen induces signaling cascades that lead to B cell activation and eventual production of high affinity antibodies. During activation, BCR organize into signaling microclusters, which are platforms for signal amplification. The physical processes underlying receptor movement and aggregation are not well understood. Here we study the dynamics of single BCRs on activated murine primary B cells using TIRF imaging and single particle tracking. The tracks obtained are analyzed using perturbation expectation-maximization (pEM) a systems-level analysis that allows the identification of different short-time diffusive states from a set of single particle tracks. We identified five different diffusive states on wild type cells, which correspond to different molecular states of the BCR. By using actin polymerization inhibitors and mutant cells lacking important actin regulators we were able to identify the BCR molecule configuration associated with each diffusive state.

  2. Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    PubMed Central

    Papa, Michelle Premazzi; de Morais, Ana Theresa Silveira; Peçanha, Ligia Maria Torres; de Arruda, Luciana Barros

    2015-01-01

    Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients. PMID:26656738

  3. The Contribution of B Cells to Renal Interstitial Inflammation

    PubMed Central

    Heller, Florian; Lindenmeyer, Maja T.; Cohen, Clemens D.; Brandt, Ulrike; Draganovici, Dan; Fischereder, Michael; Kretzler, Matthias; Anders, Hans-Joachim; Sitter, Thomas; Mosberger, Isabella; Kerjaschki, Dontscho; Regele, Heinz; Schlöndorff, Detlef; Segerer, Stephan

    2007-01-01

    Local B-cell infiltrates play a role in tissue fibrosis, neolymphangiogenesis, and renal allograft survival. We sought to characterize the B-cell infiltrates, factors involved in B-cell recruitment, and lymphangiogenesis in renal interstitial injury (ie, acute and chronic interstitial nephritis and chronic IgA nephropathy). CD20-positive B cells formed a prominent part of the interstitial infiltrating cells. Together with CD3-positive T cells, the CD20-positive B cells formed larger nodular structures. CD10-positive pre-B cells were rare, and the majority were mature CD27-positive B cells. Proliferating B cells were detected within nodular infiltrates. The level of mRNA expression of the chemokine CXCL13 was increased and correlated with CD20 mRNA in the tubulointerstitial space. CXCL13 protein was predominantly found at sites of nodular infiltrates, in association with CXCR5-positive B cells. Furthermore, sites of chronic interstitial inflammation were associated with a high number of lymphatic vessels. B-cell infiltrates form a prominent part of the interstitial infiltrates both in primary interstitial lesions and in IgA nephropathy. CXCR5-positive B cells might be recruited via the chemokine CXCL13 and seem to contribute to the formation of intrarenal lymphoid follicle-like structures. These might represent an intrarenal immune system. PMID:17255314

  4. STAT6-mediated BCL6 repression in primary mediastinal B-cell lymphoma (PMBL)

    PubMed Central

    Ritz, Olga; Rommel, Karolin; Dorsch, Karola; Kelsch, Elena; Melzner, Julia; Buck, Michaela; Leroy, Karen; Papadopoulou, Vasiliki; Wagner, Simon; Marienfeld, Ralf; Brüderlein, Silke; Lennerz, Jochen K.; Möller, Peter

    2013-01-01

    Primary mediastinal B-cell lymphoma (PMBL) is characterized by aberrant activation of JAK/STAT-signaling resulting in constitutive presence of phosphorylated STAT6 (pSTAT6). In primary PMBL samples pSTAT6 is only expressed in a sub-population of lymphoma cells in a pattern that is reminiscent of that of the BCL6 oncogene. Double-fluorescence staining was carried out to determine the association between these two proteins in ten primary PMBL cases and three available PMBL cell line models. Surprisingly, only a minute fraction of double-positive nuclei was observed, while each sample contained considerable fractions of single-positive pSTAT6 and BCL6 nuclei. The intratumoral coexistence of BCL6+/pSTAT6− and BCL6−/pSTAT6+ subpopulations suggests a negative interaction between these factors. In silico screening of the STAT6 /BCL6 promoters for DNA consensus binding sites identified five STAT-binding-sites in the BCL6 promoter. We confirmed STAT6 binding to the BCL6 promoter in vitro and in vivo by band shift / super shift assays and chromatin immunoprecipitations. Using BCL6 luciferase reporter assays, depletion of STAT6 by siRNA, and ectopic overexpression of a constitutive active STAT6 mutant, we proved that pSTAT6 is sufficient to transcriptionally repress BCL6. Recently developed small molecule inhibitors 79-6 and TG101348 that increases BCL6 target gene expression and decreases pSTAT6 levels, respectively, demonstrate that a combined targeting results in additive efficacy regarding their negative effect on cell viability. The delineated pSTAT6-mediated molecular repression mechanism links JAK/STAT to BCL6-signaling in PMBL and may carry therapeutic potential. PMID:23852366

  5. STAT6-mediated BCL6 repression in primary mediastinal B-cell lymphoma (PMBL).

    PubMed

    Ritz, Olga; Rommel, Karolin; Dorsch, Karola; Kelsch, Elena; Melzner, Julia; Buck, Michaela; Leroy, Karen; Papadopoulou, Vasiliki; Wagner, Simon; Marienfeld, Ralf; Brüderlein, Silke; Lennerz, Jochen K; Möller, Peter

    2013-07-01

    Primary mediastinal B-cell lymphoma (PMBL) is characterized by aberrant activation of JAK/STAT-signaling resulting in constitutive presence of phosphorylated STAT6 (pSTAT6). In primary PMBL samples pSTAT6 is only expressed in a sub-population of lymphoma cells in a pattern that is reminiscent of that of the BCL6 oncogene. Double-fluorescence staining was carried out to determine the association between these two proteins in ten primary PMBL cases and three available PMBL cell line models. Surprisingly, only a minute fraction of double-positive nuclei was observed, while each sample contained considerable fractions of single-positive pSTAT6 and BCL6 nuclei. The intratumoral coexistence of BCL6+/pSTAT6- and BCL6-/pSTAT6+ subpopulations suggests a negative interaction between these factors. In silico screening of the STAT6 /BCL6 promoters for DNA consensus binding sites identified five STAT-binding-sites in the BCL6 promoter. We confirmed STAT6 binding to the BCL6 promoter in vitro and in vivo by band shift / super shift assays and chromatin immunoprecipitations. Using BCL6 luciferase reporter assays, depletion of STAT6 by siRNA, and ectopic overexpression of a constitutive active STAT6 mutant, we proved that pSTAT6 is sufficient to transcriptionally repress BCL6. Recently developed small molecule inhibitors 79-6 and TG101348 that increases BCL6 target gene expression and decreases pSTAT6 levels, respectively, demonstrate that a combined targeting results in additive efficacy regarding their negative effect on cell viability. The delineated pSTAT6-mediated molecular repression mechanism links JAK/STAT to BCL6-signaling in PMBL and may carry therapeutic potential. PMID:23852366

  6. CNS accumulation of regulatory B cells is VLA-4-dependent

    PubMed Central

    Lehmann-Horn, Klaus; Sagan, Sharon A.; Winger, Ryan C.; Spencer, Collin M.; Bernard, Claude C.A.; Sobel, Raymond A.

    2016-01-01

    Objective: To investigate the role of very late antigen-4 (VLA-4) on regulatory B cells (Breg) in CNS autoimmune disease. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced in mice selectively deficient for VLA-4 on B cells (CD19cre/α4f/f) by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (p)35–55 or recombinant human (rh) MOG protein. B-cell and T-cell populations were examined by flow cytometry and immunohistochemistry. Breg were evaluated by intracellular IL-10 staining of B cells and, secondly, by coexpression of CD1d and CD5. Results: As previously reported, EAE was less severe in B-cell VLA-4-deficient vs control CD19cre mice when induced by rhMOG, a model that is B-cell-dependent and leads to efficient B-cell activation and antibody production. Paradoxically, B-cell VLA-4-deficient mice developed more severe clinical disease than control mice when EAE was induced with MOG p35-55, a B-cell-independent encephalitogen that does not efficiently activate B cells. Peripheral T-cell and humoral immune responses were not altered in B-cell VLA-4-deficient mice. In MOG p35-55-induced EAE, B-cell VLA-4 deficiency reduced CNS accumulation of B but not T cells. Breg were detected in the CNS of control mice with MOG p35-55-induced EAE. However, more severe EAE in B-cell VLA-4-deficient mice was associated with virtual absence of CNS Breg. Conclusions: Our results demonstrate that CNS accumulation of Breg is VLA-4-dependent and suggest that Breg may contribute to regulation of CNS autoimmunity in situ. These observations underscore the need to choose the appropriate encephalitogen when studying how B cells contribute to pathogenesis or regulation of CNS autoimmunity. PMID:27027096

  7. [Primary mediastinal B-cell lymphoma].

    PubMed

    Coso, D; Rey, J; Bouabdallah, R

    2010-02-01

    Primary mediastinal B-cell lymphoma (PMBL) is a clinicopathological entity among the world health organization classification of lymphoid neoplasms. PMBL often concerns young adults, and the disease remains a localized disease in the majority of cases. The outcome of patients with PMBL is variable and unlike diffuse large cell lymphomas, the international prognostic index seems to be less applicable to such disease. The combination of rituximab and chemotherapy is the gold standard treatment of patients with good prognosis features and allows high cure rates. However, high-dose chemotherapy supported by peripheral blood stem cell support is often warranted in poor-prognosis patients. The use of positrons emission tomography examination is more and more used in such situations to select the best therapeutic strategy. PMID:20207294

  8. The human intestinal B-cell response.

    PubMed

    Spencer, J; Sollid, L M

    2016-09-01

    The intestinal immune system is chronically challenged by a huge plethora of antigens derived from the lumen. B-cell responses in organized gut-associated lymphoid tissues and regional lymph nodes that are driven chronically by gut antigens generate the largest population of antibody-producing cells in the body: the gut lamina propria plasma cells. Although animal studies have provided insights into mechanisms that underpin this dynamic process, some very fundamental differences in this system appear to exist between species. Importantly, this prevents extrapolation from mice to humans to inform translational research questions. Therefore, in this review we will describe the structures and mechanisms involved in the propagation, dissemination, and regulation of this immense plasma cell population in man. Uniquely, we will seek our evidence exclusively from studies of human cells and tissues. PMID:27461177

  9. Rheumatoid factors, B cells and immunoglobulin genes.

    PubMed

    Jefferis, R

    1995-04-01

    The paradigm of self, non-self discrimination in the immune system is under review as autoreactive B or T cells are increasingly delineated within normal individuals. The products of autoreactive B cells are, mostly, polyspecific IgM antibodies of low affinity. These 'natural' antibodies include rheumatoid factors (RF) encoded by unmutated germline immunoglobulin genes. In rheumatoid arthritis (RA) the RF may be of the IgM, IgG or IgA isotype, show evidence of somatic mutation and have increased affinity; consistent with maturation of an antigen driven immune response. This response could be initiated or driven by an auto-immunogenic form of IgG or an exogenous cross-reactive antigen. Changes in galactosylation of IgG have been reported to be a valuable diagnostic and prognostic indicator in RA. Speculation that these changes may precipitate some of the disease processes is critically reviewed.

  10. Identification of the Abundant Hydroxyproline-Rich Glycoproteins in the Root Walls of Wild-Type Arabidopsis, an ext3 Mutant Line, and Its Phenotypic Revertant

    PubMed Central

    Chen, Yuning; Ye, Dening; Held, Michael A.; Cannon, Maura C.; Ray, Tui; Saha, Prasenjit; Frye, Alexandra N.; Mort, Andrew J.; Kieliszewski, Marcia J.

    2015-01-01

    Extensins are members of the cell wall hydroxyproline-rich glycoprotein (HRGP) superfamily that form covalently cross-linked networks in primary cell walls. A knockout mutation in EXT3 (AT1G21310), the gene coding EXTENSIN 3 (EXT3) in Arabidopsis Landsberg erecta resulted in a lethal phenotype, although about 20% of the knockout plants have an apparently normal phenotype (ANP). In this study the root cell wall HRGP components of wild-type, ANP and the ext3 mutant seedlings were characterized by peptide fractionation of trypsin digested anhydrous hydrogen fluoride deglycosylated wall residues and by sequencing using LC-MS/MS. Several HRGPs, including EXT3, were identified in the wild-type root walls but not in walls of the ANP and lethal mutant. Indeed the ANP walls and walls of mutants displaying the lethal phenotype possessed HRGPs, but the profiles suggest that changes in the amount and perhaps type may account for the corresponding phenotypes. PMID:27135319

  11. B-cell repertoire responses to varicella-zoster vaccination in human identical twins.

    PubMed

    Wang, Chen; Liu, Yi; Cavanagh, Mary M; Le Saux, Sabine; Qi, Qian; Roskin, Krishna M; Looney, Timothy J; Lee, Ji-Yeun; Dixit, Vaishali; Dekker, Cornelia L; Swan, Gary E; Goronzy, Jörg J; Boyd, Scott D

    2015-01-13

    Adaptive immune responses in humans rely on somatic genetic rearrangements of Ig and T-cell receptor loci to generate diverse antigen receptors. It is unclear to what extent an individual's genetic background affects the characteristics of the antibody repertoire used in responding to vaccination or infection. We studied the B-cell repertoires and clonal expansions in response to attenuated varicella-zoster vaccination in four pairs of adult identical twins and found that the global antibody repertoires of twin pair members showed high similarity in antibody heavy chain V, D, and J gene segment use, and in the length and features of the complementarity-determining region 3, a major determinant of antigen binding. These twin similarities were most pronounced in the IgM-expressing B-cell pools, but were seen to a lesser extent in IgG-expressing B cells. In addition, the degree of antibody somatic mutation accumulated in the B-cell repertoire was highly correlated within twin pair members. Twin pair members had greater numbers of shared convergent antibody sequences, including mutated sequences, suggesting similarity among memory B-cell clonal lineages. Despite these similarities in the memory repertoire, the B-cell clones used in acute responses to ZOSTAVAX vaccination were largely unique to each individual. Taken together, these results suggest that the overall B-cell repertoire is significantly shaped by the underlying germ-line genome, but that stochastic or individual-specific effects dominate the selection of clones in response to an acute antigenic stimulus. PMID:25535378

  12. The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

    PubMed

    Siegel, Andrea M; Herskowitz, Jeremy H; Speck, Samuel H

    2008-04-01

    Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a

  13. Syk Tyrosine Kinase Is Required for the Positive Selection of Immature B Cells into the Recirculating B Cell Pool

    PubMed Central

    Turner, Martin; Gulbranson-Judge, Adam; Quinn, Marian E.; Walters, Alice E.; MacLennan, Ian C.M.; Tybulewicz, Victor L.J.

    1997-01-01

    The tyrosine kinase Syk has been implicated as a key signal transducer from the B cell antigen receptor (BCR). We show here that mutation of the Syk gene completely blocks the maturation of immature B cells into recirculating cells and stops their entry into B cell follicles. Furthermore, using radiation chimeras we demonstrate that this developmental block is due to the absence of Syk in the B cells themselves. Syk-deficient B cells are shown to have the life span of normal immature B cells. If this is extended by over-expression of Bcl-2, they accumulate in the T zone and red pulp of the spleen in increased numbers, but still fail to mature to become recirculating follicular B cells. Despite this defect in maturation, Syk-deficient B cells were seen to give rise to switched as well as nonswitched splenic plasma cells. Normally only a proportion of immature B cells is recruited into the recirculating pool. Our results suggest that Syk transduces a BCR signal that is absolutely required for the positive selection of immature B cells into the recirculating B cell pool. PMID:9396770

  14. Persistence of Marek's disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

    PubMed Central

    Fynan, E; Block, T M; DuHadaway, J; Olson, W; Ewert, D L

    1992-01-01

    Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell. Images PMID:1326647

  15. The MHC class II cofactor, HLA-DM, interacts with immunoglobulin in B cells

    PubMed Central

    Ayyangar, Sashi; Jiang, Wei; Rajasekaran, Narendiran; Spura, Armin; Hessell, Ann J.; Madec, Anne-Marie; Mellins, Elizabeth D.

    2014-01-01

    B cells internalize extracellular antigen into endosomes using the immunoglobulin (Ig) component of the B cell receptor. In endosomes, antigen-derived peptides are loaded onto MHC class II proteins (MHC-II). How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC-II peptide loading, co-precipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes co-localize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble HLA-DM (sDM) directly binds the Ig Fab domain, and increases levels of free antigen released from immune complexes. Together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences. PMID:25098292

  16. Screening of abscisic acid insensative (ABI) and low phosphorous efficiency (LPE) mutants from some sequenced lines in the sorghum TILLING population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum population for Targeting Induced Local Lesion IN Genome (TILLING) was generated from BTx623 in 2005 and publicly available in 2009. After releasing to the public, this population was intensively screened by morphological observation in the field and a number of mutants with useful traits wer...

  17. Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.

    PubMed

    Vrolix, Kathleen; Fraussen, Judith; Losen, Mario; Stevens, Jo; Lazaridis, Konstantinos; Molenaar, Peter C; Somers, Veerle; Bracho, Maria Alma; Le Panse, Rozen; Stinissen, Piet; Berrih-Aknin, Sonia; Maessen, Jos G; Van Garsse, Leen; Buurman, Wim A; Tzartos, Socrates J; De Baets, Marc H; Martinez-Martinez, Pilar

    2014-08-01

    Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.

  18. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  19. Regulation of VH replacement by B cell receptor-mediated signaling in human immature B cells.

    PubMed

    Liu, Jing; Lange, Miles D; Hong, Sang Yong; Xie, Wanqin; Xu, Kerui; Huang, Lin; Yu, Yangsheng; Ehrhardt, Götz R A; Zemlin, Michael; Burrows, Peter D; Su, Kaihong; Carter, Robert H; Zhang, Zhixin

    2013-06-01

    VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 μHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.

  20. Loss of B Cells in Patients with Heterozygous Mutations in IKAROS

    PubMed Central

    Cunningham-Rundles, C.; Reichenbach, J.; Stray-Pedersen, A.; Gelfand, E.W.; Maffucci, P.; Pierce, K.R.; Abbott, J.K.; Voelkerding, K.V.; South, S.T.; Augustine, N.H.; Bush, J.S.; Dolen, W.K.; Wray, B.B.; Itan, Y.; Cobat, A.; Sorte, H.S.; Ganesan, S.; Prader, S.; Martins, T.B.; Lawrence, M.G.; Orange, J.S.; Calvo, K.R.; Niemela, J.E.; Casanova, J.-L.; Fleisher, T.A.; Hill, H.R.; Kumánovics, A.

    2016-01-01

    BACKGROUND Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.) PMID:26981933

  1. Brucella abortus-infected B cells induce osteoclastogenesis.

    PubMed

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2016-09-01

    Brucella abortus is an intracellular bacterium that establishes lifelong infections in livestock and humans although the mechanisms of its chronicity are poorly understood. Activated B cells have long lifespan and B. abortus infection activates B cells. Our results indicate that the direct infection of B cells with B. abortus induced matrix metalloproteinase-9 (MMP-9), receptor activator for NF κB ligand (RANKL), tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion. In addition, supernatants from B. abortus-infected B cells induced bone marrow-derived monocytes to undergo osteoclastogenesis. Using osteoprotegerin, RANKL's decoy receptor, we determined that RANKL is involved in osteoclastogenesis induced by supernatants from B. abortus-infected B cells. The results presented here shed light on how the interactions of B. abortus with B cells may have a role in the pathogenesis of brucellar osteoarticular disease.

  2. Dual immunoglobulin light chain B cells: Trojan horses of autoimmunity?

    PubMed

    Pelanda, Roberta

    2014-04-01

    Receptor editing, a major mechanism of B cell tolerance, can also lead to allelic inclusion at the immunoglobulin light chain loci and the development of B cells that coexpress two different immunoglobulin light chains and, therefore, two antibody specificities. Most allelically included B cells express two κ chains, although rare dual-λ cells are also observed. Moreover, these cells typically coexpress an autoreactive and a nonautoreactive antibody. Thus, allelically included B cells could operate like 'Trojan horses': expression and function of the nonautoreactive antigen receptors might promote their maturation, activation, and terminal differentiation into effector cells that also express and secrete autoantibodies. Indeed, dual-κ B cells are greatly expanded into effector B cell subsets in some autoimmune mice, thus indicating they might play an important role in disease.

  3. Primary B-cell lymphoblastic lymphoma of the testis.

    PubMed

    Tombolini, Flavia; Lacetera, Vito; Gini, Guido; Capelli, Debora; Leoni, Pietro; Montironi, Rodolfo; Galosi, Andrea Benedetto; Muzzonigro, Giovanni

    2014-12-01

    We present a rare case of primary lymphoblastic B-cell lymphoma of the testis focusing on ultrasonographic and pathological features and clinical implications. Pathological examination revealed primary testicular lymphoblastic B-cell lymphoma which was treated with adjuvant chemotherapy, including rachicentesis with administration of chemotherapy and with radiotherapy of contralateral testis. Primary testicular lymphoblastic B cell lymphoma is an aggressive disease and it is necessary a multimodal therapy (surgery, chemotherapy and radiotherapy) to prevent metastasis. PMID:25641484

  4. Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

    PubMed Central

    Wang, Jing; Tang, Shan; Wan, Zhengpeng; Gao, Yiren; Cao, Yiyun; Yi, Junyang; Si, Yanyan; Zhang, Haowen; Liu, Lei; Liu, Wanli

    2016-01-01

    Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens. PMID:26764382

  5. Long-term culture system for selective growth of human B-cell progenitors.

    PubMed Central

    Rawlings, D J; Quan, S G; Kato, R M; Witte, O N

    1995-01-01

    We describe a simple reproducible system for enrichment and long-term culture of human B-cell progenitors. Enriched CD34+ cord blood mononuclear cells are seeded onto a murine stromal cell line to establish a biphasic culture system. These cultures are characterized by transient growth of myeloid cells followed by outgrowth of cells highly enriched for early B-cell progenitors. Cultures consisting of > 90% early B-lineage cells [expressing CD10, CD19, CD38, and CD45 but lacking CD20, CD22, CD23, and surface IgM] are maintained for > 12 weeks without growth factor addition. Cells remain predominantly germ line at the immunoglobulin locus and express only low levels of cytoplasmic mu chain, terminal deoxynucleotidyltransferase, and recombination-activating gene 1 product. They are unresponsive to the pre-B-cell growth factors interleukin 7 or stem cell factor, or both, suggesting that growth support is provided by a cross-reactive murine stromal cell factor. Cultured B-cell progenitors are generated in large numbers ( > 10(8) cells from a typical cord blood specimen) suitable for use in biochemical analysis and gene-transfer studies. This system should be useful for study of normal and abnormal early human B-lymphopoiesis. Images Fig. 2 Fig. 3 Fig. 4 PMID:7533295

  6. Monoclonal B-cell lymphocytosis in healthy blood donors: an unexpectedly common finding.

    PubMed

    Shim, Youn K; Rachel, Jane M; Ghia, Paolo; Boren, Jeff; Abbasi, Fatima; Dagklis, Antonis; Venable, Geri; Kang, Jiyeon; Degheidy, Heba; Plapp, Fred V; Vogt, Robert F; Menitove, Jay E; Marti, Gerald E

    2014-02-27

    Circulating monoclonal B cells may be detected in healthy adults, a condition called monoclonal B-cell lymphocytosis (MBL). MBL has also been identified in donated blood, but no systematic study of blood donors has been reported. Using sensitive and specific laboratory methods, we detected MBL in 149 (7.1%; 95% confidence interval, 6.0% to 8.3%) of 2098 unique donors ages 45 years or older in a Midwestern US regional blood center between 2010 and 2011. Most of the 149 donors had low-count MBL, including 99 chronic lymphocytic leukemia-like (66.4%), 22 atypical (14.8%), and 19 CD5(-) (12.8%) immunophenotypes. However, 5 donors (3.4%) had B-cell clonal counts above 500 cells per µL, including 3 with 1693 to 2887 cells per µL; the clone accounted for nearly all their circulating B cells. Four donors (2.7%) had 2 distinct MBL clones. Of 51 MBL samples in which immunoglobulin heavy chain (IGH)V-D-J genotypes could be determined, 71% and 29% used IGHV3- and IGHV4-family genes, respectively. Sequencing revealed 82% with somatic hypermutation, whereas 18% had >98% germ-line identity, including 5 with entirely germ-line sequences. In conclusion, MBL prevalence is much higher in blood donors than previously reported, and although uncommon, the presence of high-count MBL warrants further investigations to define the biological fate of the transfused cells in recipients.

  7. Small Molecule Growth Inhibitors of Human Oncogenic Gammaherpesvirus Infected B-Cells

    PubMed Central

    Dzeng, Richard K.; Jha, Hem Chandra; Lu, Jie; Saha, Abhik; Banerjee, Sagarika; Robertson, Erle S.

    2014-01-01

    Epstein-Barr virus (EBV) and Kaposi’s sarcoma associated herpesvirus (KSHV) are two human gammaherpesviruses associated with a broad spectrum of B-cell lymphomas, most acutely in immuno-compromised populations. However, there are no drugs which specifically target KSHV or EBV-associated lymphomas. To identify small molecules which selectively inhibit the growth of EBV or KSHV-associated B-cell lines, we performed a fluorescence based high-throughput screen on multiple stable GFP expressing virus-infected or uninfected B-cell lines. We identified 40 initial compounds with selective growth inhibition and subsequently determined the 50% growth inhibitory concentrations (GI50) for each drug. We further examined compounds with higher specificity to explore the underlying molecular mechanisms using transcription factor analysis, as well as a sh-RNA based knockdown strategy. Our data identified ten compounds with relatively high efficacy for growth inhibition. Two novel small molecules, NSC#10010 and NSC#65381 were potent growth inhibitors for gammaherpesvirus-associated B-lymphomas through activation of both the NF-κB and c-Myc- mediated signaling pathways. These drugs can serve as potential lead compounds to expand the current therapeutic window against EBV or KSHV associated human B-cell malignancies. PMID:25306391

  8. Depletion of B cells in murine lupus: efficacy and resistance.

    PubMed

    Ahuja, Anupama; Shupe, Jonathan; Dunn, Robert; Kashgarian, Michael; Kehry, Marilyn R; Shlomchik, Mark J

    2007-09-01

    In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.

  9. The molecular biology of diffuse large B-cell lymphoma.

    PubMed

    Frick, Mareike; Dörken, Bernd; Lenz, Georg

    2011-12-01

    Diffuse large B-cell lymphoma (DLBCL) represents the most common type of malignant lymphoma. In the last few years, significant progress has been achieved in the understanding of the molecular pathogenesis of this entity. Gene expression profiling has identified three molecular DLBCL subtypes, termed germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). In this review, we summarize our current understanding of the biology of these DLBCL subtypes with a special emphasis on novel diagnostic and therapeutic approaches. PMID:23556103

  10. B-cell targeted therapeutics in clinical development

    PubMed Central

    2013-01-01

    B lymphocytes are the source of humoral immunity and are thus a critical component of the adaptive immune system. However, B cells can also be pathogenic and the origin of disease. Deregulated B-cell function has been implicated in several autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. B cells contribute to pathological immune responses through the secretion of cytokines, costimulation of T cells, antigen presentation, and the production of autoantibodies. DNA-and RNA-containing immune complexes can also induce the production of type I interferons, which further promotes the inflammatory response. B-cell depletion with the CD20 antibody rituximab has provided clinical proof of concept that targeting B cells and the humoral response can result in significant benefit to patients. Consequently, the interest in B-cell targeted therapies has greatly increased in recent years and a number of new biologics exploiting various mechanisms are now in clinical development. This review provides an overview on current developments in the area of B-cell targeted therapies by describing molecules and subpopulations that currently offer themselves as therapeutic targets, the different strategies to target B cells currently under investigation as well as an update on the status of novel therapeutics in clinical development. Emerging data from clinical trials are providing critical insight regarding the role of B cells and autoantibodies in various autoimmune conditions and will guide the development of more efficacious therapeutics and better patient selection. PMID:23566679

  11. Involvement of B cells in non-infectious uveitis

    PubMed Central

    Smith, Justine R; Stempel, Andrew J; Bharadwaj, Arpita; Appukuttan, Binoy

    2016-01-01

    Non-infectious uveitis—or intraocular inflammatory disease—causes substantial visual morbidity and reduced quality of life amongst affected individuals. To date, research of pathogenic mechanisms has largely been focused on processes involving T lymphocyte and/or myeloid leukocyte populations. Involvement of B lymphocytes has received relatively little attention. In contrast, B-cell pathobiology is a major field within general immunological research, and large clinical trials have showed that treatments targeting B cells are highly effective for multiple systemic inflammatory diseases. B cells, including the terminally differentiated plasma cell that produces antibody, are found in the human eye in different forms of non-infectious uveitis; in some cases, these cells outnumber other leukocyte subsets. Recent case reports and small case series suggest that B-cell blockade may be therapeutic for patients with non-infectious uveitis. As well as secretion of antibody, B cells may promote intraocular inflammation by presentation of antigen to T cells, production of multiple inflammatory cytokines and support of T-cell survival. B cells may also perform various immunomodulatory activities within the eye. This translational review summarizes the evidence for B-cell involvement in non-infectious uveitis, and considers the potential contributions of B cells to the development and control of the disease. Manipulations of B cells and/or their products are promising new approaches to the treatment of non-infectious uveitis. PMID:26962453

  12. Invited article: inhibition of B cell functions: implications for neurology.

    PubMed

    Dalakas, Marinos C

    2008-06-01

    B cells are involved in the pathophysiology of many neurologic diseases, either in a causative or contributory role, via production of autoantibodies, cytokine secretion, or by acting as antigen-presenting cells leading to T cell activation. B cells are clonally expanded in various CNS disorders, such as multiple sclerosis (MS), paraneoplastic CNS disorders, or stiff-person syndrome, and are activated to produce pathogenic autoantibodies in demyelinating neuropathies and myasthenia. B cell activating factor (BAFF) and a proliferating inducing ligand (APRIL), key cytokines for B cell survival, are strongly unregulated in MS brain and in muscles of inflammatory myopathies. Modulation of B cell functions using a series of monoclonal antibodies against CD20+ B cells or the molecules that increase B cell survival, such as BAFF/APRIL and their receptors BAFF-R, TACI, and BCMA, provide a rational approach to the treatment of the aforementioned neurologic disorders. In controlled studies, rituximab, a B cell-depleting monoclonal antibody, has been encouraging in MS and paraproteinemic anti-MAG demyelinating neuropathy, exerting long-lasting remissions. In uncontrolled series, benefit has been reported in several disorders. B cell depletion is a well-tolerated therapeutic option currently explored in the treatment of several autoimmune neurologic disorders.

  13. A novel mutant 10Ala/Arg together with mutant 144Ser/Arg of hepatitis B virus X protein involved in hepatitis B virus-related hepatocarcinogenesis in HepG2 cell lines.

    PubMed

    Shi, Ying; Wang, Junwei; Wang, Yuhe; Wang, Anna; Guo, Hongliang; Wei, Feili; Mehta, Sanjay R; Espitia, Stephen; Smith, Davey M; Liu, Longgen; Zhang, Yulin; Chen, Dexi

    2016-02-28

    Hepatitis B virus (HBV) infection-related hepatocellular carcinoma (HCC) represents a major health problem worldwide. HBV X (HBx) protein is the most common open reading frame that may undergo mutations, resulting in the development of HCC. This study aimed to determine specific HBx mutations that differentiate the central- and para-tumor tissues, and identify their association with HCC development. HBx gene from HCC tumor and para-tumor tissues of 47 HCC patients was amplified, sequenced and statistically analyzed. A novel combination of 2 mutations at residues 10 and 144 was identified which might play a significant role in HCC development. Expression vectors carrying HBx with the specific mutations were constructed and transfected into HepG2 and p53-null HepG2 cells. Compared to wild type (WT) and single mutation of HBx at residue 10 or 144, the 10/144 double mutations strongly up-regulated p21 expression and prolonged G1/S transition in WT- and p53-null HepG2 cells. Apoptosis was also inhibited by HBx harboring 10/44 double-mutation. Binding of 10/144 double-mutant HBx to p53 was lower than WT HBx. Conclusively, the 10/144 double mutation of HBx might play a crucial role in HCC formation.

  14. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    SciTech Connect

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  15. Effects of fuzzless cottonseed phenotype on cottonseed nutrient composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines under well-watered and water stress conditions.

    PubMed

    Bellaloui, Nacer; Turley, Rickie B

    2013-01-01

    There is no information available on the effect of fuzzless seed trait on cottonseed nutrient composition (minerals, N, S, protein, and oil) under drought stress. The objective of this research was to investigate the effect of the fuzzless seed trait on cottonseed nutrients using five sets of near-isogenic lines (NILs). Each set consists of two lines that share the same genetic background, but differ in seed fuzziness (fuzzy, F; fuzzless, N). The near isogenic lines will enable us to compare the effect of the trait without confounding the genotypic background effects. We hypothesized that since the fuzzless trait involved in fiber initiation development, and was reported to be involved in biochemical, molecular, and genetic processes, this trait may also alter cottonseed nutrient composition. Results showed that NIL sets accumulated different levels of minerals in seeds and leaves, and the fuzzless trait (N) in most of the lines altered seed and leaf mineral accumulations when compared with fuzzy lines (F) or the control line. For example, K, P, Mg, Cu, and Na concentrations in seeds were higher in MD N and STV N than in their equivalent MD F and STV F lines. Leaf concentrations of Ca, K, Mg, S, B, Cu, and Fe in MD N lines were higher than MD F line. Lower levels of nutrients in seeds and leaves were observed under water stress conditions, especially Ca, Mg, N, and B in seeds.Generally and with few exceptions, seed protein was higher in fuzzy lines than in fuzzless lines; however, seed oil was higher in fuzzless lines than in fuzzy lines. Our research demonstrated that fuzzless trait altered the composition and level of nutrients in seed and leaves in well watered and water stressed plants. Differences in protein and oil between fuzzy and fuzzless seeds may indicate alteration in nitrogen and carbon fixation and metabolism. The differential accumulation of seed nutrients in this germplasm could be used by cotton breeders to select for higher cottonseed quality

  16. Effects of fuzzless cottonseed phenotype on cottonseed nutrient composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines under well-watered and water stress conditions1

    PubMed Central

    Bellaloui, Nacer; Turley, Rickie B.

    2013-01-01

    There is no information available on the effect of fuzzless seed trait on cottonseed nutrient composition (minerals, N, S, protein, and oil) under drought stress. The objective of this research was to investigate the effect of the fuzzless seed trait on cottonseed nutrients using five sets of near-isogenic lines (NILs). Each set consists of two lines that share the same genetic background, but differ in seed fuzziness (fuzzy, F; fuzzless, N). The near isogenic lines will enable us to compare the effect of the trait without confounding the genotypic background effects. We hypothesized that since the fuzzless trait involved in fiber initiation development, and was reported to be involved in biochemical, molecular, and genetic processes, this trait may also alter cottonseed nutrient composition. Results showed that NIL sets accumulated different levels of minerals in seeds and leaves, and the fuzzless trait (N) in most of the lines altered seed and leaf mineral accumulations when compared with fuzzy lines (F) or the control line. For example, K, P, Mg, Cu, and Na concentrations in seeds were higher in MD N and STV N than in their equivalent MD F and STV F lines. Leaf concentrations of Ca, K, Mg, S, B, Cu, and Fe in MD N lines were higher than MD F line. Lower levels of nutrients in seeds and leaves were observed under water stress conditions, especially Ca, Mg, N, and B in seeds.Generally and with few exceptions, seed protein was higher in fuzzy lines than in fuzzless lines; however, seed oil was higher in fuzzless lines than in fuzzy lines. Our research demonstrated that fuzzless trait altered the composition and level of nutrients in seed and leaves in well watered and water stressed plants. Differences in protein and oil between fuzzy and fuzzless seeds may indicate alteration in nitrogen and carbon fixation and metabolism. The differential accumulation of seed nutrients in this germplasm could be used by cotton breeders to select for higher cottonseed quality

  17. Molecular and cytogenetic characterization of expanded B-cell clones from multiclonal versus monoclonal B-cell chronic lymphoproliferative disorders

    PubMed Central

    Henriques, Ana; Rodríguez-Caballero, Arancha; Criado, Ignacio; Langerak, Anton W.; Nieto, Wendy G.; Lécrevisse, Quentin; González, Marcos; Cortesão, Emília; Paiva, Artur; Almeida, Julia; Orfao, Alberto

    2014-01-01

    Chronic antigen-stimulation has been recurrently involved in the earlier stages of monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The expansion of two or more B-cell clones has frequently been reported in individuals with these conditions; potentially, such coexisting clones have a greater probability of interaction with common immunological determinants. Here, we analyzed the B-cell receptor repertoire and molecular profile, as well as the phenotypic, cytogenetic and hematologic features, of 228 chronic lymphocytic leukemia-like and non-chronic lymphocytic leukemia-like clones comparing multiclonal (n=85 clones from 41 cases) versus monoclonal (n=143 clones) monoclonal B-cell lymphocytosis, chronic lymphocytic leukemia and other B-cell chronic lymphoproliferative disorders. The B-cell receptor of B-cell clones from multiclonal cases showed a slightly higher degree of HCDR3 homology than B-cell clones from mono clonal cases, in association with unique hematologic (e.g. lower B-lymphocyte counts) and cytogenetic (e.g. lower frequency of cytogenetically altered clones) features usually related to earlier stages of the disease. Moreover, a subgroup of coexisting B-cell clones from individual multiclonal cases which were found to be phylogenetically related showed unique molecular and cytogenetic features: they more frequently shared IGHV3 gene usage, shorter HCDR3 sequences with a greater proportion of IGHV mutations and del(13q14.3), than other unrelated B-cell clones. These results would support the antigen-driven nature of such multiclonal B-cell expansions, with potential involvement of multiple antigens/epitopes. PMID:24488564

  18. A Gene Panel, Including LRP12, Is Frequently Hypermethylated in Major Types of B-Cell Lymphoma

    PubMed Central

    Bethge, Nicole; Honne, Hilde; Andresen, Kim; Hilden, Vera; Trøen, Gunhild; Liestøl, Knut; Holte, Harald; Delabie, Jan; Lind, Guro E.; Smeland, Erlend B.

    2014-01-01

    Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt's lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (BMP7, BMPER, CDH1, DUSP4 and LRP12), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes LRP12 and CDH1 showed the highest methylation frequencies (94% and 92%, respectively). BMPER (58%), DUSP4 (32%) and BMP7 (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of LRP12 and CDH1 methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma. PMID:25226156

  19. A gene panel, including LRP12, is frequently hypermethylated in major types of B-cell lymphoma.

    PubMed

    Bethge, Nicole; Honne, Hilde; Andresen, Kim; Hilden, Vera; Trøen, Gunhild; Liestøl, Knut; Holte, Harald; Delabie, Jan; Lind, Guro E; Smeland, Erlend B

    2014-01-01

    Epigenetic modifications and DNA methylation in particular, have been recognized as important mechanisms to alter gene expression in malignant cells. Here, we identified candidate genes which were upregulated after an epigenetic treatment of B-cell lymphoma cell lines (Burkitt's lymphoma, BL; Follicular lymphoma, FL; Diffuse large B-cell lymphoma, DLBCL activated B-cell like, ABC; and germinal center like, GCB) and simultaneously expressed at low levels in samples from lymphoma patients. Qualitative methylation analysis of 24 candidate genes in cell lines revealed five methylated genes (BMP7, BMPER, CDH1, DUSP4 and LRP12), which were further subjected to quantitative methylation analysis in clinical samples from 59 lymphoma patients (BL, FL, DLBCL ABC and GCB; and primary mediastinal B-cell lymphoma, PMBL). The genes LRP12 and CDH1 showed the highest methylation frequencies (94% and 92%, respectively). BMPER (58%), DUSP4 (32%) and BMP7 (22%), were also frequently methylated in patient samples. Importantly, all gene promoters were unmethylated in various control samples (CD19+ peripheral blood B cells, peripheral blood mononuclear cells and tonsils) as well as in follicular hyperplasia samples, underscoring a high specificity. The combination of LRP12 and CDH1 methylation could successfully discriminate between the vast majority of the lymphoma and control samples, emphasized by receiver operating characteristic analysis with a c-statistic of 0.999. These two genes represent promising epigenetic markers which may be suitable for monitoring of B-cell lymphoma. PMID:25226156

  20. Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient

    PubMed Central

    BANGA, J P; MOORE, J K; DUHINDAN, N; MADEC, A M; VAN ENDERT, P M; ORGIAZZI, J; ENDL, J

    2004-01-01

    We used a GAD65-specific human B–T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B–T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation. PMID:14678267

  1. ABSENCE OF SCLEROSTIN ADVERSELY AFFECTS B CELL SURVIVAL

    PubMed Central

    Cain, Corey J.; Rueda, Randell; McLelland, Bryce; Collette, Nicole M.; Loots, Gabriela G.; Manilay, Jennifer O.

    2012-01-01

    Increased osteoblast activity in sclerostin-knockout (Sost−/−) mice results in generalized hyperostosis and bones with small bone marrow cavities due to hyperactive mineralizing osteoblast populations. Hematopoietic cell fate decisions are dependent on their local microenvironment, which contains osteoblast and stromal cell populations that support both hematopoietic stem cell quiescence and facilitate B cell development. In this study, we investigated whether high bone mass environments affect B cell development via the utilization of Sost−/− mice, a model of sclerosteosis. We found the bone marrow of Sost−/− mice to be specifically depleted of B cells, due to elevated apoptosis at all B cell developmental stages. In contrast, B cell function in the spleen was normal. Sost expression analysis confirmed that Sost is primarily expressed in osteocytes and is not expressed in any hematopoietic lineage, which indicated that the B cell defects in Sost−/− mice are non-cell autonomous and this was confirmed by transplantation of wildtype (WT) bone marrow into lethally irradiated Sost−/− recipients. WT→Sost−/− chimeras displayed a reduction in B cells, whereas reciprocal Sost−/−→WT chimeras did not, supporting the idea that the Sost−/− bone environment cannot fully support normal B cell development. Expression of the pre-B cell growth stimulating factor, Cxcl12, was significantly lower in bone marrow stromal cells of Sost−/− mice while the Wnt target genes Lef-1 and Ccnd1 remained unchanged in B cells. Taken together, these results demonstrate a novel role for Sost in the regulation of bone marrow environments that support B cells. PMID:22434688

  2. Tissue-Specific B-Cell Dysfunction and Generalized Memory B-Cell Loss during Acute SIV Infection

    PubMed Central

    Peruchon, Sandrine; Chaoul, Nada; Burelout, Chantal; Delache, Benoit; Brochard, Patricia; Laurent, Pascale; Cognasse, Fabrice; Prévot, Sophie; Garraud, Olivier; Le Grand, Roger; Richard, Yolande

    2009-01-01

    Background Primary HIV-infected patients display severe and irreversible damage to different blood B-cell subsets which is not restored by highly efficient anti-retroviral therapy (HAART). Because longitudinal investigations of primary HIV-infection is limited by the availability of lymphoid organs, we studied the tissue-specific B-cell dysfunctions in acutely simian immunodeficiency virus (SIV) mac251-infected Cynomolgus macaques. Methods and Findings Experiments were performed on three groups of macaques infected for 14, 21 or 28 days and on three groups of animals treated with HAART for two-weeks either initiated at 4 h, 7 or 14 days post-infection (p.i.). We have simultaneously compared changes in B-cell phenotypes and functions and tissue organization of B-cell areas in various lymphoid organs. We showed that SIV induced a steady decline in SIgG-expressing memory (SIgD−CD27+) B-cells in spleen and lymph nodes during the first 4 weeks of infection, concomitant to selective homing/sequestration of B-cells to the small intestine and spleen. SIV non-specific Ig production was transiently increased before D14p.i., whereas SIV-specific Ig production was only detectable after D14p.i., coinciding with the presence of CD8+ T-cells and IgG-expressing plasma cells within germinal centres. Transient B-cell apoptosis on D14p.i. and commitment to terminal differentiation contributed to memory B-cell loss. HAART abrogated B-cell apoptosis, homing to the small intestine and SIV-specific Ig production but had minimal effect on early Ig production, increased B-cell proportions in spleen and loss of memory B-cells. Therefore, virus–B-cell interactions and SIV-induced inflammatory cytokines may differently contribute to early B-cell dysfunction and impaired SIV/HIV-specific antibody response. Conclusions These data establish tissue-specific impairments in B-cell trafficking and functions and a generalized and steady memory B-cell loss in secondary lymphoid organs

  3. BAFF enhances chemotaxis of primary human B cells: a particular synergy between BAFF and CXCL13 on memory B cells.

    PubMed

    Badr, Gamal; Borhis, Gwenoline; Lefevre, Eric A; Chaoul, Nada; Deshayes, Frederique; Dessirier, Valérie; Lapree, Genevieve; Tsapis, Andreas; Richard, Yolande

    2008-03-01

    B-cell-activating factor of the TNF family, (BAFF), and a proliferation-inducing ligand (APRIL) regulate B-lymphocyte survival and activation. We report that BAFF, but not APRIL, increased the chemotactic response of primary human B cells to CCL21, CXCL12, and CXCL13. The BAFF-induced increase in B-cell chemotaxis was totally abolished by blockade of BAFF-R and was strongly dependent on the activation of PI3K/AKT, NF-kappaB, and p38MAPK pathways. BAFF had similar effects on the chemotaxis of naive and memory B cells in response to CCL21 but increased more strongly that of memory B cells to CXCL13 than that of naive B cells. Our findings indicate a previously unreported role for the BAFF/BAFF-R pair in mature B-cell chemotaxis. The synergy between CXCL13 and BAFF produced by stromal cells and follicular dendritic cells may have important implications for B-cell homeostasis, the development of normal B-cell areas, and for the formation of germinal center-like follicles that may be observed in various autoimmune diseases.

  4. Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma.

    PubMed

    Iqbal, Javeed; Shen, Yulei; Huang, Xin; Liu, Yanyan; Wake, Laura; Liu, Cuiling; Deffenbacher, Karen; Lachel, Cynthia M; Wang, Chao; Rohr, Joseph; Guo, Shuangping; Smith, Lynette M; Wright, George; Bhagavathi, Sharathkumar; Dybkaer, Karen; Fu, Kai; Greiner, Timothy C; Vose, Julie M; Jaffe, Elaine; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M; Cook, James R; Tubbs, Raymond R; Armitage, James O; Weisenburger, Dennis D; Staudt, Louis M; Gascoyne, Randy D; McKeithan, Timothy W; Chan, Wing C

    2015-02-12

    We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)-DLBCL compared with activated B-cell (ABC)-DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the "gold standard" GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL. PMID:25498913

  5. Global microRNA expression profiling uncovers molecular markers for classification and prognosis in aggressive B-cell lymphoma

    PubMed Central

    Shen, Yulei; Huang, Xin; Liu, Yanyan; Wake, Laura; Liu, Cuiling; Deffenbacher, Karen; Lachel, Cynthia M.; Wang, Chao; Rohr, Joseph; Guo, Shuangping; Smith, Lynette M.; Wright, George; Bhagavathi, Sharathkumar; Dybkaer, Karen; Fu, Kai; Greiner, Timothy C.; Vose, Julie M.; Jaffe, Elaine; Rimsza, Lisa; Rosenwald, Andreas; Ott, German; Delabie, Jan; Campo, Elias; Braziel, Rita M.; Cook, James R.; Tubbs, Raymond R.; Armitage, James O.; Weisenburger, Dennis D.; Staudt, Louis M.; Gascoyne, Randy D.; McKeithan, Timothy W.; Chan, Wing C.

    2015-01-01

    We studied the global microRNA (miRNA) expression in diffuse large B-cell lymphoma (DLBCL; n = 79), Burkitt lymphoma (BL; n = 36), primary mediastinal B-cell lymphoma (PMBL; n = 12), B-cell lines (n = 11), and normal subsets of naïve B cells, centroblasts (CBs), and peripheral blood B cells along with their corresponding gene expression profiles (GEPs). The normal B-cell subsets have well-defined miRNA signatures. The CB miRNA signature was significantly associated with germinal center B-cell (GCB)–DLBCL compared with activated B-cell (ABC)–DLBCL (P = .002). We identified a 27-miRNA signature that included v-myc avian myelomatosis viral oncogene homolog (MYC) targets and enabled the differentiation of BL from DLBCL, a distinction comparable with the “gold standard” GEP-defined diagnosis. Distinct miRNA signatures were identified for DLBCL subgroups, including GCB-DLBCL, activated B-cell (ABC)-DLBCL, and PMBL. Interestingly, most of the unclassifiable-DLBCL by GEP showed a strong similarity to the ABC-DLBCL by miRNA expression profiling. Consistent results for BL and DLBCL subgroup classification were observed in formalin-fixed, paraffin-embedded tissue, making such tests practical for clinical use. We also identified predictive miRNA biomarker signatures in DLBCL, including high expression of miR-155, which is significantly associated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) treatment failure. This finding was further supported by the observation that high expression of miR-155 sensitizes cells to v-akt murine thymoma viral oncogene homolog-1 inhibitors in vitro, suggesting a novel treatment option for resistant DLBCL. PMID:25498913

  6. B Cells: The Old New Players in Reproductive Immunology

    PubMed Central

    Fettke, Franziska; Schumacher, Anne; Costa, Serban-Dan; Zenclussen, Ana Claudia

    2014-01-01

    Reproductive immunology research has long focused on T cell responses to paternal antigens and tolerance mechanisms supporting fetal well-being. The participation of B cells herein was not widely studied. Because of the fascinating immunological uniqueness of pregnancy, it is however to be expected that such pleiotropic cells play a considerable role. In fact, on the one hand B cells contribute toward pregnancy tolerance by secreting the immunomodulatory cytokine IL-10 but on the other hand can seriously harm pregnancy because of their capacity of producing autoantibodies. As for protective B cells, new evidences in mouse models arise suggesting that IL-10 producing B cells, the so-called B10 cells, help in maintaining tolerance toward semi-allogenic fetal antigens. They may be also important to fight danger signals at the fetal-maternal interface as, e.g., in the case of infections with the aim to restore the disrupted fetal tolerance. In human pregnancies, IL-10 producing B cells increase with pregnancy onset but not in the case of spontaneous abortions. In vitro, they are able to suppress TNF-α production by T cells from pregnant individuals. Their generation and functionality will be discussed throughout this review article. B cells can be deleterious to pregnancy as well. Aberrant B cell compartment is associated with obstetric pathologies. In particular, the capacity of B2 cells to produce specific autoantibodies or of B-1a B cells to secrete natural autoantibodies that can turn autoreactive will be discussed herein. PMID:25002862

  7. Are autoantibodies the targets of B-cell-directed therapy?

    PubMed

    Pisetsky, David S; Grammer, Amrie C; Ning, Tony C; Lipsky, Peter E

    2011-09-01

    B-cell-directed therapy-the use of agents that eliminate B cells or block cytokines important for B-cell function-is emerging as a promising approach to the treatment of rheumatic disease. Target diseases, including systemic lupus erythematosus (SLE), display diverse patterns of autoantibody production and aberrant activation of B cells. Despite the success of this general approach, the mechanisms by which B-cell-directed therapy ameliorates disease, and the role of autoantibodies as biomarkers of clinical response remain unclear. Importantly, although B-cell-directed therapy can reduce the production of some autoantibodies, the effects can be variable and heterogeneous, probably reflecting the critical (but ill-defined) roles of different B-cell and plasma cell populations in autoantibody production. Future studies during clinical trials of these agents are needed to define which B-cell and autoantibody populations are affected (or ought to be), and to discover informative biomarkers of clinical response that can be used to advance this therapeutic approach.

  8. A fine romance: T follicular helper cells and B cells.

    PubMed

    King, Cecile

    2011-06-24

    T follicular helper (Tfh) cells help B cells to generate affinity-matured antibodies. Three papers in this issue of Immunity (Choi et al., 2011; Kerfoot et al., 2011; Kitano et al., 2011) provide information about the reciprocal relationship between B cells and Tfh cells.

  9. DNA breaks early in replication in B cell cancers

    Cancer.gov

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  10. A fine romance: T follicular helper cells and B cells.

    PubMed

    King, Cecile

    2011-06-24

    T follicular helper (Tfh) cells help B cells to generate affinity-matured antibodies. Three papers in this issue of Immunity (Choi et al., 2011; Kerfoot et al., 2011; Kitano et al., 2011) provide information about the reciprocal relationship between B cells and Tfh cells. PMID:21703537

  11. B cells as therapeutic targets in autoimmune neurological disorders.

    PubMed

    Dalakas, Marinos C

    2008-10-01

    B cells have a fundamental role in the pathogenesis of various autoimmune neurological disorders, not only as precursors of antibody-producing cells, but also as important regulators of the T-cell activation process through their participation in antigen presentation, cytokine production, and formation of ectopic germinal centers in the intermeningeal spaces. Two B-cell trophic factors-BAFF (B-cell-activating factor) and APRIL (a proliferation-inducing ligand)-and their receptors are strongly upregulated in many immunological disorders of the CNS and PNS, and these molecules contribute to clonal expansion of B cells in situ. The availability of monoclonal antibodies or fusion proteins against B-cell surface molecules and trophic factors provides a rational approach to the treatment of autoimmune neurological diseases. This article reviews the role of B cells in autoimmune neurological disorders and summarizes the experience to date with rituximab, a B-cell-depleting monoclonal antibody against CD20, for the treatment of relapsing-remitting multiple sclerosis, autoimmune neuropathies, neuromyelitis optica, paraneoplastic neurological disorders, myasthenia gravis, and inflammatory myopathies. It is expected that ongoing controlled trials will establish the efficacy and long-term safety profile of anti-B-cell agents in several autoimmune neurological disorders, as well as exploring the possibility of a safe and synergistic effect with other immunosuppressants or immunomodulators.

  12. Therapeutic strategies targeting B-cells in multiple sclerosis.

    PubMed

    Milo, Ron

    2016-07-01

    Multiple sclerosis (MS) is a chronic inflammatory and demyelinating disease of the central nervous system (CNS) that traditionally has been considered to be mediated primarily by T-cells. Increasing evidence, however, suggests the fundamental role of B-cells in the pathogenesis of the disease. Recent strategies targeting B-cells in MS have demonstrated impressive and sometimes surprising results: B-cell depletion by monoclonal antibodies targeting the B-cell surface antigen CD20 (e.g. rituximab, ocrelizumab, ofatumumab) was shown to exert profound anti-inflammatory effect in MS with favorable risk-benefit ratio, with ocrelizumab demonstrating efficacy in both relapsing-remitting (RR) and primary-progressive (PP) MS in phase III clinical trials. Depletion of CD52 expressing T- and B-cells and monocytes by alemtuzumab resulted in impressive and durable suppression of disease activity in RRMS patients. On the other hand, strategies targeting B-cell cytokines such as atacicept resulted in increased disease activity. As our understanding of the biology of B-cells in MS is increasing, new compounds that target B-cells continue to be developed which promise to further expand the armamentarium of MS therapies and allow for more individualized therapy for patients with this complex disease.

  13. B cells do not present antigen covalently linked to microspheres.

    PubMed Central

    Galelli, A; Charlot, B; Dériaud, E; Leclerc, C

    1993-01-01

    B cells have been shown to present antigen to T cells very efficiently through their capacity to capture antigens by their membrane immunoglobulin. This direct cognate interaction of T and B cells results in the proliferation and differentiation of B cells. This concept has been established using soluble proteins. However, most of the antigens to which the immune system is exposed are included in complex particulate structures such as bacteria or parasites. The capacity of B cells to present these large and complex antigens is still unclear. To address this question we have studied the presentation by trinitrophenyl (TNP)-specific B cells of the same antigen TNP-KLH (keyhole limpet haemocyanin), either in a soluble form or covalently linked to poly(acrolein) microspheres, from 0.25 to 1.5 microns in diameter. In the presence of irradiated splenocytes or purified macrophages as a source of antigen-presenting cells (APC), KLH-specific T cells proliferated in response to soluble TNP-KLH or to TNP-KLH coupled to beads. In contrast, TNP-specific memory B cells were totally ineffective in presenting the TNP-KLH beads to KLH-specific T cells whereas they presented very efficiently soluble TNP-KLH. Similar results were obtained with the A20 B lymphoma or with lipopolysaccharide (LPS)-activated TNP-specific B cells. These results therefore indicate that B cells are unable to present large size particulate antigens such as bacteria or parasites. PMID:8509143

  14. DHA-enriched fish oil targets B cell lipid microdomains and enhances ex vivo and in vivo B cell function

    PubMed Central

    Gurzell, Eric A.; Teague, Heather; Harris, Mitchel; Clinthorne, Jonathan; Shaikh, Saame Raza; Fenton, Jenifer I.

    2013-01-01

    DHA is a n-3 LCPUFA in fish oil that generally suppresses T lymphocyte function. However, the effect of fish oil on B cell function remains relatively understudied. Given the important role of B cells in gut immunity and increasing human fish oil supplementation, we sought to determine whether DFO leads to enhanced B cell activation in the SMAD−/− colitis-prone mouse model, similar to that observed with C57BL/6 mice. This study tested the hypothesis that DHA from fish oil is incorporated into the B cell membrane to alter lipid microdomain clustering and enhance B cell function. Purified, splenic B cells from DFO-fed mice displayed increased DHA levels and diminished GM1 microdomain clustering. DFO enhanced LPS-induced B cell secretion of IL-6 and TNF-α and increased CD40 expression ex vivo compared with CON. Despite increased MHCII expression in the unstimulated ex vivo B cells from DFO-fed mice, we observed no difference in ex vivo OVA-FITC uptake in B cells from DFO or CON mice. In vivo, DFO increased lymphoid tissue B cell populations and surface markers of activation compared with CON. Finally, we investigated whether these ex vivo and in vivo observations were consistent with systemic changes. Indeed, DFO-fed mice had significantly higher plasma IL-5, IL-13, and IL-9 (Th2-biasing cytokines) and cecal IgA compared with CON. These results support the hypothesis and an emerging concept that fish oil enhances B cell function in vivo. PMID:23180828

  15. Precursor B Cells Increase in the Lung during Airway Allergic Inflammation: A Role for B Cell-Activating Factor

    PubMed Central

    Malmhäll, Carina; Rådinger, Madeleine; Ramos-Ramirez, Patricia; Lu, You; Deák, Tünde; Semitekolou, Maria; Gaga, Mina; Sjöstrand, Margareta; Lötvall, Jan; Bossios, Apostolos

    2016-01-01

    Background B cells, key cells in allergic inflammation, differentiate in the bone marrow and their precursors include pro-B, pre-B and immature B cells. Eosinophil progenitor cells increase in the lung after allergen exposure. However, the existence and possible role of B cell precursors in the lung during allergic inflammation remains elusive. Methods A BALB/c mouse model of allergic airway inflammation was utilized to perform phenotypic and quantification analyses of pro-B and pre-B cells in the lung by flow cytometry. B cell maturation factors IL-7 and B cell-activating factor (BAFF) and their receptors (CD127 and BAFFR, BCMA, TACI, respectively) were also evaluated in the lung and serum. The effect of anti-BAFF treatment was investigated both in vivo (i.p. administration of BAFF-R-Ig fusion protein) and in vitro (colony forming cell assay). Finally, BAFF levels were examined in the bronchoalveolar lavage (BAL) of asthmatic patients and healthy controls. Results Precursor pro and pre-B cells increase in the lung after allergen exposure, proliferate in the lung tissue in vivo, express markers of chemotaxis (CCR10 and CXCR4) and co-stimulation (CD40, CD86) and are resistant to apoptosis (Bax). Precursor B cells express receptors for BAFF at baseline, while after allergen challenge both their ligand BAFF and the BCMA receptor expression increases in B cell precursors. Blocking BAFFR in the lung in vivo decreases eosinophils and proliferating precursor B cells. Blocking BAFFR in bone marrow cultures in vitro reduces pre-B colony formation units. BAFF is increased in the BAL of severe asthmatics. Conclusion Our data support the concept of a BAFF-mediated role for B cell precursors in allergic airway inflammation. PMID:27513955

  16. Dynamic Epstein-Barr virus gene expression on the path to B-cell transformation.

    PubMed

    Price, Alexander M; Luftig, Micah A

    2014-01-01

    Epstein-Barr virus (EBV) is an oncogenic human herpesvirus in the γ-herpesvirinae subfamily that contains a 170-180kb double-stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B-cell compartment of the peripheral blood. EBV can be reactivated from its latent state, leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome and structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady-state viral gene expression within EBV-immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection, EBV only expressed the well-characterized latency-associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation and delayed responses in the known latency genes. This chapter summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, the inhibition of apoptosis, and innate and adaptive immune responses. PMID:24373315

  17. NK cell depletion diminish tumour-specific B cell responses.

    PubMed

    Jensen, Markus; Tawadros, Samir; Sedlacek, Hans-Harald; Schultze, Joachim L; Berthold, Frank

    2004-05-15

    Natural killer (NK) cells can exercise immediate cytotoxicity against malignant cells and thus far modulate the development of tumour directed T cell immunity. To investigate the impact of NK cells on the development of tumour directed B cell immunity mice were immunised with IMR5-75 human neuroblastoma cells with or without prior in vivo NK cell depletion. Flow cytometry analyses gave evidence for an impaired IgG response against the cells immunised with. Dissection of Th1 (IgG2a) and Th2 (IgG1) oriented B cell responses revealed Th1 responses as primarily affected, while Th2 oriented B cell responses as measured by flow cytometry and GD2 ganglioside-specific ELISA were enforced. The data reveal an unexpected impact of NK cells on the development of tumour directed B cell responses. Consequently, NK cell function has also to be taken into account when developing B cell-based cancer immunotherapy.

  18. The regulation and activation of lupus-associated B cells.

    PubMed

    Fields, Michele L; Hondowicz, Brian D; Wharton, Gina N; Adair, Brigette S; Metzgar, Michele H; Alexander, Shawn T; Caton, Andrew J; Erikson, Jan

    2005-04-01

    Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.

  19. Loss-of-Function Mutants and Overexpression Lines of the Arabidopsis Cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS Exhibit Different Defects in Prophase-I Meiocytes but Produce the Same Meiotic Products

    PubMed Central

    Wang, Yixing; Yang, Ming

    2014-01-01

    In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products. PMID:25402453

  20. Loss-of-function mutants and overexpression lines of the Arabidopsis cyclin CYCA1;2/Tardy Asynchronous Meiosis exhibit different defects in prophase-i meiocytes but produce the same meiotic products.

    PubMed

    Wang, Yixing; Yang, Ming

    2014-01-01

    In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/Tardy Asynchronous Meiosis (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products. PMID:25402453

  1. B-cell receptor signaling inhibitors for treatment of autoimmune inflammatory diseases and B-cell malignancies.

    PubMed

    Puri, Kamal D; Di Paolo, Julie A; Gold, Michael R

    2013-08-01

    B-cell receptor (BCR) signaling is essential for normal B-cell development, selection, survival, proliferation, and differentiation into antibody-secreting cells. Similarly, this pathway plays a key role in the pathogenesis of multiple B-cell malignancies. Genetic and pharmacological approaches have established an important role for the Spleen tyrosine kinase (Syk), Bruton's tyrosine kinase (Btk), and phosphatidylinositol 3-kinase isoform p110delta (PI3Kδ) in coupling the BCR and other BCRs to B-cell survival, migration, and activation. In the past few years, several small-molecule inhibitory drugs that target PI3Kδ, Btk, and Syk have been developed and shown to have efficacy in clinical trials for the treatment of several types of B-cell malignancies. Emerging preclinical data have also shown a critical role of BCR signaling in the activation and function of self-reactive B cells that contribute to autoimmune diseases. Because BCR signaling plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, inhibition of this pathway may represent a promising new strategy for treating these diseases. This review summarizes recent achievements in the mechanism of action, pharmacological properties, and clinical activity and toxicity of these BCR signaling inhibitors, with a focus on their emerging role in treating lymphoid malignancies and autoimmune disorders.

  2. Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking

    PubMed Central

    Flores-Fernández, Rocio; Blanco-Favela, Francisco; Fuentes-Pananá, Ezequiel M.; Chávez-Sánchez, Luis; Gorocica-Rosete, Patricia; Pizaña-Venegas, Alberto; Chávez-Rueda, Adriana Karina

    2016-01-01

    Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease. PMID:27314053

  3. Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

    PubMed

    Sarkar, Mohosin; Liu, Yun; Qi, Junpeng; Peng, Haiyong; Morimoto, Jumpei; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2016-04-01

    Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.

  4. Selective loss of B-cell phenotype in lymphocyte predominant Hodgkin lymphoma.

    PubMed

    Tedoldi, S; Mottok, A; Ying, J; Paterson, J C; Cui, Y; Facchetti, F; van Krieken, J H J M; Ponzoni, M; Ozkal, S; Masir, N; Natkunam, Y; Pileri, Sa; Hansmann, M-L; Mason, Dy; Tao, Q; Marafioti, T

    2007-12-01

    The neoplastic Reed-Sternberg cells characteristic of classical Hodgkin's lymphoma (cHL) are of B-cell origin but they almost always show striking loss of a range of B-cell-associated molecules. In contrast, the neoplastic cells found in lymphocyte predominant Hodgkin's lymphoma (LPHL) (L&H cells) are traditionally thought of as possessing the full repertoire of features associated with germinal centre B cells (eg BCL-6 expression, 'ongoing' Ig gene mutation). In the present paper, we report an extensive phenotypic analysis of L&H cells which revealed down-regulation of a number of markers associated with the B-cell lineage (eg CD19, CD37) and with the germinal centre maturation stage (eg PAG, LCK). The promoter methylation status of three of these down-regulated genes (CD10, CD19, and LCK) was further studied in microdissected L&H cells, and this revealed that their promoters were unmethylated. In contrast, these genes showed promoter methylation in cell lines derived from CHL. Further investigation of the mechanisms responsible for the deregulation of these molecules in L&H cells may provide new insights into the genetic abnormalities underlying LPHL. PMID:17935142

  5. Transcriptional control of MHC class II gene expression during differentiation from B cells to plasma cells.

    PubMed

    Dellabona, P; Latron, F; Maffei, A; Scarpellino, L; Accolla, R S

    1989-04-15

    In this study we investigated the molecular mechanisms responsible for the extinction of the constitutive MHC class II gene expression of human B cells on somatic cell hybridization with murine plasmocytoma cells. We found that this event is due to trans-acting suppressor functions of mouse origin pre-existing in the plasmocytoma cells and acting at transcriptional level. Transcription of the entire family of human class II genes is suppressed, including genes as DO beta for which a distinct regulation of expression in B cells had been previously demonstrated. Suppression appears specific for class II genes because in the hybrids expression of MHC class I genes of mouse is unaffected and of human only partially reduced. Interestingly, also murine invariant chain gene is expressed in both parental plasmocytoma and hybrid cells although at reduced amounts as compared to a murine class II positive B cell line. The class II negative phenotype of hybrid cells and parental plasmocytoma cells is highly stable and unaffected by treatment with protein synthesis inhibitors, suggesting that the transcriptional suppressor function is not mediated by rapid, labile turning-over proteins. Possible mechanisms responsible for transcriptional regulation of MHC class II gene expression during terminal differentiation of B cells to plasma cells are discussed. PMID:2495328

  6. Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

    PubMed

    Sarkar, Mohosin; Liu, Yun; Qi, Junpeng; Peng, Haiyong; Morimoto, Jumpei; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

    2016-04-01

    Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset. PMID:26851280

  7. Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling

    PubMed Central

    1995-01-01

    To explore the mechanism(s) by which the Syk protein tyrosine kinase participates in B cell antigen receptor (BCR) signaling, we have studied the function of various Syk mutants in B cells made Syk deficient by homologous recombination knockout. Both Syk SH2 domains were required for BCR-mediated Syk and phospholipase C (PLC)-gamma 2 phosphorylation, inositol 1,4,5-triphosphate release, and Ca2+ mobilization. A possible explanation for this requirement was provided by findings that recruitment of Syk to tyrosine-phosphorylated immunoglobulin (Ig) alpha and Ig beta requires both Syk SH2 domains. A Syk mutant in which the putative autophosphorylation site (Y518/Y519) of Syk was changed to phenylalanine was also defective in signal transduction; however, this mutation did not affect recruitment to the phosphorylated immunoreceptor family tyrosine-based activation motifs (ITAMs). These findings not only confirm that both SH2 domains are necessary for Syk binding to tyrosine-phosphorylated Ig alpha and Ig beta but indicate that this binding is necessary for Syk (Y518/519) phosphorylation after BCR ligation. This sequence of events is apparently required for coupling the BCR to most cellular protein tyrosine phosphorylation, to the phosphorylation and activation of PLC- gamma 2, and to Ca2+ mobilization. PMID:7500027

  8. Gene expression profiling of the response to interferon beta in Epstein-Barr-transformed and primary B cells of patients with multiple sclerosis.

    PubMed

    Khsheibun, Rana; Paperna, Tamar; Volkowich, Anat; Lejbkowicz, Izabella; Avidan, Nili; Miller, Ariel

    2014-01-01

    The effects of interferon-beta (IFN-β), one of the key immunotherapies used in multiple sclerosis (MS), on peripheral blood leukocytes and T cells have been extensively studied. B cells are a less abundant leukocyte type, and accordingly less is known about the B cell-specific response to IFN-β. To identify gene expression changes and pathways induced by IFN-β in B cells, we studied the in vitro response of human Epstein Barr-transformed B cells (lymphoblast cell lines-LCLs), and validated our results in primary B cells. LCLs were derived from an MS patient repository. Whole genome expression analysis identified 115 genes that were more than two-fold differentially up-regulated following IFN-β exposure, with over 50 previously unrecognized as IFN-β response genes. Pathways analysis demonstrated that IFN-β affected LCLs in a similar manner to other cell types by activating known IFN-β canonical pathways. Additionally, IFN-β increased the expression of innate immune response genes, while down-regulating many B cell receptor pathway genes and genes involved in adaptive immune responses. Novel response genes identified herein, NEXN, DDX60L, IGFBP4, and HAPLN3, B cell receptor pathway genes, CD79B and SYK, and lymphocyte activation genes, LAG3 and IL27RA, were validated as IFN-β response genes in primary B cells. In this study new IFN-β response genes were identified in B cells, with possible implications to B cell-specific functions. The study's results emphasize the applicability of LCLs for studies of human B cell drug response. The usage of LCLs from patient-based repositories may facilitate future studies of drug response in MS and other immune-mediated disorders with a B cell component.

  9. Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Hussain, Alamdar; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2013-08-01

    Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

  10. B Cells in Chronic Graft versus Host Disease

    PubMed Central

    Sarantopoulos, Stefanie; Blazar, Bruce R.; Cutler, Corey; Ritz, Jerome

    2015-01-01

    Chronic graft versus host disease (cGVHD) continues to be a common complication of allogeneic hematopoietic stem cell transplantation (HSCT). Unlike acute GVHD, which is mediated almost entirely by donor T cells, the immune pathology of cGVHD is more complex and donor B cells have also been found to play an important role. Recent studies from several laboratories have enhanced our understanding of how donor B cells contribute to this clinical syndrome and this has led to new therapeutic opportunities. Here, Dr. Sarantopoulos reviews some of the important mechanisms responsible for persistent B cell activation and loss of B cell tolerance in patients with cGVHD. Dr. Blazar describes recent studies in preclinical models that have identified novel B cell directed agents that may be effective for prevention or treatment of cGVHD. Some B cell directed therapies have already been tested in patients with cGVHD and Dr. Cutler reviews the results of these studies documenting the potential efficacy of this approach. Supported by studies mechanistic studies in patients and preclinical models, new B cell directed therapies for cGVHD will now be evaluated in clinical trials. PMID:25452031

  11. Gallium arsenide exposure impairs splenic B cell accessory function.

    PubMed

    Gondre-Lewis, Timothy A; Hartmann, Constance B; Caffrey, Rebecca E; McCoy, Kathleen L

    2003-03-01

    Gallium arsenide (GaAs) is utilized in industries for its semiconductor and optical properties. Chemical exposure of animals systemically suppresses several immune functions. The ability of splenic B cells to activate antigen-specific helper CD4(+) T cell hybridomas was assessed, and various aspects of antigen-presenting cell function were examined. GaAs-exposed murine B cells were impaired in processing intact soluble protein antigens, and the defect was antigen dependent. In contrast, B cells after exposure competently presented peptides to the T cells, which do not require processing. Cell surface expression of major histocompatibility complex (MHC) class II molecules and several costimulatory molecules on splenic B cells, which are critical for helper T cell activation, was not affected by chemical exposure. GaAs exposure also did not influence the stability of MHC class II heterodimers, suggesting that the defect may precede peptide exchange. GaAs-exposed B cells contained a normal level of aspartyl cathepsin activity; however, proteolytic activities of thiol cathepsins B and L were approximately half the control levels. Furthermore, two cleavage fragments of invariant chain, a molecular chaperone of MHC class II molecules, were increased in GaAs-exposed B cells, indicative of defective degradation. Thus, diminished thiol proteolytic activity in B cells may be responsible for their impaired antigen processing and invariant chain degradation, which may contribute to systemic immunosuppression caused by GaAs exposure.

  12. YY1 Is Required for Germinal Center B Cell Development

    PubMed Central

    Vuyyuru, Raja; Jha, Vibha; Hodewadekar, Suchita; Manser, Tim; Atchison, Michael L.

    2016-01-01

    YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction. PMID:27167731

  13. The Relationship between B-cell Epitope and Mimotope Sequences.

    PubMed

    Zhang, Chunhua; Li, Yunyun; Tang, Weina; Zhou, Zhiguo; Sun, Pingping; Ma, Zhiqiang

    2016-01-01

    B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint.

  14. Salmonella induces PD-L1 expression in B cells.

    PubMed

    Lopez-Medina, Marcela; Perez-Lopez, Araceli; Alpuche-Aranda, Celia; Ortiz-Navarrete, Vianney

    2015-10-01

    Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.

  15. The Relationship between B-cell Epitope and Mimotope Sequences.

    PubMed

    Zhang, Chunhua; Li, Yunyun; Tang, Weina; Zhou, Zhiguo; Sun, Pingping; Ma, Zhiqiang

    2016-01-01

    B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint. PMID:26715528

  16. Characterization of a rabbit germ-line VH gene that is a candidate donor for VH gene conversion in mutant Alicia rabbits.

    PubMed

    Chen, H T; Alexander, C B; Mage, R G

    1995-06-15

    Normal rabbits preferentially rearrange the 3'-most VH gene, VH1, to encode Igs with VHa allotypes, which constitute the majority of rabbit serum Igs. A gene conversion-like mechanism is employed to diversify the primary Ab repertoire. In mutant Alicia rabbits that derived from a rabbit with VHa2 allotype, the VH1 gene was deleted. Our previous studies showed that the first functional gene (VH4) or VH4-like genes were rearranged in 2- to 8-wk-old homozygous Alicia. The VH1a2-like sequences that were found in splenic mRNA from 6-wk and older Alicia rabbits still had some residues that were typical of VH4. The appearances of sequences resembling that of VH1a2 may have been caused by gene conversions that altered the sequences of the rearranged VH or there may have been rearrangement of upstream VH1a2-like genes later in development. To investigate this further, we constructed a cosmid library and isolated a VH1a2-like gene, VH12-1-6, with a sequence almost identical to VH1a2. This gene had a deleted base in the heptamer of its recombination signal sequence. However, even if this defect diminished or eliminated its ability to rearrange, the a2-like gene could have acted as a donor for gene-conversion-like alteration of rearranged VH genes. Sequence comparisons suggested that this gene or a gene like it could have acted as a donor for gene conversion in mutant Alicia and in normal rabbits.

  17. Human Memory B Cells in Healthy Gingiva, Gingivitis, and Periodontitis.

    PubMed

    Mahanonda, Rangsini; Champaiboon, Chantrakorn; Subbalekha, Keskanya; Sa-Ard-Iam, Noppadol; Rattanathammatada, Warattaya; Thawanaphong, Saranya; Rerkyen, Pimprapa; Yoshimura, Fuminobu; Nagano, Keiji; Lang, Niklaus P; Pichyangkul, Sathit

    2016-08-01

    The presence of inflammatory infiltrates with B cells, specifically plasma cells, is the hallmark of periodontitis lesions. The composition of these infiltrates in various stages of homeostasis and disease development is not well documented. Human tissue biopsies from sites with gingival health (n = 29), gingivitis (n = 8), and periodontitis (n = 21) as well as gingival tissue after treated periodontitis (n = 6) were obtained and analyzed for their composition of B cell subsets. Ag specificity, Ig secretion, and expression of receptor activator of NF-κB ligand and granzyme B were performed. Although most of the B cell subsets in healthy gingiva and gingivitis tissues were CD19(+)CD27(+)CD38(-) memory B cells, the major B cell component in periodontitis was CD19(+)CD27(+)CD38(+)CD138(+)HLA-DR(low) plasma cells, not plasmablasts. Plasma cell aggregates were observed at the base of the periodontal pocket and scattered throughout the gingiva, especially apically toward the advancing front of the lesion. High expression of CXCL12, a proliferation-inducing ligand, B cell-activating factor, IL-10, IL-6, and IL-21 molecules involved in local B cell responses was detected in both gingivitis and periodontitis tissues. Periodontitis tissue plasma cells mainly secreted IgG specific to periodontal pathogens and also expressed receptor activator of NF-κB ligand, a bone resorption cytokine. Memory B cells resided in the connective tissue subjacent to the junctional epithelium in healthy gingiva. This suggested a role of memory B cells in maintaining periodontal homeostasis. PMID:27335500

  18. B-cell memory and the persistence of antibody responses.

    PubMed Central

    MacLennan, I C; García de Vinuesa, C; Casamayor-Palleja, M

    2000-01-01

    Antigens such as viral envelope proteins and bacterial exotoxins induce responses which result in the production of neutralizing antibody. These responses persist for years and provide highly efficient defence against reinfection. During these antibody responses a proportion of participating B cells mutate the genes that encode their immunoglobulin variable regions. This can increase the affinity of the antibody, but can also induce autoreactive B cells. Selection mechanisms operate which allow the cells with high affinity for the provoking antigen to persist, while other B cells recruited into the response die. PMID:10794052

  19. BAFF: a fundamental survival factor for B cells.

    PubMed

    Mackay, Fabienne; Browning, Jeffrey L

    2002-07-01

    B-cell-activating factor of the tumour-necrosis-factor family (BAFF) enhances B-cell survival--a function that is indispensable for B-cell maturation--and has a role in enhancing immune responses. Moreover, the overexpression of BAFF results in severe autoimmune disorders in mice, and elevated serum levels of BAFF occur in some patients who have autoimmune diseases. The elucidation of the role of BAFF has set the stage for a new approach to the treatment of autoimmune disease.

  20. PI3 Kinase signals BCR dependent mature B cell survival

    PubMed Central

    Srinivasan, Lakshmi; Sasaki, Yoshiteru; Calado, Dinis Pedro; Zhang, Baochun; Paik, Ji Hye; DePinho, Ronald A.; Kutok, Jeffrey L.; Kearney, John F.; Otipoby, Kevin L.; Rajewsky, Klaus

    2009-01-01

    Summary Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role. PMID:19879843

  1. SAR3419: an anti-CD19-Maytansinoid Immunoconjugate for the treatment of B-cell malignancies.

    PubMed

    Blanc, Veronique; Bousseau, Anne; Caron, Anne; Carrez, Chantal; Lutz, Robert J; Lambert, John M

    2011-10-15

    SAR3419 is a novel anti-CD19 humanized monoclonal antibody conjugated to a maytansine derivate through a cleavable linker for the treatment of B-cell malignancies. SAR3419 combines the strengths of a high-potency tubulin inhibitor and the exquisite B-cell selectivity of an anti-CD19 antibody. The internalization and processing of SAR3419, following its binding at the surface of CD19-positive human lymphoma cell lines and xenograft models, release active metabolites that trigger cell-cycle arrest and apoptosis, leading to cell death and tumor regression. SAR3419 has also been shown to be active in different lymphoma xenograft models, including aggressive diffuse large B-cell lymphoma, resulting in complete regressions and tumor-free survival. In these models, the activity of SAR3419 compared favorably with rituximab and lymphoma standard of care chemotherapy. Two phase I trials with 2 different schedules of SAR3419 as a single agent were conducted in refractory/relapsed B-cell non-Hodgkin lymphoma. Activity was reported in both schedules, in heavily pretreated patients of both follicular and diffuse large B-cell lymphoma subtypes, with a notable lack of significant hematological toxicity, validating SAR3419 as an effective antibody-drug conjugate and opening opportunities in the future. Numerous B-cell-specific anti-CD19 biologics are available to treat B-cell non-Hodgkin lymphoma, and early phase I results obtained with SAR3419 suggest that it is a promising candidate for further development in this disease. In addition, thanks to the broad expression of CD19, SAR3419 may provide treatment options for B-cell leukemias that are often CD20-negative. PMID:22003072

  2. HIV-dependent depletion of influenza-specific memory B cells impacts B cell responsiveness to seasonal influenza immunisation

    PubMed Central

    Wheatley, Adam K.; Kristensen, Anne B.; Lay, William N.; Kent, Stephen J.

    2016-01-01

    Infection with HIV drives significant alterations in B cell phenotype and function that can markedly influence antibody responses to immunisation. Anti-retroviral therapy (ART) can partially reverse many aspects of B cell dysregulation, however complete normalisation of vaccine responsiveness is not always observed. Here we examine the effects of underlying HIV infection upon humoral immunity to seasonal influenza vaccines. Serological and memory B cell responses were assessed in 26 HIV+ subjects receiving ART and 30 healthy controls immunised with the 2015 Southern Hemisphere trivalent inactivated influenza vaccine (IIV3). Frequencies and phenotypes of influenza hemagglutinin (HA)-specific B cells were assessed by flow cytometry using recombinant HA probes. Serum antibody was measured using hemagglutination inhibition assays. Serological responses to IIV3 were comparable between HIV+ and HIV− subjects. Likewise, the activation and expansion of memory B cell populations specific for vaccine-component influenza strains was observed in both cohorts, however peak frequencies were diminished in HIV+ subjects compared to uninfected controls. Lower circulating frequencies of memory B cells recognising vaccine-component and historical influenza strains were observed in HIV+ subjects at baseline, that were generally restored to levels comparable with HIV− controls post-vaccination. HIV infection is therefore associated with depletion of selected HA-specific memory B cell pools. PMID:27220898

  3. ATM deficiency promotes development of murine B-cell lymphomas that resemble diffuse large B-cell lymphoma in humans

    PubMed Central

    Hathcock, Karen S.; Padilla-Nash, Hesed M.; Camps, Jordi; Shin, Dong-Mi; Triner, Daniel; Shaffer, Arthur L.; Maul, Robert W.; Steinberg, Seth M.; Gearhart, Patricia J.; Staudt, Louis M.; Morse, Herbert C.; Ried, Thomas

    2015-01-01

    The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M+ B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell–like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors. PMID:26400962

  4. B Cell Development in the Bone Marrow Is Regulated by Homeostatic Feedback Exerted by Mature B Cells

    PubMed Central

    Shahaf, Gitit; Zisman-Rozen, Simona; Benhamou, David; Melamed, Doron; Mehr, Ramit

    2016-01-01

    Cellular homeostasis in the B cell compartment is strictly imposed to balance cell production and cell loss. However, it is not clear whether B cell development in the bone marrow is an autonomous process or subjected to regulation by the peripheral B cell compartment. To specifically address this question, we used mice transgenic for human CD20, where effective depletion of B lineage cells is obtained upon administration of mouse anti-human CD20 antibodies, in the absence of any effect on other cell lineages and/or tissues. We followed the kinetics of B cell return to equilibrium by BrdU labeling and flow cytometry and analyzed the resulting data by mathematical modeling. Labeling was much faster in depleted mice. Compared to control mice, B cell-depleted mice exhibited a higher proliferation rate in the pro-/pre-B compartment, and higher cell death and lower differentiation in the immature B cell compartment. We validated the first result by analysis of the expression of Ki67, the nuclear protein expressed in proliferating cells, and the second using Annexin V staining. Collectively, our results suggest that B lymphopoiesis is subjected to homeostatic feedback mechanisms imposed by mature B cells in the peripheral compartment. PMID:27047488

  5. Transgelin-2 in B-Cells Controls T-Cell Activation by Stabilizing T Cell - B Cell Conjugates

    PubMed Central

    Chae, Myoung-Won; Kim, Hye-Ran; Kim, Chang-Hyun; Jun, Chang-Duk; Park, Zee-Yong

    2016-01-01

    The immunological synapse (IS), a dynamic and organized junction between T-cells and antigen presenting cells (APCs), is critical for initiating adaptive immunity. The actin cytoskeleton plays a major role in T-cell reorganization during IS formation, and we previously reported that transgelin-2, an actin-binding protein expressed in T-cells, stabilizes cortical F-actin, promoting T-cell activation in response to antigen stimulation. Transgelin-2 is also highly expressed in B-cells, although no specific function has been reported. In this study, we found that deficiency in transgelin-2 (TAGLN2-/-) in B-cells had little effect on B-cell development and activation, as measured by the expression of CD69, MHC class II molecules, and CD80/86. Nevertheless, in B-cells, transgelin-2 accumulated in the IS during the interaction with T-cells. These results led us to hypothesize that transgelin-2 may also be involved in IS stability in B-cells, thereby influencing T-cell function. Notably, we found that transgelin-2 deficiency in B-cells reduced T-cell activation, as determined by the release of IL-2 and interferon-γ and the expression of CD69. Furthermore, the reduced T-cell activation was correlated with reduced B-cell–T-cell conjugate formation. Collectively, these results suggest that actin stability in B-cells during IS formation is critical for the initiation of adaptive T-cell immunity. PMID:27232882

  6. B cells contribute to heterogeneity of IL-17 producing cells in rheumatoid arthritis and healthy controls.

    PubMed

    Schlegel, Paul Martin; Steiert, Ingeborg; Kötter, Ina; Müller, Claudia A

    2013-01-01

    Secretion of the proinflammatory cytokine Interleukin-17A (IL-17A) is the hallmark of a unique lineage of CD4 T cells designated Th17 cells, which may play a crucial role in the pathogenesis of rheumatoid arthritis (RA) and many autoimmune diseases. Recently, IL-17-producing cells other than T cells have been described, including diverse innate immune cells. Here, we show that the cellular sources of IL-17A in RA include a significant number of non-T cells. Multicolour fluorescence analysis of IL-17-expressing peripheral blood mononuclear cells (PBMC) revealed larger proportions of IL-17(+)CD3(-) non-T cells in RA patients than in healthy controls (constitutive, 13.6% vs. 8.4%, and after stimulation with PMA/ionomycin 17.4% vs. 7.9% p < 0.001 in both cases). The source of IL-17 included CD3(-)CD56(+) NK cells, CD3(-)CD14(+) myeloid cells as well as the expected CD3(+)CD4(+) Th17 cells and surprisingly a substantial number of CD3(-)CD19(+) B cells. The presence of IL-17A-expressing B cells was confirmed by specific PCR of peripheral MACS-sorted CD19(+) B cells, as well as by the analysis of different EBV-transformed B cell lines. Here we report for the first time that in addition to Th17 cells and different innate immune cells B cells also contribute to the IL-17A found in RA patients and healthy controls.

  7. Immature B Cell Egress from Bone Marrow Is SOCS3 Independent

    PubMed Central

    Pereira, João P.

    2015-01-01

    Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3fl/fl Mb1cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling. PMID:26274929

  8. Reduction of lignin levels in mutant sorghum lines developed for saccharification leads to increased production of insecticidal compounds in stalk pith

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of material for biomass that can be converted to energy sources such as ethanol is impeded by the presence of lignin that limits saccharafication. Lines of crops such as sorghum have been developed with reduced levels of lignin that have promise for use in bioenergy production due to enha...

  9. Baseline, Trend, and Normalization of Carcinoembryonic Antigen as Prognostic Factors in Epidermal Growth Factor Receptor-Mutant Nonsmall Cell Lung Cancer Patients Treated With First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

    PubMed Central

    Chen, Yu-Mu; Lai, Chien-Hao; Chang, Huang-Chih; Chao, Tung-Ying; Tseng, Chia-Cheng; Fang, Wen-Feng; Wang, Chin-Chou; Chung, Yu-Hsiu; Huang, Kuo-Tung; Chen, Hung-Cheng; Chang, Ya-Chun; Lin, Meng-Chih

    2015-01-01

    Abstract Among epidermal growth factor receptor (EGFR) mutation status unknown nonsmall cell lung cancer (NSCLC) patients, those with higher carcinoembryonic antigen (CEA) level are more likely to response to EGFR-tyrosine kinase inhibitors (TKIs) because they tend to have mutant epidermal growth factor receptor (EGFR). However, patients with higher CEA also have more tumor burden. With the above paradoxical evidence, it is prudent to understand the prognostic significance of baseline CEA in patients with EGFR-mutant NSCLC treated with first-line EGFR-TKIs. The clinical significance of the trend in CEA after treatment and the impact of CEA normalization during EGFR-TKI therapy are also unknown and potentially important. A total of 241 patients who received first-line EGFR-TKIs were included. As to baseline CEA, patients were divided into normal, low, and high baseline CEA by cut point determined by receiver operating characteristic curves. As to CEA responses, patients were divided into 3 groups accordingly to their amount of CEA change after taking TKIs. In group A, 1-month follow-up CEA level decreased more than 35% with nadir CEA normalization; in group B, 1-month follow-up CEA level decreased more than 35% without nadir CEA normalization; and in group C, 1-month follow-up CEA level decreased less than 35% or increased. Patients with higher baseline CEA levels had shorter progression-free survival (PFS) and overall survival (OS) (CEA > 32 vs 5–32 vs <5 ng/mL, PFS = 8.8 vs 11.3 vs 14.4 months, respectively, P < 0.001; OS = 17.8 vs 22.0 vs 27.9 months, respectively, P = 0.01). For trend and CEA normalization in groups A, B, and C, PFS was 14.3, 10.6, and 7.1 months, respectively (P < 0.001); OS was 29.7, 20.0, and 16.2 months, respectively (P < 0.001). Baseline, trend, and normalization of CEA levels are potential prognostic markers for patients with EGFR-mutant advanced NSCLC treated with first line EGFR-TKIs. PMID:26683939

  10. B-cell survival factors in autoimmune rheumatic disorders.

    PubMed

    Morais, Sandra A; Vilas-Boas, Andreia; Isenberg, David A

    2015-08-01

    Autoimmune rheumatic disorders have complex etiopathogenetic mechanisms in which B cells play a central role. The importance of factors stimulating B cells, notably the B-cell activating factor (BAFF) and A proliferation inducing ligand (APRIL) axis is now recognized. BAFF and APRIL are cytokines essential for B-cell proliferation and survival from the immature stages to the development of plasma cells. Their levels are increased in some subsets of patients with autoimmune disorders. Several recent biologic drugs have been developed to block this axis, namely belimumab [already licensed for systemic lupus erythematosus (SLE) treatment], tabalumab, atacicept and blisibimod. Many clinical trials to evaluate the safety and efficacy of these drugs in several autoimmune disorders are ongoing, or have been completed recently. This review updates the information on the use of biologic agents blocking BAFF/APRIL for patients with SLE, rheumatoid arthritis, Sjögren's syndrome and myositis. PMID:26288664

  11. An eruption of European B-cell biology.

    PubMed

    Cancro, Michael P

    2010-09-01

    Volcanic ash clouds disrupted the 2010 ESF/EMBO meeting on B cells and protection. Nevertheless, the delegates who did make it to Catalonia put together their own programme of talks covering a range of themes from mutualism to epigenetics.

  12. BAFF suppresses IL-15 expression in B cells.

    PubMed

    Ma, Ning; Xing, Chen; Xiao, He; He, Youdi; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Marrero, Bernadette; Wang, Yujuan; Zhang, Shengquan; Shen, Beifen; Li, Yan; Wang, Renxi

    2014-05-01

    Clinical trials have shown that BAFF inhibitors do not reduce memory B cell levels but can reduce the number of mature B cells. It remains uncertain whether BAFF affects memory-maintaining cytokines such as IL-15. We found that BAFF suppressed IL-15 expression in B cells from lupus-like or experimental allergic encephalomyelitis mice. When BAFF was blocked with atacicept-IgG, IL-15 expression was upregulated in lupus-like or experimental allergic encephalomyelitis mice. Finally, we showed that BAFF suppressed IL-15 expression in transitional 2 B cells by reducing Foxo1 expression and inducing Foxo1 phosphorylation. This study suggests that BAFF suppresses IL-15 expression in autoimmune diseases, and this opens up the possible opportunity for the clinical application of BAFF- and IL-15-specific therapeutic agents.

  13. How Follicular Dendritic Cells Shape the B-Cell Antigenome

    PubMed Central

    Kranich, Jan; Krautler, Nike Julia

    2016-01-01

    Follicular dendritic cells (FDCs) are stromal cells residing in primary follicles and in germinal centers of secondary and tertiary lymphoid organs (SLOs and TLOs). There, they play a crucial role in B-cell activation and affinity maturation of antibodies. FDCs have the unique capacity to bind and retain native antigen in B-cell follicles for long periods of time. Therefore, FDCs shape the B-cell antigenome (the sum of all B-cell antigens) in SLOs and TLOs. In this review, we discuss recent findings that explain how this stromal cell type can arise in almost any tissue during TLO formation and, furthermore, focus on the mechanisms of antigen capture and retention involved in the generation of long-lasting antigen depots displayed on FDCs. PMID:27446069

  14. Diffuse Large B-Cell Lymphoma Version 1.2016.

    PubMed

    Zelenetz, Andrew D; Gordon, Leo I; Wierda, William G; Abramson, Jeremy S; Advani, Ranjana H; Andreadis, C Babis; Bartlett, Nancy; Byrd, John C; Fayad, Luis E; Fisher, Richard I; Glenn, Martha J; Habermann, Thomas M; Lee Harris, Nancy; Hernandez-Ilizaliturri, Francisco; Hoppe, Richard T; Horwitz, Steven M; Kaminski, Mark S; Kelsey, Christopher R; Kim, Youn H; Krivacic, Susan; LaCasce, Ann S; Lunning, Matthew; Nademanee, Auayporn; Porcu, Pierluigi; Press, Oliver; Rabinovitch, Rachel; Reddy, Nishitha; Reid, Erin; Roberts, Kenneth; Saad, Ayman A; Sokol, Lubomir; Swinnen, Lode J; Vose, Julie M; Yahalom, Joachim; Zafar, Nadeem; Dwyer, Mary; Sundar, Hema

    2016-02-01

    Diffuse large B-cell lymphomas (DLBCL) are now considered a heterogeneous group of distinct molecular subtypes (germinal center B-cell DLBCL, activated B-cell DLBCL, and primary mediastinal large B-cell lymphoma (PMBL) with varied natural history and response to therapy. In addition, a subset of patients with DLBCL have concurrent MYC and/or BCL2 gene rearrangements (double-hit lymphomas; DHL) and others have a dual expression of both MYC and BCL2 proteins (double-expressing DLBCL; DEL). The standard of care for the treatment of patients with PMBL, DHL, or DEL has not been established. Adequate immunophenotyping and molecular testing (in selected circumstances) are necessary for the accurate diagnosis of different subtypes of DLBCL. The NCCN Guidelines included in this issue, part of the NCCN Guidelines for non-Hodgkin's lymphomas, address the diagnosis and management of DLBCL and its subtypes. PMID:26850490

  15. Chronic B-Cell Leukemias and Agent Orange

    MedlinePlus

    ... survivors' benefits . Research on B-cell leukemias and herbicides The Health and Medicine Division (HMD) (formally known ... sufficient evidence of an association between exposure to herbicides and chronic lymphocytic leukemia. In 2003, VA recognized ...

  16. COMPUTATION MODELING OF TCDD DISRUPTION OF B CELL TERMINAL DIFFERENTIATION

    EPA Science Inventory

    In this study, we established a computational model describing the molecular circuit underlying B cell terminal differentiation and how TCDD may affect this process by impinging upon various molecular targets.

  17. B-cell survival factors in autoimmune rheumatic disorders

    PubMed Central

    Morais, Sandra A.; Vilas-Boas, Andreia

    2015-01-01

    Autoimmune rheumatic disorders have complex etiopathogenetic mechanisms in which B cells play a central role. The importance of factors stimulating B cells, notably the B-cell activating factor (BAFF) and A proliferation inducing ligand (APRIL) axis is now recognized. BAFF and APRIL are cytokines essential for B-cell proliferation and survival from the immature stages to the development of plasma cells. Their levels are increased in some subsets of patients with autoimmune disorders. Several recent biologic drugs have been developed to block this axis, namely belimumab [already licensed for systemic lupus erythematosus (SLE) treatment], tabalumab, atacicept and blisibimod. Many clinical trials to evaluate the safety and efficacy of these drugs in several autoimmune disorders are ongoing, or have been completed recently. This review updates the information on the use of biologic agents blocking BAFF/APRIL for patients with SLE, rheumatoid arthritis, Sjögren’s syndrome and myositis. PMID:26288664

  18. Insights into the Composition and Assembly of the Membrane Arm of Plant Complex I through Analysis of Subcomplexes in Arabidopsis Mutant Lines*

    PubMed Central

    Meyer, Etienne H.; Solheim, Cory; Tanz, Sandra K.; Bonnard, Géraldine; Millar, A. Harvey

    2011-01-01

    NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a long-standing research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied. PMID:21606486

  19. Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines induced by γ-irradiation ((60)Co) of adventitious roots.

    PubMed

    Zhang, Jun-Ying; Sun, Hyeon-Jin; Song, In-Ja; Bae, Tae-Woong; Kang, Hong-Gyu; Ko, Suk-Min; Kwon, Yong-Ik; Kim, Il-Woung; Lee, Jaechun; Park, Shin-Young; Lim, Pyung-Ok; Kim, Yong Hwan; Lee, Hyo-Yeon

    2014-07-01

    An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.

  20. A Mutant with Expression Deletion of Gene Sec-1 in a 1RS.1BL Line and Its Effect on Production Quality of Wheat

    PubMed Central

    Li, Zhi; Ren, Tianheng; Yan, Benju; Tan, Feiquan; Yang, Manyu; Ren, Zhenglong

    2016-01-01

    The chromosome arm 1RS of rye (Secale cereal L.) has been used worldwide as a source of genes for agronomic and resistant improvement. However, the 1RS arm in wheat has end-use quality defects that are partially attributable to the presence of ω-secalins, which are encoded by genes at the Sec-1 locus. Various attempts in removing the Sec-1 genes from the 1RS.1BL translocation chromosome have been made. In the present study, two new primary 1RS.1BL translocation lines, T917-26 and T917-15, were developed from a cross between wheat variety “A42912” and Chinese local rye “Weining.” The lines T917-15 and T917-26 carried a pair of intact and homogeneous 1RS.1BL chromosomes. The line T917-26 also harbored an expression deletion of some genes at the Sec-1 locus, which originated from a mutation that occurred simultaneously with wheat-rye chromosome translocations. These results suggest that the accompanying mutations of the evolutionarily significant translocations are remarkable resources for plant improvement. Comparison of translocation lines with its wheat parent showed improvements in the end-use quality parameters, which included protein content (PC), water absorption (WA), sodium dodecyl sulfate sedimentation (SDSS), wet gluten (WG), dry gluten (DG) and dough stickiness (DS), whereas significant reduction in gluten index (GI) and stability time (ST) were observed. These findings indicate that 1RS in wheat has produced a higher amount of protein, although these comprised worse compositions. However, in the T917-26 line that harbored an expression deletion mutation in the Sec-1 genes, the quality parameters were markedly improved relative to its sister line, T917-15, especially for GI and DS (P < 0.05). These results indicated that expression deletion of Sec-1 genes significantly improves the end-use quality of wheat cultivars harboring the 1RS.1BL translocation. Strategies to remove the Sec-1 genes from the 1RS.1BL translocation in wheat improvement are

  1. A Mutant with Expression Deletion of Gene Sec-1 in a 1RS.1BL Line and Its Effect on Production Quality of Wheat.

    PubMed

    Li, Zhi; Ren, Tianheng; Yan, Benju; Tan, Feiquan; Yang, Manyu; Ren, Zhenglong

    2016-01-01

    The chromosome arm 1RS of rye (Secale cereal L.) has been used worldwide as a source of genes for agronomic and resistant improvement. However, the 1RS arm in wheat has end-use quality defects that are partially attributable to the presence of ω-secalins, which are encoded by genes at the Sec-1 locus. Various attempts in removing the Sec-1 genes from the 1RS.1BL translocation chromosome have been made. In the present study, two new primary 1RS.1BL translocation lines, T917-26 and T917-15, were developed from a cross between wheat variety "A42912" and Chinese local rye "Weining." The lines T917-15 and T917-26 carried a pair of intact and homogeneous 1RS.1BL chromosomes. The line T917-26 also harbored an expression deletion of some genes at the Sec-1 locus, which originated from a mutation that occurred simultaneously with wheat-rye chromosome translocations. These results suggest that the accompanying mutations of the evolutionarily significant translocations are remarkable resources for plant improvement. Comparison of translocation lines with its wheat parent showed improvements in the end-use quality parameters, which included protein content (PC), water absorption (WA), sodium dodecyl sulfate sedimentation (SDSS), wet gluten (WG), dry gluten (DG) and dough stickiness (DS), whereas significant reduction in gluten index (GI) and stability time (ST) were observed. These findings indicate that 1RS in wheat has produced a higher amount of protein, although these comprised worse compositions. However, in the T917-26 line that harbored an expression deletion mutation in the Sec-1 genes, the quality parameters were markedly improved relative to its sister line, T917-15, especially for GI and DS (P < 0.05). These results indicated that expression deletion of Sec-1 genes significantly improves the end-use quality of wheat cultivars harboring the 1RS.1BL translocation. Strategies to remove the Sec-1 genes from the 1RS.1BL translocation in wheat improvement are discussed.

  2. VISA is required for B cell expression of TLR7.

    PubMed

    Xu, Liang-Guo; Jin, Lei; Zhang, Bi-Cheng; Akerlund, Linda J; Shu, Hong-Bing; Cambier, John C

    2012-01-01

    B cells play a critical role in the initialization and development of the systemic lupus erythematosus that is dependent on the expression of the endosomal ssRNA receptor TLR7. Previous studies have established that B cell expression of TLR7 is controlled by the type I IFN secreted by plasmacytoid dendritic cells. In this article, we report that VISA, also known as MAVS, IPS-1, and CardIf, essential for RIG-I/MDA5-mediated signaling following sensing of cytosolic RNA, regulate B cell expression of TLR7 and CD23. We found that B cells from a VISA(-/-) mouse express reduced TLR7 but normal basal levels of type I IFN. We also show that although IFN-β and TLR7 agonists synergize to promote TLR7 expression in VISA(-/-) B cells, they do not fully complement the defect seen in VISA(-/-) cells. Cell transfer experiments revealed that the observed effects of VISA(-/-) are B cell intrinsic. The reduced TLR7 expression in B cells is correlated with impaired TLR7 agonist-induced upregulation of activation markers CD69 and CD86, cell proliferation, production of IFN-α, TNF, and IL-12, and NF-κB activation. Finally, studies indicate that genetic background may influence the observed phenotype of our VISA(-/-) mice, because VISA(-/-) B cells differ in CD23 and TLR7 expression when on C57BL/6 versus 129Sv-C57BL/6 background. Thus, our findings suggest an unexpected link between VISA-mediated cytosolic RLR signaling and autoimmunity.

  3. B CELL DEPLETION THERAPY EXACERBATES MURINE PRIMARY BILIARY CIRRHOSIS

    PubMed Central

    Dhirapong, Amy; Lleo, Ana; Yang, Guo-Xiang; Tsuneyama, Koichi; Dunn, Robert; Kehry, Marilyn; Packard, Thomas A.; Cambier, John C.; Liu, Fu-Tong; Lindor, Keith; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric

    2010-01-01

    Primary biliary cirrhosis (PBC) is considered a model autoimmune disease due to the clinical homogeneity of patients and the classic hallmark of anti-mitochondrial antibodies (AMAS). Indeed, the presence of AMAS is the most highly directed and specific autoantibody in autoimmune diseases. However, the contribution of B cells to the pathogenesis of PBC is unclear. Thus, although AMAs appear to interact with the biliary cell apotope and contribute to biliary pathology, there is no correlation of disease severity and titer of AMA. The recent development of well characterized mAbs specific for the B cell populations, anti-CD20 and anti-CD79, and the development of a well defined xenobiotic induced model of autoimmune cholangitis, prompted us to utilize these reagents and the model to address the contribution of B cells in the pathogenesis of murine PBC. Prior to the induction of autoimmune cholangitis, mice were treated with either anti-CD20, anti-CD79, or isotype matched control mAb and followed for B cell development, the appearance of AMAs, liver pathology and cytokine production. Results of the studies reported herein show that the in vivo depletion of B cells using either anti-CD20 or anti-CD79 led to the development of a more severe form of cholangitis than control mice which is in contrast with results from a number of other autoimmune models which have documented an important therapeutic role of B cell specific depletion. The anti-CD20/CD79 treated mice have increased liver T cell infiltrates and higher levels of pro-inflammatory cytokines. In conclusion, our results reflect a novel disease protective role of B cells in PBC and suggest that B cell depletion therapy in humans with PBC should be approached with caution. PMID:21274873

  4. Rituximab induces Interleukin-6 production by human B cells

    PubMed Central

    Jones, Jonathan D.; Hamilton, B. JoNell; Skopelja, Sladjana; Rigby, William F. C.

    2014-01-01

    Objective Rituximab (RTX), an anti-CD20 monoclonal antibody, is highly effective in the treatment of several autoimmune diseases. The mechanism by which RTX treatment improves Rheumatoid Arthritis and ANCA-Associated Vasculitis is not easily related to B cell depletion. We have shown that RTX mediates a rapid stripping of CD20 and CD19 from the human B cell through a process known as trogocytosis. We hypothesized that changes in B cell phenotype resulting from trogocytosis would diminish the ability of B cells to promote autoimmune disease. Methods Human PBMC were cultured with RTX under conditions that permitted trogocytosis. Changes in B cell phenotype and cytokine production were measured under basal and activated (IL-4/anti-CD40) conditions. The effects of RTX were characterized for their requirements for FcγR and Fc-dependent interactions. Results Trogocytosis induced a marked loss of surface CD19, IgD, CD40 and BR3, but did not alter induction of CD86 expression on purified B cells by IL-4/anti-CD40 treatment. Unexpectedly, RTX-dependent trogocytosis of normal human B cells in vitro led to a rapid upregulation of IL-6 production, with no effect on TNFα, IL-1β, INFγ, or IL-10 production. This effect was Fc-dependent and required the presence of an FcγR bearing cell. This effect involved the release of pre-formed intracellular IL-6 protein as well as marked increases in IL-6 mRNA levels. Conclusion RTX mediated trogocytosis of B cells in vitro results in acute production and release of IL-6. The nature of this effect and its relationship to acute infusion reactions seen with RTX administration remain to be determined. PMID:25080282

  5. Autoimmunity, polyclonal B-cell activation and infection.

    PubMed

    Granholm, N A; Cavallo, T

    1992-02-01

    It is widely believed that autoimmunity is an integral part of the immune system, and that genetic, immunologic, hormonal, environmental and other factors contribute to the pathogenesis of autoimmune disease. Thus, autoimmune disease may represent an abnormal expression of immune functions instead of loss of tolerance to self, and it can be organ specific or systemic in its manifestations. We review the various factors that contribute to the development of autoimmune disease; we also review the mechanisms of polyclonal B-cell activation, with emphasis on the role of infectious agents. We consider systemic lupus erythematosus in humans and in experimental animals as prototypic autoimmune disease, and we summarize data to indicate that polyclonal B-cell activation is central to the pathogenesis of systemic autoimmune disease. The effect of polyclonal B-cell activation, brought about by injections of a B-cell activator-lipopolysaccharide from Gram-negative bacteria-is sufficient to cause autoimmune disease in an immunologically normal host. In fact, autoimmune disease can be arrested if excessive polyclonal B-cell activation is suppressed; alternatively, autoimmune disease can be exacerbated if polyclonal B-cell activation is enhanced. We explore the mechanism of tissue injury when autoimmune disease is induced or exacerbated, and we consider the pathogenic roles of autoantibodies, immune complexes, complement, the blood cell carrier system, and the mononuclear phagocyte system. Although polyclonal B-cell activation may be the mechanism whereby various factors can cause or exacerbate systemic autoimmune disease, polyclonal B-cell activation may cause autoimmune disease on its own.

  6. Long Noncoding RNA Expression during Human B-Cell Development.

    PubMed

    Petri, Andreas; Dybkær, Karen; Bøgsted, Martin; Thrue, Charlotte Albæk; Hagedorn, Peter H; Schmitz, Alexander; Bødker, Julie Støve; Johnsen, Hans Erik; Kauppinen, Sakari

    2015-01-01

    Long noncoding RNAs (lncRNAs) have emerged as important regulators of diverse cellular processes, but their roles in the developing immune system are poorly understood. In this study, we analysed lncRNA expression during human B-cell development by array-based expression profiling of eleven distinct flow-sorted B-cell subsets, comprising pre-B1, pre-B2, immature, naive, memory, and plasma cells from bone marrow biopsies (n = 7), and naive, centroblast, centrocyte, memory, and plasmablast cells from tonsil tissue samples (n = 6), respectively. A remapping strategy was used to assign the array probes to 37630 gene-level probe sets, reflecting recent updates in genomic and transcriptomic databases, which enabled expression profiling of 19579 long noncoding RNAs, comprising 3947 antisense RNAs, 5277 lincRNAs, 7625 pseudogenes, and 2730 additional lncRNAs. As a first step towards inferring the functions of the identified lncRNAs in developing B-cells, we analysed their co-expression with well-characterized protein-coding genes, a method known as "guilt by association". By using weighted gene co-expression network analysis, we identified 272 lincRNAs, 471 antisense RNAs, 376 pseudogene RNAs, and 64 lncRNAs within seven sub-networks associated with distinct stages of B-cell development, such as early B-cell development, B-cell proliferation, affinity maturation of antibody, and terminal differentiation. These data provide an important resource for future studies on the functions of lncRNAs in development of the adaptive immune response, and the pathogenesis of B-cell malignancies that originate from distinct B-cell subpopulations. PMID:26394393

  7. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants.

    PubMed

    Maringer, Katherine; Saheb, Entsar; Bush, John

    2014-01-01

    Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA), and dominant negative (DN) forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell-cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  8. B cells mediate chronic allograft rejection independently of antibody production.

    PubMed

    Zeng, Qiang; Ng, Yue-Harn; Singh, Tripti; Jiang, Ke; Sheriff, Khaleefathullah A; Ippolito, Renee; Zahalka, Salwa; Li, Qi; Randhawa, Parmjeet; Hoffman, Rosemary A; Ramaswami, Balathiripurasundari; Lund, Frances E; Chalasani, Geetha

    2014-03-01

    Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

  9. Cutaneous primary B-cell lymphomas: from diagnosis to treatment*

    PubMed Central

    Lima, Margarida

    2015-01-01

    Primary cutaneous B-cell lymphomas are a heterogeneous group of mature B-cells neoplasms with tropism for the skin, whose biology and clinical course differ significantly from the equivalent nodal lymphomas. The most indolent forms comprise the primary cutaneous marginal zone and follicle center B-cell lymphomas that despite the excellent prognosis have cutaneous recurrences very commonly. The most aggressive forms include the primary cutaneous large B-cell lymphomas, consisting in two major groups: the leg type, with poor prognosis, and others, the latter representing a heterogeneous group of lymphomas from which specific entities are supposed to be individualized over time, such as intravascular large B-cell lymphomas. Treatment may include surgical excision, radiotherapy, antibiotics, corticosteroids, interferon, monoclonal antibodies and chemotherapy, depending on the type of lymphoma and on the type and location of the skin lesions. In subtypes with good prognosis is contraindicated overtreatment and in those associated with a worse prognosis the recommended therapy relies on CHOP-like regimens associated with rituximab, assisted or not with local radiotherapy. We review the primary cutaneous B-cell lymphomas, remembering the diagnostic criteria, differential diagnosis, classification, and prognostic factors and presenting the available therapies. PMID:26560215

  10. Origin of B-Cell Neoplasms in Autoimmune Disease

    PubMed Central

    Hemminki, Kari; Liu, Xiangdong; Ji, Jianguang; Försti, Asta

    2016-01-01

    Autoimmune diseases (ADs) are associated with a number of B-cell neoplasms but the associations are selective in regard to the type of neoplasm and the conferred risks are variable. So far no mechanistic bases for these differential associations have been demonstrated. We speculate that developmental origin of B-cells might propose a mechanistic rationale for their carcinogenic response to autoimmune stimuli and tested the hypothesis on our previous studies on the risks of B-cell neoplasms after any of 33 ADs. We found that predominantly germinal center (GC)-derived B-cells showed multiple associations with ADs: diffuse large B cell lymphoma associated with 15 ADs, follicular lymphoma with 7 ADs and Hodgkin lymphoma with 11 ADs. Notably, these neoplasms shared significant associations with 5 ADs (immune thrombocytopenic purpura, polymyositis/dermatomyositis, rheumatoid arthritis, Sjogren syndrome and systemic lupus erythematosis). By contrast, primarily non-GC neoplasms, acute lymphocytic leukemia, chronic lymphocytic leukemia and myeloma associated with 2 ADs only and mantle cell lymphoma with 1 AD. None of the neoplasms shared associated ADs. These data may suggest that autoimmune stimulation critically interferes with the rapid cell division, somatic hypermutation, class switch recombination and immunological selection of maturing B-cell in the GC and delivers damage contributing to transformation. PMID:27355450

  11. B cells with regulatory properties in transplantation tolerance

    PubMed Central

    Durand, Justine; Chiffoleau, Elise

    2015-01-01

    Induction of tolerance remains a major goal in transplantation. Indeed, despite potent immunosuppression, chronic rejection is still a real problem in transplantation. The humoral response is an important mediator of chronic rejection, and numerous strategies have been developed to target either B cells or plasma cells. However, the use of anti-CD20 therapy has highlighted the beneficial role of subpopulation of B cells, termed regulatory B cells. These cells have been characterized mainly in mice models of auto-immune diseases but emerging literature suggests their role in graft tolerance in transplantation. Regulatory B cells seem to be induced following inflammation to restrain excessive response. Different phenotypes of regulatory B cells have been described and are functional at various differentiation steps from immature to plasma cells. These cells act by multiple mechanisms such as secretion of immuno-suppressive cytokines interleukin-10 (IL-10) or IL-35, cytotoxicity, expression of inhibitory receptors or by secretion of non-inflammatory antibodies. Better characterization of the development, phenotype and mode of action of these cells seems urgent to develop novel approaches to manipulate the different B cell subsets and the response to the graft in a clinical setting. PMID:26722647

  12. Transcriptional analysis of the B cell germinal center reaction

    PubMed Central

    Klein, Ulf; Tu, Yuhai; Stolovitzky, Gustavo A.; Keller, Jeffrey L.; Haddad, Joseph; Miljkovic, Vladan; Cattoretti, Giorgio; Califano, Andrea; Dalla-Favera, Riccardo

    2003-01-01

    The germinal center (GC) reaction is crucial for T cell-dependent immune responses and is targeted by B cell lymphomagenesis. Here we analyzed the transcriptional changes that occur in B cells during GC transit (naïve B cells → centroblasts → centrocytes → memory B cells) by gene expression profiling. Naïve B cells, characterized by the expression of cell cycle-inhibitory and antiapoptotic genes, become centroblasts by inducing an atypical proliferation program lacking c-Myc expression, switching to a proapoptotic program, and down-regulating cytokine, chemokine, and adhesion receptors. The transition from GC to memory cells is characterized by a return to a phenotype similar to that of naïve cells except for an apoptotic program primed for both death and survival and for changes in the expression of cell surface receptors including IL-2 receptor β. These results provide insights into the dynamics of the GC reaction and represent the basis for the analysis of B cell malignancies. PMID:12604779

  13. Origin of B-Cell Neoplasms in Autoimmune Disease.

    PubMed

    Hemminki, Kari; Liu, Xiangdong; Ji, Jianguang; Försti, Asta

    2016-01-01

    Autoimmune diseases (ADs) are associated with a number of B-cell neoplasms but the associations are selective in regard to the type of neoplasm and the conferred risks are variable. So far no mechanistic bases for these differential associations have been demonstrated. We speculate that developmental origin of B-cells might propose a mechanistic rationale for their carcinogenic response to autoimmune stimuli and tested the hypothesis on our previous studies on the risks of B-cell neoplasms after any of 33 ADs. We found that predominantly germinal center (GC)-derived B-cells showed multiple associations with ADs: diffuse large B cell lymphoma associated with 15 ADs, follicular lymphoma with 7 ADs and Hodgkin lymphoma with 11 ADs. Notably, these neoplasms shared significant associations with 5 ADs (immune thrombocytopenic purpura, polymyositis/dermatomyositis, rheumatoid arthritis, Sjogren syndrome and systemic lupus erythematosis). By contrast, primarily non-GC neoplasms, acute lymphocytic leukemia, chronic lymphocytic leukemia and myeloma associated with 2 ADs only and mantle cell lymphoma with 1 AD. None of the neoplasms shared associated ADs. These data may suggest that autoimmune stimulation critically interferes with the rapid cell division, somatic hypermutation, class switch recombination and immunological selection of maturing B-cell in the GC and delivers damage contributing to transformation. PMID:27355450

  14. Altered B cell receptor signaling in human systemic lupus erythematosus

    PubMed Central

    Jenks, Scott A.; Sanz, Iñaki

    2009-01-01

    Regulation of B cell receptor signaling is essential for the development of specific immunity while retaining tolerance to self. Systemic lupus erythematosus (SLE) is characterized by a loss of B cell tolerance and the production of anti-self antibodies. Accompanying this break down in tolerance are alterations in B cell receptor signal transduction including elevated induced calcium responses and increased protein phosphorylation. Specific pathways that negatively regulate B cell signaling have been shown to be impaired in some SLE patients. These patients have reduced levels of the kinase Lyn in lipid raft microdomains and this reduction is inversely correlated with increased CD45 in lipid rafts. Function and expression of the inhibitory immunoglobulin receptor FcγRIIB is also reduced in Lupus IgM- CD27+ memory cells. Because the relative contribution of different memory and transitional B cell subsets can be abnormal in SLE patients, we believe studies targeted to well defined B cell subsets will be necessary to further our understanding of signaling abnormalities in SLE. Intracellular flow cytometric analysis of signaling is a useful approach to accomplish this goal. PMID:18723129

  15. Receptor editing and genetic variability in human autoreactive B cells.

    PubMed

    Lang, Julie; Ota, Takayuki; Kelly, Margot; Strauch, Pamela; Freed, Brian M; Torres, Raul M; Nemazee, David; Pelanda, Roberta

    2016-01-11

    The mechanisms by which B cells undergo tolerance, such as receptor editing, clonal deletion, and anergy, have been established in mice. However, corroborating these mechanisms in humans remains challenging. To study how autoreactive human B cells undergo tolerance, we developed a novel humanized mouse model. Mice expressing an anti-human Igκ membrane protein to serve as a ubiquitous neo self-antigen (Ag) were transplanted with a human immune system. By following the fate of self-reactive human κ(+) B cells relative to nonautoreactive λ(+) cells, we show that tolerance of human B cells occurs at the first site of self-Ag encounter, the bone marrow, via a combination of receptor editing and clonal deletion. Moreover, the amount of available self-Ag and the genetics of the cord blood donor dictate the levels of central tolerance and autoreactive B cells in the periphery. Thus, this model can be useful for studying specific mechanisms of human B cell tolerance and to reveal differences in the extent of this process among human populations.

  16. Characterization of two monoclonal antibodies (UCL4D12 and UCL3D3) that discriminate between human mantle zone and marginal zone B cells.

    PubMed

    Smith-Ravin, J; Spencer, J; Beverley, P C; Isaacson, P G

    1990-10-01

    Two new monoclonal antibodies (MoAbs), UCL3D3 and UCL4D12 were obtained following immunization with follicular lymphoma (UCL3D3) or low-grade primary B cell gastric lymphoma cells (UCL4D12). In normal splenic white pulp, tonsil and small intestinal Peyer's patches, UCL4D12 recognizes marginal zone B cells and a subpopulation of follicle centre cells, whereas mantle zone B cells are UCL4D12 negative. In contrast, UCL3D3 recognizes mantle zone B cells and follicular dendritic cells, but not marginal zone B cells or follicle centre B cells. Double-immunofluorescence studies showed that in the splenic white pulp, these antibodies stain reciprocally. The majority of UCL3D3+ cells are sIgM+ and sIgD+ whereas a higher proportion of UCL4D12+ cells express surface IgM (sIgM) but not surface IgD (sIgD). Less than 10% of splenic B cells express both 3D3 and 4D12 antigens. None of the cell lines tested expressed either antigen. Functional studies showed that both antigens play a role in B cell activation as the MoAbs increase the mitogenic effect of Staphylococcus aureus Cowan I on tonsil B cells. This effect was maximal at 72 h in culture. TPA activation was reduced, and no effect was observed with anti-immunoglobulin (anti mu) or CDw40 (G28.5). UCL3D3 and UCL4D12 did not show any stimulatory effect on their own. Biochemical studies show that both MoAbs recognize proteins of 80-90 kD under reducing conditions. These two MoAbs appear to recognize new B cell surface antigens which may be useful for identifying subpopulations of B cells. PMID:2208792

  17. Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function.

    PubMed

    Portugal, Silvia; Tipton, Christopher M; Sohn, Haewon; Kone, Younoussou; Wang, Jing; Li, Shanping; Skinner, Jeff; Virtaneva, Kimmo; Sturdevant, Daniel E; Porcella, Stephen F; Doumbo, Ogobara K; Doumbo, Safiatou; Kayentao, Kassoum; Ongoiba, Aissata; Traore, Boubacar; Sanz, Inaki; Pierce, Susan K; Crompton, Peter D

    2015-05-08

    Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.

  18. Lymphomagenic CARD11/BCL10/MALT1 signaling drives malignant B-cell proliferation via cooperative NF-κB and JNK activation

    PubMed Central

    Knies, Nathalie; Alankus, Begüm; Weilemann, Andre; Tzankov, Alexandar; Brunner, Kristina; Ruff, Tanja; Kremer, Marcus; Keller, Ulrich B.; Lenz, Georg; Ruland, Jürgen

    2015-01-01

    The aggressive activated B cell-like subtype of diffuse large B-cell lymphoma is characterized by aberrant B-cell receptor (BCR) signaling and constitutive nuclear factor kappa-B (NF-κB) activation, which is required for tumor cell survival. BCR-induced NF-κB activation requires caspase recruitment domain-containing protein 11 (CARD11), and CARD11 gain-of-function mutations are recurrently detected in human diffuse large B-cell lymphoma (DLBCL). To investigate the consequences of dysregulated CARD11 signaling in vivo, we generated mice that conditionally express the human DLBCL-derived CARD11(L225LI) mutant. Surprisingly, CARD11(L225LI) was sufficient to trigger aggressive B-cell lymphoproliferation, leading to early postnatal lethality. CARD11(L225LI) constitutively associated with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) to simultaneously activate the NF-κB and c-Jun N-terminal kinase (JNK) signaling cascades. Genetic deficiencies of either BCL10 or MALT1 completely rescued the phenotype, and pharmacological inhibition of JNK was, similar to NF-κB blockage, toxic to autonomously proliferating CARD11(L225LI)-expressing B cells. Moreover, constitutive JNK activity was observed in primary human activated B cell-like (ABC)-DLBCL specimens, and human ABC-DLBCL cells were also sensitive to JNK inhibitors. Thus, our results demonstrate that enforced activation of CARD11/BCL10/MALT1 signaling is sufficient to drive transformed B-cell expansion in vivo and identify the JNK pathway as a therapeutic target for ABC-DLBCL. PMID:26668357

  19. Antibodies That Block or Activate Mouse B Cell Activating Factor of the Tumor Necrosis Factor (TNF) Family (BAFF), Respectively, Induce B Cell Depletion or B Cell Hyperplasia.

    PubMed

    Kowalczyk-Quintas, Christine; Schuepbach-Mallepell, Sonia; Vigolo, Michele; Willen, Laure; Tardivel, Aubry; Smulski, Cristian R; Zheng, Timothy S; Gommerman, Jennifer; Hess, Henry; Gottenberg, Jacques-Eric; Mackay, Fabienne; Donzé, Olivier; Schneider, Pascal

    2016-09-16

    B cell activating factor of the TNF family (BAFF), also known as B lymphocyte stimulator, is a ligand required for the generation and maintenance of B lymphocytes. In this study, the ability of different monoclonal antibodies to recognize, inhibit, or activate mouse BAFF was investigated. One of them, a mouse IgG1 named Sandy-2, prevented the binding of BAFF to all of its receptors, BAFF receptor, transmembrane activator and calcium modulating ligand interactor, and B cell maturation antigen, at a stoichiometric ratio; blocked the activity of mouse BAFF on a variety of cell-based reporter assays; and antagonized the prosurvival action of BAFF on primary mouse B cells in vitro A single administration of Sandy-2 in mice induced B cell depletion within 2 weeks, down to levels close to those observed in BAFF-deficient mice. This depletion could then be maintained with a chronic treatment. Sandy-2 and a previously described rat IgG1 antibody, 5A8, also formed a pair suitable for the sensitive detection of endogenous circulating BAFF by ELISA or using a homogenous assay. Interestingly, 5A8 and Sandy-5 displayed activities opposite to that of Sandy-2 by stimulating recombinant BAFF in vitro and endogenous BAFF in vivo These tools will prove useful for the detection and functional manipulation of endogenous mouse BAFF and provide an alternative to the widely used BAFF receptor-Fc decoy receptor for the specific depletion of BAFF in mice. PMID:27451394

  20. Intrinsic differences in the initiation of B cell receptor signaling favor responses of human IgG(+) memory B cells over IgM(+) naive B cells.

    PubMed

    Davey, Angel M; Pierce, Susan K

    2012-04-01

    The acquisition of long-lived memory B cells (MBCs) is critical for the defense against many infectious diseases. Despite their importance, little is known about how Ags trigger human MBCs, even though our understanding of the molecular basis of Ag activation of B cells in model systems has advanced considerably. In this study, we use quantitative, high-resolution, live-cell imaging at the single-cell and single-molecule levels to describe the earliest Ag-driven events in human isotype-switched, IgG-expressing MBCs and compare them with those in IgM-expressing naive B cells. We show that human MBCs are more robust than naive B cells at each step in the initiation of BCR signaling, including interrogation of Ag-containing membranes, formation of submicroscopic BCR oligomers, and recruitment and activation of signaling-associated kinases. Despite their robust response to Ag, MBCs remain highly sensitive to FcγRIIB-mediated inhibition. We also demonstrate that in the absence of Ag, a portion of MBC receptors spontaneously oligomerized, and phosphorylated kinases accumulated at the membrane and speculate that heightened constitutive signaling may play a role in maintaining MBC longevity. Using high-resolution imaging, we have provided a description of the earliest events in the Ag activation of MBCs and evidence for acquired cell-intrinsic differences in the initiation of BCR signaling in human naive and MBCs.

  1. Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways.

    PubMed

    Lenz, Georg; Wright, George W; Emre, N C Tolga; Kohlhammer, Holger; Dave, Sandeep S; Davis, R Eric; Carty, Shannon; Lam, Lloyd T; Shaffer, A L; Xiao, Wenming; Powell, John; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D; Connors, Joseph M; Campo, Elias; Jaffe, Elaine S; Delabie, Jan; Smeland, Erlend B; Rimsza, Lisa M; Fisher, Richard I; Weisenburger, Dennis D; Chan, Wing C; Staudt, Louis M

    2008-09-01

    Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by high-resolution, genome-wide copy number analysis coupled with gene-expression profiling. Of 272 recurrent chromosomal aberrations that were associated with gene-expression alterations, 30 were used differentially by the DLBCL subtypes (P < 0.006). An amplicon on chromosome 19 was detected in 26% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs. A highly up-regulated gene in this amplicon was SPIB, which encodes an ETS family transcription factor. Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines but not to GCB DLBCL, PMBL, or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL. Deletion of the INK4a/ARF tumor suppressor locus and trisomy 3 also occurred almost exclusively in ABC DLBCLs and was associated with inferior outcome within this subtype. FOXP1 emerged as a potential oncogene in ABC DLBCL that was up-regulated by trisomy 3 and by more focal high-level amplifications. In GCB DLBCL, amplification of the oncogenic mir-17-92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent, but these events did not occur in ABC DLBCL. Together, these data provide genetic evidence that the DLBCL subtypes are distinct diseases that use different oncogenic pathways. PMID:18765795

  2. Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways

    PubMed Central

    Lenz, Georg; Wright, George W.; Emre, N. C. Tolga; Kohlhammer, Holger; Dave, Sandeep S.; Davis, R. Eric; Carty, Shannon; Lam, Lloyd T.; Shaffer, A. L.; Xiao, Wenming; Powell, John; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D.; Connors, Joseph M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Rimsza, Lisa M.; Fisher, Richard I.; Weisenburger, Dennis D.; Chan, Wing C.; Staudt, Louis M.

    2008-01-01

    Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by high-resolution, genome-wide copy number analysis coupled with gene-expression profiling. Of 272 recurrent chromosomal aberrations that were associated with gene-expression alterations, 30 were used differentially by the DLBCL subtypes (P < 0.006). An amplicon on chromosome 19 was detected in 26% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs. A highly up-regulated gene in this amplicon was SPIB, which encodes an ETS family transcription factor. Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines but not to GCB DLBCL, PMBL, or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL. Deletion of the INK4a/ARF tumor suppressor locus and trisomy 3 also occurred almost exclusively in ABC DLBCLs and was associated with inferior outcome within this subtype. FOXP1 emerged as a potential oncogene in ABC DLBCL that was up-regulated by trisomy 3 and by more focal high-level amplifications. In GCB DLBCL, amplification of the oncogenic mir-17–92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent, but these events did not occur in ABC DLBCL. Together, these data provide genetic evidence that the DLBCL subtypes are distinct diseases that use different oncogenic pathways. PMID:18765795

  3. BCR-crosslinking induces a transcription of protein phosphatase component G5PR that is required for mature B-cell survival

    SciTech Connect

    Huq Ronny, Faisal Mahmudul; Igarashi, Hideya; Sakaguchi, Nobuo . E-mail: nobusaka@kaiju.medic.kumamoto-u.ac.jp

    2006-02-03

    BCR-crosslinking triggers activation-induced cell death (AICD) selectively in the restricted stage of B-cell differentiation. We examined the transcription of a protein phosphatase subunit G5PR in immature and mature B-cells, because absence of this factor augmented cell sensitivity to AICD, associated with increased activation of JNK and Bim. BCR-crosslinking-induced G5pr transcription in AICD-resistant mature splenic IgM{sup lo}IgD{sup hi} B-cells but not in AICD susceptible immature IgM{sup hi}IgD{sup lo} B-cells. Thus, G5pr induction correlated with the prevention of AICD; High in mature splenic CD23{sup hi} B-cells but low in immature B-cells of neonatal mice, sub-lethally irradiated mice, or xid mice. Lack of G5pr upregulation was associated with the prolonged activation of JNK. The G5pr cDNA transfection protected an immature B-cell line WEHI-231 from BCR-mediated AICD. The differential expression of G5PR might be responsible for the antigen-dependent selection of B-cells.

  4. Epstein-Barr virus infection in vitro can rescue germinal center B cells with inactivated immunoglobulin genes.

    PubMed

    Chaganti, Sridhar; Bell, Andrew I; Pastor, Noelia Begue; Milner, Anne E; Drayson, Mark; Gordon, John; Rickinson, Alan B

    2005-12-15

    Immunoglobulin genotyping of Epstein-Barr virus (EBV)-positive posttransplantation lymphoproliferative disease has suggested that such lesions often arise from atypical post-germinal center B cells, in some cases carrying functionally inactivated immunoglobulin genes. To investigate whether EBV can rescue cells that are failed products of the somatic hypermutation process occurring in germinal centers (GCs), we isolated GC cells from tonsillar cell suspensions and exposed them to EBV in vitro. Screening more than 100 EBV-transformed cell lines of GC origin identified 6 lines lacking surface immunoglobulin, a phenotype never seen among lines derived from circulating naive or memory B cells. Furthermore, 3 of the 6 surface immunoglobulin-negative GC lines carried inactivating mutations in the immunoglobulin H (IgH) variable gene sequence. The ability of EBV to rescue aberrant products of the germinal center reaction in vitro strengthens the probability that a parallel activity contributes to EBV's lymphomagenic potential in vivo.

  5. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

    PubMed Central

    Maringer, Katherine; Saheb, Entsar; Bush, John

    2014-01-01

    Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA), and dominant negative (DN) forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses. PMID:25157910

  6. Isolation of a mutant MDBK cell line resistant to bovine viral diarrhea virus infection due to a block in viral entry.

    PubMed

    Flores, E F; Donis, R O

    1995-04-20

    A cell line, termed CRIB, resistant to infection with bovine viral diarrhea virus (BVDV) has been derived from the MDBK bovine kidney cell line. CRIB cells were obtained by selection and cloning of cells surviving infection with a highly cytolytic BVDV strain. CRIB cells contain no detectable infectious or defective BVDV as ascertained by cocultivation, animal inoculation, indirect immunofluorescence, Western immunoblot, Northern hybridization, and RNA PCR. Inoculation of CRIB cells with 24 cytopathic and noncytopathic BVDV strains does not result in expression of viral genes or amplification of input virus. Karyotype and isoenzyme analyses demonstrated that CRIB are genuine bovine cells. CRIB cells are as susceptible as the parental MDBK cells to 10 other bovine viruses, indicating that these cells do not have a broad defect blocking viral replication. Transfection of CRIB cells with BVDV RNA or virus inoculation in the presence of polyethylene-glycol results in productive infection, indicating that the defect of CRIB cells is at the level of virus entry. CRIB cells are the first bovine cells reported to be resistant to BVDV infection in vitro and may be a useful tool for studying the early interactions of pestiviruses with host cells.

  7. Safety and Tolerability Study of PCI-32765 in B Cell Lymphoma and Chronic Lymphocytic Leukemia

    ClinicalTrials.gov

    2016-04-26

    B-cell Chronic Lymphocytic Leukemia; Small Lymphocytic Lymphoma; Diffuse Well-differentiated Lymphocytic Lymphoma; B Cell Lymphoma; Follicular Lymphoma,; Mantle Cell Lymphoma; Non-Hodgkin's Lymphoma; Waldenstrom Macroglobulinemia; Burkitt Lymphoma; B-Cell Diffuse Lymphoma

  8. GCN5 is essential for IRF-4 gene expression followed by transcriptional activation of Blimp-1 in immature B cells.

    PubMed

    Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo

    2014-03-01

    During B-cell differentiation, the gene expression of B-cell differentiation-related transcription factors must be strictly controlled by epigenetic mechanisms including histone acetylation and deacetylation, to complete the differentiation pathway. GCN5, one of the most important histone acetyltransferases, is involved in epigenetic events for transcriptional regulation through alterations in the chromatin structure. In this study, by analyzing the homozygous DT40 mutants GCN5(-/-), generated with gene targeting techniques, we found that GCN5 was necessary for transcriptional activation of IRF-4, an essential transcription factor for plasma cell differentiation. GCN5 deficiency caused drastic decreases in both the mRNA and the protein levels of Blimp-1 and IRF-4. The ectopic expression of Blimp-1 and IRF-4 suggests that IRF-4, but not Blimp-1, is the target gene of GCN5 in immature B cells. Moreover, a chromatin immunoprecipitation assay showed that GCN5 bound to the IRF-4 gene around its 5'-flanking region and acetylated H3K9 residues within chromatin surrounding the region in vivo, suggesting that gene expression of IRF-4 is certainly regulated by GCN5. These results reveal that GCN5 is essential for IRF-4 gene expression, followed by transcriptional activation of Blimp-1, and plays a key role in epigenetic regulation of B-cell differentiation.

  9. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting.

    PubMed

    Chen, Fengjiao; Wang, Ying; Yuan, Yilin; Zhang, Wei; Ren, Zijian; Jin, Yong; Liu, Xiaorui; Xiong, Qiang; Chen, Qin; Zhang, Manling; Li, Xiaokang; Zhao, Lihua; Li, Ze; Wu, Zhaoqiang; Zhang, Yanfei; Hu, Feifei; Huang, Juan; Li, Rongfeng; Dai, Yifan

    2015-08-20

    Generating B cell-deficient mutant is the first step to produce human antibody repertoires in large animal models. In this study, we applied the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system to target the JH region of the pig IgM heavy chain gene which is crucial for B cell development and differentiation. Transfection of IgM-targeting Cas9 plasmid in primary porcine fetal fibroblasts (PFFs) enabled inducing gene knock out (KO) in up to 53.3% of colonies analyzed, a quarter of which harbored biallelic modification, which was much higher than that of the traditional homologous recombination (HR). With the aid of somatic cell nuclear transfer (SCNT) technology, three piglets with the biallelic IgM heavy chain gene mutation were produced. The piglets showed no antibody-producing B cells which indicated that the biallelic mutation of the IgM heavy chain gene effectively knocked out the function of the IgM and resulted in a B cell-deficient phenotype. Our study suggests that the CRISPR/Cas9 system combined with SCNT technology is an efficient genome-editing approach in pigs.

  10. Pivotal advance: CEACAM1 is a negative coreceptor for the B cell receptor and promotes CD19-mediated adhesion of B cells in a PI3K-dependent manner.

    PubMed

    Lobo, Elizabeth O; Zhang, Zhifang; Shively, John E

    2009-08-01

    Upon antigen binding, the BCR transduces a signal culminating in proliferation or in AICD of the B cell. Coreceptor engagement and subsequent modification of the BCR signal pathway are mechanisms that guide the B cell to its appropriate fate. For example, in the absence of coreceptor engagement, anti-sIgM antibodies induce apoptosis in the human Daudi B cell lymphoma cell line. ITIM-bearing B cell coreceptors that potentially may act as negative coreceptors include FcRgammaIIb, CD22, CD72, and CEACAM1 (CD66a). Although the role of CEACAM1 as an inhibitory coreceptor in T cells has been established, its role in B cells is poorly defined. We show that anti-sIgM antibody and PI3K inhibitor LY294002-induced apoptosis are reduced significantly in CEACAM1 knock-down clones compared with WT Daudi cells and that anti-sIgM treatment induced CEACAM1 tyrosine phosphorylation and association with SHP-1 in WT cells. In contrast, treatment of WT Daudi cells with anti-CD19 antibodies does not induce apoptosis and has reduced tyrosine phosphorylation and SHP-1 recruitment to CEACAM1. Thus, similar to its function in T cells, CEACAM1 may act as an inhibitory B cell coreceptor, most likely through recruitment of SHP-1 and inhibition of a PI3K-promoted activation pathway. Activation of B cells by anti-sIgM or anti-CD19 antibodies also leads to cell aggregation that is promoted by CEACAM1, also in a PI3K-dependent manner.

  11. Dissecting the contribution of EBNA3C domains important for EBV-induced B-cell growth and proliferation

    PubMed Central

    Lu, Jie; Prasad Aj, Mahadesh; Banerjee, Shuvomoy; Robertson, Erle S.

    2015-01-01

    Epstein-Barr virus (EBV) is an oncogenic gammaherpes virus which is linked to pathogenesis of several human lymphatic malignancies. The EBV essential latent antigen EBNA3C is critical for efficient conversion of primary human B-lymphocytes to lymphoblastic cell lines and for continued LCL growth. EBNA3C, an EBV latent antigen with oncogenic potential can bind and regulate the functions of a wide range of cellular transcription factors. In our current reverse genetics study, we deleted the full length EBNA3C, and independently the RBP-Jκ and Nm23-H1 binding sites within EBNA3C using BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the EBV EBNA3C open reading frame (ORF) and more specifically the residues 621–675 which binds Nm23H1 and SUMO-1 showed a significant reduction in the ability of the cells to proliferate. Furthermore, they exhibited lower infectivity of human peripheral blood mononuclear cells (PBMCs). We also showed that recombinant EBV with deletions of the EBNA3C ORF, as well as a recombinant with residues 621–675 within EBNA3C ORF deleted had diminished abilities to activate CD40. Our study also revealed that the full length (1–992) and 621–675 aa deletions of EBNA3C when compared to wild type EBV infected PBMCs had differential expression patterns for the phosphorylation of MAP kinases specifically p38, JNK and ERK. Regulation of β-catenin also differed among wild type and EBNA3C deleted mutants. These temporal differences in signaling activities of these recombinant viruses in PBMCs is likely important in defining their functional importance in EBV-mediated B-cell transformation. PMID:26336822

  12. Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination

    PubMed Central

    Svensson, Mattias N. D.; Andersson, Karin M. E.; Wasén, Caroline; Nurkkala-Karlsson, Merja; Jonsson, Ing-Marie; Brisslert, Mikael; Bemark, Mats; Bokarewa, Maria I.

    2015-01-01

    Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6+ germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1. PMID:26627255

  13. Anti-apoptotic role and clinical relevance of neurotrophins in diffuse large B-cell lymphomas

    PubMed Central

    Dubanet, Lydie; Bentayeb, Hafidha; Petit, Barbara; Olivrie, Agnès; Saada, Sofiane; de la Cruz-Morcillo, Miguel A; Lalloué, Fabrice; Gourin, Marie-Pierre; Bordessoule, Dominique; Faumont, Nathalie; Delage-Corre, Manuela; Fauchais, Anne-Laure; Jauberteau, Marie-Odile; Troutaud, Danielle

    2015-01-01

    Background: Diffuse large B-cell lymphoma (DLBCL) is a fatal malignancy that needs to identify new targets for additional therapeutic options. This study aimed to clarify the clinical and biological significance of endogenous neurotrophin (nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF)) in DLBCL biopsy samples and cell lines. Methods: We analysed expression of NGF, BDNF, and their receptors (Trk, p75NTR) in 51 biopsies and cell lines by immunohistochemistry, immunofluorescence, and western blotting. To investigate the biological role of BDNF/TrkB/p75NTR axis, effects of neurotrophin signalling inhibition were determined on tumour cell survival and vascular endothelial growth factor (VEGF) secretion. The pharmacological pan-Trk inhibitor K252a was used for in vitro and in vivo studies. Results: A BDNF/TrkB axis was expressed in all biopsies, which was independent of the germinal centre B-cell (GCB)/non-GCB profile. p75NTR, TrkB, and BDNF tumour scores were significantly correlated and high NGF expression was significantly associated with MUM1/IRF4, and the non-GCB subtype. Diffuse large B-cell lymphoma cell lines co-expressed neurotrophins and their receptors. The full-length TrkB receptor was found in all cell lines, which was also phosphorylated at Tyr-817. p75NTR was associated to Trk and not to its cell death co-receptor sortilin. In vitro, inhibition of neurotrophin signalling induced cell apoptosis. K252a caused cell apoptosis, decreased VEGF secretion, and potentiated rituximab effect, notably in less rituximab-sensitive cells. In vivo, K252a significantly reduced tumour growth and potentiated the effects of rituximab in a GCB-DLBCL xenograft model. Conclusions: This work argues for a pro-survival role of endogenous neurotrophins in DLBCLs and inhibition of Trk signalling might be a potential treatment strategy for rituximab resistant subgroups. PMID:26284337

  14. TIM-1 signaling in B cells regulates antibody production

    SciTech Connect

    Ma, Juan; Usui, Yoshihiko; Takeda, Kazuyoshi; Harada, Norihiro; Yagita, Hideo; Okumura, Ko; Akiba, Hisaya

    2011-03-11

    Highlights: {yields} TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. {yields} Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. {yields} TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3{sup +} anti-CD28-stimulated CD4{sup +} T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

  15. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

    PubMed

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C A; Woll, Petter S; Jacobsen, Sten Eirik W; Sitnicka, Ewa

    2016-07-14

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.

  16. The role of B-cell receptor inhibitors in the treatment of patients with chronic lymphocytic leukemia

    PubMed Central

    Wiestner, Adrian

    2015-01-01

    Chronic lymphocytic leukemia is a malignancy of mature auto-reactive B cells. Genetic and functional studies implicate B-cell receptor signaling as a pivotal pathway in its pathogenesis. Full B-cell receptor activation requires tumor-microenvironment interactions in lymphoid tissues. Spleen tyrosine kinase, Bruton’s tyrosine kinase, and the phosphatidylinositol 3-kinase (PI3K) δ isoform are essential for B-cell receptor signal transduction but also mediate the effect of other pathways engaged in chronic lymphocytic leukemia cells in the tissue-microenvironment. Orally bioavailable inhibitors of spleen tyrosine kinase, Bruton’s tyrosine kinase, or PI3Kδ, induce high rates of durable responses. Ibrutinib, a covalent inhibitor of Bruton’s tyrosine kinase, and idelalisib, a selective inhibitor of PI3Kδ, have obtained regulatory approval in chronic lymphocytic leukemia. Ibrutinib and idelalisib are active in patients with high-risk features, achieving superior disease control in difficult-to-treat patients than prior best therapy, making them the preferred agents for chronic lymphocytic leukemia with TP53 aberrations and for patients resistant to chemoimmunotherapy. In randomized trials, both ibrutinib, versus ofatumumab, and idelalisib in combination with rituximab, versus placebo with rituximab improved survival in relapsed/refractory chronic lymphocytic leukemia. Responses to B-cell receptor inhibitors are mostly partial, and within clinical trials treatment is continued until progression or occurrence of intolerable side effects. Ibrutinib and idelalisib are, overall, well tolerated; notable adverse events include increased bruising and incidence of atrial fibrillation on ibrutinib and colitis, pneumonitis and transaminase elevations on idelalisib. Randomized trials investigate the role of B-cell receptor inhibitors in first-line therapy and the benefit of combinations. This review discusses the biological basis for targeted therapy of chronic lymphocytic

  17. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice

    PubMed Central

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C. A.; Woll, Petter S.; Jacobsen, Sten Eirik W.

    2016-01-01

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  18. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis.

    PubMed

    Park, Youn-Hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG→TCG) at codon 47 and the codon 72 polymorphism (CGC→CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer.

  19. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

    PubMed Central

    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-01-01

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals-induced carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the mechanistic studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. PMID:26091798

  20. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis.

    PubMed

    Park, Youn-Hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG→TCG) at codon 47 and the codon 72 polymorphism (CGC→CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. PMID:26091798

  1. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

    PubMed

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C A; Woll, Petter S; Jacobsen, Sten Eirik W; Sitnicka, Ewa

    2016-07-14

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  2. B-cell-targeted therapy for systemic lupus erythematosus.

    PubMed

    Sabahi, Ramin; Anolik, Jennifer H

    2006-01-01

    Systemic lupus erythematosus (SLE) is a complex disease characterised by numerous autoantibodies and clinical involvement in multiple organ systems. The immunological events triggering the onset of clinical manifestations have not yet been fully defined, but a central role for B cells in the pathogenesis of this disease has more recently gained prominence as a result of research in both mice and humans. Both antibody-dependent and -independent mechanisms of B cells are important in SLE. Autoantibodies contribute to autoimmunity by multiple mechanisms, including immune complex-mediated type III hypersensitivity reactions, type II antibody-dependent cytotoxicity, and by instructing innate immune cells to produce pathogenic cytokines such as interferon-alpha, tumour necrosis factor and interleukin-1. Suggested autoantibody-independent B-cell functions include antigen presentation, T-cell activation and polarisation, and dendritic-cell modulation. Several of these functions are mediated by the ability of B cells to produce immunoregulatory cytokines, chemokines and lymphangiogenic growth factors, and by their critical contribution to lymphoid tissue development and organisation, including the development of ectopic tertiary lymphoid tissue. Given the large body of evidence implicating abnormalities in the B-cell compartment in SLE, a recent therapeutic focus has been to develop interventions that target the B-cell compartment by multiple mechanisms.Rituximab, a mouse-human chimeric monoclonal antibody against CD20 that specifically depletes B cells, has been studied the most extensively. Although promising open-label data await confirmation in ongoing multicentre placebo-controlled trials, a number of preliminary conclusions can be drawn. The adequacy of peripheral B-cell depletion depends on achieving high and sustained serum rituximab concentrations, pharmacokinetics that can be varied with treatment dose and factors that may affect drug clearance, such as human anti

  3. The Role of Latently Infected B Cells in CNS Autoimmunity

    PubMed Central

    Márquez, Ana Citlali; Horwitz, Marc Steven

    2015-01-01

    The onset of multiple sclerosis (MS) is caused by both genetic and environmental factors. Among the environmental factors, it is believed that previous infection with Epstein–Barr virus (EBV) may contribute in the development of MS. EBV has been associated with other autoimmune diseases, such as systemic lupus erythematous, and cancers like Burkitt’s lymphoma. EBV establishes a life-long latency in B cells with occasional reactivation of the virus throughout the individual’s life. The role played by B cells in MS pathology has been largely studied, yet is not clearly understood. In MS patients, Rituximab, a novel treatment that targets CD20+ B cells, has proven to have successful results in diminishing the number of relapses in remitting relapsing MS; however, the mechanism of how this drug acts has not been clearly established. In this review, we analyze the evidence of how B cells latently infected with EBV might be altering the immune system response and helping in the development of MS. We will also discuss how animal models, such as experimental autoimmune encephalomyelitis (EAE) and murine gammaherpesvirus-68 (γHV-68), can be used as powerful tools in the study of the relationship between EBV, MS, and B cells. PMID:26579121

  4. TRAF3 deficiency promotes metabolic reprogramming in B cells

    PubMed Central

    Mambetsariev, Nurbek; Lin, Wai W.; Wallis, Alicia M.; Stunz, Laura L.; Bishop, Gail A.

    2016-01-01

    The adaptor protein TNF receptor-associated factor 3 (TRAF3) is a critical regulator of B lymphocyte survival. B cell-specific TRAF3 deficiency results in enhanced viability and is associated with development of lymphoma and multiple myeloma. We show that TRAF3 deficiency led to induction of two proteins important for glucose metabolism, Glut1 and Hexokinase 2 (HXK2). This was associated with increased glucose uptake. In the absence of TRAF3, anaerobic glycolysis and oxidative phosphorylation were increased in B cells without changes in mitochondrial mass or reactive oxygen species. Chemical inhibition of glucose metabolism or glucose deprivation substantially attenuated the enhanced survival of TRAF3-deficient B cells, with a decrease in the pro-survival protein Mcl-1. Changes in Glut1 and Mcl-1 levels, glucose uptake and B cell number in the absence of TRAF3 were all dependent upon NF-κB inducing kinase (NIK). These results indicate that TRAF3 deficiency suffices to metabolically reprogram B cells, a finding that improves our understanding of the role of TRAF3 as a tumor suppressor, and suggests potential therapeutic strategies. PMID:27752131

  5. Long noncoding RNAs in B-cell development and activation

    PubMed Central

    Brazão, Tiago F.; Johnson, Jethro S.; Müller, Jennifer; Heger, Andreas; Ponting, Chris P.

    2016-01-01

    Long noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia. PMID:27381906

  6. Target sequence accessibility limits activation-induced cytidine deaminase activity in primary mediastinal B-cell lymphoma.

    PubMed

    Popov, Sergey W; Moldenhauer, Gerhard; Wotschke, Beate; Brüderlein, Silke; Barth, Thomas F; Dorsch, Karola; Ritz, Olga; Möller, Peter; Leithäuser, Frank

    2007-07-15

    Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in activated B lymphocytes and is potentially implicated in genomic instability of B-cell malignancies. For unknown reasons, B-cell neoplasms often lack SHM and CSR in spite of high AID expression. Here, we show that primary mediastinal B-cell lymphoma (PMBL), an immunoglobulin (Ig)-negative lymphoma that possesses hypermutated, class-switched Ig genes, expresses high levels of AID with an intact primary structure but does not do CSR in 14 of 16 cases analyzed. Absence of CSR coincided with low Ig germ-line transcription, whereas high level germ-line transcription was observed only in those two cases with active CSR. Interleukin-4/CD40L costimulation induced CSR and a marked up-regulation of germ-line transcription in the PMBL-derived cell line MedB-1. In the PMBL cell line Karpas 1106P, CSR was not inducible and germ-line transcription remained low on stimulation. However, Karpas 1106P, but not MedB-1, had ongoing SHM of the Ig gene and BCL6. These genes were transcribed in Karpas 1106P, whereas transcription was undetectable or low in MedB-1 cells. Thus, accessibility of the target sequences seems to be a major limiting factor for AID-dependent somatic gene diversification in PMBL. PMID:17638864

  7. Bruton's Tyrosine Kinase Regulates the Activation of Gene Rearrangements at the λ Light Chain Locus in Precursor B Cells in the Mouse

    PubMed Central

    Dingjan, Gemma M.; Middendorp, Sabine; Dahlenborg, Katarina; Maas, Alex; Grosveld, Frank; Hendriks, Rudolf W.

    2001-01-01

    Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an ∼50% reduction in the frequency of immunoglobulin (Ig) λ light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant BtkE41K showed increased λ usage. As the κ/λ ratio is dependent on (a) the level and kinetics of κ and λ locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of λ usage. Crossing 3-83μδ autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig+ B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced λ usage. An intrinsic defect in λ locus recombination was further supported by the finding in Btk-deficient mice of reduced λ usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the λ locus for V(D)J recombination in pre-B cells. PMID:11369788

  8. A Computational Study of the Effects of Syk Activity on B Cell Receptor Signaling Dynamics

    PubMed Central

    McGee, Reginald L.; Krisenko, Mariya O.; Geahlen, Robert L.; Rundell, Ann E.; Buzzard, Gregery T.

    2015-01-01

    The kinase Syk is intricately involved in early signaling events in B cells and is required for proper response when antigens bind to B cell receptors (BCRs). Experiments using an analog-sensitive version of Syk (Syk-AQL) have better elucidated its role, but have not completely characterized its behavior. We present a computational model for BCR signaling, using dynamical systems, which incorporates both wild-type Syk and Syk-AQL. Following the use of sensitivity analysis to identify significant reaction parameters, we screen for parameter vectors that produced graded responses to BCR stimulation as is observed experimentally. We demonstrate qualitative agreement between the model and dose response data for both mutant and wild-type kinases. Analysis of our model suggests that the level of NF-κB activation, which is reduced in Syk-AQL cells relative to wild-type, is more sensitive to small reductions in kinase activity than Erkp activation, which is essentially unchanged. Since this profile of high Erkp and reduced NF-κB is consistent with anergy, this implies that anergy is particularly sensitive to small changes in catalytic activity. Also, under a range of forward and reverse ligand binding rates, our model of Erkp and NF-κB activation displays a dependence on a power law affinity: the ratio of the forward rate to a non-unit power of the reverse rate. This dependence implies that B cells may respond to certain details of binding and unbinding rates for ligands rather than simple affinity alone. PMID:26525178

  9. DOCK2 regulates cell proliferation through Rac and ERK activation in B cell lymphoma

    SciTech Connect

    Wang, Lei; Nishihara, Hiroshi; Kimura, Taichi; Kato, Yasutaka; Tanino, Mishie; Nishio, Mitsufumi; Obara, Masato; Endo, Tomoyuki; Koike, Takao; Tanaka, Shinya

    2010-04-23

    DOCK2; a member of the CDM protein family, regulates cell motility and cytokine production through the activation of Rac in mammalian hematopoietic cells and plays a pivotal role in the modulation of the immune system. Here we demonstrated the alternative function of DOCK2 in hematopoietic tumor cells, especially in terms of its association with the tumor progression. Immunostaining for DOCK2 in 20 cases of human B cell lymphoma tissue specimens including diffuse large B cell lymphoma and follicular lymphoma revealed the prominent expression of DOCK2 in all of the lymphoma cells. DOCK2-knockdown (KD) of the B cell lymphoma cell lines, Ramos and Raji, using the lentiviral shRNA system presented decreased cell proliferation compared to the control cells. Furthermore, the tumor formation of DOCK2-KD Ramos cell in nude mice was significantly abrogated. Western blotting analysis and pull-down assay using GST-PAK-RBD kimeric protein suggested the presence of DOCK2-Rac-ERK pathway regulating the cell proliferation of these lymphoma cells. This is the first report to clarify the prominent role of DOCK2 in hematopoietic malignancy.

  10. Antibody response to a T-dependent antigen requires B cell expression of complement receptors

    PubMed Central

    1996-01-01

    Several lines of evidence indicate that antibody responses to T- dependent antigens require complement receptors expressed on either B lymphocytes or follicular dendritic cells. We have used RAG-2 deficient blastocyst complementation to create mice specifically lacking B cell complement receptors. Despite normal expression of complement receptor 1 (CR1[CD35]) and CR2 (CD21) on follicular dendritic cells, these mice have a profound defect in their capacity to mount a T-dependent antibody response. This is the first direct demonstration in vivo that B cell expression of complement receptors is required for a humoral immune response. This is the first direct demonstration in vivo that B cell expression of complement receptors is required for a humoral immune response. This suggests that CD21 and/or CD35 on B lymphocytes may be required for cellular activation, adsorptive endocytosis of antigen, recruitment to germinal centers, and/or protection from apoptosis during the humoral response to T-dependent antigens. PMID:8666942

  11. Role of EBNA-3 Family Proteins in EBV Associated B-cell Lymphomagenesis

    PubMed Central

    Bhattacharjee, Shaoni; Ghosh Roy, Shatadru; Bose, Priyanka; Saha, Abhik

    2016-01-01

    Epstein-Barr virus (EBV) is highly ubiquitous in human population and establishes a lifelong asymptomatic infection within the infected host unless the immune system is compromised. Following initial infection in the oropharyngeal epithelial cells, EBV primarily infects naive B-lymphocytes and develops a number of B-cell lymphomas particularly in immune-deficient individuals. In vitro, EBV can also infect and subsequently transform quiescent B-lymphocytes into continuously proliferating lymphoblastoid cell lines (LCLs) resembling EBV-induced lymphoproliferative disorders in which a subset of latent transcripts are detected. Genetic studies revealed that EBNA-3 family comprising of three adjacent genes in the viral genome—EBNA-3A and -3C, but not -3B, are critical for B-cell transformation. Nevertheless, all three proteins appear to significantly contribute to maintain the overall proliferation and viability of transformed cells, suggesting a critical role in lymphoma development. Apart from functioning as important viral transcriptional regulators, EBNA-3 proteins associate with many cellular proteins in different signaling networks, providing a suitable platform for lifelong survival of the virus and concurrent lymphoma development in the infected host. The chapter describes the function of each these EBV nuclear antigen 3 proteins employed by the virus as a means to understand viral pathogenesis of several EBV-associated B-cell malignancies. PMID:27092119

  12. Anti-cancer activity of withaferin A in B-cell lymphoma.

    PubMed

    McKenna, M K; Gachuki, B W; Alhakeem, S S; Oben, K N; Rangnekar, V M; Gupta, R C; Bondada, S

    2015-01-01

    Withaferin A (WA), a withanolide from the plant, Ashwagandha (Withania somnifera) used in Ayurvedic medicine, has been found to be valuable in the treatment of several medical ailments. WA has been found to have anticancer activity against various solid tumors, but its effects on hematological malignancies have not been studied in detail. WA strongly inhibited the survival of several human and murine B cell lymphoma cell lines. Additionally, in vivo studies with syngeneic-graft lymphoma cells suggest that WA inhibits the growth of tumor but does not affect other proliferative tissues. We demonstrate that WA inhibits the efficiency of NF-κB nuclear translocation in diffuse large B cell lymphomas and found that WA treatment resulted in a significant decrease in protein levels involved in B cell receptor signaling and cell cycle regulation. WA inhibited the activity of heat shock protein (Hsp) 90 as reflected by a sharp increase in Hsp70 expression levels. Hence, we propose that the anti-cancer effects of WA in lymphomas are likely due to its ability to inhibit Hsp90 function and subsequent reduction of critical kinases and cell cycle regulators that are clients of Hsp90. PMID:26020511

  13. A comprehensive review of lenalidomide in B-cell non-Hodgkin lymphoma

    PubMed Central

    Arora, Mili; Gowda, Sonia; Tuscano, Joseph

    2016-01-01

    Lenalidomide, an immunomodulatory drug that the US Food and Drug Administration (FDA) approved for the treatment of multiple myeloma, 5q- myelodysplasia and mantle-cell lymphoma (MCL), has encouraging efficacy in other B-cell malignancies. Its unique mechanism of action is in part due to altering the tumor microenvironment and potentiating the activity of T and natural-killer (NK) cells. Impressive clinical activity and excellent tolerability allows broad applicability. Lenalidomide has been used in a wide range of B-cell malignancies for years, but in 2013, the FDA marked its approval as a single agent only in relapsed/refractory mantle-cell lymphoma. Perhaps most impressive is the efficacy of lenalidomide when combined with monoclonal antibodies. Impressive efficacy and toxicity profiles with the combination of lenalidomide and rituximab in B-cell lymphomas in both the upfront and relapsed/refractory setting may allow a shift in our current treatment paradigm in both indolent and aggressive non-Hodgkin lymphoma (NHL). This review will summarize the current data in the relapsed/refractory and front-line setting of NHL with single-agent lenalidomide as well as its use in combination with other agents. PMID:27493711

  14. A comprehensive review of lenalidomide in B-cell non-Hodgkin lymphoma.

    PubMed

    Arora, Mili; Gowda, Sonia; Tuscano, Joseph

    2016-08-01

    Lenalidomide, an immunomodulatory drug that the US Food and Drug Administration (FDA) approved for the treatment of multiple myeloma, 5q- myelodysplasia and mantle-cell lymphoma (MCL), has encouraging efficacy in other B-cell malignancies. Its unique mechanism of action is in part due to altering the tumor microenvironment and potentiating the activity of T and natural-killer (NK) cells. Impressive clinical activity and excellent tolerability allows broad applicability. Lenalidomide has been used in a wide range of B-cell malignancies for years, but in 2013, the FDA marked its approval as a single agent only in relapsed/refractory mantle-cell lymphoma. Perhaps most impressive is the efficacy of lenalidomide when combined with monoclonal antibodies. Impressive efficacy and toxicity profiles with the combination of lenalidomide and rituximab in B-cell lymphomas in both the upfront and relapsed/refractory setting may allow a shift in our current treatment paradigm in both indolent and aggressive non-Hodgkin lymphoma (NHL). This review will summarize the current data in the relapsed/refractory and front-line setting of NHL with single-agent lenalidomide as well as its use in combination with other agents. PMID:27493711

  15. Dysregulation of system xc(-) expression induced by mutant huntingtin in a striatal neuronal cell line and in R6/2 mice.

    PubMed

    Frederick, Natalie M; Bertho, Julie; Patel, Kishan K; Petr, Geraldine T; Bakradze, Ekaterina; Smith, Sylvia B; Rosenberg, Paul A

    2014-10-01

    Oxidative stress has been implicated in the pathogenesis of Huntington's disease (HD), however, the origin of the oxidative stress is unknown. System xc(-) plays a role in the import of cystine to synthesize the antioxidant glutathione. We found in the STHdh(Q7/Q7) and STHdh(Q111/Q111) striatal cell lines, derived from neuronal precursor cells isolated from knock-in mice containing 7 or 111 CAG repeats in the huntingtin gene, that there is a decrease in system xc(-) function. System xc(-) is composed of two proteins, the substrate specific transporter, xCT, and an anchoring protein, CD98. The decrease in function in system xc(-) that we observed is caused by a decrease in xCT mRNA and protein expression in the STHdh(Q111/Q111) cells. In addition, we found a decrease in protein and mRNA expression in the transgenic R6/2 HD mouse model at 6weeks of age. STHdh(Q111/Q111) cells have lower basal levels of GSH and higher basal levels of ROS. Acute inhibition of system xc(-) causes greater increase in oxidative stress in the STHdh(Q111/Q111) cells than in the STHdh(Q7/Q7) cells. These results suggest that a defect in the regulation of xCT may be involved in the pathogenesis of HD by compromising xCT expression and increasing susceptibility to oxidative stress.

  16. An analysis of B cell selection mechanisms in germinal centers.

    PubMed

    Meyer-Hermann, Michael E; Maini, Philip K; Iber, Dagmar

    2006-09-01

    Affinity maturation of antibodies during immune responses is achieved by multiple rounds of somatic hypermutation and subsequent preferential selection of those B cells that express B cell receptors with improved binding characteristics for the antigen. The mechanism underlying B cell selection has not yet been defined. By employing an agent-based model, we show that for physiologically reasonable parameter values affinity maturation can be driven by competition for neither binding sites nor antigen--even in the presence of competing secreted antibodies. Within the tested mechanisms, only clonal competition for T cell help or a refractory time for the interaction of centrocytes with follicular dendritic cells is found to enable affinity maturation while generating the experimentally observed germinal centre characteristics and tolerating large variations in the initial antigen density. PMID:16707510

  17. Novel Therapies for Aggressive B-Cell Lymphoma

    PubMed Central

    Foon, Kenneth A.; Takeshita, Kenichi; Zinzani, Pier L.

    2012-01-01

    Aggressive B-cell lymphoma (BCL) comprises a heterogeneous group of malignancies, including diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, and mantle cell lymphoma (MCL). DLBCL, with its 3 subtypes, is the most common type of lymphoma. Advances in chemoimmunotherapy have substantially improved disease control. However, depending on the subtype, patients with DLBCL still exhibit substantially different survival rates. In MCL, a mature B-cell lymphoma, the addition of rituximab to conventional chemotherapy regimens has increased response rates, but not survival. Burkitt lymphoma, the most aggressive BCL, is characterized by a high proliferative index and requires more intensive chemotherapy regimens than DLBCL. Hence, there is a need for more effective therapies for all three diseases. Increased understanding of the molecular features of aggressive BCL has led to the development of a range of novel therapies, many of which target the tumor in a tailored manner and are summarized in this paper. PMID:22536253

  18. The role of B cells and autoantibodies in neuropsychiatric lupus.

    PubMed

    Wen, Jing; Stock, Ariel D; Chalmers, Samantha A; Putterman, Chaim

    2016-09-01

    The central nervous system manifestations of SLE (neuropsychiatric lupus, NPSLE) occur frequently, though are often difficult to diagnose and treat. Symptoms of NPSLE can be quite diverse, including chronic cognitive and emotional manifestations, as well as acute presentations, such as stroke and seizures. Although the pathogenesis of NPSLE has yet to be well characterized, B-cell mediated damage is believed to be an important contributor. B-cells and autoantibodies may traverse the blood brain barrier promoting an inflammatory environment consisting of glia activation, neurodegeneration, and consequent averse behavioral outcomes. This review will evaluate the various suggested roles of B-cells and autoantibodies in NPSLE, as well as therapeutic modalities targeting these pathogenic mediators.

  19. FOXP1 suppresses immune response signatures and MHC class II expression in activated B-cell-like diffuse large B-cell lymphomas

    PubMed Central

    Brown, P J; Wong, K K; Felce, S L; Lyne, L; Spearman, H; Soilleux, E J; Pedersen, L M; Møller, M B; Green, T M; Gascoyne, D M; Banham, A H

    2016-01-01

    The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P<0.05). FOXP1 knockdown in ABC-DLBCL cells led to increased cell-surface expression of HLA-DRA and CD74. In R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL patients (n=150), reduced HLA-DRA (<90% frequency) expression correlated with inferior overall survival (P=0.0003) and progression-free survival (P=0.0012) and with non-GCB subtype stratified by the Hans, Choi or Visco–Young algorithms (all P<0.01). In non-GCB DLBCL cases with <90% HLA-DRA, there was an inverse correlation with the frequency (P=0.0456) and intensity (P=0.0349) of FOXP1 expression. We propose that FOXP1 represents a novel regulator of genes targeted by the class II MHC transactivator CIITA (MHC II and CD74) and therapeutically targeting the FOXP1 pathway may improve antigen presentation and immune surveillance in high-risk DLBCL patients. PMID:26500140

  20. High-efficiency Generation of Multiple Short Noncoding RNA in B-cells and B-cell-derived Extracellular Vesicles

    PubMed Central

    Almanza, Gonzalo; Zanetti, Maurizio

    2015-01-01

    Short noncoding (snc)RNAs are important new players in the landscape of biologics with therapeutic potential. Recently, we reported on a new method for the synthesis and delivery of snc RNA in B-cells transfected with plasmid DNA. Here using the same approach, we demonstrate that B-cells can be programmed for the enforced biogenesis and synchronous release of multiple sncRNAs. Our data show that this goal is feasible and that multiple sncRNA are released in the extracellular compartment in amounts comparable to those from B-cells programmed to express and secrete one scnRNA only. Furthermore, we found that the cargo of extracellular vescicles (EVs) isolated from programmed B-cells is remarkably enriched for multiple sncRNA. On average, we found that the content of multiple sncRNAs in EVs is 3.6 copynumber/EV. Collectively, we demonstrate that B-cells can be easily programmed toward the synthesis and release of multiple sncRNAs, including sncRNA-laden EVs, efficiently and specifically. PMID:26670278

  1. Successful differentiation to T cells, but unsuccessful B-cell generation, from B-cell-derived induced pluripotent stem cells.

    PubMed

    Wada, Haruka; Kojo, Satoshi; Kusama, Chie; Okamoto, Naoki; Sato, Yorino; Ishizuka, Bunpei; Seino, Ken-ichiro

    2011-01-01

    Forced expression of certain transcription factors in somatic cells results in generation of induced pluripotent stem (iPS) cells, which differentiate into various cell types. We investigated T-cell and B-cell lineage differentiation from iPS cells in vitro. To evaluate the impact of iPS cell source, murine splenic B-cell-derived iPS (B-iPS) cells were generated after retroviral transduction of four transcription factors (Oct4, Sox2, Klf4 and c-Myc). B-iPS cells were identical to embryonic stem (ES) cells and mouse embryonic fibroblast (MEF)-derived iPS cells in morphology, ES cell marker expression as well as teratoma and chimera mouse formation. Both B-iPS and MEF-derived iPS cells differentiated into lymphocytes in OP9 co-culture systems. Both efficiently differentiated into T-cell lineage that produced IFN-γ on T-cell receptor stimulation. However, iPS cells including B-iPS cells were relatively resistant to B-cell lineage differentiation. One of the reasons of the failure of B-cell lineage differentiation seemed due to a defect of Pax5 expression in the differentiated cells. Therefore, current in vitro differentiation systems using iPS cells are sufficient for inducing T-cell but not B-cell lineage. PMID:21135032

  2. Engagement of major histocompatibility complex class I and class II molecules up-regulates intercellular adhesion of human B cells via a CD11/CD18-independent mechanism.

    PubMed

    Alcover, A; Juillard, V; Acuto, O

    1992-02-01

    We have studied the role of major histocompatibility complex (MHC) molecules in the regulation of intercellular adhesion of human B cells. We found that molecules able to bind to MHC class II molecules, such as monoclonal antibodies or staphylococcal enterotoxins, induced rapid and sustained homotypic adhesion of Epstein-Barr virus (EBV)-transformed B cell lines as well as peripheral blood B lymphocytes. Moreover, anti-MHC class I monoclonal antibodies also stimulated intercellular adherence. Adhesion induced upon MHC engagement was faster and stronger than that triggered by phorbol esters. It needed active metabolism, but divalent cations were not required. Monoclonal antibodies directed against LFA-1 (CD11a/CD18) or its ligand ICAM-1 (CD54) did not inhibit MHC class II-induced homotypic adhesion of various EBV-transformed B cell lines, nor of a variant of the B cell line Raji expressing very low LFA-1 surface levels. Moreover, EBV-transformed B cells from a severe lymphocyte adhesion deficiency patient, lacking surface CD11/CD18, also aggregated in response to anti-MHC class I or class II monoclonal antibodies. Together these data indicate that engagement of MHC molecules may transduce signals to B cells resulting in up-regulation of intercellular adhesion, via an LFA-1-independent mechanism. This may play a role in the stabilization of T cell/antigen-presenting cell conjugates at the moment of antigen recognition.

  3. Statin-induced changes in gene expression in EBV-transformed and native B-cells.

    PubMed

    Bolotin, Eugene; Armendariz, Angela; Kim, Kyungpil; Heo, Seok-Jin; Boffelli, Dario; Tantisira, Kelan; Rotter, Jerome I; Krauss, Ronald M; Medina, Marisa W

    2014-03-01

    Human lymphoblastoid cell lines (LCLs), generated through Epstein-Barr Virus (EBV) transformation of B-lymphocytes (B-cells), are a commonly used model system for identifying genetic influences on human diseases and on drug responses. We have previously used LCLs to examine the cellular effects of genetic variants that modulate the efficacy of statins, the most prescribed class of cholesterol-lowering drugs used for the prevention and treatment of cardiovascular disease. However, statin-induced gene expression differences observed in LCLs may be influenced by their transformation, and thus differ from those observed in native B-cells. To assess this possibility, we prepared LCLs and purified B-cells from the same donors, and compared mRNA profiles after 24 h incubation with simvastatin (2 µm) or sham buffer. Genes involved in cholesterol metabolism were similarly regulated between the two cell types under both the statin and sham-treated conditions, and the statin-induced changes were significantly correlated. Genes whose expression differed between the native and transformed cells were primarily implicated in cell cycle, apoptosis and alternative splicing. We found that ChIP-seq signals for MYC and EBNA2 (an EBV transcriptional co-activator) were significantly enriched in the promoters of genes up-regulated in the LCLs compared with the B-cells, and could be involved in the regulation of cell cycle and alternative splicing. Taken together, the results support the use of LCLs for the study of statin effects on cholesterol metabolism, but suggest that drug effects on cell cycle, apoptosis and alternative splicing may be affected by EBV transformation. This dataset is now uploaded to GEO at the accession number GSE51444.

  4. C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells.

    PubMed

    Di Stefano, Bruno; Sardina, Jose Luis; van Oevelen, Chris; Collombet, Samuel; Kallin, Eric M; Vicent, Guillermo P; Lu, Jun; Thieffry, Denis; Beato, Miguel; Graf, Thomas

    2014-02-13

    CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM). However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial-mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a 'path-breaker' as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications. PMID:24336202

  5. Innate immune control of EBV-infected B cells by invariant natural killer T cells.

    PubMed

    Chung, Brian K; Tsai, Kevin; Allan, Lenka L; Zheng, Dong Jun; Nie, Johnny C; Biggs, Catherine M; Hasan, Mohammad R; Kozak, Frederick K; van den Elzen, Peter; Priatel, John J; Tan, Rusung

    2013-10-10

    Individuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.

  6. Regulation of AID, the B-cell genome mutator.

    PubMed

    Keim, Celia; Kazadi, David; Rothschild, Gerson; Basu, Uttiya

    2013-01-01

    The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis.

  7. Bovine T-lymphocyte lines reactive with Brucella abortus.

    PubMed

    Smith, R; Kapatsa, J C; Rosenbaum, B A; Adams, L G

    1990-04-01

    Bovine T-cell lines reactive with Brucella abortus were established by repeated stimulation with B abortus and mitomycin C-treated autologous antigen-presenting cells. Representative results were obtained, using 33 cell lines from 14 cows. Cultures responded to the virulent laboratory strain 2308, the vaccine strain 19, and the rough mutant strain RB51 in thymidine-incorporation assays. The cells in these cultures required antigen-presenting cells for their response to B abortus. Autologous antigen-presenting cells were optimal for most lines tested, although some T-cell lines could respond to B abortus in the presence of some, but not all, allogeneic antigen-presenting cells. The cell lines expressed cell surface markers characteristics of activated bovine T cels. Of the cell lines tested for expression of cluster-determinant (CD) 4 and CD8 cell surface antigens, no cells in any cultures expressed the bovine CD8 equivalent, but all cultures included CD4+ cells in variable amounts. Some cell lines consisted of up to 50% CD2+CD4-CD8- cells. None of the cell lines tested expressed surface immunoglobulin or other bovine B-cell markers. Thus, these long-term cell lines appear to include 2 T-lymphocyte subsets: the helper/inducer subset and a second subset expressing a phenotype similar to major histocompatibility complex-unrestricted cytolytic cells in other species.

  8. Surrogate light chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by marginal zone B cells.

    PubMed

    Ren, Weicheng; Grimsholm, Ola; Bernardi, Angelina I; Höök, Nina; Stern, Anna; Cavallini, Nicola; Mårtensson, Inga-Lill

    2015-04-01

    Selection of the primary antibody repertoire takes place in pro-/pre-B cells, and subsequently in immature and transitional B cells. At the first checkpoint, μ heavy (μH) chains assemble with surrogate light (SL) chain into a precursor B-cell receptor. In mice lacking SL chain, μH chain selection is impaired, and serum autoantibody levels are elevated. However, whether the development of autoantibody-producing cells is due to an inability of the resultant B-cell receptors to induce central and/or peripheral B-cell tolerance or other factors is unknown. Here, we show that receptor editing is defective, and that a higher proportion of BM immature B cells are prone to undergoing apoptosis. Furthermore, transitional B cells are also more prone to undergoing apoptosis, with a stronger selection pressure to enter the follicular B-cell pool. Those that enter the marginal zone (MZ) B-cell pool escape selection and survive, possibly due to the B-lymphopenia and elevated levels of B-cell activating factor. Moreover, the MZ B cells are responsible for the elevated IgM anti-dsDNA antibody levels detected in these mice. Thus, the SL chain is required for central and peripheral B-cell tolerance and inhibits anti-DNA antibody production by MZ B cells.

  9. AUY922 effectively targets against activated B cell subtype of diffuse large B-cell lymphoma and low-grade lymphoma cells harboring genetic alteration-associated nuclear factor-κB activation.

    PubMed

    Tsai, Hui-Jen; Shih, Neng-Yao; Kuo, Sung-Hsin; Cheng, Ann-Lii; Lin, Hui-You; Chen, Tsai-Yun; Chang, Kung-Chao; Lin, Sheng-Fung; Chang, Jeffrey S; Chen, Li-Tzong

    2015-01-01

    Recurrent genetic alterations that are frequently observed in some low-grade lymphomas, such as activated B cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL) and mucosa-associated lymphoid tissue type lymphoma (MALT lymphoma) are usually associated with nuclear factor-κB (NF-κB) activation and confer resistance to therapy. In this study, we investigated the therapeutic efficacy and molecular mechanisms of AUY922, a novel Hsp90 inhibitor, in representative cell lines OCI-Ly3 (ABC-DLBCL) and MA-1 (a low-grade lymphoma cell line with t(14;18)/IgH-MALT1translocation) to explore its potential use in the treatment of refractory B-cell lymphoma. Our results showed that AUY922 effectively induced growth inhibition and apoptosis of OCI-Ly3 and MA-1 cells, which were accompanied by down-regulation of the expression levels of NF-κB and Bcl-2 family proteins, as well as molecules of multiple signaling pathways involving cell proliferation, growth and survival. The growth inhibitory effect of AUY922 was further confirmed in a mouse xenograft model. These findings indicate the potential use of AUY922 in B cell lymphomas.

  10. Very Rapid and Efficient Generation of Induced Pluripotent Stem Cells from Mouse Pre-B Cells.

    PubMed

    Di Stefano, Bruno; Graf, Thomas

    2016-01-01

    One of the major obstacles in generating induced pluripotent stem (iPS) cells suitable for therapeutic application is the low efficiency of the process and the long time required, with many iPS lines acquiring genomic aberrations. In this chapter we describe a highly efficient iPS reprogramming system based on the transient expression in pre-B cells of the transcription factor C/EBPα, followed by the induction of the four Yamanaka factors (OSKM). In addition, the process is very rapid, yielding Oct4 positive cells within 2 days and Nanog-positive iPS cell colonies within a week.

  11. FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma.

    PubMed

    Xie, Linka; Ritz, Olga; Leithäuser, Frank; Guan, Hanfeng; Färbinger, Johanna; Weitzer, Clarissa D; Gehringer, Franziska; Bruederlein, Silke; Holzmann, Karlheinz; Vogel, Marion J; Möller, Peter; Wirth, Thomas; Ushmorov, Alexey

    2014-07-30

    Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative Med-B1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL. PMID:24977668

  12. FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma

    PubMed Central

    Leithäuser, Frank; Guan, Hanfeng; Färbinger, Johanna; Weitzer, Clarissa D.; Gehringer, Franziska; Brüderlein, Silke; Holzmann, Karlheinz; Vogel, Marion J.; Möller, Peter; Wirth, Thomas; Ushmorov, Alexey

    2014-01-01

    Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative MedB-1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL. PMID:24977668

  13. Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

    PubMed Central

    Liu, Ta-Ming; Woyach, Jennifer A.; Zhong, Yiming; Lozanski, Arletta; Lozanski, Gerard; Dong, Shuai; Strattan, Ethan; Lehman, Amy; Zhang, Xiaoli; Jones, Jeffrey A.; Flynn, Joseph; Andritsos, Leslie A.; Maddocks, Kami; Jaglowski, Samantha M.; Blum, Kristie A.; Byrd, John C.; Dubovsky, Jason A.

    2015-01-01

    Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinib’s capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2R665W confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance. PMID:25972157

  14. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

    SciTech Connect

    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit

  15. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas.

    PubMed

    Kluiver, Joost; Poppema, Sibrand; de Jong, Debora; Blokzijl, Tjasso; Harms, Geert; Jacobs, Susan; Kroesen, Bart-Jan; van den Berg, Anke

    2005-10-01

    In a previous study we demonstrated high expression of the non-coding BIC gene in the vast majority of Hodgkin's lymphomas (HLs). Evidence suggesting that BIC is a primary microRNA transcript containing the mature microRNA-155 (miR-155) as part of a RNA hairpin is now accumulating. We therefore analysed HL cell lines and tissue samples to determine whether miR-155 is also expressed in HL. High levels of miR-155 could be demonstrated, indicating that BIC is processed into a microRNA in HL. Most non-HL subtypes were negative for BIC as determined by RNA-ISH. However, in diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBL), significant percentages of positive tumour cells were observed in 12/18 and 8/8 cases. A higher proportion of tumour cells were positive for BIC in DLBCL with activated B cell-like phenotype than in DLBCL with germinal centre B cell-like phenotype. Differential BIC expression was confirmed by qRT-PCR analysis. Northern blot analysis showed expression of miR-155 in all DLBCL and PMBL derived cell lines and tissue samples analysed. In summary, we demonstrate expression of primary microRNA BIC and its derivative miR-155 in HL, PMBL and DLBCL. PMID:16041695

  16. The Histological Classification of Diffuse Large B-cell Lymphomas

    PubMed Central

    Xie, Yi; Pittaluga, Stefania; Jaffe, Elaine S.

    2015-01-01

    Diffuse large B cell lymphomas (DLBCLs) are aggressive B-cell neoplasms with considerable clinical, biologic and pathologic diversity, in part reflecting the functional diversity of the B-cell system and multiple pathways of transformation. In recent years, the advent of new high-throughput genomic technologies has provided new insights into the biology of DLBCL, leading to the identification of distinct molecular identities and novel pathogenetic pathways. This increasing complexity had led to an expanding number of entities in the WHO classification. Using a multi-modality approach, the updated 2008 classification delineated some new subgroups, including DLBCLs associated with particular age groups or specific anatomic sites, as well as two borderline categories: tumors at the interface between classical Hodgkin lymphoma (cHL) and DLBCL as well as between Burkitt Lymphoma (BL) and DLBCL. This article reviews the histopathologic features of the various aggressive B-cell lymphoma subtypes included in the 2008 classification, with emphasis on some of the new entities as well as areas of diagnostic challenge. PMID:25805585

  17. Diffuse Large B Cell Lymphoma Mimicking Granulomatosis with Polyangiitis

    PubMed Central

    Horowitz, Netanel; Ben-Itzhak, Ofer; Braun-Moscovici, Yolanda

    2016-01-01

    In a patient with systemic multiorgan disease with overlapping features, the differential diagnosis included infectious diseases, malignancies, and systemic autoimmune or inflammatory diseases. We present an unusual case of a young male with B cell lymphoma who presented with symptoms mimicking systemic vasculitis and review the existing literature. PMID:27293945

  18. 324 Facility B-Cell quality process plan

    SciTech Connect

    Carlson, J.L.

    1998-06-10

    B-Cell is currently being cleaned out (i.e., removal of equipment, fixtures and residual radioactive materials) and deactivated. TPA Milestone M-89-02 dictates that all mixed waste and equipment be removed from B-Cell by 5/31/99. The following sections describe the major activities that remain for completion of the TPA milestone. This includes: (1) Size Reduce Tank 119 and Miscellaneous Equipment. This activity is the restart of hotwork in B-Cell to size reduce the remainder of Tank 119 and other miscellaneous pieces of equipment into sizes that can be loaded into a grout container. This activity also includes the process of preparing the containers for shipment from the cell. The specific activities and procedures used are detailed in a table. (2) Load and Ship Low-Level Waste. This activity covers the process of taking a grouted LLW container from B-Cell and loading it into the cask in the REC airlock and Cask Handling Area (CHA) for shipment to the LLBG. The detailed activities and procedures for this part of cell cleanout are included in second table.

  19. Likelihood-Based Inference of B Cell Clonal Families

    PubMed Central

    Ralph, Duncan K.

    2016-01-01

    The human immune system depends on a highly diverse collection of antibody-making B cells. B cell receptor sequence diversity is generated by a random recombination process called “rearrangement” forming progenitor B cells, then a Darwinian process of lineage diversification and selection called “affinity maturation.” The resulting receptors can be sequenced in high throughput for research and diagnostics. Such a collection of sequences contains a mixture of various lineages, each of which may be quite numerous, or may consist of only a single member. As a step to understanding the process and result of this diversification, one may wish to reconstruct lineage membership, i.e. to cluster sampled sequences according to which came from the same rearrangement events. We call this clustering problem “clonal family inference.” In this paper we describe and validate a likelihood-based framework for clonal family inference based on a multi-hidden Markov Model (multi-HMM) framework for B cell receptor sequences. We describe an agglomerative algorithm to find a maximum likelihood clustering, two approximate algorithms with various trade-offs of speed versus accuracy, and a third, fast algorithm for finding specific lineages. We show that under simulation these algorithms greatly improve upon existing clonal family inference methods, and that they also give significantly different clusters than previous methods when applied to two real data sets. PMID:27749910

  20. Regulatory roles of B cells in infectious diseases.

    PubMed

    Fillatreau, Simon

    2016-01-01

    B lymphocytes provide essential mechanisms of protection against infectious diseases. The secretion of specific antibodies by long-lived plasma cells is thought to account for the improved resistance afforded by most successful vaccines against pathogens. Accordingly, a goal in vaccine development is to induce potent B cell responses in order to drive the efficient formation of long-lived antibody-secreting cells. However, the roles of activated B cells are complex in infectious diseases. It was recently observed that activated B cells could also negatively regulate host defence mechanisms, both during primary infection and, after vaccination, upon secondary challenge, via mechanisms involving their production of the anti-inflammatory cytokines interleukin (IL)-10 and IL-35. Remarkably, the B cells expressing IL-10 and IL-35 in vivo were distinct subsets of IgMhiCD19+CD138hi antibody-secreting cells. A better understanding of the diverse roles of these distinct antibody-secreting cell subsets in immunity and immunological memory, as well as of the signals controlling their generation, might help the rational development of better prophylactic and therapeutic vaccines. PMID:27586794

  1. Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures.

    PubMed

    Cox, Kara S; Tang, Aimin; Chen, Zhifeng; Horton, Melanie S; Yan, Hao; Wang, Xin-Min; Dubey, Sheri A; DiStefano, Daniel J; Ettenger, Andrew; Fong, Rachel H; Doranz, Benjamin J; Casimiro, Danilo R; Vora, Kalpit A

    2016-01-01

    Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.

  2. Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures

    PubMed Central

    Cox, Kara S.; Tang, Aimin; Chen, Zhifeng; Horton, Melanie S.; Yan, Hao; Wang, Xin-Min; Dubey, Sheri A.; DiStefano, Daniel J.; Ettenger, Andrew; Fong, Rachel H.; Doranz, Benjamin J.; Casimiro, Danilo R.; Vora, Kalpit A.

    2016-01-01

    Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization. PMID:26491897

  3. Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures.

    PubMed

    Cox, Kara S; Tang, Aimin; Chen, Zhifeng; Horton, Melanie S; Yan, Hao; Wang, Xin-Min; Dubey, Sheri A; DiStefano, Daniel J; Ettenger, Andrew; Fong, Rachel H; Doranz, Benjamin J; Casimiro, Danilo R; Vora, Kalpit A

    2016-01-01

    Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization. PMID:26491897

  4. KLF4 is a tumor suppressor in B-cell non-Hodgkin lymphoma and in classic Hodgkin lymphoma.

    PubMed

    Guan, Hanfeng; Xie, Linka; Leithäuser, Frank; Flossbach, Lucia; Möller, Peter; Wirth, Thomas; Ushmorov, Alexey

    2010-09-01

    The transcription factor KLF4 may act both as an oncogene and a tumor suppressor in a tissue-depending manner. In T- and pre-B-cell lymphoma, KLF4 was found to act as tumor suppressor. We found the KLF4 promoter methylated in B-cell lymphoma cell lines and in primary cases of B-cell lymphomas, namely, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and in classic Hodgkin lymphoma (cHL) cases. Promoter hypermethylation was associated with silencing of KLF4 expression. Conditional overexpression of KLF4 in Burkitt lymphoma cell lines moderately retarded proliferation, via cell-cycle arrest in G(0)/G(1). In the cHL cell lines, KLF4 induced massive cell death that could partially be inhibited with Z-VAD.fmk. A quantitative reverse-transcribed polymerase chain reaction array revealed KLF4 target genes, including the proapoptotic gene BAK1. Using an shRNA-mediated knock-down approach, we found that BAK1 is largely responsible for KLF4-induced apoptosis. In addition, we found that KLF4 negatively regulates CXCL10, CD86, and MSC/ABF-1 genes. These genes are specifically up-regulated in HRS cells of cHL and known to be involved in establishing the cHL phenotype. We conclude that epigenetic silencing of KLF4 in B-cell lymphomas and particularly in cHL may favor lymphoma survival by loosening cell-cycle control and protecting from apoptosis. PMID:20519630

  5. Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties.

    PubMed

    Brandtzaeg, Per; Johansen, Finn-Eirik

    2005-08-01

    Mucosal antibody defense depends on a complex cooperation between local B cells and secretory epithelia. Mucosa-associated lymphoid tissue gives rise to B cells with striking J-chain expression that are seeded to secretory effector sites. Such preferential homing constitutes the biological basis for local production of polymeric immunoglobulin A (pIgA) and pentameric IgM with high affinity to the epithelial pIg receptor that readily can export these antibodies to the mucosal surface. This ultimate functional goal of mucosal B-cell differentiation appears to explain why the J chain is also expressed by IgG- and IgD-producing plasma cells (PCs) occurring at secretory tissue sites; these immunocytes may be considered as 'spin-offs' from early effector clones that through class switch are on their way to pIgA production. Abundant evidence supports the notion that intestinal PCs are largely derived from B cells initially activated in gut-associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists concerning the relative importance of M cells, major histocompatibility complex class II-expressing epithelial cells, and professional antigen-presenting cells for the uptake, processing, and presentation of luminal antigens in GALT to accomplish the extensive and sustained priming and expansion of mucosal B cells. Likewise, it is unclear how the germinal center reaction in GALT so strikingly can promote class switch to IgA and expression of J chain. Although B-cell migration from GALT to the intestinal lamina propria is guided by rather well-defined adhesion molecules and chemokines/chemokine receptors, the cues directing preferential homing to different segments of the gut require better definition. This is even more so for the molecules involved in homing of mucosal B cells to secretory effector sites beyond the gut, and in this respect, the role of Waldever's ring (including the palatine tonsils and adenoids) as a regional inductive tissue needs further

  6. Control of Viremia Enables Acquisition of Resting Memory B Cells with Age and Normalization of Activated B Cell Phenotypes in HIV-Infected Children

    PubMed Central

    Muema, Daniel M.; Macharia, Gladys N.; Hassan, Amin S.; Mwaringa, Shalton M.; Fegan, Greg W.; Berkley, James A.; Urban, Britta C.

    2015-01-01

    HIV affects the function of all lymphocyte populations, including B cells. Phenotypic and functional defects of B cells in HIV-infected adults have been well characterized, but defects in children have not been studied to the same extent. We determined the proportion of B cell subsets and frequencies of Ag-specific memory B cells in peripheral blood from HIV-infected children and healthy controls, using flow cytometry and B cell ELISPOT, respectively. In addition, we measured the quantities and avidities of plasma Abs against various Ags by ELISA. We also determined plasma levels of BAFF and expression of BAFF receptors on B cells. Children with high HIV viremia had increased proportions of activated mature B cells, tissue-like memory B cells and plasmablasts, and low proportions of naive B cells when compared with community controls and children with low HIV viremia, similar to adults infected with HIV. HIV-infected groups had lower proportions of resting memory B cells than did community controls. Notably, high HIV viremia prevented the age-dependent accumulation of class-switched resting memory B cells. HIV-infected children, regardless of the level of viremia, showed lower quantities and avidities of IgG and lower frequencies of memory B cells against Expanded Program on Immunization vaccines. The HIV-infected children had an altered BAFF profile that could have affected their B cell compartment. Therefore, B cell defects in HIV-infected children are similar to those seen in HIV-infected adults. However, control of HIV viremia is associated with normalization of activated B cell subsets and allows age-dependent accumulation of resting memory B cells. PMID:26116511

  7. Control of Viremia Enables Acquisition of Resting Memory B Cells with Age and Normalization of Activated B Cell Phenotypes in HIV-Infected Children.

    PubMed

    Muema, Daniel M; Macharia, Gladys N; Hassan, Amin S; Mwaringa, Shalton M; Fegan, Greg W; Berkley, James A; Nduati, Eunice W; Urban, Britta C

    2015-08-01

    HIV affects the function of all lymphocyte populations, including B cells. Phenotypic and functional defects of B cells in HIV-infected adults have been well characterized, but defects in children have not been studied to the same extent. We determined the proportion of B cell subsets and frequencies of Ag-specific memory B cells in peripheral blood from HIV-infected children and healthy controls, using flow cytometry and B cell ELISPOT, respectively. In addition, we measured the quantities and avidities of plasma Abs against various Ags by ELISA. We also determined plasma levels of BAFF and expression of BAFF receptors on B cells. Children with high HIV viremia had increased proportions of activated mature B cells, tissue-like memory B cells and plasmablasts, and low proportions of naive B cells when compared with community controls and children with low HIV viremia, similar to adults infected with HIV. HIV-infected groups had lower proportions of resting memory B cells than did community controls. Notably, high HIV viremia prevented the age-dependent accumulation of class-switched resting memory B cells. HIV-infected children, regardless of the level of viremia, showed lower quantities and avidities of IgG and lower frequencies of memory B cells against Expanded Program on Immunization vaccines. The HIV-infected children had an altered BAFF profile that could have affected their B cell compartment. Therefore, B cell defects in HIV-infected children are similar to those seen in HIV-infected adults. However, control of HIV viremia is associated with normalization of activated B cell subsets and allows age-dependent accumulation of resting memory B cells.

  8. Murine Gammaherpesvirus 68 Reactivation from B Cells Requires IRF4 but Not XBP-1

    PubMed Central

    Matar, Caline G.; Rangaswamy, Udaya Shankari; Wakeman, Brian S.; Iwakoshi, Neal

    2014-01-01

    ABSTRACT Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well-established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate early BZLF-1 or BRLF1/gene 50 promoters of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as interleukin-10 (IL-10) production, both of which are dependent on M2 induction of interferon regulatory factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation, and consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members or the involvement of other plasma cell-specific transcription factors. Regardless, these findings

  9. Activation of the Endoplasmic Reticulum Stress-Associated Transcription Factor X Box-Binding Protein-1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive B-Cell Lymphomas with Prognostic Implications

    PubMed Central

    Balague, Olga; Mozos, Ana; Martinez, Daniel; Hernandez, Luis; Colomo, Lluis; Mate, Jose Luis; Teruya-Feldstein, Julie; Lin, Oscar; Campo, Elias; Lopez-Guillermo, Armando; Martinez, Antonio

    2009-01-01

    X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4+/Bcl-6−/Pax-5− B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas. PMID:19389935

  10. Translational Mini-Review Series on B cell subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies.

    PubMed

    Vossenkämper, A; Lutalo, P M K; Spencer, J

    2012-01-01

    Systemic lupus erythematosus (SLE) and Sjögren's syndrome are autoimmune disorders which are characterized by a disturbed B cell homeostasis which leads ultimately to dysfunction of various organs. One of the B cell subsets that appear in abnormal numbers is the population of transitional B cells, which is increased in the blood of patients with SLE and Sjögren's syndrome. Transitional B cells are newly formed B cells. In mice, transitional B cells undergo selection checks for unwanted specificity in the bone marrow and the spleen in order to eliminate autoreactive B cells from the circulating naive B cell population. In humans, the exact anatomical compartments and mechanisms of the specificity check-points for transitional B cells remain unclear, but appear to be defective in SLE and Sjögren's syndrome. This review aims to highlight the current understanding of transitional B cells and their defects in the two disorders before and after B cell-targeted therapies.

  11. B cell autophagy mediates TLR7-dependent autoimmunity and inflammation

    PubMed Central

    Weindel, Chi G; Richey, Lauren J; Bolland, Silvia; Mehta, Abhiruchi J; Kearney, John F; Huber, Brigitte T

    2015-01-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5f/f). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines. PMID:26120731

  12. Inducible resistance to Fas-mediated apoptosis in B cells.

    PubMed

    Rothstein, T L

    2000-12-01

    Apoptosis produced in B cells through Fas (APO-1, CD95) triggering is regulated by signals derived from other surface receptors: CD40 engagement produces upregulation of Fas expression and marked susceptibility to Fas-induced cell death, whereas antigen receptor engagement, or IL-4R engagement, inhibits Fas killing and in so doing induces a state of Fas-resistance, even in otherwise sensitive, CD40-stimulated targets. Surface immunoglobulin and IL-4R utilize at least partially distinct pathways to produce Fas-resistance that differentially depend on PKC and STAT6, respectively. Further, surface immunoglobulin signaling for inducible Fas-resistance bypasses Btk, requires NF-kappaB, and entails new macromolecular synthesis. Terminal effectors of B cell Fas-resistance include the known anti-apoptotic gene products, Bcl-xL and FLIP, and a novel anti-apoptotic gene that encodes FAIM (Fas Apoptosis Inhibitory Molecule). faim was identified by differential display and exists in two alternatively spliced forms; faim-S is broadly expressed, but faim-L expression is tissue-specific. The FAIM sequence is highly evolu- tionarily conserved, suggesting an important role for this molecule throughout phylogeny. Inducible resistance to Fas killing is hypothesized to protect foreign antigen-specific B cells during potentially hazardous interactions with FasL-bearing T cells, whereas autoreactive B cells fail to become Fas-resistant and are deleted via Fas-dependent cytotoxicity. Inadvertent or aberrant acquisition of Fas-resistance may permit autoreactive B cells to escape Fas deletion, and malignant lymphocytes to impede anti-tumor immunity.

  13. B cell autophagy mediates TLR7-dependent autoimmunity and inflammation.

    PubMed

    Weindel, Chi G; Richey, Lauren J; Bolland, Silvia; Mehta, Abhiruchi J; Kearney, John F; Huber, Brigitte T

    2015-01-01

    Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, defined by loss of B cell self-tolerance that results in production of antinuclear antibodies (ANA) and chronic inflammation. While the initiating events in lupus development are not well defined, overexpression of the RNA-recognizing toll-like receptor (TLR)7 has been linked to SLE in humans and mice. We postulated that autophagy plays an essential role in TLR7 activation of B cells for the induction of SLE by delivering RNA ligands to the endosomes, where this innate immune receptor resides. To test this hypothesis, we compared SLE development in Tlr7 transgenic (Tg) mice with or without B cell-specific ablation of autophagy (Cd19-Cre Atg5(f/f)). We observed that in the absence of B cell autophagy the 2 hallmarks of SLE, ANA and inflammation, were eliminated, thus curing these mice of lupus. This was also evident in the significantly extended survival of the autophagy-deficient mice compared to Tlr7.1 Tg mice. Furthermore, glomerulonephritis was ameliorated, and the serum levels of inflammatory cytokines in the knockout (KO) mice were indistinguishable from those of control mice. These data provide direct evidence that B cells require TLR7-dependent priming through an autophagy-dependent mechanism before autoimmunity is induced, thereafter involving many cell types. Surprisingly, hyper-IgM production persisted in Tlr7.1 Tg mice in the absence of autophagy, likely involving a different activation pathway than the production of autoantibodies. Furthermore, these mice still presented with anemia, but responded with a striking increase in extramedullary hematopoiesis (EMH), possibly due to the absence of pro-inflammatory cytokines.

  14. Light chain editing in kappa-deficient animals: a potential mechanism of B cell tolerance

    PubMed Central

    1994-01-01

    The genetic organization of the kappa and lambda light chain loci permits multiple, successive rearrangement attempts at each allele. Multiple rearrangements allow autoreactive B cells to escape clonal deletion by editing their surface receptors. Editing may also facilitate efficient B cell production by salvaging cells with nonproductive light chain (L chain) rearrangements. To study receptor editing of kappa L chains, we have characterized B cells from mice hemizygous for the targeted inactivation of kappa (JCkD/wt) which have an anti-DNA heavy chain transgene, 3H9. Hybridomas from JCkD/wt mice exhibited an increased frequency of rearrangements to downstream Jk segments (such as Jk5) compared with most surveys from normal mice, consistent with receptor editing by sequential kappa locus rearrangements in JCkD/wt. We observed an even higher frequency of rearrangements to Jk5 in 3H9 JCkD/wt animals compared with nontransgenic JCkD/wt, consistent with editing of autoreactive kappa in 3H9 JCkD/wt. We also recovered a large number of 3H9 JCkD/wt lines with Vk12/13-Jk5 rearrangements and could demonstrate by PCR and Southern analysis that up to three quarters of these lines underwent multiple kappa rearrangements. To investigate editing at the lambda locus, we used homozygous kappa-deficient animals (JCkD/JCkD and 3H9 JCkD/JCkD). The frequencies of V lambda 1 and V lambda 2 rearrangements among splenic hybridomas in 3H9 JCkD/JCkD were reduced by 75% whereas V lambda X was increased 5-10-fold, compared with nontransgenic JCkD/JCkD animals. This indicates that V lambda 1 and V lambda 2 are negatively regulated in 3H9 JCkD/JCkD, consistent with earlier studies that showed that the 3H9 heavy chain, in combination with lambda 1 binds DNA. As successive lambda rearrangements to V lambda X do not inactivate V lambda 1, the consequence of lambda editing in 3H9 JCkD/JCkD would be failed allelic exclusion at lambda. However, analysis of 18 3H9 JCkD/JCkD hybridomas with V lambda 1

  15. Genetic and Physical Interaction of the B-Cell SLE-Associated Genes BANK1 and BLK

    PubMed Central

    Castillejo-López, Casimiro; Delgado-Vega, Angélica M.; Wojcik, Jerome; Kozyrev, Sergey V.; Thavathiru, Elangovan; Wu, Ying-Yu; Sánchez, Elena; Pöllmann, David; López-Egido, Juan R.; Fineschi, Serena; Domínguez, Nicolás; Lu, Rufei; James, Judith A.; Merrill, Joan T.; Kelly, Jennifer A.; Kaufman, Kenneth M.; Moser, Kathy; Gilkeson, Gary; Frostegård, Johan; Pons-Estel, Bernardo A.; D’Alfonso, Sandra; Witte, Torsten; Callejas, José Luis; Harley, John B.; Gaffney, Patrick; Martin, Javier; Guthridge, Joel M.; Alarcón-Riquelme, Marta E.

    2012-01-01

    Objectives Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis. Methods We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK. Results Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary naïve B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies. Conclusions Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway. PMID:21978998

  16. Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

    PubMed Central

    Randall, T D; Lund, F E; Brewer, J W; Aldridge, C; Wall, R; Corley, R B

    1993-01-01

    Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6. Images PMID:8321200

  17. Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function

    PubMed Central

    Portugal, Silvia; Tipton, Christopher M; Sohn, Haewon; Kone, Younoussou; Wang, Jing; Li, Shanping; Skinner, Jeff; Virtaneva, Kimmo; Sturdevant, Daniel E; Porcella, Stephen F; Doumbo, Ogobara K; Doumbo, Safiatou; Kayentao, Kassoum; Ongoiba, Aissata; Traore, Boubacar; Sanz, Inaki; Pierce, Susan K; Crompton, Peter D

    2015-01-01

    Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity. DOI: http://dx.doi.org/10.7554/eLife.07218.001 PMID:25955968

  18. Aberrant immunoglobulin class switch recombination and switch translocations in activated B cell-like diffuse large B cell lymphoma.

    PubMed

    Lenz, Georg; Nagel, Inga; Siebert, Reiner; Roschke, Anna V; Sanger, Warren; Wright, George W; Dave, Sandeep S; Tan, Bruce; Zhao, Hong; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D; Campo, Elias; Jaffe, Elaine S; Smeland, Erlend B; Fisher, Richard I; Kuehl, W Michael; Chan, Wing C; Staudt, Louis M

    2007-03-19

    To elucidate the mechanisms underlying chromosomal translocations in diffuse large B cell lymphoma (DLBCL), we investigated the nature and extent of immunoglobulin class switch recombination (CSR) in these tumors. We used Southern blotting to detect legitimate and illegitimate CSR events in tumor samples of the activated B cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subgroups of DLBCL. The frequency of legitimate CSR was lower in ABC DLBCL than in GCB DLBCL and PMBL. In contrast, ABC DLBCL had a higher frequency of internal deletions within the switch mu (Smu) region compared with GCB DLBCL and PMBL. ABC DLBCLs also had frequent deletions within Sgamma and other illegitimate switch recombinations. Sequence analysis revealed ongoing Smu deletions within ABC DLBCL tumor clones, which were accompanied by ongoing duplications and activation-induced cytidine deaminase-dependent somatic mutations. Unexpectedly, short fragments derived from multiple chromosomes were interspersed within Smu in one case. These findings suggest that ABC DLBCLs have abnormalities in the regulation of CSR that could predispose to chromosomal translocations. Accordingly, aberrant switch recombination was responsible for translocations in ABC DLBCLs involving BCL6, MYC, and a novel translocation partner, SPIB. PMID:17353367

  19. Transcription of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene occurs before induction of the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in B cells.

    PubMed

    Schlager, S; Speck, S H; Woisetschläger, M

    1996-06-01

    The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with

  20. Ibrutinib Before and After Stem Cell Transplant in Treating Patients With Relapsed or Refractory Diffuse Large B-cell Lymphoma

    ClinicalTrials.gov

    2016-10-27

    Activated B-Cell-Like Diffuse Large B-Cell Lymphoma; B-Cell Lymphoma, Unclassifiable, With Features Intermediate Between Diffuse Large B-Cell Lymphoma and Burkitt Lymphoma; Recurrent Diffuse Large B-Cell Lymphoma; Refractory Diffuse Large B-Cell Lymphoma

  1. Expansion of Activated Peripheral Blood Memory B Cells in Rheumatoid Arthritis, Impact of B Cell Depletion Therapy, and Biomarkers of Response

    PubMed Central

    Adlowitz, Diana G.; Barnard, Jennifer; Biear, Jamie N.; Cistrone, Christopher; Owen, Teresa; Wang, Wensheng; Palanichamy, Arumugam; Ezealah, Ezinma; Campbell, Debbie; Wei, Chungwen; Looney, R. John; Sanz, Inaki; Anolik, Jennifer H.

    2015-01-01

    Although B cell depletion therapy (BCDT) is effective in a subset of rheumatoid arthritis (RA) patients, both mechanisms and biomarkers of response are poorly defined. Here we characterized abnormalities in B cell populations in RA and the impact of BCDT in order to elucidate B cell roles in the disease and response biomarkers. In active RA patients both CD27+IgD- switched memory (SM) and CD27-IgD- double negative memory (DN) peripheral blood B cells contained significantly higher fractions of CD95+ and CD21- activated cells compared to healthy controls. After BCD the predominant B cell populations were memory, and residual memory B cells displayed a high fraction of CD21- and CD95+ compared to pre-depletion indicating some resistance of these activated populations to anti-CD20. The residual memory populations also expressed more Ki-67 compared to pre-treatment, suggesting homeostatic proliferation in the B cell depleted state. Biomarkers of clinical response included lower CD95+ activated memory B cells at depletion time points and a higher ratio of transitional B cells to memory at reconstitution. B cell function in terms of cytokine secretion was dependent on B cell subset and changed with BCD. Thus, SM B cells produced pro-inflammatory (TNF) over regulatory (IL10) cytokines as compared to naïve/transitional. Notably, B cell TNF production decreased after BCDT and reconstitution compared to untreated RA. Our results support the hypothesis that the clinical and immunological outcome of BCDT depends on the relative balance of protective and pathogenic B cell subsets established after B cell depletion and repopulation. PMID:26047509

  2. N-terminally truncated FOXP1 protein expression and alternate internal FOXP1 promoter usage in normal and malignant B cells

    PubMed Central

    Brown, Philip J.; Gascoyne, Duncan M.; Lyne, Linden; Spearman, Hayley; Felce, Suet Ling; McFadden, Nora; Chakravarty, Probir; Barrans, Sharon; Lynham, Steven; Calado, Dinis P.; Ward, Malcolm; Banham, Alison H.

    2016-01-01

    Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5′ non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation. PMID:27056922

  3. The Transcription Factor Bach2 Is Phosphorylated at Multiple Sites in Murine B Cells but a Single Site Prevents Its Nuclear Localization.

    PubMed

    Ando, Ryo; Shima, Hiroki; Tamahara, Toru; Sato, Yoshihiro; Watanabe-Matsui, Miki; Kato, Hiroki; Sax, Nicolas; Motohashi, Hozumi; Taguchi, Keiko; Yamamoto, Masayuki; Nio, Masaki; Maeda, Tatsuya; Ochiai, Kyoko; Muto, Akihiko; Igarashi, Kazuhiko

    2016-01-22

    The transcription factor Bach2 regulates the immune system at multiple points, including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells are still unclear. Here, we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of the B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in the mature B cell line BAL17. Among the sites identified, serine 535 (Ser-535) was critical for the regulation of Bach2 because a single mutation of Ser-535 abolished cytoplasmic accumulation of Bach2, promoting its nuclear accumulation in pre-B cells, whereas Ser-509 played an auxiliary role. Bach2 repressor activity was enhanced by the Ser-535 mutation in B cells. These results suggest that the PI3K-Akt-mTOR pathway inhibits Bach2 by both repressing its expression and inducing its phosphorylation in B cells. PMID:26620562

  4. Modifying akt signaling in B-cell chronic lymphocytic leukemia cells.

    PubMed

    Hofbauer, Sebastian W; Piñón, Josefina D; Brachtl, Gabriele; Haginger, Lucia; Wang, Wei; Jöhrer, Karin; Tinhofer, Ingeborg; Hartmann, Tanja Nicole; Greil, Richard

    2010-09-15

    Emerging evidence suggests that the survival of B-cell chronic lymphocytic leukemia (CLL) cells is dependent on microenvironmental influences such as antigenic stimulation and support by stromal cells. Akt, also known as protein kinase B, is a central component in prosurvival signaling downstream of these events. We investigated the role of Akt and its modulation by the protooncogene T-cell leukemia 1a (Tcl1a) in the survival pathways of primary CLL samples and CLL-derived prolymphocytic cell lines MEC-1 and MEC-2. Akt activation was increased by the protective presence of human bone marrow stromal cells and B-cell receptor mimicking signals but antagonized by direct Akt blockade with the novel specific inhibitor AiX, with preferential apoptosis induction in CLL cells with an unmutated immunoglobulin status, which predicts poor clinical outcome. In addition, we found a direct interaction of Akt with Tcl1a in an endogenous coimmunoprecipitation assay. Confirming the critical role of Tcl1a in modulating Akt signaling, Akt activation was enhanced by overexpressing Tcl1a in CLL. In contrast, decreasing Tcl1a levels by small interfering RNA reduced Akt activation in the fludarabine-insensitive CLL cell line MEC-2 and sensitized the malignant cells to fludarabine treatment. In summary, our data reveal a significant role for the Akt-Tcl1a axis in CLL survival and propose a further evaluation of this interplay for targeting chemoresistance phenomena.

  5. Deletion of genes encoding PU.1 and Spi-B in B cells impairs differentiation and induces pre-B cell acute lymphoblastic leukemia.

    PubMed

    Sokalski, Kristen M; Li, Stephen K H; Welch, Ian; Cadieux-Pitre, Heather-Anne T; Gruca, Marek R; DeKoter, Rodney P

    2011-09-01

    The E26 transformation-specific (Ets) transcription factor PU.1 is required to generate lymphoid progenitor cells from hematopoietic stem cells, but it is not required to generate B cells from committed B-cell lineage progenitors. We hypothesized that PU.1 function in B-cell differentiation is complemented by the related Ets transcription factor Spi-B. To test this hypothesis, mice were generated lacking both PU.1 and Spi-B in the B-cell lineage. Unlike mice lacking PU.1 or Spi-B, mice deficient in both PU.1 and Spi-B in the B-cell lineage had reduced frequencies of B cells as well as impaired B-cell differentiation. Strikingly, all PU.1 and Spi-B-deficient mice developed pre-B cell acute lymphoblastic leukemia before 30 weeks of age. Pre-B cells accumulated in the thymus resulting in massive thymic enlargement and dyspnea. These findings demonstrate that PU.1 and Spi-B are essential transcriptional regulators of B-cell differentiation as well as novel tumor suppressors in the B-cell lineage.

  6. Coriolus versicolor mushroom polysaccharides exert immunoregulatory effects on mouse B cells via membrane Ig and TLR-4 to activate the MAPK and NF-κB signaling pathways.

    PubMed

    Yang, Shu-fa; Zhuang, Tai-feng; Si, Yan-mei; Qi, Ke-yan; Zhao, Juan

    2015-03-01

    This study aimed to characterize the immunopotentiating effects and immune receptors for Coriolus versicolor mushroom polysaccharides (CVP), a Chinese medicinal fungus that exerts anti-tumor activities by enhancing host immunity. Proliferation assays were used to determine whether CVP could activate splenocytes. Flow cytometry analysis and IgM and IgG detection were used to characterize CVP-binding cells. Immune receptors were analyzed in immunoprecipitation and western blot assays. The downstream signaling pathways were identified by western blotting or immunostaining. CVP significantly stimulated the proliferation of mouse splenocytes. Fluorescence-labeled CVP (fl-CVP) selectively stained mouse B cells, but not T cells. CVP induced the production of IgM and IgG1 with or without exogenous IL-4. Membrane Ig (B cell antigen-receptor, BCR) was identified as a CVP-binding protein in immunoprecipitation and western blot experiments. CVP-induced B cell proliferation could be significantly inhibited by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ). Phosphorylation of ERK-1/2 and p38 MAPK were clearly increased in a time-dependent manner, as was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP stimulation. Together, we demonstrate that CVP can bind and induce B cell activation using membrane Ig and TLR-4 as potential immune receptors. CVP activates mouse B cells through the MAPK and NF-κB signaling pathways.

  7. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve.

    PubMed

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O; Marasco, Wayne A

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of 'universal' influenza vaccine strategies.

  8. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

    PubMed Central

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M. Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O.; Marasco, Wayne A.

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies. PMID:27619409

  9. DUSP4 deficiency caused by promoter hypermethylation drives JNK signaling and tumor cell survival in diffuse large B cell lymphoma

    PubMed Central

    Schmid, Corina A.; Robinson, Mark D.; Scheifinger, Nicole A.; Müller, Sebastian; Cogliatti, Sergio; Tzankov, Alexandar

    2015-01-01

    The epigenetic dysregulation of tumor suppressor genes is an important driver of human carcinogenesis. We have combined genome-wide DNA methylation analyses and gene expression profiling after pharmacological DNA demethylation with functional screening to identify novel tumor suppressors in diffuse large B cell lymphoma (DLBCL). We find that a CpG island in the promoter of the dual-specificity phosphatase DUSP4 is aberrantly methylated in nodal and extranodal DLBCL, irrespective of ABC or GCB subtype, resulting in loss of DUSP4 expression in 75% of >200 examined cases. The DUSP4 genomic locus is further deleted in up to 13% of aggressive B cell lymphomas, and the lack of DUSP4 is a negative prognostic factor in three independent cohorts of DLBCL patients. Ectopic expression of wild-type DUSP4, but not of a phosphatase-deficient mutant, dephosphorylates c-JUN N-terminal kinase (JNK) and induces apoptosis in DLBCL cells. Pharmacological or dominant-negative JNK inhibition restricts DLBCL survival in vitro and in vivo and synergizes strongly with the Bruton’s tyrosine kinase inhibitor ibrutinib. Our results indicate that DLBCL cells depend on JNK signaling for survival. This finding provides a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, ideally in synthetic lethal combinations with inhibitors of chronic active B cell receptor signaling. PMID:25847947

  10. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve.

    PubMed

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O; Marasco, Wayne A

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of 'universal' influenza vaccine strategies. PMID:27619409

  11. Significance of p27 Immunostaining in B-Cell Neoplasm.

    PubMed

    El-Kerdany, Tahany A; Shams Eldin El Telbany, Manal A; Esmaeel, Manal M; Mahmoud, Hanan M

    2016-08-01

    P27 is an important cell cycle regulatory protein. Many reports have validated the utility of p27 as a prognostic marker in different human cancers and to prove its prognostic role in B-cell neoplasm; 80 newly diagnosed B-cell neoplasm patients with mean age of 46.6 years recruited from Hematology/Oncology Unit of Ain Shams University Hospitals during the period from January 2008 till June 2010 were studied for their p27 immunostaining results which showed that all cases of chronic lymphocytic leukemia (CLL) were positive for p27, whereas all mantly cell lymphoma cases were negative for it. There was significantly higher p27 positivity in CLL cases compared with non-Hodgkin lymphoma and that indolent cases showed significantly higher rate of positivity when compared with aggressive and highly aggressive cases. So, we can use this marker to differentiate CLL and mantly cell lymphoma in cases of confusion.

  12. 324 Facility B-cell quality process plan

    SciTech Connect

    Carlson, J.L.

    1998-07-29

    B-Cell is currently being cleaned out (i.e., removal of equipment, fixtures and residual radioactive materials) and deactivated. TPA Milestone M-89-02 dictates that all mixed waste and equipment be removed from B-Cell by 5/31/99. The following sections describe the major activities that remain for completion of the TPA milestone. These include: Size Reduce Tank 119 and Miscellaneous Equipment; Load and Ship Low-Level Waste; Remove and Size Reduce the 1B Rack; Collect Dispersible Material from Cell Floor; Remove and Size Reduce the 2A Rack; Size Reduce the 1A Rack; Load and Ship Mixed Waste to PUREX Tunnels; and Move Spent Fuel to A-Cell;

  13. The NF-κB subunit c-Rel regulates Bach2 tumour suppressor expression in B-cell lymphoma

    PubMed Central

    Hunter, J E; Butterworth, J A; Zhao, B; Sellier, H; Campbell, K J; Thomas, H D; Bacon, C M; Cockell, S J; Gewurz, B E; Perkins, N D

    2016-01-01

    The REL gene, encoding the NF-κB subunit c-Rel, is frequently amplified in B-cell lymphoma and functions as a tumour-promoting transcription factor. Here we report the surprising result that c-rel–/– mice display significantly earlier lymphomagenesis in the c-Myc driven, Eμ-Myc model of B-cell lymphoma. c-Rel loss also led to earlier onset of disease in a separate TCL1-Tg-driven lymphoma model. Tumour reimplantation experiments indicated that this is an effect intrinsic to the Eμ-Myc lymphoma cells but, counterintuitively, c-rel–/– Eμ-Myc lymphoma cells were more sensitive to apoptotic stimuli. To learn more about why loss of c-Rel led to earlier onset of disease, microarray gene expression analysis was performed on B cells from 4-week-old, wild-type and c-rel–/– Eμ-Myc mice. Extensive changes in gene expression were not seen at this age, but among those transcripts significantly downregulated by the loss of c-Rel was the B-cell tumour suppressor BTB and CNC homology 2 (Bach2). Quantitative PCR and western blot analysis confirmed loss of Bach2 in c-Rel mutant Eμ-Myc tumours at both 4 weeks and the terminal stages of disease. Moreover, Bach2 expression was also downregulated in c-rel–/– TCL1-Tg mice and RelA Thr505Ala mutant Eμ-Myc mice. Analysis of wild-type Eμ-Myc mice demonstrated that the population expressing low levels of Bach2 exhibited the earlier onset of lymphoma seen in c-rel–/– mice. Confirming the relevance of these findings to human disease, analysis of chromatin immunoprecipitation sequencing data revealed that Bach2 is a c-Rel and NF-κB target gene in transformed human B cells, whereas treatment of Burkitt's lymphoma cells with inhibitors of the NF-κB/IκB kinase pathway or deletion of c-Rel or RelA resulted in loss of Bach2 expression. These data reveal a surprising tumour suppressor role for c-Rel in lymphoma development explained by regulation of Bach2 expression, underlining the context-dependent complexity of NF

  14. CGCI Investigators Reveal Comprehensive Landscape of Diffuse Large B-Cell Lymphoma (DLBCL) Genomes | Office of Cancer Genomics

    Cancer.gov

    Researchers from British Columbia Cancer Agency used whole genome sequencing to analyze 40 DLBCL cases and 13 cell lines in order to fill in the gaps of the complex landscape of DLBCL genomes. Their analysis, “Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing,” was published online in Blood on May 22. The authors are Ryan Morin, Marco Marra, and colleagues.  

  15. Are T cells at the origin of B cell lymphomas?

    PubMed

    Meyer-Hermann, Michael E

    2007-02-21

    Lymphoma pathogenesis is at least in some cases related to transformed B cells (BCs) arising from germinal centre reactions (GCRs). In this article possible deregulations of GCRs are investigated using in silico simulations. It is found that the final differentiation of BCs as regulated by helper T cells (TCs) is the best candidate mechanism for such a deregulation. This shifts the paradigm of BC lymphoma pathogenesis from BC transformations to an emphasized role of TC-BC interactions. PMID:17070849

  16. Early alterations of B cells in patients with septic shock

    PubMed Central

    2013-01-01

    Introduction It has recently been proposed that B lymphocytes are involved in sepsis pathogenesis. The goal of this study is to investigate potential abnormalities in a subset distribution and activation of circulating B lymphocytes in patients with septic shock. Methods This observational prospective study was conducted in a medical-surgical ICU. All patients with septic shock were eligible for inclusion. B-cell phenotypes (CD19+CD69+, CD19+CD23+, CD19+CD5+, CD19+CD80, CD19+CD86+, CD19+CD40 and CD19+CD95+) were assessed by quantitative flow cytometry upon admission to the ICU and 3, 7, 14 and 28 d later. Results Fifty-two patients were included. Thirty-six healthy volunteers matched for age and sex were used as controls. The patients had lymphopenia that was maintained during 28 d of follow-up. In patients with septic shock who died, the percentage of CD19+CD23+ was lower during the 7 d of follow-up than it was in survival patients. Moreover, the percentage of CD80+ and CD95+ expression on B cells was higher in patients who died than in survivors. Receiver operating characteristic curve analysis showed that a CD19+CD23+ value of 64.6% at ICU admission enabled discrimination between survivors and nonsurvivors with a sensitivity of 90.9% and a specificity of 80.0% (P = 0.0001). Conclusions Patients with septic shock who survive and those who don't have different patterns of abnormalities in circulating B lymphocytes. At ICU admission, a low percentage of CD23+ and a high of CD80+ and CD95+ on B cells were associated with increased mortality of patients with septic shock. Moreover, a drop in circulating B cells persisted during 28 d of ICU follow-up. PMID:23721745

  17. Molecular characterization of primary mediastinal B cell lymphoma.

    PubMed

    Tsang, P; Cesarman, E; Chadburn, A; Liu, Y F; Knowles, D M

    1996-06-01

    Primary mediastinal B cell lymphoma (PMBL) is a diffuse large B cell lymphoma (DLCL) postulated to arise from noncirculating thymic B lymphocytes. Because of its distinctive clinical and morphological features and putative unique cellular origin, PMBL is generally considered a distinct clinicopathological entity. Little is known, however, about the molecular characteristics of PMBL. Therefore, we analyzed 16 PMBLs for molecular alterations involving the bcl-1, bcl-2, bcl-6, c-myc, H-ras, K-ras, N-ras, and p53 genes and for Epstein-Barr virus infection, which are commonly involved in lymphoid neoplasia. Employing a combination of Southern blotting and/or polymerase chain reaction and single-strand conformation polymorphism assays, we detected genetic alterations in 7 of the 16 (44%) PMBLs. Whereas the bcl-6 gene is rearranged in up to 45% of DLCLs, rearrangement of the bcl-6 gene was detected in only 1 of these 16 (6%) PMBLS. Point mutations of the 5' noncoding region of the c-myc gene were demonstrated in 3 other cases (19%), although c-myc gene rearrangements were not seen by Southern blotting. Missense point mutations of the p53 gene were identified in 3 additional PMBLs (19%). Alterations of the bcl-1, bcl-2, or ras genes and evidence of Epstein-Barr virus infection were not observed. In conclusion, a variety of molecular lesions occur in PMBLs and may be involved in their pathogenesis. This molecular genetic pattern bears little resemblance to that known for other B cell malignancies, including DLCL. In particular, the infrequent occurrence of bcl-6 gene rearrangement in PMBLs distinguishes them from other DLCLs of B cell origin, suggesting that PMBLs do not represent a distinct subtype of DLCL. PMID:8669486

  18. Molecular characterization of primary mediastinal B cell lymphoma.

    PubMed Central

    Tsang, P.; Cesarman, E.; Chadburn, A.; Liu, Y. F.; Knowles, D. M.

    1996-01-01

    Primary mediastinal B cell lymphoma (PMBL) is a diffuse large B cell lymphoma (DLCL) postulated to arise from noncirculating thymic B lymphocytes. Because of its distinctive clinical and morphological features and putative unique cellular origin, PMBL is generally considered a distinct clinicopathological entity. Little is known, however, about the molecular characteristics of PMBL. Therefore, we analyzed 16 PMBLs for molecular alterations involving the bcl-1, bcl-2, bcl-6, c-myc, H-ras, K-ras, N-ras, and p53 genes and for Epstein-Barr virus infection, which are commonly involved in lymphoid neoplasia. Employing a combination of Southern blotting and/or polymerase chain reaction and single-strand conformation polymorphism assays, we detected genetic alterations in 7 of the 16 (44%) PMBLs. Whereas the bcl-6 gene is rearranged in up to 45% of DLCLs, rearrangement of the bcl-6 gene was detected in only 1 of these 16 (6%) PMBLS. Point mutations of the 5' noncoding region of the c-myc gene were demonstrated in 3 other cases (19%), although c-myc gene rearrangements were not seen by Southern blotting. Missense point mutations of the p53 gene were identified in 3 additional PMBLs (19%). Alterations of the bcl-1, bcl-2, or ras genes and evidence of Epstein-Barr virus infection were not observed. In conclusion, a variety of molecular lesions occur in PMBLs and may be involved in their pathogenesis. This molecular genetic pattern bears little resemblance to that known for other B cell malignancies, including DLCL. In particular, the infrequent occurrence of bcl-6 gene rearrangement in PMBLs distinguishes them from other DLCLs of B cell origin, suggesting that PMBLs do not represent a distinct subtype of DLCL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8669486

  19. [Significance of regulatory B cells in nosogenesis of immune thrombocytopenia].

    PubMed

    Li, Xin; Wang, Fang; Ding, Kai Yang; Dai, Lan

    2014-04-01

    This study was aimed to investigate the role of regulatory B cells (Breg) in pathogenesis of immune thrombocytopenia (ITP) and its clinical significance. A total of 35 ITP patients and 20 normal controls were enrolled in this study. The expression of CD19(+)CD24(hi)CD38(hi) B cells was detected by flow cytometry and the expression of IL-10 mRNA and TGF-β1 mRNA was assayed by RT-PCR. The results indicated that the expression level of CD19(+)CD24(hi)CD38(hi) B cells in peripheral blood of newly diagnosed ITP patients was obviously lower than that in normal controls (P < 0.05); the expression level of CD19(+)CD24(hi)CD38(hi) B cells in ITP patients with increased platelet count after treatment was higher than that before treatment (P < 0.05); the expression level of IL-10 mRNA in newly diagnosed ITP patients was significantly lower than that the in normal controls (P < 0.05), the expression level of TGF-β1 mRNA in newly diagnosed ITP patients increases as compared with normal controls (P < 0.05), after treatment with DXM the expression of IL-10 mRNA was enhanced, the expression of TGF-β1 mRNA was reduced as compared with expression level before treatment (P < 0.05). It is concluded that the Breg cells may play an important role in the pathogenesis of ITP via humoral immunity and its regulation of T lymphocytes.

  20. Large anaplastic spinal B-cell lymphoma in a cat.

    PubMed

    Flatland, Bente; Fry, Michael M; Newman, Shelley J; Moore, Peter F; Smith, Joanne R; Thomas, William B; Casimir, Roslyn H

    2008-12-01

    A 5-year-old female spayed domestic shorthair cat was presented for evaluation of tetraparesis. The neurologic lesion was localized to the cervical spinal segment (C1-C6). A left axillary mass was identified, and the results of fine needle aspiration cytology indicated malignant round cell neoplasia of possible histiocytic origin. The cells were large, had marked anisocytosis and anisokaryosis, occasional bi- and multinucleation, and cytoplasmic vacuolation. Euthanasia was performed due to the poor prognosis associated with severe, progressive neurologic signs and a malignant neoplasm. Postmortem examination revealed spinal cord compression and an extradural mass at the C1-C2 spinal segment, with neoplastic cells in the adjacent vertebral bodies, surrounding skeletal muscle, left axillary lymph node, and bone marrow from the right femur. The initial histologic diagnosis was anaplastic sarcoma, but immunohistochemical results indicated the cells were CD20+ and CD45R+ and CD3-, compatible with a diagnosis of B-cell lymphoma. CD79a staining was nonspecific and uninterpretable. Weak to moderate CD18 positivity and E-cadherin positivity were also observed. Clonality of the B-cell population could not be demonstrated using PCR testing for antigen receptor gene rearrangement. To the authors' knowledge, this is the first reported case of a feline spinal anaplastic B-cell lymphoma exhibiting bi- and multinucleated cells. The prognostic significance of this cell morphology and immunophenotype is unknown.

  1. APOBEC3 enzymes restrict marginal zone B cells

    PubMed Central

    Beck-Engeser, Gabriele B.; Winkelmann, Rebecca; Wheeler, Matthew L.; Shansab, Maryam; Yu, Philipp; Wünsche, Sarah; Walchhütter, Anja; Metzner, Mirjam; Vettermann, Christian; Eilat, Dan; DeFranco, Anthony; Jäck, Hans-Martin; Wabl, Matthias

    2016-01-01

    In general, a long-lasting immune response to viruses is achieved when they are infectious and replication-competent. In the mouse, the neutralizing antibody response to Friend murine leukemia virus is contributed by an allelic form of the enzyme Apobec3 (abbreviated A3). This is counterintuitive, because A3 directly controls viremia before the onset of adaptive anti-viral immune responses. It suggests that A3 also affects the antibody response directly. Here we studied the relative size of cell populations of the adaptive immune system as a function of A3 activity. We created a transgenic mouse that expresses all seven human A3 enzymes (hA3) and compared it to wild-type and mouse A3 (mA3)-deficient mice. A3 enzymes decreased the number of marginal zone (MZ) B cells, but not the number of follicular B or T cells. When mA3 was knocked out, the retroelement hitchhiker-1 and sialyl transferases encoded by genes close to it were overexpressed three and two orders of magnitude, respectively. We suggest that A3 shifts the balance, from the fast antibody response mediated by MZ B cells with little affinity maturation, to a more sustained germinal center B-cell response, which drives affinity maturation and, thereby, a better neutralizing response. PMID:25501566

  2. Adaptive Response of T and B Cells in Atherosclerosis.

    PubMed

    Ketelhuth, Daniel F J; Hansson, Göran K

    2016-02-19

    Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of cholesterol-containing lipoproteins, particularly low-density lipoprotein, in the artery wall. In the arterial intima, lipoprotein components that are generated through oxidative, lipolytic, and proteolytic activities lead to the formation of several danger-associated molecular patterns, which can activate innate immune cells as well as vascular cells. Moreover, self- and non-self-antigens, such as apolipoprotein B-100 and heat shock proteins, can contribute to vascular inflammation by triggering the response of T and B cells locally. This process can influence the initiation, progression, and stability of plaques. Substantial clinical and experimental data support that the modulation of adaptive immune system may be used for treating and preventing atherosclerosis. This may lead to the development of more selective and less harmful interventions, while keeping host defense mechanisms against infections and tumors intact. Approaches such as vaccination might become a realistic option for cardiovascular disease, especially if they can elicit regulatory T and B cells and the secretion of atheroprotective antibodies. Nevertheless, difficulties in translating certain experimental data into new clinical therapies remain a chall