The bradykinin B2 receptor in the early immune response against Listeria infection.

PubMed

Kaman, Wendy E; Wolterink, Arthur F W M; Bader, Michael; Boele, Linda C L; van der Kleij, Desiree

2009-02-01

The endogenous danger signal bradykinin was recently found implicated in the development of immunity against parasites via dendritic cells. We here report an essential role of the B(2) (B(2)R) bradykinin receptor in the early immune response against Listeria infection. Mice deficient in B(2)R (B(2)R(-/-) mice) were shown to suffer from increased hepatic bacterial burden and concomitant dramatic weight loss during infection with Listeria monocytogenes. Levels of cytokines known to play a pivotal role in the early phase immune response against L. monocytogenes, IL-12p70 and IFN-gamma, were reduced in B(2)R(-/-) mice. To extend these findings to the human system, we show that bradykinin potentiates the production of IL-12p70 in human monocyte-derived dendritic cells. Thus, we show that bradykinin and the B(2)R play a role in early innate immune functions during bacterial infection.

Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm.

PubMed

Chen, B; Teng, Jiawen; Liu, Hongwei; Pan, X; Zhou, Y; Huang, Shu; Lai, Mowen; Bian, Guohui; Mao, Bin; Sun, Wencui; Zhou, Qiongxiu; Yang, Shengyong; Nakahata, Tatsutoshi; Ma, Feng

2017-08-01

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models. © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Acquired initiating mutations in early hematopoietic cells of CLL patients.

PubMed

Damm, Frederik; Mylonas, Elena; Cosson, Adrien; Yoshida, Kenichi; Della Valle, Véronique; Mouly, Enguerran; Diop, M'boyba; Scourzic, Laurianne; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Kikushige, Yoshikane; Davi, Frederick; Lambert, Jérôme; Gautheret, Daniel; Merle-Béral, Hélène; Sutton, Laurent; Dessen, Philippe; Solary, Eric; Akashi, Koichi; Vainchenker, William; Mercher, Thomas; Droin, Nathalie; Ogawa, Seishi; Nguyen-Khac, Florence; Bernard, Olivier A

2014-09-01

Appropriate cancer care requires a thorough understanding of the natural history of the disease, including the cell of origin, the pattern of clonal evolution, and the functional consequences of the mutations. Using deep sequencing of flow-sorted cell populations from patients with chronic lymphocytic leukemia (CLL), we established the presence of acquired mutations in multipotent hematopoietic progenitors. Mutations affected known lymphoid oncogenes, including BRAF, NOTCH1, and SF3B1. NFKBIE and EGR2 mutations were observed at unexpectedly high frequencies, 10.7% and 8.3% of 168 advanced-stage patients, respectively. EGR2 mutations were associated with a shorter time to treatment and poor overall survival. Analyses of BRAF and EGR2 mutations suggest that they result in deregulation of B-cell receptor (BCR) intracellular signaling. Our data propose disruption of hematopoietic and early B-cell differentiation through the deregulation of pre-BCR signaling as a phenotypic outcome of CLL mutations and show that CLL develops from a pre-leukemic phase. The origin and pathogenic mechanisms of CLL are not fully understood. The current work indicates that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. It advocates for abnormalities in early B-cell differentiation as a phenotypic convergence of the diverse acquired mutations observed in CLL. ©2014 American Association for Cancer Research.

Bruton’s tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib

PubMed Central

Aalipour, Amin

2014-01-01

Aberrant signaling of the B-cell receptor pathway has been linked to the development and maintenance of B-cell malignancies. Bruton’s tyrosine kinase (BTK), a protein early in this pathway, has emerged as a new therapeutic target in a variety of such malignancies. Ibrutinib, the most clinically advanced small molecule inhibitor of BTK, has demonstrated impressive tolerability and activity in a range of B-cell lymphomas which led to its recent approval for relapsed mantle cell lymphoma and chronic lymphocytic leukemia. This review focuses on the preclinical and clinical development of ibrutinib and discusses its therapeutic potential. PMID:25360238

Hypomorphic Rag1 mutations alter the pre-immune repertoire at early stages of lymphoid development.

PubMed

Ott de Bruin, Lisa M; Bosticardo, Marita; Barbieri, Alessandro; Lin, Sherry G; Rowe, Jared H; Poliani, Pietro L; Ching, Kimberly; Eriksson, Daniel; Landegren, Nils; Kämpe, Olle; Manis, John P; Notarangelo, Luigi D

2018-05-09

Hypomorphic RAG1 mutations allowing residual T and B cell development have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (CID-G/AI) and abnormalities of the peripheral T and B cell repertoire. To examine how hypomorphic Rag1 mutations affect the earliest stages of lymphocyte development, we used CRISPR/Cas9 to generate mouse models with equivalent mutations found in patients with CID-G/AI. Immunological characterization showed partial development of T and B lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with CID-G/AI. By using high throughput sequencing, we identified marked skewing of Igh V and Trb V gene usage in early progenitors, with a bias for productive Igh and Trb rearrangements after selection occurred and increased apoptosis of B cell progenitors. Rearrangement at the Igk locus was impaired, and polyreactive IgM antibodies were detected. This study provides novel insights in how hypomorphic Rag1 mutations alter the primary repertoire of T and B cells, setting the stage for immune dysregulation frequently seen in patients. Copyright © 2018 American Society of Hematology.

Higher B-cell activating factor levels at birth are positively associated with maternal dairy farm exposure and negatively related to allergy development.

PubMed

Lundell, Anna-Carin; Hesselmar, Bill; Nordström, Inger; Adlerberth, Ingegerd; Wold, Agnes E; Rudin, Anna

2015-10-01

A high proportion of circulating immature/naive CD5(+) B cells during early infancy is a risk factor for allergy development. B-cell activating factor (BAFF) is an important cytokine for B-cell maturation. We sought to investigate whether BAFF levels are related to environmental exposures during pregnancy and early childhood and whether BAFF levels are associated with postnatal B-cell maturation and allergic disease. In the FARMFLORA study, including both farming and nonfarming families, we measured BAFF levels in plasma from mothers and their children at birth and at 1, 4, 18, and 36 months of age. Infants' blood samples were also analyzed for B-cell numbers and proportions of CD5(+) and CD27(+) B cells. Allergic disease was clinically evaluated at 18 and 36 months of age. Circulating BAFF levels were maximal at birth, and farmers' children had higher BAFF levels than nonfarmers' children. Higher BAFF levels at birth were positively associated with proportions of CD27(+) memory B cells among farmers' children and inversely related to proportions of CD5(+) immature/naive B cells among nonfarmers' children. Children with allergic disease at 18 months of age had lower cord blood BAFF levels than nonallergic children. At birth, girls had higher BAFF levels and lower proportions of CD5(+) B cells than boys. Farm exposure during pregnancy appears to induce BAFF production in the newborn child, and high neonatal BAFF levels were associated with more accelerated postnatal B-cell maturation, which lend further strength to the role of B cells in the hygiene hypothesis. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Specification of posterior midbrain region in zebrafish neuroepithelium.

PubMed

Miyagawa, T; Amanuma, H; Kuroiwa, A; Takeda, H

1996-04-01

The developing vertebrate nervous system displays a pronounced anterior-posterior (A-P) pattern, but the mechanism that generates this pattern is poorly understood. We examined through cell-transplantation experiments, when and how the cells in the zebrafish posterior midbrain acquire regional specificity along the A-P axis as shown by pax[b] gene expression. Labelled donor cells from the presumptive midbrain region at various stages were transplanted into more anterior part of unlabelled host embryos of the same developmental stage, and the expression of pax[b] in the donor cells were examined by in situ hybridization. The results indicated that, in the cells from the presumptive midbrain region, expression of pax[b] was determined as early as the 55%-epiboly (6.5 h, early gastrulation) when the underlying hypoblastic layer reached the presumptive midbrain region. We also found that when transplanted heterotopically, anterior, but not posterior, hypoblast cells induced expression of pax[b] in the overlying ectoderm. Expression of a midbrain specific gene is determined during early gastrulation and the hypoblastic layer plays an important role in this determination process.

PLU-1/JARID1B/KDM5B is required for embryonic survival and contributes to cell proliferation in the mammary gland and in ER+ breast cancer cells.

PubMed

Catchpole, Steven; Spencer-Dene, Bradley; Hall, Debbie; Santangelo, Samantha; Rosewell, Ian; Guenatri, Mounia; Beatson, Richard; Scibetta, Angelo G; Burchell, Joy M; Taylor-Papadimitriou, Joyce

2011-05-01

The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER+ breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER+ breast cancer.

Antibody repertoire diversification through VH gene replacement in mice cloned from an IgA plasma cell.

PubMed

Kumar, Rashmi; Bach, Martina P; Mainoldi, Federica; Maruya, Mikako; Kishigami, Satoshi; Jumaa, Hassan; Wakayama, Teruhiko; Kanagawa, Osami; Fagarasan, Sidonia; Casola, Stefano

2015-02-03

In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe VHQ52(NT); Vκgr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell. In VHQ52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52(NT) animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre-B-cell receptor signaling, and involved predominantly one adjacent VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.

A Gammaherpesvirus Bcl-2 Ortholog Blocks B Cell Receptor-Mediated Apoptosis and Promotes the Survival of Developing B Cells In Vivo

PubMed Central

Coleman, Carrie B.; McGraw, Jennifer E.; Feldman, Emily R.; Roth, Alexa N.; Keyes, Lisa R.; Grau, Katrina R.; Cochran, Stephanie L.; Waldschmidt, Thomas J.; Liang, Chengyu; Forrest, J. Craig; Tibbetts, Scott A.

2014-01-01

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the recurrent infection and vBcl-2-mediated survival of developing B cells. PMID:24516386

Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans.

PubMed

Evans, Erica K; Tester, Richland; Aslanian, Sharon; Karp, Russell; Sheets, Michael; Labenski, Matthew T; Witowski, Steven R; Lounsbury, Heather; Chaturvedi, Prasoon; Mazdiyasni, Hormoz; Zhu, Zhendong; Nacht, Mariana; Freed, Martin I; Petter, Russell C; Dubrovskiy, Alex; Singh, Juswinder; Westlin, William F

2013-08-01

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.

Effect of increased HoxB4 on human megakaryocytic development

PubMed Central

Zhong, Yiming; Sullenbarger, Brent; Lasky, Larry C.

2010-01-01

In order to ex vivo produce clinically useful quantity of platelets, we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis; however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data indicate that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating Tpo R and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine. PMID:20599537

Sevoflurane represses the self-renewal ability by regulating miR-7a,7b/Klf4 signalling pathway in mouse embryonic stem cells.

PubMed

Wang, Qimin; Li, Guifeng; Li, Baolin; Chen, Qiu; Lv, Dongdong; Liu, Jiaying; Ma, Jieyu; Sun, Nai; Yang, Longqiu; Fei, Xuejie; Song, Qiong

2016-10-01

Sevoflurane is a frequently-used clinical inhalational anaesthetic and can cause toxicity to embryos during foetal development. Embryonic stem cells (ESCs) are derived from the inner cell mass of blastospheres and can be used as a useful model of early development. Here, we found that sevoflurane significantly influenced self-renewal ability of mESCs on stemness maintenance and cell proliferation. The cell cycle was arrested via G1 phase delay. We further found that sevoflurane upregulated expression of miR-7a,7b to repress self-renewal. Next we performed rescue experiments and found that after adding miR-7a,7b inhibitor into mESCs treated with sevoflurane, its influence on self-renewal could be blocked. Further we identified stemness factor Klf4 as the direct target of miR-7a,7b. Overexpression of Klf4 restored self-renewal ability repressed by miR-7a,7b or sevoflurane. In this work, we determined that sevoflurane repressed self-renewal ability by regulating the miR-7a,7b/Klf4 signalling pathway in mESCs. Our study demonstrated molecular mechanism underlying the side effects of sevoflurane during early development, laying the foundation for studies on safe usage of inhalational anaesthetic during non-obstetric surgery. © 2016 John Wiley & Sons Ltd.

Late-onset Epstein-Barr virus-related disease in acute leukemia patients after haploidentical hematopoietic stem cell transplantation is associated with impaired early recovery of T and B lymphocytes.

PubMed

Liu, Jiangying; Yan, Chenhua; Zhang, Chunli; Xu, Lanping; Liu, Yanrong; Huang, Xiaojun

2015-10-01

Epstein-Barr virus-related disease (EBVD) is a serious clinical complication in patients who have undergone haploidentical hematopoietic stem cell transplantation (haploHSCT). Some recipients develop EBVD relatively late after haploHSCT, and most of these patients suffer a poor outcome. This retrospective cohort study characterized the early adaptive immune recovery of patients with acute leukemia presenting with EBVD more than 100 d after haploHSCT. Patients with acute leukemia who received haploHSCT and developed EBVD 100 d later (n = 8) were compared with a matched control group without EBVD (n = 24) with regard to peripheral WBC, lymphocytes, and neutrophils (at 30, 60, and 90 d) and recoveries of B and T lymphocytes (at 30 and 90 d, via immunophenotyping/flow cytometry). Ninety days after haploHSCT, the median values of WBCs and lymphocytes, and the recoveries of CD19(+) B cells and CD4(+) , CD8(+) , and CD4(+) CD45RO(+) T cells, were significantly lower in patients who developed EBVD, relative to the control group. These results suggest a significant association between deficient early recovery of B and T lymphocytes and the development of late-onset EBVD after haploHSCT. Our observation could facilitate clinical intervention and the improvement of overall survival of patients undergoing haploHSCT. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Argonaute-1 functions as a mitotic regulator by controlling Cyclin B during Drosophila early embryogenesis.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Bag, Indira; Hunt, Clayton R; Ramaiah, M Janaki; Pandita, Tej K; Bhadra, Utpal; Pal-Bhadra, Manika

2014-02-01

The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. In this study, we established evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. We identified involvement of 2 novel embryonic miRNAs--miR-981 and miR--317-for spatiotemporal regulation of cyclin B. In summary, our results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (mei-41 and grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, our results have established a novel role of Ago-1 as a regulator of the cell cycle.

Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors.

PubMed

Mega, Tiziana; Lupia, Michela; Amodio, Nicola; Horton, Sarah J; Mesuraca, Maria; Pelaggi, Daniela; Agosti, Valter; Grieco, Michele; Chiarella, Emanuela; Spina, Raffaella; Moore, Malcolm A S; Schuringa, Jan Jacob; Bond, Heather M; Morrone, Giovanni

2011-07-01

Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.

Analysis of gene expression and Ig transcription in PU.1/Spi-B-deficient progenitor B cell lines.

PubMed

Schweitzer, Brock L; DeKoter, Rodney P

2004-01-01

A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1(-/-)Spi-B(-/-) IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1(-/-)Spi-B(-/-) pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Igkappa transcription are induced after IL-7 withdrawal of wild-type or PU.1(-/-)Spi-B(-/-) pro-B cells. In contrast, we found that Iglambda transcription is reduced in PU.1(-/-)Spi-B(-/-) pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Iglambda, but not IgH or Igkappa, transcription, is dependent on PU.1 and/or Spi-B. The PU.1(-/-)Spi-B(-/-) pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.

Nuclear lamins during gametogenesis, fertilization and early development

NASA Technical Reports Server (NTRS)

Maul, G. G.; Schatten, G.

1986-01-01

The distribution of lamins (described by Gerace, 1978, as major proteins of nuclear envelope) during gametogenesis, fertilization, and early development was investigated in germ cells of a mouse (Mus musculus), an echinoderm (Lytechinus variegatus), and the surf clam (Spisula solidissima) was investigated in order to determine whether the differences detected could be correlated with differences in the function of cells in these stages of the germ cells. In order to monitor the behavior of lamins, the gametes and embryos were labeled with antibodies to lamins A, C, and B extracted from autoimmune sera of patients with scleroderma and Lupus erythematosus. Results indicated that lamin B could be identified in nuclear envelopes on only those nuclei where chromatin is attached and where RNA synthesis takes place.

Macrophage Functions in Early Dissemination and Dormancy of Breast Cancer

DTIC Science & Technology

2016-09-01

mammary gland development 17,18, 69   arguing that normal mammary epithelial cells cooperate with these innate immune cells 70   for invasive... cells lacking 218     11   lymphoid and granulocytic markers (Supplementary Fig.3B). viSNE plots 30 of myelo-219   monocytic cells (Fig.5A...macrophages are actively recruited by pre-malignant ErbB2 overexpressing cancer cells and that these intra-epithelial macrophages then produce factors

Ly6d marks the earliest stage of B-cell specification and identifies the branchpoint between B-cell and T-cell development

PubMed Central

Inlay, Matthew A.; Bhattacharya, Deepta; Sahoo, Debashis; Serwold, Thomas; Seita, Jun; Karsunky, Holger; Plevritis, Sylvia K.; Dill, David L.; Weissman, Irving L.

2009-01-01

Common lymphoid progenitors (CLPs) clonally produce both B- and T-cell lineages, but have little myeloid potential in vivo. However, some studies claim that the upstream lymphoid-primed multipotent progenitor (LMPP) is the thymic seeding population, and suggest that CLPs are primarily B-cell-restricted. To identify surface proteins that distinguish functional CLPs from B-cell progenitors, we used a new computational method of Mining Developmentally Regulated Genes (MiDReG). We identified Ly6d, which divides CLPs into two distinct populations: one that retains full in vivo lymphoid potential and produces more thymocytes at early timepoints than LMPP, and another that behaves essentially as a B-cell progenitor. PMID:19833765

A Special Connection between γδ T Cells and Natural Antibodies?

PubMed Central

Born, Willi K.; Huang, Yafei; Zeng, Wanjiang; Torres, Raul M.; O’Brien, Rebecca L.

2017-01-01

Natural antibodies (NAbs) play an important role in early host defense, autophagy and tissue remodeling, and in immune regulation. They arise spontaneously (without specific immunization), and are already present at birth. NAbs are produced by B1 B cells, MZ B cells and other B cell types. They include all major Ig subclasses but IgM antibodies are prevalent, especially early in development. NAbs may be poly-specific, recognize particular auto-antigens, or detect neo-determinants such as those exposed during apoptosis or generated by oxidation. NAbs do not require cognate T cell help but depend on soluble mediators produced by T cells. Our recent studies suggest that γδ T cells may have a special relationship with NAbs, and play a prominent role in their regulation, in part through the fine-tuning of IL-4-levels. The spontaneously activated state of these cells likely enables their cytokine production and other functions in the absence of external stimulation. Ontogenetically, the earlier arising γδ T cells are better positioned than αβ T cells to shape the developing repertoire of NAbs. Intriguingly, ligand specificities of NAbs and γδ T cell receptors appear to be overlapping, perhaps allowing γδ cognate help for certain NAb specificities. Via NAbs, γδ T cells could exert a regulatory influence on numerous processes in health and disease. PMID:27235134

A Special Connection between γδ T Cells and Natural Antibodies?

PubMed

Born, Willi K; Huang, Yafei; Zeng, Wanjiang; Torres, Raul M; O'Brien, Rebecca L

2016-12-01

Natural antibodies (NAbs) play an important role in early host defense, autophagy and tissue remodeling, and in immune regulation. They arise spontaneously (without specific immunization), and are already present at birth. NAbs are produced by B1 B cells, MZ B cells and other B cell types. They include all major Ig subclasses but IgM antibodies are prevalent, especially early in development. NAbs may be poly-specific, recognize particular auto-antigens, or detect neo-determinants such as those exposed during apoptosis or generated by oxidation. NAbs do not require cognate T cell help but depend on soluble mediators produced by T cells. Our recent studies suggest that γδ T cells may have a special relationship with NAbs, and play a prominent role in their regulation, in part through the fine-tuning of IL-4 levels. The spontaneously activated state of these cells likely enables their cytokine production and other functions in the absence of external stimulation. Ontogenetically, the earlier arising γδ T cells are better positioned than αβ T cells to shape the developing repertoire of NAbs. Intriguingly, ligand specificities of NAbs and γδ T cell receptors appear to be overlapping, perhaps allowing γδ cognate help for certain NAb specificities. Via NAbs, γδ T cells could exert a regulatory influence on numerous processes in health and disease.

B-1a transitional cells are phenotypically distinct and are lacking in mice deficient in IκBNS.

PubMed

Pedersen, Gabriel K; Àdori, Monika; Khoenkhoen, Sharesta; Dosenovic, Pia; Beutler, Bruce; Karlsson Hedestam, Gunilla B

2014-09-30

B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93(+)IgM(+)CD5(+)) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93(+)IgM(+)CD5(-) cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS.

Boron deficiency inhibits root growth by controlling meristem activity under cytokinin regulation.

PubMed

Poza-Viejo, Laura; Abreu, Isidro; González-García, Mary Paz; Allauca, Paúl; Bonilla, Ildefonso; Bolaños, Luis; Reguera, María

2018-05-01

Significant advances have been made in the last years trying to identify regulatory pathways that control plant responses to boron (B) deficiency. Still, there is a lack of a deep understanding of how they act regulating growth and development under B limiting conditions. Here, we analyzed the impact of B deficit on cell division leading to root apical meristem (RAM) disorganization. Our results reveal that inhibition of cell proliferation under the regulatory control of cytokinins (CKs) is an early event contributing to root growth arrest under B deficiency. An early recovery of QC46:GUS expression after transferring B-deficient seedlings to control conditions revealed a role of B in the maintenance of QC identity whose loss under deficiency occurred at later stages of the stress. Additionally, the D-type cyclin CYCD3 overexpressor and triple mutant cycd3;1-3 were used to evaluate the effect on mitosis inhibition at the G1-S boundary. Overall, this study supports the hypothesis that meristem activity is inhibited by B deficiency at early stages of the stress as it does cell elongation. Likewise, distinct regulatory mechanisms seem to take place depending on the severity of the stress. The results presented here are key to better understand early signaling responses under B deficiency. Copyright © 2018 Elsevier B.V. All rights reserved.

Neutrophil mediated IFN activation in the bone marrow alters B cell development in human and murine SLE1

PubMed Central

Palanichamy, Arumugam; Bauer, Jason W; Yalavarthi, Srilakshmi; Meednu, Nida; Barnard, Jennifer; Owen, Teresa; Cistrone, Christopher; Bird, Anna; Rabinovich, Alfred; Nevarez, Sarah; Knight, Jason S.; Dedrick, Russell; Rosenberg, Alexander; Wei, Chungwen; Rangel-Moreno, Javier; Liesveld, Jane; Sanz, Inaki; Baechler, Emily; Kaplan, Mariana J.; Anolik, Jennifer H

2014-01-01

Inappropriate activation of type I interferon (IFN) plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). Here we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood (PB) and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre B cells suggesting an inhibition in early B cell development and an expansion of B cells at the transitional (T2) stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type I IFN receptor. BM neutrophils were abundant, responsive to and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil mediated IFN activation and alterations in B cell ontogeny and selection. PMID:24379124

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagata, Takayuki; Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575; Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp

Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examinedmore » the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.« less

Targeting Vaccine-Induced Extrafollicular Pathway of B Cell Differentiation Improves Rabies Postexposure Prophylaxis

PubMed Central

Haley, Shannon L.; Tzvetkov, Evgeni P.; Meuwissen, Samantha; Plummer, Joseph R.

2017-01-01

ABSTRACT Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP. IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated here, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality. PMID:28148792

Targeting Vaccine-Induced Extrafollicular Pathway of B Cell Differentiation Improves Rabies Postexposure Prophylaxis.

PubMed

Haley, Shannon L; Tzvetkov, Evgeni P; Meuwissen, Samantha; Plummer, Joseph R; McGettigan, James P

2017-04-15

Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP. IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated here, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality. Copyright © 2017 American Society for Microbiology.

IL17A Regulates Tumor Latency and Metastasis in Lung Adeno and Squamous SQ.2b and AD.1 Cancer.

PubMed

You, Ran; DeMayo, Francesco J; Liu, Jian; Cho, Sung-Nam; Burt, Bryan M; Creighton, Chad J; Casal, Roberto F; Lazarus, Donald R; Lu, Wen; Tung, Hui-Ying; Yuan, Xiaoyi; Hill-McALester, Andrea; Kim, Myunghoo; Perusich, Sarah; Cornwell, Loraine; Rosen, Daniel; Song, Li-Zhen; Paust, Silke; Diehl, Gretchen; Corry, David; Kheradmand, Farrah

2018-04-13

Somatic mutations can promote malignant transformation of airway epithelial cells and induce inflammatory responses directed against resultant tumors. Tumor-infiltrating T lymphocytes (TIL) in early-stage non-small cell lung cancer (NSCLC) secrete distinct proinflammatory cytokines, but the contribution of these TILs to tumor development and metastasis remains unknown. We show here that TILs in early-stage NSCLC are biased toward IL17A expression (Th17) when compared with adjacent tumor-free tissue, whereas Th17 cells are decreased in tumor infiltrating locoregional lymph nodes in advanced NSCLC. Mice in which Pten and Smad4 ( Pts4 d/d ) are deleted from airway epithelial cells develop spontaneous tumors, that share genetic signatures with squamous- (SQ.2b), and adeno- (AD.1) subtypes of human NSCLC. Pts4 d/d mice globally lacking in IL17a ( Pts4 d/d Il17a -/- ) showed decreased tumor latency and increased metastasis. Th17 cells were required for recruitment of CD103 + dendritic cells, and adoptive transfer of IL17a -sufficient CD4 + T cells reversed early tumor development and metastasis in Pts4 d/d Il17a -/- mice. Together, these findings support a key role for Th17 cells in TILs associated with the Pts4 d/d model of NSCLC and suggest therapeutic and biomarker strategies for human SQ2b and AD1 lung cancer. Cancer Immunol Res; 1-13. ©2018 AACR. ©2018 American Association for Cancer Research.

Distinct expression patterns for type II topoisomerases IIA and IIB in the early foetal human telencephalon.

PubMed

Harkin, Lauren F; Gerrelli, Dianne; Gold Diaz, Diana C; Santos, Chloe; Alzu'bi, Ayman; Austin, Caroline A; Clowry, Gavin J

2016-03-01

TOP2A and TOP2B are type II topoisomerase enzymes that have important but distinct roles in DNA replication and RNA transcription. Recently, TOP2B has been implicated in the transcription of long genes in particular that play crucial roles in neural development and are susceptible to mutations contributing to neurodevelopmental conditions such as autism and schizophrenia. This study maps their expression in the early foetal human telencephalon between 9 and 12 post-conceptional weeks. TOP2A immunoreactivity was restricted to cell nuclei of the proliferative layers of the cortex and ganglionic eminences (GE), including the ventricular zone and subventricular zone (SVZ) closely matching expression of the proliferation marker KI67. Comparison with sections immunolabelled for NKX2.1, a medial GE (MGE) marker, and PAX6, a cortical progenitor cell and lateral GE (LGE) marker, revealed that TOP2A-expressing cells were more abundant in MGE than the LGE. In the cortex, TOP2B is expressed in cell nuclei in both proliferative (SVZ) and post-mitotic compartments (intermediate zone and cortical plate) as revealed by comparison with immunostaining for PAX6 and the post-mitotic neuron marker TBR1. However, co-expression with KI67 was rare. In the GE, TOP2B was also expressed by proliferative and post-mitotic compartments. In situ hybridisation studies confirmed these patterns of expression, except that TOP2A mRNA is restricted to cells in the G2/M phase of division. Thus, during early development, TOP2A is likely to have a role in cell proliferation, whereas TOP2B is expressed in post-mitotic cells and may be important in controlling expression of long genes even at this early stage. © 2015 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells

PubMed Central

1993-01-01

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD- deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell- independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen- driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens. PMID:8418208

Base-Resolution Analysis of DNA Methylation Patterns Downstream of Dnmt3a in Mouse Naïve B Cells.

PubMed

Duncan, Christopher G; Kondilis-Mangum, Hrisavgi D; Grimm, Sara A; Bushel, Pierre R; Chrysovergis, Kaliopi; Roberts, John D; Tyson, Frederick L; Merrick, B Alex; Wade, Paul A

2018-03-02

The DNA methyltransferase, Dnmt3a , is dynamically regulated throughout mammalian B cell development and upon activation by antigenic stimulation. Dnmt3a inactivation in hematopoietic stem cells has been shown to drive B cell-related malignancies, including chronic lymphocytic leukemia, and associates with specific DNA methylation patterns in transformed cells. However, while it is clear that inactivation of Dnmt3a in hematopoietic stem cells has profound functional effects, the consequences of Dnmt3a inactivation in cells of the B lineage are unclear. To assess whether loss of Dnmt3a at the earliest stages of B cell development lead to DNA methylation defects that might impair function, we selectively inactivated Dnmt3a early in mouse B cell development and then utilized whole genome bisulfite sequencing to generate base-resolution profiles of Dnmt3a +/+ and Dnmt3a -/- naïve splenic B cells. Overall, we find that global methylation patterns are largely consistent between Dnmt3a +/+ and Dnmt3a -/- naïve B cells, indicating a minimal functional effect of DNMT3A in mature B cells. However, loss of Dnmt3a induced 449 focal DNA methylation changes, dominated by loss-of-methylation events. Regions found to be hypomethylated in Dnmt3a -/- naïve splenic B cells were enriched in gene bodies of transcripts expressed in B cells, a fraction of which are implicated in B cell-related disease. Overall, the results from this study suggest that factors other than Dnmt3a are the major drivers for methylome maintenance in B cell development. Copyright © 2018 Duncan et al.

Corrected and Republished from: BCL11A Is a Critical Component of a Transcriptional Network That Activates Recombinase Activating Gene Expression and V(D)J Recombination

PubMed Central

Lee, Baeck-Seung; Lee, Bum-Kyu; Iyer, Vishwanath R.; Sleckman, Barry P.; Shaffer, Arthur L.; Ippolito, Gregory C.

2017-01-01

ABSTRACT Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination. PMID:29038163

Core binding factors are necessary for natural killer cell development and cooperate with Notch signaling during T-cell specification

PubMed Central

Guo, Yalin; Maillard, Ivan; Chakraborti, Sankhamala; Rothenberg, Ellen V.

2008-01-01

CBFβ is the non-DNA binding subunit of the core binding factors (CBFs). Mice with reduced CBFβ levels display profound, early defects in T-cell but not B-cell development. Here we show that CBFβ is also required at very early stages of natural killer (NK)–cell development. We also demonstrate that T-cell development aborts during specification, as the expression of Gata3 and Tcf7, which encode key regulators of T lineage specification, is substantially reduced, as are functional thymic progenitors. Constitutively active Notch or IL-7 signaling cannot restore T-cell expansion or differentiation of CBFβ insufficient cells, nor can overexpression of Runx1 or CBFβ overcome a lack of Notch signaling. Therefore, the ability of the prethymic cell to respond appropriately to Notch is dependent on CBFβ, and both signals converge to activate the T-cell developmental program. PMID:18390836

Expression of Essential B Cell Development Genes in Horses with Common Variable Immunodeficiency

PubMed Central

Tallmadge, R.L.; Such, K.A.; Miller, K.C.; Matychak, M.B.; Felippe, M.J.B.

2012-01-01

Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p < 0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p < 0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients. PMID:22464097

Morphologic and Functional Effects of Gamma Secretase Inhibition on Splenic Marginal Zone B Cells

PubMed Central

de Vera Mudry, Maria Cristina; Regenass-Lechner, Franziska; Ozmen, Laurence; Altmann, Bernd; Festag, Matthias; Singer, Thomas; Müller, Lutz; Jacobsen, Helmut; Flohr, Alexander

2012-01-01

The γ-secretase complex is a promising target in Alzheimer's disease because of its role in the amyloidogenic processing of β-amyloid precursor protein. This enzyme also catalyzes the cleavage of Notch receptor, resulting in the nuclear translocation of intracellular Notch where it modulates gene transcription. Notch signaling is essential in cell fate decisions during embryogenesis, neuronal differentiation, hematopoiesis, and development of T and B cells, including splenic marginal zone (MZ) B cells. This B cell compartment participates in the early phases of the immune response to blood-borne bacteria and viruses. Chronic treatment with the oral γ-secretase inhibitor RO4929097 resulted in dose-dependent decreased cellularity (atrophy) of the MZ of rats and mice. Significant decreases in relative MZ B-cell numbers of RO4929097-treated animals were confirmed by flow cytometry. Numbers of MZ B cells reverted to normal after a sufficient RO4929097-free recovery period. Functional characterization of the immune response in relation to RO4929097-related MZ B cell decrease was assessed in mice vaccinated with inactivated vesicular stomatitis virus (VSV). Compared with the immunosuppressant cyclosporin A, RO4929097 caused only mild and reversible delayed early neutralizing IgM and IgG responses to VSV. Thus, the functional consequence of MZ B cell decrease on host defense is comparatively mild. PMID:23316412

Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis

PubMed Central

Meyer-Bahlburg, Almut; Becker-Herman, Shirly; Humblet-Baron, Stephanie; Khim, Socheath; Weber, Michele; Bouma, Gerben; Thrasher, Adrian J.; Batista, Facundo D.

2008-01-01

To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp+ murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp+ human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease. PMID:18687984

Deficiency of transcription factor RelB perturbs myeloid and DC development by hematopoietic-extrinsic mechanisms

PubMed Central

Briseño, Carlos G.; Gargaro, Marco; Durai, Vivek; Davidson, Jesse T.; Theisen, Derek J.; Anderson, David A.; Novack, Deborah V.; Murphy, Theresa L.; Murphy, Kenneth M.

2017-01-01

RelB is an NF-κB family transcription factor activated in the noncanonical pathway downstream of NF-κB–inducing kinase (NIK) and TNF receptor family members including lymphotoxin-β receptor (LTβR) and CD40. Early analysis suggested that RelB is required for classical dendritic cell (cDC) development based on a severe reduction of cDCs in Relb−/− mice associated with profound myeloid expansion and perturbations in B and T cells. Subsequent analysis of radiation chimeras generated from wild-type and Relb−/− bone marrow showed that RelB exerts cell-extrinsic actions on some lineages, but it has remained unclear whether the impact of RelB on cDC development is cell-intrinsic or -extrinsic. Here, we reevaluated the role of RelB in cDC and myeloid development using a series of radiation chimeras. We found that there was no cell-intrinsic requirement for RelB for development of most cDC subsets, except for the Notch2- and LTβR-dependent subset of splenic CD4+ cDC2s. These results identify a relatively restricted role of RelB in DC development. Moreover, the myeloid expansion in Relb−/− mice resulted from hematopoietic-extrinsic actions of RelB. This result suggests that there is an unrecognized but critical role for RelB within the nonhematopoietic niche that controls normal myelopoiesis. PMID:28348230

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Yiming; Program in Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH; Sullenbarger, Brent

Research highlights: {yields} HoxB4 overexpression in human TF1 cells increased the expression of CD61 and CD41a. {yields} HoxB4 fusion protein enhanced megakaryocytic development of CD34{sup +} cord blood cells. {yields} Ectopic HoxB4 increased Tpo receptor expression and decreased c-Myb expression. {yields} HoxB4 RNA silencing increased c-Myb expression and decreased Fli-1 expression. -- Abstract: In order to produce clinically useful quantities of platelets ex vivo we may need to firstly enhance early self-renewal of hematopoietic stem cells (HSCs) and/or megakaryocyte (Mk) progenitors. The homeodomain transcription factor HoxB4 has been shown to be an important regulator of stem cell renewal and hematopoiesis;more » however, its effect on megakaryopoiesis is unclear. In this study, we investigated the effect of HoxB4 overexpression or RNA silencing on megakaryocytic development in the human TF1 progenitor cell line; we then used recombinant tPTD-HoxB4 fusion protein to study the effect of exogenous HoxB4 on megakaryocytic development of human CD34 positively-selected cord blood cells. We found that ectopic HoxB4 in TF1 cells increased the antigen expression of CD61and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), Scl-1, Cyclin D1, Fog-1 and Fli-1 while it decreased c-Myb expression. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant tPTD-HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 while it inhibited c-Myb expression. Our data suggest that increased HoxB4 enhanced early megakaryocytic development in human TF1 cells and CD34 positively-selected cord blood cells primarily by upregulating TpoR and Fli-1 expression and downregulating c-Myb expression. Increasing HoxB4 expression or adding recombinant HoxB4 protein might be a way to expand Mks for the production of platelets for use in transfusion medicine.« less

Discovering sparse transcription factor codes for cell states and state transitions during development

PubMed Central

Furchtgott, Leon A; Melton, Samuel; Menon, Vilas; Ramanathan, Sharad

2017-01-01

Computational analysis of gene expression to determine both the sequence of lineage choices made by multipotent cells and to identify the genes influencing these decisions is challenging. Here we discover a pattern in the expression levels of a sparse subset of genes among cell types in B- and T-cell developmental lineages that correlates with developmental topologies. We develop a statistical framework using this pattern to simultaneously infer lineage transitions and the genes that determine these relationships. We use this technique to reconstruct the early hematopoietic and intestinal developmental trees. We extend this framework to analyze single-cell RNA-seq data from early human cortical development, inferring a neocortical-hindbrain split in early progenitor cells and the key genes that could control this lineage decision. Our work allows us to simultaneously infer both the identity and lineage of cell types as well as a small set of key genes whose expression patterns reflect these relationships. DOI: http://dx.doi.org/10.7554/eLife.20488.001 PMID:28296636

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

PubMed

Kurzer, Jason H; Weinberg, Olga K

2018-05-25

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

Murine Visceral Leishmaniasis: IgM and Polyclonal B-Cell Activation Lead to Disease Exacerbation

PubMed Central

Deak, Eszter; Jayakumar, Asha; Wing Cho, Ka; Goldsmith-Pestana, Karen; Dondji, Blaise; Lambris, John D.; McMahon-Pratt, Diane

2010-01-01

In visceral leishmaniasis, the draining lymph node (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with L. infantum. Although not unexpected, at early times post-infection there is a marked B cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JHD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JHD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JHD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to wild type BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5aR (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-γ). Overall these studies indicate that polyclonal B cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention. PMID:20213734

Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner.

PubMed

Liu, Rui; King, Ashleigh; Bouillet, Philippe; Tarlinton, David M; Strasser, Andreas; Heierhorst, Jörg

2018-01-01

The proapoptotic BH3-only protein BIM ( Bcl2l11 ) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre ) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre ) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim -deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro , conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc -driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions.

Proapoptotic BIM Impacts B Lymphoid Homeostasis by Limiting the Survival of Mature B Cells in a Cell-Autonomous Manner

PubMed Central

Liu, Rui; King, Ashleigh; Bouillet, Philippe; Tarlinton, David M.; Strasser, Andreas; Heierhorst, Jörg

2018-01-01

The proapoptotic BH3-only protein BIM (Bcl2l11) plays key roles in the maintenance of multiple hematopoietic cell types. In mice, germline knockout or conditional pan-hematopoietic deletion of Bim results in marked splenomegaly and significantly increased numbers of B cells. However, it has remained unclear whether these abnormalities reflect the loss of cell-intrinsic functions of BIM within the B lymphoid lineage and, if so, which stages in the lifecycle of B cells are most impacted by the loss of BIM. Here, we show that B lymphoid-specific conditional deletion of Bim during early development (i.e., in pro-B cells using Mb1-Cre) or during the final differentiation steps (i.e., in transitional B cells using Cd23-Cre) led to a similar >2-fold expansion of the mature follicular B cell pool. Notably, while the expansion of mature B cells was quantitatively similar in conditional and germline Bim-deficient mice, the splenomegaly was significantly attenuated after B lymphoid-specific compared to global Bim deletion. In vitro, conditional loss of Bim substantially increased the survival of mature B cells that were refractory to activation by lipopolysaccharide. Finally, we also found that conditional deletion of just one Bim allele by Mb1-Cre dramatically accelerated the development of Myc-driven B cell lymphoma, in a manner that was comparable to the effect of germline Bim heterozygosity. These data indicate that, under physiological conditions, BIM regulates B cell homeostasis predominantly by limiting the life span of non-activated mature B cells, and that it can have additional effects on developing B cells under pathological conditions. PMID:29623080

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation[S

PubMed Central

Allende, Maria L.; Cook, Emily K.; Larman, Bridget C.; Nugent, Adrienne; Brady, Jacqueline M.; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J.

2018-01-01

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses. PMID:29358305

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation.

PubMed

Allende, Maria L; Cook, Emily K; Larman, Bridget C; Nugent, Adrienne; Brady, Jacqueline M; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J; Proia, Richard L

2018-03-01

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB -corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB -corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB -corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.

Early increase of peripheral B cell levels in kidney transplant recipients with CMV infection or reactivation.

PubMed

Besançon-Watelet, C; De March, A K; Renoult, E; Kessler, M; Béné, M C; Faure, G C; Sarda, M N

2000-02-15

Cytomegalovirus (CMV) infection or reactivation is a frequent complication of renal transplantation. Diagnosis of these conditions relies on the detection of circulating antigen, or of specific IgM and/or IgG, which develop over several weeks. Evocative clinical features may be detected earlier, but lack specificity. Rapid and early changes in the partition of lymphocyte subsets could be an additional indication of pending CMV infection. A systematic follow-up of peripheral B lymphocytes identified immunophenotypically by the determination of surface immunoglobulins (sIg), performed in 97 kidney transplant recipients, allowed to identify transient increases apparently predictive of CMV primo-infection or reactivation over the next 3 months. To better define the nature of these B cells, an extended investigation was performed for 14 prospective patients. In addition to surface Ig, membrane CD19, HLA-DR, and CD80 expression were explored. The cytoplasmic presence of mu, kappa, and lambda chains was also examined. B cell function was investigated using the ELISPOT technique, which allows an enumeration of the populations of IgG, IgA, and IgM secreting B cells. Retrospective analysis of the clinical outcome of the cohort of 97 patients evidenced that early transient increases in B cell levels were significantly (P<0.0001) associated with CMV infection. The same trend was noted in the smaller series of patients who benefited from a more extensive investigation of B cells, 10 of whom presented clinical or biological signs of CMV infection. Mature B cells, expressing surface Ig, CD19, DR, and CD80 are those presenting transient increases. No significant variation of preB (cmu+/kappalambda-) or activated (spot-forming) cells was evidenced in these patients. Individual examination of each patient's immune reconstitution profile allows to detect transient peaks of mature B cell during the initial immunosuppressive therapy, that appear to be predictive of oncoming CMV infection or reactivation.

Rituximab does not reset defective early B cell tolerance checkpoints

PubMed Central

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

2015-01-01

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

The Spectrum of Epstein-Barr Virus-Associated Lymphoproliferative Disease in Korea: Incidence of Disease Entities by Age Groups

PubMed Central

Cho, Eun-Yoon; Kim, Ki-Hyun; Kim, Won-Seog; Yoo, Keon Hee; Koo, Hong-Hoe

2008-01-01

This study is to identify the spectrum of Epstein-Barr virus (EBV)-positive lymphoproliferative diseases (LPD) and relationships between these diseases in Korea. The EBV status and clinicopathology of 764 patients, including acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV (CAEBV) infections, B-LPD arising in chronic latent EBV infection, T & natural killer (NK) cell non-Hodgkin's lymphomas (NHL), B-NHLs, and Hodgkin's lymphomas (HD), were analyzed. T or NK cell NHLs were the most common forms of EBV-positive NHLs (107/167, 64%); among these, nasal-type NK/T cell lymphomas were the most common (89/107, 83%). According to the age, Burkitt's lymphoma was the most common in early childhood; in teenagers, chronic (active) EBV infection-associated LPD was the most common type. The incidence of NK/T cell lymphoma began to increase from the twenties and formed the major type of EBV-associated tumor throughout life. Diffuse large B cell lymphoma formed the major type in the sixties and seventies. In conclusion, primary infections in early childhood are complicated by the development of CAEBV infections that are main predisposing factors for EBV-associated T or NK cell malignancies in young adults. In old patients, decreased immunity associated with old age and environmental cofactors may provoke the development of peripheral T cell lymphoma, unspecified, and diffuse large B cell lymphoma. PMID:18436998

The spectrum of Epstein-Barr virus-associated lymphoproliferative disease in Korea: incidence of disease entities by age groups.

PubMed

Cho, Eun-Yoon; Kim, Ki-Hyun; Kim, Won-Seog; Yoo, Keon Hee; Koo, Hong-Hoe; Ko, Young-Hyeh

2008-04-01

This study is to identify the spectrum of Epstein-Barr virus (EBV)-positive lymphoproliferative diseases (LPD) and relationships between these diseases in Korea. The EBV status and clinicopathology of 764 patients, including acute EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV (CAEBV) infections, B-LPD arising in chronic latent EBV infection, T & natural killer (NK) cell non-Hodgkin's lymphomas (NHL), B-NHLs, and Hodgkin's lymphomas (HD), were analyzed. T or NK cell NHLs were the most common forms of EBV-positive NHLs (107/167, 64%); among these, nasal-type NK/T cell lymphomas were the most common (89/107, 83%). According to the age, Burkitt's lymphoma was the most common in early childhood; in teenagers, chronic (active) EBV infection-associated LPD was the most common type. The incidence of NK/T cell lymphoma began to increase from the twenties and formed the major type of EBV-associated tumor throughout life. Diffuse large B cell lymphoma formed the major type in the sixties and seventies. In conclusion, primary infections in early childhood are complicated by the development of CAEBV infections that are main predisposing factors for EBV-associated T or NK cell malignancies in young adults. In old patients, decreased immunity associated with old age and environmental cofactors may provoke the development of peripheral T cell lymphoma, unspecified, and diffuse large B cell lymphoma.

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.

Restricted T cell receptor repertoire in CLL-like monoclonal B cell lymphocytosis and early stage CLL.

PubMed

Blanco, Gonzalo; Vardi, Anna; Puiggros, Anna; Gómez-Llonín, Andrea; Muro, Manuel; Rodríguez-Rivera, María; Stalika, Evangelia; Abella, Eugenia; Gimeno, Eva; López-Sánchez, Manuela; Senín, Alicia; Calvo, Xavier; Abrisqueta, Pau; Bosch, Francesc; Ferrer, Ana; Stamatopoulos, Kostas; Espinet, Blanca

2018-01-01

Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor β chain (TRB) repertoire of the CD4 + and CD8 + T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4 + T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related.

Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection.

PubMed

Price, Alexander M; Dai, Joanne; Bazot, Quentin; Patel, Luv; Nikitin, Pavel A; Djavadian, Reza; Winter, Peter S; Salinas, Cristina A; Barry, Ashley Perkins; Wood, Kris C; Johannsen, Eric C; Letai, Anthony; Allday, Martin J; Luftig, Micah A

2017-04-20

Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis.

Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection

PubMed Central

Muir, Roshell; Metcalf, Talibah; Tardif, Virginie; Takata, Hiroshi; Phanuphak, Nittaya; Kroon, Eugene; Colby, Donn J.; Trichavaroj, Rapee; Valcour, Victor; Robb, Merlin L.; Michael, Nelson L.; Ananworanich, Jintanat; Trautmann, Lydie; Haddad, Elias K.

2016-01-01

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory. PMID:27463374

Nicotine prevents the apoptosis induced by menadione in human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Tao; Lu Heng; Shang Xuan

Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role ofmore » anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-{kappa}B was up-regulated. Interference analysis of NF-{kappa}B in A549 cells showed that knock down of NF-{kappa}B resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-{kappa}B inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer.« less

The Syk kinase as a therapeutic target in leukemia and lymphoma.

PubMed

Efremov, Dimitar G; Laurenti, Luca

2011-05-01

The B-cell receptor (BCR) delivers antigen-dependent and -independent signals that have been implicated in the pathogenesis of several common B-cell malignancies. Agents that can efficiently block BCR signaling have recently been developed and are currently being evaluated as novel targeted therapies. Among these, agents that inhibit the Syk kinase appear particularly promising in preclinical and early clinical studies. The manuscript provides an overview of recent findings that implicate Syk and the BCR signaling pathway in the pathogenesis of several common lymphoid malignancies. It outlines preclinical and early clinical experiences with the Syk inhibitor fostamatinib disodium (R788) and discusses various options for further clinical development of this compound. Inhibitors of Syk or other components of the BCR signaling pathway are emerging as an exciting novel class of agents for the treatment of common B-cell malignancies. Future efforts should focus on defining the disease entities that are most likely to benefit from these agents, although considerable evidence is already available to pursue such studies in patients with chronic lymphocytic leukemia. Combinations with chemo-immunotherapy, treatment of early-stage disease and consolidation therapy should all be explored and could lead to the development of novel therapeutic approaches with improved efficacy, tolerability and toxicity profiles.

Dietary Restriction and Fasting Arrest B and T Cell Development and Increase Mature B and T Cell Numbers in Bone Marrow

PubMed Central

Shushimita, Shushimita; de Bruijn, Marjolein J. W.; de Bruin, Ron W. F.; IJzermans, Jan N. M.; Hendriks, Rudi W.; Dor, Frank J. M. F.

2014-01-01

Dietary restriction (DR) delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many “neuronal and surgery related damaging phenomena” such as Parkinson’s disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN). Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3+ T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined. PMID:24504160

Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development

PubMed Central

Stengel, Kristy R.; Barnett, Kelly R.; Wang, Jing; Liu, Qi; Hodges, Emily; Hiebert, Scott W.; Bhaskara, Srividya

2017-01-01

Histone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed Hdac3 conditional knockout mice with Mb1-Cre knockin animals to delete Hdac3 in early progenitor B cells. The spleens of Hdac3F/−Mb1-Cre+/− mice were virtually devoid of mature B cells, and B220+CD43+ B-cell progenitors accumulated within the bone marrow. Quantitative deep sequencing of the Ig heavy chain locus from B220+CD43+ populations identified a defect in VHDJH recombination with a severe reduction in productive rearrangements, which directly corresponded to the loss of pre-B cells from Hdac3Δ/− bone marrow. For Hdac3Δ/− B cells that did show productive VDJ rearrangement, there was significant skewing toward the incorporation of proximal VH gene segments and a corresponding reduction in distal VH gene segment use. Although transcriptional effects within these loci were modest, Hdac3Δ/− progenitor cells displayed global changes in chromatin structure that likely hindered effective distal V-DJ recombination. Reintroduction of wild-type Hdac3 restored normal B-cell development, whereas an Hdac3 point mutant lacking deacetylase activity failed to complement this defect. Thus, the deacetylase activity of Hdac3 is required for the generation of mature B cells. PMID:28739911

Development of the expressed Ig CDR-H3 repertoire is marked by focusing of constraints in length, amino acid use, and charge that are first established in early B cell progenitors.

PubMed

Ivanov, Ivaylo I; Schelonka, Robert L; Zhuang, Yingxin; Gartland, G Larry; Zemlin, Michael; Schroeder, Harry W

2005-06-15

To gain insight into the mechanisms that regulate the development of the H chain CDR3 (CDR-H3), we used the scheme of Hardy to sort mouse bone marrow B lineage cells into progenitor, immature, and mature B cell fractions, and then performed sequence analysis on V(H)7183-containing Cmu transcripts. The essential architecture of the CDR-H3 repertoire observed in the mature B cell fraction F was already established in the early pre-B cell fraction C. These architectural features include V(H) gene segment use preference, D(H) family usage, J(H) rank order, predicted structures of the CDR-H3 base and loop, and the amino acid composition and average hydrophobicity of the CDR-H3 loop. With development, the repertoire was focused by eliminating outliers to what appears to be a preferred repertoire in terms of length, amino acid composition, and average hydrophobicity. Unlike humans, the average length of CDR-H3 increased during development. The majority of this increase came from enhanced preservation of J(H) sequence. This was associated with an increase in the prevalence of tyrosine. With an accompanying increase in glycine, a shift in hydrophobicity was observed in the CDR-H3 loop from near neutral in fraction C (-0.08 +/- 0.03) to mild hydrophilic in fraction F (-0.17 +/- 0.02). Fundamental constraints on the sequence and structure of CDR-H3 are thus established before surface IgM expression.

Leaky phenotype of X-linked agammaglobulinaemia in a Japanese family

PubMed Central

Kaneko, H; Kawamoto, N; Asano, T; Mabuchi, Y; Horikoshi, H; Teramoto, T; JIN-RONG; Matsui, E; Kondo, M; Fukao, T; Kasahara, K; Kondo, N

2005-01-01

X-linked agammaglobulinaemia (XLA) is an inherited immunodeficiency that is caused by a block in early B-cell differentiation. Whereas early B precursors in the bone marrow are present in substantial numbers, XLA-affected individuals have dramatically reduced numbers of circulating mature B cells, plasma cells and immunoglobulins of all isotypes. We report on a Japanese family with 3 XLA patients, in whom the serum immunoglobulin levels and number of B cells showed a significant difference among them in spite of harbouring the same splice donor site mutation in the BTK gene. We developed concise method for detection of this mutation, which is helpful for discovering the carrier. Patient 2 showed a significant serum immunoglobulin levels of all isotypes, including allergen-specific IgE. Expression of a normal and truncated size BTK gene was detected in patient 2′s peripheral blood mononuclear cells (PBMCs). Expression of BTK protein was also detected in some B cells. These results suggest that the leaky phenotype in patient 2 was caused in part by the expression of a normal BTK gene transcript. The increased frequency of infection with age expanded the number of B cells with normal BTK gene expression and produced the serum immunoglobulin, including IgE. PMID:15932514

Molecular cloning and expression of rat and mouse B61 gene: implications on organogenesis.

PubMed

Takahashi, H; Ikeda, T

1995-09-07

ECK is a member of EPH receptor protein-tyrosine kinase subfamily and human B61 has been identified as the ligand for ECK recently. In order to better understand the roles of B61-ECK signalling pathway in mammalian development, we have cloned rat and mouse B61 cDNA and examined the expression pattern during rat development. Sequence analysis has revealed that there is a considerable degree of identity among rat, mouse and human B61 (98.0% between rat and mouse, 86.3% between rat and human in amino acid level). Examination of B61 mRNA expression by in situ hybridization analysis revealed tight association of B61 with endothelial cells at an early stage and epithelial cells in various tissues including lung, kidney, intestine, skin at later stage of organogenesis. In the developing skeletal system, B61 is expressed in periosteum, perichondrium and hypertrophic chondrocytes and osteoblasts. In the developing nervous system, expression of B61 is restricted in the neurons of dorsal root ganglia. These expression profiles of B61 in epithelial cells of various organs, developing skeletal system and dorsal root ganglia match those of ECK. Our data suggest that B61 plays pivotal roles in organogenesis, especially vasculogenesis/angiogenesis and epithelial cell proliferation/differentiation.

Maize early endosperm growth and development: from fertilization through cell type differentiation.

PubMed

Leroux, Brian M; Goodyke, Austin J; Schumacher, Katelyn I; Abbott, Chelsi P; Clore, Amy M; Yadegari, Ramin; Larkins, Brian A; Dannenhoffer, Joanne M

2014-08-01

• Given the worldwide economic importance of maize endosperm, it is surprising that its development is not the most comprehensively studied of the cereals. We present detailed morphometric and cytological descriptions of endosperm development in the maize inbred line B73, for which the genome has been sequenced, and compare its growth with four diverse Nested Association Mapping (NAM) founder lines.• The first 12 d of B73 endosperm development were described using semithin sections of plastic-embedded kernels and confocal microscopy. Longitudinal sections were used to compare endosperm length, thickness, and area.• Morphometric comparison between Arizona- and Michigan-grown B73 showed a common pattern. Early endosperm development was divided into four stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. We observed tightly synchronous nuclear divisions in the coenocyte, elucidated that the onset of cellularization was coincident with endosperm size, and identified a previously undefined cell type (basal intermediate zone, BIZ). NAM founders with small mature kernels had larger endosperms (0-6 d after pollination) than lines with large mature kernels.• Our B73-specific model of early endosperm growth links developmental events to relative endosperm size, while accounting for diverse growing conditions. Maize endosperm cellularizes through alveolation, then random partitioning of the central vacuole. This unique cellularization feature of maize contrasts with the smaller endosperms of Arabidopsis, barley, and rice that strictly cellularize through repeated alveolation. NAM analysis revealed differences in endosperm size during early development, which potentially relates to differences in timing of cellularization across diverse lines of maize. © 2014 Botanical Society of America, Inc.

Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

PubMed Central

Pan, Xuan; Jones, Morgan; Jiang, Jie; Zaprazna, Kristina; Yu, Duonan; Pear, Warren; Maillard, Ivan; Atchison, Michael L.

2012-01-01

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs. PMID:22292011

Early pathologic findings and long-term improvement in anti-Ma2-associated encephalitis.

PubMed

Blumenthal, D T; Salzman, K L; Digre, K B; Jensen, R L; Dunson, W A; Dalmau, J

2006-07-11

A 67-year-old man sequentially developed anti-Ma2-associated paraneoplastic encephalitis (PNE) and contralateral herpes simplex encephalitis (HSE). Brain biopsy 1 month before HSE revealed extensive infiltrates of T cells, B cells, and plasma cells. Most T cells expressed the cytotoxic granule-associated protein TIA-1 and the membranolytic protein granzyme-B. Although recovery was thought to be unlikely, treatment of the PNE with corticosteroids and resection of the associated lung cancer resulted in dramatic improvement for 21 months.

Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway.

PubMed

Hagn, Magdalena; Jahrsdörfer, Bernd

2012-11-01

B cells are generally believed to operate as producers of high affinity antibodies to defend the body against microorganisms, whereas cellular cytotoxicity is considered as an exclusive prerogative of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). In conflict with this dogma, recent studies have demonstrated that the combination of interleukin-21 (IL-21) and B-cell receptor (BCR) stimulation enables B cells to produce and secrete the active form of the cytotoxic serine protease granzyme B (GrB). Although the production of GrB by B cells is not accompanied by that of perforin as in the case of many other GrB-secreting cells, recent findings suggest GrB secretion by B cells may play a significant role in early antiviral immune responses, in the regulation of autoimmune responses, and in cancer immunosurveillance. Here, we discuss in detail how GrB-secreting B cells may influence a variety of immune processes. A better understanding of the role that GrB-secreting B cells are playing in the immune system may allow for the development and improvement of novel immunotherapeutic approaches against infectious, autoimmune and malignant diseases.

JNK-associated scattered growth of YD-10B oral squamous carcinoma cells while maintaining the epithelial phenotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Gayoung; Kim, Hyun-Man

Cell scattering of epithelial carcinoma cancer cells is one of the critical event in tumorigenesis. Cells losing epithelial cohesion detach from aggregated epithelial cell masses and may migrate to fatal organs through metastasis. The present study investigated the molecular mechanism by which squamous cell carcinoma cells grow scattered at the early phase of transformation while maintaining the epithelial phenotype. We studied YD-10B cells, which are established from human oral squamous cell carcinoma, because the cells grow scattered without the development of E-cadherin junctions (ECJs) under routine culture conditions despite the high expression of functional E-cadherin. The functionality of their E-cadherinmore » was demonstrated in that YD-10B cells developed ECJs, transiently or persistently, when they were cultured on substrates coated with a low amount of fibronectin or to confluence. The phosphorylation of JNK was up-regulated in YD-10B cells compared with that in human normal oral keratinocyte cells or human squamous cell carcinoma cells, which grew aggregated along with well-organized ECJs. The suppression of JNK activity induced the aggregated growth of YD-10B cells concomitant with the development of ECJs. These results indicate for the first time that inherently up-regulated JNK activity induces the scattered growth of the oral squamous cell carcinoma cells through down-regulating the development of ECJ despite the expression of functional E-cadherin, a hallmark of the epithelial phenotype. - Highlights: • JNK dissociates YD-10B oral squamous cell carcinoma cells. • JNK suppresses the development of E-cadherin junctions of oral carcinoma cells. • Suppression of JNK activity reverses the scattered growth of oral carcinoma cells.« less

Phenotypic Tfh development promoted by CXCR5-controlled re-localization and IL-6 from radiation-resistant cells.

PubMed

Chen, Xin; Ma, Weiwei; Zhang, Tingxin; Wu, Longyan; Qi, Hai

2015-11-01

How follicular T-helper (Tfh) cells develop is incompletely understood. We find that, upon antigen exposure in vivo, both naïve and antigen-experienced T cells sequentially upregulate CXCR5 and Bcl6 within the first 24 h, relocate to the T-B border, and give rise to phenotypic Bcl6(+)CXCR5(+) Tfh cells before the first cell division. CXCR5 upregulation is more dependent on ICOS costimulation than that of Bcl6, and early Bcl6 induction requires T-cell expression of CXCR5 and, presumably, relocation toward the follicle. This early and rapid upregulation of CXCR5 and Bcl6 depends on IL-6 produced by radiation-resistant cells. These results suggest that a Bcl6(hi)CXCR5(hi) phenotype does not automatically define a Tfh lineage but might reflect a state of antigen exposure and non-commitment to terminal effector fates and that niches in the T-B border and/or the follicle are important for optimal Bcl6 induction and maintenance.

The Rice Basic Helix-Loop-Helix Transcription Factor TDR INTERACTING PROTEIN2 Is a Central Switch in Early Anther Development[C][W

PubMed Central

Fu, Zhenzhen; Yu, Jing; Cheng, Xiaowei; Zong, Xu; Xu, Jie; Chen, Mingjiao; Li, Zongyun; Zhang, Dabing; Liang, Wanqi

2014-01-01

In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell–like features undergo cell divisions and differentiation to produce the anther, the male reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetal programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetal programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell–type determination. PMID:24755456

B3GNT3 Expression Is a Novel Marker Correlated with Pelvic Lymph Node Metastasis and Poor Clinical Outcome in Early-Stage Cervical Cancer

PubMed Central

Niu, Chunhao; Song, Libing; Zhang, Yanna

2015-01-01

Background The β1,3-N-acetylglucosaminyltransferase-3 gene (B3GNT3) encodes a member of the B3GNT family that functions as the backbone structure of dimeric sialyl-Lewis A and is involved in L-selectin ligand biosynthesis, lymphocyte homing and lymphocyte trafficking. B3GNT3 has been implicated as an important element in the development of certain cancers. However, the characteristics of B3GNT3 in the development and progression of cancer remain largely unknown. Thus, our study aimed to investigate the expression pattern and the prognostic value of B3GNT3 in patients with early-stage cervical cancer. Methods The mRNA and protein levels of B3GNT3 expression were examined in eight cervical cancer cell lines and ten paired cervical cancer tumors, using real-time PCR and western blotting, respectively. Immunohistochemistry (IHC) was used to analyze B3GNT3 protein expression in paraffin-embedded tissues from 196 early-stage cervical cancer patients. Statistical analyses were applied to evaluate the association between B3GNT3 expression scores and clinical parameters, as well as patient survival. Results B3GNT3 expression was significantly upregulated in cervical cancer cell lines and lesions compared with normal cells and adjacent noncancerous cervical tissues. In the 196 cases of tested early-stage cervical cancer samples, the B3GNT3 protein level was positively correlated with high risk TYPES of human papillomavirus (HPV) infection (P = 0.026), FIGO stage (P < 0.001), tumor size (P = 0.025), tumor recurrence (P = 0.004), vital status (P < 0.001), concurrent chemotherapy and radiotherapy (P = 0.016), lymphovascular space involvement (P = 0.003) and most importantly, lymph node metastasis (P = 0.003). Patients with high B3GNT3 expression had a shorter overall survival (OS) and disease-free survival (DFS) compared with those with low expression of this protein. Multivariate analysis suggested that B3GNT3 expression is an independent prognostic indicator for cervical cancer patients. Conclusions Our study demonstrated that elevated B3GNT3 expression is associated with pelvic lymph node metastasis and poor outcome in early-stage cervical cancer patients. B3GNT3 may be a novel prognostic marker and therapeutic target for the treatment of cervical cancer. PMID:26709519

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

PubMed

Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

2015-11-01

Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry. Copyright © 2015 Elsevier Inc. All rights reserved.

The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish

PubMed Central

Gokey, Jason J.; Dasgupta, Agnik; Amack, Jeffrey D.

2015-01-01

Asymmetric fluid flows generated by motile cilia in a transient ‘organ of asymmetry’ are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H+-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer’s vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures—neuromasts and olfactory placodes—suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry. PMID:26254189

African Trypanosomes Undermine Humoral Responses and Vaccine Development: Link with Inflammatory Responses?

PubMed Central

Stijlemans, Benoit; Radwanska, Magdalena; De Trez, Carl; Magez, Stefan

2017-01-01

African trypanosomosis is a debilitating disease of great medical and socioeconomical importance. It is caused by strictly extracellular protozoan parasites capable of infecting all vertebrate classes including human, livestock, and game animals. To survive within their mammalian host, trypanosomes have evolved efficient immune escape mechanisms and manipulate the entire host immune response, including the humoral response. This report provides an overview of how trypanosomes initially trigger and subsequently undermine the development of an effective host antibody response. Indeed, results available to date obtained in both natural and experimental infection models show that trypanosomes impair homeostatic B-cell lymphopoiesis, B-cell maturation and survival and B-cell memory development. Data on B-cell dysfunctioning in correlation with parasite virulence and trypanosome-mediated inflammation will be discussed, as well as the impact of trypanosomosis on heterologous vaccine efficacy and diagnosis. Therefore, new strategies aiming at enhancing vaccination efficacy could benefit from a combination of (i) early parasite diagnosis, (ii) anti-trypanosome (drugs) treatment, and (iii) anti-inflammatory treatment that collectively might allow B-cell recovery and improve vaccination. PMID:28596768

Human Embryonic Stem Cell-Derived Mesenchymal Stromal Cells Decrease the Development of Severe Experimental Autoimmune Uveitis in B10.RIII Mice.

PubMed

Qin, Yu; Chan, Ann M; Chang, Yu-Ling; Matynia, Anna; Kouris, Nicholas A; Kimbrel, Erin A; Ashki, Negin; Parikh, Sachin; Gorin, Michael B; Lanza, Robert; Levinson, Ralph D; Gordon, Lynn K

2017-09-15

We investigated the effect of exogenously administered human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) in experimental autoimmune uveitis (EAU) in B10.RIII mice, a murine model of severe uveitis. B10.RIII mice were immunized with an uveitogenic peptide, and intraperitoneal injections of 5 million hESC-MSCs per animal were given on the same day. Behavioral light sensitivity assays, histological evaluation, cytokine production, and regulatory T cells were analyzed at the peak of the disease. Histological and behavioral evidence demonstrated that early systemic treatment with hESC-MSCs decreases the development of severe EAU in B10.RIII mice. hESC-MSCs suppress Th17 and upregulate Th1 and Th2 responses as well as IL-2 and GM-CSF in splenocytes from hESC-MSC-treated mice. MSCs that originate from hESC decrease the development of severe EAU in B10.RIII mice, likely through systemic immune modulation. Further investigation is needed to determine any potential effect on active EAU.

B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism

PubMed Central

Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

2011-01-01

Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. PMID:21235532

B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism.

PubMed

Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

2011-03-01

Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

De novo variants in EBF3 are associated with hypotonia, developmental delay, intellectual disability, and autism

PubMed Central

Tanaka, Akemi J.; Cho, Megan T.; Willaert, Rebecca; Retterer, Kyle; Zarate, Yuri A.; Bosanko, Katie; Stefans, Vikki; Oishi, Kimihiko; Williamson, Amy; Wilson, Golder N.; Basinger, Alice; Barbaro-Dieber, Tina; Ortega, Lucia; Sorrentino, Susanna; Gabriel, Melissa K.; Anderson, Ilse J.; Sacoto, Maria J. Guillen; Schnur, Rhonda E.; Chung, Wendy K.

2017-01-01

Using whole-exome sequencing, we identified seven unrelated individuals with global developmental delay, hypotonia, dysmorphic facial features, and an increased frequency of short stature, ataxia, and autism with de novo heterozygous frameshift, nonsense, splice, and missense variants in the Early B-cell Transcription Factor Family Member 3 (EBF3) gene. EBF3 is a member of the collier/olfactory-1/early B-cell factor (COE) family of proteins, which are required for central nervous system (CNS) development. COE proteins are highly evolutionarily conserved and regulate neuronal specification, migration, axon guidance, and dendritogenesis during development and are essential for maintaining neuronal identity in adult neurons. Haploinsufficiency of EBF3 may affect brain development and function, resulting in developmental delay, intellectual disability, and behavioral differences observed in individuals with a deleterious variant in EBF3. PMID:29162653

Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny

PubMed Central

Kumar, Satyendra; Wuerffel, Robert; Achour, Ikbel; Lajoie, Bryan; Sen, Ranjan; Dekker, Job; Feeney, Ann J.; Kenter, Amy L.

2013-01-01

V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome. PMID:24240234

Functional silencing is initiated and maintained in immature anti-insulin B cells.

PubMed

Henry, Rachel A; Acevedo-Suárez, Carlos A; Thomas, James W

2009-03-15

Mechanisms of B cell tolerance act during development in the bone marrow and periphery to eliminate or restrict autoreactive clones to prevent autoimmune disease. B cells in the spleens of mice that harbor anti-insulin BCR transgenes (125Tg) are maintained in a functionally silenced or anergic state by endogenous hormone, but it is not clear when and where anergy is induced. An in vitro bone marrow culture system was therefore used to probe whether small protein hormones, a critical class of autoantigens, could interact with the BCR to induce anergy early during B cell development. Upon exposure to insulin, anti-insulin (125Tg) immature B cells show similar hallmarks of anergy as those observed in mature splenic B cells. These include BCR down-regulation, impaired proliferative responses to anti-CD40, and diminished calcium mobilization upon stimulation with BCR-dependent and independent stimuli. Inhibition of calcineurin also results in reduced immature B cell proliferation in a similar manner, suggesting a potential mechanism through which reduced intracellular calcium mobilization may be altering cellular proliferation. Signs of impairment appear after short-term exposure to insulin, which are reversible upon Ag withdrawal. This suggests that a high degree of functional plasticity is maintained at this stage and that constant Ag engagement is required to maintain functional inactivation. These findings indicate that tolerance observed in mature, splenic 125Tg B cells is initiated by insulin in the developing B cell compartment and thus highlight an important therapeutic window for the prevention of insulin autoimmunity.

Salicylate-based anti-inflammatory drugs inhibit the early lesion of diabetic retinopathy.

PubMed

Zheng, Ling; Howell, Scott J; Hatala, Denise A; Huang, Kun; Kern, Timothy S

2007-02-01

It has been previously reported that aspirin inhibited the development of diabetic retinopathy in diabetic animals, raising the possibility that anti-inflammatory drugs may have beneficial effects on diabetic retinopathy. To further explore this, we compared effects of oral consumption of three different salicylate-based drugs (aspirin, sodium salicylate, and sulfasalazine) on the development of early stages of diabetic retinopathy in rats. These three drugs differ in their ability to inhibit cyclooxygenase but share an ability to inhibit nuclear factor-kappaB (NF-kappaB). Diabetes of 9-10 months duration significantly increased the number of TUNEL (transferase-mediated dUTP nick-end labeling)-positive capillary cells and acellular (degenerate) capillaries in the retinal vasculature, and all three salicylate-based drugs inhibited this cell death and formation of acellular capillaries without altering the severity of hyperglycemia. In short-term diabetes (2-4 months), all three salicylates inhibited the diabetes-induced loss of neuronal cells from the ganglion cell layer. Oral aspirin (as a representative of the salicylate family) inhibited diabetes-induced increase in NF-kappaB DNA-binding affinity in electrophoretic mobility shift assay and transcription factor array in nuclear extract isolated from whole retina. All three salicylates inhibited the diabetes-induced translocation of p50 (a subunit of NF-kappaB) into nuclei of retinal vascular endothelial cells of the isolated retinal vasculature, as well as of p50 and p65 into nuclei of cells in the ganglion cell layer and inner nuclear layer on whole-retinal sections. Sulfasalazine (also as a representative of the salicylates) inhibited the diabetes-induced upregulation of several inflammatory gene products, which are regulated by NF-kappaB, including vascular cell adhesion molecule, intracellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 in whole-retinal lysate. Salicylates, in doses administrated in our experiments, inhibited NF-kappaB and perhaps other transcription factors in the retina, were well tolerated, and offered new tools to investigate and inhibit the development of diabetic retinopathy.

Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3 and 3-B-3(-) Chondroitin Sulphate Motifs Are Morphogenetic Markers Of Tissue Development.

PubMed

Hayes, Anthony J; Smith, Susan M; Caterson, Bruce; Melrose, James

2018-06-11

This study reviewed the occurrence of chondroitin sulphate (CS) motifs 4-C-3, 7-D-4 and 3-B-3(-) which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulphation motifs 7-D-4, 4-C-3 and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Patterns of B-lymphocyte gene expression elicited by lipopolysaccharide mitogen.

PubMed Central

Janossy, G; Snajdr, J; Simak-Ellis, M

1976-01-01

When large proportions of B lymphocytes from the murine spleen are stimulated in vitro by bacterial lipopolysaccharide (LPS) B lymphoblasts with small amounts of intracellular immunoglobulin (Ig) and plasmablasts with large amounts of intracellular Ig concomitantly proliferate. It is likely that B lymphocytes are heterogeneous and LPS activates B cells to express their predetermined functional capacity since bromodeoxyuridine does not inhibit the initiation of Ig synthesis in plasmablasts, and Ig synthesis starts before these cells complete their first mitosis. The results suggest that LPS is a potent polyclonal activator (of a B-cell subset) but it is not a differentiation factor in the sense that it is unable to determine whether its target cell develops extensive endoplasmic reticulum or follows a different pathway. The results do not exclude that modulation of B cells' genetic programming might take place during T cell-dependent B-lymphocyte activation. The observed B-cell heterogeneity offers a possible explanation for the concomitant emergence of B memory cells and antibody producers during the early phase of immune responses in vivo. Images Figure 3 Figure 5 Figure 7 Figure 8 PMID:1088414

Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy

PubMed Central

Sauer, Aisha V.; Morbach, Henner; Brigida, Immacolata; Ng, Yen-Shing; Aiuti, Alessandro; Meffre, Eric

2012-01-01

Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions. PMID:22622038

Compound 49b Reduces Inflammatory Markers and Apoptosis after Ocular Blast Injury

DTIC Science & Technology

2014-09-01

drug, Compound 49b, have anti-apoptotic and anti-inflammatory properties in retinal endothelial cells and in a diabetic retinopathy model [7, 10...plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations. Mol Vis, 2009; 15: 1418-28. 12...in other retinal damage models, specifically the streptozotocin- induced type 1 diabetic retinopathy model and retinal endothelial cells cultured in

The Shigella flexneri OspB effector: an early immunomodulator.

PubMed

Ambrosi, Cecilia; Pompili, Monica; Scribano, Daniela; Limongi, Dolores; Petrucca, Andrea; Cannavacciuolo, Sonia; Schippa, Serena; Zagaglia, Carlo; Grossi, Milena; Nicoletti, Mauro

2015-01-01

Through the action of the type three secretion system (T3SS) Shigella flexneri delivers several effectors into host cells to promote cellular invasion, multiplication and to exploit host-cell signaling pathways to modulate the host innate immune response. Although much progress has been made in the understanding of many type III effectors, the molecular and cellular mechanism of the OspB effector is still poorly characterized. In this study we present new evidence that better elucidates the role of OspB as pro-inflammatory factor at very early stages of infection. Indeed, we demonstrate that, during the first hour of infection, OspB is required for full activation of ERK1/2 and p38 MAPKs and the cytosolic phospholipase A(2) (cPLA(2)). Activation of cPLA(2) ultimately leads to the production and secretion of PMN chemoattractant metabolite(s) uncoupled with release of IL-8. Moreover, we also present evidence that OspB is required for the development of the full and promptly inflammatory reaction characteristic of S. flexneri wild-type infection in vivo. Based on OspB and OspF similarity (both effectors share similar transcription regulation, temporal secretion into host cells and nuclear localization) we hypothesized that OspB and OspF effectors may form a pair aimed at modulating the host cell response throughout the infection process, with opposite effects. A model is presented to illustrate how OspB activity would promote S. flexneri invasion and bacterial dissemination at early critical phases of infection. Copyright © 2014 Elsevier GmbH. All rights reserved.

Artificial Induction of Sox21 Regulates Sensory Cell Formation in the Embryonic Chicken Inner Ear

PubMed Central

Freeman, Stephen D.; Daudet, Nicolas

2012-01-01

During embryonic development, hair cells and support cells in the sensory epithelia of the inner ear derive from progenitors that express Sox2, a member of the SoxB1 family of transcription factors. Sox2 is essential for sensory specification, but high levels of Sox2 expression appear to inhibit hair cell differentiation, suggesting that factors regulating Sox2 activity could be critical for both processes. Antagonistic interactions between SoxB1 and SoxB2 factors are known to regulate cell differentiation in neural tissue, which led us to investigate the potential roles of the SoxB2 member Sox21 during chicken inner ear development. Sox21 is normally expressed by sensory progenitors within vestibular and auditory regions of the early embryonic chicken inner ear. At later stages, Sox21 is differentially expressed in the vestibular and auditory organs. Sox21 is restricted to the support cell layer of the auditory epithelium, while it is enriched in the hair cell layer of the vestibular organs. To test Sox21 function, we used two temporally distinct gain-of-function approaches. Sustained over-expression of Sox21 from early developmental stages prevented prosensory specification, and abolished the formation of both hair cells and support cells. However, later induction of Sox21 expression at the time of hair cell formation in organotypic cultures of vestibular epithelia inhibited endogenous Sox2 expression and Notch activity, and biased progenitor cells towards a hair cell fate. Interestingly, Sox21 did not promote hair cell differentiation in the immature auditory epithelium, which fits with the expression of endogenous Sox21 within mature support cells in this tissue. These results suggest that interactions among endogenous SoxB family transcription factors may regulate sensory cell formation in the inner ear, but in a context-dependent manner. PMID:23071561

Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4.

PubMed

Ito, Akira; Nosrat, Christopher A

2009-09-01

Taste buds and the peripheral nerves innervating them are two important components of the peripheral gustatory system. They require appropriate connections for the taste system to function. Neurotrophic factors play crucial roles in the innervation of peripheral sensory organs and tissues. Both brain-derived neurotrophic factor (BDNF) null-mutated and neurotrophin-4 (NT-4) null-mutated mice exhibit peripheral gustatory deficits. BDNF and NT-4 bind to a common high affinity tyrosine kinase receptor, TrkB (NTRK-2), and a common p75 neurotrophin receptor (NGFR). We are currently using a transgenic mouse model to study peripheral taste system development and innervation in the absence of both TrkB ligands. We show that taste cell progenitors express taste cell markers during early stages of taste bud development in both BDNF(-/-)xNT-4(-/-) and wild-type mice. At early embryonic stages, taste bud progenitors express Troma-1, Shh, and Sox2 in all mice. At later stages, lack of innervation becomes a prominent feature in BDNF(-/-)xNT-4(-/-) mice leading to a decreasing number of fungiform papillae and morphologically degenerating taste cells. A total loss of vallate taste cells also occurs in postnatal transgenic mice. Our data indicate an initial independence but a later permissive and essential role for innervation in taste bud development and maintenance.

Discrete β-adrenergic mechanisms regulate early and late erythropoiesis in erythropoietin-resistant anemia.

PubMed

Hasan, Shirin; Mosier, Michael J; Szilagyi, Andrea; Gamelli, Richard L; Muthumalaiappan, Kuzhali

2017-10-01

Anemia of critical illness is resistant to exogenous erythropoietin. Packed red blood cells transfusions is the only treatment option, and despite related cost and morbidity, there is a need for alternate strategies. Erythrocyte development can be divided into erythropoietin-dependent and erythropoietin-independent stages. We have shown previously that erythropoietin-dependent development is intact in burn patients and the erythropoietin-independent early commitment stage, which is regulated by β1/β2-adrenergic mechanisms, is compromised. Utilizing the scald burn injury model, we studied erythropoietin-independent late maturation stages and the effect of β1/β2, β-2, or β-3 blockade in burn mediated erythropoietin-resistant anemia. Burn mice were randomized to receive daily injections of propranolol (nonselective β1/β2 antagonist), nadolol (long-acting β1/β2 antagonist), butoxamine (selective β2 antagonist), or SR59230A (selective β3 antagonist) for 6 days after burn. Total bone marrow cells were characterized as nonerythroid cells, early and late erythroblasts, nucleated orthochromatic erythroblasts and enucleated reticulocyte subsets using CD71, Ter119, and Syto-16 by flow cytometry. Multipotential progenitors were probed for MafB expressing cells. Although propranolol improved early and late erythroblasts, only butoxamine and selective β3-antagonist administrations were positively reflected in the peripheral blood hemoglobin and red blood cells count. While burn impeded early commitment and late maturation stages, β1/β2 antagonism increased the early erythroblasts through commitment stages via β2 specific MafB regulation. β3 antagonism was more effective in improving overall red blood cells through late maturation stages. The study unfolds novel β2 and β3 adrenergic mechanisms orchestrating erythropoietin resistant anemia after burn, which impedes both the early commitment stage and the late maturation stages, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

RhoB-dependent modulation of postendocytic traffic in polarized Madin-Darby canine kidney cells.

PubMed

Rondanino, Christine; Rojas, Raul; Ruiz, Wily G; Wang, Exing; Hughey, Rebecca P; Dunn, Kenneth W; Apodaca, Gerard

2007-07-01

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.

De novo variants in EBF3 are associated with hypotonia, developmental delay, intellectual disability, and autism.

PubMed

Tanaka, Akemi J; Cho, Megan T; Willaert, Rebecca; Retterer, Kyle; Zarate, Yuri A; Bosanko, Katie; Stefans, Vikki; Oishi, Kimihiko; Williamson, Amy; Wilson, Golder N; Basinger, Alice; Barbaro-Dieber, Tina; Ortega, Lucia; Sorrentino, Susanna; Gabriel, Melissa K; Anderson, Ilse J; Sacoto, Maria J Guillen; Schnur, Rhonda E; Chung, Wendy K

2017-11-01

Using whole-exome sequencing, we identified seven unrelated individuals with global developmental delay, hypotonia, dysmorphic facial features, and an increased frequency of short stature, ataxia, and autism with de novo heterozygous frameshift, nonsense, splice, and missense variants in the Early B-cell Transcription Factor Family Member 3 ( EBF3 ) gene. EBF3 is a member of the collier/olfactory-1/early B-cell factor (COE) family of proteins, which are required for central nervous system (CNS) development. COE proteins are highly evolutionarily conserved and regulate neuronal specification, migration, axon guidance, and dendritogenesis during development and are essential for maintaining neuronal identity in adult neurons. Haploinsufficiency of EBF3 may affect brain development and function, resulting in developmental delay, intellectual disability, and behavioral differences observed in individuals with a deleterious variant in EBF3 . © 2017 Tanaka et al.; Published by Cold Spring Harbor Laboratory Press.

Bruton tyrosine kinase inhibitors: a promising novel targeted treatment for B cell lymphomas

PubMed Central

Aalipour, Amin; Advani, Ranjana H.

2015-01-01

Summary Constitutive or aberrant signalling of the B cell receptor signalling cascade has been implicated in the propagation and maintenance of a variety of B cell malignancies. Small molecule inhibitors of Bruton tyrosine kinase (BTK), a protein early in this cascade and specifically expressed in B cells, have emerged as a new class of targeted agents. There are several BTK inhibitors, including ONO-WG-307, LFM-A13, dasatinib, CC-292, and PCI-32765 (ibrutinib), in preclinical and/or clinical development of which ibrutinib is currently in phase III trials. Recent clinical data suggest significant activity of ibrutinib as a first in class oral inhibitor of BTK. This review provides an overview of ongoing clinical studies of BTK inhibitors. PMID:24111579

Zebrafish Foxi1 provides a neuronal ground state during inner ear induction preceding the Dlx3b/4b-regulated sensory lineage.

PubMed

Hans, Stefan; Irmscher, Anne; Brand, Michael

2013-05-01

Vertebrate inner ear development is a complex process that involves the induction of a common territory for otic and epibranchial precursors and their subsequent segregation into otic and epibranchial cell fates. In zebrafish, the otic-epibranchial progenitor domain (OEPD) is induced by Fgf signaling in a Foxi1- and Dlx3b/4b-dependent manner, but the functional differences of Foxi1 and Dlx3b/4b in subsequent cell fate specifications within the developing inner ear are poorly understood. Based on pioneer tracking (PioTrack), a novel Cre-dependent genetic lineage tracing method, and genetic data, we show that the competence to embark on a neuronal or sensory fate is provided sequentially and very early during otic placode induction. Loss of Foxi1 prevents neuronal precursor formation without affecting hair cell specification, whereas loss of Dlx3b/4b inhibits hair cell but not neuronal precursor formation. Consistently, in Dlx3b/4b- and Sox9a-deficient b380 mutants almost all otic epithelial fates are absent, including sensory hair cells, and the remaining otic cells adopt a neuronal fate. Furthermore, the progenitors of the anterior lateral line ganglia also arise from the OEPD in a Foxi1-dependent manner but are unaffected in the absence of Dlx3b/4b or in b380 mutants. Thus, in addition to otic fate Foxi1 provides neuronal competence during OEPD induction prior to and independently of the Dlx3b/4b-mediated sensory fate of the developing inner ear.

Chronic lymphocytic leukemia.

PubMed

Kay, Neil E; Hamblin, Terry J; Jelinek, Diane F; Dewald, Gordon W; Byrd, John C; Farag, Sherif; Lucas, Margaret; Lin, Thomas

2002-01-01

This update of early stage B-cell chronic lymphocytic leukemia (B-CLL) embraces current information on the diagnosis, biology, and intervention required to more fully develop algorithms for management of this disease. Emphasis on early stage is based on the rapid advancement in our understanding of the disease parameters and our increasing ability to predict for a given early stage patient whether there is a need for more aggressive management. In Section I, Dr. Terry Hamblin addresses the nature of the disease, accurate diagnostic procedures, evidence for an early "preclinical" phase, the use of newer prognostic features to distinguish who will be likely to progress or not, and whether it is best to watch or treat early stage disease. In Section II, Dr. Neil Kay and colleagues address the biologic aspects of the disease and how they may relate to disease progression. Review of the newer insights into gene expression, recurring genetic defects, role of cytokines/autocrine pathways, and the interaction of the CLL B cell with the microenvironment are emphasized. The relationship of these events to both trigger disease progression and as opportunities for future therapeutic intervention even in early stage disease is also considered. In Section III, Dr. John Byrd and colleagues review the historical and now current approaches to management of the previously untreated progressive B-CLL patient. They discuss what decision tree could be used in the initial decision to treat a given patient. The use of single agents versus newer combination approaches such as chemoimmunotherapy are discussed here. In addition, the place of marrow transplant and some of the newer antibodies available for treatment of B-CLL are considered. Finally, a challenge to utilize our growing knowledge of the biology of B-CLL in the early stage B-CLL is proffered.

Targeted Deletion of the Gene Encoding the La Autoantigen (Sjögren's Syndrome Antigen B) in B Cells or the Frontal Brain Causes Extensive Tissue Loss

PubMed Central

Gaidamakov, Sergei; Maximova, Olga A.; Chon, Hyongi; Blewett, Nathan H.; Wang, Hongsheng; Crawford, Amanda K.; Day, Amanda; Tulchin, Natalie; Crouch, Robert J.; Morse, Herbert C.; Blitzer, Robert D.

2014-01-01

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage. We developed a conditional La allele and used it in mice that express Cre recombinase in either B cell progenitors or the forebrain. B cell Mb1Cre La-deleted mice produce no B cells. Consistent with αCamKII Cre, which induces deletion in hippocampal CA1 cells in the third postnatal week and later throughout the neocortex, brains develop normally in La-deleted mice until ∼5 weeks and then lose a large amount of forebrain cells and mass, with evidence of altered pre-tRNA processing. The data indicate that La is required not only in proliferating cells but also in nondividing postmitotic cells. Thus, La is essential in different cell types and required for normal development of various tissue types. PMID:24190965

Protective cellular responses to Burkholderia mallei infection.

PubMed

Rowland, Caroline A; Lever, M Stephen; Griffin, Kate F; Bancroft, Gregory J; Lukaszewski, Roman A

2010-10-01

Burkholderia mallei is a Gram-negative bacillus causing the disease glanders in humans. During intraperitoneal infection, BALB/c mice develop a chronic disease characterised by abscess formation where mice normally die up to 70 days post-infection. Although cytokine responses have been investigated, cellular immune responses to B. mallei infection have not previously been characterised. Therefore, the influx and activation status of splenic neutrophils, macrophages and T cells was examined during infection. Gr-1+ neutrophils and F4/80+ macrophages infiltrated the spleen 5 h post-infection and an increase in activated macrophages, neutrophils and T cells occurred by 24 h post-infection. Mice depleted of Gr-1+ cells were acutely susceptible to B. mallei infection, succumbing to the infection 5 days post-infection. Mice depleted of both CD4 and CD8 T cells did not succumb to the infection until 14 days post-infection. Infected μMT (B cell) and CD28 knockout mice did not differ from wildtype mice whereas iNOS-2 knockout mice began to succumb to the infection 30 days post-infection. The data presented suggests that Gr-1+ cells, activated early in B. mallei infection, are essential for controlling the early, innate response to B. mallei infection and T cells or nitric oxide are important during the later stages of infection. Crown Copyright © 2010. Published by Elsevier SAS. All rights reserved.

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 utilization of B cell depletion therapy in autoimmune diseases and the need for new therapeutic approaches in SLE, a better understanding of human B cell subsets and the balance of pathogenic and regulatory functions is of the essence.

Regulation of VH Replacement by B Cell Receptor (BCR)-mediated Signaling in Human Immature B Cells

PubMed Central

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

2013-01-01

VH replacement provides a unique RAG-mediated recombination mechanism to edit non-functional IgH genes or IgH genes encoding self reactive B cell receptors (BCRs) and contributes to the diversification of antibody repertoire in mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. Here we show that crosslinking 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 co-stimulation, presumably through activation of the PI3 kinase pathway. These results show for the first time that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments. PMID:23630348

CD19(+)CD21(low) B cells and patients at risk for NIH-defined chronic graft-versus-host disease with bronchiolitis obliterans syndrome.

PubMed

Kuzmina, Zoya; Krenn, Katharina; Petkov, Ventzislav; Körmöczi, Ulrike; Weigl, Roman; Rottal, Arno; Kalhs, Peter; Mitterbauer, Margit; Ponhold, Lothar; Dekan, Gerhard; Greinix, Hildegard T; Pickl, Winfried F

2013-03-07

Bronchiolitis obliterans syndrome (BOS), pathognomonic for chronic graft-versus-host disease (cGVHD) of the lung, is a progressive and often fatal complication after allogeneic hematopoietic cell transplantation (HCT). Biomarkers for the prediction and diagnosis of BOS are urgently needed to improve patients' prognosis. We prospectively evaluated B-cell subpopulations and B-cell activating factor (BAFF) in 136 patients (46 BOS, 41 no cGVHD, 49 cutaneous cGVHD) to define novel biomarkers for early diagnosis of National Institutes of Health-defined BOS diagnosed a median of 11 mo after HCT. Patients with newly diagnosed BOS had significantly higher percentages of CD19(+)CD21(low) B cells (25.5 versus 6.6%, P < .0001), BAFF (7.3 versus 3.5 ng/mL, P = .02), and BAFF/CD19(+) ratio (0.18 versus 0.02 ng/10(3) CD19(+) B cells, P 5 .007) compared with patients without cGVHD. The area under the receiver operating curve for CD19(+)CD21(low) B cells was 0.97 (95% confidence interval, 0.94-0.99) and a cutoff point >9% was optimal for diagnosing BOS in patients with first drop of pulmonary function tests with a sensitivity of 96% and a negative predictive value of 94%. Thus, elevated levels of CD19(+)CD21(low) B cells are a potential novel biomarker for HCT patients at risk for developing BOS at an early stage and could allow improvement of patient outcome.

Transcriptional response of peripheral lymphocytes to early fibrosarcoma: a model system for cancer detection based on hybridization signatures.

PubMed

Marques, Márcia M C; Junta, Cristina M; Zárate-Blades, Carlos R; Sakamoto-Hojo, Elza Tiemi; Donadi, Eduardo A; Passos, Geraldo A S

2009-07-01

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer.

Expression of growth hormone and its transcription factor, Pit-1, in early bovine development.

PubMed

Joudrey, E M; Lechniak, D; Petrik, J; King, W A

2003-03-01

During bovine embryogenesis, bovine growth hormone (bGH) contributes to proliferation, differentiation, and modulation of embryo metabolism. Pituitary-specific transcription factor-1 (Pit-1) is a transcription factor that binds to promoters of GH, prolactin (PRL), and thyroid-stimulating hormone-beta (TSHbeta) encoding genes. A polymorphism in the fifth exon of the bGH gene resulting in a leucine (Leu) to valine (Val) substitution provides an Alu I restriction site when the Leu allele is present. To determine the onset of embryonic expression of the bGH gene, oocytes derived from ovaries homozygous for Leu alleles were fertilized in vitro with spermatozoa obtained from a Val homozygote. For each developmental stage examined, three separate pools of embryos composed of approximately 100 cell samples underwent RNA isolation, reverse transcription to cDNA, and amplification by nested PCR (nPCR). Bovine GH gene transcripts were identified at 2- to 4-cell (n = 162), 8- to 16-cell (n = 73), morulae (n = 51), and blastocyst (n = 15) stages. Likewise, transcripts for Pit-1 were detected at 2-cell (n = 125), 4-cell (n = 114), 8-cell (n = 56), 12-to-32-cell (n = 32), morulae (n = 68), and blastocyst (n = 14) stages. After digestion with Alu1, bGH cDNA was genotyped by restriction fragment length polymorphism (RFLP) analysis. Bovine GH mRNA was present in all pools of stages examined. Both Leu and Val alleles (maternal and paternal) were only detected in pools of embryos that had reached 8- to 16-cell stage. Results suggest that transcription of the bGH gene begins at the 8- to 16-cell stage in bovine embryos, possibly under control of the transcription factor, Pit-1, and that RFLP analysis of the bGH gene can be used to determine parental origin of transcripts in early embryonic development. Copyright 2003 Wiley-Liss, Inc.

The early cellular signatures of protective immunity induced by live viral vaccination.

PubMed

Kohler, Siegfried; Bethke, Nicole; Böthe, Matthias; Sommerick, Sophie; Frentsch, Marco; Romagnani, Chiara; Niedrig, Matthias; Thiel, Andreas

2012-09-01

Here, we have used primary vaccination of healthy donors with attenuated live yellow fever virus 17D (YFV-17D) as a model to study the generation of protective immunity. In short intervals after vaccination, we analyzed the induction of YFV-17D specific T- and B-cell immunity, bystander activation, dendritic cell subsets, changes in serum cytokine levels, and YFV-17D-specific antibodies. We show activation of innate immunity and a concomitant decline of numbers of peripheral blood T and B cells. An early peak of antigen-specific T cells at day 2, followed by mobilization of innate immune cells, preceded the development of maximal adaptive immunity against YFV-17D at day 14 after vaccination. Interestingly, potent adaptive immunity as measured by high titers of neutralizing YFV-17D-specific antibodies, correlated with early activation and recruitment of YFV-17D-specific CD4(+) T cells and higher levels of sIL-6R. Thus our data might provide new insights into the interplay of innate and adaptive immunity for the induction of protective immunity. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Micromere lineages in the glossiphoniid leech Helobdella

NASA Technical Reports Server (NTRS)

Huang, Francoise Z.; Kang, Dongmin; Ramirez-Weber, Felipe-Andres; Bissen, Shirley T.; Weisblat, David A.

2002-01-01

In leech embryos, segmental mesoderm and ectoderm arise from teloblasts by lineages that are already relatively well characterized. Here, we present data concerning the early divisions and the definitive fate maps of the micromeres, a group of 25 small cells that arise during the modified spiral cleavage in leech (Helobdella robusta) and contribute to most of the nonsegmental tissues of the adult. Three noteworthy results of this work are as follows. (1) The c"' and dm' clones (3d and 3c in traditional nomenclature) give rise to a hitherto undescribed network of fibers that run from one end of the embryo to the other. (2) The clones of micromeres b" and b"' (2b and 3b in traditional nomenclature) die in normal development; the b" clone can be rescued to assume the normal c" fate if micromere c" or its clone are ablated in early development. (3) Two qualitative differences in micromere fates are seen between H. robusta (Sacramento) and another Helobdella sp. (Galt). First, in Helobdella sp. (Galt), the clone of micromere b" does not normally die, and contributes a subset of the cells arising exclusively from c" in H. robusta (Sacramento). Second, in Helobdella sp. (Galt), micromere c"' makes no definitive contribution, whereas micromere dm' gives rise to cells equivalent to those arising from c"' and dm' in H. robusta (Sacramento).

Reviewing the current evidence supporting early B-cells as the cellular origin of Merkel cell carcinoma.

PubMed

Sauer, C M; Haugg, A M; Chteinberg, E; Rennspiess, D; Winnepenninckx, V; Speel, E-J; Becker, J C; Kurz, A K; Zur Hausen, A

2017-08-01

Merkel cell carcinoma (MCC) is a highly malignant skin cancer characterized by early metastases and poor survival. Although MCC is a rare malignancy, its incidence is rapidly increasing in the U.S. and Europe. The discovery of the Merkel cell polyomavirus (MCPyV) has enormously impacted our understanding of its etiopathogenesis and biology. MCCs are characterized by trilinear differentiation, comprising the expression of neuroendocrine, epithelial and B-lymphoid lineage markers. To date, it is generally accepted that the initial assumption of MCC originating from Merkel cells (MCs) is unlikely. This is owed to their post-mitotic character, absence of MCPyV in MCs and discrepant protein expression pattern in comparison to MCC. Evidence from mouse models suggests that epidermal/dermal stem cells might be of cellular origin in MCC. The recently formulated hypothesis of MCC originating from early B-cells is based on morphology, the consistent expression of early B-cell lineage markers and the finding of clonal immunoglobulin chain rearrangement in MCC cells. In this review we elaborate on the cellular ancestry of MCC, the identification of which could pave the way for novel and more effective therapeutic regimens. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia

PubMed Central

Paszkiewicz, Paulina J.; Fräßle, Simon P.; Srivastava, Shivani; Sommermeyer, Daniel; Hudecek, Michael; Sadelain, Michel; Liu, Lingfeng; Jensen, Michael C.; Riddell, Stanley R.; Busch, Dirk H.

2016-01-01

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies. PMID:27760047

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia.

PubMed

Paszkiewicz, Paulina J; Fräßle, Simon P; Srivastava, Shivani; Sommermeyer, Daniel; Hudecek, Michael; Drexler, Ingo; Sadelain, Michel; Liu, Lingfeng; Jensen, Michael C; Riddell, Stanley R; Busch, Dirk H

2016-11-01

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.

Trkb signaling in pericytes is required for cardiac microvessel stabilization.

PubMed

Anastasia, Agustin; Deinhardt, Katrin; Wang, Shiyang; Martin, Laura; Nichol, Donna; Irmady, Krithi; Trinh, Jasmine; Parada, Luis; Rafii, Shahin; Hempstead, Barbara L; Kermani, Pouneh

2014-01-01

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

Trkb Signaling in Pericytes Is Required for Cardiac Microvessel Stabilization

PubMed Central

Wang, Shiyang; Martin, Laura; Nichol, Donna; Irmady, Krithi; Trinh, Jasmine; Parada, Luis; Rafii, Shahin; Hempstead, Barbara L.; Kermani, Pouneh

2014-01-01

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb −/−) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb−/− embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development. PMID:24498100

Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis.

PubMed

Dash, Surjya Narayan; Lehtonen, Eero; Wasik, Anita A; Schepis, Antonino; Paavola, Jere; Panula, Pertti; Nelson, W James; Lehtonen, Sanna

2014-04-01

The conserved septin family of filamentous small GTPases plays important roles in mitosis, cell migration and cell morphogenesis by forming scaffolds and diffusion barriers. Recent studies in cultured cells in vitro indicate that a septin complex of septin 2, 7 and 9 is required for ciliogenesis and cilia function, but septin function in ciliogenesis in vertebrate organs in vivo is not understood. We show that sept7b is expressed in ciliated cells in different tissues during early zebrafish development. Knockdown of sept7b by using morpholino antisense oligonucleotides caused misorientation of basal bodies and cilia, reduction of apical actin and the shortening of motile cilia in Kupffer's vesicle and pronephric tubules. This resulted in pericardial and yolk sac edema, body axis curvature and hydrocephaly. Notably, in sept7b morphants we detected strong left-right asymmetry defects in the heart and lateral plate mesoderm (situs inversus), reduced fluid flow in the kidney, the formation of kidney cysts and loss of glomerular filtration barrier function. Thus, sept7b is essential during zebrafish development for pronephric function and ciliogenesis, and loss of expression of sept7b results in defects that resemble human ciliopathies.

Compare the Difference of B-cell Epitopes of EgAgB1 and EgAgB3 Proteins Selected through Bioinformatic Analysis

NASA Astrophysics Data System (ADS)

An, Mengting; Zhang, Fengbo; Zhu, Yuejie; Zhao, Xiao; Ding, Jianbing

2018-01-01

Cystic echinococcosis, as a zoonosis, seriously endangers humans and animals, so early diagnosis of this disease is particularly important. Therefore, this study is to predict B-cell epitopes of EgAgB1 and EgAgB3 proteins by bioinformatics software. B-cell epitopes of EgAgB1 and EgAgB3 proteins are predicted using DNAStar and IEDB software. The results suggest that there are two potential B-cell epitopes in EgAgB1, which located in the 8-15 and 31-37 amino acid residue segments. And two potential B-cell epitopes in EgAgB2, located in the 20∼27 and 47∼53 amino acid residue segments. This study predicted the B-cell epitopes of EgAgB1 and EgAgB3 proteins, which laid the foundation for the early diagnosis of Cystic echinococcosis.

Development of venetoclax for therapy of lymphoid malignancies.

PubMed

Zhu, Huayuan; Almasan, Alexandru

2017-01-01

B-cell lymphoma-2 (BCL-2) family dysfunction and impairment of apoptosis are common in most B-cell lymphoid malignancies. Venetoclax (Venclexta™, formerly ABT-199, GDC-0199) is a highly selective BCL-2 inhibitor, which mimics its BCL-2 homology 3-domain to induce apoptosis. It was approved for treatment of previously treated chronic lymphocytic leukemia (CLL) patients with 17p deletion early in 2016. It has also been in clinical trials for other B-cell lymphoid malignancies. Unlike the other recently approved targeted agents idelalisib and ibrutinib, so far there has been no relapse reported in some patients. Also, unlike the other targeted agents, it is effective against tumor cells that reside in the blood marrow. Despite its promising outcome in CLL, preclinical data have already uncovered mechanistic insights underlying venetoclax resistance, such as upregulation of MCL-1 or BCL-xL expression and protective signaling from the microenvironment. In this review, we describe the role of the BCL-2 family in the pathogenesis of B-cell lymphoid malignancies, the development of venetoclax, and its current clinical outcome in CLL and other B-cell malignancies. We also discuss the resistance mechanisms that develop following venetoclax therapy, potential strategies to overcome them, and how this knowledge can be translated into clinical applications.

Development of venetoclax for therapy of lymphoid malignancies

PubMed Central

Zhu, Huayuan; Almasan, Alexandru

2017-01-01

B-cell lymphoma-2 (BCL-2) family dysfunction and impairment of apoptosis are common in most B-cell lymphoid malignancies. Venetoclax (Venclexta™, formerly ABT-199, GDC-0199) is a highly selective BCL-2 inhibitor, which mimics its BCL-2 homology 3-domain to induce apoptosis. It was approved for treatment of previously treated chronic lymphocytic leukemia (CLL) patients with 17p deletion early in 2016. It has also been in clinical trials for other B-cell lymphoid malignancies. Unlike the other recently approved targeted agents idelalisib and ibrutinib, so far there has been no relapse reported in some patients. Also, unlike the other targeted agents, it is effective against tumor cells that reside in the blood marrow. Despite its promising outcome in CLL, preclinical data have already uncovered mechanistic insights underlying venetoclax resistance, such as upregulation of MCL-1 or BCL-xL expression and protective signaling from the microenvironment. In this review, we describe the role of the BCL-2 family in the pathogenesis of B-cell lymphoid malignancies, the development of venetoclax, and its current clinical outcome in CLL and other B-cell malignancies. We also discuss the resistance mechanisms that develop following venetoclax therapy, potential strategies to overcome them, and how this knowledge can be translated into clinical applications. PMID:28331288

Local bone marrow renin-angiotensin system in the genesis of leukemia and other malignancies.

PubMed

Haznedaroglu, I C; Malkan, U Y

2016-10-01

The existence of a local renin-angiotensin system (RAS) specific to the hematopoietic bone marrow (BM) microenvironment had been proposed two decades ago. Most of the RAS molecules including ACE, ACE2, AGT, AGTR1, AGTR2, AKR1C4, AKR1D1, ANPEP, ATP6AP2, CMA1, CPA3, CTSA, CTSD, CTSG, CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, DPP3, EGFR, ENPEP, GPER, HSD11B1, HSD11B2, IGF2R, KLK1, LNPEP, MAS1, MME, NR3C1, NR3C2, PREP, REN, RNPEP, and THOP1 are locally present in the BM microenvironment. Local BM RAS peptides control the hematopoietic niche, myelopoiesis, erythropoiesis, thrombopoiesis and the development of other cellular lineages. Local BM RAS is important in hematopoietic stem cell biology and microenvironment. Angiotensin II regulates the proliferation, differentiation, and engraftment of hematopoietic stem cells. Activation of Mas receptor or ACE2 promotes proliferation of CD34+ cells. BM contains a progenitor that expresses renin throughout development. Angiotensin II attenuates the migration and proliferation of CD34+ Cells and promotes the adhesion of both MNCs and CD34+ cells. Renin cells in hematopoietic organs are precursor B cells. The renin cell requires RBP-J to differentiate. Mutant renin-expressing hematopoietic precursors can cause leukemia. Deletion of RBP-J in the renin-expressing progenitors enriches the precursor B-cell gene programme. Mutant cells undergo a neoplastic transformation, and mice develop a highly penetrant B-cell leukemia with multi-organ infiltration and early death. Many biological conditions during the development and function of blood cells are mediated by RAS, such as apoptosis, cellular proliferation, intracellular signaling, mobilization, angiogenesis, and fibrosis. The aim of this paper is to review recent developments regarding the actions of local BM RAS in the genesis of leukemia and other malignancies molecules.

Affinity of antigen encounter and other early B-cell signals determine B-cell fate

PubMed Central

Benson, Micah J; Erickson, Loren D; Gleeson, Michael W; Noelle, Randolph J

2010-01-01

Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway. PMID:17433651

Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

PubMed

Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

2012-12-01

The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

Independent regulation of the two Pax5 alleles during B-cell development.

PubMed

Nutt, S L; Vambrie, S; Steinlein, P; Kozmik, Z; Rolink, A; Weith, A; Busslinger, M

1999-04-01

The developmental control genes of the Pax family are frequently associated with mouse mutants and human disease syndromes. The function of these transcription factors is sensitive to gene dosage, as mutation of one allele or a modest increase in gene number results in phenotypic abnormalities. Pax5 has an important role in B-cell and midbrain development. By following the expression of individual Pax5 alleles at the single-cell level, we demonstrate here that Pax5 is subject to allele-specific regulation during B-lymphopoiesis. Pax5 is predominantly transcribed from only one allele in early progenitors and mature B cells, whereas it switches to a biallelic transcription mode in immature B cells. The allele-specific regulation of Pax5 is stochastic, reversible, independent of parental origin and correlates with synchronous replication, in contrast with imprinted and other monoallelically expressed genes. As a consequence, B-lymphoid tissues are mosaics with respect to the transcribed Pax5 allele, and thus mutation of one allele in heterozygous mice results in deletion of the cell population expressing the mutant allele due to loss of Pax5 function at the single-cell level. Similar allele-specific regulation may be a common mechanism causing the haploinsufficiency and frequent association of other Pax genes with human disease.

Enhanced UV-B radiation during pupal stage reduce body mass and fat content, while increasing deformities, mortality and cell death in female adults of solitary bee Osmia bicornis.

PubMed

Wasielewski, Oskar; Wojciechowicz, Tatiana; Giejdasz, Karol; Krishnan, Natraj

2015-08-01

The effects of enhanced UV-B radiation on the oogenesis and morpho-anatomical characteristics of the European solitary red mason bee Osmia bicornis L. (Hymenoptera: Megachilidae) were tested under laboratory conditions. Cocooned females in the pupal stage were exposed directly to different doses (0, 9.24, 12.32, and 24.64 kJ/m(2) /d) of artificial UV-B. Our experiments revealed that enhanced UV-B radiation can reduce body mass and fat body content, cause deformities and increase mortality. Following UV exposure at all 3 different doses, the body mass of bees was all significantly reduced compared to the control, with the highest UV dose causing the largest reduction. Similarly, following UV-B radiation, in treated groups the fat body index decreased and the fat body index was the lowest in the group receiving the highest dose of UV radiation. Mortality and morphological deformities, between untreated and exposed females varied considerably and increased with the dose of UV-B radiation. Morphological deformities were mainly manifested in the wings and mouthparts, and occurred more frequently with an increased dose of UV. Cell death was quantified by the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (DNA fragmentation) during early stages of oogenesis of O. bicornis females. The bees, after UV-B exposure exhibited more germarium cells with fragmented DNA. The TUNEL test indicated that in germarium, low doses of UV-B poorly induced the cell death during early development. However, exposure to moderate UV-B dose increased programmed cell death. In females treated with the highest dose of UV-B the vast majority of germarium cells were TUNEL-positive. © 2014 Institute of Zoology, Chinese Academy of Sciences.

Generation of Bayesian prediction models for OATP-mediated drug-drug interactions based on inhibition screen of OATP1B1, OATP1B1∗15 and OATP1B3.

PubMed

van de Steeg, E; Venhorst, J; Jansen, H T; Nooijen, I H G; DeGroot, J; Wortelboer, H M; Vlaming, M L H

2015-04-05

Human organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are important hepatic uptake transporters. Early assessment of OATP1B1/1B3-mediated drug-drug interactions (DDIs) is therefore important for successful drug development. A promising approach for early screening and prediction of DDIs is computational modeling. In this study we aimed to generate a rapid, single Bayesian prediction model for OATP1B1, OATP1B1∗15 and OATP1B3 inhibition. Besides our previously generated HEK-OATP1B1 and HEK-OATP1B1∗15 cells, we now generated and characterized HEK-OATP1B3 cells. Using these cell lines we investigated the inhibitory potential of 640 FDA-approved drugs from a commercial library (10μM) on the uptake of [(3)H]-estradiol-17β-d-glucuronide (1μM) by OATP1B1, OATP1B1∗15, and OATP1B3. Using a cut-off of ⩾60% inhibition, 8% and 7% of the 640 drugs were potent OATP1B1 and OATP1B1∗15 inhibitors, respectively. Only 1% of the tested drugs significantly inhibited OATP1B3, which was not sufficient for Bayesian modeling. Modeling of OATP1B1 and OATP1B1∗15 inhibition revealed that presence of conjugated systems and (hetero)cycles with acceptor/donor atoms in- or outside the ring enhance the probability of a molecule binding these transporters. The overall performance of the model for OATP1B1 and OATP1B1∗15 was ⩾80%, including evaluation with a true external test set. Our Bayesian classification model thus represents a fast, inexpensive and robust means of assessing potential binding of new chemical entities to OATP1B1 and OATP1B1∗15. As such, this model may be used to rank compounds early in the drug development process, helping to avoid adverse effects in a later stage due to inhibition of OATP1B1 and/or OATP1B1∗15. Copyright © 2015 Elsevier B.V. All rights reserved.

Current status and perspectives of chimeric antigen receptor modified T cells for cancer treatment.

PubMed

Wang, Zhenguang; Guo, Yelei; Han, Weidong

2017-12-01

Chimeric antigen receptor (CAR) is a recombinant immunoreceptor combining an antibody-derived targeting fragment with signaling domains capable of activating cells, which endows T cells with the ability to recognize tumor-associated surface antigens independent of the expression of major histocompatibility complex (MHC) molecules. Recent early-phase clinical trials of CAR-modified T (CAR-T) cells for relapsed or refractory B cell malignancies have demonstrated promising results (that is, anti-CD19 CAR-T in B cell acute lymphoblastic leukemia (B-ALL)). Given this success, broadening the clinical experience of CAR-T cell therapy beyond hematological malignancies has been actively investigated. Here we discuss the basic design of CAR and review the clinical results from the studies of CAR-T cells in B cell leukemia and lymphoma, and several solid tumors. We additionally discuss the major challenges in the further development and strategies for increasing anti-tumor activity and safety, as well as for successful commercial translation.

A role for B cells in the development of T cell helper function in a malaria infection in mice

PubMed Central

Langhorne, Jean; Cross, Caroline; Seixas, Elsa; Li, Ching; von der Weid, Thierry

1998-01-01

B cell knockout mice are unable to clear a primary erythrocytic infection of Plasmodium chabaudi chabaudi. However, the early acute infection is controlled to some extent, giving rise to a chronic relapsing parasitemia that can be reduced either by drug treatment or by adoptive transfer of B cells. Similar to mice rendered B-cell deficient by lifelong treatment with anti-μ antibodies, B cell knockout mice (μMT) retain a predominant CD4+ Th1-like response to malarial antigens throughout a primary infection. This contrasts with the response seen in control C57BL/6 mice in which the CD4+ T-cell response has switched to that characteristic of Th2 cells at the later stages of infection, manifesting efficient help for specific antibodies in vitro and interleukin 4 production. Both chloroquine and adoptive transfer of immune B cells reduced parasite load. However, the adoptive transfer of B cells resulted in a Th2 response in recipient μMT mice, as indicated by a relative increase in the precursor frequency of helper cells for antibody production. These data support the idea that B cells play a role in the regulation of CD4+ T subset responses. PMID:9465085

Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression

PubMed Central

Sungalee, Stéphanie; Mamessier, Emilie; Morgado, Ester; Grégoire, Emilie; Brohawn, Philip Z.; Morehouse, Christopher A.; Jouve, Nathalie; Monvoisin, Céline; Menard, Cédric; Debroas, Guilhaume; Faroudi, Mustapha; Mechin, Violaine; Navarro, Jean-Marc; Drevet, Charlotte; Eberle, Franziska C.; Chasson, Lionel; Baudimont, Fannie; Mancini, Stéphane J.; Tellier, Julie; Picquenot, Jean-Michel; Kelly, Rachel; Vineis, Paolo; Ruminy, Philippe; Chetaille, Bruno; Jaffe, Elaine S.; Schiff, Claudine; Hardwigsen, Jean; Tice, David A.; Higgs, Brandon W.; Tarte, Karin; Nadel, Bertrand; Roulland, Sandrine

2014-01-01

It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)+ memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation–induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)+ precursors and shapes the systemic presentation of FL patients. PMID:25384217

Epitope-Specific Evolution of Human B Cell Responses to Borrelia burgdorferi VlsE Protein from Early to Late Stages of Lyme Disease.

PubMed

Jacek, Elzbieta; Tang, Kevin S; Komorowski, Lars; Ajamian, Mary; Probst, Christian; Stevenson, Brian; Wormser, Gary P; Marques, Adriana R; Alaedini, Armin

2016-02-01

Most immunogenic proteins of Borrelia burgdorferi, the causative agent of Lyme disease, are known or expected to contain multiple B cell epitopes. However, the kinetics of the development of human B cell responses toward the various epitopes of individual proteins during the course of Lyme disease has not been examined. Using the highly immunogenic VlsE as a model Ag, we investigated the evolution of humoral immune responses toward its immunodominant sequences in 90 patients with a range of early to late manifestations of Lyme disease. The results demonstrate the existence of asynchronous, independently developing, Ab responses against the two major immunogenic regions of the VlsE molecule in the human host. Despite their strong immunogenicity, the target epitopes were inaccessible to Abs on intact spirochetes, suggesting a lack of direct immunoprotective effect. These observations document the association of immune reactivity toward specific VlsE sequences with different phases of Lyme disease, demonstrating the potential use of detailed epitope mapping of Ags for staging of the infection, and offer insights regarding the pathogen's possible immune evasion mechanisms. Copyright © 2016 by The American Association of Immunologists, Inc.

TopBP1 deficiency impairs V(D)J recombination during lymphocyte development

PubMed Central

Kim, Jieun; Kyu Lee, Sung; Jeon, Yoon; Kim, Yehyun; Lee, Changjin; Ho Jeon, Sung; Shim, Jaegal; Kim, In-Hoo; Hong, Seokmann; Kim, Nayoung; Lee, Ho; Seong, Rho Hyun

2014-01-01

TopBP1 was initially identified as a topoisomerase II-β-binding protein and it plays roles in DNA replication and repair. We found that TopBP1 is expressed at high levels in lymphoid tissues and is essential for early lymphocyte development. Specific abrogation of TopBP1 expression resulted in transitional blocks during early lymphocyte development. These defects were, in major part, due to aberrant V(D)J rearrangements in pro-B cells, double-negative and double-positive thymocytes. We also show that TopBP1 was located at sites of V(D)J rearrangement. In TopBP1-deficient cells, γ-H2AX foci were found to be increased. In addition, greater amount of γ-H2AX product was precipitated from the regions where TopBP1 was localized than from controls, indicating that TopBP1 deficiency results in inefficient DNA double-strand break repair. The developmental defects were rescued by introducing functional TCR αβ transgenes. Our data demonstrate a novel role for TopBP1 as a crucial factor in V(D)J rearrangement during the development of B, T and iNKT cells. PMID:24442639

NASA Pilot and Researcher Prepare for a Solar Cell Calibration Flight

NASA Image and Video Library

1964-04-21

Pilot Earle Boyer and researcher Henry Brandhorst prepare for a solar cell calibration flight in a Martin B-57B Canberra at the National Aeronautics and Space Administration (NASA) Lewis Research Center. Lewis was in the early stages of decades-long energy conversion and space power research effort. Brandhorst, a member of the Chemistry and Energy Conversion Division, led a team of Lewis researchers in a quest to develop new power sources to sustain spacecraft in orbit. Solar cells proved to be an important source of energy, but researchers discovered that their behavior varied at different atmospheric levels. Their standardization and calibration were critical. Brandhorst initiated a standardized way to calibrate solar cells in the early 1960s using the B-57B aircraft. The pilots would take the aircraft up into the troposphere and open the solar cell to the sunlight. The aircraft would steadily descend while instruments recorded how much energy was being captured by the solar cell. From this data, Brandhorst could determine the estimated power for a particular solar cell at any altitude. Pilot Earle Boyer joined NASA Lewis in October 1962. He had flown Convair F-102 Delta Dagger fighters in the Air Force and served briefly in the National Guard before joining the Langley Research Center. Boyer was only at Langley a few months before he transferred to Cleveland. He flew the B-57B, a Convair F-106 Delta Dart, Gulfstream G-1 with an experimental turboprop, Learjet and many other aircraft over the next 32 years at Lewis.

The Membrane Skeleton Controls Diffusion Dynamics and Signaling through the B Cell Receptor

PubMed Central

Treanor, Bebhinn; Depoil, David; Gonzalez-Granja, Aitor; Barral, Patricia; Weber, Michele; Dushek, Omer; Bruckbauer, Andreas; Batista, Facundo D.

2010-01-01

Summary Early events of B cell activation after B cell receptor (BCR) triggering have been well characterized. However, little is known about the steady state of the BCR on the cell surface. Here, we simultaneously visualize single BCR particles and components of the membrane skeleton. We show that an ezrin- and actin-defined network influenced steady-state BCR diffusion by creating boundaries that restrict BCR diffusion. We identified the intracellular domain of Igβ as important in mediating this restriction in diffusion. Importantly, alteration of this network was sufficient to induce robust intracellular signaling and concomitant increase in BCR mobility. Moreover, by using B cells deficient in key signaling molecules, we show that this signaling was most probably initiated by the BCR. Thus, our results suggest the membrane skeleton plays a crucial function in controlling BCR dynamics and thereby signaling, in a way that could be important for understanding tonic signaling necessary for B cell development and survival. PMID:20171124

Buccal Cell Cytokeratin 14 Correlates with Multiple Blood Biomarkers of Alzheimer's Disease Risk.

PubMed

Leifert, Wayne R; Nguyen, Tori; Rembach, Alan; Martins, Ralph; Rainey-Smith, Stephanie; Masters, Colin L; Ames, David; Rowe, Christopher C; Macaulay, S Lance; François, Maxime; Fenech, Michael F

2015-01-01

Mild cognitive impairment (MCI) may reflect early stages of neurodegenerative disorders such as Alzheimer's disease (AD). Our hypothesis was that cytokeratin 14 (CK14) expression could be used with blood-based biomarkers such as homocysteine, vitamin B12, and folate to identify individuals with MCI or AD from the Australian Imaging, Biomarkers and Lifestyle (AIBL) flagship study of aging. Buccal cells from 54 individuals were analyzed by a newly developed method that is rapid, automated, and quantitative for buccal cell CK14 expression levels. CK14 was negatively correlated with plasma Mg²⁺ and LDL, while positively correlated with vitamin B12, red cell hematocrit/volume, and basophils in the MCI group and positively correlated with insulin and vitamin B12 in the AD group. The combined biomarker panel (CK14 expression, plasma vitamin B12, and homocysteine) was significantly lower in the MCI (p = 0.003) and AD (p = 0.0001) groups compared with controls. Receiver-operating characteristic curves yielded area under the curve (AUC) values of 0.829 for the MCI (p = 0.002) group and 0.856 for the AD (p = 0.0003) group. These complex associations of multiple related parameters highlight the differences between the MCI and AD cohorts and possibly an underlying metabolic pathology associated with the development of early memory impairment. The changes in buccal cell CK14 expression observed in this pilot study supports previous results suggesting the peripheral biomarkers and metabolic changes are not restricted to brain pathology alone in MCI and AD and could prove useful as a potential biomarker in identifying individuals with an increased risk of developing MCI and eventually AD.

THE RELATION BETWEEN DNA SYNTHESIS AND CHROMOSOME STRUCTURE AS RESOLVED BY X-RAY DAMAGE

PubMed Central

Evans, H. J.; Savage, J. R. K.

1963-01-01

Vicia faba root tip cells were treated for short periods with tritiated thymidine, either immediately before or after exposure of roots to x-rays, and autoradiograph preparations were analysed in an attempt to test the hypothesis that chromatid type (B') aberrations are induced only in those chromosome regions that have synthesized DNA prior to x-irradiation, whereas chromosome type (B'') aberrations are induced only in unduplicated chromosome regions. Studying the relation between presence or absence of label at loci involved in aberrations, in cells irradiated at different development stages, and the pattern of labelling in cells carrying both types of aberration leads to the conclusion that B'' aberrations are induced only in unreplicated chromosome regions. Following replication, only B' aberrations are induced, but these aberrations are also induced in chromosome regions preparing to incorporate DNA. It is suggested that the doubled response of the chromosome to x-rays prior to DNA incorporation might reflect a physical separation of replicating units prior to replication. The aberration yields in damaged cells which were irradiated in G 1 S, and early G 2 were in the ratio of 1.0:2.0:3.2. The data indicate that the increased yield of B' in early G 2 relative to S cells may be a consequence of changes in the spatial distribution of the chromosomes within the nucleus. PMID:14064107

Delayed transition to new cell fates during cellular reprogramming

PubMed Central

Cheng, Xianrui; Lyons, Deirdre C.; Socolar, Joshua E. S.; McClay, David R.

2014-01-01

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues. PMID:24780626

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program

PubMed Central

Hamey, Fiona K.; Errami, Youssef

2017-01-01

Differentiation of lineage-committed cells from multipotent progenitors requires the establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased toward overproduction of pro–B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell–programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumor formation. PMID:28899870

ECR-MAPK regulation in liver early development.

PubMed

Zhao, Xiu-Ju; Zhuo, Hexian

2014-01-01

Early growth is connected to a key link between embryonic development and aging. In this paper, liver gene expression profiles were assayed at postnatal day 22 and week 16 of age. Meanwhile another independent animal experiment and cell culture were carried out for validation. Significance analysis of microarrays, qPCR verification, drug induction/inhibition assays, and metabonomics indicated that alpha-2u globulin (extracellular region)-socs2 (-SH2-containing signals/receptor tyrosine kinases)-ppp2r2a/pik3c3 (MAPK signaling)-hsd3b5/cav2 (metabolism/organization) plays a vital role in early development. Taken together, early development of male rats is ECR and MAPK-mediated coordination of cancer-like growth and negative regulations. Our data represent the first comprehensive description of early individual development, which could be a valuable basis for understanding the functioning of the gene interaction network of infant development.

In vivo detection of peripherin-specific autoreactive B cells during type 1 diabetes pathogenesis1

PubMed Central

Garabatos, Nahir; Alvarez, Raimon; Carrillo, Jorge; Carrascal, Jorge; Izquierdo, Cristina; Chapman, Harold D.; Presa, Maximiliano; Mora, Conchi; Serreze, David V.; Verdaguer, Joan; Stratmann, Thomas

2014-01-01

Summary Autoreactive B cells are essential for the pathogenesis of type 1 diabetes. The genesis and dynamics of autoreactive B cells remain unknown. Here, we analyzed the immune response in the NOD mouse model to the neuronal protein peripherin (PRPH), a target antigen of islet-infiltrating B cells. PRPH autoreactive B cells recognized a single linear epitope of this protein, in contrast to the multiple epitope recognition commonly observed during autoreactive B cell responses. Autoantibodies to this epitope were also detected in the disease-resistant NOR and C57BL/6 strains. To specifically detect the accumulation of these B cells, we developed a novel approach, octameric peptide display, to follow the dynamics and localization of anti-PRPH B cell during disease progression. Before extended insulitis established, anti-PRPH B cells preferentially accumulated in the peritoneum. Anti-PRPH B cells were likewise detected in C57BL/6 mice, albeit at lower frequencies. As disease unfolded in NOD mice, anti-PRPH B cells invaded the islets and increased in number at the peritoneum of diabetic but not pre-diabetic mice. Isotype switched B cells were only detected in the peritoneum. Anti-PRPH B cells represent a heterogeneous population composed of both B1 and B2 subsets. In the spleen, anti-PRPH B cell were predominantly in the follicular subset. Therefore, anti-PRPH B cells represent a heterogeneous population that is generated early in life but proliferates as diabetes establishes. These findings on the temporal and spatial progression of autoreactive B cells should be relevant for our understanding of B cell function in diabetes pathogenesis. PMID:24610011

Development of Burkholderia mallei and pseudomallei vaccines.

PubMed

Silva, Ediane B; Dow, Steven W

2013-01-01

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-γ and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit vaccines have typically provided less robust immunity, but are safer to administer to a wider variety of people, including immune compromised individuals because they do not reactivate or cause disease. The challenges facing B. mallei and B. pseudomalllei vaccine development include identification of broadly protective antigens, design of efficient vaccine delivery and adjuvant systems, and a better understanding of the correlates of protection from both acute and chronic infection.

Development of Burkholderia mallei and pseudomallei vaccines

PubMed Central

Silva, Ediane B.; Dow, Steven W.

2013-01-01

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative bacteria that cause glanders and melioidosis, respectively. Inhalational infection with either organism can result in severe and rapidly fatal pneumonia. Inoculation by the oral and cutaneous routes can also produce infection. Chronic infection may develop after recovery from acute infection with both agents, and control of infection with antibiotics requires prolonged treatment. Symptoms for both meliodosis and glanders are non-specific, making diagnosis difficult. B. pseudomallei can be located in the environment, but in the host, B. mallei and B. psedomallei are intracellular organisms, and infection results in similar immune responses to both agents. Effective early innate immune responses are critical to controlling the early phase of the infection. Innate immune signaling molecules such as TLR, NOD, MyD88, and pro-inflammatory cytokines such as IFN-γ and TNF-α play key roles in regulating control of infection. Neutrophils and monocytes are critical cells in the early infection for both microorganisms. Both monocytes and macrophages are necessary for limiting dissemination of B. pseudomallei. In contrast, the role of adaptive immune responses in controlling Burkholderia infection is less well understood. However, T cell responses are critical for vaccine protection from Burkholderia infection. At present, effective vaccines for prevention of glanders or meliodosis have not been developed, although recently development of Burkholderia vaccines has received renewed attention. This review will summarize current and past approaches to develop B. mallei and B. pseudomalllei vaccines, with emphasis on immune mechanisms of protection and the challenges facing the field. At present, immunization with live attenuated bacteria provides the most effective and durable immunity, and it is important therefore to understand the immune correlates of protection induced by live attenuated vaccines. Subunit vaccines have typically provided less robust immunity, but are safer to administer to a wider variety of people, including immune compromised individuals because they do not reactivate or cause disease. The challenges facing B. mallei and B. pseudomalllei vaccine development include identification of broadly protective antigens, design of efficient vaccine delivery and adjuvant systems, and a better understanding of the correlates of protection from both acute and chronic infection. PMID:23508691

Dengue Virus Activates Polyreactive, Natural IgG B Cells after Primary and Secondary Infection

PubMed Central

Toh, Ying Xiu; Flamand, Marie; Devi, Shamala; Koh, Mickey B.; Hibberd, Martin L.; Ooi, Eng Eong; Low, Jenny G.; Leo, Yee Sin; Gu, Feng; Fink, Katja

2011-01-01

Background Dengue virus is transmitted by mosquitoes and has four serotypes. Cross-protection to other serotypes lasting for a few months is observed following infection with one serotype. There is evidence that low-affinity T and/or B cells from primary infections contribute to the severe syndromes often associated with secondary dengue infections. such pronounced immune-mediated enhancement suggests a dengue-specific pattern of immune cell activation. This study investigates the acute and early convalescent B cell response leading to the generation of cross-reactive and neutralizing antibodies following dengue infection. Methodology/Principal Findings We assayed blood samples taken from dengue patients with primary or secondary infection during acute disease and convalescence and compared them to samples from patients presenting with non-dengue related fever. Dengue induced massive early plasmablast formation, which correlated with the appearance of polyclonal, cross-reactive IgG for both primary and secondary infection. Surprisingly, the contribution of IgG to the neutralizing titer 4–7 days after fever onset was more than 50% even after primary infection. Conclusions/Significance Poly-reactive and virus serotype cross-reactive IgG are an important component of the innate response in humans during both primary and secondary dengue infection, and “innate specificities” seem to constitute part of the adaptive response in dengue. While of potential importance for protection during secondary infection, cross-reactive B cells will also compete with highly neutralizing B cells and possibly interfere with their development. PMID:22216280

Early Peritoneal Immune Response during Echinococcus granulosus Establishment Displays a Biphasic Behavior

PubMed Central

Mourglia-Ettlin, Gustavo; Marqués, Juan Martín; Chabalgoity, José Alejandro; Dematteis, Sylvia

2011-01-01

Background Cystic echinococcosis is a worldwide distributed helminth zoonosis caused by the larval stage of Echinococcus granulosus. Human secondary cystic echinococcosis is caused by dissemination of protoscoleces after accidental rupture of fertile cysts and is due to protoscoleces ability to develop into new metacestodes. In the experimental model of secondary cystic echinococcosis mice react against protoscoleces producing inefficient immune responses, allowing parasites to develop into cysts. Although the chronic phase of infection has been analyzed in depth, early immune responses at the site of infection establishment, e.g., peritoneal cavity, have not been well studied. Because during early stages of infection parasites are thought to be more susceptible to immune attack, this work focused on the study of cellular and molecular events triggered early in the peritoneal cavity of infected mice. Principal Findings Data obtained showed disparate behaviors among subpopulations within the peritoneal lymphoid compartment. Regarding B cells, there is an active molecular process of plasma cell differentiation accompanied by significant local production of specific IgM and IgG2b antibodies. In addition, peritoneal NK cells showed a rapid increase with a significant percentage of activated cells. Peritoneal T cells showed a substantial increase, with predominance in CD4+ T lymphocytes. There was also a local increase in Treg cells. Finally, cytokine response showed local biphasic kinetics: an early predominant induction of Th1-type cytokines (IFN-γ, IL-2 and IL-15), followed by a shift toward a Th2-type profile (IL-4, IL-5, IL-6, IL-10 and IL-13). Conclusions Results reported here open new ways to investigate the involvement of immune effectors players in E. granulosus establishment, and also in the sequential promotion of Th1- toward Th2-type responses in experimental secondary cystic echinococcosis. These data would be relevant for designing rational therapies based on stimulation of effective responses and blockade of evasion mechanisms. PMID:21912714

In Utero Exposure to Exosomal and B-Cell Alloantigens Lessens Alloreactivity of Recipients' Lymphocytes Rather than Confers Allograft Tolerance.

PubMed

Chen, Jeng-Chang; Ou, Liang-Shiou; Chan, Cheng-Chi; Kuo, Ming-Ling; Tseng, Li-Yun; Chang, Hsueh-Ling

2018-01-01

According to actively acquired tolerance, antigen exposure before full immune development in fetal or early neonatal life will cause tolerance to this specific antigen. In this study, we aimed to examine whether allogeneic tolerance could be elicited by in utero exposure to surface MHC antigens of allogenic cells or soluble form of MHC exosomes. Gestational day 14 FVB/N fetuses were subjected to intraperitoneal injection of allogeneic major histocompatibility complex (MHC) exosomes or highly enriched B-cells. Postnatally, the recipients were examined for the immune responses to donor alloantigens by lymphocyte proliferative reactions and skin transplantation. In utero exposure to allogeneic MHC exosomes abolished the alloreactivity of recipients' lymphocytes to the alloantigens, but could not confer skin allograft tolerance. In utero transplantation of highly enriched allogeneic B-cells generated low-level B-cell chimerism in the recipients. However, it only extended the survivals of skin allograft by a few days despite the lack of donor-specific alloreactivity of recipients' lymphocyte. Thus, an early in utero contact with exosomal or B-cell alloantigens did not lead to full skin tolerance but rather, at best, only to delayed skin rejection in the presence of microchimerism made by B-cell inocula. These results argued against the theory of actively acquired tolerance, and implicated that in utero exposure to marrow cells in previous studies was a unique model of allo-tolerance induction that involved the establishment of significant hematopoietic chimerism. Taken together with the discovery of in utero sensitization to ovalbumin in our previous studies, the immunological consequences of fetal exposure to foreign antigens might vary according to the type or nature of antigens introduced.

Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.

PubMed

Kochenderfer, James N; Rosenberg, Steven A

2013-05-01

Most B-cell malignancies express CD19, and a majority of patients with B-cell malignancies are not cured by current standard therapies. Chimeric antigen receptors (CARs) are fusion proteins consisting of antigen recognition moieties and T-cell activation domains. T cells can be genetically modified to express CARs, and adoptive transfer of anti-CD19 CAR T cells is now being tested in clinical trials. Effective clinical treatment with anti-CD19 CAR T cells was first reported in 2010 after a patient with advanced-stage lymphoma treated at the NCI experienced a partial remission of lymphoma and long-term eradication of normal B cells. Additional patients have subsequently obtained long-term remissions of advanced-stage B-cell malignancies after infusions of anti-CD19 CAR T cells. Long-term eradication of normal CD19(+) B cells from patients receiving infusions of anti-CD19 CAR T cells demonstrates the potent antigen-specific activity of these T cells. Some patients treated with anti-CD19 CAR T cells have experienced acute adverse effects, which were associated with increased levels of serum inflammatory cytokines. Although anti-CD19 CAR T cells are at an early stage of development, the potent antigen-specific activity observed in patients suggests that infusions of anti-CD19 CAR T cells might become a standard therapy for some B-cell malignancies.

Marek's disease virus infection of phagocytes: a de novo in vitro infection model.

PubMed

Chakraborty, Pankaj; Vervelde, Lonneke; Dalziel, Robert G; Wasson, Peter S; Nair, Venugopal; Dutia, Bernadette M; Kaiser, Pete

2017-05-01

Marek's disease virus (MDV) is an alphaherpesvirus that induces T-cell lymphomas in chickens. Natural infections in vivo are caused by the inhalation of infected poultry house dust and it is presumed that MDV infection is initiated in the macrophages from where the infection is passed to B cells and activated T cells. Virus can be detected in B and T cells and macrophages in vivo, and both B and T cells can be infected in vitro. However, attempts to infect macrophages in vitro have not been successful. The aim of this study was to develop a model for infecting phagocytes [macrophages and dendritic cells (DCs)] with MDV in vitro and to characterize the infected cells. Chicken bone marrow cells were cultured with chicken CSF-1 or chicken IL-4 and chicken CSF-2 for 4 days to produce macrophages and DCs, respectively, and then co-cultured with FACS-sorted chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing EGFP. Infected phagocytes were identified and sorted by FACS using EGFP expression and phagocyte-specific mAbs. Detection of MDV-specific transcripts of ICP4 (immediate early), pp38 (early), gB (late) and Meq by RT-PCR provided evidence for MDV replication in the infected phagocytes. Time-lapse confocal microscopy was also used to demonstrate MDV spread in these cells. Subsequent co-culture of infected macrophages with CEFs suggests that productive virus infection may occur in these cell types. This is the first report of in vitro infection of phagocytic cells by MDV.

Ultraviolet B irradiation induces expansion of intraepithelial tumor cells in a tissue model of early cancer progression.

PubMed

Mudgil, Adarsh V; Segal, Nadav; Andriani, Frank; Wang, Youai; Fusenig, Norbert E; Garlick, Jonathan A

2003-07-01

Ultraviolet B irradiation is thought to enable skin cancer progression as clones of genetically damaged keratinocytes escape apoptosis and expand at the expense of adjacent normal cells. Mechanisms through which potentially malignant cells in human skin undergo clonal expansion, however, are not well understood. The goal of this study was to characterize the role of ultraviolet B irradiation on the intraepithelial expansion of early stage human tumor cells in organotypic skin cultures. To accomplish this, we have studied the effect of ultraviolet B irradiation on organotypic cultures that were fabricated by mixing normal human keratinocytes with beta-galactosidase-marked, intraepithelial tumor cells (HaCaT-ras, clone II-4), which bear mutations in both p53 alleles and harbor an activated H-ras oncogene. We found that when organotypic mixtures were exposed to an ultraviolet B dose of 50 mJ per cm2, intraepithelial tumor cells underwent a significant degree of proliferative expansion compared to nonirradiated cultures. To understand this response, organotypic cultures of nor-mal keratinocytes were exposed to ultraviolet B and showed a dose-dependent increase in numbers of sunburn cells and TUNEL-positive cells although their proliferation was suppressed. In contrast, neither the apoptotic nor the proliferative response of II-4 cells was altered by ultraviolet B in organotypic cultures. The differential response of these cell types suggested that II-4 cells were resistant to ultraviolet-B-induced alterations, which allowed these intraepithelial tumor cells to gain a selective growth and survival advantage relative to neighboring normal cells. These findings demonstrate that ultraviolet B exposure can induce the intraepithelial expansion of apoptosis-resistant, p53-mutant, and ras-activated keratinocytes, suggesting that this agent can act to promote the early stages of epithelial carcinogenesis.

Development of the neonatal B and T cell repertoire in swine: implications for comparative and veterinary immunology.

PubMed

Butler, John E; Sinkora, Marek; Wertz, Nancy; Holtmeier, Wolfgang; Lemke, Caitlin D

2006-01-01

Birth in all higher vertebrates is at the center of the critical window of development in which newborns transition from dependence on innate immunity to dependence on their own adaptive immunity, with passive maternal immunity bridging this transition. Therefore we have studied immunological development through fetal and early neonatal life. In swine, B cells appear earlier in fetal development than T cells. B cell development begins in the yolk sac at the 20th day of gestation (DG20), progresses to fetal liver at DG30 and after DG45 continues in bone marrow. The first wave of developing T cells is gammadelta cells expressing a monomorphic Vdelta rearrangement. Thereafter, alphabeta T cells predominate and at birth, at least 19 TRBV subgroups are expressed, 17 of which appear highly homologous with those in humans. In contrast to the T cell repertoire and unlike humans and mice, the porcine pre-immune VH (IGHV-D-J) repertoire is highly restricted, depending primarily on CDR3 for diversity. The V-KAPPA (IGKV-J) repertoire and apparently also the V-LAMBDA (IGLV-J) repertoire, are also restricted. Diversification of the pre-immune B cell repertoire of swine and the ability to respond to both T-dependent and T-independent antigen depends on colonization of the gut after birth in which colonizing bacteria stimulate with Toll-like receptor ligands, especially bacterial DNA. This may explain the link between repertoire diversification and the anatomical location of primary lymphoid tissue like the ileal Peyers patches. Improper development of adaptive immunity can be caused by infectious agents like the porcine reproductive and respiratory syndrome virus that causes immune dysregulation resulting in immunological injury and autoimmunity.

Pediatric precursor B acute lymphoblastic leukemia: are T helper cells the missing link in the infectious etiology theory?

PubMed

Bürgler, Simone; Nadal, David

2017-12-01

Precursor B acute lymphoblastic leukemia (BCP-ALL), the most common childhood malignancy, arises from an expansion of malignant B cell precursors in the bone marrow. Epidemiological studies suggest that infections or immune responses to infections may promote such an expansion and thus BCP-ALL development. Nevertheless, a specific pathogen responsible for this process has not been identified. BCP-ALL cells critically depend on interactions with the bone marrow microenvironment. The bone marrow is also home to memory T helper (Th) cells that have previously expanded during an immune response in the periphery. In secondary lymphoid organs, Th cells can interact with malignant cells of mature B cell origin, while such interactions between Th cells and malignant immature B cell in the bone marrow have not been described yet. Nevertheless, literature supports a model where Th cells-expanded during an infection in early childhood-migrate to the bone marrow and support BCP-ALL cells as they support normal B cells. Further research is required to mechanistically confirm this model and to elucidate the interaction pathways between leukemia cells and cells of the tumor microenvironment. As benefit, targeting these interactions could be included in current treatment regimens to increase therapeutic efficiency and to reduce relapses.

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling.

PubMed

Povey, Jane F; O'Malley, Christopher J; Root, Tracy; Martin, Elaine B; Montague, Gary A; Feary, Marc; Trim, Carol; Lang, Dietmar A; Alldread, Richard; Racher, Andrew J; Smales, C Mark

2014-08-20

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements. Copyright © 2014 Elsevier B.V. All rights reserved.

Testicular receptor 2, Nr2c1, is associated with stem cells in the developing olfactory epithelium and other cranial sensory and skeletal structures.

PubMed

Baker, Jennifer L; Wood, Bernard; Karpinski, Beverly A; LaMantia, Anthony-S; Maynard, Thomas M

2016-01-01

Comparative genomic analysis of the nuclear receptor family suggests that the testicular receptor 2, Nr2c1, undergoes positive selection in the human-chimpanzee clade based upon a significant increase in nonsynonymous compared to synonymous substitutions. Previous in situ analyses of Nr2c1 lacked the temporal range and spatial resolution necessary to characterize cellular expression of this gene from early to mid gestation, when many nuclear receptors are key regulators of tissue specific stem or progenitor cells. Thus, we asked whether Nr2c1 protein is associated with stem cell populations in the mid-gestation mouse embryo. Nr2c1 is robustly expressed in the developing olfactory epithelium. Its expression in the olfactory epithelium shifts from multiple progenitor classes at early stages to primarily transit amplifying cells later in olfactory epithelium development. In the early developing central nervous system, Nr2c1 is limited to the anterior telencephalon/olfactory bulb anlagen, coincident with Nestin-positive neuroepithelial stem cells. Nr2c1 is also seen in additional cranial sensory specializations including cells surrounding the mystacial vibrissae, the retinal pigment epithelium and Scarpa's ganglion. Nr2c1 was also detected in a subset of mesenchymal cells in developing teeth and cranial bones. The timing and distribution of embryonic expression suggests that Nr2c1 is primarily associated with the early genesis of mammalian cranial sensory neurons and craniofacial skeletal structures. Thus, Nr2c1 may be a candidate for mediating parallel adaptive changes in cranial neural sensory specializations such as the olfactory epithelium, retina and mystacial vibrissae and in non-neural craniofacial features including teeth. Copyright © 2015 Elsevier B.V. All rights reserved.

MYCN Promotes the Expansion of Phox2B-Positive Neuronal Progenitors to Drive Neuroblastoma Development

PubMed Central

Alam, Goleeta; Cui, Hongjuan; Shi, Huilin; Yang, Liqun; Ding, Jane; Mao, Ling; Maltese, William A.; Ding, Han-Fei

2009-01-01

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B+ neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin+ cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B+ neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development. PMID:19608868

Environmental and T cell-intrinsic factors limit the expansion of neonatal follicular T helper cells but may be circumvented by specific adjuvants.

PubMed

Mastelic, Béatris; Kamath, Arun T; Fontannaz, Paola; Tougne, Chantal; Rochat, Anne-Françoise; Belnoue, Elodie; Combescure, Christophe; Auderset, Floriane; Lambert, Paul-Henri; Tacchini-Cottier, Fabienne; Siegrist, Claire-Anne

2012-12-15

Follicular Th (T(FH)) cells have emerged as a new Th subset providing help to B cells and supporting their differentiation into long-lived plasma cells or memory B cells. Their differentiation had not yet been investigated following neonatal immunization, which elicits delayed and limited germinal center (GC) responses. We demonstrate that neonatal immunization induces CXCR5(high)PD-1(high) CD4(+) T(FH) cells that exhibit T(FH) features (including Batf, Bcl6, c-Maf, ICOS, and IL-21 expression) and are able to migrate into the GCs. However, neonatal T(FH) cells fail to expand and to acquire a full-blown GC T(FH) phenotype, as reflected by a higher ratio of GC T(FH)/non-GC CD4(+) T cells in immunized adults than neonates (3.8 × 10(-3) versus 2.2 × 10(-3), p = 0.01). Following the adoptive transfer of naive adult OT-II CD4(+) T cells, OT-II T(FH) cells expand in the vaccine-draining lymph nodes of immunized adult but not infant recipients, whereas naive 2-wk-old CD4(+) OT-II cells failed to expand in adult hosts, reflecting the influence of both environmental and T cell-intrinsic factors. Postponing immunization to later in life increases the number of T(FH) cells in a stepwise manner, in direct correlation with the numbers of GC B cells and plasma cells elicited. Remarkably, adjuvantation with CpG oligonucleotides markedly increased GC T(FH) and GC B cell neonatal responses, up to adult levels. To our knowledge, this is the first demonstration that the T(FH) cell development limits early life GC responses and that adjuvants/delivery systems supporting T(FH) differentiation may restore adultlike early life GC B cell responses.

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

Discrete roles and bifurcation of PTEN signaling and mTORC1-mediated anabolic metabolism underlie IL-7–driven B lymphopoiesis

PubMed Central

Zeng, Hu; Yu, Mei; Tan, Haiyan; Li, Yuxin; Su, Wei; Shi, Hao; Dhungana, Yogesh; Guy, Cliff; Neale, Geoffrey; Cloer, Caryn; Peng, Junmin; Wang, Demin; Chi, Hongbo

2018-01-01

Interleukin-7 (IL-7) drives early B lymphopoiesis, but the underlying molecular circuits remain poorly understood, especially how Stat5 (signal transducer and activator of transcription 5)–dependent and Stat5-independent pathways contribute to this process. Combining transcriptome and proteome analyses and mouse genetic models, we show that IL-7 promotes anabolic metabolism and biosynthetic programs in pro-B cells. IL-7–mediated activation of mTORC1 (mechanistic target of rapamycin complex 1) supported cell proliferation and metabolism in a Stat5-independent, Myc-dependent manner but was largely dispensable for cell survival or Rag1 and Rag2 gene expression. mTORC1 was also required for Myc-driven lymphomagenesis. PI3K (phosphatidylinositol 3-kinase) and mTORC1 had discrete effects on Stat5 signaling and independently controlled B cell development. PI3K was actively suppressed by PTEN (phosphatase and tensin homolog) in pro-B cells to ensure proper IL-7R expression, Stat5 activation, heavy chain rearrangement, and cell survival, suggesting the unexpected bifurcation of the classical PI3K-mTOR signaling. Together, our integrative analyses establish IL-7R–mTORC1–Myc and PTEN-mediated PI3K suppression as discrete signaling axes driving B cell development, with differential effects on IL-7R–Stat5 signaling. PMID:29399633

TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.

PubMed

Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf

2017-06-01

Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

PubMed Central

Grigoryev, Yevgeniy A.; Kurian, Sunil M.; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L.; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M.; Kantor, Aaron B.; Marsh, Christopher; Salomon, Daniel R.

2010-01-01

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant. PMID:20976225

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

PubMed

Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

2010-10-14

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

Specific requirement of the chromatin modifier mSin3B in cell cycle exit and cellular differentiation

PubMed Central

David, Gregory; Grandinetti, Kathryn B.; Finnerty, Patricia M.; Simpson, Natalie; Chu, Gerald C.; DePinho, Ronald A.

2008-01-01

The Sin3-histone deacetylase (HDAC) corepressor complex is conserved from yeast to humans. Mammals possess two highly related Sin3 proteins, mSin3A and mSin3B, which serve as scaffolds tethering HDAC enzymatic activity, and numerous sequence-specific transcription factors to enable local chromatin regulation at specific gene targets. Despite broad overlapping expression of mSin3A and mSin3B, mSin3A is cell-essential and vital for early embryonic development. Here, genetic disruption of mSin3B reveals a very different phenotype characterized by the survival of cultured cells and lethality at late stages of embryonic development with defective differentiation of multiple lineages—phenotypes that are strikingly reminiscent of those associated with loss of retinoblastoma family members or E2F transcriptional repressors. Additionally, we observe that, whereas mSin3B−/− cells cycle normally under standard growth conditions, they show an impaired ability to exit the cell cycle with limiting growth factors. Correspondingly, mSin3B interacts physically with the promoters of known E2F target genes, and its deficiency is associated with derepression of these gene targets in vivo. Together, these results reveal a critical role for mSin3B in the control of cell cycle exit and terminal differentiation in mammals and establish contrasting roles for the mSin3 proteins in the growth and development of specific lineages. PMID:18332431

A parapoxviral virion protein inhibits NF-κB signaling early in infection

PubMed Central

Khatiwada, Sushil; Delhon, Gustavo; Nagendraprabhu, Ponnuraj; Chaulagain, Sabal; Luo, Shuhong; Diel, Diego G.; Flores, Eduardo F.

2017-01-01

Poxviruses have evolved unique proteins and mechanisms to counteract the nuclear factor κB (NF-κB) signaling pathway, which is an essential regulatory pathway of host innate immune responses. Here, we describe a NF-κB inhibitory virion protein of orf virus (ORFV), ORFV073, which functions very early in infected cells. Infection with ORFV073 gene deletion virus (OV-IA82Δ073) led to increased accumulation of NF-κB essential modulator (NEMO), marked phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, IκBα and NF-κB subunit p65 (NF-κB-p65), and to early nuclear translocation of NF-κB-p65 in virus-infected cells (≤ 30 min post infection). Expression of ORFV073 alone was sufficient to inhibit TNFα induced activation of the NF-κB signaling in uninfected cells. Consistent with observed inhibition of IKK complex activation, ORFV073 interacted with the regulatory subunit of the IKK complex NEMO. Infection of sheep with OV-IA82Δ073 led to virus attenuation, indicating that ORFV073 is a virulence determinant in the natural host. Notably, ORFV073 represents the first poxviral virion-associated NF-κB inhibitor described, highlighting the significance of viral inhibition of NF-κB signaling very early in infection. PMID:28787456

Kinetics of B cell responses to Plasmodium falciparum erythrocyte membrane protein 1 in Ghanaian women naturally exposed to malaria parasites.

PubMed

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F; Barfod, Lea; Hviid, Lars

2014-06-01

Naturally acquired protective immunity to Plasmodium falciparum malaria takes years to develop. It relies mainly on Abs, particularly IgG specific for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins on the infected erythrocyte surface. It is only partially understood why acquisition of clinical protection takes years to develop, but it probably involves a range of immune-evasive parasite features, not least of which are PfEMP1 polymorphism and clonal variation. Parasite-induced subversion of immunological memory and expansion of "atypical" memory B cells may also contribute. In this first, to our knowledge, longitudinal study of its kind, we measured B cell subset composition, as well as PfEMP1-specific Ab levels and memory B cell frequencies, in Ghanaian women followed from early pregnancy up to 1 y after delivery. Cell phenotypes and Ag-specific B cell function were assessed three times during and after pregnancy. Levels of IgG specific for pregnancy-restricted, VAR2CSA-type PfEMP1 increased markedly during pregnancy and declined after delivery, whereas IgG levels specific for two PfEMP1 proteins not restricted to pregnancy did not. Changes in VAR2CSA-specific memory B cell frequencies showed typical primary memory induction among primigravidae and recall expansion among multigravidae, followed by contraction postpartum in all. No systematic changes in the frequencies of memory B cells specific for the two other PfEMP1 proteins were identified. The B cell subset analysis confirmed earlier reports of high atypical memory B cell frequencies among residents of P. falciparum-endemic areas, and indicated an additional effect of pregnancy. Our study provides new knowledge regarding immunity to P. falciparum malaria and underpins efforts to develop PfEMP1-based vaccines against this disease. Copyright © 2014 by The American Association of Immunologists, Inc.

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development.

PubMed

Vogt, Edgar J; Meglicki, Maciej; Hartung, Kristina Ilka; Borsuk, Ewa; Behr, Rüdiger

2012-12-01

The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Novel Bruton’s tyrosine kinase inhibitors currently in development

PubMed Central

D’Cruz, Osmond J; Uckun, Fatih M

2013-01-01

Bruton’s tyrosine kinase (Btk) is intimately involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Btk is overexpressed and constitutively active in several B-lineage lymphoid malignancies. Btk has emerged as a new antiapoptotic molecular target for treatment of B-lineage leukemias and lymphomas. Preclinical and early clinical results indicate that Btk inhibitors may be useful in the treatment of leukemias and lymphomas. PMID:23493945

Comprehensive single cell-resolution analysis of the role of chromatin regulators in early C. elegans embryogenesis.

PubMed

Krüger, Angela V; Jelier, Rob; Dzyubachyk, Oleh; Zimmerman, Timo; Meijering, Erik; Lehner, Ben

2015-02-15

Chromatin regulators are widely expressed proteins with diverse roles in gene expression, nuclear organization, cell cycle regulation, pluripotency, physiology and development, and are frequently mutated in human diseases such as cancer. Their inhibition often results in pleiotropic effects that are difficult to study using conventional approaches. We have developed a semi-automated nuclear tracking algorithm to quantify the divisions, movements and positions of all nuclei during the early development of Caenorhabditis elegans and have used it to systematically study the effects of inhibiting chromatin regulators. The resulting high dimensional datasets revealed that inhibition of multiple regulators, including F55A3.3 (encoding FACT subunit SUPT16H), lin-53 (RBBP4/7), rba-1 (RBBP4/7), set-16 (MLL2/3), hda-1 (HDAC1/2), swsn-7 (ARID2), and let-526 (ARID1A/1B) affected cell cycle progression and caused chromosome segregation defects. In contrast, inhibition of cir-1 (CIR1) accelerated cell division timing in specific cells of the AB lineage. The inhibition of RNA polymerase II also accelerated these division timings, suggesting that normal gene expression is required to delay cell cycle progression in multiple lineages in the early embryo. Quantitative analyses of the dataset suggested the existence of at least two functionally distinct SWI/SNF chromatin remodeling complex activities in the early embryo, and identified a redundant requirement for the egl-27 and lin-40 MTA orthologs in the development of endoderm and mesoderm lineages. Moreover, our dataset also revealed a characteristic rearrangement of chromatin to the nuclear periphery upon the inhibition of multiple general regulators of gene expression. Our systematic, comprehensive and quantitative datasets illustrate the power of single cell-resolution quantitative tracking and high dimensional phenotyping to investigate gene function. Furthermore, the results provide an overview of the functions of essential chromatin regulators during the early development of an animal. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional Dicer Is Necessary for Appropriate Specification of Radial Glia during Early Development of Mouse Telencephalon

PubMed Central

Nowakowski, Tomasz Jan; Mysiak, Karolina Sandra; Pratt, Thomas; Price, David Jonathan

2011-01-01

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon. PMID:21826226

Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon.

PubMed

Nowakowski, Tomasz Jan; Mysiak, Karolina Sandra; Pratt, Thomas; Price, David Jonathan

2011-01-01

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1⁻/⁻ dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon.

An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

PubMed Central

Tugizov, Sharof; Maidji, Ekaterina; Xiao, Jianqiao; Pereira, Lenore

1999-01-01

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway. PMID:10482621

ADAM13 Induces Cranial Neural Crest by Cleaving Class B Ephrins and Regulating Wnt Signaling

PubMed Central

Wei, Shuo; Xu, Guofeng; Bridges, Lance C.; Williams, Phoebe; White, Judith M.; DeSimone, Douglas W.

2010-01-01

SUMMARY The cranial neural crest (CNC) are multipotent embryonic cells that contribute to craniofacial structures and other cells and tissues of the vertebrate head. During embryogenesis, CNC is induced at the neural plate boundary through the interplay of several major signaling pathways. Here we report that the metalloproteinase activity of ADAM13 is required for early induction of CNC in Xenopus. In both cultured cells and X. tropicalis embryos, membrane-bound Ephrins (Efns) B1 and B2 were identified as substrates for ADAM13. ADAM13 upregulates canonical Wnt signaling and early expression of the transcription factor snail2, whereas EfnB1 inhibits the canonical Wnt pathway and snail2 expression. We propose that by cleaving class B Efns, ADAM13 promotes canonical Wnt signaling and early CNC induction. PMID:20708595

Correlation of karyotype and immunophenotype in childhood acute lymphoblastic leukemia; experience at the National Cancer Institute, Cairo University, Egypt.

PubMed

Hamouda, Faiza; El-Sissy, Azza H; Radwan, Ashraf K; Hussein, Hany; Gadallah, Farida H; Al-Sharkawy, Nahla; Sedhom, Eman; Ebeid, Emad; Salem, Shereen I

2007-06-01

To identify chromosomal pattern among the major immunophenotypic subgroups in Egyptian children with ALL, and its correlation with clinical presentation and disease free survival. Cytogenetic and immunophenotypic analysis were done for all patients. Patients received ALL-PNCI-III/98 chemotherapy protocol used at NCI, Cairo University. The frequency of pseudodiploidy and normal karyotype in the whole group was 42.9% and 33.3% respectively. The frequency of pseudodiploidy was 36.8% in CALLA positive early pre B, 30.7% in pre B cases, 71.4% in T cell cases and 100% in mature B cell cases. At 12 months, DFS was 50% for pseudodiploid group having pre B phenotype, compared to 16.6% for pseudodiploid group with CALLA positive early pre B ALL. Sixteen percent of the studied cases showed T cell phenotype, 71.4% of them showed pseudodiploid karyotype, all of them had high risk features. Hyperdiploidy was found in 31.5% of CALLA positive early pre B cases and was associated with favorable prognostic features and DFS of 66.6% at 12 months. Hyperdiploidy of >50 chromosome represented 62.5% of hyperdipoid cases, 80% of them were CALLA positive early pre B ALL carrying good risk features. Fifty percent of normal karyotypic patients showed pre B phenotype, while 42.8% showed CALLA positive early pre B ALL. Their age, TLC, DFS, were almost comparable. CALLA early pre B phenotype has a positive impact on chromosomal pattern having best outcome among patients with hyperdiploidy. The Pseudodiploid karyotype carries a better outcome with pre B phenotype.

[Patient with B-CLL with a history of unrelated hematopoietic cells donation--retrospective analysis of CLL development and implication for the recipient].

PubMed

Jindra, P; Žejšková, L; Peková, S; Navrátilová, J; Schutzová, M; Vokurka, S; Koza, V

2012-01-01

Donor cell leukemia (DCL) is a relatively rare but well documented complication of hematopoietic stem cell transplantation. So far, publications described only DCL arising de novo in the recipient. In this study, we describe a case of chronic lymphocytic leukemia (B-CLL) developing in a volunteer unrelated donor from the Czech National Marrow Donors Registry (CNMDR) several years after donation. From archival DNA sample, we have retrospectively found that subclinical CLL clone was already present at the time of donation but early death of recipient prevented eventual development of DCL. This case documents well the long period between detection of B-CLL clone and full development of clinical-laboratory symptomatology. The medical and ethical questions posed by an isolated case of detection of hematological malignancy present either only in the donor or only in the recipient are discussed. The case demonstrates the increasing risk of development of various forms of DCL and thus highlights the need for long-term monitoring of stem cell donor, not only in terms of health of donor but also in terms of potential risks for the recipient.

Estrogen and Cytochrome P450 1B1 Contribute to Both Early- and Late-Stage Head and Neck Carcinogenesis

PubMed Central

Shatalova, Ekaterina G.; Klein-Szanto, Andres J.P.; Devarajan, Karthik; Cukierman, Edna; Clapper, Margie L.

2010-01-01

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common type of cancer in the U.S. The goal of this study was to evaluate the contribution of estrogens to the development of HNSCCs. Various cell lines derived from early- and late-stage head and neck lesions were used to: characterize the expression of estrogen synthesis and metabolism genes, including cytochrome P450 (CYP)1B1, examine the effect of estrogen on gene expression and evaluate the role of CYP1B1 and/or estrogen in cell motility, proliferation and apoptosis. Estrogen metabolism genes (CYP1B1, CYP1A1, catechol-o-methyltransferase, UDP-glucuronosyltransferase 1A1, and glutathione-S-transferase P1) and estrogen receptor (ER)β were expressed in cell lines derived from both premalignant (MSK-Leuk1) and malignant (HNSCC) lesions. Exposure to estrogen induced CYP1B1 2.3 to 3.6 fold relative to vehicle-treated controls (P=0.0004) in MSK-Leuk1 cells but not in HNSCC cells. CYP1B1 knockdown by shRNA reduced the migration and proliferation of MSK-Leuk1 cells by 57% and 45%, respectively. Exposure of MSK-Leuk1 cells to estrogen inhibited apoptosis by 26%, while supplementation with the antiestrogen fulvestrant restored estrogen-dependent apoptosis. Representation of the estrogen pathway in human head and neck tissues from 128 patients was examined using tissue microarrays. The majority of the samples exhibited immunohistochemical staining for ERβ (91.9%), CYP1B1 (99.4%) and 17β-estradiol (88.4%). CYP1B1 and ERβ were elevated in HNSCCs relative to normal epithelium (P=0.024 and 0.008, respectively). These data provide novel insight into the mechanisms underlying head and neck carcinogenesis and facilitate the identification new targets for chemopreventive intervention. PMID:21205741

Proteomic analysis on the alteration of protein expression in the early-stage placental villous tissue of electromagnetic fields associated with cell phone exposure.

PubMed

Luo, Qiong; Jiang, Ying; Jin, Min; Xu, Jian; Huang, He-Feng

2013-09-01

To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified- procollagen-proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

PubMed

Aït Ghezali, Lamia; Arbabian, Atousa; Roudot, Hervé; Brouland, Jean-Philippe; Baran-Marszak, Fanny; Salvaris, Evelyn; Boyd, Andrew; Drexler, Hans G; Enyedi, Agnes; Letestu, Remi; Varin-Blank, Nadine; Papp, Bela

2017-06-26

Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. We show that E2A-PBX1 + leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Immunohistochemical Markers of Neural Progenitor Cells in the Early Embryonic Human Cerebral Cortex

PubMed Central

Vinci, L.; Ravarino, A.; Fanos, V.; Naccarato, A.G.; Senes, G.; Gerosa, C.; Bevilacqua, G.; Faa, G.; Ambu, R.

2016-01-01

The development of the human central nervous system represents a delicate moment of embryogenesis. The purpose of this study was to analyze the expression of multiple immunohistochemical markers in the stem/progenitor cells in the human cerebral cortex during the early phases of development. To this end, samples from cerebral cortex were obtained from 4 human embryos of 11 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including GFAP, WT1, Nestin, Vimentin, CD117, S100B, Sox2, PAX2, PAX5, Tβ4, Neurofilament, CD44, CD133, Synaptophysin and Cyclin D1. Our study shows the ability of the different immunohistochemical markers to evidence different zones of the developing human cerebral cortex, allowing the identification of the multiple stages of differentiation of neuronal and glial precursors. Three important markers of radial glial cells are evidenced in this early gestational age: Vimentin, Nestin and WT1. Sox2 was expressed by the stem/progenitor cells of the ventricular zone, whereas the postmitotic neurons of the cortical plate were immunostained by PAX2 and NSE. Future studies are needed to test other important stem/progenitor cells markers and to better analyze differences in the immunohistochemical expression of these markers during gestation. PMID:26972711

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development.

PubMed

Webb, Sarah E; Miller, Andrew L

2006-11-01

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming pathways known to modulate early developmental events (such as the Wnt/Ca(2+)pathway; [T.A. Westfall, B. Hjertos, D.C. Slusarski, Requirement for intracellular calcium modulation in zebrafish dorsal-ventral patterning, Dev. Biol. 259 (2003) 380-391]).

Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development.

PubMed

Phillips, Bryan T; Kwon, Hye-Joo; Melton, Colt; Houghtaling, Paul; Fritz, Andreas; Riley, Bruce B

2006-06-15

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high-throughput sequencing.

PubMed

Xiong, X R; Lan, D L; Li, J; Zi, X D; Li, M Y

2016-12-01

Small RNA represents several unique non-coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II-arrested stage (M II) and then sequenced using small RNA high-throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR-342 has the largest fold change (9.25-fold). Six highly expressed miRNAs (let-7i, miR-10b, miR-10c, miR-143, miR-146b and miR-148) were validated by real-time quantitative PCR (RT-qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy-five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division. © 2016 Blackwell Verlag GmbH.

NF-κB dynamics show digital activation and analog information processing in cells

NASA Astrophysics Data System (ADS)

Tay, Savas; Hughey, Jake; Lee, Timothy; Lipniacki, Tomasz; Covert, Markus; Quake, Stephen

2010-03-01

Cells operate in ever changing environments using extraordinary communication capabilities. Cell-to-cell communication is mediated by signaling molecules that form spatiotemporal concentration gradients, which requires cells to respond to a wide range of signal intensities. We used high-throughput microfluidic cell culture, quantitative gene expression analysis and mathematical modeling to investigate how single mammalian cells respond to different concentrations of the signaling molecule TNF-α via the transcription factor NF-κB. We measured NF-κB activity in thousands of live cells under TNF-α doses covering four orders of magnitude. In contrast to population studies, the activation is a stochastic, switch-like process at the single cell level with fewer cells responding at lower doses. The activated cells respond fully and express early genes independent of the TNF-α concentration, while only high dose stimulation results in the expression of late genes. Cells also encode a set of analog parameters such as the NF-κB peak intensity, response time and number of oscillations to modulate the outcome. We developed a stochastic model that reproduces both the digital and analog dynamics as well as the gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-α induced NF-κB signaling in various types of cells.

c-Myc Represses Transcription of Epstein-Barr Virus Latent Membrane Protein 1 Early after Primary B Cell Infection.

PubMed

Price, Alexander M; Messinger, Joshua E; Luftig, Micah A

2018-01-15

Recent evidence has shown that the Epstein-Barr virus (EBV) oncogene LMP1 is not expressed at high levels early after EBV infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased 50-fold between 7 days postinfection and the LCL state. Metabolic labeling of nascent transcribed mRNA indicated that this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to that in infected B cells early after infection. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B cell infection directly repress LMP1 transcription. IMPORTANCE EBV is a highly successful pathogen that latently infects more than 90% of adults worldwide and is also causally associated with a number of B cell malignancies. During the latent life cycle, EBV expresses a set of viral oncoproteins and noncoding RNAs with the potential to promote cancer. Critical among these is the viral latent membrane protein LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here we show that transcription of the LMP1 gene can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV oncogenes will allow us to better treat cancers that rely on these viral products for survival. Copyright © 2018 American Society for Microbiology.

Interleukin-21 regulates expression of key Epstein-Barr virus oncoproteins, EBNA2 and LMP1, in infected human B cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Konforte, Danijela; Department of Immunology, University of Toronto, Toronto, M5S 1A8; Simard, Nathalie

Epstein-Barr virus (EBV) persists for the life of the host by accessing the long-lived memory B cell pool. It has been proposed that EBV uses different combinations of viral proteins, known as latency types, to drive infected B cells to make the transition from resting B cells to memory cells. This process is normally antigen-driven. A major unresolved question is what factors coordinate expression of EBV latency proteins. We have recently described novel type III latency EBV{sup +} B cell lines (OCI-BCLs) that were induced to differentiate into late plasmablasts/early plasma cells in culture with interleukin-21 (IL-21), mimicking normal Bmore » cell development. The objective of this study was to determine whether IL-21-mediated signals also regulate the expression of key EBV latent proteins during this window of development. Here we show that IL-21-reduced gene and protein expression of growth-transforming EBV nuclear antigen 2 (EBNA2) in OCI-BCLs. By contrast, the expression of CD40-like, latent membrane protein 1 (LMP1) strongly increased in these cells suggesting an EBNA2-independent mode of regulation. Same results were also observed in Burkitt's lymphoma line Jijoye and B95-8 transformed lymphoblastoid cell lines. The effect of IL-21 on EBNA2 and LMP1 expression was attenuated by a pharmacological JAK inhibitor indicating involvement of JAK/STAT signalling in this process. Our study also shows that IL-21 induced transcription of ebna1 from the viral Q promoter (Qp)« less

149 HCV AND lymphoma: Genetic and epigenetic factors

PubMed Central

Zignego, AL; Gragnani, L; Fognani, E; Piluso, A

2014-01-01

Over 180 million people worldwide are chronically infected with the hepatitis C virus (HCV). HCV infection is a major cause for hepatocellular carcinoma (HCC), moreover the association with B-cell lymphoproliferative disorders (LPDs) like mixed cryoglobulinemia (MC) or B-cell non-Hodgkin lymphoma (B-NHL) is undisputed. The mechanisms by which HCV contributes to LPD development are still poorly understood. Available data suggest that the viral infection may induce LPDs through a multifactorial and multistep process that involves the sustained activation of B cells, the abnormal and prolonged B cell survival, and genetic and/or epigenetic factors. Concerning genetic factors, different authors reported an association between specific HLA clusters or B-cell activating factor promoter genotype and a higher risk of developing MC and lymphoma. In addition, the results of a large, ongoing genome wide association study (GWAS) will probably allow the identification of specific genetic profile of HCV patients with LPDs. Furthermore, microRNAs (miRNAs) can give a major contribution to the pathogenesis of several neoplastic, lymphoproliferative diseases and it is conceivable their involvement in the pathogenesis of HCV-related LPDs. We recently showed that specific miRNAs were differently modulated in PBMCs from HCV patients who developed MC and/or NHL. In addition, HCV patients who developed HCC, showed a differential miRNAs regulation. In conclusion, available data suggest that the genetic/epigenetic analysis of HCV-related cancerogenesis is of great usefulness in both the pathogenetic and clinical/translational areas possibly allowing the definition of diagnostic/prognostic markers for early detection of lymphatic or hepatic cancer.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas.

PubMed

Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

2014-01-01

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season.

Testicular histology and germ cell cytology during spermatogenesis in the Mississippi map turtle, Graptemys pseudogeographica kohnii, from Northeast Arkansas

PubMed Central

Lancaster, Kelsey; Trauth, Stanley E; Gribbins, Kevin M

2014-01-01

The testicular histology and cytology of spermatogenesis in Graptemys pseudogeographica kohnii were examined using specimens collected between July 1996 and May 2004 from counties in northeastern Arkansas. A histological examination of the testes and germ cell cytology indicates a postnuptial testicular cycle of spermatogenesis and a major fall spermiation event. The majority of the germ cell populations in May and June specimens are represented by resting spermatogonia, type A spermatogonia, type B spermatogonia, pre-leptotene spermatocytes, and numerous Sertoli cell nuclei near the basement membrane. The start of proliferation is evident as spermatogonia in metaphase are present near the basal lamina and many of these germ cells have entered meiosis in June seminiferous tubules. Major spermatogenic events occur in the June and July specimens and result in an increased height of the seminiferous epithelium and increased diameter of the seminiferous tubules. The germ cell population during this time is represented by spermatogonia (type A, B, and resting), hypertrophic cells, large populations of early primary spermatocytes, and early round spermatids. By September, the major germ cell population has progressed past meiosis with abundant round and early elongating spermatids dominating the seminiferous epithelium. October seminiferous epithelia are marked by a decreas in height and mature spermatozoa fill the luminal space. Round and elongating spermatids constitute the largest portion of the germ cell population. Following the spermiation event, the testes enter a period of quiescence that lasts till the next spermatogenic cycle, which begins in the subsequent spring. Based on the cytological development of the seminiferous tubules revealed by our study, Graptemys pseudogeographica kohnii demonstrates a temporal germ cell development strategy similar to other temperate reptiles. A single major generation of germ cells progresses through spermatogenesis each year resulting in a single spermiation event with sperm stored within the epididymis until the next spring mating season. PMID:26413408

Cell wall α-1,3-glucan prevents α-amylase adsorption onto fungal cell in submerged culture of Aspergillus oryzae.

PubMed

Zhang, Silai; Sato, Hiroki; Ichinose, Sakurako; Tanaka, Mizuki; Miyazawa, Ken; Yoshimi, Akira; Abe, Keietsu; Shintani, Takahiro; Gomi, Katsuya

2017-07-01

We have previously reported that α-amylase (Taka-amylase A, TAA) activity disappears in the later stage of submerged Aspergillus oryzae culture as a result of TAA adsorption onto the cell wall. Chitin, one of the major components of the cell wall, was identified as a potential factor that facilitates TAA adsorption. However, TAA adsorption only occurred in the later stage of cultivation, although chitin was assumed to be sufficiently abundant in the cell wall regardless of the submerged culture period. This suggested the presence a factor that inhibits TAA adsorption to the cell wall in the early stage of cultivation. In the current study, we identified α-1,3-glucan as a potential inhibiting factor for TAA adsorption. We constructed single, double, and triple disruption mutants of three α-1,3-glucan synthase genes (agsA, agsB, and agsC) in A. oryzae. Growth characteristics and cell wall component analysis of these disruption strains showed that AgsB plays a major role in α-1,3-glucan synthesis. In the ΔagsB mutant, TAA was adsorbed onto the mycelium in all stages of cultivation (early and later), and the ΔagsB mutant cell walls had a significantly high capacity for TAA adsorption. Moreover, the α-1,3-glucan content of the cell wall prepared from the wild-type strain in the later stage of cultivation was markedly reduced compared with that in the early stage. These results suggest that α-1,3-glucan is a potential inhibiting factor for TAA adsorption onto the cell wall component, chitin, in the early stage of submerged culture in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Neurotrophins Acting Via TRKB Receptors Activate the JAGGED1-NOTCH2 Cell-Cell Communication Pathway to Facilitate Early Ovarian Development

PubMed Central

Dorfman, Mauricio D.; Kerr, Bredford; Garcia-Rudaz, Cecilia; Paredes, Alfonso H.; Dissen, Gregory A.

2011-01-01

Tropomyosin-related kinase (TRK) receptor B (TRKB) mediates the supportive actions of neurotrophin 4/5 and brain-derived neurotrophic factor on early ovarian follicle development. Absence of TRKB receptors reduces granulosa cell (GC) proliferation and delays follicle growth. In the present study, we offer mechanistic insights into this phenomenon. DNA array and quantitative PCR analysis of ovaries from TrkB-null mice revealed that by the end of the first week of postnatal life, Jagged1, Hes1, and Hey2 mRNA abundance is reduced in the absence of TRKB receptors. Although Jagged1 encodes a NOTCH receptor ligand, Hes1 and Hey2 are downstream targets of the JAGGED1-NOTCH2 signaling system. Jagged1 is predominantly expressed in oocytes, and the abundance of JAGGED1 is decreased in TrkB−/− oocytes. Lack of TRKB receptors also resulted in reduced expression of c-Myc, a NOTCH target gene that promotes entry into the cell cycle, but did not alter the expression of genes encoding core regulators of cell-cycle progression. Selective restoration of JAGGED1 synthesis in oocytes of TrkB−/− ovaries via lentiviral-mediated transfer of the Jagged1 gene under the control of the growth differentiation factor 9 (Gdf9) promoter rescued c-Myc expression, GC proliferation, and follicle growth. These results suggest that neurotrophins acting via TRKB receptors facilitate early follicle growth by supporting a JAGGED1-NOTCH2 oocyte-to-GC communication pathway, which promotes GC proliferation via a c-MYC-dependent mechanism. PMID:22028443

Hepatic dendritic cell subsets in the mouse.

PubMed

Jomantaite, Ieva; Dikopoulos, Nektarios; Kröger, Andrea; Leithäuser, Frank; Hauser, Hansjörg; Schirmbeck, Reinhold; Reimann, Jörg

2004-02-01

The CD11c(+) cell population in the non-parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c(+) DC population from immunocompetent or immunodeficient [recombinase-activating gene-1 (RAG1)(-/-)] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220(+) CD11c(int) subset of 'plasmacytoid' DC, and a B220(-) CD11c(+) DC subset. The latter DC population could be subdivided into a major, immature (CD40(lo) CD80(lo) CD86(lo) MHC class II(lo)) CD11c(int) subset, and a minor, mature (CD40(hi) CD80(hi) CD86(hi) MHC class II(hi)) CD11c(hi) subset. Stimulated B220(+) but not B220(-) DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220(-) DC three- to fourfold within 18 h post-injection, and up-regulated their surface expression of activation marker, while it contracted the B220(+) DC population. Early in virus infection, the hepatic B220(+) DC subset expanded, and both, the B220(+) as well as B220(-) DC populations in the liver matured. In vitro, B220(-) but not B220(+) DC primed CD4(+) or CD8(+)T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220(+) and B220(-) subsets in CD11c(+) DC populations freshly isolated from the mouse liver.

Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies.

PubMed

Bermejo, Daniela A; Amezcua Vesely, María C; Khan, Mahmood; Acosta Rodríguez, Eva V; Montes, Carolina L; Merino, Maria C; Toellner, Kai Michael; Mohr, Elodie; Taylor, Dale; Cunningham, Adam F; Gruppi, Adriana

2011-01-01

Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas' disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B-cell activation is associated with hypergammaglobulinaemia and delayed parasite-specific antibody response. In the present study we analysed the development of a B-cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post-infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138(+) B220(+) plasma cells in EF foci, red pulp and T-cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B-cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite-specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138(+) B220(+) cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B-cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.

MicroRNA miR-125b induces senescence in human melanoma cells.

PubMed

Glud, Martin; Manfé, Valentina; Biskup, Edyta; Holst, Line; Dirksen, Anne Marie Ahlburg; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T; Gniadecki, Robert

2011-06-01

MicroRNAs (miRNAs) are small noncoding RNA molecules involved in gene regulation. Aberrant expression of miRNA has been associated with the development or progression of several diseases, including cancer. In a previous study, we found that the expression of miRNA-125b (miR-125b) was two-fold lower in malignant melanoma producing lymph node micrometastases than in nonmetastasizing tumors. To get further insight into the functional role of miR-125b, we assessed whether its overexpression or silencing affects apoptosis, proliferation, or senescence in melanoma cell lines. We showed that overexpression of miR-125b induced typical senescent cell morphology, including increased cytoplasmatic/nucleus ratio and intensive cytoplasmatic β-galactosidase expression. In contrast, inhibition of miR-125b resulted in 30-35% decreased levels of spontaneous apoptosis. We propose that downregulation of miR-125b in an early cutaneous malignant melanoma can contribute to the increased metastatic capability of this tumor.

B-1 phagocytes: the myeloid face of B-1 cells.

PubMed

Popi, Ana Flavia

2015-12-01

The relationship between malignant B cells and macrophages has long been established. Furthermore, evolutionary studies have demonstrated that B cells from early vertebrates have both phagocytic and antibody production capabilities. In addition to their lymphoid nature, B-1 cells retain several myeloid characteristics. Various reports have demonstrated that B-1 cells can differentiate into phagocytes. However, descriptions of B-1 cells as a novel phagocyte cell member are rarely found in the literature. This review aims to present the available data regarding B-1 cell-derived phagocytes and also discusses how their existence might be relevant to hematopoiesis and immune responses. © 2015 New York Academy of Sciences.

B cells in the spotlight: innocent bystanders or major players in the pathogenesis of type 1 diabetes.

PubMed

Silveira, Pablo A; Grey, Shane T

2006-01-01

It has long been established that type 1 diabetes (T1D) is a T cell-mediated autoimmune disease, with CD4+ and CD8+ T cells being largely responsible for the destruction of beta cells within the pancreatic islets of Langerhans. Although autoantibodies specific for islet cell proteins are regularly detected in individuals with T1D and can be utilized as effective markers for predicting the onset of disease, they are not believed to be directly pathogenic to beta cells. Thus, activation of autoantibody-secreting B cells has long been regarded as a secondary consequence of the ongoing self-reactive T cell response. However, recently, studies in the nonobese diabetic mouse model of disease have demonstrated that B cells are an important component in the development of T1D by virtue of their ability to act as the preferential antigen presenting cell population required for efficient expansion of diabetogenic CD4+ T cells. Furthermore, autoantibodies might also be responsible for mediating early beta cell pathogenesis in this model.

Cellular and Molecular Level Responses After Radiofrequency Radiation Exposure, Alone or in Combination with X-rays or Chemicals

DTIC Science & Technology

1992-07-27

cell surface marker CD22 , which plays a role in early B-cell activation, is present within the cytoplasm of all B- cells, but expressed only on the...surface of a subpopulation of those cells. CD22 is an activation receptor associated with cell proliferation of small resting B-cells, and acts as an...adhesion molecule mediating the binding of B-cells to other hematopoietic cells (Stamenkovic & Seed, 1990). The CD22 surface glycoprotein is the putative

Rgs13 constrains early B cell responses and limits germinal center sizes.

PubMed

Hwang, Il-Young; Hwang, Kyung-Sun; Park, Chung; Harrison, Kathleen A; Kehrl, John H

2013-01-01

Germinal centers (GCs) are microanatomic structures that develop in secondary lymphoid organs in response to antigenic stimulation. Within GCs B cells clonally expand and their immunoglobulin genes undergo class switch recombination and somatic hypermutation. Transcriptional profiling has identified a number of genes that are prominently expressed in GC B cells. Among them is Rgs13, which encodes an RGS protein with a dual function. Its canonical function is to accelerate the intrinsic GTPase activity of heterotrimeric G-protein α subunits at the plasma membrane, thereby limiting heterotrimeric G-protein signaling. A unique, non-canonical function of RGS13 occurs following translocation to the nucleus, where it represses CREB transcriptional activity. The functional role of RGS13 in GC B cells is unknown. To create a surrogate marker for Rgs13 expression and a loss of function mutation, we inserted a GFP coding region into the Rgs13 genomic locus. Following immunization GFP expression rapidly increased in activated B cells, persisted in GC B cells, but declined in newly generated memory B and plasma cells. Intravital microscopy of the inguinal lymph node (LN) of immunized mice revealed the rapid appearance of GFP(+) cells at LN interfollicular regions and along the T/B cell borders, and eventually within GCs. Analysis of WT, knock-in, and mixed chimeric mice indicated that RGS13 constrains extra-follicular plasma cell generation, GC size, and GC B cell numbers. Analysis of select cell cycle and GC specific genes disclosed an aberrant gene expression profile in the Rgs13 deficient GC B cells. These results indicate that RGS13, likely acting at cell membranes and in nuclei, helps coordinate key decision points during the expansion and differentiation of naive B cells.

Activation of the Early B-Cell-Specific mb-1 (Ig-α) Gene by Pax-5 Is Dependent on an Unmethylated Ets Binding Site

PubMed Central

Maier, Holly; Colbert, Jeff; Fitzsimmons, Daniel; Clark, Dawn R.; Hagman, James

2003-01-01

Methylation of cytosine in CpG dinucleotides promotes transcriptional repression in mammals by blocking transcription factor binding and recruiting methyl-binding proteins that initiate chromatin remodeling. Here, we use a novel cell-based system to show that retrovirally expressed Pax-5 protein activates endogenous early B-cell-specific mb-1 genes in plasmacytoma cells, but only when the promoter is hypomethylated. CpG methylation does not directly affect binding of the promoter by Pax-5. Instead, methylation of an adjacent CpG interferes with assembly of ternary complexes comprising Pax-5 and Ets proteins. In electrophoretic mobility shift assays, recruitment of Ets-1 is blocked by methylation of the Ets site (5′CCGGAG) on the antisense strand. In transfection assays, selective methylation of a single CpG within the Pax-5-dependent Ets site greatly reduces mb-1 promoter activity. Prior demethylation of the endogenous mb-1 promoter is required for its activation by Pax-5 in transduced cells. Although B-lineage cells have only unmethylated mb-1 genes and do not modulate methylation of the mb-1 promoter during development, other tissues feature high percentages of methylated alleles. Together, these studies demonstrate a novel DNA methylation-dependent mechanism for regulating transcriptional activity through the inhibition of DNA-dependent protein-protein interactions. PMID:12612069

A Cell-Based Approach to Early Pancreatic Cancer Detection

DTIC Science & Technology

2016-10-01

Award Number: W81XWH-15-1-0457 TITLE: A Cell -Based Approach to Early Pancreatic Cancer Detection PRINCIPAL INVESTIGATOR: Dr. Ben Stanger...SUBTITLE 5a. CONTRACT NUMBER A Cell -Based Approach to Early Pancreatic Cancer Detection 5b. GRANT NUMBER W81XWH-15-1-0457 5c. PROGRAM ELEMENT NUMBER 6...tumor cells from mouse blood by depleting the sample of white blood cells (WBCs). Furthermore, the RNA profile of these cells can be assessed by

Preclinical Immunomodulation by the Probiotic Bifidobacterium breve M-16V in Early Life.

PubMed

Rigo-Adrover, Maria Del Mar; Franch, Àngels; Castell, Margarida; Pérez-Cano, Francisco José

2016-01-01

This study aimed to investigate the effect of supplementation with the probiotic Bifidobacterium breve M-16V on the maturation of the intestinal and circulating immune system during suckling. In order to achieve this purpose, neonatal Lewis rats were supplemented with the probiotic strain from the 6th to the 18th day of life. The animals were weighed during the study, and faecal samples were obtained and evaluated daily. On day 19, rats were euthanized and intestinal wash samples, mesenteric lymph node (MLN) cells, splenocytes and intraepithelial lymphocytes (IEL) were obtained. The probiotic supplementation in early life did not modify the growth curve and did not enhance the systemic immune maturation. However, it increased the proportion of cells bearing TLR4 in the MLN and IEL, and enhanced the percentage of the integrin αEβ7+ and CD62L+ cells in the MLN and that of the integrin αEβ7+ cells in the IEL, suggesting an enhancement of the homing process of naïve T lymphocytes to the MLN, and the retention of activated lymphocytes in the intraepithelial compartment. Interestingly, B. breve M-16V enhanced the intestinal IgA synthesis. In conclusion, supplementation with the probiotic strain B. breve M-16V during suckling improves the development of mucosal immunity in early life.

Zebrafish atoh1 genes: classic proneural activity in the inner ear and regulation by Fgf and Notch.

PubMed

Millimaki, Bonny B; Sweet, Elly M; Dhason, Mary S; Riley, Bruce B

2007-01-01

Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. We have addressed these issues in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development.

Acute and regressive scleroderma concomitant to an acute CMV primary infection.

PubMed

Goulabchand, Radjiv; Khellaf, Lakhdar; Forestier, Amandine; Costes, Valerie; Foulongne, Vincent; le Quellec, Alain; Guilpain, Philippe

2014-12-01

To describe the pathophysiological mechanisms involving cytomegalovirus (CMV) primary infection and natural killer (NK) cell expansion in the development of localized scleroderma. A 43-year-old woman presented acute erythematous discoloration and skin thickening concerning face, neck, trunk, abdomen, and the four limbs, predominantly in proximal areas. Our case did not respond to systemic sclerosis criteria diagnosis. However, skin and muscle biopsy revealed early scleroderma associated with capillary thrombi, and tissue infiltration with NK cells (CD56+/Granzyme B). Scleroderma was attributed to CMV primary infection responsible for cytolytic hepatitis (7-fold over the limit) and circulating NK cell excess. After 6 months of prednisone and a 2-year follow-up, a complete resolution of symptoms was observed. Our observation suggests a potential triggering role of CMV primary infection in the development of scleroderma. Histological features from our observation addresses the role of CMV and NK cells in the development of endothelial damage and fibrotic process. Copyright © 2014 Elsevier B.V. All rights reserved.

Six3 cooperates with Hedgehog signaling to specify ventral telencephalon by promoting early expression of Foxg1a and repressing Wnt signaling.

PubMed

Carlin, Dan; Sepich, Diane; Grover, Vandana K; Cooper, Michael K; Solnica-Krezel, Lilianna; Inbal, Adi

2012-07-01

Six3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/β-catenin pathway.

Six3 cooperates with Hedgehog signaling to specify ventral telencephalon by promoting early expression of Foxg1a and repressing Wnt signaling

PubMed Central

Carlin, Dan; Sepich, Diane; Grover, Vandana K.; Cooper, Michael K.; Solnica-Krezel, Lilianna; Inbal, Adi

2012-01-01

Six3 exerts multiple functions in the development of anterior neural tissue of vertebrate embryos. Whereas complete loss of Six3 function in the mouse results in failure of forebrain formation, its hypomorphic mutations in human and mouse can promote holoprosencephaly (HPE), a forebrain malformation that results, at least in part, from abnormal telencephalon development. However, the roles of Six3 in telencephalon patterning and differentiation are not well understood. To address the role of Six3 in telencephalon development, we analyzed zebrafish embryos deficient in two out of three Six3-related genes, six3b and six7, representing a partial loss of Six3 function. We found that telencephalon forms in six3b;six7-deficient embryos; however, ventral telencephalic domains are smaller and dorsal domains are larger. Decreased cell proliferation or excess apoptosis cannot account for the ventral deficiency. Instead, six3b and six7 are required during early segmentation for specification of ventral progenitors, similar to the role of Hedgehog (Hh) signaling in telencephalon development. Unlike in mice, we observe that Hh signaling is not disrupted in embryos with reduced Six3 function. Furthermore, six3b overexpression is sufficient to compensate for loss of Hh signaling in isl1- but not nkx2.1b-positive cells, suggesting a novel Hh-independent role for Six3 in telencephalon patterning. We further find that Six3 promotes ventral telencephalic fates through transient regulation of foxg1a expression and repression of the Wnt/β-catenin pathway. PMID:22736245

Macrophage-Mediated Glial Cell Elimination in the Postnatal Mouse Cochlea

PubMed Central

Brown, LaShardai N.; Xing, Yazhi; Noble, Kenyaria V.; Barth, Jeremy L.; Panganiban, Clarisse H.; Smythe, Nancy M.; Bridges, Mary C.; Zhu, Juhong; Lang, Hainan

2017-01-01

Hearing relies on the transmission of auditory information from sensory hair cells (HCs) to the brain through the auditory nerve. This relay of information requires HCs to be innervated by spiral ganglion neurons (SGNs) in an exclusive manner and SGNs to be ensheathed by myelinating and non-myelinating glial cells. In the developing auditory nerve, mistargeted SGN axons are retracted or pruned and excessive cells are cleared in a process referred to as nerve refinement. Whether auditory glial cells are eliminated during auditory nerve refinement is unknown. Using early postnatal mice of either sex, we show that glial cell numbers decrease after the first postnatal week, corresponding temporally with nerve refinement in the developing auditory nerve. Additionally, expression of immune-related genes was upregulated and macrophage numbers increase in a manner coinciding with the reduction of glial cell numbers. Transient depletion of macrophages during early auditory nerve development, using transgenic CD11bDTR/EGFP mice, resulted in the appearance of excessive glial cells. Macrophage depletion caused abnormalities in myelin formation and transient edema of the stria vascularis. Macrophage-depleted mice also showed auditory function impairment that partially recovered in adulthood. These findings demonstrate that macrophages contribute to the regulation of glial cell number during postnatal development of the cochlea and that glial cells play a critical role in hearing onset and auditory nerve maturation. PMID:29375297

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth.

PubMed

Radchuk, Volodymyr; Weier, Diana; Radchuk, Ruslana; Weschke, Winfriede; Weber, Hans

2011-01-01

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.

Centrosome Hypertrophy Induced by p53 Mutations Leads to Tumor Aneuploidy

DTIC Science & Technology

2001-06-01

and Early Development (Palazzo, RE, ed). Academic Press. In Press. Manuscripts Submitted 1. Antonino B. D’Assoro, Susan L. Barrett, Christopher Folk...cytoskeleton of the tumor cells. A A Lingle DAMD17-98-1-8122 10 APPENDICES Appendix 1 - Manuscripts Submitted 1. Antonino B. D’Assoro, Susan L. Barrett...Aggressive Phenotypes In Breast Cancer Antonino B. D’Assorol, Susan L. Barrett’, Christopher Folk’, Vivian C. Negron’, Kelly Boeneman’, Robert Busby’, Clark

Early suppression of adipocyte lipid turnover induces immunometabolic modulation in cancer cachexia syndrome.

PubMed

Henriques, Felipe Santos; Sertié, Rogério Antônio Laurato; Franco, Felipe Oliveira; Knobl, Pamela; Neves, Rodrigo Xavier; Andreotti, Sandra; Lima, Fabio Bessa; Guilherme, Adilson; Seelaender, Marilia; Batista, Miguel Luiz

2017-05-01

Cancer cachexia is a multifactorial syndrome characterized by body weight loss, atrophy of adipose tissue (AT) and systemic inflammation. However, there is limited information regarding the mechanisms of immunometabolic response in AT from cancer cachexia. Male Wistar rats were inoculated with 2 × 10 7 of Walker 256 tumor cells [tumor bearing (TB) rats]. The mesenteric AT (MeAT) was collected on d 0, 4, 7 (early stage), and 14 (cachexia stage) after tumor cell injection. Surgical biopsies for MeAT were obtained from patients who had gastrointestinal cancer with cachexia. Lipolysis showed an early decrease in glycerol release in TB d 4 (TB4) rats in relation to the control, followed by a 6-fold increase in TB14 rats, whereas de novo lipogenesis was markedly lower in the incorporation of glucose into fatty acids in TB14 rats during the development of cachexia. CD11b and CD68 were positive in TB7 and TB14 rats, respectively. In addition, we found cachexia stage results similar to those of animals in MeAT from patients: an increased presence of CD68 + , iNOS2 + , TNFα + , and HSL + cells. In summary, translational analysis of MeAT from patients and an animal model of cancer cachexia enabled us to identify early disruption in Adl turnover and subsequent inflammatory response during the development of cancer cachexia.-Henriques, F. S., Sertié, R. A. L., Franco, F. O., Knobl, P., Neves, R. X., Andreotti, S., Lima, F. B., Guilherme, A., Seelaender, M., Batista, M. L., Jr. Early suppression of adipocyte lipid turnover induces immunometabolic modulation in cancer cachexia syndrome. © FASEB.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

PubMed

Ebert, Allison D; Shelley, Brandon C; Hurley, Amanda M; Onorati, Marco; Castiglioni, Valentina; Patitucci, Teresa N; Svendsen, Soshana P; Mattis, Virginia B; McGivern, Jered V; Schwab, Andrew J; Sareen, Dhruv; Kim, Ho Won; Cattaneo, Elena; Svendsen, Clive N

2013-05-01

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology. Copyright © 2013 Elsevier B.V. All rights reserved.

Emerging pulmonary vasculature lacks fate specification.

PubMed

Schwarz, Margaret A; Caldwell, Lauren; Cafasso, Danielle; Zheng, Haihua

2009-01-01

Lung morphogenesis requires precise coordination between branching morphogenesis and vascularization to generate distal airways capable of supporting respiration at the cell-cell interface. The specific origins and types of blood vessels that initially form in the lung, however, remain obscure. Herein, we definitively show that during the early phases of lung development [i.e., embryonic day (E) 11.5], functional vessels, replete with blood flow, are restricted to the mesenchyme, distal to the epithelium. However, by day E14.5, and in response to epithelial-derived VEGF signals, functional vessels extend from the mesenchyme to the epithelial interface. Moreover, these vessels reside adjacent to multipotent mesenchymal stromal cells that likely play a regulatory role in this process. As well as and distinct from the systemic vasculature, immunostaining for EphrinB2 and EphB4 revealed that arterial and venous identity is not distinguishable in emergent pulmonary vasculature. Collectively, this study provides evidence that lung vascularization initially originates in the mesenchyme, distal to the epithelium, and that arterial-venous specification does not exist in the early lung. At a mechanistic level, we show that basilar epithelial VEGF prompts endothelial cells to move toward the epithelium where they undergo morphogenesis during the proliferative, canalicular stage. Thus our findings challenge existing notions of vascular origin and identity during development.

How the early sporulation sigma factor sigmaF delays the switch to late development in Bacillus subtilis.

PubMed

Karmazyn-Campelli, Céline; Rhayat, Lamya; Carballido-López, Rut; Duperrier, Sandra; Frandsen, Niels; Stragier, Patrick

2008-03-01

Sporulation in Bacillus subtilis is a primitive differentiation process involving two cell types, the forespore and the mother cell. Each cell implements two successive transcription programmes controlled by specific sigma factors. We report that activity of sigma(G), the late forespore sigma factor, is kept in check by Gin, the product of csfB, a gene controlled by sigma(F), the early forespore sigma factor. Gin abolishes sigma(G) transcriptional activity when sigma(G) is artificially synthesized during growth, but has no effect on sigma(F). Gin interacts strongly with sigma(G) but not with sigma(F) in a yeast two-hybrid experiment. The absence of Gin allows sigma(G) to be active during sporulation independently of the mother-cell development to which it is normally coupled. Premature sigma(G) activity leads to the formation of slow-germinating spores, and complete deregulation of sigma(G) synthesis is lethal when combined with gin inactivation. Gin allows sigma(F) to delay the switch to the late forespore transcription programme by preventing sigma(G) to take over before the cell has reached a critical stage of development. A similar strategy, following a completely unrelated route, is used by the mother cell.

Early detection of disease program: Evaluation of the cellular immune response

NASA Technical Reports Server (NTRS)

Criswell, B. S.; Knight, V.; Martin, R. R.; Kasel, J. A.

1974-01-01

The early cellular responses of specific components of the leukocyte and epithelial cell populations to foreign challenges of both an infectious and noninfectious character were evaluated. Procedures for screening potential flight crews were developed, documented, and tested on a control population. Methods for preparing suitable populations of lymphocytes, polymorphonuclear leukocytes, macrophages, and epithelial cells were first established and evaluated. Epithelial cells from viral infected individuals were screened with a number of anti-viral antisera. This procedure showed the earliest indication of disease as well as providing a specific diagnosis to the physicians. Both macrophages and polymorphonuclear leukocytes were studied from normal individuals, smokers, and patients with viral infections. Newer techniques enabling better definition of lymphocyte subpopulations were then developed, namely the E and EAC rosette procedures for recognition of T (thymus-derived) and B (bone-marrow-derived) lymphocyte subpopulations. Lymphocyte and lymphocyte subpopulation response to multiple mitogens have been evaluated.

Provisional matrix: A role for versican and hyaluronan.

PubMed

Wight, Thomas N

2017-07-01

Hyaluronan and versican are extracellular matrix (ECM) components that are enriched in the provisional matrices that form during the early stages of development and disease. These two molecules interact to create pericellular "coats" and "open space" that facilitate cell sorting, proliferation, migration, and survival. Such complexes also impact the recruitment of leukocytes during development and in the early stages of disease. Once thought to be inert components of the ECM that help hold cells together, it is now quite clear that they play important roles in controlling cell phenotype, shaping tissue response to injury and maintaining tissue homeostasis. Conversion of hyaluronan-/versican-enriched provisional matrix to collagen-rich matrix is a "hallmark" of tissue fibrosis. Targeting the hyaluronan and versican content of provisional matrices in a variety of diseases including, cardiovascular disease and cancer, is becoming an attractive strategy for intervention. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased proliferation of late-born retinal progenitor cells by gestational lead exposure delays rod and bipolar cell differentiation.

PubMed

Chaney, Shawnta Y; Mukherjee, Shradha; Giddabasappa, Anand; Rueda, Elda M; Hamilton, W Ryan; Johnson, Jerry E; Fox, Donald A

2016-01-01

Studies of neuronal development in the retina often examine the stages of proliferation, differentiation, and synaptic development, albeit independently. Our goal was to determine if a known neurotoxicant insult to a population of retinal progenitor cells (RPCs) would affect their eventual differentiation and synaptic development. To that end, we used our previously published human equivalent murine model of low-level gestational lead exposure (GLE). Children and animals with GLE exhibit increased scotopic electroretinogram a- and b-waves. Adult mice with GLE exhibit an increased number of late-born RPCs, a prolonged period of RPC proliferation, and an increased number of late-born rod photoreceptors and rod and cone bipolar cells (BCs), with no change in the number of late-born Müller glial cells or early-born neurons. The specific aims of this study were to determine whether increased and prolonged RPC proliferation alters the spatiotemporal differentiation and synaptic development of rods and BCs in early postnatal GLE retinas compared to control retinas. C57BL/6N mouse pups were exposed to lead acetate via drinking water throughout gestation and until postnatal day 10, which is equivalent to the human gestation period for retinal neurogenesis. RT-qPCR, immunohistochemical analysis, and western blots of well-characterized, cell-specific genes and proteins were performed at embryonic and early postnatal ages to assess rod and cone photoreceptor differentiation, rod and BC differentiation and synaptic development, and Müller glial cell differentiation. Real-time quantitative PCR (RT-qPCR) with the rod-specific transcription factors Nrl , Nr2e3 , and Crx and the rod-specific functional gene Rho , along with central retinal confocal studies with anti-recoverin and anti-rhodopsin antibodies, revealed a two-day delay in the differentiation of rod photoreceptors in GLE retinas. Rhodopsin immunoblots supported this conclusion. No changes in glutamine synthetase gene or protein expression, a marker for late-born Müller glial cells, were observed in the developing retinas. In the retinas from the GLE mice, anti-PKCα, - Chx10 (Vsx2) and -secretagogin antibodies revealed a two- to three-day delay in the differentiation of rod and cone BCs, whereas the expression of the proneural and BC genes Otx2 and Chx10 , respectively, increased. In addition, confocal studies of proteins associated with functional synapses (e.g., vesicular glutamate transporter 1 [VGluT1], plasma membrane calcium ATPase [PMCA], transient receptor potential channel M1 [TRPM1], and synaptic vesicle glycoprotein 2B [SV2B]) revealed a two-day delay in the formation of the outer and inner plexiform layers of the GLE retinas. Moreover, several markers revealed that the initiation of the differentiation and intensity of the labeling of early-born cells in the retinal ganglion cell and inner plexiform layers were not different in the control retinas. Our combined gene, confocal, and immunoblot findings revealed that the onset of rod and BC differentiation and their subsequent synaptic development is delayed by two to three days in GLE retinas. These results suggest that perturbations during the early proliferative stages of late-born RPCs fated to be rods and BCs ultimately alter the coordinated time-dependent progression of rod and BC differentiation and synaptic development. These GLE effects were selective for late-born neurons. Although the molecular mechanisms are unknown, alterations in soluble neurotrophic factors and/or their receptors are likely to play a role. Since neurodevelopmental delays and altered synaptic connectivity are associated with neuropsychiatric and behavioral disorders as well as cognitive deficits, future work is needed to determine if similar effects occur in the brains of GLE mice and whether children with GLE experience similar delays in retinal and brain neuronal differentiation and synaptic development.

Functional capacities of human IgM memory B cells in early inflammatory responses and secondary germinal center reactions.

PubMed

Seifert, Marc; Przekopowitz, Martina; Taudien, Sarah; Lollies, Anna; Ronge, Viola; Drees, Britta; Lindemann, Monika; Hillen, Uwe; Engler, Harald; Singer, Bernhard B; Küppers, Ralf

2015-02-10

The generation and functions of human peripheral blood (PB) IgM(+)IgD(+)CD27(+) B lymphocytes with somatically mutated IgV genes are controversially discussed. We determined their differential gene expression to naive B cells and to IgM-only and IgG(+) memory B cells. This analysis revealed a high similarity of IgM(+)(IgD(+))CD27(+) and IgG(+) memory B cells but also pointed at distinct functional capacities of both subsets. In vitro analyses revealed a tendency of activated IgM(+)IgD(+)CD27(+) B cells to migrate to B-cell follicles and undergo germinal center (GC) B-cell differentiation, whereas activated IgG(+) memory B cells preferentially showed a plasma cell (PC) fate. This observation was supported by reverse regulation of B-cell lymphoma 6 and PR domain containing 1 and differential BTB and CNC homology 1, basic leucine zipper transcription factor 2 expression. Moreover, IgM(+)IgD(+)CD27(+) B lymphocytes preferentially responded to neutrophil-derived cytokines. Costimulation with catecholamines, carcinoembryonic antigen cell adhesion molecule 8 (CEACAM8), and IFN-γ caused differentiation of IgM(+)IgD(+)CD27(+) B cells into PCs, induced class switching to IgG2, and was reproducible in cocultures with neutrophils. In conclusion, this study substantiates memory B-cell characteristics of human IgM(+)IgD(+)CD27(+) B cells in that they share typical memory B-cell transcription patterns with IgG(+) post-GC B cells and show a faster and more vigorous restimulation potential, a hallmark of immune memory. Moreover, this work reveals a functional plasticity of human IgM memory B cells by showing their propensity to undergo secondary GC reactions upon reactivation, but also by their special role in early inflammation via interaction with immunomodulatory neutrophils.

Maternal Serum B-Cell Activating Factor Levels: Candidate Early Biomarker for Hypertensive Disorders of Pregnancy.

PubMed

Stohl, Hindi E; Lee, Richard H; Manetta, Joseph; Kikly, Kristine; Korst, Lisa M; Stohl, William

2017-11-01

Hypertensive disorders of pregnancy are a leading cause of maternal and perinatal morbidity and mortality. Early suppression of B-cell lymphopoiesis is necessary for a normal pregnancy. Dysregulation of factors critical to B-cell survival may result in pregnancy complications, including hypertension. In this prospective observational study at a single medical center, serum levels of BAFF (B-cell activating factor) were measured in pregnant participants at each trimester, at delivery, and postpartum and in nonpregnant controls at a single time point. Comparisons were made between nonpregnant and pregnant subjects and between time periods of pregnancy. First-trimester serum BAFF levels were further tested for association with hypertensive disorders of pregnancy. The study included 149 healthy pregnant women, 25 pregnant women with chronic hypertension, and 48 nonpregnant controls. Median first-trimester serum BAFF level (ng/mL) for healthy women (0.90) was lower than median serum BAFF levels for women with chronic hypertension (0.96; P =0.013) and controls (1.00; P =0.002). Serum BAFF levels steadily declined throughout pregnancy, with the median second-trimester level lower than the corresponding first-trimester level (0.77; P =0.003) and the median third-trimester level lower than the corresponding second-trimester level (0.72; P =0.025). The median first-trimester serum BAFF level was elevated in women who subsequently developed hypertension compared with women who remained normotensive (1.02 versus 0.85; P =0.012), with the area under the receiver operating characteristic curve being 0.709. First-trimester serum BAFF level may be an early and clinically useful predictor of hypertensive disorders of pregnancy. © 2017 American Heart Association, Inc.

A c-Myc and surface CD19 signaling amplification loop promotes B cell lymphoma development and progression in mice.

PubMed

Poe, Jonathan C; Minard-Colin, Veronique; Kountikov, Evgueni I; Haas, Karen M; Tedder, Thomas F

2012-09-01

Malignant B cells responding to external stimuli are likely to gain a growth advantage in vivo. These cells may therefore maintain surface CD19 expression to amplify transmembrane signals and promote their expansion and survival. To determine whether CD19 expression influences this process, Eμ-Myc transgenic (c-Myc(Tg)) mice that develop aggressive and lethal B cell lymphomas were made CD19 deficient (c-Myc(Tg)CD19⁻/⁻). Compared with c-Myc(Tg) and c-Myc(Tg)CD19⁺/⁻ littermates, the median life span of c-Myc(Tg)CD19⁻/⁻ mice was prolonged by 81-83% (p < 0.0001). c-Myc(Tg)CD19⁻/⁻ mice also lived 42% longer than c-Myc(Tg) littermates following lymphoma detection (p < 0.01). Tumor cells in c-Myc(Tg) and c-Myc(Tg)CD19⁻/⁻ mice were B lineage derived, had a similar phenotype with a large blastlike appearance, invaded multiple lymphoid tissues, and were lethal when adoptively transferred into normal recipient mice. Importantly, reduced lymphomagenesis in c-Myc(Tg)CD19⁻/⁻ mice was not due to reductions in early B cell numbers prior to disease onset. In mechanistic studies, constitutive c-Myc expression enhanced CD19 expression and phosphorylation on active sites. Reciprocally, CD19 expression in c-Myc(Tg) B cells enhanced c-Myc phosphorylation at regulatory sites, sustained higher c-Myc protein levels, and maintained a balance of cyclin D2 expression over that of cyclin D3. These findings define a new and novel c-Myc:CD19 regulatory loop that positively influences B cell transformation and lymphoma progression.

Years in Cologne.

PubMed

Rajewsky, Klaus

2013-01-01

This review describes the building and scientific activity of the Immunology Department at the Institute for Genetics in Cologne, cofounded by Max Delbrück in post-World War II Germany. The protagonist, a child of Russian emigrants, became interested in antibodies as a postdoc at the Pasteur Institute in Paris and a proponent of the antigen-bridge model of T-B cell collaboration during his early time in Cologne. He was challenged by the gap between cellular immunology and molecular genetics and profited from the advances of the latter as well as postwar economic growth in Germany. The Immunology Department became a place, and little universe in itself, where young scientists from all over the world came together to study cellular and molecular mechanisms of antibody formation. This included work on normal and malignant B cells in the human, particularly the origin of Hodgkin lymphoma, but the main focus was on B cell development and homeostasis, the germinal center reaction, and immunological memory, developing recombinase-assisted and conditional gene targeting in mice as a main technical tool.

YY1 Controls Immunoglobulin Class Switch Recombination and Nuclear Activation-Induced Deaminase Levels

PubMed Central

Zaprazna, Kristina

2012-01-01

Activation-induced deaminase (AID) is an enzyme required for class switch recombination (CSR) and somatic hypermutation (SHM), processes that ensure antibody maturation and expression of different immunoglobulin isotypes. AID function is tightly regulated by tissue- and stage-specific expression, nuclear localization, and protein stability. Transcription factor YY1 is crucial for early B cell development, but its function at late B cell stages is unknown. Here, we show that YY1 conditional knockout in activated splenic B cells interferes with CSR. Knockout of YY1 did not affect B cell proliferation, transcription of the AID and IgM genes, or levels of various switch region germ line transcripts. However, we show that YY1 physically interacts with AID and controls the accumulation of nuclear AID, at least in part, by increasing nuclear AID stability. We show for the first time that YY1 plays a novel role in CSR and controls nuclear AID protein levels. PMID:22290437

Induction of Encephalitis in Rhesus Monkeys Infused with Lymphocryptovirus-Infected B-Cells Presenting MOG34–56 Peptide

PubMed Central

Haanstra, Krista G.; Wubben, Jacqueline A. M.; Jonker, Margreet; Hart, Bert A. ‘t.

2013-01-01

The overlapping epidemiology of multiple sclerosis (MS) and Epstein-Barr virus (EBV), the increased risk to develop MS after infectious mononucleosis (IM) and the localization of EBV-infected B-cells within the MS brain suggest a causal link between EBV and MS. However, the underlying mechanism is unknown. We hypothesize that EBV-infected B-cells are capable of eliciting a central nervous system (CNS) targeting autoimmune reaction. To test this hypothesis we have developed a novel experimental model in rhesus monkeys of IM-like disease induced by infusing autologous B-lymphoblastoid cells (B-LCL). Herpesvirus papio (HVP) is a lymphocryptovirus related to EBV and was used to generate rhesus monkey B-LCL. Three groups of five animals were included; each group received three intravenous infusions of B-LCL that were either pulsed with the encephalitogenic self peptide MOG34–56 (group A), a mimicry peptide (981–1003) of the major capsid protein of cytomegalovirus (CMVmcp981–1003; group B) or the citrullinated MOG34–56 (cMOG34–56; group C). Groups A and B received on day 98 a single immunization with MOG34–56 in incomplete Freund’s adjuvant (IFA). Group C monkeys were euthanized just prior to day 98 without booster immunization. We observed self-peptide-specific proliferation of T-cells, superimposed on similar strong proliferation of CD3+CD8+ T-cells against the B-LCL as observed in IM. The brains of several monkeys contained perivascular inflammatory lesions of variable size, comprising CD3+ and CD68+ cells. Moreover, clusters of CD3+ and CD20+ cells were detected in the meninges. The only evident clinical sign was substantial loss of bodyweight (>15%), a symptom observed both in early autoimmune encephalitis and IM. In conclusion, this model suggests that EBV-induced B-LCL can elicit a CNS targeting inflammatory (auto)immune reaction. PMID:23977076

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

PubMed

Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest

PubMed Central

Gascoyne, Duncan M.; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E.; Croucher, Peter I.; Banham, Alison H.

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology. PMID:26034982

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

PubMed

Marroquin Belaunzaran, Osiris; Kleber, Sascha; Schauer, Stefan; Hausmann, Martin; Nicholls, Flora; Van den Broek, Maries; Payeli, Sravan; Ciurea, Adrian; Milling, Simon; Stenner, Frank; Shaw, Jackie; Kollnberger, Simon; Bowness, Paul; Petrausch, Ulf; Renner, Christoph

2015-01-01

HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders. The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry. HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules. HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats

PubMed Central

Marroquin Belaunzaran, Osiris; Kleber, Sascha; Schauer, Stefan; Hausmann, Martin; Nicholls, Flora; Van den Broek, Maries; Payeli, Sravan; Ciurea, Adrian; Milling, Simon; Stenner, Frank; Shaw, Jackie; Kollnberger, Simon; Bowness, Paul; Petrausch, Ulf; Renner, Christoph

2015-01-01

Objectives HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272–specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders. Methods The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry. Results HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules. Conclusion HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders. PMID:26125554

Inhibition of tumor necrosis factor-{alpha}-induced expression of adhesion molecules in human endothelial cells by the saponins derived from roots of Platycodon grandiflorum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Ji Young; Kim, Dong Hee; Kim, Hyung Gyun

2006-01-15

Adhesion molecules play an important role in the development of atherogenesis and are produced by endothelial cells after being stimulated with various inflammatory cytokines. This study examined the effect of saponins that were isolated from the roots of Platycodon grandiflorum A. DC (Campanulaceae), Changkil saponins (CKS), on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. CKS significantly inhibited the TNF{alpha}-induced increase in monocyte adhesion to endothelial cells as well as decreased the protein and mRNA expression levels of vascular adhesion molecule-1 and intercellular cell adhesion molecule-1 on endothelial cells. Furthermore, CKS significantly inhibited themore » TNF{alpha}-induced production of intracellular reactive oxygen species (ROS) and activation of NF-{kappa}B by preventing I{kappa}B degradation and inhibiting I{kappa}B kinase activity. Overall, CKS has anti-atherosclerotic and anti-inflammatory activity, which is least in part the result of it reducing the cytokine-induced endothelial adhesion to monocytes by inhibiting intracellular ROS production, NF-{kappa}B activation, and cell adhesion molecule expression in endothelial cells.« less

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

PubMed Central

Kaji, Tomohiro; Ishige, Akiko; Hikida, Masaki; Taka, Junko; Hijikata, Atsushi; Kubo, Masato; Nagashima, Takeshi; Takahashi, Yoshimasa; Kurosaki, Tomohiro; Okada, Mariko; Ohara, Osamu

2012-01-01

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell–dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell–dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen. PMID:23027924

Manipulating memory CD8 T cell numbers by timed enhancement of IL-2 signals1

PubMed Central

Kim, Marie T.; Kurup, Samarchith P.; Starbeck-Miller, Gabriel R.; Harty, John T.

2016-01-01

Due to the growing burden of tumors and chronic infections, manipulating CD8 T cell responses for clinical use has become an important goal for immunologists. Here, we show that dendritic cell (DC) immunization coupled with relatively early (days 1–3) or late (days 4–6) administration of enhanced IL-2-signals both increase peak effector CD8 T cell numbers, but only early IL-2 signals enhance memory numbers. IL-2 signals delivered at relatively late time points drive terminal differentiation, marked Bim mediated contraction and do not increase memory T cell numbers. In contrast, early IL-2 signals induce effector cell metabolic profiles more conducive to memory formation. Of note, down-regulation of CD80 and CD86 was observed on DCs in vivo following early IL-2 treatment. Mechanistically, early IL-2 treatment enhanced CTLA-4 expression on regulatory T (Treg) cells, and CTLA-4 blockade alongside IL-2 treatment in vivo prevented the decrease in CD80 and CD86, supporting a cell-extrinsic role of CTLA-4 in down-regulating B7-ligand expression on DCs. Finally, DC immunization followed by early IL-2 treatment and αCTLA-4 blockade resulted in lower memory CD8 T cell numbers compared to the DC + early IL-2 treatment group. These data suggest that curtailed signaling through the B7-CD28 co-stimulatory axis during CD8 T cell activation limits terminal differentiation and preserves memory CD8 T cell formation and thus, should be considered in future T cell vaccination strategies. PMID:27439516

Adjuvant Effect of Killed Propionibacterium acnes on Mouse Peritoneal B-1 Lymphocytes and Their Early Phagocyte Differentiation

PubMed Central

Mussalem, Juliana Sekeres; Squaiella-Baptistão, Carla Cristina; Teixeira, Daniela; Yendo, Tatiana Mina; Thies, Felipe Garutti; Popi, Ana Flavia; Mariano, Mario; Longo-Maugéri, Ieda

2012-01-01

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses. PMID:22448280

Adjuvant effect of killed Propionibacterium acnes on mouse peritoneal B-1 lymphocytes and their early phagocyte differentiation.

PubMed

Mussalem, Juliana Sekeres; Squaiella-Baptistão, Carla Cristina; Teixeira, Daniela; Yendo, Tatiana Mina; Thies, Felipe Garutti; Popi, Ana Flavia; Mariano, Mario; Longo-Maugéri, Ieda

2012-01-01

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses.

Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination

PubMed Central

Brooks, Jill M.; Long, Heather M.; Tierney, Rose J.; Shannon-Lowe, Claire; Leese, Alison M.; Fitzpatrick, Martin; Taylor, Graham S.; Rickinson, Alan B.

2016-01-01

Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three “first wave” proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that “first wave” antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design. PMID:27096949

VAV1-Cre mediated hematopoietic deletion of CBL and CBL-B leads to JMML-like aggressive early-neonatal myeloproliferative disease

PubMed Central

An, Wei; Mohapatra, Bhopal C.; Zutshi, Neha; Bielecki, Timothy A.; Goez, Benjamin T.; Luan, Haitao; Iseka, Fany; Mushtaq, Insha; Storck, Matthew D.; Band, Vimla; Band, Hamid

2016-01-01

CBL and CBL-B ubiquitin ligases play key roles in hematopoietic stem cell homeostasis and their aberrations are linked to leukemogenesis. Mutations of CBL, often genetically-inherited, are particularly common in Juvenile Myelomonocytic Leukemia (JMML), a disease that manifests early in children. JMML is fatal unless corrected by bone marrow transplant, which is effective in only half of the recipients, stressing the need for animal models that recapitulate the key clinical features of this disease. However, mouse models established so far only develop hematological malignancy in adult animals. Here, using VAV1-Cre-induced conditional CBL/CBL-B double knockout (DKO) in mice, we established an animal model that exhibits a neonatal myeloproliferative disease (MPD). VAV1-Cre induced DKO mice developed a strong hematological phenotype at postnatal day 10, including severe leukocytosis and hepatomegaly, bone marrow cell hypersensitivity to cytokines including GM-CSF, and rapidly-progressive disease and invariable lethality. Interestingly, leukemic stem cells were most highly enriched in neonatal liver rather than bone marrow, which, along with the spleen and thymus, were hypo-cellular. Nonetheless, transplantation assays showed that both DKO bone marrow and liver cells can initiate leukemic disease in the recipient mice with seeding of both spleen and bone marrow. Together, our results support the usefulness of the new hematopoietic-specific CBL/CBL-B double KO animal model to study JMML-related pathogenesis and to further understand the function of CBL family proteins in regulating fetal and neonatal hematopoiesis. To our knowledge, this is the first mouse model that exhibits neonatal MPD in infancy, by day 10 of postnatal life. PMID:27449297

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

PubMed

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

Epstein–Barr virus-associated lymphomas

PubMed Central

Shannon-Lowe, Claire; Rickinson, Alan B.

2017-01-01

Epstein–Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host. This article is part of the themed issue ‘Human oncogenic viruses’. PMID:28893938

Epstein-Barr virus-associated lymphomas.

PubMed

Shannon-Lowe, Claire; Rickinson, Alan B; Bell, Andrew I

2017-10-19

Epstein-Barr virus (EBV), originally discovered through its association with Burkitt lymphoma, is now aetiologically linked to a remarkably wide range of lymphoproliferative lesions and malignant lymphomas of B-, T- and NK-cell origin. Some occur as rare accidents of virus persistence in the B lymphoid system, while others arise as a result of viral entry into unnatural target cells. The early finding that EBV is a potent B-cell growth transforming agent hinted at a simple oncogenic mechanism by which this virus could promote lymphomagenesis. In reality, the pathogenesis of EBV-associated lymphomas involves a complex interplay between different patterns of viral gene expression and cellular genetic changes. Here we review recent developments in our understanding of EBV-associated lymphomagenesis in both the immunocompetent and immunocompromised host.This article is part of the themed issue 'Human oncogenic viruses'. © 2017 The Authors.

IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis.

PubMed

Zhang, Yu; Davis, Celestia; Shah, Sapana; Hughes, Daniel; Ryan, James C; Altomare, Diego; Peña, Maria Marjorette O

2017-01-01

Liver metastasis is the major cause of death from colorectal cancer (CRC). Understanding its mechanisms is necessary for timely diagnosis and development of effective therapies. Interleukin-33 (IL-33) is an IL-1 cytokine family member that uniquely functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or an early danger signal. Its role in invoking type 2 immune response has been established; however, it has contrasting roles in tumor development and metastasis. We identified IL-33 as a potently upregulated cytokine in a highly metastatic murine CRC cell line and examined its role in tumor growth and metastasis to the liver. IL-33 was transgenically expressed in murine and human adenocarcinoma and carcinoma cell lines and their growth and spontaneous metastasis to the liver were assessed in orthotopic models of CRC in wild-type C57Bl/6 and Il33 knockout mice. The results showed that increased expression of IL-33 in CRC cells enhanced their tumor take, growth, and liver metastasis. Tumor- rather than host-derived IL-33 induced the enhanced recruitment of CD11b + GR1 + and CD11b + F4/80 + myeloid cells to remodel the tumor microenvironment by increased expression of mobilizing cytokines, and tumor angiogenesis by activating endothelial cells. IL-33 expression was elevated in patient tumor tissues, induced early in adenoma development, and activated by pro-inflammatory cytokines derived from the tumor microenvironment. The data suggest that tumor-derived IL-33 modulates the tumor microenvironment to potently promote colon carcinogenesis and liver metastasis, underscoring its potential as a therapeutic target. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Embryological origin of the endocardium and derived valve progenitor cells: from developmental biology to stem cell-based valve repair.

PubMed

Pucéat, Michel

2013-04-01

The cardiac valves are targets of both congenital and acquired diseases. The formation of valves during embryogenesis (i.e., valvulogenesis) originates from endocardial cells lining the myocardium. These cells undergo an endothelial-mesenchymal transition, proliferate and migrate within an extracellular matrix. This leads to the formation of bilateral cardiac cushions in both the atrioventricular canal and the outflow tract. The embryonic origin of both the endocardium and prospective valve cells is still elusive. Endocardial and myocardial lineages are segregated early during embryogenesis and such a cell fate decision can be recapitulated in vitro by embryonic stem cells (ESC). Besides genetically modified mice and ex vivo heart explants, ESCs provide a cellular model to study the early steps of valve development and might constitute a human therapeutic cell source for decellularized tissue-engineered valves. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. Copyright © 2012 Elsevier B.V. All rights reserved.

Concanavalin A-mediated polyclonal helper assay of normal thymocytes and its use for the analysis of ontogeny.

PubMed

Kina, T; Nishikawa, S; Amagai, T; Katsura, Y

1987-01-01

A concanavalin A (Con A)-mediated polyclonal helper assay system was established by using the thymus cells or splenic T cells as a source of helper T cells. When splenic B cells were cocultured with thymus cells or splenic Lyt-2- T cells in the presence of an optimal dose of Con A, B Cells were polyclonally activated and differentiated into immunoglobulin-secreting cells. This Con A-mediated helper activity was completely inhibited by the addition of alpha-methyl-D-mannoside and could not be substituted by culture supernatant of Con A-stimulated thymocytes or splenic T cells. Almost all the activity of the thymus cells was carried by peanut agglutinin low binding population. Genetic restriction between T and B cells was not observed in this helper function. In ontogeny, Con A-mediated helper activity in the thymus was first detected at a few days after birth and reached the adult level at about 1 week of age. The polyclonal helper assay system developed in the present study provides a sensitive system to analyse the helper function of thymus cells and also to delineate the early phase of the differentiation of helper T cell population.

Early onset of puberty and early ovarian failure in CYP7B1 knockout mice.

PubMed

Omoto, Yoko; Lathe, Richard; Warner, Margaret; Gustafsson, Jan-Ake

2005-02-22

CYP7B1 is the enzyme responsible for hydroxylation and termination of the estrogenic actions of the androgen metabolite, 5alpha-androstane-3beta, 17beta-diol (3betaAdiol). 3betaAdiol is estrogenic in ERalpha or ERbeta positive cells only if they do not express CYP7B1. In this study we show that female CYP7B1(-/-) mice experience early onset of growth of the uterus and mammary glands and commence estrus cycles 2 days earlier than their wild-type littermates. Adult mammary glands and uteri appear to be under continuous estrogenic stimulation. We conclude that, by cell-specific regulation of the estrogenicity of 3betaAdiol, CYP7B1 performs two major tasks: (i) it allows 3betaAdiol to have growth inhibitory effects through ERbeta and (ii) it permits estradiol-specific activation of estrogen receptors by protection of certain cells from the estrogenic effects of 3betaAdiol. When CYP7B1 is inactivated, 3betaAdiol activates estrogen receptors indiscriminately, and the overall effect is prolonged and inappropriate exposure to estrogen.

Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE

PubMed Central

Wu, Yee Ling; Teichmann, Sarah A.

2017-01-01

Noncoding transcripts originating upstream of the immunoglobulin constant region (I transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of Iε and Iγ1 transcription in response to interleukin 4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. In this study, we combine novel mouse reporters and single-cell RNA sequencing to reveal the heterogeneity in IL-4–induced I transcription. We identify an early population of cells expressing Iε but not Iγ1 and demonstrate that early Iε transcription leads to switching to IgE and occurs at lower activation levels than Iγ1. Our results reveal how probabilistic transcription with a lower activation threshold for Iε directs the early choice of IgE versus IgG1, a key physiological response against parasitic infestations and a mediator of allergy and asthma. PMID:27994069

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

PubMed Central

Wei, Shuo; Xu, Guofeng; Bridges, Lance C.; Williams, Phoebe; Nakayama, Takuya; Shah, Anoop; Grainger, Robert M.; White, Judith M.; DeSimone, Douglas W.

2012-01-01

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in X. tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus. PMID:22227340

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection.

PubMed

Weiss, Eric R; Lamers, Susanna L; Henderson, Jennifer L; Melnikov, Alexandre; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Nusbaum, Chad; Luzuriaga, Katherine

2018-01-15

Over 90% of the world's population is persistently infected with Epstein-Barr virus. While EBV does not cause disease in most individuals, it is the common cause of acute infectious mononucleosis (AIM) and has been associated with several cancers and autoimmune diseases, highlighting a need for a preventive vaccine. At present, very few primary, circulating EBV genomes have been sequenced directly from infected individuals. While low levels of diversity and low viral evolution rates have been predicted for double-stranded DNA (dsDNA) viruses, recent studies have demonstrated appreciable diversity in common dsDNA pathogens (e.g., cytomegalovirus). Here, we report 40 full-length EBV genome sequences obtained from matched oral wash and B cell fractions from a cohort of 10 AIM patients. Both intra- and interpatient diversity were observed across the length of the entire viral genome. Diversity was most pronounced in viral genes required for establishing latent infection and persistence, with appreciable levels of diversity also detected in structural genes, including envelope glycoproteins. Interestingly, intrapatient diversity declined significantly over time ( P < 0.01), and this was particularly evident on comparison of viral genomes sequenced from B cell fractions in early primary infection and convalescence ( P < 0.001). B cell-associated viral genomes were observed to converge, becoming nearly identical to the B95.8 reference genome over time (Spearman rank-order correlation test; r = -0.5589, P = 0.0264). The reduction in diversity was most marked in the EBV latency genes. In summary, our data suggest independent convergence of diverse viral genome sequences toward a reference-like strain within a relatively short period following primary EBV infection. IMPORTANCE Identification of viral proteins with low variability and high immunogenicity is important for the development of a protective vaccine. Knowledge of genome diversity within circulating viral populations is a key step in this process, as is the expansion of intrahost genomic variation during infection. We report full-length EBV genomes sequenced from the blood and oral wash of 10 individuals early in primary infection and during convalescence. Our data demonstrate considerable diversity within the pool of circulating EBV strains, as well as within individual patients. Overall viral diversity decreased from early to persistent infection, particularly in latently infected B cells, which serve as the viral reservoir. Reduction in B cell-associated viral genome diversity coincided with a convergence toward a reference-like EBV genotype. Greater convergence positively correlated with time after infection, suggesting that the reference-like genome is the result of selection. Copyright © 2018 American Society for Microbiology.

Memory B lymphocytes determine repertoire oligoclonality early after haematopoietic stem cell transplantation

PubMed Central

OMAZIC, B; LUNDKVIST, I; MATTSSON, J; PERMERT, J; NÄSMAN-BJÖRK, I

2003-01-01

The objective of this study was to investigate if oligoclonality of the Ig repertoire post-haematopoietic stem cell transplantation (HSCT) is restricted to memory B lymphocytes or if it is a general property among B lymphocytes. As a measure of B lymphocyte repertoire diversity, we have analysed size distribution of polymerase chain reaction (PCR) amplified Ig H complementarity determining region 3 (CDR3) in naive and memory B lymphocytes isolated from patients before HSCT and at 3, 6 and 12 months after HSCT as well as from healthy controls. We demonstrate a limited variation of the IgH CDR3 repertoire in the memory B lymphocyte population compared to the naive B cell population. This difference was significant at 3 and 6 months post-HSCT. Compared to healthy controls there is a significant restriction of the memory B lymphocyte repertoire at 3 months after HSCT, but not of the naive B lymphocyte repertoire. Twelve months after HSCT, the IgH CDR3 repertoire in both memory and naive B lymphocytes are as diverse as in healthy controls. Thus, our findings suggest a role for memory B cells in the restriction of the oligoclonal B cell repertoire observed early after HSCT, which may be of importance when considering reimmunization of transplanted patients. PMID:12974769

Obligatory Requirement for Antibody in Recovery from a Primary Poxvirus Infection

PubMed Central

Chaudhri, Geeta; Panchanathan, Vijay; Bluethmann, Horst; Karupiah, Gunasegaran

2006-01-01

To understand the correlates of protective immunity against primary variola virus infection in humans, we have used the well-characterized mousepox model. This is an excellent surrogate small-animal model for smallpox in which the disease is caused by infection with the closely related orthopoxvirus, ectromelia virus. Similarities between the two infections include virus replication and transmission, aspects of pathology, and development of pock lesions. Previous studies using ectromelia virus have established critical roles for cytokines and effector functions of CD8 T cells in the control of acute stages of poxvirus infection. Here, we have used mice deficient in B cells to demonstrate that B-cell function is also obligatory for complete virus clearance and recovery of the host. In the absence of B cells, virus persists and the host succumbs to infection, despite the generation of CD8 T-cell responses. Intriguingly, transfer of naive B cells or ectromelia virus-immune serum to B-cell-deficient mice with established infection allowed these animals to clear virus and fully recover. In contrast, transfer of ectromelia virus-immune CD8 T cells was ineffective. Our data show that mice deficient in CD8 T-cell function die early in infection, whereas those deficient in B cells or antibody production die much later, indicating that B-cell function becomes critical after the effector phase of the CD8 T-cell response to infection subsides. Strikingly, our results show that antibody prevents virus from seeding the skin and forming pock lesions, which are important for virus transmission between hosts. PMID:16775322

The miR-23a~27a~24-2 microRNA cluster buffers transcription and signaling pathways during hematopoiesis

PubMed Central

Kurkewich, Jeffrey L.; Klopfenstein, Nathan; Wood, Christian; Boucher, Austin

2017-01-01

MicroRNA cluster mirn23a has previously been shown to promote myeloid development at the expense of lymphoid development in overexpression and knockout mouse models. This polarization is observed early in hematopoietic development, with an increase in common lymphoid progenitors (CLPs) and a decrease in all myeloid progenitor subsets in adult bone marrow. The pool size of multipotential progenitors (MPPs) is unchanged; however, in this report we observe by flow cytometry that polarized subsets of MPPs are changed in the absence of mirn23a. Additionally, in vitro culture of MPPs and sorted MPP transplants showed that these cells have decreased myeloid and increased lymphoid potential in vitro and in vivo. We investigated the mechanism by which mirn23a regulates hematopoietic differentiation and observed that mirn23a promotes myeloid development of hematopoietic progenitors through regulation of hematopoietic transcription factors and signaling pathways. Early transcription factors that direct the commitment of MPPs to CLPs (Ikzf1, Runx1, Satb1, Bach1 and Bach2) are increased in the absence of mirn23a miRNAs as well as factors that commit the CLP to the B cell lineage (FoxO1, Ebf1, and Pax5). Mirn23a appears to buffer transcription factor levels so that they do not stochastically reach a threshold level to direct differentiation. Intriguingly, mirn23a also inversely regulates the PI3 kinase (PI3K)/Akt and BMP/Smad signaling pathways. Pharmacological inhibitor studies, coupled with dominant active/dominant negative biochemical experiments, show that both signaling pathways are critical to mirn23a’s regulation of hematopoietic differentiation. Lastly, consistent with mirn23a being a physiological inhibitor of B cell development, we observed that the essential B cell transcription factor EBF1 represses expression of mirn23a. In summary, our data demonstrates that mirn23a regulates a complex array of transcription and signaling pathways to modulate adult hematopoiesis. PMID:28704388

Expression of germline markers in three species of amphioxus supports a preformation mechanism of germ cell development in cephalochordates

PubMed Central

2013-01-01

Background In a previous study, we showed that the cephalochordate amphioxus Branchiostoma floridae has localized maternal transcripts of conserved germ cell markers Vasa and Nanos in its early embryos. These results provided strong evidence to support a preformation mechanism for primordial germ cell (PGC) development in B. floridae. Results In this study, we further characterize the expression of B. floridae homologs of Piwi and Tudor, which play important roles in germline development in diverse metazoan animals. We show that maternal mRNA of one of the identified Piwi-like homologs, Bf-Piwil1, also colocalizes with Vasa in the vegetal germ plasm and has zygotic expression in both the putative PGCs and the tail bud, suggesting it may function in both germline and somatic stem cells. More interestingly, one Tudor family gene, Bf-Tdrd7, is only expressed maternally and colocalizes with Vasa in germ plasm, suggesting that it may function exclusively in germ cell specification. To evaluate the conservation of the preformation mechanism among amphioxus species, we further analyze Vasa, Nanos, Piwil1, and Tdrd7 expression in two Asian amphioxus species, B. belcheri and B. japonicum. Their maternal transcripts all localize in similar patterns to those seen in B. floridae. In addition, we labeled putative PGCs with Vasa antibody to trace their dynamic distribution in developing larvae. Conclusions We identify additional germ plasm components in amphioxus and demonstrate the molecular distinction between the putative germline stem cells and somatic stem cells. Moreover, our results suggest that preformation may be a conserved mechanism for PGC specification among Branchiostoma species. Our Vasa antibody staining results suggest that after the late neurula stage, amphioxus PGCs probably proliferate with the tail bud cells during posterior elongation and are deposited near the forming myomere boundaries. Subsequently, these PGCs would concentrate at the ventral tip of the myoseptal walls to form the gonad anlagen. PMID:23777831

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage

PubMed Central

Cao, Zubing; Carey, Timothy S.; Ganguly, Avishek; Wilson, Catherine A.; Paul, Soumen; Knott, Jason G.

2015-01-01

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. PMID:25858457

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

PubMed Central

Arzumanyan, Alla; Kulathinal, Rob J.; Blain, Stacy W.; Holcombe, Randall F.; Mahajna, Jamal; Marino, Maria; Martinez-Chantar, Maria L.; Nawroth, Roman; Sanchez-Garcia, Isidro; Sharma, Dipali; Saxena, Neeraj K.; Singh, Neetu; Vlachostergios, Panagiotis J.; Guo, Shanchun; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Niccolai, Elena; Amin, Amr; Ashraf, S. Salman; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Aquilano, Katia; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Nowsheen, Somaira

2016-01-01

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). This data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression. PMID:25892662

Dynamic expression of transcription factor Brn3b during mouse cranial nerve development

PubMed Central

Sajgo, Szilard; Ali, Seid; Popescu, Octavian; Badea, Tudor Constantin

2015-01-01

During development transcription factor combinatorial codes define a large variety of morphologically and physiologically distinct neurons. Such a combinatorial code has been proposed for the differentiation of projection neurons of the somatic and visceral components of cranial nerves. It is possible that individual neuronal cell types are not specified by unique transcription factors, but rather emerge through the intersection of their expression domains. Brn3a, Brn3b and Brn3c, in combination with each other and/or transcription factors of other families, can define subgroups of Retinal Ganglion Cells (RGC), Spiral and Vestibular Ganglia, inner ear and vestibular hair cell neurons in the vestibuloacoustic system, and groups of somatosensory neurons in the Dorsal Root Ganglia (DRG). In the present study we investigated the expression and potential role of the Brn3b transcription factor in cranial nerves and associated nuclei of the brainstem. We report the dynamic expression of Brn3b in the somatosensory component of cranial nerves II, V, VII and VIII and visceromotor nuclei of nerves VII, IX, X, as well as other brainstem nuclei during different stages of development into adult stage. We find that genetically identified Brn3bKO RGC axons show correct but delayed pathfinding during the early stages of embryonic development. However loss of Brn3b does not affect the anatomy of the other cranial nerves normally expressing this transcription factor. PMID:26356988

Preclinical Immunomodulation by the Probiotic Bifidobacterium breve M-16V in Early Life

PubMed Central

Rigo-Adrover, Maria del Mar; Franch, Àngels; Castell, Margarida; Pérez-Cano, Francisco José

2016-01-01

This study aimed to investigate the effect of supplementation with the probiotic Bifidobacterium breve M-16V on the maturation of the intestinal and circulating immune system during suckling. In order to achieve this purpose, neonatal Lewis rats were supplemented with the probiotic strain from the 6th to the 18th day of life. The animals were weighed during the study, and faecal samples were obtained and evaluated daily. On day 19, rats were euthanized and intestinal wash samples, mesenteric lymph node (MLN) cells, splenocytes and intraepithelial lymphocytes (IEL) were obtained. The probiotic supplementation in early life did not modify the growth curve and did not enhance the systemic immune maturation. However, it increased the proportion of cells bearing TLR4 in the MLN and IEL, and enhanced the percentage of the integrin αEβ7+ and CD62L+ cells in the MLN and that of the integrin αEβ7+ cells in the IEL, suggesting an enhancement of the homing process of naïve T lymphocytes to the MLN, and the retention of activated lymphocytes in the intraepithelial compartment. Interestingly, B. breve M-16V enhanced the intestinal IgA synthesis. In conclusion, supplementation with the probiotic strain B. breve M-16V during suckling improves the development of mucosal immunity in early life. PMID:27820846

The generation and analyses of a novel combination of recombinant adenovirus vaccines targeting three tumor antigens as an immunotherapeutic

PubMed Central

Gabitzsch, Elizabeth S.; Tsang, Kwong Yok; Palena, Claudia; David, Justin M.; Fantini, Massimo; Kwilas, Anna; Rice, Adrian E.; Latchman, Yvette; Hodge, James W.; Gulley, James L.; Madan, Ravi A.; Heery, Christopher R.; Balint, Joseph P.

2015-01-01

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no “antigenic competition” in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies. PMID:26374823

Transient protective effect of B-vitamins in experimental epilepsy in the mouse brain.

PubMed

Rabie, Tamer; Mühlhofer, Wolfgang; Bruckner, Thomas; Schwab, Anna; Bauer, Alexander T; Zimmermann, Manfred; Bonke, Dieter; Marti, Hugo H; Schenkel, Johannes

2010-05-01

The regulation of programmed cell death in the nervous system of vertebrates is a complex mechanism aimed to remove superfluous or damaged cells. Epileptic seizures can lead to an activation of pathways resulting in neuronal cell death. B-vitamins might have a neuroprotective potential reducing cell death following appropriate stimulation. Here, the role of the B-vitamins B(1) (thiamine), B(6) (pyridoxine), and B(12) (cobalamine) was investigated in a mouse model of experimental epilepsy induced by kainate. B-vitamin pre-treated animals showed a significantly reduced epileptic score during the first 15 min after kainate injection. The molecular response to kainate showed a bi-phased time course with early induction of Bcl-2 expression within 12 h and a second induction after 7 days of kainate exposure. B-vitamin pre-treatment resulted in significant higher Bcl-2 expression in control animals (no kainate) and at 12 h within the early phase. Bcl-2 expression was not affected by B-vitamins within the second phase. BAX expression was not significantly influenced during the whole experiment. Three days after kainate stimulation, the number of TdT-mediated dUTP-biotin nick end labeling-positive cells in the hippocampal region was lower in B-vitamin-treated animals. Therefore, B-vitamin pre-treatment may attenuate the response to epileptic stimulation.

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

PubMed

Duivenvoorden, Hendrika M; Rautela, Jai; Edgington-Mitchell, Laura E; Spurling, Alex; Greening, David W; Nowell, Cameron J; Molloy, Timothy J; Robbins, Elizabeth; Brockwell, Natasha K; Lee, Cheok Soon; Chen, Maoshan; Holliday, Anne; Selinger, Cristina I; Hu, Min; Britt, Kara L; Stroud, David A; Bogyo, Matthew; Möller, Andreas; Polyak, Kornelia; Sloane, Bonnie F; O'Toole, Sandra A; Parker, Belinda S

2017-12-01

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

PubMed Central

2011-01-01

Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome. PMID:22111588

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

PubMed

Xu, Ning; Shen, Sylvie; Dolnikov, Alla

2017-04-01

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Using the direct-injection model of early uveal melanoma hepatic metastasis to identify TPS as a potentially useful serum biomarker.

PubMed

Barak, Vivian; Frenkel, Shahar; Valyi-Nagy, Klara; Leach, Lu; Apushkin, Marsha A; Lin, Amy Y; Kalickman, Inna; Baumann, Nikola A; Pe'er, Jacob; Maniotis, Andrew J; Folberg, Robert

2007-10-01

To develop a method to screen for serum biomarkers of early hepatic metastasis from uveal melanoma. Cytokeratin 18 (TPS) was identified from gene expression profiles as protein generated by highly invasive uveal melanoma cells. Sera were collected from two groups of 15 SCID mice 2 weeks after injection of either tissue culture medium or MUM2B human metastatic uveal melanoma cells into the mouse liver. Serum TPS levels were assayed in 53 healthy human controls, 64 uveal melanoma patients who were disease free for at least 10 years, and 37 patients with metastatic uveal melanoma. After 2 weeks, small hepatic nodules (0.1-2.8 mm; mean, 0.80 mm) developed in 11 of 15 mice injected with MUM2B cells. Serum TPS levels in media-injected mice (84.7 U/L) were substantially lower than levels in MUM2B-injected mice (601 mug/L). TPS levels were significantly higher (P < 0.0001) in patients with metastatic uveal melanoma (139.63 +/- 22.20) than in healthy controls (54.23 +/- 0.01) or in patients free of disease (69.29 +/- 9.76). Significant differences were found between TPS levels before and after the development of hepatic metastases (P < 0.01), and serum TPS levels became elevated in four patients at least 6 months before the detection of hepatic metastases by abdominal ultrasonography. The direct-injection model of uveal melanoma in the mouse liver may be used to screen for potential serum biomarkers of metastatic uveal melanoma.

HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling.

PubMed

Huang, Qingsong; Niu, Zhiguo; Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

2017-08-01

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target.

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation.

PubMed

Reddy, Jay P; Peddibhotla, Sirisha; Bu, Wen; Zhao, Jing; Haricharan, Svasti; Du, Yi-Chieh Nancy; Podsypanina, Katrina; Rosen, Jeffrey M; Donehower, Larry A; Li, Yi

2010-02-23

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

Haploinsufficiency in tumor predisposition syndromes: altered genomic transcription in morphologically normal cells heterozygous for VHL or TSC mutation

PubMed Central

Peri, Suraj; Caretti, Elena; Tricarico, Rossella; Devarajan, Karthik; Cheung, Mitchell; Sementino, Eleonora; Menges, Craig W.; Nicolas, Emmanuelle; Vanderveer, Lisa A.; Howard, Sharon; Conrad, Peggy; Crowell, James A.; Campbell, Kerry S.; Ross, Eric A.; Godwin, Andrew K.; Yeung, Anthony T.; Clapper, Margie L.; Uzzo, Robert G.; Henske, Elizabeth P.; Ricketts, Christopher J.; Vocke, Cathy D.; Linehan, W. Marston; Testa, Joseph R.; Bellacosa, Alfonso; Kopelovich, Levy; Knudson, Alfred G.

2017-01-01

Tumor suppressor genes and their effector pathways have been identified for many dominantly heritable cancers, enabling efforts to intervene early in the course of disease. Our approach on the subject of early intervention was to investigate gene expression patterns of morphologically normal one-hit cells before they become hemizygous or homozygous for the inherited mutant gene which is usually required for tumor formation. Here, we studied histologically non-transformed renal epithelial cells from patients with inherited disorders that predispose to renal tumors, including von Hippel-Lindau (VHL) disease and Tuberous Sclerosis (TSC). As controls, we studied histologically normal cells from non-cancerous renal epithelium of patients with sporadic clear cell renal cell carcinoma (ccRCC). Gene expression analyses of VHLmut/wt or TSC1/2mut/wt versus wild-type (WT) cells revealed transcriptomic alterations previously implicated in the transition to precancerous renal lesions. For example, the gene expression changes in VHLmut/wt cells were consistent with activation of the hypoxia response, associated, in part, with the Warburg effect. Knockdown of any remaining VHL mRNA using shRNA induced secondary expression changes, such as activation of NF?B and interferon pathways, that are fundamentally important in the development of RCC. We posit that this is a general pattern of hereditary cancer predisposition, wherein haploinsufficiency for VHL or TSC1/2, or potentially other tumor susceptibility genes, is sufficient to promote development of early lesions, while cancer results from inactivation of the remaining normal allele. The gene expression changes identified here are related to the metabolic basis of renal cancer and may constitute suitable targets for early intervention. PMID:27682873

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

PubMed

Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

2000-12-01

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

Epidermal cell-shape regulation and subpopulation kinetics during butyrate-induced terminal maturation of normal and SV40-transformed human keratinocytes: epithelial models of differentiation therapy.

PubMed

Staiano-Coico, L; Steinberg, M; Higgins, P J

1990-10-15

Recent data indicate that malignant human epidermal cells may be appropriate targets for sodium butyrate (NaB)-mediated differentiation therapy. The response of pre- and post-crisis populations of SV40-transformed human keratinocytes (SVKs) to this differentiation-inducing agent was assessed, therefore, within the framework of NaB-directed normal human keratinocyte (NHK) maturation. NaB augmented cornified envelope (CE) production in NHK and pre-crisis SVK cultures; the time-course and efficiency of induced maturation were similar in the 2 cell systems. In NHKs, the percentage of amplifying ("B" substate) cells decreased with time in NaB correlating with increases in both "C" stage keratinocytes and CEs. The latter formed over one or 2 layers of nucleated basal-like cells. Inductions were accompanied by immediate cell cycle blocks (in both the G1 and G2/M phases), reorganization within the actin cytoskeleton, and transient early increases in cellular actin content. Increased NHK and pre-crisis SVK cytoskeletal-associated actin reached a maximum approximately 48 hr after NaB addition and preceded development of CEs. The CE precursors, thus, probably reside in the "B" substate. Post-crisis SVKs, in contrast, were refractive to NaB-induced terminal maturation or cell-cycle perturbation, failed to initiate actin filament rearrangements, and retained a basal cell-like phenotype. Stable transformation of human SVKs in post-crisis phase, therefore, appears to be associated with loss of maturation "competence" within the "B" keratinocyte subpopulation.

B cell and T cell immunity in the female genital tract: potential of distinct mucosal routes of vaccination and role of tissue-associated dendritic cells and natural killer cells.

PubMed

Anjuère, F; Bekri, S; Bihl, F; Braud, V M; Cuburu, N; Czerkinsky, C; Hervouet, C; Luci, C

2012-10-01

The female genital mucosa constitutes the major port of entry of sexually transmitted infections. Most genital microbial pathogens represent an enormous challenge for developing vaccines that can induce genital immunity that will prevent their transmission. It is now established that long-lasting protective immunity at mucosal surfaces has to involve local B-cell and T-cell effectors as well as local memory cells. Mucosal immunization constitutes an attractive way to generate systemic and genital B-cell and T-cell immune responses that can control early infection by sexually transmitted pathogens. Nevertheless, no mucosal vaccines against sexually transmitted infections are approved for human use. The mucosa-associated immune system is highly compartmentalized and the selection of any particular route or combinations of routes of immunization is critical when defining vaccine strategies against genital infections. Furthermore, mucosal surfaces are complex immunocompetent tissues that comprise antigen-presenting cells and also innate immune effectors and non-immune cells that can act as 'natural adjuvants' or negative immune modulators. The functions of these cells have to be taken into account when designing tissue-specific antigen-delivery systems and adjuvants. Here, we will discuss data that compare different mucosal routes of immunization to generate B-cell and T-cell responses in the genital tract, with a special emphasis on the newly described sublingual route of immunization. We will also summarize data on the understanding of the effector and induction mechanisms of genital immunity that may influence the development of vaccine strategies against genital infections. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

The importance of the Non Obese Diabetic (NOD) mouse model in autoimmune diabetes

PubMed Central

Pearson, James A; Wong, F. Susan; Wen, Li

2016-01-01

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the pancreatic infiltration of immune cells resulting in T cell-mediated destruction of the insulin-producing beta cells. The successes of the Non Obese Diabetic (NOD) mouse model have come in multiple forms including identifying key genetic and environmental risk factors e.g. Idd loci and effects of microorganisms including the gut microbiota, respectively, and how they may contribute to disease susceptibility and pathogenesis. Furthermore, the NOD model also provides insights into the roles of the innate immune cells as well as the B cells in contributing to the T cell-mediated disease. Unlike many autoimmune disease models, the NOD mouse develops spontaneous disease and has many similarities to human T1D. Through exploiting these similarities many targets have been identified for immune-intervention strategies. Although many of these immunotherapies did not have a significant impact on human T1D, they have been shown to be effective in the NOD mouse in early stage disease, which is not equivalent to trials in newly-diagnosed patients with diabetes. However, the continued development of humanized NOD mice would enable further clinical developments, bringing T1D research to a new translational level. Therefore, it is the aim of this review to discuss the importance of the NOD model in identifying the roles of the innate immune system and the interaction with the gut microbiota in modifying diabetes susceptibility. In addition, the role of the B cells will also be discussed with new insights gained through B cell depletion experiments and the impact on translational developments. Finally, this review will also discuss the future of the NOD mice and the development of humanized NOD mice, providing novel insights into human T1D. PMID:26403950

c-Myc-Induced Survivin Is Essential for Promoting the Notch-Dependent T Cell Differentiation from Hematopoietic Stem Cells

PubMed Central

Haque, Rizwanul; Song, Jianyong; Haque, Mohammad; Lei, Fengyang; Sandhu, Praneet; Ni, Bing; Zheng, Songguo; Fang, Deyu; Yang, Jin-Ming; Song, Jianxun

2017-01-01

Notch is indispensable for T cell lineage commitment, and is needed for thymocyte differentiation at early phases. During early stages of T cell development, active Notch prevents other lineage potentials including B cell lineage and myeloid cell (e.g., dendritic cell) lineage. Nevertheless, the precise intracellular signaling pathways by which Notch promotes T cell differentiation remain unclear. Here we report that the transcription factor c-Myc is a key mediator of the Notch signaling–regulated T cell differentiation. In a well-established in vitro differentiation model of T lymphocytes from hematopoietic stem cells, we showed that Notch1 and 4 directly promoted c-Myc expression; dominant-negative (DN) c-Myc inhibited early T cell differentiation. Moreover, the c-Myc expression activated by Notch signaling increased the expression of survivin, an inhibitor of apoptosis (IAP) protein. We further demonstrated that over-expression of c-Myc increased the abundance of survivin and the T cell differentiation thereof, whereas dn c-Myc reduced survivin levels and concomitantly retarded the differentiation. The c-Myc–dependent survivin induction is functionally germane, because Notch-dependent T cell differentiation was canceled by the depletion of survivin. These results identify both c-Myc and survivin as important mediators of the Notch signaling–regulated differentiation of T lymphocytes from hematopoietic stem cells. PMID:28272325

The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.

PubMed

Liao, W; Bisgrove, B W; Sawyer, H; Hug, B; Bell, B; Peters, K; Grunwald, D J; Stainier, D Y

1997-01-01

The zebrafish cloche mutation affects both the endothelial and hematopoietic lineages at a very early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W., Zon, L. I. and Fishman, M. C. (1995). Development 121, 3141-3150). The most striking vascular phenotype is the absence of endocardial cells from the heart. Microscopic examination of mutant embryos reveals the presence of endothelial-like cells in the lower trunk and tail regions while head vessels appear to be missing, indicating a molecular diversification of the endothelial lineage. Cell transplantation experiments show that cloche acts cell-autonomously within the endothelial lineage. To analyze further the role of cloche in regulating endothelial cell differentiation, we have examined the expression of flk-1 and tie, two receptor tyrosine kinase genes expressed early and sequentially in the endothelial lineage. In wild-type fish, flk-1-positive cells are found throughout the embryo and differentiate to form the nascent vasculature. In cloche mutants, flk-1-positive cells are found only in the lower trunk and tail regions, and this expression is delayed as compared to wild-type. Unlike the flk-1-positive cells in wild-type embryos, those in cloche mutants do not go on to express tie, suggesting that their differentiation is halted at an early stage. We also find that the cloche mutation is not linked to flk-1. These data indicate that cloche affects the differentiation of all endothelial cells and that it acts at a very early stage, either by directly regulating flk-1 expression or by controlling the differentiation of cells that normally develop to express flk-1. cloche mutants also have a blood deficit and their hematopoietic tissues show no expression of the hematopoietic transcription factor genes GATA-1 or GATA-2 at early stages. Because the appearance of distinct levels of flk-1 expression is delayed in cloche mutants, we examined GATA-1 expression at late embryonic stages and found some blood cell differentiation that appears to be limited to the region lined by the flk-1-expressing cells. The spatial restriction of blood in the ventroposterior-most region of cloche mutant embryos may be indicative of a ventral source of signal(s) controlling hematopoietic differentiation. In addition, the restricted colocalization of blood and endothelium in cloche mutants suggests that important interactions occur between these two lineages during normal development.

Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells

PubMed Central

Hahne, Michael; Combe, Bernard; Morel, Jacques; Daien, Claire I.

2017-01-01

B cells can have a regulatory role, mainly mediated by interleukin 10 (IL-10). IL-10 producing B cells (B10 cells) cells remain to be better characterized. Annexin V binds phosphatidylserine (PS), which is externalized during apoptosis. Previous works suggested that B10 cells are apoptotic cells since they bind Annexin V. Others showed that Annexin V binding could also be expressed on viable B cells. We aimed to explore if PS exposure can be a marker of B10 cells and if PS exposure has a functional role on B cell IL-10 production in healthy subjects. We found that B10 cells were significantly more often Annexin V+ than IL-10 non-producing B cells. After CpG activation, Annexin V+ B cells differentiated more often into B10 cells than Annexin Vneg B cells. Cell death and early apoptosis were similar between Annexin V+ and Annexin Vneg B cells. PS blockage, using biotinylated AnV and glyburide, decreased B10 cell differentiation. This study showed that B10 cells have an increased PS exposure independently of any apoptotic state. B cells exposing PS differentiate more into B10 cells whereas PS blockage inhibits B10 cells generation. These results strongly suggest a link between PS exposure and B10 cells. PMID:28072868

Rab4b controls an early endosome sorting event by interacting with the γ-subunit of the clathrin adaptor complex 1.

PubMed

Perrin, Laura; Laura, Perrin; Lacas-Gervais, Sandra; Sandra, Lacas-Gervais; Gilleron, Jérôme; Jérôme, Gilleron; Ceppo, Franck; Franck, Ceppo; Prodon, François; François, Prodon; Benmerah, Alexandre; Alexandre, Benmerah; Tanti, Jean-François; Jean-François, Tanti; Cormont, Mireille; Mireille, Cormont

2013-11-01

The endocytic pathway is essential for cell homeostasis and numerous small Rab GTPases are involved in its control. The endocytic trafficking step controlled by Rab4b has not been elucidated, although recent data suggested it could be important for glucose homeostasis, synaptic homeostasis or adaptive immunity. Here, we show that Rab4b is required for early endosome sorting of transferrin receptors (TfRs) to the recycling endosomes, and we identified the AP1γ subunit of the clathrin adaptor AP-1 as a Rab4b effector and key component of the machinery of early endosome sorting. We show that internalised transferrin (Tf) does not reach Vamp3/Rab11 recycling endosomes in the absence of Rab4b, whereas it is rapidly recycled back to the plasma membrane. By contrast, overexpression of Rab4b leads to the accumulation of internalised Tf within AP-1- and clathrin-coated vesicles. These vesicles are poor in early and recycling endocytic markers except for TfR and require AP1γ for their formation. Furthermore, the targeted overexpression of the Rab4b-binding domain of AP1γ to early endosome upon its fusion with FYVE domains inhibited the interaction between Rab4b and endogenous AP1γ, and perturbed Tf traffic. We thus proposed that the interaction between early endocytic Rab4b and AP1γ could allow the budding of clathrin-coated vesicles for subsequent traffic to recycling endosomes. The data also uncover a novel type of endosomes, characterised by low abundance of either early or recycling endocytic markers, which could potentially be generated in cell types that naturally express high level of Rab4b.

CEACAM1 induces B-cell survival and is essential for protective antiviral antibody production

PubMed Central

Khairnar, Vishal; Duhan, Vikas; Maney, Sathish Kumar; Honke, Nadine; Shaabani, Namir; Pandyra, Aleksandra A.; Seifert, Marc; Pozdeev, Vitaly; Xu, Haifeng C.; Sharma, Piyush; Baldin, Fabian; Marquardsen, Florian; Merches, Katja; Lang, Elisabeth; Kirschning, Carsten; Westendorf, Astrid M.; Häussinger, Dieter; Lang, Florian; Dittmer, Ulf; Küppers, Ralf; Recher, Mike; Hardt, Cornelia; Scheffrahn, Inka; Beauchemin, Nicole; Göthert, Joachim R.; Singer, Bernhard B.; Lang, Philipp A.; Lang, Karl S.

2015-01-01

B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1−/− mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1−/− mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses. PMID:25692415

Vaccine-specific antibody secreting cells are a robust early marker of LAIV-induced B-cell response in ferrets.

PubMed

Cherukuri, Anu; Servat, Esteban; Woo, Jennifer

2012-01-05

Currently, a robust set of immune correlates for live attenuated influenza vaccine (LAIV) efficacy in humans has not been fully elucidated. The serum hemagglutination inhibition (HAI) assay has been historically used to measure humoral immune responses to injectable inactivated influenza vaccination. However, serum antibody titers do not reliably reflect the complete mechanism of action of LAIV, which is an intranasally delivered vaccine and is expected to induce local mucosal and cellular immune responses in addition to humoral immune responses. Therefore, we designed a study to evaluate potential immune correlates of LAIV vaccination in the ferret animal model of influenza infection. Ferrets were vaccinated with increasing doses of LAIV and four weeks later challenged with a homologous wild-type (wt) H1N1 strain. Humoral immune responses measured following LAIV vaccination included HAI, serum antibodies and antibody secreting cells (ASC); and the responses were found to correlate with the dose level of LAIV administered in this model. Protection from wt virus challenge was determined by measuring inhibition of wt viral replication in nasal washes and in lung tissue. Results demonstrated that LAIV doses ≥ 5.0 log(10) Plaque Forming Units (PFU) elicited vaccine-specific IgG and IgA ASC frequencies and induced complete protection in the lungs. Further, we developed a novel model utilizing seropositive older ferrets to demonstrate that in the background of previous wt influenza infection LAIV induces a robust vaccine-specific B-cell response even in the absence of serum antibody response, a result that suggests that effector B-cell responses generated by LAIV are not inhibited by prior viral exposure. Finally, we demonstrated that LAIV elicits strain-specific memory B-cell responses that are measurable in a background of wt influenza infections. Taken together, results from these studies identified the antigen-specific ASC frequency as a useful early biomarker of LAIV-induced B-cell immune response. Copyright © 2011 Elsevier Ltd. All rights reserved.

The SCL gene specifies haemangioblast development from early mesoderm.

PubMed

Gering, M; Rodaway, A R; Göttgens, B; Patient, R K; Green, A R

1998-07-15

The SCL gene encodes a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of all haematopoietic lineages. SCL is also expressed in endothelial cells, but its function is not essential for specification of endothelial progenitors and the role of SCL in endothelial development is obscure. We isolated the zebrafish SCL homologue and show that it was co-expressed in early mesoderm with markers of haematopoietic, endothelial and pronephric progenitors. Ectopic expression of SCL mRNA in zebrafish embryos resulted in overproduction of common haematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue. Notochord and neural tube formation were unaffected. These results provide the first evidence that SCL specifies formation of haemangioblasts, the proposed common precursor of blood and endothelial lineages. Our data also underline the striking similarities between the role of SCL in haematopoiesis/vasculogenesis and the function of other bHLH proteins in muscle and neural development.

The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila.

PubMed

Li, Michelle A; Alls, Jeffrey D; Avancini, Rita M; Koo, Karen; Godt, Dorothea

2003-11-01

Interactions between somatic and germline cells are critical for the normal development of egg and sperm. Here we show that the gene traffic jam (tj) produces a soma-specific factor that controls gonad morphogenesis and is required for female and male fertility. tj encodes the only large Maf factor in Drosophila melanogaster, an orthologue of the atypical basic Leu zipper transcription factors c-Maf and MafB/Kreisler in vertebrates. Expression of tj occurs in somatic gonadal cells that are in direct contact with germline cells throughout development. In tj mutant gonads, somatic cells fail to inter-mingle and properly envelop germline cells, causing an early block in germ cell differentiation. In addition, tj mutant somatic cells show an increase in the level of expression for several adhesion molecules. We propose that tj is a critical modulator of the adhesive properties of somatic cells, facilitating germline-soma interactions that are essential for germ cell differentiation.

Beneficial Effects of cART Initiated during Primary and Chronic HIV-1 Infection on Immunoglobulin-Expression of Memory B-Cell Subsets

PubMed Central

Pensieroso, Simone; Tolazzi, Monica; Chiappetta, Stefania; Nozza, Silvia; Lazzarin, Adriano; Tambussi, Giuseppe; Scarlatti, Gabriella

2015-01-01

Introduction During HIV-1 infection the B-cell compartment undergoes profound changes towards terminal differentiation, which are only partially restored by antiretroviral therapy (cART). Materials and Methods To investigate the impact of infection as early as during primary HIV-1 infection (PHI) we assessed distribution of B-cell subsets in 19 PHI and 25 chronic HIV-1-infected (CHI) individuals before and during 48 weeks of cART as compared to healthy controls (n = 23). We also analysed Immunoglobulin-expression of memory B-cell subsets to identify alterations in Immunoglobulin-maturation. Results Determination of B-cell subsets at baseline showed that total and Naive B-cells were decreased whereas Activated Memory (AM), Tissue-like Memory (TLM) B-cells and Plasma cells were increased in both PHI and CHI patients. After 4 weeks of cART total B-cells increased, while AM, TLM B-cells and Plasma cells decreased, although without reaching normal levels in either group of individuals. This trend was maintained until week 48, though only total B-cells normalized in both PHI and CHI. Resting Memory (RM) B-cells were preserved since baseline. This subset remained stable in CHI, while was expanded by an early initiation of cART during PHI. Untreated CHI patients showed IgM-overexpression at the expenses of switched (IgM-IgD-) phenotypes of the memory subsets. Interestingly, in PHI patients a significant alteration of Immunoglobulin-expression was evident at BL in TLM cells, and after 4 weeks, despite treatment, in AM and RM subsets. After 48 weeks of therapy, Immunoglobulin-expression of AM and RM almost normalized, but remained perturbed in TLM cells in both groups. Conclusions In conclusion, aberrant activated and exhausted B-cell phenotypes rose already during PHI, while most of the alterations in Ig-expression seen in CHI appeared later, despite 4 weeks of effective cART. After 48 weeks of cART B-cell subsets distribution improved although without full normalization, while Immunoglobulin-expression normalized among AM and RM, remaining perturbed in TLM B-cells of PHI and CHI. PMID:26474181

Beneficial Effects of cART Initiated during Primary and Chronic HIV-1 Infection on Immunoglobulin-Expression of Memory B-Cell Subsets.

PubMed

Pogliaghi, Manuela; Ripa, Marco; Pensieroso, Simone; Tolazzi, Monica; Chiappetta, Stefania; Nozza, Silvia; Lazzarin, Adriano; Tambussi, Giuseppe; Scarlatti, Gabriella

2015-01-01

During HIV-1 infection the B-cell compartment undergoes profound changes towards terminal differentiation, which are only partially restored by antiretroviral therapy (cART). To investigate the impact of infection as early as during primary HIV-1 infection (PHI) we assessed distribution of B-cell subsets in 19 PHI and 25 chronic HIV-1-infected (CHI) individuals before and during 48 weeks of cART as compared to healthy controls (n = 23). We also analysed Immunoglobulin-expression of memory B-cell subsets to identify alterations in Immunoglobulin-maturation. Determination of B-cell subsets at baseline showed that total and Naive B-cells were decreased whereas Activated Memory (AM), Tissue-like Memory (TLM) B-cells and Plasma cells were increased in both PHI and CHI patients. After 4 weeks of cART total B-cells increased, while AM, TLM B-cells and Plasma cells decreased, although without reaching normal levels in either group of individuals. This trend was maintained until week 48, though only total B-cells normalized in both PHI and CHI. Resting Memory (RM) B-cells were preserved since baseline. This subset remained stable in CHI, while was expanded by an early initiation of cART during PHI. Untreated CHI patients showed IgM-overexpression at the expenses of switched (IgM-IgD-) phenotypes of the memory subsets. Interestingly, in PHI patients a significant alteration of Immunoglobulin-expression was evident at BL in TLM cells, and after 4 weeks, despite treatment, in AM and RM subsets. After 48 weeks of therapy, Immunoglobulin-expression of AM and RM almost normalized, but remained perturbed in TLM cells in both groups. In conclusion, aberrant activated and exhausted B-cell phenotypes rose already during PHI, while most of the alterations in Ig-expression seen in CHI appeared later, despite 4 weeks of effective cART. After 48 weeks of cART B-cell subsets distribution improved although without full normalization, while Immunoglobulin-expression normalized among AM and RM, remaining perturbed in TLM B-cells of PHI and CHI.

γ-Tocopherol supplementation of allergic female mice augments development of CD11c+CD11b+ dendritic cells in utero and allergic inflammation in neonates

PubMed Central

Abdala-Valencia, Hiam; Soveg, Frank

2016-01-01

γ-Tocopherol increases responses to allergen challenge in allergic adult mice, but it is not known whether γ-tocopherol regulates the development of allergic disease. Development of allergic disease often occurs early in life. In clinical studies and animal models, offspring of allergic mothers have increased responsiveness to allergen challenge. Therefore, we determined whether γ-tocopherol augments development of allergic responses in offspring of allergic female mice. Allergic female mice were supplemented with γ-tocopherol starting at mating. The pups from allergic mothers developed allergic lung responses, whereas pups from saline-treated mothers did not respond to allergen challenge. The γ-tocopherol supplementation of allergic female mice increased the numbers of eosinophils twofold in the pup bronchoalveolar lavage and lungs after allergen challenge. There was also about a twofold increase in pup lung CD11b+ subsets of CD11c+ dendritic cells and in numbers of these dendritic cells expressing the transcription factor IRF4. There was no change in several CD11b− dendritic cell subsets. Furthermore, maternal supplementation with γ-tocopherol increased the number of fetal liver CD11b+CD11c+ dendritic cells twofold in utero. In the pups, γ-tocopherol increased lung expression of the inflammatory mediators CCL11, amphiregulin, activin A, and IL-5. In conclusion, maternal supplementation with γ-tocopherol increased fetal development of subsets of dendritic cells that are critical for allergic responses and increased development of allergic responses in pups from allergic mothers. These results have implications for supplementation of allergic mothers with γ-tocopherol in prenatal vitamins. PMID:26801566

[In vitro development and chimeric efficiency of mouse-porcine interspecies chimeric embryos in different culture systems].

PubMed

Wang, Ying; Ren, Jilong; Song, Yuran; Hai, Tang; Zhou, Qi; Liu, Zhonghua

2016-07-25

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human's, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.

The Crucial Role of Early Mitochondrial Injury in L-Lysine-Induced Acute Pancreatitis

PubMed Central

Biczó, György; Hegyi, Péter; Dósa, Sándor; Shalbuyeva, Natalia; Berczi, Sándor; Sinervirta, Riitta; Hracskó, Zsuzsanna; Siska, Andrea; Kukor, Zoltán; Jármay, Katalin; Venglovecz, Viktória; Varga, Ilona S.; Iványi, Béla; Alhonen, Leena; Wittmann, Tibor; Gukovskaya, Anna; Takács, Tamás

2011-01-01

Abstract Aims Large doses of intraperitoneally injected basic amino acids, L-arginine, or L-ornithine, induce acute pancreatitis in rodents, although the mechanisms mediating pancreatic toxicity remain unknown. Another basic amino acid, L-lysine, was also shown to cause pancreatic acinar cell injury. The aim of the study was to get insight into the mechanisms through which L-lysine damages the rat exocrine pancreas, in particular to characterize the kinetics of L-lysine-induced mitochondrial injury, as well as the pathologic responses (including alteration of antioxidant systems) characteristic of acute pancreatitis. Results We showed that intraperitoneal administration of 2 g/kg L-lysine induced severe acute necrotizing pancreatitis. L-lysine administration caused early pancreatic mitochondrial damage that preceded the activation of trypsinogen and the proinflammatory transcription factor nuclear factor-κB (NF-κB), which are commonly thought to play an important role in the development of acute pancreatitis. Our data demonstrate that L-lysine impairs adenosine triphosphate synthase activity of isolated pancreatic, but not liver, mitochondria. Innovation and Conclusion Taken together, early mitochondrial injury caused by large doses of L-lysine may lead to the development of acute pancreatitis independently of pancreatic trypsinogen and NF-κB activation. PMID:21644850

CD4 T Follicular Helper Cells and HIV Infection: Friends or Enemies?

PubMed

Moukambi, Félicien; Rodrigues, Vasco; Fortier, Yasmina; Rabezanahary, Henintsoa; Borde, Chloé; Krust, Bernard; Andreani, Guadalupe; Silvestre, Ricardo; Petrovas, Constantinos; Laforge, Mireille; Estaquier, Jérôme

2017-01-01

Follicular T helper (Tfh) cells, a subset of CD4 T lymphocytes, are essential for memory B cell activation, survival, and differentiation and assist B cells in the production of antigen-specific antibodies. Work performed in recent years pointed out the importance of Tfh cells in the context of HIV and SIV infections. The importance of tissue distribution of Tfh is also an important point since their frequency differs between peripheral blood and lymph nodes compared to the spleen, the primary organ for B cell activation, and differentiation. Our recent observations indicated an early and profound loss of splenic Tfh cells. The role of transcriptional activator and repressor factors that control Tfh differentiation is also discussed in the context of HIV/SIV infection. Because Tfh cells are important for B cell differentiation and antibody production, accelerating the Tfh responses early during HIV/SIV infection could be promising as novel immunotherapeutic approach or alternative vaccine strategies. However, because Tfh cells are infected during the HIV/SIV infection and represent a reservoir, this may interfere with HIV vaccine strategy. Thus, Tfh represent the good and bad guys during HIV infection.

CD4 T Follicular Helper Cells and HIV Infection: Friends or Enemies?

PubMed Central

Moukambi, Félicien; Rodrigues, Vasco; Fortier, Yasmina; Rabezanahary, Henintsoa; Borde, Chloé; Krust, Bernard; Andreani, Guadalupe; Silvestre, Ricardo; Petrovas, Constantinos; Laforge, Mireille; Estaquier, Jérôme

2017-01-01

Follicular T helper (Tfh) cells, a subset of CD4 T lymphocytes, are essential for memory B cell activation, survival, and differentiation and assist B cells in the production of antigen-specific antibodies. Work performed in recent years pointed out the importance of Tfh cells in the context of HIV and SIV infections. The importance of tissue distribution of Tfh is also an important point since their frequency differs between peripheral blood and lymph nodes compared to the spleen, the primary organ for B cell activation, and differentiation. Our recent observations indicated an early and profound loss of splenic Tfh cells. The role of transcriptional activator and repressor factors that control Tfh differentiation is also discussed in the context of HIV/SIV infection. Because Tfh cells are important for B cell differentiation and antibody production, accelerating the Tfh responses early during HIV/SIV infection could be promising as novel immunotherapeutic approach or alternative vaccine strategies. However, because Tfh cells are infected during the HIV/SIV infection and represent a reservoir, this may interfere with HIV vaccine strategy. Thus, Tfh represent the good and bad guys during HIV infection. PMID:28265271

Ultrastructural changes of goat corpus luteum during the estrous cycle.

PubMed

Jiang, Yi-Fan; Hsu, Meng-Chieh; Cheng, Chiung-Hsiang; Tsui, Kuan-Hao; Chiu, Chih-Hsien

2016-07-01

The present study was designed to study the ultrastructure of goat corpora lutea (CL, n=10) and structural changes as related to steroidogenic functions during the estrous cycle. The reproduction status of goats was estimated by analyzing serum progesterone concentrations. The CL at various stages was surgically collected. To characterize ultrastructural features associated with steroidogenesis, tissue and cellular structures were studied. Blood supplies were examined based on features of the endothelial cells and capillary structures in the CL. Activated endothelial cells and developing vessels were observed in the early stage, whereas mature endothelial cells, accumulating extracellular matrix fibers, and stabilized vessels were observed in the middle and late stages of assessment. In the late stage of assessment, shrunken goat luteal cells scattered around the capillaries were detected and formed circular regression areas. Features of autophagy and luteal cell apoptosis were noted. In large luteal cells, steroidogenic organelles were present, including microvillar channels, endoplasmic reticulum, and mitochondria. Conformational changes in the endoplasmic reticulum and increased mitochondria with tubular cristae were observed in the early-middle CL transitions. In contrast, mitochondria swelled and the cristae transformed to the lamellar type in the late stage, suggesting that organelle plasticity could contribute to steroidogenesis in goat CL. In conclusion, results suggest angiogenesis occurs in early developing CL and programmed cell death occurred in the late stage of CL assessment in the present study. Structures and quantiles of steroidogenic organelles are correlated with the steroidogenic functions in goats. Copyright © 2016 Elsevier B.V. All rights reserved.

Notch ligands Delta1 and Jagged1 transmit distinct signals to T-cell precursors

PubMed Central

Lehar, Sophie M.; Dooley, James; Farr, Andrew G.; Bevan, Michael J.

2009-01-01

Signaling through the Notch pathway plays an essential role in inducing T-lineage commitment and promoting the maturation of immature thymocytes. Using an in vitro culture system, we show that 2 different classes of Notch ligands, Jagged1 or Delta1, transmit distinct signals to T-cell progenitors. OP9 stromal cells expressing either Jagged1 or Delta1 inhibit the differentiation of DN1 thymocytes into the B-cell lineage, but only the Delta1-expressing stromal cells promote the proliferation and maturation of T-cell progenitors through the early double-negative (DN) stages of thymocyte development. Whereas the majority of bone marrow-derived stem cells do not respond to Jagged1 signals, T-cell progenitors respond to Jagged1 signals during a brief window of their development between the DN1 and DN3 stages of thymic development. During these stages, Jagged1 signals can influence the differentiation of immature thymocytes along the natural killer (NK) and γδ T-cell lineages. PMID:15486060

Blinatumomab for the treatment of acute lymphoblastic leukemia.

PubMed

Kaplan, Jason B; Grischenko, Marina; Giles, Francis J

2015-12-01

Acute lymphoblastic leukemia (ALL) is a potentially fatal disease that involves clonal expansion of early lymphoid progenitor cells. Much of drug development for ALL treatment involves targeting antigens of the clonal cell surface. Blinatumomab belongs to an emerging class of anti-cancer therapeutics referred to as bispecific T-cell engaging antibodies. The Food and Drug Administration approved its use in relapsed or refractory adult Philadelphia chromosome-negative B-cell precursor ALL in December of 2014. Blinatumomab contains both an anti-CD3 and anti-CD19 arm, allowing for the juxtaposition of CD3+ T-cells to malignant CD19+ B-cells, thereby resulting in granzyme- and perforin-mediated B-cell apoptosis. Preclinical studies suggest that blinatumomab's efficacy is related to the effector-to-target ratio and to the difference between its affinity for CD19 and CD3. Preclinical and early phase clinical studies have allowed for the characterization of the pharmacokinetics of blinatumomab, including the determination of its short half-life. The metabolic pathway has not been fully characterized but is thought to be similar to that of other antibodies. Phase I and II studies led to the identification of an ideal stepwise dose, involving long-term continuous intravenous infusion (CIVI), to optimize its efficacy and reduce the risk of certain toxicities. A high remission rate and duration were noted among a relapsed/refractory population of patients. The results of clinical trials have identified cytokine release syndrome and neurotoxicity, among others, as serious drug-related toxicities, leading to the institution of a Risk Evaluation and Mitigation Strategy. Blinatumomab represents a significant addition to the treatment options for ALL, but it is not without its limitations, of which are its short-half life, necessitating long-term CIVI, and the eventual emergence of CD19-negative clones. Continual development of the agent involves assessing its role in the frontline setting and in combination with chemotherapy.

The Deadly Dance of B Cells with Trypanosomatids.

PubMed

Silva-Barrios, Sasha; Charpentier, Tania; Stäger, Simona

2018-02-01

B cells are notorious actors for the host's protection against several infectious diseases. So much so that early vaccinology seated its principles upon their long-term protective antibody secretion capabilities. Indeed, there are many examples of acute infectious diseases that are combated by functional humoral responses. However, some chronic infectious diseases actively induce immune deregulations that often lead to defective, if not deleterious, humoral immune responses. In this review we summarize how Leishmania and Trypanosoma spp. directly manipulate B cell responses to induce polyclonal B cell activation, hypergammaglobulinemia, low-specificity antibodies, limited B cell survival, and regulatory B cells, contributing therefore to immunopathology and the establishment of persistent infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

Early onset of puberty and early ovarian failure in CYP7B1 knockout mice

PubMed Central

Omoto, Yoko; Lathe, Richard; Warner, Margaret; Gustafsson, Jan-Åke

2005-01-01

CYP7B1 is the enzyme responsible for hydroxylation and termination of the estrogenic actions of the androgen metabolite, 5α-androstane-3β, 17β-diol (3βAdiol). 3βAdiol is estrogenic in ERα or ERβ positive cells only if they do not express CYP7B1. In this study we show that female CYP7B1–/– mice experience early onset of growth of the uterus and mammary glands and commence estrus cycles 2 days earlier than their wild-type littermates. Adult mammary glands and uteri appear to be under continuous estrogenic stimulation. We conclude that, by cell-specific regulation of the estrogenicity of 3βAdiol, CYP7B1 performs two major tasks: (i) it allows 3βAdiol to have growth inhibitory effects through ERβ and (ii) it permits estradiol-specific activation of estrogen receptors by protection of certain cells from the estrogenic effects of 3βAdiol. When CYP7B1 is inactivated, 3βAdiol activates estrogen receptors indiscriminately, and the overall effect is prolonged and inappropriate exposure to estrogen. PMID:15710898

CD22 is required for formation of memory B cell precursors within germinal centers.

PubMed

Chappell, Craig P; Draves, Kevin E; Clark, Edward A

2017-01-01

CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens.

CD22 is required for formation of memory B cell precursors within germinal centers

PubMed Central

Chappell, Craig P.; Draves, Kevin E.

2017-01-01

CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens. PMID:28346517

Human IL-21 and IL-21R deficiencies: two novel entities of primary immunodeficiency.

PubMed

Kotlarz, Daniel; Ziętara, Natalia; Milner, Joshua D; Klein, Christoph

2014-12-01

This review highlights the recent identification of human interleukin-21 (IL-21) and interleukin-21 receptor (IL-21R) deficiencies as novel entities of primary immunodeficiency. We recently described the first patients with IL-21R deficiency who had cryptosporidial infections associated with chronic cholangitis and liver disease. All IL-21R-deficient patients suffered from recurrent respiratory tract infections. Immunological work-up revealed impaired B cell proliferation and immunoglobulin class-switch, reduced T cell effector functions, and variable natural killer cell dysfunctions. Recently, these findings have been extended by the discovery of one patient with a mutation in the IL21 gene. This patient predominantly manifested with very early onset inflammatory bowel disease and recurrent respiratory infections. Laboratory examination showed reduced circulating B cells and impaired B cell class-switch. Human IL-21 and IL-21R deficiencies cause severe, primary immunodeficiency reminiscent of common variable immunodeficiency. Early diagnosis is critical to prevent life-threatening complications, such as secondary liver failure. In view of the critical role of IL-21 in controlling immune homeostasis, early hematopoietic stem cell transplantation might be considered as therapeutic intervention in affected children.

Cyclophilin B facilitates the replication of Orf virus.

PubMed

Zhao, Kui; Li, Jida; He, Wenqi; Song, Deguang; Zhang, Ximu; Zhang, Di; Zhou, Yanlong; Gao, Feng

2017-06-15

Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID 50 ) assay and qRT-PCR detection. In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

In vitro Reactivity to Implant Metals Demonstrates a Person Dependent Association with both T-Cell and B-Cell Activation

PubMed Central

Hallab, Nadim James; Caicedo, Marco; Epstein, Rachael; McAllister, Kyron; Jacobs, Joshua J

2009-01-01

Hypersensitivity to metallic implants remains relatively unpredictable and poorly understood. We initially hypothesized that metal-induced lymphocyte proliferation responses to soluble metal challenge (ions) are mediated exclusively by early T-cell activation (not B-cells), typical of a Delayed-Type-Hypersensitivity response. We tested this by comparing proliferation (6-days) of primary lymphocytes with early T-cell and B-cell activation (48-hours) in three groups of subjects likely to demonstrate elevated metal-reactivity: Group 1(n=12) history of metal-sensitivity with no implant; Group 2a(n=6) well performing metal-on-metal THRs, and Group 2b(n=20) subjects with poorly performing metal-on-polymer total joint arthroplasties (TJA). Group 1 showed 100%(12/12) metal reactivity (Stimulation Index>2) to Ni. Group 2a&2b were 83%(5/6) and 75%(15/22) metal reactive (to Co, Cr or Ni) respectively. Of the n=32 metal reactive subjects to Co, Cr or Ni (SI>2), n=22/32 demonstrated >2-fold elevations in % of T-cell or B-cell activation (CD25+,CD69+) to metal challenge compared to untreated control. 18/22 metal-activated subjects demonstrated an exclusively T-cell or B-cell activation response to metal challenge, where 6/18 demonstrated exclusively B-cell activation and 12/18 demonstrated a T-cell only response, as measured by surface activation markers CD25+ and CD69+. However, there was no direct correlation (R2<0.1) between lymphocyte proliferation and % T-cell or B-cell activation (CD25+:CD69+). Proliferation assays (LTT) showed greater ability to detect metal reactivity than did subject-dependent results of flow-cytometry analysis of T-cell or B-cell activation. The high incidence of lymphocyte reactivity and activation, indicate that more complex than initially hypothesized immune responses may contribute to the etiology of debris induced osteolysis in metal-sensitive individuals. PMID:19235773

Innate lymphoid cells, precursors and plasticity.

PubMed

Gronke, Konrad; Kofoed-Nielsen, Michael; Diefenbach, Andreas

2016-11-01

Innate lymphoid cells (ILC) have only recently been recognized as a separate entity of the lymphoid lineage. Their subpopulations share common characteristics in terms of early development and major transcriptional circuitry with their related cousins of the T cell world. It is currently hypothesized that ILCs constitute an evolutionary older version of the lymphoid immune system. They are found at all primary entry points for pathogens such as mucosal surfaces of the lung and gastrointestinal system, the skin and the liver, which is the central contact point for pathogens that breach the intestinal barrier and enter the circulation. There, ILC contribute to the first line defense as well as to organ homeostasis. However, ILC are not only involved in classical defense tasks, but also contribute to the organogenesis of lymphoid organs as well as tissue remodeling and even stem cell regeneration. ILC may, therefore, implement different functions according to their emergence in ontogeny, their development and their final tissue location. We will review here their early development from precursors of the fetal liver and the adult bone marrow as well as their late plasticity in adaptation to their environment. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species.

PubMed

De Milito, Angelo; Iessi, Elisabetta; Logozzi, Mariantonia; Lozupone, Francesco; Spada, Massimo; Marino, Maria Lucia; Federici, Cristina; Perdicchio, Maurizio; Matarrese, Paola; Lugini, Luana; Nilsson, Anna; Fais, Stefano

2007-06-01

Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI.

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells.

PubMed

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G(0)/G(1) phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. Copyright © 2012 Elsevier B.V. All rights reserved.

Early low-grade gastric MALToma rarely transforms into diffuse large cell lymphoma or progresses beyond the stomach and regional lymph nodes.

PubMed

Liu, Ting-Yun; Dei, Pei-Han; Kuo, Sung-Hsin; Lin, Chung-Wu

2010-06-01

Gastric mucosa-associated lymphoid tissue lymphoma (MALToma) usually presents at an early stage involving only the stomach and/or regional lymph nodes. Although a sequential transformation from low-grade gastric MALToma (GM) to high-grade GM to secondary diffuse large B-cell lymphoma (DLBCL) is commonly assumed, documented cases of transformation are rare. We aim to determine the frequency of transformation. We identified 55 early low-grade GMs, 18 early high-grade GMs, and 13 advanced GMs at the National Taiwan University Hospital from 1995 to 2005. The median follow-up time was 59 months. We found that only one early low-grade GM and two early high-grade GMs transformed into secondary DLBCLs and progressed outside the stomach and regional lymph nodes. Significantly, we identified 13 low-grade GMs that were refractory to Helicobacter eradication therapy or relapsed after initial response. All 13 cases had been followed-up for at least 3 years without development of secondary DLBCLs. The frequency of transformation for early low-grade GM was less than 2% (1/55). Although two lymphoma-unrelated mortalities were identified, none of the 55 patients with early-low grade GMs died of the disease. Compared with chronic lymphocytic leukemia, which has a 16% transformation rate and a median transformation time of 24 months, we conclude that early low-grade GM rarely transforms into secondary DLBCL or progresses beyond the stomach. Without transformation or progression, patients with early low-grade GM rarely die of the disease and should be treated conservatively. Copyright (c) 2010 Formosan Medical Association & Elsevier. Published by Elsevier B.V. All rights reserved.

E2f1–3 Are Critical for Myeloid Development*

PubMed Central

Trikha, Prashant; Sharma, Nidhi; Opavsky, Rene; Reyes, Andres; Pena, Clarissa; Ostrowski, Michael C.; Roussel, Martine F.; Leone, Gustavo

2011-01-01

Hematopoietic development involves the coordinated activity of differentiation and cell cycle regulators. In current models of mammalian cell cycle control, E2f activators (E2f1, E2f2, and E2f3) are portrayed as the ultimate transcriptional effectors that commit cells to enter and progress through S phase. Using conditional gene knock-out strategies, we show that E2f1–3 are not required for the proliferation of early myeloid progenitors. Rather, these E2fs are critical for cell survival and proliferation at two distinct steps of myeloid development. First, E2f1–3 are required as transcriptional repressors for the survival of CD11b+ myeloid progenitors, and then they are required as activators for the proliferation of CD11b+ macrophages. In bone marrow macrophages, we show that E2f1–3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation. Together, these findings expose dual functions for E2f1–3 at distinct stages of myeloid development in vivo, first as repressors in cell survival and then as activators in cell proliferation. In summary, this work places E2f1–3 in a specific signaling cascade that is critical for myeloid development in vivo. PMID:21115501

High-throughput sequencing of the T cell receptor β gene identifies aggressive early-stage mycosis fungoides.

PubMed

de Masson, Adele; O'Malley, John T; Elco, Christopher P; Garcia, Sarah S; Divito, Sherrie J; Lowry, Elizabeth L; Tawa, Marianne; Fisher, David C; Devlin, Phillip M; Teague, Jessica E; Leboeuf, Nicole R; Kirsch, Ilan R; Robins, Harlan; Clark, Rachael A; Kupper, Thomas S

2018-05-09

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor β gene ( TCRB ) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

CARs and other T cell therapies for MM: The clinical experience.

PubMed

Danhof, Sophia; Hudecek, Michael; Smith, Eric L

2018-06-01

Harnessing the endogenous immune system to eliminate malignant cells has long been an intriguing approach. After considerable success in the treatment of B-cell acute lymphoblastic leukemia, chimeric antigen receptor (CAR)-modified T cells have entered early clinical evaluation in the field of multiple myeloma (MM). The choice of suitable non-CD19 target antigens is challenging and a variety of myeloma-associated surface molecules have been under preclinical investigation. Most recent clinical protocols have focused on targeting B-cell maturation antigen (BCMA), and early results are promising. The trials differ in receptor constructs, patient selection, dosing strategies and conditioning chemotherapy and will thus pave the way to eventually define the optimal parameters. Other sources for autologous T-cell therapy of MM include affinity-enhanced T-cell receptor-modified cells and marrow infiltrating lymphocytes. In summary, adoptive T-cell transfer for the treatment of MM is still in its infancy, but if early response rates indicate durability, will be a paradigm changing therapeutic modality for the treatment of MM. Copyright © 2018. Published by Elsevier Ltd.

Development of Technologies for Early Detection and Stratification of Breast Cancer

DTIC Science & Technology

2014-10-01

cancer. Similar screening has been done in urine samples with CYR61 and LCN2 (Figure 2a-b). The Moses lab provided breast cancer urine samples and two ...diseased samples, while CYR61 appears to have two different populations whereas diseased urine samples have lower levels of the protein present. More... system for eDAR so that each isolated cell can be deposited into a well of a 96-well plate for high-throughput downstream single-cell digital

The pathogenic role of persistent milk signaling in mTORC1- and milk-microRNA-driven type 2 diabetes mellitus.

PubMed

Melnik, Bodo C

2015-01-01

Milk, the secretory product of the lactation genome, promotes growth of the newborn mammal. Milk delivers insulinotropic amino acids, thus maintains a molecular crosstalk with the pancreatic β-cell of the milk recipient. Homeostasis of β-cells and insulin production depend on the appropriate magnitude of mTORC1 signaling. mTORC1 is activated by branched-chain amino acids (BCAAs), glutamine, and palmitic acid, abundant nutrient signals of cow´s milk. Furthermore, milk delivers bioactive exosomal microRNAs. After milk consumption, bovine microRNA-29b, a member of the diabetogenic microRNA-29- family, reaches the systemic circulation and the cells of the milk consumer. MicroRNA-29b downregulates branchedchain α-ketoacid dehydrogenase, a potential explanation for increased BCAA serum levels, the metabolic signature of insulin resistance and type 2 diabetes mellitus (T2DM). In non-obese diabetic mice, microRNA-29b downregulates the antiapoptotic protein Mcl-1, which leads to early β-cell death. In all mammals except Neolithic humans, milk-driven mTORC1 signaling is physiologically restricted to the postnatal period. In contrast, chronic hyperactivated mTORC1 signaling has been associated with the development of age-related diseases of civilization including T2DM. Notably, chronic hyperactivation of mTORC1 enhances endoplasmic reticulum stress that promotes apoptosis. In fact, hyperactivated β-cell mTORC1 signaling induced early β-cell apoptosis in a mouse model. The EPIC-InterAct Study demonstrated an association between milk consumption and T2DM in France, Italy, United Kingdom, Germany, and Sweden. In contrast, fermented milk products and cheese exhibit an inverse correlation. Since the early 1950´s, refrigeration technology allowed widespread consumption of fresh pasteurized milk, which facilitates daily intake of bioactive bovine microRNAs. Persistent uptake of cow´s milk-derived microRNAs apparently transfers an overlooked epigenetic diabetogenic program that should not reach the human food chain.

Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

PubMed

Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

2016-06-15

Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

The transcription factor, T-bet, primes intestine transplantation rejection and is associated with disrupted mucosal homeostasis.

PubMed

Ranganathan, Sarangarajan; Ashokkumar, Chethan; Ningappa, Mylarappa; Schmitt, Lori; Higgs, Brandon W; Sindhi, Rakesh

2015-04-01

The transcription factor, t-bet, promotes inflammatory polarization and intestinal homing of many inflammatory cells. In previous studies, the t-bet and granulysin genes were upregulated in peripheral blood before and after intestine transplantation (ITx) rejection, but not during rejection, possibly because of sequestration in allograft mucosa. Mucosal sequestration of t-bet and granulysin may also explain the presence of inflammatory CD14+ monocyte-derived macrophages (MDM) and immunoglobulin G+ B-cell lineage cells, and loss of mature non-inflammatory CD138+ plasma cells in allograft mucosa during ITx rejection in these previous studies. T-bet-stained and granulysin-stained cells, MDM and CD138+ plasma cells were evaluated with immunohistochemistry in serial biopsies from 17 children, in whom changes in MDM and CD138+ plasma cells were observed previously. T-bet-positive mucosal cells were significantly higher in postperfusion (P = 0.035) and early posttransplant biopsies (P = 0.016) among rejectors, compared with nonrejectors. T-bet-positive cell counts per high-power field (hpf) were (a) positively correlated with MDM counts/hpf in postperfusion (Spearman r = 0.73; P = 0.01) and early posttransplant biopsies (r = 0.54, r = 0.046), and (b) negatively correlated with CD138+B-/pre-plasma cells in early posttransplant biopsies (r = 0.63, P = 0.038). T-bet expression in CD14+ monocytes, CD19+B cells, and several other leukocyte subsets was higher in random blood samples from two rejectors, compared with those from five normal human subjects and three nonrejectors. Scant granulysin-stained mucosal cells precluded additional evaluation of this cytotoxin and its role in ITx rejection. The transcription factor, t-bet, primes ITx rejection, and associates with disrupted homeostatic relationships between innate and adaptive immune cells in the allograft mucosa during rejection.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

PubMed

Kimber, S J; Sneddon, S F; Bloor, D J; El-Bareg, A M; Hawkhead, J A; Metcalfe, A D; Houghton, F D; Leese, H J; Rutherford, A; Lieberman, B A; Brison, D R

2008-05-01

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

PubMed Central

Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

2017-01-01

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target. PMID:28881635

Antibody-Independent Control of γ-Herpesvirus Latency via B Cell Induction of Anti-Viral T Cell Responses

PubMed Central

McClellan, Kelly B; Gangappa, Shivaprakash; Speck, Samuel H; Virgin, Herbert W.

2006-01-01

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine γ-herpesvirus 68 (γHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell−/− mice during the early phase of γHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell−/− mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of γ-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection. PMID:16789842

miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification.

PubMed

Nissan, Xavier; Denis, Jérôme Alexandre; Saidani, Manoubia; Lemaitre, Gilles; Peschanski, Marc; Baldeschi, Christine

2011-08-15

The molecular mechanisms controlling the differentiation of human basal keratinocyte stem cells towards the epidermis are well characterized, whereas the earliest process leading to the specification of embryonic stem cells into keratinocytes is still not well understood. MicroRNAs are regulators of many cellular events, but evidence for microRNA acting on the differentiation of human embryonic stem cells into a specific lineage has been elusive. By using our recent protocol for obtaining functional keratinocytes from hESC, we attempted to analyze the role of microRNAs in the early stages of epidermal differentiation. Thus, we identified a set of 5 microRNAs, namely miR-200a, miR-200b, miR-203, miR-205 and miR-429, that are specifically overexpressed during the early stages of the differentiation process. Interestingly, our functional analyses revealed an instrumental role of miR-203, which had been previously shown to play a key role during the formation of the pluristratified epidermis by basal keratinocyte stem cells, in the early keratinocyte commitment. These results highlight the determinant and unique role of miR-203 during the entire process of epidermal development by extending its spectrum of action from the early commitment of embryonic stem cells to ultimate differentiation of the organ. Copyright © 2011 Elsevier Inc. All rights reserved.

The Pseudopod System for Axon-Glia Interactions: Stimulation and Isolation of Schwann Cell Protrusions that Form in Response to Axonal Membranes.

PubMed

Poitelon, Yannick; Feltri, M Laura

2018-01-01

In the peripheral nervous system, axons dictate the differentiation state of Schwann cells. Most of this axonal influence on Schwann cells is due to juxtacrine interactions between axonal transmembrane molecules (e.g., the neuregulin growth factor) and receptors on the Schwann cell (e.g., the ErbB2/ErbB3 receptor). The fleeting nature of this interaction together with the lack of synchronicity in the development of the Schwann cell population limits our capability to study this phenomenon in vivo. Here we present a simple Boyden Chamber-based method to study this important cell-cell interaction event. We isolate the early protrusions of Schwann cells that are generated in response to juxtacrine stimulation by sensory neuronal membranes. This method is compatible with a large array of current biochemical analyses and provides an effective approach to study biomolecules that are differentially localized in Schwann cell protrusions and cell bodies in response to axonal signals. A similar approach can be extended to different kinds of cell-cell interactions.

A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

PubMed Central

Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

2014-01-01

In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 μM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ∼300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 μM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (μTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings. PMID:24967604

The TEL-AML1 fusion protein of acute lymphoblastic leukemia modulates IRF3 activity during early B-cell differentiation.

PubMed

de Laurentiis, A; Hiscott, J; Alcalay, M

2015-12-03

The t(12;21) translocation is the most common genetic rearrangement in childhood acute lymphoblastic leukemia (ALL) and gives rise to the TEL-AML1 fusion gene. Many studies on TEL-AML1 describe specific properties of the fusion protein, but a thorough understanding of its function is lacking. We exploited a pluripotent hematopoietic stem/progenitor cell line, EML1, and generated a cell line (EML-TA) stably expressing the TEL-AML1 fusion protein. EML1 cells differentiate to mature B-cells following treatment with IL7; whereas EML-TA display an impaired differentiation capacity and remain blocked at an early stage of maturation. Global gene expression profiling of EML1 cells at different stages of B-lymphoid differentiation, compared with EML-TA, identified the interferon (IFN)α/β pathway as a primary target of repression by TEL-AML1. In particular, expression and phosphorylation of interferon-regulatory factor 3 (IRF3) was decreased in EML-TA cells; strikingly, stable expression of IRF3 restored the capacity of EML-TA cells to differentiate into mature B-cells. Similarly, IRF3 silencing in EML1 cells by siRNA was sufficient to block B-lymphoid differentiation. The ability of TEL-AML1 to block B-cell differentiation and downregulate the IRF3-IFNα/β pathway was confirmed in mouse and human primary hematopoietic precursor cells (Lin- and CD34+ cells, respectively), and in a patient-derived cell line expressing TEL-AML1 (REH). Furthermore, treatment of TEL-AML1 expressing cells with IFNα/β was sufficient to overcome the maturation block. Our data provide new insight on TEL-AML1 function and may offer a new therapeutic opportunity for B-ALL.

Plasmodium falciparum-Infected Erythrocytes Induce Granzyme B by NK Cells through Expression of Host-Hsp70

PubMed Central

Böttger, Evelyn; Multhoff, Gabriele; Kun, Jürgen F. J.; Esen, Meral

2012-01-01

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo. PMID:22438997

Plasmodium falciparum-infected erythrocytes induce granzyme B by NK cells through expression of host-Hsp70.

PubMed

Böttger, Evelyn; Multhoff, Gabriele; Kun, Jürgen F J; Esen, Meral

2012-01-01

In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

PubMed

de Andres, María C; Perez-Pampin, Eva; Calaza, Manuel; Santaclara, Francisco J; Ortea, Ignacio; Gomez-Reino, Juan J; Gonzalez, Antonio

2015-08-29

DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results. Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR). Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression. Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

Zebrafish Lmx1b.1 and Lmx1b.2 are required for maintenance of the isthmic organizer.

PubMed

O'Hara, F Patrick; Beck, Ernestine; Barr, Lauren K; Wong, Lily L; Kessler, Daniel S; Riddle, Robert D

2005-07-01

The mesencephalic and metencephalic region (MMR) of the vertebrate central nervous system develops in response to signals produced by the isthmic organizer (IsO). We have previously reported that the LIM homeobox transcription factor Lmx1b is expressed within the chick IsO, where it is sufficient to maintain expression of the secreted factor wnt1. In this paper, we show that zebrafish express two Lmx1b orthologs, lmx1b.1 and lmx1b.2, in the rostral IsO, and demonstrate that these genes are necessary for key aspects of MMR development. Simultaneous knockdown of Lmx1b.1 and Lmx1b.2 using morpholino antisense oligos results in a loss of wnt1, wnt3a, wnt10b, pax8 and fgf8 expression at the IsO, leading ultimately to programmed cell death and the loss of the isthmic constriction and cerebellum. Single morpholino knockdown of either Lmx1b.1 or Lmx1b.2 has no discernible effect on MMR development. Maintenance of lmx1b.1 and lmx1b.2 expression at the isthmus requires the function of no isthmus/pax2.1, as well as Fgf signaling. Transient misexpression of Lmx1b.1 or Lmx1b.2 during early MMR development induces ectopic wnt1 and fgf8 expression in the MMR, as well as throughout much of the embryo. We propose that Lmx1b.1- and Lmx1b.2-mediated regulation of wnt1, wnt3a, wnt10b, pax8 and fgf8 maintains cell survival in the isthmocerebellar region.

Acute myeloid/T-lymphoblastic leukaemia (AMTL): a distinct category of acute leukaemias with common pathogenesis in need of improved therapy.

PubMed

Gutierrez, Alejandro; Kentsis, Alex

2018-03-01

Advances in the classification of acute leukaemias have led to improved outcomes for a substantial fraction of patients. However, chemotherapy resistance remains a major problem for specific subsets of acute leukaemias. Here, we propose that a molecularly distinct subtype of acute leukaemia with shared myeloid and T cell lymphoblastic features, which we term acute myeloid/T-lymphoblastic leukaemia (AMTL), is divided across 3 diagnostic categories owing to variable expression of markers deemed to be defining of myeloid and T-lymphoid lineages, such as myeloperoxidase and CD3. This proposed diagnostic group is supported by (i) retained myeloid differentiation potential during early T cell lymphoid development, (ii) recognition that some cases of acute myeloid leukaemia (AML) harbour hallmarks of T cell development, such as T-cell receptor gene rearrangements and (iii) common gene mutations in subsets of AML and T cell acute lymphoblastic leukaemia (T-ALL), including WT1, PHF6, RUNX1 and BCL11B. This proposed diagnostic entity overlaps with early T cell precursor (ETP) T-ALL and T cell/myeloid mixed phenotype acute leukaemias (MPALs), and also includes a subset of leukaemias currently classified as AML with features of T-lymphoblastic development. The proposed classification of AMTL as a distinct entity would enable more precise prospective diagnosis and permit the development of improved therapies for patients whose treatment is inadequate with current approaches. © 2018 John Wiley & Sons Ltd.

Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early and chronic hidradenitis suppurativa lesions.

PubMed

van der Zee, H H; de Ruiter, L; Boer, J; van den Broecke, D G; den Hollander, J C; Laman, J D; Prens, E P

2012-01-01

Current insight into the histopathological course of events during disease progression in hidradenitis suppurativa (HS) is fragmentary. To identify histological alterations and leucocyte subsets in normal-appearing perilesional skin, and early and chronic HS lesions. In this observational study we examined eight perilesional skin samples, and six early and 10 chronic prototypic HS lesions, as well as skin samples from four healthy donors using in situ immunostaining. Perilesional skin showed mild psoriasiform hyperplasia and follicular plugging as well as a low-grade influx of tryptase-positive mast cells, CD3+ T cells, CD138+ plasma cells and factor XIIIa+ dendritic cells. In early HS lesions, neutrophilic abscess formation and influx of mainly macrophages, monocytes and dendritic cells predominated. In chronic disease, the infiltrate expanded with markedly increased frequencies of CD20+ and CD79a+ B cells and CD138+ plasma cells. As in early lesions, free keratin fibres were detected in the dermis and within giant cells. Single detached keratinocytes and strands of follicular epithelium were observed in the dermis, the latter frequently expressing Ki67, indicative of active proliferation. Psoriasiform hyperplasia, follicular plugging and low-grade leucocytic infiltration are already present in normal-appearing perilesional skin. Keratin fibres in the dermis are associated with clinical disease. Early lesions are characterized by neutrophilic abscess formation and influx of mainly histiocytes, and chronic lesions mainly by expansion of B cells and plasma cells in 'pseudo' follicles. Proliferating strands of follicular epithelium may initiate fistula formation. Mast cells are increased in all stages of HS including perilesional skin. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif*

PubMed Central

Siponen, Marina I.; Wisniewska, Magdalena; Lehtiö, Lari; Johansson, Ida; Svensson, Linda; Raszewski, Grzegorz; Nilsson, Lennart; Sigvardsson, Mikael; Berglund, Helena

2010-01-01

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family. PMID:20592035

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation

PubMed Central

Reddy, Jay P.; Peddibhotla, Sirisha; Bu, Wen; Zhao, Jing; Haricharan, Svasti; Du, Yi-Chieh Nancy; Podsypanina, Katrina; Rosen, Jeffrey M.; Donehower, Larry A.; Li, Yi

2010-01-01

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention. PMID:20133707

Two distinct auto-regulatory loops operate at the PU.1 locus in B cells and myeloid cells

PubMed Central

Leddin, Mathias; Perrod, Chiara; Hoogenkamp, Maarten; Ghani, Saeed; Assi, Salam; Heinz, Sven; Wilson, Nicola K.; Follows, George; Schönheit, Jörg; Vockentanz, Lena; Mosammam, Ali M.; Chen, Wei; Tenen, Daniel G.; Westhead, David R.; Göttgens, Berthold

2011-01-01

The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type–specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements. PMID:21239694

Detection of early changes in lung cell cytology by flow-systems analysis techniques. [Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steinkamp, J.A.; Wilson, J.S.; Svitra, Z.V.

1980-03-01

Ongoing experiments designed to develop automated flow-analysis methods for assaying damage to pulmonary lavage cells in experimental animals exposed by inhalation to environmental pollutants are summarized. Pulmonary macrophages were characterized on their ability to phagocytize polystyrene latex fluorescent spheres. Lung cells consisting primarily of macrophages and leukocytes were analyzed for fluorescence (phagocytosis of spheres) and size using flow cytometric methods. Studies also concentrated on combining phagocytosis with other cellular parameters (DNA content, cell viability, and B-glucuronidase activity). As baseline studies are completed in normal animals, experimental animals will be exposed to gaseous and particulate environmental pollutants. (ERB

Reference spectra from squamous epithelium and connective tissue allow whole section proteomics analysis.

PubMed

Roesch-Ely, Mariana; Schnölzer, Martina; Nees, Matthias; Plinkert, Peter K; Bosch, Franz X

2010-01-01

We reasoned that micro-dissection of tumour cells for protein expression studies should be omitted since tumour-stroma interactions are an important part of the biology of solid tumours. To study such interactions in head and neck squamous cell carcinoma (HNSCC) development, we generated reference protein spectra for normal squamous epithelium and connective tissue by SELDI-TOF-MS. Calgranulins A and B, Annexin1 and Histone H4 were found to be strongly enriched in the epithelium. The alpha-defensins 1-3 and the haemoglobin subunits were identified in the connective tissue. Tumour-distant epithelia, representing early pre-malignant lesions, showed up-regulated expression of the stromal alpha-defensins, whereas the epithelial Annexin 1 was down-regulated. Thus, tumour microenvironment interactions occur very early in the carcinogenic process. These data demonstrate that omitting micro-dissection is actually beneficial for studying changes in protein expression during development and progression of solid tumours.

Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1.

PubMed

Schwenk, Robert; Banania, Glenna; Epstein, Judy; Kim, Yohan; Peters, Bjoern; Belmonte, Maria; Ganeshan, Harini; Huang, Jun; Reyes, Sharina; Stryhn, Anette; Ockenhouse, Christian F; Buus, Soren; Richie, Thomas L; Sedegah, Martha

2013-10-29

Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans. Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated. Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity. The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.

Whole-genome fingerprint of the DNA methylome during human B cell differentiation.

PubMed

Kulis, Marta; Merkel, Angelika; Heath, Simon; Queirós, Ana C; Schuyler, Ronald P; Castellano, Giancarlo; Beekman, Renée; Raineri, Emanuele; Esteve, Anna; Clot, Guillem; Verdaguer-Dot, Néria; Duran-Ferrer, Martí; Russiñol, Nuria; Vilarrasa-Blasi, Roser; Ecker, Simone; Pancaldi, Vera; Rico, Daniel; Agueda, Lidia; Blanc, Julie; Richardson, David; Clarke, Laura; Datta, Avik; Pascual, Marien; Agirre, Xabier; Prosper, Felipe; Alignani, Diego; Paiva, Bruno; Caron, Gersende; Fest, Thierry; Muench, Marcus O; Fomin, Marina E; Lee, Seung-Tae; Wiemels, Joseph L; Valencia, Alfonso; Gut, Marta; Flicek, Paul; Stunnenberg, Hendrik G; Siebert, Reiner; Küppers, Ralf; Gut, Ivo G; Campo, Elías; Martín-Subero, José I

2015-07-01

We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.

Characterization of Betula platyphylla gene transcripts associated with early development of male inflorescence.

PubMed

Xing, Lei; Liu, Xue-Mei

2012-02-01

Birch (Betula platyphylla), an eminent tree species in Northeast and Inner Mongolia of China, has been widely used in architecture, furniture, and paper making in recent years. In order to retrieve genes involved in early development of B. platyphylla male inflorescence, RNA populations extracted from early and late developmental stage were analyzed by cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique. Following amplification of 256 pairs of primer combinations, ~7000 fragments were generated, of which 350 transcripts expressing more in early stage than late. Of 350 specific transcripts, 198 clear and reproducible electrophoresis bands were retrieved and sequenced successfully, 74 of them (37%) showing significant homologies to known genes after GO annotation. Majority of the predicted gene products were involved in metabolism (24.56%), cellular process (27.19%), response to stimulus (11.4%) and cell growth (8.7%). Transcripts ME56, ME108, ME206 and ME310, representing metabolism, cellular process, response to stimulus and cell growth, respectively, were selected for further study to validate cDNA-AFLP expression patterns via RT-PCR and qRT-PCR analysis. RT-PCR and qRT-PCR expression pattern results were consistent with cDNA-AFLP analysis results.

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

PubMed

Vasseur, Romain; Skrypek, Nicolas; Duchêne, Belinda; Renaud, Florence; Martínez-Maqueda, Daniel; Vincent, Audrey; Porchet, Nicole; Van Seuningen, Isabelle; Jonckheere, Nicolas

2015-12-01

The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

The differential expression of EphB2 and EphB4 receptor kinases in normal bladder and in transitional cell carcinoma of the bladder.

PubMed

Li, Xiuqing; Choi, Wesley W; Yan, Rui; Yu, Haiyang; Krasnoperov, Valery; Kumar, S Ram; Schuckman, Anne; Klumpp, David J; Pan, Chong-Xian; Quinn, David; Gill, Inderbir S; Gill, Parkash S; Liu, Ren

2014-01-01

Effective treatment of transitional cell carcinoma (TCC) of the bladder requires early diagnosis. Identifying novel molecular markers in TCC would guide the development of diagnostic and therapeutic targets. Ephrins mediate signals via tyrosine kinase activity that modulates diverse physiologic and developmental processes, and ephrins are increasingly implicated in carcinogenesis. The aim of our study was to examine the differential regulation of EphB4 and EphB2 in normal bladder and in TCC of the bladder in 40 patients undergoing radical cystectomy for curative intent. Immunostaining and Western blotting revealed that normal urothelium expresses EphB2 (20 of 24 cases, 83% of the time) not EphB4 (0 of 24 cases, 0%). In sharp contrast, TCC specimens show loss of EphB2 expression (0 of 34 cases, 0%) and gain of EphB4 expression (32 of 34, 94%). Furthermore, EphB4 signal strength statistically correlated with higher tumor stage, and trended toward the presence of carcinoma in situ (CIS). These results are confirmed by analysis of normal urothelial and tumor cell lines. EphB2 is not a survival factor in normal urothelium, while EphB4 is a survival factor in TCC. Treatment of bladder tumor xenograft with an EphB4 inhibitor sEphB4-HSA leads to 62% tumor regression and complete remission when combined with Bevacizumab. Furthermore, tissue analysis revealed that sEphB4-HSA led to increased apoptosis, decreased proliferation, and reduced vessel density, implicating direct tumor cell targeting as well as anti-angiogenesis effect. In summary loss of EphB2 and gain of EphB4 expression represents an inflection point in the development, growth and possibly progression of TCC. Therapeutic compounds targeting EphB4 have potential for diagnosing and treating TCC.

CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients

PubMed Central

Turtle, Cameron J.; Hanafi, Laïla-Aïcha; Berger, Carolina; Gooley, Theodore A.; Cherian, Sindhu; Hudecek, Michael; Sommermeyer, Daniel; Melville, Katherine; Pender, Barbara; Budiarto, Tanya M.; Robinson, Emily; Steevens, Natalia N.; Chaney, Colette; Soma, Lorinda; Chen, Xueyan; Li, Daniel; Cao, Jianhong; Heimfeld, Shelly; Jensen, Michael C.; Riddell, Stanley R.; Maloney, David G.

2016-01-01

BACKGROUND. T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR–T cell products were prepared from unselected T cells. METHODS. We conducted a clinical trial to evaluate CD19 CAR–T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy. RESULTS. The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR–T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR–T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell–mediated anti-CAR transgene product immune responses developed after CAR–T cell infusion in some patients, limited CAR–T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR–T cell persistence and disease-free survival. CONCLUSION. Immunotherapy with a CAR–T cell product of defined composition enabled identification of factors that correlated with CAR–T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR–T cell dosing strategies that mitigated toxicity and improved disease-free survival. TRIAL REGISTRATION. ClinicalTrials.gov NCT01865617. FUNDING. R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation. PMID:27111235

The paired domain-containing nuclear factor pax[b] is expressed in specific commissural interneurons in zebrafish embryos.

PubMed

Mikkola, I; Fjose, A; Kuwada, J Y; Wilson, S; Guddal, P H; Krauss, S

1992-10-01

The zebrafish paired box (Pax) genes are expressed in the early neural tube and are thought to be transcription factors that regulate the differentiation of cells in the central nervous system (CNS). The protein product of one of these Pax genes, pax[b], is detectable as a nuclear antigen in all the regions of the embryo that transcribe the gene including the posterior midbrain, the nephritic primordium, the Wolffian duct, the optic stalk, and, in specific neurons, in the hindbrain and spinal cord. The timing and pattern of axonal outgrowth by the early pax[b]-positive neurons suggest that they are the commissural secondary ascending (CoSA) interneurons in the spinal cord; the primary commissural interneurons (MiD2c and MiD3c) in hindbrain rhombomeres mi2 and mi3; and a previously unclassified set of commissural interneurons that we termed the commissural caudalrhombomere ascending (CoCaA) interneurons in the caudal hindbrain. In contrast, the Mauthner interneurons do not express pax[b] early in development. Shortly after the appearance of the first pax[b]-positive interneurons, additional nuclei adjacent to the first pax[b]-positive interneurons become pax[b] positive. This pattern of expression suggests that the pax[b] protein may be involved in determining the identity of specific commissural interneurons.

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

PubMed

Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Schimmer, Simone; Brandau, Sven; Altenhoff, Petra; Sparwasser, Tim; Dittmer, Ulf

2013-02-01

The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination

PubMed Central

HWANG, JOYCE K.; ALT, FREDERICK W.; YEAP, LENG-SIEW

2015-01-01

The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination. PMID:26104555

Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GM-CSF, and LIF on the development of bovine embryos produced in vitro.

PubMed

Neira, J A; Tainturier, D; Peña, M A; Martal, J

2010-03-15

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P<0.05) on Day 8 after in vitro fertilization and similar results to use of SOF+10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-beta1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer. Copyright 2010 Elsevier Inc. All rights reserved.

Targeting B7x and B7 H3 as New Immunotherapies for Prostate Cancer

DTIC Science & Technology

2015-09-01

andMelnick, A. M. (2013) EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell 23...symptoms, signs, or laboratoryLigands from the B7 family bind to receptors of the CD28 family, which regulate early T cell activation in lymphoid organs...was first identified in lymphoid cells lines induced to undergo programmed cell death [17]. Later reports noted that PD-1 is expressed on acti- vated

Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes.

PubMed

Reed-Geaghan, Erin G; Wright, Margaret C; See, Lauren A; Adelman, Peter C; Lee, Kuan Hsien; Koerber, H Richard; Maricich, Stephen M

2016-04-13

The extent to which the skin instructs peripheral somatosensory neuron maturation is unknown. We studied this question in Merkel cell-neurite complexes, where slowly adapting type I (SAI) neurons innervate skin-derived Merkel cells. Transgenic mice lacking Merkel cells had normal dorsal root ganglion (DRG) neuron numbers, but fewer DRG neurons expressed the SAI markers TrkB, TrkC, and Ret. Merkel cell ablation also decreased downstream TrkB signaling in DRGs, and altered the expression of genes associated with SAI development and function. Skin- and Merkel cell-specific deletion of Bdnf during embryogenesis, but not postnatal Bdnf deletion or Ntf3 deletion, reproduced these results. Furthermore, prototypical SAI electrophysiological signatures were absent from skin regions where Bdnf was deleted in embryonic Merkel cells. We conclude that BDNF produced by Merkel cells during a precise embryonic period guides SAI neuron development, providing the first direct evidence that the skin instructs sensory neuron molecular and functional maturation. Peripheral sensory neurons show incredible phenotypic and functional diversity that is initiated early by cell-autonomous and local environmental factors found within the DRG. However, the contribution of target tissues to subsequent sensory neuron development remains unknown. We show that Merkel cells are required for the molecular and functional maturation of the SAI neurons that innervate them. We also show that this process is controlled by BDNF signaling. These findings provide new insights into the regulation of somatosensory neuron development and reveal a novel way in which Merkel cells participate in mechanosensation. Copyright © 2016 the authors 0270-6474/16/364362-15$15.00/0.

Screening of Variations in CD22 Gene in Children with B-Precursor Acute Lymphoblastic Leukemia.

PubMed

Aslar Oner, Deniz; Akin, Dilara Fatma; Sipahi, Kadir; Mumcuoglu, Mine; Ezer, Ustun; Kürekci, A Emin; Akar, Nejat

2016-09-01

CD22 is expressed on the surface of B-cell lineage cells from the early progenitor stage of pro-B cell until terminal differentiation to mature B cells. It plays a role in signal transduction and as a regulator of B-cell receptor signaling in B-cell development. We aimed to screen exons 9-14 of the CD22 gene, which is a mutational hot spot region in B-precursor acute lymphoblastic leukemia (pre-B ALL) patients, to find possible genetic variants that could play role in the pathogenesis of pre-B ALL in Turkish children. This study included 109 Turkish children with pre-B ALL who were diagnosed at Losante Hospital for Children with Leukemia. Genomic DNA was extracted from both peripheral blood and bone marrow leukocytes. Gene amplification was performed with PCR, and all samples were screened for the variants by single strand conformation polymorphism. Samples showing band shifts were sequenced on an automated sequencer. In our patient group a total of 9 variants were identified in the CD22 gene by sequencing: a novel variant in intron 10 (T2199G); a missense variant in exon 12; 5 intronic variants between exon 12 and intron 13; a novel intronic variant (C2424T); and a synonymous in exon 13. Thirteen of 109 children (11.9%) carried the T2199G novel intronic variant located in intron 10, and 17 of 109 children (15.6%) carried the C2424T novel intronic variant. Novel variants in the CD22 gene in children with pre-B ALL in Turkey that are not present, in the Human Gene Mutation Database or NCBI SNP database, were found.

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 to susceptible trout. Copyright © 2014 Elsevier Ltd. All rights reserved.

ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development.

PubMed

Lopez, M; Oettgen, P; Akbarali, Y; Dendorfer, U; Libermann, T A

1994-05-01

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments.

PubMed Central

Nutt, S L; Morrison, A M; Dörfler, P; Rolink, A; Busslinger, M

1998-01-01

The Pax-5 gene codes for the transcription factor BSAP which is essential for the progression of adult B lymphopoiesis beyond an early progenitor (pre-BI) cell stage. Although several genes have been proposed to be regulated by BSAP, CD19 is to date the only target gene which has been genetically confirmed to depend on this transcription factor for its expression. We have now taken advantage of cultured pre-BI cells of wild-type and Pax-5 mutant bone marrow to screen a large panel of B lymphoid genes for additional BSAP target genes. Four differentially expressed genes were shown to be under the direct control of BSAP, as their expression was rapidly regulated in Pax-5-deficient pre-BI cells by a hormone-inducible BSAP-estrogen receptor fusion protein. The genes coding for the B-cell receptor component Ig-alpha (mb-1) and the transcription factors N-myc and LEF-1 are positively regulated by BSAP, while the gene coding for the cell surface protein PD-1 is efficiently repressed. Distinct regulatory mechanisms of BSAP were revealed by reconstituting Pax-5-deficient pre-BI cells with full-length BSAP or a truncated form containing only the paired domain. IL-7 signalling was able to efficiently induce the N-myc gene only in the presence of full-length BSAP, while complete restoration of CD19 synthesis was critically dependent on the BSAP protein concentration. In contrast, the expression of the mb-1 and LEF-1 genes was already reconstituted by the paired domain polypeptide lacking any transactivation function, suggesting that the DNA-binding domain of BSAP is sufficient to recruit other transcription factors to the regulatory regions of these two genes. In conclusion, these loss- and gain-of-function experiments demonstrate that BSAP regulates four newly identified target genes as a transcriptional activator, repressor or docking protein depending on the specific regulatory sequence context. PMID:9545244

Comparative proteomic analysis of Populus trichocarpa early stem from primary to secondary growth.

PubMed

Liu, Jinwen; Hai, Guanghui; Wang, Chong; Cao, Shenquan; Xu, Wenjing; Jia, Zhigang; Yang, Chuanping; Wang, Jack P; Dai, Shaojun; Cheng, Yuxiang

2015-08-03

Wood is derived from the secondary growth of tree stems. In this study, we investigated the global changes of protein abundance in Populus early stems using a proteomic approach. Morphological and histochemical analyses revealed three typical stages during Populus early stems, which were the primary growth stage, the transition stage from primary to secondary growth and the secondary growth stage. A total of 231 spots were differentially abundant during various growth stages of Populus early stems. During Populus early stem lignifications, 87 differential spots continuously increased, while 49 spots continuously decreased. These two categories encompass 58.9% of all differential spots, which suggests significant molecular changes from primary to secondary growth. Among 231 spots, 165 unique proteins were identified using LC-ESI-Q-TOF-MS, which were classified into 14 biological function groups. The proteomic characteristics indicated that carbohydrate metabolism, oxido-reduction, protein degradation and secondary cell wall metabolism were the dominantly occurring biochemical processes during Populus early stem development. This study helps in elucidating biochemical processes and identifies potential wood formation-related proteins during tree early stem development. It is a comprehensive proteomic investigation on tree early stem development that, for the first time, reveals the overall molecular networks that occur during Populus early stem lignifications. Copyright © 2015. Published by Elsevier B.V.

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

PubMed Central

Robinson, Amanda R.; Kwek, Swee Sen; Kenney, Shannon C.

2012-01-01

The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells. PMID:22346751

Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth.

PubMed

Ng, Jovyn K T; Schröder, Roswitha; Sutherland, Paul W; Hallett, Ian C; Hall, Miriam I; Prakash, Roneel; Smith, Bronwen G; Melton, Laurence D; Johnston, Jason W

2013-11-19

There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation. 'Scifresh' (slow softening) and 'Royal Gala' (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. 'Scifresh' apples showed reduced loss of firmness and greater dry matter accumulation compared with 'Royal Gala' during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in 'Scifresh' were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of 'Scifresh' showed larger, more angular shaped cells than 'Royal Gala', with less airspaces and denser tissue. Stronger cell adhesion in ripe 'Scifresh' resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in 'Royal Gala'. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in 'Scifresh'. Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process.

Cell wall structures leading to cultivar differences in softening rates develop early during apple (Malus x domestica) fruit growth

PubMed Central

2013-01-01

Background There is a paucity of information regarding development of fruit tissue microstructure and changes in the cell walls during fruit growth, and how these developmental processes differ between cultivars with contrasting softening behaviour. In this study we compare two apple cultivars that show different softening rates during fruit development and ripening. We investigate whether these different softening behaviours manifest themselves late during ethylene-induced softening in the ripening phase, or early during fruit expansion and maturation. Results ‘Scifresh’ (slow softening) and ‘Royal Gala’ (rapid softening) apples show differences in cortical microstructure and cell adhesion as early as the cell expansion phase. ‘Scifresh’ apples showed reduced loss of firmness and greater dry matter accumulation compared with ‘Royal Gala’ during early fruit development, suggesting differences in resource allocation that influence tissue structural properties. Tricellular junctions in ‘Scifresh’ were rich in highly-esterified pectin, contributing to stronger cell adhesion and an increased resistance to the development of large airspaces during cell expansion. Consequently, mature fruit of ‘Scifresh’ showed larger, more angular shaped cells than ‘Royal Gala’, with less airspaces and denser tissue. Stronger cell adhesion in ripe ‘Scifresh’ resulted in tissue fracture by cell rupture rather than by cell-to-cell-separation as seen in ‘Royal Gala’. CDTA-soluble pectin differed in both cultivars during development, implicating its involvement in cell adhesion. Low pectin methylesterase activity during early stages of fruit development coupled with the lack of immuno-detectable PG was associated with increased cell adhesion in ‘Scifresh’. Conclusions Our results indicate that cell wall structures leading to differences in softening rates of apple fruit develop early during fruit growth and well before the induction of the ripening process. PMID:24252512

Early growth response 2 and Egr3 are unique regulators in immune system.

PubMed

Taefehshokr, Sina; Key, Yashar Azari; Khakpour, Mansour; Dadebighlu, Pourya; Oveisi, Amin

2017-01-01

The immune system is evolved to defend the body against pathogens and is composed of thousands of complicated and intertwined pathways, which are highly controlled by processes such as transcription and repression of cellular genes. Sometimes the immune system malfunctions and a break down in self-tolerance occurs. This lead to the inability to distinguish between self and non-self and cause attacks on host tissues, a condition also known as autoimmunity, which can result in chronic debilitating diseases. Early growth response genes are family of transcription factors comprising of four members, Egr1, Egr2, Egr3 and Egr4. All of which contain three cyc2-His2 zinc fingers. Initially, Egr2 function was identified in the regulation of peripheral nerve myelination, hindbrain segmentation. Egr3, on the other hand, is highly expressed in muscle spindle development. Egr2 and Egr3 are induced due to the antigen stimulation and this signaling is implemented through the B and T cell receptors in the adaptive immunity. T cell receptor signaling plays a key role in Egr 2 and 3 expressions via their interaction with NFAT molecules. Egr 2 and 3 play a crucial role in regulation of the immune system and their involvement in B and T cell activation, anergy induction and preventing the autoimmune disease has been investigated. The deficiency of these transcription factors has been associated to deficient Cbl-b expression, a resistant to anergy phenotype, and expression of effector and activated T cells.

Differential response of cell-cycle and cell-expansion regulators to heat stress in apple (Malus domestica) fruitlets.

PubMed

Flaishman, Moshe A; Peles, Yuval; Dahan, Yardena; Milo-Cochavi, Shira; Frieman, Aviad; Naor, Amos

2015-04-01

Temperature is one of the most significant factors affecting physiological and biochemical aspects of fruit development. Current and progressing global warming is expected to change climate in the traditional deciduous fruit tree cultivation regions. In this study, 'Golden Delicious' trees, grown in a controlled environment or commercial orchard, were exposed to different periods of heat treatment. Early fruitlet development was documented by evaluating cell number, cell size and fruit diameter for 5-70 days after full bloom. Normal activities of molecular developmental and growth processes in apple fruitlets were disrupted under daytime air temperatures of 29°C and higher as a result of significant temporary declines in cell-production and cell-expansion rates, respectively. Expression screening of selected cell cycle and cell expansion genes revealed the influence of high temperature on genetic regulation of apple fruitlet development. Several core cell-cycle and cell-expansion genes were differentially expressed under high temperatures. While expression levels of B-type cyclin-dependent kinases and A- and B-type cyclins declined moderately in response to elevated temperatures, expression of several cell-cycle inhibitors, such as Mdwee1, Mdrbr and Mdkrps was sharply enhanced as the temperature rose, blocking the cell-cycle cascade at the G1/S and G2/M transition points. Moreover, expression of several expansin genes was associated with high temperatures, making them potentially useful as molecular platforms to enhance cell-expansion processes under high-temperature regimes. Understanding the molecular mechanisms of heat tolerance associated with genes controlling cell cycle and cell expansion may lead to the development of novel strategies for improving apple fruit productivity under global warming. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Plasmablasts and plasma cells: reconsidering teleost immune system organization.

PubMed

Ye, Jianmin; Kaattari, Ilsa; Kaattari, Stephen

2011-12-01

Comparative immunologists have expended extensive efforts in the characterization of early fish B cell development; however, analysis of the post-antigen induction stages of antibody secreting cell (ASC) differentiation has been limited. In contrast, work with murine ASCs has resolved the physically and functionally distinct cells known as plasmablasts, the short-lived plasma cells and long-lived plasma cells. Teleost ASCs are now known to also possess comparable subpopulations, which can greatly differ in such basic functions as lifespan, antigen sensitivity, antibody secretion rate, differentiative potential, and distribution within the body. Understanding the mechanisms by which these subpopulations are produced and distributed is essential for both basic understanding in comparative immunology and practical vaccine engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.

Partial pneumonectomy of telomerase null mice carrying shortened telomeres initiates cell growth arrest resulting in a limited compensatory growth response

PubMed Central

Jackson, Sha-Ron; Lee, Jooeun; Reddy, Raghava; Williams, Genevieve N.; Kikuchi, Alexander; Freiberg, Yael; Warburton, David

2011-01-01

Telomerase mutations and significantly shortened chromosomal telomeres have recently been implicated in human lung pathologies. Natural telomere shortening is an inevitable consequence of aging, which is also a risk factor for development of lung disease. However, the impact of shortened telomeres and telomerase dysfunction on the ability of lung cells to respond to significant challenge is still largely unknown. We have previously shown that lungs of late generation, telomerase null B6.Cg-Terctm1Rdp mice feature alveolar simplification and chronic stress signaling at baseline, a phenocopy of aged lung. To determine the role telomerase plays when the lung is challenged, B6.Cg-Terctm1Rdp mice carrying shortened telomeres and wild-type controls were subjected to partial pneumonectomy. We found that telomerase activity was strongly induced in alveolar epithelial type 2 cells (AEC2) of the remaining lung immediately following surgery. Eighty-six percent of wild-type animals survived the procedure and exhibited a burst of early compensatory growth marked by upregulation of proliferation, stress response, and DNA repair pathways in AEC2. In B6.Cg-Terctm1Rdp mice carrying shortened telomeres, response to pneumonectomy was characterized by decreased survival, diminished compensatory lung growth, attenuated distal lung progenitor cell response, persistent DNA damage, and cell growth arrest. Overall, survival correlated strongly with telomere length. We conclude that functional telomerase and properly maintained telomeres play key roles in both long-term survival and the early phase of compensatory lung growth following partial pneumonectomy. PMID:21460122

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence.

PubMed

Perruche, Sylvain; Marandin, Aliette; Kleinclauss, François; Angonin, Régis; Fresnay, Stéphanie; Baron, Marie Hélène; Tiberghien, Pierre; Saas, Philippe

2006-02-27

Use of a reduced-intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a coexistence of both host and donor hematopoietic cells may influence posttransplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced-intensity conditioning transplantation (RICT), autoantibody production, and chronic GVHD (cGVHD)-related pathology. Chimerism state, circulating anticardiolipin, and antidouble stranded DNA autoantibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. We observed a novel association between mixed chimerism state, high levels of pathogenic IgG autoantibodies, and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of autoantibodies. Recipient B cell persistence may contribute to the frequency and/or severity of cGVHD after RICT.

Gap-Junctional communication between developing Drosophila muscles is essential for their normal development.

PubMed

Todman, M G; Baines, R A; Stebbings, L A; Davies, J A; Bacon, J P

1999-01-01

Recent experiments have demonstrated that a family of proteins, known as the innexins, are structural components of invertebrate gap junctions. The shaking-B (shak-B) locus of Drosophila encodes two members of this emerging family, Shak-B(lethal) and Shak-B(neural). This study focuses on the role of Shak-B gap junctions in the development of embryonic and larval muscle. During embryogenesis, shak-B transcripts are expressed in a subset of the somatic muscles; expression is strong in ventral oblique muscles (VO4-6) but only weak in ventral longitudinals (VL3 and 4). Carboxyfluorescein injected into VO4 of wild-type early stage 16 embryos spreads, via gap junctions, to label adjacent muscles, including VL3 and 4. In shak-B2 embryos (in which the shak-B(neural) function is disrupted), dye injected into VO4 fails to spread into other muscles. In the first instar larva, when dye coupling between muscles is no longer present, another effect of the shak-B2 mutation is revealed by whole-cell voltage clamp. In a calcium-free saline, only two voltage-activated potassium currents are present in wild-type muscles; a fast IA and a slow IK current. In shak-B2 larvae, these two currents are significantly reduced in magnitude in VO4 and 5, but remain normal in VL3. Expression of shak-B(neural) in a shak-B2 background fully rescues both dye coupling in embryonic muscle and whole-cell currents in first instar VO4 and 5. Our observations show that Shak-B(neural) is one of a set of embryonic gap-junction proteins, and that it is required for the normal temporal development of potassium currents in some larval muscles.

The distinct role of strand-specific miR-514b-3p and miR-514b-5p in colorectal cancer metastasis.

PubMed

Ren, Lin-Lin; Yan, Ting-Ting; Shen, Chao-Qin; Tang, Jia-Yin; Kong, Xuan; Wang, Ying-Chao; Chen, Jinxian; Liu, Qiang; He, Jie; Zhong, Ming; Chen, Hao-Yan; Hong, Jie; Fang, Jing-Yuan

2018-06-07

The abnormal expression of microRNAs (miRNAs) in colorectal cancer (CRC) progression has been widely investigated. It was reported that the same hairpin RNA structure could generate mature products from each strand, termed 5p and 3p, which binds different target mRNAs. Here, we explored the expression, functions, and mechanisms of miR-514b-3p and miR-514b-5p in CRC cells and tissues. We found that miR-514b-3p was significantly down-regulated in CRC samples, and the ratio of miR-514b-3p/miR-514b-5p increased from advanced CRC, early CRC to matched normal colorectal tissues. Follow-up functional experiments illustrated that miR-514b-3p and miR-514b-5p had distinct effects through interacting with different target genes: MiR-514b-3p reduced CRC cell migration, invasion and drug resistance through increasing epithelial marker and decreasing mesenchymal marker expressions, conversely, miR-514b-5p exerted its pro-metastatic properties in CRC by promoting EMT progression. MiR-514b-3p overexpressing CRC cells developed tumors more slowly in mice compared with control cells, however, miR-514b-5p accelerated tumor metastasis. Overall, our data indicated that though miR-514b-3p and miR-514b-5p were transcribed from the same RNA hairpin, each microRNA has distinct effect on CRC metastasis.

Dynamic mathematical modeling of IL13-induced signaling in Hodgkin and primary mediastinal B-cell lymphoma allows prediction of therapeutic targets.

PubMed

Raia, Valentina; Schilling, Marcel; Böhm, Martin; Hahn, Bettina; Kowarsch, Andreas; Raue, Andreas; Sticht, Carsten; Bohl, Sebastian; Saile, Maria; Möller, Peter; Gretz, Norbert; Timmer, Jens; Theis, Fabian; Lehmann, Wolf-Dieter; Lichter, Peter; Klingmüller, Ursula

2011-02-01

Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.

Toll-like receptor 4-positive macrophages protect mice from Pasteurella pneumotropica-induced pneumonia

NASA Technical Reports Server (NTRS)

Hart, Marcia L.; Mosier, Derek A.; Chapes, Stephen K.

2003-01-01

This study investigates Toll-like receptor 4 (TLR4)-positive macrophages in early recognition and clearance of pulmonary bacteria. TLR4 is a trans-membrane receptor that is the primary recognition molecule for lipopolysaccharide of gram-negative bacteria. The TLR4(Lps-del) mouse strains C57BL10/ScN (B10) and STOCK Abb(tm1) TLR4(Lps-del) Slc11a1(s)(B10 x C2D) are susceptible to pulmonary infections and develop pneumonia when naturally or experimentally infected by the opportunistic bacterium Pasteurella pneumotropica. Since these mice have the TLR4(Lps-del) genotype, we hypothesized that reconstitution of mice with TLR4-positive macrophages would provide resistance to this bacterium. A cultured macrophage cell line (C2D macrophages) and bone marrow cells from C2D mice were adoptively transferred to B10 and B10 x C2D mice by intraperitoneal injection. C2D macrophages increased B10 and B10 x C2D mouse resistance to P. pneumotropica. In C2D-recipient mice there was earlier transcription of tumor necrosis factor alpha and chemokines JE and macrophage inflammatory protein 2 (MIP-2) in the lungs of B10 and B10 x C2D mice, and there was earlier transcription of KC and MIP-1alpha in B10 x C2D mice. In addition, the course of inflammation following experimental Pasteurella challenge was altered in C2D recipients. C2D macrophages also protected B10 x C2D mice, which lack CD4(+) T cells. These data indicate that macrophages are critical for pulmonary immunity and can provide host resistance to P. pneumotropica. This study indicates that TLR4-positive macrophages are important for early recognition and clearance of pulmonary bacterial infections.

Differential role of gp130-dependent STAT and Ras signalling for haematopoiesis following bone-marrow transplantation.

PubMed

Kroy, Daniela C; Hebing, Lisa; Sander, Leif E; Gassler, Nikolaus; Erschfeld, Stephanie; Sackett, Sara; Galm, Oliver; Trautwein, Christian; Streetz, Konrad L

2012-01-01

Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(ΔMx)), or to selectively disrupt gp130-dependent Ras (gp130(ΔMxRas)) or STAT signalling (gp130(ΔMxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. BM derived from gp130 deficient donor mice (gp130(ΔMx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(ΔMxRas) and gp130(ΔMxSTAT) donor BM. BMT of gp130(ΔMxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(ΔMxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages.

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage.

PubMed

Cao, Zubing; Carey, Timothy S; Ganguly, Avishek; Wilson, Catherine A; Paul, Soumen; Knott, Jason G

2015-05-01

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. © 2015. Published by The Company of Biologists Ltd.

The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

PubMed Central

Chu, Bing; Yao, Feng; Cheng, Cheng; Wu, Yang; Mei, Yanli; Li, Xuejie; Liu, Yan; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

2014-01-01

During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica. PMID:24404204

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early onset autoinflammatory syndrome

PubMed Central

Zhou, Qing; Wang, Hongying; Schwartz, Daniella M.; Stoffels, Monique; Park, Yong Hwan; Zhang, Yuan; Yang, Dan; Demirkaya, Erkan; Takeuchi, Masaki; Tsai, Wanxia Li; Lyons, Jonathan J.; Yu, Xiaomin; Ouyang, Claudia; Chen, Celeste; Chin, David T.; Zaal, Kristien; Chandrasekharappa, Settara C.; Hanson, Eric P.; Yu, Zhen; Mullikin, James C.; Hasni, Sarfaraz A.; Wertz, Ingrid; Ombrello, Amanda K.; Stone, Deborah L.; Hoffmann, Patrycja; Jones, Anne; Barham, Beverly K.; Leavis, Helen L.; van Royen-Kerkof, Annet; Sibley, Cailin; Batu, Ezgi D.; Gül, Ahmet; Siegel, Richard M.; Boehm, Manfred; Milner, Joshua D.; Ozen, Seza; Gadina, Massimo; Chae, JaeJin; Laxer, Ronald M.; Kastner, Daniel L.; Aksentijevich, Ivona

2016-01-01

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity1. Herein we describe a new syndrome caused by high penetrance heterozygous germline mutations in the NFκB regulatory protein TNFAIP3 (A20) in six unrelated families with early onset systemic inflammation. The syndrome resembles Behçet’s disease (BD), which is typically considered a polygenic disorder with onset in early adulthood2. A20 is a potent inhibitor of the NFκB signaling pathway3. TNFAIP3 mutant truncated proteins are likely to act by haploinsufficiency since they do not exert a dominant-negative effect in overexpression experiments. Patients’ cells show increased degradation of IκBα and nuclear translocation of NFκB p65, and increased expression of NFκB-mediated proinflammatory cytokines. A20 restricts NFκB signals via deubiquitinating (DUB) activity. In cells expressing the mutant A20 protein, there is defective removal of K63-linked ubiquitin from TRAF6, NEMO, and RIP1 after TNF stimulation. NFκB-dependent pro-inflammatory cytokines are potential therapeutic targets for these patients. PMID:26642243

The dlx5a/dlx6a Genes Play Essential Roles in the Early Development of Zebrafish Median Fin and Pectoral Structures

PubMed Central

Heude, Églantine; Shaikho, Sarah; Ekker, Marc

2014-01-01

The Dlx5 and Dlx6 genes encode homeodomain transcription factors essential for the proper development of limbs in mammalian species. However, the role of their teleost counterparts in fin development has received little attention. Here, we show that dlx5a is an early marker of apical ectodermal cells of the pectoral fin buds and of the median fin fold, but also of cleithrum precursor cells during pectoral girdle development. We propose that early median fin fold establishment results from the medial convergence of dlx5a-expressing cells at the lateral edges of the neural keel. Expression analysis also shows involvement of dlx5a during appendage skeletogenesis. Using morpholino-mediated knock down, we demonstrate that disrupted dlx5a/6a function results in pectoral fin agenesis associated with misexpression of bmp4, fgf8a, and1 and msx genes. In contrast, the median fin fold presents defects in mesenchymal cell migration and actinotrichia formation, whereas the initial specification seems to occur normally. Our results demonstrate that the dlx5a/6a genes are essential for the induction of pectoral fin outgrowth, but are not required during median fin fold specification. The dlx5a/6a knock down also causes a failure of cleithrum formation associated with a drastic loss of runx2b and col10a1 expression. The data indicate distinct requirements for dlx5a/6a during median and pectoral fin development suggesting that initiation of unpaired and paired fin formation are not directed through the same molecular mechanisms. Our results refocus arguments on the mechanistic basis of paired appendage genesis during vertebrate evolution. PMID:24858471

Chemotherapy alters the increased numbers of myeloid-derived suppressor and regulatory T cells in children with acute lymphoblastic leukemia.

PubMed

Salem, Mohamed Labib; El-Shanshory, Mohamed R; Abdou, Said H; Attia, Mohamed S; Sobhy, Shymaa M; Zidan, Mona F; Zidan, Abdel-Aziz A

2018-04-01

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. The precise mechanism behind the relapse in this disease is not clearly known. One possible mechanism could be the accumulation of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (T regs ) which we and others have reported to mediate suppression of anti-tumor immune responses. In this study, we aimed to analyze the numbers of these cells in a population of B-ALL pediatric patients. Peripheral blood samples withdrawn from B-ALL pediatric patients (n = 45 before, during and after the induction phase of chemotherapy. Using multi parametric flow cytometric analysis. MDSCs were identified as Lin - HLA-DR - CD33 + CD11b + ; and T reg cells were defined as CD4 + CD25 + CD127 -/low . Early diagnosed B-ALL patients showed significant increases in the numbers of MDSCs and T regs as compared to healthy volunteers. During induction of chemotherapy, however, the patients showed higher and lower numbers of MDSCs and T reg cells, respectively as compared to early diagnosed patients (i.e., before chemotherapy). After induction of chemotherapy, the numbers of MDSCs and T reg cells showed higher increases and decreases, respectively as compared to the numbers in patients during chemotherapy. Our results indicate that B-ALL patients harbor high numbers of both MDSCs and T regs cells. This pilot study opens a new avenue to investigate the mechanism mediating the emergence of these cells on larger number of B-ALL patients at different treatment stages.

Mesodermal Fgf10b cooperates with other Fgfs during induction of otic and epibranchial placodes in zebrafish.

PubMed Central

Maulding, Kirstin; Padanad, Mahesh S.; Dong, Jennifer; Riley, Bruce B.

2015-01-01

Background Vertebrate otic and epibranchial placodes develop in close proximity in response to localized Fgf signaling. Although less is known about epibranchial induction, the process of otic induction in highly conserved, with important roles for Fgf3 and Fgf8 reported in all species examined. Fgf10 is also critical for otic induction in mouse, but the only zebrafish ortholog examined to date, fgf10a, is not expressed early enough to play such a role. A second zebrafish ortholog, fgf10b, has not been previously examined. Results We find that zebrafish fgf10b is expressed at tailbud stage in paraxial cephalic mesoderm beneath prospective epibranchial tissue, lateral to the developing otic placode. Knockdown of fgf10b does not affect initial otic induction but impairs subsequent accumulation of otic cells. Formation of epibranchial placodes and ganglia are also moderately impaired. Combinatorial disruption of fgf10b and fgf3 exacerbates the deficiency of otic cells and eliminates epibranchial induction entirely. Disruption of fgf10b and fgf24 also strongly reduces, but does not eliminate, epibranchial induction. Conclusions fgf10b participates in a late phase of otic induction and, in combination with fgf3, is especially critical for epibranchial induction. PMID:24677486

Mesodermal Fgf10b cooperates with other fibroblast growth factors during induction of otic and epibranchial placodes in zebrafish.

PubMed

Maulding, Kirstin; Padanad, Mahesh S; Dong, Jennifer; Riley, Bruce B

2014-10-01

Vertebrate otic and epibranchial placodes develop in close proximity in response to localized fibroblast growth factor (Fgf) signaling. Although less is known about epibranchial induction, the process of otic induction in highly conserved, with important roles for Fgf3 and Fgf8 reported in all species examined. Fgf10 is also critical for otic induction in mouse, but the only zebrafish ortholog examined to date, fgf10a, is not expressed early enough to play such a role. A second zebrafish ortholog, fgf10b, has not been previously examined. We find that zebrafish fgf10b is expressed at tailbud stage in paraxial cephalic mesoderm beneath prospective epibranchial tissue, lateral to the developing otic placode. Knockdown of fgf10b does not affect initial otic induction but impairs subsequent accumulation of otic cells. Formation of epibranchial placodes and ganglia are also moderately impaired. Combinatorial disruption of fgf10b and fgf3 exacerbates the deficiency of otic cells and eliminates epibranchial induction entirely. Disruption of fgf10b and fgf24 also strongly reduces, but does not eliminate, epibranchial induction. fgf10b participates in a late phase of otic induction and, in combination with fgf3, is especially critical for epibranchial induction. Copyright © 2014 Wiley Periodicals, Inc.

Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis

PubMed Central

Echtler, Katrin; Konrad, Ildiko; Lorenz, Michael; Schneider, Simon; Hofmaier, Sebastian; Plenagl, Florian; Stark, Konstantin; Czermak, Thomas; Tirniceriu, Anca; Eichhorn, Martin; Walch, Axel; Enders, Georg; Massberg, Steffen; Schulz, Christian

2017-01-01

Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases. PMID:28253287

Platelet GPIIb supports initial pulmonary retention but inhibits subsequent proliferation of melanoma cells during hematogenic metastasis.

PubMed

Echtler, Katrin; Konrad, Ildiko; Lorenz, Michael; Schneider, Simon; Hofmaier, Sebastian; Plenagl, Florian; Stark, Konstantin; Czermak, Thomas; Tirniceriu, Anca; Eichhorn, Martin; Walch, Axel; Enders, Georg; Massberg, Steffen; Schulz, Christian

2017-01-01

Platelets modulate the process of cancer metastasis. However, current knowledge on the direct interaction of platelets and tumor cells is mostly based on findings obtained in vitro. We addressed the role of the platelet fibrinogen receptor glycoprotein IIb (integrin αIIb) for experimental melanoma metastasis in vivo. Highly metastatic B16-D5 melanoma cells were injected intravenously into GPIIb-deficient (GPIIb-/-) or wildtype (WT) mice. Acute accumulation of tumor cells in the pulmonary vasculature was assessed in real-time by confocal videofluorescence microscopy. Arrest of tumor cells was dramatically reduced in GPIIb-/- mice as compared to WT. Importantly, we found that mainly multicellular aggregates accumulated in the pulmonary circulation of WT, instead B16-D5 aggregates were significantly smaller in GPIIb-/- mice. While pulmonary arrest of melanoma was clearly dependent on GPIIb in this early phase of metastasis, we also addressed tumor progression 10 days after injection. Inversely, and unexpectedly, we found that melanoma metastasis was now increased in GPIIb-/- mice. In contrast, GPIIb did not regulate local melanoma proliferation in a subcutaneous tumor model. Our data suggest that the platelet fibrinogen receptor has a differential role in the modulation of hematogenic melanoma metastasis. While platelets clearly support early steps in pulmonary metastasis via GPIIb-dependent formation of platelet-tumor-aggregates, at a later stage its absence is associated with an accelerated development of melanoma metastases.

Diverse Profiles of Ricin-Cell Interactions in the Lung Following Intranasal Exposure to Ricin

PubMed Central

Sapoznikov, Anita; Falach, Reut; Mazor, Ohad; Alcalay, Ron; Gal, Yoav; Seliger, Nehama; Sabo, Tamar; Kronman, Chanoch

2015-01-01

Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication. PMID:26593946

Diverse profiles of ricin-cell interactions in the lung following intranasal exposure to ricin.

PubMed

Sapoznikov, Anita; Falach, Reut; Mazor, Ohad; Alcalay, Ron; Gal, Yoav; Seliger, Nehama; Sabo, Tamar; Kronman, Chanoch

2015-11-17

Ricin, a plant-derived exotoxin, inhibits protein synthesis by ribosomal inactivation. Due to its wide availability and ease of preparation, ricin is considered a biothreat, foremost by respiratory exposure. We examined the in vivo interactions between ricin and cells of the lungs in mice intranasally exposed to the toxin and revealed multi-phasic cell-type-dependent binding profiles. While macrophages (MΦs) and dendritic cells (DCs) displayed biphasic binding to ricin, monophasic binding patterns were observed for other cell types; epithelial cells displayed early binding, while B cells and endothelial cells bound toxin late after intoxication. Neutrophils, which were massively recruited to the intoxicated lung, were refractive to toxin binding. Although epithelial cells bound ricin as early as MΦs and DCs, their rates of elimination differed considerably; a reduction in epithelial cell counts occurred late after intoxication and was restricted to alveolar type II cells only. The differential binding and cell-elimination patterns observed may stem from dissimilar accessibility of the toxin to different cells in the lung and may also reflect unequal interactions of the toxin with different cell-surface receptors. The multifaceted interactions observed in this study between ricin and the various cells of the target organ should be considered in the future development of efficient post-exposure countermeasures against ricin intoxication.

Dynamic Epstein-Barr Virus Gene Expression on the Path to B-Cell Transformation

PubMed Central

Price, Alexander M.; Luftig, Micah A.

2016-01-01

Epstein-Barr Virus is an oncogenic human herpesvirus in the γ-herpesvirinae sub-family that contains a 170–180 kb 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 as well as 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 as well as delayed responses in the known latency genes. This review 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, inhibition of apoptosis, and control of innate and adaptive immune responses. PMID:24373315

Reducing FLI1 Levels in the MRL/lpr Lupus Mouse Model Impacts T Cell Function by Modulating Glycosphingolipid Metabolism

PubMed Central

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A.; Basher, Fahmin; Roddy, Patrick O.; Siskind, Leah J.; Nietert, Paul J.; Nowling, Tamara K.

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1 +/+ or Fli1 +/- T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1 +/- lupus T cells compared to animals receiving Fli1 +/+ lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1 +/- T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1 +/+ T cells. Moreover, the Fli1 +/- T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1 +/+ T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus. PMID:24040398

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

PubMed

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A; Basher, Fahmin; Roddy, Patrick O; Siskind, Leah J; Nietert, Paul J; Nowling, Tamara K

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs.

PubMed

Ghosh, Sukla; Roy, Stéphane; Séguin, Carl; Bryant, Susan V; Gardiner, David M

2008-05-01

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.

MicroRNA Responses to the Genotoxic Carcinogens Aflatoxin B1 and Benzo[a]pyrene in Human HepaRG Cells.

PubMed

Marrone, April K; Tryndyak, Volodymyr; Beland, Frederick A; Pogribny, Igor P

2016-02-01

Recent advances in toxicogenomics present an opportunity to develop new in vitro testing methodologies to identify human carcinogens. We have investigated microRNA expression responses to the treatment of human liver HepaRG cells with the human genotoxic carcinogens aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P), and the structurally similar compounds aflatoxin B2 (AFB2) and benzo[e]pyrene (B[e]P) that exhibit minimal carcinogenic potential. We demonstrate that treatment of HepaRG cells with AFB1 or B[a]P resulted in specific changes in the expression of miRNAs as compared with their non-carcinogenic analogues, particularly in a marked over-expression of miR-410. An additional novel finding is the dose- and time-dependent inhibition of miR-122 in AFB1-treated HepaRG cells. Mechanistically, the AFB1-induced down-regulation of miR-122 was attributed to inhibition of the HNF4A/miR-122 regulatory pathway. These results demonstrate that HepaRG cells can be used to investigate miRNA responses to xenobiotic exposure, and illustrate the existence of early non-genotoxic events, in addition to a well-established genotoxic mode of action changes, in the mechanism of AFB1 and B[a]P carcinogenicity. Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.

NRF2 Orchestrates the Metabolic Shift during Induced Pluripotent Stem Cell Reprogramming

PubMed Central

Hawkins, Kate E.; Joy, Shona; Delhove, Juliette M.K.M.; Kotiadis, Vassilios N.; Fernandez, Emilio; Fitzpatrick, Lorna M.; Whiteford, James R.; King, Peter J.; Bolanos, Juan P.; Duchen, Michael R.; Waddington, Simon N.; McKay, Tristan R.

2016-01-01

Summary The potential of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine is vast, but current methodologies remain inefficient. Understanding the cellular mechanisms underlying iPSC reprogramming, such as the metabolic shift from oxidative to glycolytic energy production, is key to improving its efficiency. We have developed a lentiviral reporter system to assay longitudinal changes in cell signaling and transcription factor activity in living cells throughout iPSC reprogramming of human dermal fibroblasts. We reveal early NF-κB, AP-1, and NRF2 transcription factor activation prior to a temporal peak in hypoxia inducible factor α (HIFα) activity. Mechanistically, we show that an early burst in oxidative phosphorylation and elevated reactive oxygen species generation mediates increased NRF2 activity, which in turn initiates the HIFα-mediated glycolytic shift and may modulate glucose redistribution to the pentose phosphate pathway. Critically, inhibition of NRF2 by KEAP1 overexpression compromises metabolic reprogramming and results in reduced efficiency of iPSC colony formation. PMID:26904936

IL-10 and NOS2 Modulate Antigen-Specific Reactivity and Nerve Infiltration by T Cells in Experimental Leprosy

PubMed Central

Hagge, Deanna A.; Scollard, David M.; Ray, Nashone A.; Marks, Vilma T.; Deming, Angelina T.; Spencer, John S.; Adams, Linda B.

2014-01-01

Background Although immunopathology dictates clinical outcome in leprosy, the dynamics of early and chronic infection are poorly defined. In the tuberculoid region of the spectrum, Mycobacterium leprae growth is restricted yet a severe granulomatous lesion can occur. The evolution and maintenance of chronic inflammatory processes like those observed in the leprosy granuloma involve an ongoing network of communications via cytokines. IL-10 has immunosuppressive properties and IL-10 genetic variants have been associated with leprosy development and reactions. Methodology/Principal Findings The role of IL-10 in resistance and inflammation in leprosy was investigated using Mycobacterium leprae infection of mice deficient in IL-10 (IL-10−/−), as well as mice deficient in both inducible nitric oxide synthase (NOS2−/−) and IL-10 (10NOS2−/−). Although a lack of IL-10 did not affect M. leprae multiplication in the footpads (FP), inflammation increased from C57Bl/6 (B6)

Sequential EMT-MET induces neuronal conversion through Sox2

PubMed Central

He, Songwei; Chen, Jinlong; Zhang, Yixin; Zhang, Mengdan; Yang, Xiao; Li, Yuan; Sun, Hao; Lin, Lilong; Fan, Ke; Liang, Lining; Feng, Chengqian; Wang, Fuhui; Zhang, Xiao; Guo, Yiping; Pei, Duanqing; Zheng, Hui

2017-01-01

Direct neuronal conversion can be achieved with combinations of small-molecule compounds and growth factors. Here, by studying the first or induction phase of the neuronal conversion induced by defined 5C medium, we show that the Sox2-mediated switch from early epithelial–mesenchymal transition (EMT) to late mesenchymal–epithelial transition (MET) within a high proliferation context is essential and sufficient for the conversion from mouse embryonic fibroblasts (MEFs) to TuJ+ cells. At the early stage, insulin and basic fibroblast growth factor (bFGF)-induced cell proliferation, early EMT, the up-regulation of Stat3 and Sox2, and the subsequent activation of neuron projection. Up-regulated Sox2 then induced MET and directed cells towards a neuronal fate at the late stage. Inhibiting either stage of this sequential EMT-MET impaired the conversion. In addition, Sox2 could replace sequential EMT-MET to induce a similar conversion within a high proliferation context, and its functions were confirmed with other neuronal conversion protocols and MEFs reprogramming. Therefore, the critical roles of the sequential EMT-MET were implicated in direct cell fate conversion in addition to reprogramming, embryonic development and cancer progression. PMID:28580167

Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

PubMed Central

Macedo, Ana Catarina Lunz; Isaac, Lourdes

2016-01-01

The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques

PubMed Central

Moukambi, Félicien; Rabezanahary, Henintsoa; Rodrigues, Vasco; Racine, Gina; Robitaille, Lynda; Krust, Bernard; Andreani, Guadalupe; Soundaramourty, Calayselvy; Silvestre, Ricardo; Laforge, Mireille; Estaquier, Jérôme

2015-01-01

Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs) during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs) during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies. PMID:26640894

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

PubMed

Sun, Jiali; Huye, Leslie E; Lapteva, Natalia; Mamonkin, Maksim; Hiregange, Manasa; Ballard, Brandon; Dakhova, Olga; Raghavan, Darshana; Durett, April G; Perna, Serena K; Omer, Bilal; Rollins, Lisa A; Leen, Ann M; Vera, Juan F; Dotti, Gianpietro; Gee, Adrian P; Brenner, Malcolm K; Myers, Douglas G; Rooney, Cliona M

2015-01-01

Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro. We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

PubMed

den Hartog, Gerco; Jacobino, Shamir; Bont, Louis; Cox, Linda; Ulfman, Laurien H; Leusen, Jeanette H W; van Neerven, R J Joost

2014-01-01

Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins. To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV. ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated. bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV. The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

PubMed

Leerberg, Dena M; Sano, Kaori; Draper, Bruce W

2017-09-01

The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

Increased metastatic potential of tumor cells in von Willebrand factor-deficient mice.

PubMed

Terraube, V; Pendu, R; Baruch, D; Gebbink, M F B G; Meyer, D; Lenting, P J; Denis, C V

2006-03-01

The key role played by von Willebrand factor (VWF) in platelet adhesion suggests a potential implication in various pathologies, where this process is involved. In cancer metastasis development, tumor cells interact with platelets and the vessel wall to extravasate from the circulation. As a potential mediator of platelet-tumor cell interactions, VWF could influence this early step of tumor spread and therefore play a role in cancer metastasis. To investigate whether VWF is involved in metastasis development. In a first step, we characterized the interaction between murine melanoma cells B16-BL6 and VWF in vitro. In a second step, an experimental metastasis model was used to compare the formation of pulmonary metastatic foci in C57BL/6 wild-type and VWF-null mice following the injection of B16-BL6 cells or Lewis lung carcinoma cells. In vitro adhesion assays revealed that VWF is able to promote a dose-dependent adhesion of B16-BL6 cells via its Arg-Gly-Asp (RGD) sequence. In the experimental metastasis model, we found a significant increase in the number of pulmonary metastatic foci in VWF-null mice compared with the wild-type mice, a phenotype that could be corrected by restoring VWF plasma levels. We also showed that increased survival of the tumor cells in the lungs during the first 24 h in the absence of VWF was the cause of this increased metastasis. These findings suggest that VWF plays a protective role against tumor cell dissemination in vivo. Underlying mechanisms remain to be investigated.

bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship.

PubMed

Strasser, A; Harris, A W; Cory, S

1991-11-29

Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

PubMed

Chien, Yu; Cheng, Wei-Cheng; Wu, Menq-Rong; Jiang, Si-Tse; Shen, Che-Kun James; Chung, Bon-chu

2013-10-01

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

C8orf46 homolog encodes a novel protein Vexin that is required for neurogenesis in Xenopus laevis.

PubMed

Moore, Kathryn B; Logan, Mary A; Aldiri, Issam; Roberts, Jacqueline M; Steele, Michael; Vetter, Monica L

2018-05-01

Neural basic helix-loop helix (bHLH) transcription factors promote progenitor cell differentiation by activation of downstream target genes that coordinate neuronal differentiation. Here we characterize a neural bHLH target gene in Xenopus laevis, vexin (vxn; previously sbt1), that is homologous to human c8orf46 and is conserved across vertebrate species. C8orf46 has been implicated in cancer progression, but its function is unknown. Vxn is transiently expressed in differentiating progenitors in the developing central nervous system (CNS), and is required for neurogenesis in the neural plate and retina. Its function is conserved, since overexpression of either Xenopus or mouse vxn expands primary neurogenesis and promotes early retinal cell differentiation in cooperation with neural bHLH factors. Vxn protein is localized to the cell membrane and the nucleus, but functions in the nucleus to promote neural differentiation. Vxn inhibits cell proliferation, and works with the cyclin-dependent kinase inhibitor p27Xic1 (cdkn1b) to enhance neurogenesis and increase levels of the proneural protein Neurog2. We propose that vxn provides a key link between neural bHLH activity and execution of the neurogenic program. Copyright © 2018 Elsevier Inc. All rights reserved.

Kinin-B2 receptor expression and activity during differentiation of embryonic rat neurospheres.

PubMed

Martins, Antonio H; Alves, Janaína M; Trujillo, Cleber A; Schwindt, Telma T; Barnabé, Gabriela F; Motta, Fabiana L T; Guimaraes, Alessander O; Casarini, Dulce E; Mello, Luiz E; Pesquero, João B; Ulrich, Henning

2008-04-01

Neural progenitor cells were isolated from rat fetal telencephalon and proliferate as neurospheres in the presence of EGF, FGF-2, and heparin. In the absence of these growth factors, neurospheres differentiate into neurons, astrocytes, and oligodendrocytes. Using an embryonal carcinoma cell line as in vitro differentiation model, we have already demonstrated the presence of an autocrine loop system between kinin-B2 receptor activity and secretion of its ligand bradykinin (BK) as prerequisites for final neuronal differentiation (Martins et al., J Biol Chem 2005; 280: 19576-19586). The aim of this study was to verify the activity of the kallikrein-kinin system (KKS) during neural progenitor cell differentiation. Immunofluorescence studies and flow cytometry analysis revealed increases in glial fibrillary acidic protein and beta-3 tubulin expression and decrease in the number of nestin-positive cells along neurospheres differentiation, indicating the transition of neural progenitor cells to astrocytes and neurons. Kinin-B2 receptor expression and activity, secretion of BK into the medium, and presence of high-molecular weight kininogen suggest the participation of the KKS in neurosphere differentiation. Functional kinin-B2 receptors and BK secretion indicate an autocrine loop during neurosphere differentiation to neurons, astrocytes, and oligodendrocytes, reflecting events occurring during early brain development. (c) 2008 International Society for Analytical Cytology.

Profile of epratuzumab and its potential in the treatment of systemic lupus erythematosus

PubMed Central

Al Rayes, Hanan; Touma, Zahi

2014-01-01

Management of systemic lupus erythematosus (SLE) represents a fascinating, emerging field. Research has recently provided us with a better understanding of the immunologic alterations of SLE, leading to the creation of immunomodulatory agents designed to disrupt specific cell targets and pro-inflammatory pathways. Despite the improvement in the prognosis of SLE in the last 50 years with the use of immunosuppressive therapy such as cyclophosphamide and mycophenolate mofetil, cytotoxicity remains a major complication of these medications and the need for more specific targeted immunotherapy is increasing. Early recognition and treatment of SLE with targeted immunotherapy has the potential to improve quality of life and reduce the risk of disease flare-ups and complications. In this review, we will explore the role of B-cells in the pathogenesis of SLE highlighting current insights into SLE development and management. In addition, we will discuss epratuzumab’s role in the treatment of SLE. Epratuzumab is a humanized anti-CD22 monoclonal antibody that targets CD22 on B-cell and its role in B-cell modulation, migration, function, and inhibition of B-cell receptor signaling. Epratuzumab is currently in a Phase III study evaluating its efficacy in the management of moderate to severe SLE. All published trials on epratuzumab have shown great promise with safe profiles. PMID:25429203

Bordetella pertussis modulates human macrophage defense gene expression.

PubMed

Valdez, Hugo Alberto; Oviedo, Juan Marcos; Gorgojo, Juan Pablo; Lamberti, Yanina; Rodriguez, Maria Eugenia

2016-08-01

Bordetella pertussis, the etiological agent of whooping cough, still causes outbreaks. We recently found evidence that B. pertussis can survive and even replicate inside human macrophages, indicating that this host cell might serve as a niche for persistence. In this work, we examined the interaction of B. pertussis with a human monocyte cell line (THP-1) that differentiates into macrophages in culture in order to investigate the host cell response to the infection and the mechanisms that promote that intracellular survival. To that end, we investigated the expression profile of a selected number of genes involved in cellular bactericidal activity and the inflammatory response during the early and late phases of infection. The bactericidal and inflammatory response of infected macrophages was progressively downregulated, while the number of THP-1 cells heavily loaded with live bacteria increased over time postinfection. Two of the main toxins of B. pertussis, pertussis toxin (Ptx) and adenylate cyclase (CyaA), were found to be involved in manipulating the host cell response. Therefore, failure to express either toxin proved detrimental to the development of intracellular infections by those bacteria. Taken together, these results support the relevance of host defense gene manipulation to the outcome of the interaction between B. pertussis and macrophages. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Valency and density matter: Deciphering impacts of immunogen structures on immune responses against a tumor associated carbohydrate antigen using synthetic glycopolymers.

PubMed

Qin, Qian; Yin, Zhaojun; Wu, Xuanjun; Haas, Karen M; Huang, Xuefei

2016-09-01

For successful carbohydrate based anti-cancer vaccines, it is critical that B cells are activated to secret antibodies targeting the tumor associated carbohydrate antigens (TACAs). Despite the availability of many TACA based constructs, systematic understanding of the effects of structural features on anti-glycan antibody responses is lacking. In this study, a series of defined synthetic glyco-polymers bearing a representative TACA, i.e., the Thomsen-nouveau (Tn) antigen, have been prepared to probe the induction of early B cell activation and antibody production via a T cell independent mechanism. Valency and density of the antigen in the polymers turned out to be critical. An average of greater than 6 Tn per chain was needed to induce antibody production. Glycopolymers with 40 antigens per chain and backbone molecular weight of 450 kDa gave the strongest stimulation to B cells in vitro, which correlated well with its in vivo activity. Deviations from the desired valency and density led to decreased antibody production or even antigen specific B cell non-responsiveness. These findings provide important insights on how to modulate anti-TACA immune responses facilitating the development of TACA based anti-cancer vaccines using glycopolymers. Copyright © 2016 Elsevier Ltd. All rights reserved.

H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blackwell, J.M.; Hale, C.; Roberts, M.B.

1985-01-01

The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(10M) (H-2b, H-11b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(= B10) (H-2b, H-11a) and B10.D2/n (H-2d, H-11a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncuremore » phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage neishmanial activity in vivo even when suppressor T cells are not generated. Elucidation of this characteristically different noncuring/nonhealing phenotye may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.« less

Bovine embryo induces an anti-inflammatory response in uterine epithelial cells and immune cells in vitro: possible involvement of interferon tau as an intermediator

PubMed Central

TALUKDER, Anup K.; YOUSEF, Mohamed S.; RASHID, Mohammad B.; AWAI, Kensuke; ACOSTA, Tomas J.; SHIMIZU, Takashi; OKUDA, Kiyoshi; SHIMADA, Masayuki; IMAKAWA, Kazuhiko; MIYAMOTO, Akio

2017-01-01

Recent observations suggest that the bovine uterus starts to react to the early embryo immediately after its arrival from the oviduct. The present study aimed to investigate the effect of the early developing embryo on the immune-related gene profile in bovine uterine epithelial cells (BUECs) in vitro, and to further examine the impact of conditioned media (CM), either from embryo-BUEC co-culture or embryo culture alone, on gene expression in peripheral blood mononuclear cells (PBMCs). First, BUECs were co-cultured with morulae (n = 10) for D5-D9 (D0 = IVF), and gene expression in BUECs was analyzed. Subsequently, PBMCs were cultured in CM from embryo-BUEC co-culture or D5-D9 embryo culture, and gene expression was evaluated. In BUECs, the embryo induced interferon (IFN)-stimulated genes (ISGs: ISG15, OAS1, and MX2), a key factor for IFN-signaling (STAT1), and type-1 IFN receptors (IFNAR1 and IFNAR2), with suppression of NFkB2, NFkBIA and pro-inflammatory cytokines (TNFA and IL1B). The embryo also stimulated PTGES and PGE2 secretion in BUECs. In PBMCs, both CM from embryo-BUEC co-culture and embryo culture alone induced ISGs, STAT1 and TGFB1, while suppressing TNFA and IL17. Similarly, interferon tau (IFNT) at 100 pg/ml suppressed NFkB2, TNFA and IL1B in BUECs, and also stimulated TGFB1 and suppressed TNFA in PBMCs. Our findings suggest that the bovine embryo, in the first four days in the uterus (D5-D9), starts to induce an anti-inflammatory response in epithelial cells and in immune cells. IFNT is likely to act as one of the intermediators for induction of the anti-inflammatory response in the bovine uterus. PMID:28603222

A novel protein kinase D inhibitor attenuates early events of experimental pancreatitis in isolated rat acini.

PubMed

Thrower, Edwin C; Yuan, Jingzhen; Usmani, Ashar; Liu, Yannan; Jones, Courtney; Minervini, Samantha N; Alexandre, Martine; Pandol, Stephen J; Guha, Sushovan

2011-01-01

Novel protein kinase C isoforms (PKC δ and ε) mediate early events in acute pancreatitis. Protein kinase D (PKD/PKD1) is a convergent point of PKC δ and ε in the signaling pathways triggered through CCK or cholinergic receptors and has been shown to activate the transcription factor NF-κB in acute pancreatitis. For the present study we hypothesized that a newly developed PKD/PKD1 inhibitor, CRT0066101, would prevent the initial events leading to pancreatitis. We pretreated isolated rat pancreatic acinar cells with CRT0066101 and a commercially available inhibitor Gö6976 (10 μM). This was followed by stimulation for 60 min with high concentrations of cholecystokinin (CCK, 0.1 μM), carbachol (CCh, 1 mM), or bombesin (10 μM) to induce initial events of pancreatitis. PKD/PKD1 phosphorylation and activity were measured as well as zymogen activation, amylase secretion, cell injury and NF-κB activation. CRT0066101 dose dependently inhibited secretagogue-induced PKD/PKD1 activation and autophosphorylation at Ser-916 with an IC(50) ∼3.75-5 μM but had no effect on PKC-dependent phosphorylation of the PKD/PKD1 activation loop (Ser-744/748). Furthermore, CRT0066101 reduced secretagogue-induced zymogen activation and amylase secretion. Gö6976 reduced zymogen activation but not amylase secretion. Neither inhibitor affected basal zymogen activation or secretion. CRT0066101 did not affect secretagogue-induced cell injury or changes in cell morphology, but it reduced NF-κB activation by 75% of maximal for CCK- and CCh-stimulated acinar cells. In conclusion, CRT0066101 is a potent and specific PKD family inhibitor. Furthermore, PKD/PKD1 is a potential mediator of zymogen activation, amylase secretion, and NF-κB activation induced by a range of secretagogues in pancreatic acinar cells.

The pre-B cell receptor: turning autoreactivity into self-defense.

PubMed

Vettermann, Christian; Jäck, Hans-Martin

2010-05-01

The first step in establishing the antibody repertoire in humans and mice is the rearrangement of immunoglobulin heavy chain (HC) genes in early B lineage cells. These cells then assemble microHCs with surrogate light chains (SLC) into a pre-B cell receptor (pre-BCR). We propose that the pre-BCR has evolved from an ancient autoreactive BCR, since the SLC is an autoreactive entity that binds to the pre-BCR itself and to other self-antigens. Abrogation of autoreactivity in the SLC diminishes pre-BCR signaling and impairs the clonal expansion of pre-B cells producing functional microHCs. Since SLC expression is restricted to pre-B cells, the autoreactivity encoded by the pre-BCR can be utilized to pre-select the antibody repertoire, while simultaneously avoiding the formation of autoreactive B lymphocytes. Copyright 2010 Elsevier Ltd. All rights reserved.

Bovine lactoferricin B induces apoptosis of human gastric cancer cell line AGS by inhibition of autophagy at a late stage.

PubMed

Pan, W-R; Chen, P-W; Chen, Y-L S; Hsu, H-C; Lin, C-C; Chen, W-J

2013-01-01

Gastric cancer is one of the most common malignant cancers, with poor prognosis and high mortality rates worldwide. Therefore, development of an effective therapeutic method without side effects is an urgent need. It has been reported that cationic antimicrobial peptides can selectively bind to negatively charged prokaryotic and cancer cell membranes and exert cytotoxicity without causing severe drug resistance. In the current study, we prepared a series of peptide fragments derived from bovine lactoferrin and evaluated their anticancer potency toward the gastric cancer cell line AGS. Cell viability assay revealed that a 25-AA peptide fragment, lactoferricin B25 (LFcinB25), exhibited the most potent anticancer capability against AGS cells. Lactoferricin B25 selectively inhibited AGS cell growth in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) value of 64 μM. Flow cytometry showed a notable increment of the sub-G1 populations of the cell cycle, indicating the induction of apoptosis by LFcinB25. Western blot analysis further revealed that upon LFcinB25 treatment for 2 to 6h, apoptosis-related caspases-3, 7, 8, 9, and poly(ADP-ribose) polymerase (PARP) were cleaved and activated, whereas autophagy-related LC3-II and beclin-1 were concomitantly increased. Thus, both apoptosis and autophagy are involved in the early stage of LFcinB25-induced cell death of AGS cells. However, upon treatment with LFcinB25 for 12 to 24h, LC3-II began to decrease, whereas cleaved beclin-1 increased in a time-dependent manner, suggesting that consecutive activation of caspases cleaved beclin-1 to inhibit autophagy, thus enhancing apoptosis at the final stage. These findings provide support for future application of LFcinB25 as a potential therapeutic agent for gastric cancer. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Collaboration of Antibody and Inflammation in Clearance of Rabies Virus from the Central Nervous System

PubMed Central

Hooper, D. Craig; Morimoto, Kinjiro; Bette, Michael; Weihe, Eberhard; Koprowski, Hilary; Dietzschold, Bernhard

1998-01-01

To investigate the involvement of various cellular and humoral aspects of immunity in the clearance of rabies virus from the central nervous system, (CNS), we studied the development of clinical signs and virus clearance from the CNS in knockout mice lacking either B and T cells, CD8+ cytotoxic T cells, B cells, alpha/beta interferon (IFN-α/β) receptors, IFN-γ receptors, or complement components C3 and C4. Following intranasal infection with the attenuated rabies virus CVS-F3, normal adult mice of different genetic backgrounds developed a transient disease characterized by loss of body weight and appetite depression which peaked at 13 days postinfection (p.i.). While these animals had completely recovered by day 21 p.i., mice lacking either B and T cells or B cells alone developed a progressive disease and succumbed to infection. Mice lacking either CD8+ T cells, IFN receptors, or complement components C3 and C4 showed no significant differences in the development of clinical signs by comparison with intact counterparts having the same genetic background. However, while infectious virus and viral RNA could be detected in normal control mice only until day 8 p.i., in all of the gene knockout mice studied except those lacking C3 and C4, virus infection persisted through day 21 p.i. Analysis of rabies virus-specific antibody production together with histological assessment of brain inflammation in infected animals revealed that clearance of CVS-F3 by 21 days p.i. correlated with both a strong inflammatory response in the CNS early in the infection (day 8 p.i.), and the rapid (day 10 p.i.) production of significant levels of virus-neutralizing antibody (VNA). These studies confirm that rabies VNA is an absolute requirement for clearance of an established rabies virus infection. However, for the latter to occur in a timely fashion, collaboration between VNA and inflammatory mechanisms is necessary. PMID:9557653

Expression of the SNARE Protein SNAP-23 Is Essential for Cell Survival

PubMed Central

Kaul, Sunil; Mittal, Sharad K.; Feigenbaum, Lionel; Kruhlak, Michael J.; Roche, Paul A.

2015-01-01

Members of the SNARE-family of proteins are known to be key regulators of the membrane-membrane fusion events required for intracellular membrane traffic. The ubiquitously expressed SNARE protein SNAP-23 regulates a wide variety of exocytosis events and is essential for mouse development. Germline deletion of SNAP-23 results in early embryonic lethality in mice, and for this reason we now describe mice and cell lines in which SNAP-23 can be conditionally-deleted using Cre-lox technology. Deletion of SNAP-23 in CD19-Cre expressing mice prevents B lymphocyte development and deletion of SNAP-23 using a variety of T lymphocyte-specific Cre mice prevents T lymphocyte development. Acute depletion of SNAP-23 in mouse fibroblasts leads to rapid apoptotic cell death. These data highlight the importance of SNAP-23 for cell survival and describe a mouse in which specific cell types can be eliminated by expression of tissue-specific Cre-recombinase. PMID:25706117

Localization of basic fibroblast growth factor binding sites in the chick embryonic neural retina.

PubMed

Cirillo, A; Arruti, C; Courtois, Y; Jeanny, J C

1990-12-01

We have investigated the localization of basic fibroblast growth factor (bFGF) binding sites during the development of the neural retina in the chick embryo. The specificity of the affinity of bFGF for its receptors was assessed by competition experiments with unlabelled growth factor or with heparin, as well as by heparitinase treatment of the samples. Two different types of binding sites were observed in the neural retina by light-microscopic autoradiography. The first type, localized mainly to basement membranes, was highly sensitive to heparitinase digestion and to competition with heparin. It was not developmentally regulated. The second type of binding site, resistant to heparin competition, appeared to be associated with retinal cells from the earliest stages studied (3-day-old embryo, stages 21-22 of Hamburger and Hamilton). Its distribution was found to vary during embryonic development, paralleling layering of the neural retina. Binding of bFGF to the latter sites was observed throughout the retinal neuroepithelium at early stages but displayed a distinct pattern at the time when the inner and outer plexiform layers were formed. During the development of the inner plexiform layer, a banded pattern of bFGF binding was observed. These bands, lying parallel to the vitreal surface, seemed to codistribute with the synaptic bands existing in the inner plexiform layer. The presence of intra-retinal bFGF binding sites whose distribution varies with embryonic development suggests a regulatory mechanism involving differential actions of bFGF on neural retinal cells.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

PubMed Central

Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

2016-01-01

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis. PMID:26493868

The effects of upaB deletion and the double/triple deletion of upaB, aatA, and aatB genes on pathogenicity of avian pathogenic Escherichia coli.

PubMed

Zhu-Ge, Xiang-Kai; Pan, Zi-Hao; Tang, Fang; Mao, Xiang; Hu, Lin; Wang, Shao-Hui; Xu, Bin; Lu, Cheng-Ping; Fan, Hong-Jie; Dai, Jian-Jun

2015-12-01

Autotransporters (ATs) are associated with pathogenesis of Avian Pathogenic Escherichia coli (APEC). The molecular characterization of APEC ATs can provide insights about their relevance to APEC pathogenesis. Here, we characterized a conventional autotransporter UpaB in APEC DE205B genome. The upaB existed in 41.9 % of 236 APEC isolates and was predominantly associated with ECOR B2 and D. Our studies showed that UpaB mediates the DE205B adhesion in DF-1 cells, and enhances autoaggregation and biofilm formation of fimbria-negative E. coli AAEC189 (MG1655Δfim) in vitro. Deletion of upaB of DE205B attenuates the virulence in duck model and early colonization in the duck lungs during APEC systemic infection. Furthermore, double and triple deletion of upaB, aatA, and aatB genes cumulatively attenuated DE205B adhesion in DF-1 cells, accompanying with decreased 50 % lethal dose (LD50) in duck model and the early colonization in the duck lungs. However, DE205BΔupaB/ΔaatA/ΔaatB might "compensate" the influence of gene deletion by upregulating the expression of fimbrial adhesin genes yqiL, yadN, and vacuolating autotransporter vat during early colonization of APEC. Finally, we demonstrated that vaccination with recombinant UpaB, AatA, and AatB proteins conferred protection against colisepticemia caused by DE205B infection in duck model.

Abacavir-Reactive Memory T Cells Are Present in Drug Naïve Individuals

PubMed Central

Lucas, Andrew; Lucas, Michaela; Strhyn, Anette; Keane, Niamh M.; McKinnon, Elizabeth; Pavlos, Rebecca; Moran, Ellen M.; Meyer-Pannwitt, Viola; Gaudieri, Silvana; D’Orsogna, Lloyd; Kalams, Spyros; Ostrov, David A.; Buus, Søren; Peters, Bjoern; Mallal, Simon; Phillips, Elizabeth

2015-01-01

Background Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population. Methods To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling. Results Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells. Conclusions We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection. PMID:25674793

Abacavir-reactive memory T cells are present in drug naïve individuals.

PubMed

Lucas, Andrew; Lucas, Michaela; Strhyn, Anette; Keane, Niamh M; McKinnon, Elizabeth; Pavlos, Rebecca; Moran, Ellen M; Meyer-Pannwitt, Viola; Gaudieri, Silvana; D'Orsogna, Lloyd; Kalams, Spyros; Ostrov, David A; Buus, Søren; Peters, Bjoern; Mallal, Simon; Phillips, Elizabeth

2015-01-01

Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population. To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling. Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells. We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.

Detection of survivin, carcinoembryonic antigen and ErbB2 level in oral squamous cell carcinoma patients.

PubMed

Li, Shu-Xia; Yang, Yan-Qi; Jin, Li-Jian; Cai, Zhi-Gang; Sun, Zheng

2016-01-01

The aim of this study was to detect the survivin, carcinoembryonic antigen (CEA) and ErbB2 in the saliva, serum and local tumor-exfoliated cells of oral squamous cell carcinoma (OSCC) patients, for providing reliable tumor markers for the early detection of oral malignant cancer. The saliva, serum, and local tumor-exfoliated cell samples of 26 OSCC patients without chemotherapy and 10 non-cancer patients were collected in Department of Oral and Maxillofacial Surgery, School of Stomatology, Peking University. The contents of survivin, CEA and ErbB2 using were detected usingenzyme-linked immunosorbent assay. The survivin and CEA levels in saliva and local tumor-exfoliated cells of OSCC patients were significantly higher than those in the non-cancer patients (P < 0.05), but there was no significant difference in the content of the above factors in the serum sample between two groups. There was no significant difference in the ErbB2 content in the saliva, serum or local tumor-exfoliated cells between two groups. Survivin and CEA levels are significantly increased in the saliva and local tumor-exfoliated cells in OSCC patients, and they can be used as reliable markers for the early detection of oral malignant cancer.

IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection

PubMed Central

Sebina, Ismail; James, Kylie R.; Soon, Megan S. F.; Best, Shannon E.; Montes de Oca, Marcela; Amante, Fiona H.; Thomas, Bryce S.; Beattie, Lynette; Souza-Fonseca-Guimaraes, Fernando; Smyth, Mark J.; Hertzog, Paul J.; Hill, Geoffrey R.; Engwerda, Christian R.

2016-01-01

Parasite-specific antibodies protect against blood-stage Plasmodium infection. However, in malaria-endemic regions, it takes many months for naturally-exposed individuals to develop robust humoral immunity. Explanations for this have focused on antigenic variation by Plasmodium, but have considered less whether host production of parasite-specific antibody is sub-optimal. In particular, it is unclear whether host immune factors might limit antibody responses. Here, we explored the effect of Type I Interferon signalling via IFNAR1 on CD4+ T-cell and B-cell responses in two non-lethal murine models of malaria, P. chabaudi chabaudi AS (PcAS) and P. yoelii 17XNL (Py17XNL) infection. Firstly, we demonstrated that CD4+ T-cells and ICOS-signalling were crucial for generating germinal centre (GC) B-cells, plasmablasts and parasite-specific antibodies, and likewise that T follicular helper (Tfh) cell responses relied on B cells. Next, we found that IFNAR1-signalling impeded the resolution of non-lethal blood-stage infection, which was associated with impaired production of parasite-specific IgM and several IgG sub-classes. Consistent with this, GC B-cell formation, Ig-class switching, plasmablast and Tfh differentiation were all impaired by IFNAR1-signalling. IFNAR1-signalling proceeded via conventional dendritic cells, and acted early by limiting activation, proliferation and ICOS expression by CD4+ T-cells, by restricting the localization of activated CD4+ T-cells adjacent to and within B-cell areas of the spleen, and by simultaneously suppressing Th1 and Tfh responses. Finally, IFNAR1-deficiency accelerated humoral immune responses and parasite control by boosting ICOS-signalling. Thus, we provide evidence of a host innate cytokine response that impedes the onset of humoral immunity during experimental malaria. PMID:27812214

MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias

PubMed Central

Calin, George Adrian; Liu, Chang-Gong; Sevignani, Cinzia; Ferracin, Manuela; Felli, Nadia; Dumitru, Calin Dan; Shimizu, Masayoshi; Cimmino, Amelia; Zupo, Simona; Dono, Mariella; Dell'Aquila, Marie L.; Alder, Hansjuerg; Rassenti, Laura; Kipps, Thomas J.; Bullrich, Florencia; Negrini, Massimo; Croce, Carlo M.

2004-01-01

Little is known about the expression levels or function of micro-RNAs (miRNAs) in normal and neoplastic cells, although it is becoming clear that miRNAs play important roles in the regulation of gene expression during development [Ambros, V. (2003) Cell 113, 673–676; McManus, M. T. (2003) Semin. Cancer Biol. 13, 253–258]. We now report the genomewide expression profiling of miRNAs in human B cell chronic lymphocytic leukemia (CLL) by using a microarray containing hundreds of human precursor and mature miRNA oligonucleotide probes. This approach allowed us to identify significant differences in miRNome expression between CLL samples and normal CD5+ B cells; data were confirmed by Northern blot analyses and real-time RT-PCR. At least two distinct clusters of CLL samples can be identified that were associated with the presence or absence of Zap-70 expression, a predictor of early disease progression. Two miRNA signatures were associated with the presence or absence of mutations in the expressed Ig variableregion genes or with deletions at 13q14, respectively. These data suggest that miRNA expression patterns have relevance to the biological and clinical behavior of this leukemia. PMID:15284443

A Critical Role for the TLR4/TRIF Pathway in Allogeneic Hematopoietic Cell Rejection by Innate Immune Cells

PubMed Central

Xu, Hong; Yan, Jun; Zhu, Ziqiang; Hussain, Lala-Rukh; Huang, Yiming; Ding, Chuanlin; Bozulic, Larry D.; Wen, Yujie; Ildstad, Suzanne T.

2013-01-01

We show for the first time that signaling through the TLR4/TRIF pathway plays a critical role in allogeneic bone marrow cell (BMC) rejection. This appears to be unique to BMC as organ allografts are rejected mainly via MyD88 signaling. Using T or T/B cell-deficient mice, we found that BMC allorejection occurred early before T cell activation and was T and B cell-independent, suggesting an effector role for innate immune cells in BMC rejection. We further demonstrated the innate immune signaling in BMC allorejection by showing superior engraftment in mice deficient in TRIF or TLR4 but not MyD88 or TLR3. The restored cytotoxicity in TRIF deficient recipients transferred with wildtype F4/80+ or NK1.1+ cells suggests TRIF signaling dependence on macrophages or NK cells in early BMC rejection. Production of the proinflammatory cytokine IL-6 and TRIF relevant chemokine MCP-1 was significantly increased early after bone marrow transplantation. In vivo specific depletion of macrophages or NK innate immune cells in combination with anti-CD154/rapamycin resulted in additive-enhanced allogeneic engraftment. The requirement for irradiation was completely eliminated when both macrophages and NK cells were depleted in combination with anti-CD154/rapamycin to target T and B cells, supporting the hypothesis that two barriers involving innate and adaptive immunity exist in mediating rejection of allogeneic BMC. In summary, our results clearly demonstrate a previously unappreciated role for innate immunity in BMC allorejection via signaling through a unique MyD88-independent TLR4/TRIF mechanism. These findings may have direct clinical impact on strategies for conditioning recipients for stem cell transplantation. PMID:23146386

Glioma cell VEGFR-2 confers resistance to chemotherapeutic and antiangiogenic treatments in PTEN-deficient glioblastoma

PubMed Central

Blaes, Jonas; Osswald, Matthias; Rübmann, Petra; Milford, David; Urban, Severino; Jestaedt, Leonie; Heiland, Sabine; Bendszus, Martin; Hertenstein, Anne; Pfenning, Philipp-Niclas; de Almodóvar, Carmen Ruiz; Wick, Antje; Winkler, Frank; von Deimling, Andreas; Platten, Michael; Wick, Wolfgang; Weiler, Markus

2015-01-01

Loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for tumor cell-specific expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a subgroup prone to develop evasive resistance towards antiangiogenic treatments. Immunohistochemical analysis of human tumor tissues showed VEGFR-2 expression in glioma cells in 19% of specimens examined, mainly in the infiltration zone. Glioma cell VEGFR-2 positivity was restricted to PTEN-deficient tumor specimens. PTEN overexpression reduced VEGFR-2 expression in vitro, as well as knock-down of raptor or rictor. Genetic interference with VEGFR-2 revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in glioma cells. VEGFR-2-dependent cellular effects were concomitant with activation of 'kappa-light-chain-enhancer’ of activated B-cells, protein kinase B, and N-myc downstream regulated gene 1. Two-photon in vivo microscopy revealed that expression of VEGFR-2 in glioma cells hampers antiangiogenesis. Bevacizumab induces a proinvasive response in VEGFR-2-positive glioma cells. Patients with PTEN-negative glioblastomas had a shorter survival after initiation of bevacizumab therapy compared with PTEN-positive glioblastomas. Conclusively, expression of VEGFR-2 in glioma cells indicates an aggressive glioblastoma subgroup developing early resistance to temozolomide or bevacizumab. Loss of PTEN may serve as a biomarker identifying those tumors upfront by routine neuropathological methods. PMID:25682871

Tonsillar CD56brightNKG2A+ NK cells restrict primary Epstein-Barr virus infection in B cells via IFN-γ.

PubMed

Jud, Aurelia; Kotur, Monika; Berger, Christoph; Gysin, Claudine; Nadal, David; Lünemann, Anna

2017-01-24

Natural killer (NK) cells constitute the first line of defense against viruses and cancers cells. Epstein-Barr virus (EBV) was the first human virus to be directly implicated in carcinogenesis, and EBV infection is associated with a broad spectrum of B cell lymphomas. How NK cells restrict EBV-associated oncogenesis is not understood. Here, we investigated the efficacies and mechanisms of distinct NK cell subsets from tonsils, the portal of entry of EBV, in limiting EBV infection in naïve, germinal center-associated and memory B cells. We found that CD56bright and NKG2A expression sufficiently characterizes the potent anti-EBV capacity of tonsillar NK cells. We observed restriction of EBV infection in B cells as early as 18 hours after infection. The restriction was most efficient in naïve B cells and germinal center-associated B cells, the B cell subsets that exhibited highest susceptibility to EBV infection in vitro. IFN-γ release by and partially NKp44 engagement of CD56bright and NKG2A positive NK cells mediated the restriction that eventually inhibited B-cell transformation. Thus, harnessing CD56brightNKG2A+ NK cell function might be promising to improve treatment strategies that target EBV-associated B cell lymphomas.

SoxNeuro orchestrates central nervous system specification and differentiation in Drosophila and is only partially redundant with Dichaete

PubMed Central

2014-01-01

Background Sox proteins encompass an evolutionarily conserved family of transcription factors with critical roles in animal development and stem cell biology. In common with vertebrates, the Drosophila group B proteins SoxNeuro and Dichaete are involved in central nervous system development, where they play both similar and unique roles in gene regulation. Sox genes show extensive functional redundancy across metazoans, but the molecular basis underpinning functional compensation mechanisms at the genomic level are currently unknown. Results Using a combination of genome-wide binding analysis and gene expression profiling, we show that SoxNeuro directs embryonic neural development from the early specification of neuroblasts through to the terminal differentiation of neurons and glia. To address the issue of functional redundancy and compensation at a genomic level, we compare SoxNeuro and Dichaete binding, identifying common and independent binding events in wild-type conditions, as well as instances of compensation and loss of binding in mutant backgrounds. Conclusions We find that early aspects of group B Sox functions in the central nervous system, such as stem cell maintenance and dorsoventral patterning, are highly conserved. However, in contrast to vertebrates, we find that Drosophila group B1 proteins also play prominent roles during later aspects of neural morphogenesis. Our analysis of the functional relationship between SoxNeuro and Dichaete uncovers evidence for redundant and independent functions for each protein, along with unexpected examples of compensation and interdependency, thus providing new insights into the general issue of transcription factor functional redundancy. PMID:24886562

[In vitro early detection of amyloid plaques in Alzheimer's disease by Pittsburgh compound B-modified magnetic nanoparticles].

PubMed

Zeng, J Q; Wu, J Q; Li, M H; Wang, P J

2017-11-07

Objective: To construct magnetic nanoparticles targeting β-amyloid (Aβ) plaques, the pathological biomarker of Alzheimer's disease (AD) and to study their binding capability in vitro . Methods: Superparamagnetic nanoparticles Mn(0.6)Zn(0.4)Fe(2)O(4) (MZF) were coated with amphiphilic star-block copolymeric micelles and modified with Aβ-specific probe Pittsburgh compound B (PiB) to construct a novel magnetic nanoparticle MZF-PiB, which specifically targeted amyloid plaques. Transmission electron microscope was used to study the morphological features of MZF-PiB. Superparamagnetism of MZF-PiB was assessed by its r(2) relaxation rate by using 3.0 T MRI scanner. Cytotoxic test was applied to determine biosafety of MZF-PiB nanoparticles in differentiated human neuroblastoma cells (SH-SY5Y) and Madin-Darby canine kidney (MDCK). In vitro binding tests were conducted via immunohistochemistry on 6-month old AD mice brain sections. Differences of cell viability between groups were compared with one-way analysis of variance. Results: MZF-PiB nanoparticles were successfully constructed. Transmission electron microscope images showed that the nanoparticles were about 100 nm in size. The r(2) relaxation rate was 163.11 mMS(-1). No differences were found in cell viability of SH-SY5Y and MDCK incubated with MZF-PiB suspension for 24 h or 48 h when compared with those of untreated cells ( F =2.336, 2.539, 0.293, 1.493, all P >0.05). In vitro binding tests indicated that the MZF-PiB were specifically bound to amyloid plaques. The smallest size of detected plaques was 27 μm. Conclusion: PiB-modified nanoparticles targeting Aβ are biologically safe and highly superparamagnetic, possessing the capability to detect amyloid plaques early in vitro and the potential for early diagnosis of AD.

The effect of ultraviolet radiation on choroidal melanocytes and melanoma cell lines: cell survival and matrix metalloproteinase production.

PubMed

Lai, Kenneth; Di Girolamo, Nick; Conway, Robert M; Jager, Martine J; Madigan, Michele C

2007-05-01

Ultraviolet radiation (UVR) can induce DNA damage and regulate the expression of factors important for tumour growth and metastasis, including matrix metalloproteinases (MMPs). Epidemiological studies suggest that chronic UVR exposure, especially during early adulthood, may be a risk factor in patients with choroidal melanoma. However, the effects of UV(R)-B on human choroidal melanocyte survival and growth are unknown. In this study, we investigated if UV(R)-B affected the in vitro survival, growth and MMP production of choroidal melanocytes and melanoma cells. Cultures of primary choroidal melanocytes and melanoma cell lines (OCM-1 and OCM-8) were exposed to UV(R)-B (0-30 mJ/cm(2)). The cell morphology and growth were examined, and cell viability was assessed using an MTT assay. Gelatin zymography was used to assess the enzymatic activity for MMP-2 and -9 in conditioned media following UV(R)-B treatment. UV(R)-B > or =20 mJ/cm(2) was cytotoxic for choroidal melanocytes. Cytotoxic doses of 5 to 10 mJ/cm(2) were found for OCM-8 and OCM-1 melanoma cell lines. Low levels of UV(R)-B (2.5 and 3.5 mJ/cm(2)) significantly reduced melanoma cell viability after 48 h, although melanocyte viability was not affected by doses of UV(R)-B <10 mJ/cm(2). Conditioned media from melanoma cells and melanocytes displayed pro-MMP-2 activity independent of UV(R)-B. Control and UV(R)-B-treated OCM-1 cells secreted active MMP-2 up to 72 h. Pro-MMP-9 activity was seen from 36 h for control and UV(R)-B-treated OCM-1 and OCM-8 cells. Melanocytes appeared more resistant to physiological doses of UV(R)-B than melanoma cells; the potential of melanocytes to initially survive DNA damage following UV(R)-B exposure may be relevant to the subsequent transformation of melanocytes to melanomas. Although UV(R)-B did not induce the production and/or activation of MMP-2 and -9 in melanocytes or melanoma cells, we are currently investigating whether DNA damage-response genes such as p53 and p21 can be regulated following UVR exposure, and whether they are important for choroidal melanoma development.

CLB5-dependent activation of late replication origins in S. cerevisiae.

PubMed

Donaldson, A D; Raghuraman, M K; Friedman, K L; Cross, F R; Brewer, B J; Fangman, W L

1998-08-01

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Early detection of disease program: Evaluation of the cellular immune response

NASA Technical Reports Server (NTRS)

Criswell, B. S.; Knight, V.; Martin, R. R.; Kasel, J. A.

1975-01-01

Surfaces of normal, cultured, and mitogen-stimulated mouse lymphoid cells were examined by scanning electron microscopy (SEM). Lymphocytes with smooth, highly villous and intermediate surfaces were observed in cell suspensions from both spleens and thymuses of normal mice and from spleens of congenitally athymic (nude) mice. Several strain-specific surface features were noted, including the spine-like appearance of microvilli on C57B1/6 lymphocytes. Although thymus cell suspensions contained somewhat more smooth cells than did spleen cell preparations, lymphocyte derivation could not be inferred from SEM examination. Studies of cells stimulated with mitogenic agents for thymus-derived lymphocytes (concanavalin A) or for bone marrow-derived lymphocytes (lipopolysaccharide) suggested that, in the mouse, development of a complex villous surface is a general concomitant of lymphocyte activation and transformation.

Developmental exposure to 2,3,7,8 tetrachlorodibenzo-p-dioxin attenuates capacity of hematopoietic stem cells to undergo lymphocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahrenhoerster, Lori S.; Tate, Everett R.; Lakatos, Peter A.

The process of hematopoiesis, characterized by long-term self-renewal and multi-potent lineage differentiation, has been shown to be regulated in part by the ligand-activated transcription factor known as the aryl hydrocarbon receptor (AHR). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous contaminant and the most potent AHR agonist, also modulates regulation of adult hematopoietic stem and progenitor cell (HSC/HPC) homeostasis. However, the effect of developmental TCDD exposure on early life hematopoiesis has not been fully explored. Given the inhibitory effects of TCDD on hematopoiesis and lymphocyte development, we hypothesized that in utero exposure to TCDD would alter the functional capacity of fetal HSC/HPCs to completemore » lymphocyte differentiation. To test this hypothesis, we employed a co-culture system designed to facilitate the maturation of progenitor cells to either B or T lymphocytes. Furthermore, we utilized an innovative limiting dilution assay to precisely quantify differences in lymphocyte differentiation between HSC/HPCs obtained from fetuses of dams exposed to 3 μg/kg TCDD or control. We found that the AHR is transcribed in yolk sac hematopoietic cells and is transcriptionally active as early as gestational day (GD) 7.5. Furthermore, the number of HSC/HPCs present in the fetal liver on GD 14.5 was significantly increased in fetuses whose mothers were exposed to TCDD throughout pregnancy. Despite this increase in HSC/HPC cell number, B and T lymphocyte differentiation is decreased by approximately 2.5 fold. These findings demonstrate that inappropriate developmental AHR activation in HSC/HPCs adversely impacts lymphocyte differentiation and may have consequences for lymphocyte development in the bone marrow and thymus later in life.« less

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

PubMed

Spicer, S Timothy; Tran, Giang T; Killingsworth, Murray C; Carter, Nicole; Power, David A; Paizis, Kathy; Boyd, Rochelle; Hodgkinson, Suzanne J; Hall, Bruce M

2007-07-01

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

The Pathogenic Role of Persistent Milk Signaling in mTORC1- and Milk-MicroRNA-Driven Type 2 Diabetes Mellitus

PubMed Central

Melnik, Bodo C

2015-01-01

Milk, the secretory product of the lactation genome, promotes growth of the newborn mammal. Milk delivers insulinotropic amino acids, thus maintains a molecular crosstalk with the pancreatic β-cell of the milk recipient. Homeostasis of β-cells and insulin production depend on the appropriate magnitude of mTORC1 signaling. mTORC1 is activated by branched-chain amino acids (BCAAs), glutamine, and palmitic acid, abundant nutrient signals of cow´s milk. Furthermore, milk delivers bioactive exosomal microRNAs. After milk consumption, bovine microRNA-29b, a member of the diabetogenic microRNA-29-family, reaches the systemic circulation and the cells of the milk consumer. MicroRNA-29b downregulates branched-chain α-ketoacid dehydrogenase, a potential explanation for increased BCAA serum levels, the metabolic signature of insulin resistance and type 2 diabetes mellitus (T2DM). In non-obese diabetic mice, microRNA-29b downregulates the anti-apoptotic protein Mcl-1, which leads to early β-cell death. In all mammals except Neolithic humans, milk-driven mTORC1 signaling is physiologically restricted to the postnatal period. In contrast, chronic hyperactivated mTORC1 sig-naling has been associated with the development of age-related diseases of civilization including T2DM. Notably, chronic hyperactivation of mTORC1 enhances endoplasmic reticulum stress that promotes apoptosis. In fact, hyperactivated β-cell mTORC1 signaling induced early β-cell apoptosis in a mouse model. The EPIC-InterAct Study demonstrated an association between milk consumption and T2DM in France, Italy, United Kingdom, Germany, and Sweden. In contrast, fermented milk products and cheese exhibit an inverse correlation. Since the early 1950´s, refrigeration technology allowed widespread consumption of fresh pasteurized milk, which facilitates daily intake of bioactive bovine microRNAs. Persistent uptake of cow´s milk-derived microRNAs apparently transfers an overlooked epigenetic diabetogenic program that should not reach the human food chain. PMID:25587719

Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish.

PubMed

Akimenko, M A; Johnson, S L; Westerfield, M; Ekker, M

1995-02-01

To study the genetic regulation of growth control and pattern formation during fin development and regeneration, we have analysed the expression of four homeobox genes, msxA, msxB, msxC and msxD in zebrafish fins. The median fin fold, which gives rise to the unpaired fins, expresses these four msx genes during development. Transcripts of the genes are also present in cells of the presumptive pectoral fin buds. The most distal cells, the apical ectodermal ridge of the paired fins and the cleft and flanking cells of the median fin fold express all these msx genes with the exception of msxC. Mesenchymal cells underlying the most distal cells express all four genes. Expression of the msx genes in the fin fold and fin buds is transient and, by 3 days after fertilization, msx expression in the median fin fold falls below levels detectable by in situ hybridization. Although the fins of adult zebrafish normally have levels of msx transcripts undetectable by in situ hybridization, expression of all four genes is strongly reinduced during regeneration of both paired and unpaired fins. Induction of msx gene expression in regenerating caudal fins occurs as early as 30 hours postamputation. As the blastema forms, the levels of expression increase and reach a maximum between the third and fifth days. Then, msx expression progressively declines and disappears by day 12 when the caudal fin has grown back to its normal size. In the regenerating fin, the blastema cells that develop at the tip of each fin ray express msxB and msxC. Cells of the overlying epithelium express msxA and msxD, but do not express msxB or msxC. Amputations at various levels along the proximodistal axis of the fin suggest that msxB expression depends upon the position of the blastema, with cells of the rapidly proliferating proximal blastema expressing higher levels than the cells of the less rapidly proliferating distal blastema. Expression of msxC and msxD is independent of the position of the blastema cell along this axis. Our results suggest distinct roles for each of the four msx genes during fin development and regeneration and differential regulation of their expression.

Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3.

PubMed

Jaleco, A C; Stegmann, A P; Heemskerk, M H; Couwenberg, F; Bakker, A Q; Weijer, K; Spits, H

1999-10-15

Transgenic and gene targeted mice have contributed greatly to our understanding of the mechanisms underlying B-cell development. We describe here a model system that allows us to apply molecular genetic techniques to the analysis of human B-cell development. We constructed a retroviral vector with a multiple cloning site connected to a gene encoding green fluorescent protein by an internal ribosomal entry site. Human CD34(+)CD38(-) fetal liver cells, cultured overnight in a combination of stem cell factor and interleukin-7 (IL-7), could be transduced with 30% efficiency. We ligated the gene encoding the dominant negative helix loop helix (HLH) factor Id3 that inhibits many enhancing basic HLH transcription factors into this vector. CD34(+)CD38(-) FL cells were transduced with Id3-IRES-GFP and cultured with the murine stromal cell line S17. In addition, we cultured the transduced cells in a reaggregate culture system with an SV-transformed human fibroblast cell line (SV19). It was observed that overexpression of Id3 inhibited development of B cells in both culture systems. B-cell development was arrested at a stage before expression of the IL-7Ralpha. The development of CD34(+)CD38(-) cells into CD14(+) myeloid cells in the S17 system was not inhibited by overexpression of Id3. Moreover, Id3(+) cells, although inhibited in their B-cell development, were still able to develop into natural killer (NK) cells when cultured in a combination of Flt-3L, IL-7, and IL-15. These findings confirm the essential role of bHLH factors in B-cell development and demonstrate the feasibility of retrovirus-mediated gene transfer as a tool to genetically modify human B-cell development.

Dynamic pre-BCR homodimers fine-tune autonomous survival signals in B cell precursor acute lymphoblastic leukemia

PubMed Central

Erasmus, M. Frank; Matlawska-Wasowska, Ksenia; Kinjyo, Ichiko; Mahajan, Avanika; Winter, Stuart S.; Xu, Li; Horowitz, Michael; Lidke, Diane S.; Wilson, Bridget S.

2017-01-01

The pre-B cell receptor (pre-BCR) is an immature form of the BCR critical for early B lymphocyte development. It is composed of the membrane-bound immunoglobulin (Ig) heavy chain, surrogate light chain components, and the signaling subunits Igα and Igβ. We developed monovalent Quantum Dot (QD)-labeled probes specific for Igβ to study the behavior of pre-BCRs engaged in autonomous, ligand-independent signaling in live B cells. Single-particle tracking revealed that QD-labeled pre-BCRs engaged in transient, but frequent, homotypic interactions. Receptor motion was correlated at short separation distances, consistent with the formation of dimers and higher-order oligomers. Repeated encounters between diffusing pre-BCRs appeared to reflect transient co-confinement in plasma membrane domains. In human B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, we showed that frequent, short-lived, homotypic pre-BCR interactions stimulated survival signals, including expression of BCL6, which encodes a transcriptional repressor. These survival signals were blocked by inhibitory monovalent antigen-binding antibody fragments (Fabs) specific for the surrogate light chain components of the pre-BCR or by inhibitors of the tyrosine kinases Lyn and Syk. For comparison, we evaluated pre-BCR aggregation mediated by dimeric galectin-1, which has binding sites for carbohydrate and for the λ5 component of the surrogate light chain. Galectin-1 binding resulted in the formation of large, highly immobile pre-BCR aggregates, which was partially relieved by the addition of lactose to prevent the crosslinking of galectin-BCR complexes to other glycosylated membrane components. Analysis of the pre-BCR and its signaling partners suggested that they could be potential targets for combination therapy in BCP-ALL. PMID:27899526

Novel method for screening of enteric film coatings properties with magnetic resonance imaging.

PubMed

Dorożyński, Przemysław; Jamróz, Witold; Niwiński, Krzysztof; Kurek, Mateusz; Węglarz, Władysław P; Jachowicz, Renata; Kulinowski, Piotr

2013-11-18

The aim of the study is to present the concept of novel method for fast screening of enteric coating compositions properties without the need of preparation of tablets batches for fluid bed coating. Proposed method involves evaluation of enteric coated model tablets in specially designed testing cell with application of MRI technique. The results obtained in the testing cell were compared with results of dissolution studies of mini-tablets coated in fluid bed apparatus. The method could be useful in early stage of formulation development for screening of film coating properties that will shorten and simplify the development works. Copyright © 2013 Elsevier B.V. All rights reserved.

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.

Switched Memory B Cells Are Increased in Oligoarticular and Polyarticular Juvenile Idiopathic Arthritis and Their Change Over Time Is Related to Response to Tumor Necrosis Factor Inhibitors.

PubMed

Marasco, Emiliano; Aquilani, Angela; Cascioli, Simona; Moneta, Gian Marco; Caiello, Ivan; Farroni, Chiara; Giorda, Ezio; D'Oria, Valentina; Marafon, Denise Pires; Magni-Manzoni, Silvia; Carsetti, Rita; De Benedetti, Fabrizio

2018-04-01

To investigate whether abnormalities in B cell subsets in patients with juvenile idiopathic arthritis (JIA) correlate with clinical features and response to treatment. A total of 109 patients diagnosed as having oligoarticular JIA or polyarticular JIA were enrolled in the study. B cell subsets in peripheral blood and synovial fluid were analyzed by flow cytometry. Switched memory B cells were significantly increased in patients compared to age-matched healthy controls (P < 0.0001). When patients were divided according to age at onset of JIA, in patients with early-onset disease (presenting before age 6 years) the expansion in switched memory B cells was more pronounced than that in patients with late-onset disease and persisted throughout the disease course. In longitudinal studies, during methotrexate (MTX) treatment, regardless of the presence or absence of active disease, the number of switched memory B cells increased significantly (median change from baseline 36% [interquartile range {IQR} 15, 66]). During treatment with MTX plus tumor necrosis factor inhibitors (TNFi), in patients maintaining disease remission, the increase in switched memory B cells was significantly lower than that in patients who experienced active disease (median change from baseline 4% [IQR -6, 32] versus 41% [IQR 11, 73]; P = 0.004). The yearly rate of increases in switched memory B cells was 1.5% in healthy controls, 1.2% in patients who maintained remission during treatment with MTX plus TNFi, 4.7% in patients who experienced active disease during treatment with MTX plus TNFi, and ~4% in patients treated with MTX alone. Switched memory B cells expand during the disease course at a faster rate in JIA patients than in healthy children. This increase is more evident in patients with early-onset JIA. TNFi treatment inhibits this increase in patients who achieve and maintain remission, but not in those with active disease. © 2018, American College of Rheumatology.

Cloning and analysis of fetal ovary microRNAs in cattle.

PubMed

Tripurani, Swamy K; Xiao, Caide; Salem, Mohamed; Yao, Jianbo

2010-07-01

Ovarian folliculogenesis and early embryogenesis are complex processes, which require tightly regulated expression and interaction of a multitude of genes. Small endogenous RNA molecules, termed microRNAs (miRNAs), are involved in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs in bovine oocytes/ovaries, a bovine fetal ovary miRNA library was constructed. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs present in the miRBase database and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. The precursor sequences for 14 of the new 15 miRNAs as well as the novel miRNA were identified from the bovine genome database and their hairpin structures were predicted. Expression analysis of the 58 miRNAs in fetal ovaries in comparison to somatic tissue pools identified 8 miRNAs predominantly expressed in fetal ovaries. Further analysis of the eight miRNAs in germinal vesicle (GV) stage oocytes identified two miRNAs (bta-mir424 and bta-mir-10b), that are highly abundant in GV oocytes. Both miRNAs show similar expression patterns during oocyte maturation and preimplantation development of bovine embryos, being abundant in GV and MII stage oocytes, as well as in early stage embryos (until 16-cell stage). The amount of the novel miRNA is relatively small in oocytes and early cleavage embryos but greater in blastocysts, suggesting a role of this miRNA in blastocyst cell differentiation. Copyright 2010 Elsevier B.V. All rights reserved.

B cells and B cell products-helping to restore cellular immunity?

PubMed

Cascalho, Marilia; Platt, Jeffrey L

2006-01-01

T cells that provide vital protection against tumors, viruses and intracellular bacteria are thought to develop independently of B cells. However, recent discoveries suggest that development of T cells depends on B cells. One way B cells promote T cell development is by providing diverse peptides that may promote positive selection of thymocytes. Diverse peptides and B cells help in diversification of the T cell receptor repertoire and may decrease cross-reactivity in the mature T cell compartment. These new insights may provide the basis for the design of novel therapeutics.

Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis.

PubMed

Yang, Shun-Min; Chan, Yi-Lin; Hua, Kuo-Feng; Chang, Jia-Ming; Chen, Hui-Ling; Tsai, Yung-Jen; Hsu, Yu-Juei; Chao, Louis Kuoping; Feng-Ling, Yang; Tsai, Yu-Ling; Wu, Shih-Hsiung; Wang, Yih-Fuh; Tsai, Change-Ling; Chen, Ann; Ka, Shuk-Man

2014-08-01

Inflammatory reactions and oxidative stress are implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS), a common chronic kidney disease with relatively poor prognosis and unsatisfactory treatment regimens. Previously, we showed that osthole, a coumarin compound isolated from the seeds of Cnidium monnieri, can inhibit reactive oxygen species generation, NF-κB activation, and cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages. In this study, we further evaluated its renoprotective effect in a mouse model of accelerated FSGS (acFSGS), featuring early development of proteinuria, followed by impaired renal function, glomerular epithelial cell hyperplasia lesions (a sensitive sign that precedes the development of glomerular sclerosis), periglomerular inflammation, and glomerular hyalinosis/sclerosis. The results show that osthole significantly prevented the development of the acFSGS model in the treated group of mice. The mechanisms involved in the renoprotective effects of osthole on the acFSGS model were mainly a result of an activated Nrf2-mediated antioxidant pathway in the early stage (proteinuria and ischemic collapse of the glomeruli) of acFSGS, followed by a decrease in: (1) NF-κB activation and COX-2 expression as well as PGE2 production, (2) podocyte injury, and (3) apoptosis. Our data support that targeting the Nrf2 antioxidant pathway may justify osthole being established as a candidate renoprotective compound for FSGS. Copyright © 2014 Elsevier Inc. All rights reserved.

Uneven colonization of the lymphoid periphery by T cells that undergo early TCR{alpha} rearrangements.

PubMed

Hendricks, Deborah W; Fink, Pamela J

2009-04-01

A sparse population of thymocytes undergoes TCRalpha gene rearrangement early in development, before the double-positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRalpha rearrangements are restricted to the double-negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2(fl/fl) mice, and the TCRalphabeta(+) T cells that develop are limited in their TCRalpha repertoire, preferentially using early rearranging Valpha genes. In the gut, the Thy-1(+)TCRalphabeta(+) intraepithelial lymphocyte (IEL) compartment is surprisingly intact, whereas the Thy-1(-)TCRalphabeta(+) subset is almost completely absent. Thus, T cells expressing a TCRalpha repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2(fl/fl) T cell progenitors cannot compete with wild-type T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRalpha rearrangements. In the absence of wild-type competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1(+) TCRalphabeta(+) T cell pool from the limited population derived from Cre(+) RAG2(fl/fl) progenitors.

Uneven colonization of the lymphoid periphery by T cells that undergo early TCRα rearrangements1

PubMed Central

Hendricks, Deborah W.; Fink, Pamela J.

2009-01-01

A sparse population of thymocytes undergoes TCRα gene rearrangement early in development, before the double positive stage. The potential of these cells to contribute to the peripheral T cell pool is unknown. To examine the peripheral T cell compartment expressing a repertoire biased to early TCR gene rearrangements, we developed a mouse model in which TCRα rearrangements are restricted to the double negative stage of thymocyte development. These mice carry floxed RAG2 alleles and a Cre transgene driven by the CD4 promoter. As expected, conventional T cell development is compromised in such Cre(+) RAG2fl/fl mice, and the TCRαβ+ T cells that develop are limited in their TCRα repertoire, preferentially utilizing early-rearranging Vα genes. In the gut, the Thy-1+TCRαβ+ intraepithelial lymphocyte (IEL) compartment is surprisingly intact, while the Thy-1−TCRαβ+ subset is almost completely absent. Thus, T cells expressing a TCRα repertoire that is the product of early gene rearrangements can preferentially populate distinct IEL compartments. Despite this capacity, Cre(+) RAG2fl/fl T cell progenitors cannot compete with wild-type (WT) T cell progenitors in mixed bone marrow chimeras, suggesting that in normal mice, there is only a small contribution to the peripheral T cell pool by cells that have undergone early TCRα rearrangements. In the absence of WT competitors, aggressive homeostatic proliferation in the IEL compartment can promote a relatively normal Thy-1+ TCRαβ+ T cell pool from the limited population derived from Cre(+) RAG2fl/fl progenitors. PMID:19299725

Impact of PCOS on early embryo cleavage kinetics.

PubMed

Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L

2014-04-01

This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Neonatal microbial colonization in mice promotes prolonged dominance of CD11b(+)Gr-1(+) cells and accelerated establishment of the CD4(+) T cell population in the spleen.

PubMed

Kristensen, Matilde B; Metzdorff, Stine B; Bergström, Anders; Damlund, Dina S M; Fink, Lisbeth N; Licht, Tine R; Frøkiær, Hanne

2015-09-01

To assess the microbial influence on postnatal hematopoiesis, we examined the role of early life microbial colonization on the composition of leukocyte subsets in the neonatal spleen. A high number of CD11b(+)Gr-1(+) splenocytes present perinatally was sustained for a longer period in conventionally colonized (CONV) mice than in mono-colonized (MC) and germfree (GF) mice, and the CD4(+) T cell population established faster in CONV mice. At the day of birth, compared to GF mice, the expression of Cxcl2 was up-regulated and Arg1 down-regulated in livers of CONV mice. This coincided with lower abundance of polylobed cells in the liver of CONV mice. An earlier peak in the expression of the genes Tjp1, Cdh1, and JamA in intestinal epithelial cells of CONV mice indicated an accelerated closure of the epithelial barrier. In conclusion, we have identified an important microbiota-dependent neonatal hematopoietic event, which we suggest impacts the subsequent development of the T cell population in the murine spleen.

Analysis of epothilone B-induced cell death in normal ovarian cells.

PubMed

Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

2013-12-01

We have investigated the mode of cell death induced by a new microtubule-stabilizing agent, epothilone B (EpoB, patupilone), and a clinically used medicine, paclitaxel (PTX), in normal ovarian cells. Using fluorescence microscopy, polyacrylamide gel electrophoresis preceding Western blot analysis, as well as spectrofluorimetric and colorimetric detection, we demonstrate that, compared to EpoB, PTX induced high time-dependent morphological and biochemical changes typical of apoptosis. Induction of apoptosis followed an early increase in p53 levels. Apoptosis reached its maximum at 24-48 h. At the same time, there was a significant increase in caspase-9 and -3 activity and PARP fragmentation, which suggests that an intrinsic path was involved. Apoptosis in MM14 cells was increased more by PTX than EpoB, and also induced more necrosis responsible for inflammation (1.4-fold) than EpoB. © 2013 International Federation for Cell Biology.

Cytokine markers of B lymphocytes in minor salivary gland infiltrates in Sjögren's syndrome.

PubMed

Navarro-Mendoza, Erika P; Aguirre-Valencia, David; Posso-Osorio, Iván; Correa-Forero, Shirley Vanessa; Torres-Cutiva, Daniel-Felipe; Loaiza, Diana; Tobón, Gabriel J

2018-05-03

Sjögren's syndrome (SS) is a chronic autoimmune disorder characterised by the clinical presence of sicca syndrome. SS compromises the dysfunction of exocrine glands due to the presence of focal, mononuclear cell infiltrates that surround the ducts and replace the secretory units. Abnormal expression of different cytokines and chemokines such as B-cell activating factor, CXC Motif Chemokine Ligand 13, interleukin 6 (IL-6), IL-22, and FMS-like tyrosine kinase 3 ligand as well as that of their corresponding receptors has been implicated in the inflammatory process. The severity of glandular infiltration has been suggested to be associated with the presence of extra-glandular systemic manifestations, contributing to a clinical spectrum of the most severe disease. This review describes several cytokines and chemokines associated with B lymphocytes expressed in the minor salivary gland, their chemical structures, and their roles in SS as possible early predictors of lymphoma development and disease progression. Copyright © 2018. Published by Elsevier B.V.

Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis

PubMed Central

Nishimori, Asami; Okagawa, Tomohiro; Maekawa, Naoya; Goto, Shinya; Ikebuchi, Ryoyo; Nakahara, Ayako; Chiba, Yuzumi; Ikeda, Masaho; Murata, Shiro; Ohashi, Kazuhiko

2017-01-01

ABSTRACT Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection. PMID:28659325

Boron Induces Early Matrix Mineralization via Calcium Deposition and Elevation of Alkaline Phosphatase Activity in Differentiated Rat Bone Marrow Mesenchymal Stem Cells

PubMed Central

Movahedi Najafabadi, Bent-al-hoda; Abnosi, Mohammad Hussein

2016-01-01

Objective Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular nature of differentiated rat bone marrow mesenchymal stem cells (BMSCs). Materials and Methods In this experimental study, BMSCs were extracted and expanded to the 3rdpassage, then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) complemented with osteogenic media as well as 6 ng/ml and 6 µg/ml of BA. After 5, 10, 15 and 21 days the viability and the level of mineralization was determined using MTT assay and alizarin red respectively. In addition, the morphology, nuclear diameter and cytoplasmic area of the cells were studied with the help of fluorescent dye. The concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) as well as sodium and potassium levels were also evaluated using commercial kits and a flame photometer respectively. Results Although 6 µg/ml of BA was found to be toxic, a concentration of 6 ng/ml increased the osteogenic ability of the cell significantly throughout the treatment. In addition it was observed that B treatment caused the early induction of matrix mineralization compared to controls. Conclusion Although more investigation is required, we suggest the prescription of a very low concentration of B in the form of BA or foods containing BA, in groups at high risk of osteoporosis or in the case of bone fracture. PMID:27054120

Transient maintenance in bioreactor improves health of neuronal cells.

PubMed

Di Loreto, Silvia; Sebastiani, Pierluigi; Benedetti, Elisabetta; Zimmitti, Vincenzo; Caracciolo, Valentina; Amicarelli, Fernanda; Cimini, Annamaria; Adorno, Domenico

2006-01-01

To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, -9, and -3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is coupled with a level of activated nuclear factor-kappaB (NF-kappaB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.

Cineromycin B isolated from Streptomyces cinerochromogenes inhibits adipocyte differentiation of 3T3-L1 cells via Krüppel-like factors 2 and 3.

PubMed

Matsuo, Hirotaka; Kondo, Yoshiyuki; Kawasaki, Takashi; Imamura, Nobutaka

2015-08-15

3T3-L1 cells are preadipocytes and often used as a model for cellular differentiation to adipocytes; however, the mechanism of this differentiation is not completely understood even in these model cells. In this study, we sought to identify a unique anti-adipogenesis agent from microorganisms and to examine its mechanism of action to gain knowledge and create a tool and/or seed compound for anti-obesity drug discovery research. Screening for anti-adipogenesis agents from microorganisms was performed using a 3T3-L1 cell differentiation system, and an active compound was isolated. The inhibitory mechanism of the compound was investigated by measuring the expression of key regulators using quantitative real-time PCR and Western blot analysis. The compound with anti-adipogenic activity in 3T3-L1 cells was identified as cineromycin B. Cineromycin B at 50 μg/mL suppressed intracellular lipid accumulation and the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα), which are master regulators of adipocyte differentiation. Further investigations showed that cineromycin B increased significantly the mRNA expression of two negative regulators of adipocyte differentiation, Krüppel-like factor (KLF) 2 and KLF3, at an early stage of the differentiation. The results of siRNA transfection experiments indicated that cineromycin B is a unique adipocyte differentiation inhibitor, acting mainly via upregulation of KLF2 and KLF3, and these KLFs may play a role in the early stage of differentiation. Cineromycin B inhibited adipocyte differentiation in 3T3-L1 cells mainly via upregulation of KLF2 and KLF3 mRNA expression at an early stage of the differentiation. Copyright © 2015. Published by Elsevier Inc.

B-cell polyclonal activation and Epstein-Barr viral abortive lytic cycle are two key features in acute infectious mononucleosis.

PubMed

Al Tabaa, Yassine; Tuaillon, Edouard; Jeziorski, Eric; Ouedraogo, David Eric; Bolloré, Karine; Rubbo, Pierre-Alain; Foulongne, Vincent; Rodière, Michel; Vendrell, Jean-Pierre

2011-09-01

Acute infectious mononucleosis (AIM) is generally associated with a large EBV B cell reservoir cells and an intense B-cell polyclonal activation whereas the number of quiescent EBV-infected memory B cells in chronically EBV-infected healthy controls is very low. To evaluate the extent and functionality of ex vivo B-cell polyclonal activation, quantify the EBV DNA integrated in B cells, enumerate the functional EBV DNA reservoir in B cells and circulating B cells spontaneously secreting EBV antigens in AIM. Circulating B cells and B cells differentiating into plamablasts and plasma cells, early (BZLF1)- and late viral antigen (gp350)-secreting-cells (SCs) were enumerated in six AIM patients and seven healthy EBV carriers. In vitro B-cell polyclonal activation induced 8000-24,000 BZLF1- and 1000-3000gp350-SCs/10(6) B cells, respectively. These data suggest that only 11.1-19.5% of cells expressing BZLF1 synthesized gp350 and so completed the EBV-lytic cycle. Furthermore, circulating spontaneous BZLF1- and gp350-SCs that reflect ongoing viral replication were rare (20-120 and 10-30/10(6) B cells, respectively), and their low numbers contrasted with the high levels of circulating plasma cells (1.1-10.2% of CD19(+) B cells). The in vivo terminal-B-cell differentiation into plasma cells could unmask EBV B-cell reservoir to specific cytotoxic T-cell response and combined with a predominant abortive functional-EBV-reservoir, strongly contribute to rapid decay of cellular EBV reservoir in AIM. Copyright © 2011 Elsevier B.V. All rights reserved.

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease.

PubMed

Zhou, Qing; Wang, Hongying; Schwartz, Daniella M; Stoffels, Monique; Park, Yong Hwan; Zhang, Yuan; Yang, Dan; Demirkaya, Erkan; Takeuchi, Masaki; Tsai, Wanxia Li; Lyons, Jonathan J; Yu, Xiaomin; Ouyang, Claudia; Chen, Celeste; Chin, David T; Zaal, Kristien; Chandrasekharappa, Settara C; P Hanson, Eric; Yu, Zhen; Mullikin, James C; Hasni, Sarfaraz A; Wertz, Ingrid E; Ombrello, Amanda K; Stone, Deborah L; Hoffmann, Patrycja; Jones, Anne; Barham, Beverly K; Leavis, Helen L; van Royen-Kerkof, Annet; Sibley, Cailin; Batu, Ezgi D; Gül, Ahmet; Siegel, Richard M; Boehm, Manfred; Milner, Joshua D; Ozen, Seza; Gadina, Massimo; Chae, JaeJin; Laxer, Ronald M; Kastner, Daniel L; Aksentijevich, Ivona

2016-01-01

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB-mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB-dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

PubMed Central

Koshiba, Taichi; Kobayashi, Masaru; Matoh, Toru

2009-01-01

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD. PMID:19054807

Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins.

PubMed

Tarusawa, Etsuko; Sanbo, Makoto; Okayama, Atsushi; Miyashita, Toshio; Kitsukawa, Takashi; Hirayama, Teruyoshi; Hirabayashi, Takahiro; Hasegawa, Sonoko; Kaneko, Ryosuke; Toyoda, Shunsuke; Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu; Hirabayashi, Masumi; Yagi, Takeshi; Yoshimura, Yumiko

2016-12-02

The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections. We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity. Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.

Transcriptomic and anatomical complexity of primary, seminal, and crown roots highlight root type-specific functional diversity in maize (Zea mays L.)

PubMed Central

Tai, Huanhuan; Lu, Xin; Opitz, Nina; Marcon, Caroline; Paschold, Anja; Lithio, Andrew; Nettleton, Dan; Hochholdinger, Frank

2016-01-01

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across all root types and highlighted the unique transcriptome of seminal roots. While functions in cell remodeling and cell wall formation were prominent in primary and crown roots, stress-related genes and transcriptional regulators were over-represented in seminal roots, suggesting functional specialization of the different root types. Dynamic expression of lignin biosynthesis genes and histochemical staining suggested diversification of cell wall lignification among the three root types. Our findings highlight a cost-efficient anatomical structure and a unique expression profile of seminal roots of the maize inbred line B73 different from primary and crown roots. PMID:26628518

Excessive amounts of mu heavy chain block B-cell development.

PubMed

Zhu, Lingqiao; Chang, Cheong-Hee; Dunnick, Wesley

2011-09-01

Antigen-independent B-cell development occurs in several stages that depend on the expression of Ig heavy and light chain. We identified a line of mice that lacked mature B cells in the spleen. This mouse line carried approximately 11 copies of a transgene of the murine heavy chain constant region locus, and B-lineage cells expressed excessive amounts of the intracellular μ heavy chain. B-cell development failed in the bone marrow at the pro/pre B-cell transition, and examination of other lines with various copy numbers of the same transgene suggested that deficiencies in B-cell development increased with increased transgene copy number. Expression of a transgenic (Tg) light chain along with the Tg μ heavy chain led to minimal rescue of B-cell development in the bone marrow and B cells in the spleen. There are several potential mechanisms for the death of pro/pre B cells as a consequence of excess heavy chain expression.

Effect of exogenous transforming growth factor β1 (TGF-β1) on early bovine embryo development.

PubMed

Barrera, Antonio D; García, Elina V; Miceli, Dora C

2018-06-08

SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses.

PubMed

Balázs, Mercedesz; Martin, Flavius; Zhou, Tong; Kearney, John

2002-09-01

Marginal zone (MZ) and B1 B lymphocytes participate jointly in the early immune response against T-independent (TI) particulate antigens. Here we show that blood-derived neutrophil granulocytes and CD11c(lo) immature dendritic cells (DC) are the primary cells that efficiently capture and transport particulate bacteria to the spleen. In a systemic infection, CD11c(lo) DC, but not neutrophils, provide critical survival signals, which can be inhibited by TACI-Fc, to antigen-specific MZ B cells and promote their differentiation into IgM-secreting plasmablasts. In a local TI response, peritoneal cavity macrophages provide similar support to B1 B-derived Ag-specific blasts. In the absence of soluble TACI ligands, Ag-activated MZ- and B1-derived blasts lack survival signals and undergo apoptosis, resulting in severely impaired antibody responses.

Analysis of Dibenzothiophene Desulfurization in a Recombinant Pseudomonas putida Strain▿

PubMed Central

Calzada, Javier; Zamarro, María T.; Alcón, Almudena; Santos, Victoria E.; Díaz, Eduardo; García, José L.; Garcia-Ochoa, Felix

2009-01-01

Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization. PMID:19047400

Redefining Langerhans Cell Histiocytosis as a Myeloid Dysplasia and Identifying B | Division of Cancer Prevention

Cancer.gov

DESCRIPTION (provided by applicant): Redefining Langerhans Cell Histiocytosis as a Myeloid Dysplasia and Identifying Biomarkers for Early Detection and Risk Assessment. This application addresses Program Announcement PA-09-197: Biomarkers for Early Detection of Hematopoietic Malignancies (R01). The overall aim of this project is to identify novel biomarkers that may be used to

Somatostatin protects photoreceptor cells against high glucose-induced apoptosis.

PubMed

Arroba, Ana I; Mazzeo, Aurora; Cazzoni, Daniele; Beltramo, Elena; Hernández, Cristina; Porta, Massimo; Simó, Rafael; Valverde, Ángela M

2016-01-01

Many cellular and molecular studies in experimental animals and early retinal function tests in patients with diabetic retinopathy (DR) have shown that retinal neurodegeneration is an early event in the pathogenesis of the disease. Somatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina: SST levels are decreased in parallel to retinal neurodegeneration in early stages of DR. In this study, we characterized the induction of apoptosis (programmed cell death) in a 661W photoreceptor-like cell line cultured under high glucose (HG) conditions and the effect of SST. A 661W photoreceptor-like cell line and retinal explants from 10-week-old male C57BL/6 mice were cultured under HG conditions and treated with SST. Hyperglycemia significantly reduced the cellular viability by increasing the percentage of apoptotic cells, and this effect was ameliorated by SST (p˂0.05). Activation of caspase-8 by hyperglycemia was found in the 661W cells and retinal explants and decreased in the presence of SST (p˂0.05). Moreover, we detected activation of calpain-2 associated with hyperglycemia-induced cell death, as well as increased protein tyrosine phosphatase 1B (PTP1B) protein levels; both had a pattern of cleavage that was absent in the presence of SST (p˂0.05). Treatment of the 661W cells and retinal explants with SST for 24 h increased the phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR; tyrosine 1165/1166) and protein kinase B (Akt; serine 473), suggesting this survival signaling is activated in the neuroretina by SST (p˂0.05). This study has provided new mechanistic insights first into the involvement of calpain-2 and PTP1B in the loss of cell survival and increased caspase-8-dependent apoptosis induced by hyperglycemia in photoreceptor cells and second, on the protective effect of SST against apoptosis by the enhancement of IGF-IR-mediated Akt phosphorylation.

Somatostatin protects photoreceptor cells against high glucose–induced apoptosis

PubMed Central

Mazzeo, Aurora; Cazzoni, Daniele; Beltramo, Elena; Hernández, Cristina; Porta, Massimo; Simó, Rafael; Valverde, Ángela M.

2016-01-01

Purpose Many cellular and molecular studies in experimental animals and early retinal function tests in patients with diabetic retinopathy (DR) have shown that retinal neurodegeneration is an early event in the pathogenesis of the disease. Somatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina: SST levels are decreased in parallel to retinal neurodegeneration in early stages of DR. In this study, we characterized the induction of apoptosis (programmed cell death) in a 661W photoreceptor-like cell line cultured under high glucose (HG) conditions and the effect of SST. Methods A 661W photoreceptor-like cell line and retinal explants from 10-week-old male C57BL/6 mice were cultured under HG conditions and treated with SST. Results Hyperglycemia significantly reduced the cellular viability by increasing the percentage of apoptotic cells, and this effect was ameliorated by SST (p˂0.05). Activation of caspase-8 by hyperglycemia was found in the 661W cells and retinal explants and decreased in the presence of SST (p˂0.05). Moreover, we detected activation of calpain-2 associated with hyperglycemia-induced cell death, as well as increased protein tyrosine phosphatase 1B (PTP1B) protein levels; both had a pattern of cleavage that was absent in the presence of SST (p˂0.05). Treatment of the 661W cells and retinal explants with SST for 24 h increased the phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR; tyrosine 1165/1166) and protein kinase B (Akt; serine 473), suggesting this survival signaling is activated in the neuroretina by SST (p˂0.05). Conclusions This study has provided new mechanistic insights first into the involvement of calpain-2 and PTP1B in the loss of cell survival and increased caspase-8-dependent apoptosis induced by hyperglycemia in photoreceptor cells and second, on the protective effect of SST against apoptosis by the enhancement of IGF-IR-mediated Akt phosphorylation. PMID:28050125

Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors.

PubMed

Hans, Stefan; Liu, Dong; Westerfield, Monte

2004-10-01

The vertebrate inner ear arises from an ectodermal thickening, the otic placode, that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to Fgf signals from adjacent tissues and express two highly related paired box transcription factors Pax2a and Pax8 in the developing placode. We show that compromising the functions of both Pax2a and Pax8 together blocks zebrafish ear development, leaving only a few residual otic cells. This suggests that Pax2a and Pax8 are the main effectors downstream of Fgf signals. Our results further provide evidence that pax8 expression and pax2a expression are regulated by two independent factors, Foxi1 and Dlx3b, respectively. Combined loss of both factors eliminates all indications of otic specification. We suggest that the Foxi1-Pax8 pathway provides an early 'jumpstart' of otic specification that is maintained by the Dlx3b-Pax2a pathway.

Adipose Extracellular Matrix/Stromal Vascular Fraction Gel Secretes Angiogenic Factors and Enhances Skin Wound Healing in a Murine Model.

PubMed

Sun, Mingliang; He, Yunfan; Zhou, Tao; Zhang, Pan; Gao, Jianhua; Lu, Feng

2017-01-01

Mesenchymal stem cells are an attractive cell type for cytotherapy in wound healing. The authors recently developed a novel, adipose-tissue-derived, injectable extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel) for stem cell therapy. This study was designed to assess the therapeutic effects of ECM/SVF-gel on wound healing and potential mechanisms. ECM/SVF-gel was prepared for use in nude mouse excisional wound healing model. An SVF cell suspension and phosphate-buffered saline injection served as the control. The expression levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and monocyte chemotactic protein-1 (MCP-1) in ECM/SVF-gel were analyzed at different time points. Angiogenesis (tube formation) assays of ECM/SVF-gel extracts were evaluated, and vessels density in skin was determined. The ECM/SVF-gel extract promoted tube formation in vitro and increased the expression of the angiogenic factors VEGF and bFGF compared with those in the control. The expression of the inflammatory chemoattractant MCP-1 was high in ECM/SVF-gel at the early stage and decreased sharply during the late stage of wound healing. The potent angiogenic effects exerted by ECM/SVF-gel may contribute to the improvement of wound healing, and these effects could be related to the enhanced inflammatory response in ECM/SVF-gel during the early stage of wound healing.

Utility of LRF/Pokemon and NOTCH1 Protein Expression in the Distinction of Nodular Lymphocyte-Predominant Hodgkin Lymphoma and Classical Hodgkin Lymphoma

PubMed Central

Bohn, Olga; Maeda, Takahiro; Filatov, Alexander; Lunardi, Andrea; Pandolfi, Pier Paolo; Teruya-Feldstein, Julie

2014-01-01

Classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) are considered separate entities with different prognosis and treatment. However, morphologic features can be similar and immunohistochemical studies are essential in the distinction; thus, determination of additional biomarkers is of utmost importance. LRF/Pokemon is a protooncogene, an interacting partner co-expressed with BCL6 in germinal centers and highly expressed in diffuse large B-cell lymphoma and follicular lymphoma. Conversely, loss of the LRF gene in mouse hematopoietic stem cells results in complete block of early B cell development with concomitant Notch derepression, indicating its critical role in B versus T cell fate decision at the hematopoietic stem cell stage. For the first time, we show that LRF/Pokemon is predominantly expressed in NLPHL cases as is BCL6 with low to absent NOTCH1 protein expression; while Hodgkin Reed-Sternberg (HRS) cells in CHL show low to absent BCL6 and LRF/Pokemon expression with higher NOTCH1 expression. We illustrate a potential functional interaction between LRF and BCL6 in NLPHL pathogenesis, and differential expression of LRF/Pokemon and NOTCH1 proteins in CHL thus showing differential expression, making for an additional diagnostic marker and therapeutic target. PMID:24326827

Utility of LRF/Pokemon and NOTCH1 protein expression in the distinction between nodular lymphocyte-predominant Hodgkin lymphoma and classical Hodgkin lymphoma.

PubMed

Bohn, Olga; Maeda, Takahiro; Filatov, Alexander; Lunardi, Andrea; Pandolfi, Pier Paolo; Teruya-Feldstein, Julie

2014-02-01

Classical Hodgkin lymphoma (CHL) and nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) are considered separate entities with different prognosis and treatment. However, morphologic features can be similar and immunohistochemical studies are essential in the distinction; thus, determination of additional biomarkers is of utmost importance. LRF/Pokemon is a proto-oncogene, an interacting partner co-expressed with BCL6 in germinal centers and highly expressed in diffuse large B-cell lymphoma and follicular lymphoma. Conversely, loss of the LRF gene in mouse hematopoietic stem cells results in complete block of early B cell development with concomitant Notch de-repression, indicating its critical role in B versus T cell fate decision at the hematopoietic stem cell stage. For the first time, we show that LRF/Pokemon is predominantly expressed in NLPHL cases as is BCL6 with low to absent NOTCH1 protein expression; while Hodgkin Reed-Sternberg (HRS) cells in CHL show low to absent BCL6 and LRF/Pokemon expression with higher NOTCH1 expression. We illustrate a potential functional interaction between LRF and BCL6 in NLPHL pathogenesis, and differential expression of LRF/Pokemon and NOTCH1 proteins in CHL thus showing differential expression, making for an additional diagnostic marker and therapeutic target.

Association of mixed hematopoietic chimerism with elevated circulating autoantibodies and chronic graft-versus-host disease occurrence

PubMed Central

Perruche, Sylvain; Marandin, Aliette; Kleinclauss, François M.; Angonin, Régis; Fresnay, Stéphanie; Baron, Marie Hélène; Tiberghien, Pierre; Saas, Philippe

2006-01-01

Background Use of a reduced intensity conditioning regimen before an allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such a co-existence of both host and donor hematopoietic cells may influence post-transplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease (GVHD) as well as the intensity of the graft-versus-leukemia effect. Here we evaluated the relation between chimerism state after reduced intensity conditioning transplantation (RICT), auto-antibody production and chronic GVHD (cGVHD)-related pathology. Methods Chimerism state, circulating anti-cardiolipin and anti-double stranded DNA auto-antibody (Ab) titers as well as occurrence of cGVHD-like lesions were investigated in a murine RICT model. Results We observed a novel association between mixed chimerism state, high levels of pathogenic IgG auto-Abs and subsequent development of cGVHD-like lesions. Furthermore, we found that the persistence of host B cells, but not dendritic cell origin or subset, was a factor associated with the appearance of cGVHD-like lesions. The implication of host B cells was confirmed by a host origin of auto-Abs. Conclusions Recipient B cell persistence may therefore contribute to the frequency and/or severity of cGVHD after RICT. PMID:16495806

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

PubMed

Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

2016-11-01

Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

Distinct roles of DNMT1-dependent and DNMT1-independent methylation patterns in the genome of mouse embryonic stem cells.

PubMed

Li, Zhiguang; Dai, Hongzheng; Martos, Suzanne N; Xu, Beisi; Gao, Yang; Li, Teng; Zhu, Guangjing; Schones, Dustin E; Wang, Zhibin

2015-06-02

DNA methylation patterns are initiated by de novo DNA methyltransferases DNMT3a/3b adding methyl groups to CG dinucleotides in the hypomethylated genome of early embryos. These patterns are faithfully maintained by DNMT1 during DNA replication to ensure epigenetic inheritance across generations. However, this two-step model is based on limited data. We generated base-resolution DNA methylomes for a series of DNMT knockout embryonic stem cells, with deep coverage at highly repetitive elements. We show that DNMT1 and DNMT3a/3b activities work complementarily and simultaneously to establish symmetric CG methylation and CHH (H = A, T or C) methylation. DNMT3a/3b can add methyl groups to daughter strands after each cycle of DNA replication. We also observe an unexpected division of labor between DNMT1 and DNMT3a/3b in suppressing retrotransposon long terminal repeats and long interspersed elements, respectively. Our data suggest that mammalian cells use a specific CG density threshold to predetermine methylation levels in wild-type cells and the magnitude of methylation reduction in DNMT knockout cells. Only genes with low CG density can be induced or, surprisingly, suppressed in the hypomethylated genome. Lastly, we do not find any association between gene body methylation and transcriptional activity. We show the concerted actions of DNMT enzymes in the establishment and maintenance of methylation patterns. The finding of distinct roles of DNMT1-dependent and -independent methylation patterns in genome stability and regulation of transcription provides new insights for understanding germ cell development, neuronal diversity, and transgenerational epigenetic inheritance and will help to develop next-generation DNMT inhibitors.

The roles of ERAS during cell lineage specification of mouse early embryonic development.

PubMed

Zhao, Zhen-Ao; Yu, Yang; Ma, Huai-Xiao; Wang, Xiao-Xiao; Lu, Xukun; Zhai, Yanhua; Zhang, Xiaoxin; Wang, Haibin; Li, Lei

2015-08-01

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development. © 2015 The Authors.

A microRNA, mir133b, suppresses melanopsin expression mediated by failure dopaminergic amacrine cells in RCS rats.

PubMed

Li, Yaochen; Li, Chunshi; Chen, Zhongshan; He, Jianrong; Tao, Zui; Yin, Zheng Qin

2012-03-01

The photopigment melanopsin and melanopsin-containing RGCs (mRGCs or ipRGCs) represent a brand-new and exciting direction in the field of visual field. Although the melanopsin is much less sensitive to light and has far less spatial resolution, mRGCs have the unique ability to project to brain areas by the retinohypothalamic tract (RHT) and communicate directly with the brain. Unfortunately, melanopsin presents lower expression levels in many acute and chronic retinal diseases. The molecular mechanisms underlying melanopsin expression are not yet really understood. MicroRNAs play important roles in the control of development. Most importantly, the link of microRNA biology to a diverse set of cellular processes, ranging from proliferation, apoptosis and malignant transformation to neuronal development and fate specification is emerging. We employed Royal College of Surgeon (RCS) rats as animal model to investigate the underlying molecular mechanism regulating melanopsin expression using a panel of miRNA by quantitative real-time reverse transcription polymerase chain reaction. We identified a microRNA, mir133b, that is specifically expressed in retinal dopaminergic amacrine cells as well as markedly increased expression at early stage during retinal degeneration in RCS rats. The overexpression of mir133b downregulates the important transcription factor Pitx3 expression in dopaminergic amacrine cells in RCS rats retinas and makes amacrine cells stratification deficit in IPL. Furthermore, deficient dopaminergic amacrine cells presented decreased TH expression and dopamine production, which lead to a failure to direct mRGCs dendrite to stratify and enter INL and lead to the reduced correct connections between amacrine cells and mRGCs. Our study suggested that overexpression of mir133b and downregulated Pitx3 suppress maturation and function of dopaminergic amacrine cells, and overexpression of mir133b decreased TH and D2 receptor expression as well as dopamine production, which finally resulted in reduced melanopsin expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Evolving Identification of Blood Cells Associated with Clinically Isolated Syndrome: Importance of Time since Clinical Presentation and Diagnostic MRI

PubMed Central

Trend, Stephanie; Jones, Anderson P.; Geldenhuys, Sian; Byrne, Scott N.; Fabis-Pedrini, Marzena J.; Nolan, David; Booth, David R.; Carroll, William M.; Lucas, Robyn M.; Kermode, Allan G.; Hart, Prue H.

2017-01-01

It is not clear how the profile of immune cells in peripheral blood differs between patients with clinically isolated syndrome (CIS) and healthy controls (HC). This study aimed to identify a CIS peripheral blood signature that may provide clues for potential immunomodulatory approaches early in disease. Peripheral blood mononuclear cells (PBMCs) were collected from 18 people with CIS, 19 HC and 13 individuals with other demyelinating conditions (ODC) including multiple sclerosis (MS). Individuals with CIS separated into two groups, namely those with early (≤14 days post-diagnostic magnetic resonance imaging (MRI); n = 6) and late (≥27 days; n = 12) blood sampling. Transitional B cells were increased in the blood of CIS patients independently of when blood was taken. However, there were two time-dependent effects found in the late CIS group relative to HC, including decreased CD56bright NK cells, which correlated significantly with time since MRI, and increased CD141+ myeloid dendritic cell (mDC2) frequencies. Higher CD1c+ B cells and lower non-classical monocyte frequencies were characteristic of more recent demyelinating disease activity (ODC and early CIS). Analysing cell populations by time since symptoms (subjective) and diagnostic MRI (objective) may contribute to understanding CIS. PMID:28617321

Evolving Identification of Blood Cells Associated with Clinically Isolated Syndrome: Importance of Time since Clinical Presentation and Diagnostic MRI.

PubMed

Trend, Stephanie; Jones, Anderson P; Geldenhuys, Sian; Byrne, Scott N; Fabis-Pedrini, Marzena J; Nolan, David; Booth, David R; Carroll, William M; Lucas, Robyn M; Kermode, Allan G; Hart, Prue H

2017-06-15

It is not clear how the profile of immune cells in peripheral blood differs between patients with clinically isolated syndrome (CIS) and healthy controls (HC). This study aimed to identify a CIS peripheral blood signature that may provide clues for potential immunomodulatory approaches early in disease. Peripheral blood mononuclear cells (PBMCs) were collected from 18 people with CIS, 19 HC and 13 individuals with other demyelinating conditions (ODC) including multiple sclerosis (MS). Individuals with CIS separated into two groups, namely those with early (≤14 days post-diagnostic magnetic resonance imaging (MRI); n = 6) and late (≥27 days; n = 12) blood sampling. Transitional B cells were increased in the blood of CIS patients independently of when blood was taken. However, there were two time-dependent effects found in the late CIS group relative to HC, including decreased CD56bright NK cells, which correlated significantly with time since MRI, and increased CD141+ myeloid dendritic cell (mDC2) frequencies. Higher CD1c+ B cells and lower non-classical monocyte frequencies were characteristic of more recent demyelinating disease activity (ODC and early CIS). Analysing cell populations by time since symptoms (subjective) and diagnostic MRI (objective) may contribute to understanding CIS.

Membrane remodeling, an early event in benzo[alpha]pyrene-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tekpli, Xavier; Rissel, Mary; Huc, Laurence

2010-02-15

Benzo[alpha]pyrene (B[alpha]P) often serves as a model for mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs). Our previous work suggested a role of membrane fluidity in B[alpha]P-induced apoptotic process. In this study, we report that B[alpha]P modifies the composition of cholesterol-rich microdomains (lipid rafts) in rat liver F258 epithelial cells. The cellular distribution of the ganglioside-GM1 was markedly changed following B[alpha]P exposure. B[alpha]P also modified fatty acid composition and decreased the cholesterol content of cholesterol-rich microdomains. B[alpha]P-induced depletion of cholesterol in lipid rafts was linked to a reduced expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). Aryl hydrocarbon receptor (AhR) and B[alpha]P-related H{submore » 2}O{sub 2} formation were involved in the reduced expression of HMG-CoA reductase and in the remodeling of membrane microdomains. The B[alpha]P-induced membrane remodeling resulted in an intracellular alkalinization observed during the early phase of apoptosis. In conclusion, B[alpha]P altered the composition of plasma membrane microstructures through AhR and H{sub 2}O{sub 2} dependent-regulation of lipid biosynthesis. In F258 cells, the B[alpha]P-induced membrane remodeling was identified as an early apoptotic event leading to an intracellular alkalinization.« less

Cell-Autonomous Control of IL-7 Response Revealed In a Novel Stage of Precursor B Cells

PubMed Central

Sandoval, Gabriel J.; Graham, Daniel B.; Bhattacharya, Deepta; Sleckman, Barry P.; Xavier, Ramnik J.; Swat, Wojciech

2013-01-01

During early stages of B-lineage differentiation in bone marrow, signals emanating from IL-7 receptor and pre-B cell receptor (pre-BCR) are thought to synergistically induce proliferative expansion of progenitor cells. Paradoxically, loss of pre-BCR signaling components is associated with leukemia in both mice and humans. Exactly how progenitor B cells perform the task of balancing proliferative burst dependent on IL-7 with the termination of IL-7 signals and the initiation of LC gene rearrangement remains to be elucidated. In this report, we provide genetic and functional evidence that the cessation of IL-7 response of pre-B cells is controlled via a cell-autonomous mechanism that operates at a discreet developmental transition inside Fraction C’ (Large Pre-BII) marked by transient expression of c-Myc. Our data indicates that pre-BCR cooperates with IL-7R in expanding pre-B cell pool, but it is also critical to control differentiation program shutting off c-Myc gene in large pre-B cells. PMID:23420891

Early time evolution of negative ion clouds and electron density depletions produced during electron attachment chemical release experiments

NASA Technical Reports Server (NTRS)

Scales, W. A.; Bernhardt, P. A.; Ganguli, G.

1994-01-01

Two-dimensional electrostatic particle-in-cell simulations are used to study the early time evolution of electron depletions and negative ion clouds produced during electron attachment chemical releases in the ionosphere. The simulation model considers the evolution in the plane perpendicular to the magnetic field and a three-species plasma that contains electrons, positive ions, and also heavy negative ions that result as a by-product of the electron attachment reaction. The early time evolution (less than the negative ion cyclotron period) of the system shows that a negative charge surplus initially develops outside of the depletion boundary as the heavy negative ions move across the boundary. The electrons are initially restricted from moving into the depletion due to the magnetic field. An inhomogenous electric field develops across the boundary layer due to this charge separation. A highly sheared electron flow velocity develops in the depletion boundary due to E x B and Delta-N x B drifts that result from electron density gradients and this inhomogenous electric field. Structure eventually develops in the depletion boundary layer due to low-frequency electrostatic waves that have growth times shorter than the negative ion cyclotron period. It is proposed that these waves are most likely produced by the electron-ion hybrid instability that results from sufficiently large shears in the electron flow velocity.

Assessment of trichloroethylene (TCE) exposure in murine strains genetically-prone and non-prone to develop autoimmune disease.

PubMed

Keil, Deborah E; Peden-Adams, Margie M; Wallace, Stacy; Ruiz, Phillip; Gilkeson, Gary S

2009-04-01

There is increasing laboratory and epidemiologic evidence relating exposure to trichloroethylene (TCE) with autoimmune disease including scleroderma and lupus. New Zealand Black/New Zealand White (NZBWF1) and B6C3F1 mice were exposed to TCE (0, 1, 400 or 14,000 ppb) via drinking water for 27 or 30 weeks, respectively. NZBWF1 mice spontaneously develop autoimmune disease while B6C3F1 mice, a standard strain used in immunotoxicology testing, are not genetically prone to develop autoimmune disease. During the TCE exposure period, serum levels of total IgG, and autoantibodies (anti-ssDNA, -dsDNA, and -glomerular antigen [GA]) were monitored. At the termination of the study, renal pathology, natural killer (NK) cell activity, total IgG levels, autoantibody production, T-cell activation, and lymphocytic proliferative responses were evaluated. TCE did not alter NK cell activity, or T- and B-cell proliferation in either strain. Numbers of activated T-cells (CD4+/CD44+) were increased in the B6C3F1 mice but not in the NZBWF1 mice. Renal pathology, as indicated by renal score, was significantly increased in the B6C3F1, but not in the NZBWF1 mice. Serum levels of autoantibodies to dsDNA and ssDNA were increased at more time points in B6C3F1, as compared to the NZBWF1 mice. Anti-GA autoantibodies were increased by TCE treatment in early stages of the study in NZBWF1 mice, but by 23 weeks of age, control levels were comparable to those of TCE-exposed animals. Serum levels anti-GA autoantibodies in B6C3F1 were not affected by TCE exposure. Overall, these data suggest that TCE did not contribute to the progression of autoimmune disease in autoimmune-prone mice during the period of 11-36 weeks of age, but rather lead to increased expression of markers associated with autoimmune disease in a non-genetically prone mouse strain.

New Insights into the Pathogenesis of Pancreatitis

PubMed Central

Sah, Raghuwansh P.; Dawra, Rajinder K.; Saluja, Ashok K.

2014-01-01

Purpose of review In this article, we review important advances in our understanding of the mechanisms of pancreatitis. Recent Findings The relative contribution of intra-pancreatic trypsinogen activation and NFκB activation, the two major early independent cellular events in the etiology of pancreatitis, have been investigated using novel genetic models. Trypsinogen activation has traditionally held the spotlight for many decades as it is believed to be the central pathogenic event of pancreatitis However, recent experimental evidence points to the role of trypsin activation in early acinar cell damage but not in the inflammatory response of acute pancreatitis through NFκB activation. Further, chronic pancreatitis in the caerulein model develops independently of typsinogen activation. Sustained activation of the NFκB pathway, but not persistent intra-acinar expression of active trypsin, was shown to result in chronic pancreatitis. Calcineurin-NFAT signaling was shown to mediate downstream effects of pathologic rise in intracellular calcium. IL-6 was identified as a key cytokine mediating pancreatitis-associated lung injury. Summary Recent advances challenge the long-believed trypsin-centered understanding of pancreatitis. It is becoming increasingly clear that activation of intense inflammatory signaling mechanisms in acinar cells is crucial to the pathogenesis of pancreatitis, which may explain the strong systemic inflammatory response in pancreatitis. PMID:23892538

Overexpression of IL-7R alpha provides a competitive advantage during early T-cell development.

PubMed

Laouar, Yasmina; Crispe, I Nicholas; Flavell, Richard A

2004-03-15

Critical checkpoints controlling early thymic T-cell development and homeostasis are set by the proper signaling function of the interleukin 7 receptor (IL-7R) and the pre-T-cell antigen receptor. Although alpha beta T-cell development is observed in IL-7- and IL-7R alpha-deficient mice, the number of thymocytes is significantly reduced, implying a role for the IL-7R in controlling the size of the thymic T-cell compartment. Here, we report the overexpression of IL-7R alpha that occurs in the early T-cell compartment from AKR/J mice, animals that are highly susceptible to the spontaneous development of thymoma. Increased IL-7R alpha was revealed by surface staining, and increased IL-7R alpha mRNA was documented by using reverse transcriptase-polymerase chain reaction (RT-PCR). This resulted in increased survival of AKR/J early thymocytes, shown by the decreased frequency of TUNEL(+) (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate [dUTP]-fluorescein nick end labeling) cells. In an in vivo thymocyte repopulation model, AKR/J thymocytes had a selective advantage over healthy thymocytes. This advantage occurred at early stages of T-cell development. Our findings support the model that overexpression of growth factor receptors can contribute to proliferation and malignancy.

Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation

PubMed Central

Liu, Congxiao; Chen, Benny J.; DeOliveira, Divinomar; Sempowski, Gregory D.; Chao, Nelson J.

2010-01-01

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma–null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34+ human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks), human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses, as documented by the presence of CD4+ CD8+ T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation, human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses, the chimerism was weak and the human hematopoietic lineage development was frequently incomplete. PMID:20833978

In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination

DTIC Science & Technology

2015-12-01

was interbred with Nkx2.1-CreERT2 knock-in mouse strain, containing a tamoxifen -inducible lineage specific Cre recombinase, to generate adult lung...KrasG12D(L/-); Lkb1(L/L) mice 7 days after tamoxifen injections show that KrasG12D mice develop isolated tumors one week after the end of tamoxifen ...SA2a-b) (timeframe months 6-10 by Drs Kathuria, Cao, Ramirez, and Kotton). 1. First we will induce tumors by tamoxifen injection of Kras G12D /Lkb1L/L

West nile virus infections suppress early viral RNA synthesis and avoid inducing the cell stress granule response.

PubMed

Courtney, S C; Scherbik, S V; Stockman, B M; Brinton, M A

2012-04-01

West Nile virus (WNV) recently became endemic in the United States and is a significant cause of human morbidity and mortality. Natural WNV strain infections do not induce stress granules (SGs), while W956IC (a lineage 2/1 chimeric WNV infectious clone) virus infections produce high levels of early viral RNA and efficiently induce SGs through protein kinase R (PKR) activation. Additional WNV chimeric viruses made by replacing one or more W956IC genes with the lineage 1 Eg101 equivalent in the W956IC backbone were analyzed. The Eg-NS4b+5, Eg-NS1+3+4a, and Eg-NS1+4b+5 chimeras produced low levels of viral RNA at early times of infection and inefficiently induced SGs, suggesting the possibility that interactions between viral nonstructural proteins and/or between viral nonstructural proteins and cell proteins are involved in suppressing early viral RNA synthesis and membrane remodeling during natural WNV strain infections. Detection of exposed viral double-stranded RNA (dsRNA) in W956IC-infected cells suggested that the enhanced early viral RNA synthesis surpassed the available virus-induced membrane protection and allowed viral dsRNA to activate PKR.

Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

PubMed

Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

2009-12-29

Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells

PubMed Central

Gabriele, Morena; Del Prato, Stefano; Pucci, Laura

2017-01-01

Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS) orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs), bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG), on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER) stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation. PMID:28386305

IgG1 B cell receptor signaling is inhibited by CD22 and promotes the development of B cells whose survival is less dependent on Igα/β

PubMed Central

Waisman, Ari; Kraus, Manfred; Seagal, Jane; Ghosh, Snigdha; Melamed, Doron; Song, Jian; Sasaki, Yoshiteru; Classen, Sabine; Lutz, Claudia; Brombacher, Frank; Nitschke, Lars; Rajewsky, Klaus

2007-01-01

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) γ1 or μ heavy chains. Progenitor cells expressing γ1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro– to pre–B cell transition. Accordingly, γ1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Igα cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca2+ response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca2+ response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the γ1 chain can exert a unique signaling function that can partially replace that of the Igα/β heterodimer in B cell maintenance and may contribute to memory B cell physiology. PMID:17420268

Life sciences flight hardware development for the International Space Station

NASA Astrophysics Data System (ADS)

Kern, V. D.; Bhattacharya, S.; Bowman, R. N.; Donovan, F. M.; Elland, C.; Fahlen, T. F.; Girten, B.; Kirven-Brooks, M.; Lagel, K.; Meeker, G. B.; Santos, O.

During the construction phase of the International Space Station (ISS), early flight opportunities have been identified (including designated Utilization Flights, UF) on which early science experiments may be performed. The focus of NASA's and other agencies' biological studies on the early flight opportunities is cell and molecular biology; with UF-1 scheduled to fly in fall 2001, followed by flights 8A and UF-3. Specific hardware is being developed to verify design concepts, e.g., the Avian Development Facility for incubation of small eggs and the Biomass Production System for plant cultivation. Other hardware concepts will utilize those early research opportunities onboard the ISS, e.g., an Incubator for sample cultivation, the European Modular Cultivation System for research with small plant systems, an Insect Habitat for support of insect species. Following the first Utilization Flights, additional equipment will be transported to the ISS to expand research opportunities and capabilities, e.g., a Cell Culture Unit, the Advanced Animal Habitat for rodents, an Aquatic Facility to support small fish and aquatic specimens, a Plant Research Unit for plant cultivation, and a specialized Egg Incubator for developmental biology studies. Host systems (Figure 1A, B), e.g., a 2.5 m Centrifuge Rotor (g-levels from 0.01-g to 2-g) for direct comparisons between μg and selectable g levels, the Life Sciences Glove☐ for contained manipulations, and Habitat Holding Racks (Figure 1B) will provide electrical power, communication links, and cooling to the habitats. Habitats will provide food, water, light, air and waste management as well as humidity and temperature control for a variety of research organisms. Operators on Earth and the crew on the ISS will be able to send commands to the laboratory equipment to monitor and control the environmental and experimental parameters inside specific habitats. Common laboratory equipment such as microscopes, cryo freezers, radiation dosimeters, and mass measurement devices are also currently in design stages by NASA and the ISS international partners.

Hepatitis B virus X protein shifts human hepatic transforming growth factor (TGF)-beta signaling from tumor suppression to oncogenesis in early chronic hepatitis B.

PubMed

Murata, Miki; Matsuzaki, Koichi; Yoshida, Katsunori; Sekimoto, Go; Tahashi, Yoshiya; Mori, Shigeo; Uemura, Yoshiko; Sakaida, Noriko; Fujisawa, Junichi; Seki, Toshihito; Kobayashi, Kazuki; Yokote, Koutaro; Koike, Kazuhiko; Okazaki, Kazuichi

2009-04-01

Hepatitis B virus X (HBx) protein is suspected to participate in oncogenesis during chronic hepatitis B progression. Transforming growth factor beta (TGF-beta) signaling involves both tumor suppression and oncogenesis. TGF-beta activates TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). Reversible shifting of Smad3-mediated signaling between tumor suppression and oncogenesis in HBx-expressing hepatocytes indicated that TbetaRI-dependent pSmad3C transmitted a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promoted cell growth. We used immunostaining, immunoblotting, and in vitro kinase assay to compare pSmad3L- and pSmad3C-mediated signaling in biopsy specimens representing chronic hepatitis, cirrhosis, or hepatocellular carcinoma (HCC) from 90 patients chronically infected with hepatitis B virus (HBV) with signaling in liver specimens from HBx transgenic mice. In proportion to plasma HBV DNA levels, early chronic hepatitis B specimens showed prominence of pSmad3L in hepatocytic nuclei. HBx-activated JNK/pSmad3L/c-Myc oncogenic pathway was enhanced, while the TbetaRI/pSmad3C/p21(WAF1) tumor-suppressive pathway was impaired as human and mouse HBx-associated hepatocarcinogenesis progressed. Of 28 patients with chronic hepatitis B who showed strong oncogenic pSmad3L signaling, six developed HCC within 12 years; only one of 32 patients showing little pSmad3L developed HCC. In contrast, seven of 30 patients with little Smad3C phosphorylation developed HCC, while no patient who retained hepatocytic tumor-suppressive pSmad3C developed HCC within 12 years. HBx shifts hepatocytic TGF-beta signaling from the tumor-suppressive pSmad3C pathway to the oncogenic pSmad3L pathway in early carcinogenic process. Hepatocytic pSmad3L and pSmad3C assessment in HBV-infected liver specimens should prove clinically useful for predicting risk of HCC.

Developmental stage-specific regulation of the circadian clock by temperature in zebrafish.

PubMed

Lahiri, Kajori; Froehlich, Nadine; Heyd, Andreas; Foulkes, Nicholas S; Vallone, Daniela

2014-01-01

The circadian clock enables animals to adapt their physiology and behaviour in anticipation of the day-night cycle. Light and temperature represent two key environmental timing cues (zeitgebers) able to reset this mechanism and so maintain its synchronization with the environmental cycle. One key challenge is to unravel how the regulation of the clock by zeitgebers matures during early development. The zebrafish is an ideal model for studying circadian clock ontogeny since the process of development occurs ex utero in an optically transparent chorion and many tools are available for genetic analysis. However, the role played by temperature in regulating the clock during zebrafish development is poorly understood. Here, we have established a clock-regulated luciferase reporter transgenic zebrafish line (Tg (-3.1) per1b::luc) to study the effects of temperature on clock entrainment. We reveal that under complete darkness, from an early developmental stage onwards (48 to 72 hpf), exposure to temperature cycles is a prerequisite for the establishment of self-sustaining rhythms of zfper1b, zfaanat2, and zfirbp expression and also for circadian cell cycle rhythms. Furthermore, we show that following the 5-9 somite stage, the expression of zfper1b is regulated by acute temperature shifts.

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

PubMed Central

Blangy, A; Léopold, P; Vidal, F; Rassoulzadegan, M; Cuzin, F

1991-01-01

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development. Images PMID:1766880

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers.

PubMed

Shlien, Adam; Campbell, Brittany B; de Borja, Richard; Alexandrov, Ludmil B; Merico, Daniele; Wedge, David; Van Loo, Peter; Tarpey, Patrick S; Coupland, Paul; Behjati, Sam; Pollett, Aaron; Lipman, Tatiana; Heidari, Abolfazl; Deshmukh, Shriya; Avitzur, Na'ama; Meier, Bettina; Gerstung, Moritz; Hong, Ye; Merino, Diana M; Ramakrishna, Manasa; Remke, Marc; Arnold, Roland; Panigrahi, Gagan B; Thakkar, Neha P; Hodel, Karl P; Henninger, Erin E; Göksenin, A Yasemin; Bakry, Doua; Charames, George S; Druker, Harriet; Lerner-Ellis, Jordan; Mistry, Matthew; Dvir, Rina; Grant, Ronald; Elhasid, Ronit; Farah, Roula; Taylor, Glenn P; Nathan, Paul C; Alexander, Sarah; Ben-Shachar, Shay; Ling, Simon C; Gallinger, Steven; Constantini, Shlomi; Dirks, Peter; Huang, Annie; Scherer, Stephen W; Grundy, Richard G; Durno, Carol; Aronson, Melyssa; Gartner, Anton; Meyn, M Stephen; Taylor, Michael D; Pursell, Zachary F; Pearson, Christopher E; Malkin, David; Futreal, P Andrew; Stratton, Michael R; Bouffet, Eric; Hawkins, Cynthia; Campbell, Peter J; Tabori, Uri

2015-03-01

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

The contribution of B-cell proliferation to spleen enlargement in Babesia microti-infected mice.

PubMed Central

Inchley, C J

1987-01-01

Flow cytofluorimetric analysis showed that B-cell proliferation makes a major contribution to the enlargement and increased cellularity of the spleen, which are characteristic of Babesia microti infections in mice. Expansion of the B-cell population was accompanied by modulation of the cell surface, which affected most B lymphocytes, and which was detected as a reduction in the density of surface immunoglobulin. This effect was noted as early as Day 7, shortly after the appearance of parasites in the circulation and the onset of gross spleen changes. In contrast to the results for B cells, the frequency of splenic T cells declined, and when the data were transformed into absolute numbers it became clear that only limited T-cell proliferation had occurred. There was no evidence to suggest that the balance of T-cell subsets was shifted in favour of suppressor T cells. The relationships of these results to reports of immunosuppression by this parasite are discussed. Images Figure 2 Figure 5 PMID:3493207

Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765).

PubMed

Burger, Jan A; Buggy, Joseph J

2013-11-01

Over the past 3 years, ibrutinib (PCI-32765) has emerged as a breakthrough in targeted therapy for patients with certain types of B cell malignancies. Early stage clinical trials found ibrutinib to be particularly active in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), providing the rationale for ongoing phase 3 trials. In contrast to conventional chemo-immunotherapy, ibrutinib is not myelosuppressive, and responses are not affected by disease features that predict failure to respond to or short remission durations after chemo-immunotherapy, such as del17p. In CLL, ibrutinib characteristically causes an early redistribution of tissue-resident CLL cells into the blood, with rapid resolution of enlarged lymph nodes, along with a surge in lymphocytosis. Later, after weeks to months of continuous ibrutinib therapy, the growth- and survival-inhibitory activities of ibrutinib result in the normalization of lymphocyte counts and remissions in a majority of patients. This review discusses the discovery, preclinical and clinical development of ibrutinib, its pathophysiological basis, and outlines perspectives for future use of ibrutinib.

DNA double-strand breaks and Aurora B mislocalization induced by exposure of early mitotic cells to H2O2 appear to increase chromatin bridges and resultant cytokinesis failure.

PubMed

Cho, Min-Guk; Ahn, Ju-Hyun; Choi, Hee-Song; Lee, Jae-Ho

2017-07-01

Aneuploidy, an abnormal number of chromosomes that is a hallmark of cancer cells, can arise from tetraploid/binucleated cells through a failure of cytokinesis. Reactive oxygen species (ROS) have been implicated in various diseases, including cancer. However, the nature and role of ROS in cytokinesis progression and related mechanisms has not been clearly elucidated. Here, using time-lapse analysis of asynchronously growing cells and immunocytochemical analyses of synchronized cells, we found that hydrogen peroxide (H 2 O 2 ) treatment at early mitosis (primarily prometaphase) significantly induced cytokinesis failure. Cytokinesis failure and the resultant formation of binucleated cells containing nucleoplasmic bridges (NPBs) seemed to be caused by increases in DNA double-strand breaks (DSBs) and subsequent unresolved chromatin bridges. We further found that H 2 O 2 induced mislocalization of Aurora B during mitosis. All of these effects were attenuated by pretreatment with N-acetyl-L-cysteine (NAC) or overexpression of Catalase. Surprisingly, the PARP inhibitor PJ34 also reduced H 2 O 2 -induced Aurora B mislocalization and binucleated cell formation. Results of parallel experiments with etoposide, a topoisomerase IIα inhibitor that triggers DNA DSBs, suggested that both DNA DSBs and Aurora B mislocalization contribute to chromatin bridge formation. Aurora B mislocalization also appeared to weaken the "abscission checkpoint". Finally, we showed that KRAS-induced binucleated cell formation appeared to be also H 2 O 2 -dependent. In conclusion, we propose that a ROS, mainly H 2 O 2 increases binucleation through unresolved chromatin bridges caused by DNA damage and mislocalization of Aurora B, the latter of which appears to augment the effect of DNA damage on chromatin bridge formation. Copyright © 2017 Elsevier Inc. All rights reserved.

Early-stage detection of VE-cadherin during endothelial differentiation of human mesenchymal stem cells using SPR biosensor.

PubMed

Fathi, Farzaneh; Rezabakhsh, Aysa; Rahbarghazi, Reza; Rashidi, Mohammad-Reza

2017-10-15

Surface plasmon resonance (SPR) biosensors are most commonly applied for real-time dynamic analysis and measurement of interactions in bio-molecular studies and cell-surface analysis without the need for labeling processes. Up to present, SPR application in stem cell biology and biomedical sciences was underused. Herein, a very simple and sensitive method was developed to evaluate human mesenchymal stem cells trans-differentiation to endothelial lineage of over a period of 14 days based on VE-cadherin biomarker. The SPR signals increased with the increase of the amount of VE-cadherin expression on the cell surface during cell differentiation process. The method was able to detect ≈27 cells permm 2 . No significant effect was observed on the cell viability during the cell attachment to the surface of immune-reactive biochips and during the SPR analysis. Using this highly sensitive SPR method, it was possible to sense the early stage of endothelial differentiation on day 3 in label-free form, whereas flow cytometry and fluorescent microscopy methods were found unable to detect the cell differentiation at the same time. Therefore, the proposed method can rapidly and accurately detect cell differentiation in live cells and label-free manner without any need of cell breakage and has great potential for both diagnostic and experimental approaches. Copyright © 2017. Published by Elsevier B.V.

Challenging tumour immunological techniques that help to track cancer stem cells in malignant melanomas and other solid tumours.

PubMed

Kotlan, Beatrix; Plotar, Vanda; Eles, Klara; Horvath, Szabolcs; Balatoni, Timea; Csuka, Orsolya; Újhelyi, Mihaly; Sávolt, Ákos; Szollar, Andras; Vamosi-Nagy, Istvan; Toth, Laszlo; Farkas, Emil; Toth, Jozsef; Kasler, Miklos; Liszkay, Gabriella

2018-03-01

The arsenal of questions and answers about the minor cancer initiating cancer stem cell (CSC) population put responsible for cancer invasiveness and metastases, has left with an unsolved puzzle. Specific aims of a complex project were partly focused on revealing new biomarkers of cancer. We designed and set up novel techniques to facilitate the detection of cancerous cells. As a novel approach, we investigated B cells infiltrating breast carcinomas and melanomas (TIL-B) in terms of their tumour antigen binding potential. By developing the TIL-B phage display technology we provide here a new technology for the specific detection of highly tumour-associated antigens. Single chain Fv (scFv) antibody fragment phage ELISA, immunofluorescence (IF) FACS analysis, chamber slide technique with IF confocal laser microscopy and immunohistochemistry (IHC) in paraffin-embedded tissue sections were set up and standardized. We showed strong tumour-associated disialylated glycosphingolipid expression levels on various cancer cells using scFv antibody fragments, generated previously by uniquely invasive breast carcinoma TIL-B phage display library technology. We report herein a novel strategy to obtain antibody fragments of human origin that recognise tumour-associated ganglioside antigens. Our investigations have the power to detect privileged molecules in cancer progression, invasiveness, and metastases. The technical achievements of this study are being harnessed for early diagnostics and effective cancer therapeutics.

Cutting Edge: 2B4-Mediated Coinhibition of CD4+ T Cells Underlies Mortality in Experimental Sepsis.

PubMed

Chen, Ching-Wen; Mittal, Rohit; Klingensmith, Nathan J; Burd, Eileen M; Terhorst, Cox; Martin, Greg S; Coopersmith, Craig M; Ford, Mandy L

2017-09-15

Sepsis is a leading cause of death in the United States, but the mechanisms underlying sepsis-induced immune dysregulation remain poorly understood. 2B4 (CD244, SLAM4) is a cosignaling molecule expressed predominantly on NK cells and memory CD8 + T cells that has been shown to regulate T cell function in models of viral infection and autoimmunity. In this article, we show that 2B4 signaling mediates sepsis lymphocyte dysfunction and mortality. 2B4 expression is increased on CD4 + T cells in septic animals and human patients at early time points. Importantly, genetic loss or pharmacologic inhibition of 2B4 significantly increased survival in a murine cecal ligation and puncture model. Further, CD4-specific conditional knockouts showed that 2B4 functions on CD4 + T cell populations in a cell-intrinsic manner and modulates adaptive and innate immune responses during sepsis. Our results illuminate a novel role for 2B4 coinhibitory signaling on CD4 + T cells in mediating immune dysregulation. Copyright © 2017 by The American Association of Immunologists, Inc.

T-Helper 17 Cell Cytokine Responses in Lyme Disease Correlate With Borrelia burgdorferi Antibodies During Early Infection and With Autoantibodies Late in the Illness in Patients With Antibiotic-Refractory Lyme Arthritis

PubMed Central

Sulka, Katherine B.; Pianta, Annalisa; Crowley, Jameson T.; Arvikar, Sheila L.; Anselmo, Anthony; Sadreyev, Ruslan; Steere, Allen C.

2017-01-01

Abstract Background. Control of Lyme disease is attributed predominantly to innate and adaptive T-helper 1 cell (TH1) immune responses, whereas the role of T-helper 17 cell (TH17) responses is less clear. Here we characterized these inflammatory responses in patients with erythema migrans (EM) or Lyme arthritis (LA) to elucidate their role early and late in the infection. Methods. Levels of 21 cytokines and chemokines, representative of innate, TH1, and TH17 immune responses, were assessed by Luminex in acute and convalescent sera from 91 EM patients, in serum and synovial fluid from 141 LA patients, and in serum from 57 healthy subjects. Antibodies to Borrelia burgdorferi or autoantigens were measured by enzyme-linked immunosorbent assay. Results. Compared with healthy subjects, EM patients had significantly higher levels of innate, TH1, and TH17-associated mediators (P ≤ .05) in serum. In these patients, the levels of inflammatory mediators, particularly TH17-associated cytokines, correlated directly with B. burgdorferi immunoglobulin G antibodies (P ≤ .02), suggesting a beneficial role for these responses in control of early infection. Late in the disease, in patients with LA, innate and TH1-associated mediators were often >10-fold higher in synovial fluid than serum. In contrast, the levels of TH17-associated mediators were more variable, but correlated strongly with autoantibodies to endothelial cell growth factor, matrix metalloproteinase 10, and apolipoprotein B-100 in joints of patients with antibiotic-refractory LA, implying a shift in TH17 responses toward an autoimmune phenotype. Conclusions. Patients with Lyme disease often develop pronounced TH17 immune responses that may help control early infection. However, late in the disease, excessive TH17 responses may be disadvantageous by contributing to autoimmune responses associated with antibiotic-refractory LA. PMID:28077518

Expression of voltage-activated calcium channels in the early zebrafish embryo.

PubMed

Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

2009-05-01

Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

Pleural innate response activator B cells protect against pneumonia via a GM-CSF-IgM axis

PubMed Central

Chousterman, Benjamin G.; Hilgendorf, Ingo; Robbins, Clinton S.; Theurl, Igor; Gerhardt, Louisa M.S.; Iwamoto, Yoshiko; Quach, Tam D.; Ali, Muhammad; Chen, John W.; Rothstein, Thomas L.; Nahrendorf, Matthias; Weissleder, Ralph

2014-01-01

Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF–dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity. PMID:24821911

Alternation of histone and DNA methylation in human atherosclerotic carotid plaques.

PubMed

Greißel, A; Culmes, M; Napieralski, R; Wagner, E; Gebhard, H; Schmitt, M; Zimmermann, A; Eckstein, H-H; Zernecke, A; Pelisek, J

2015-08-01

Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development.

Zebrafish pax8 is required for otic placode induction and plays a redundant role with Pax2 genes in the maintenance of the otic placode.

PubMed

Mackereth, Melinda D; Kwak, Su-Jin; Fritz, Andreas; Riley, Bruce B

2005-01-01

Vertebrate Pax2 and Pax8 proteins are closely related transcription factors hypothesized to regulate early aspects of inner ear development. In zebrafish and mouse, Pax8 expression is the earliest known marker of otic induction, and Pax2 homologs are expressed at slightly later stages of placodal development. Analysis of compound mutants has not been reported. To facilitate analysis of zebrafish pax8, we completed sequencing of the entire gene, including the 5' and 3' UTRs. pax8 transcripts undergo complex alternative splicing to generate at least ten distinct isoforms. Two different subclasses of pax8 splice isoforms encode different translation initiation sites. Antisense morpholinos (MOs) were designed to block translation from both start sites, and four additional MOs were designed to target different exon-intron boundaries to block splicing. Injection of MOs, individually and in various combinations, generated similar phenotypes. Otic induction was impaired, and otic vesicles were small. Regional ear markers were expressed correctly, but hair cell production was significantly reduced. This phenotype was strongly enhanced by simultaneously disrupting either of the co-inducers fgf3 or fgf8, or another early regulator, dlx3b, which is thought to act in a parallel pathway. In contrast, the phenotype caused by disrupting foxi1, which is required for pax8 expression, was not enhanced by simultaneously disrupting pax8. Disrupting pax8, pax2a and pax2b did not further impair otic induction relative to loss of pax8 alone. However, the amount of otic tissue gradually decreased in pax8-pax2a-pax2b-deficient embryos such that no otic tissue was detectable by 24 hours post-fertilization. Loss of otic tissue did not correlate with increased cell death, suggesting that otic cells dedifferentiate or redifferentiate as other cell type(s). These data show that pax8 is initially required for normal otic induction, and subsequently pax8, pax2a and pax2b act redundantly to maintain otic fate.

Ibrutinib: A paradigm shift in management of CLL

PubMed Central

Badar, Talha; Burger, Jan A; Wierda, William G; O'Brien, Susan

2016-01-01

1. Summary B-cell receptor (BCR) signaling plays a vital role in B-cell malignancies; Bruton's Tyrosine Kinase (BTK) is a critical mediator of this signaling. BCR signaling, either constitutively or following antigen binding, leads to activation of several downstream pathways involved in cell survival, proliferation, and migration. The efficacy observed in studies of the BTK inhibitor, ibrutinib, confirms that BCR signaling is critical for the growth of B-cell malignancies. Ibrutinib characteristically induces redistribution of malignant B-cells from tissues into the peripheral blood, and rapid resolution of adenopathy. Further, ibrutinib therapy results in normalization of lymphocyte counts and improvement in cytopenias. Ibrutinib has been shown to have an excellent safety profile and does not cause myelosuppression. Early data from combination studies of ibrutinib with anti-CD20 monoclonal antibodies have shown more rapid responses compared to those seen with ibrutinib monotherapy. Current data strongly support continued clinical evaluation of Ibrutinib in B-cell malignancies. PMID:25387837

Ibrutinib: a paradigm shift in management of CLL.

PubMed

Badar, Talha; Burger, Jan A; Wierda, William G; O'Brien, Susan

2014-12-01

B-cell receptor (BCR) signaling plays a vital role in B-cell malignancies; Bruton tyrosine kinase is a critical mediator of this signaling. BCR signaling, either constitutively or following antigen binding, leads to activation of several downstream pathways involved in cell survival, proliferation and migration. The efficacy observed in studies of the Bruton tyrosine kinase inhibitor, ibrutinib, confirms that BCR signaling is critical for the growth of B-cell malignancies. Ibrutinib characteristically induces redistribution of malignant B cells from tissues into the peripheral blood and rapid resolution of adenopathy. Furthermore, ibrutinib therapy results in normalization of lymphocyte counts and improvement in cytopenias. Ibrutinib has been shown to have an excellent safety profile and does not cause myelosuppression. Early data from combination studies of ibrutinib with anti-CD20 monoclonal antibodies have shown more rapid responses compared to those seen with ibrutinib monotherapy. Current data strongly support continued clinical evaluation of ibrutinib in B-cell malignancies.

Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

PubMed

Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

2012-11-01

Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

Immune predispositions for cytomegalovirus retinitis in AIDS. The HNRC Group.

PubMed Central

Schrier, R D; Freeman, W R; Wiley, C A; McCutchan, J A

1995-01-01

CMV retinitis develops in approximately 28-35% of all AIDS patients at later stages of disease, often leading to blindness. To determine whether the subset of AIDS patients who developed CMV retinitis (CMV-R) were immunologically predisposed, T cell proliferation responses to CMV were examined prospectively in an HIV infected, HLA typed, longitudinal study population. Individuals who developed CMV-R had significantly lower T cell proliferation responses to CMV, both early and late in disease, compared to CD4 matched controls who have not developed CMV-R. Since HLA proteins influence T-cell recognition, phenotypes of 21 CMV-R patients were examined to determine whether certain HLA alleles were associated with low immune response and predisposed AIDS patients to CMV-R. HLA DR7 and B44 were at increased (nearly twice the expected) frequency in those with CMV-R. The combined association of either B44, 51 or DR7 with CMV-R was highly significant (P = .008, relative risk of CMV-R = 15) with correction for multiple comparisons. Low immune responses were twice as frequent in those with (61%) compared to those without (30%) predisposing alleles. Thus, AIDS patients with immunogenetically related hyporesponsiveness to CMV antigens may be at increased risk of retinitis. PMID:7706482

Functional analysis of lysosomes during mouse preimplantation embryo development.

PubMed

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

2013-01-01

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth.

PubMed

Bitko, Vira; Shulyayeva, Olena; Mazumder, Barsanjit; Musiyenko, Alla; Ramaswamy, Murali; Look, Dwight C; Barik, Sailen

2007-02-01

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.

Nonstructural Proteins of Respiratory Syncytial Virus Suppress Premature Apoptosis by an NF-κB-Dependent, Interferon-Independent Mechanism and Facilitate Virus Growth▿

PubMed Central

Bitko, Vira; Shulyayeva, Olena; Mazumder, Barsanjit; Musiyenko, Alla; Ramaswamy, Murali; Look, Dwight C.; Barik, Sailen

2007-01-01

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-κB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway. PMID:17151097

A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

PubMed Central

Lozano-Velasco, Estefanía; Vallejo, Daniel; Esteban, Francisco J.; Doherty, Chris; Hernández-Torres, Francisco; Franco, Diego

2015-01-01

The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5+ satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. PMID:26055324

The cell cycle of early mammalian embryos: lessons from genetic mouse models.

PubMed

Artus, Jérôme; Babinet, Charles; Cohen-Tannoudji, Michel

2006-03-01

Genes coding for cell cycle components predicted to be essential for its regulation have been shown to be dispensable in mice, at the whole organism level. Such studies have highlighted the extraordinary plasticity of the embryonic cell cycle and suggest that many aspects of in vivo cell cycle regulation remain to be discovered. Here, we discuss the particularities of the mouse early embryonic cell cycle and review the mutations that result in cell cycle defects during mouse early embryogenesis, including deficiencies for genes of the cyclin family (cyclin A2 and B1), genes involved in cell cycle checkpoints (Mad2, Bub3, Chk1, Atr), genes involved in ubiquitin and ubiquitin-like pathways (Uba3, Ubc9, Cul1, Cul3, Apc2, Apc10, Csn2) as well as genes the function of which had not been previously ascribed to cell cycle regulation (Cdc2P1, E4F and Omcg1).

Manipulating the tumor microenvironment ex vivo for enhanced expansion of tumor-infiltrating lymphocytes for adoptive cell therapy

PubMed Central

Chacon, Jessica Ann; Sarnaik, Amod A; Chen, Jie Qing; Creasy, Caitlin; Kale, Charuta; Robinson, John; Weber, Jeffrey; Hwu, Patrick; Pilon-Thomas, Shari; Radvanyi, Laszlo

2014-01-01

Purpose Cultured tumor fragments from melanoma metastases have been used for years as a source of tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy. The expansion of tumor-reactive CD8+ T cells with IL-2 in these early cultures is critical in generating clinically active TIL infusion products, with a population of activated 4-1BB CD8+ T cells recently found to constitute the majority of tumor-specific T cells. Experimental Design We used an agonistic anti-4-1BB antibody added during the initial tumor fragment cultures to provide in situ 4-1BB co-stimulation. Results We found that addition of an agonistic anti-4-1BB antibody could activate 4-1BB signaling within early cultured tumor fragments and accelerated the rate of memory CD8+ TIL outgrowth that were highly enriched for melanoma antigen specificity. This was associated with NFκB activation and the induction of T-cell survival and memory genes, as well as enhanced IL-2 responsiveness, in the CD8+ T cells in the fragments and emerging from the fragments. Early provision of 4-1BB co-stimulation also affected the dendritic cells (DC) by activating NFκB in DC and promoting their maturation inside the tumor fragments. Blocking HLA class I prevented the enhanced outgrowth of CD8+ T cells with anti-4-1BB, suggesting that an ongoing HLA class I-mediated antigen presentation in early tumor fragment cultures plays a role in mediating tumor-specific CD8+ TIL outgrowth. Conclusions Our results highlight a previously unrecognized concept in TIL adoptive cell therapy that the tumor microenvironment can be dynamically regulated in the initial tumor fragment cultures to regulate the types of T cells expanded and their functional characteristics. PMID:25472998

Advances and highlights in allergen immunotherapy: On the way to sustained clinical and immunologic tolerance.

PubMed

Berings, Margot; Karaaslan, Cagatay; Altunbulakli, Can; Gevaert, Philippe; Akdis, Mübeccel; Bachert, Claus; Akdis, Cezmi A

2017-11-01

Allergen immunotherapy (AIT) is an effective treatment strategy for allergic diseases and has been used for more than 100 years. In recent years, however, the expectations on concepts, conduct, statistical evaluation, and reporting have developed significantly. Products have undergone dose-response and confirmative studies in adults and children to provide evidence for the optimal dosage, safety, and efficacy of AIT vaccines using subcutaneous and sublingual delivery pathways in large patient cohorts, ensuring solid conclusions to be drawn from them for the advantage of patients and societies alike. Those standards should be followed today, and products answering to them should be preferred over others lacking optimization and proof of efficacy and safety. Molecular and cellular mechanisms of AIT include early mast cell and basophil desensitization effects, regulation of T- and B-cell responses, regulation of IgE and IgG 4 production, and inhibition of responses from eosinophils, mast cells, and basophils in the affected tissues. There were many developments to improve vaccination strategies, demonstration of new molecules involved in molecular mechanisms, and demonstration of new biomarkers for AIT during the last few years. The combination of probiotics, vitamins, and biological agents with AIT is highlighting current advances. Development of allergoids and recombinant and hypoallergenic vaccines to skew the immune response from IgE to IgG 4 and regulation of dendritic cell, mast cell, basophil, innate lymphoid cell, T-cell, and B-cell responses to allergens are also discussed in detail. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Early activation of teleost B cells in response to rhabdovirus infection.

PubMed

Abós, Beatriz; Castro, Rosario; González Granja, Aitor; Havixbeck, Jeffrey J; Barreda, Daniel R; Tafalla, Carolina

2015-02-01

To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM(+)) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM(+) cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM(+) cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM(+) cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM(+) cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM(+) cell proliferation, a consistent effect on IgM(+) cell survival was detected. Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects on B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-κB and increased IgM(+) cell survival. Interestingly, VHSV activated B lymphocytes toward an antigen-presenting profile, suggesting an important role of IgM(+) cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Spatiotemporal dynamics of OCT4 protein localization during preimplantation development in mice.

PubMed

Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Kobayashi, Hisato; Umezawa, Akihiro; Akutsu, Hidenori

2016-11-01

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming. © 2016 Society for Reproduction and Fertility.

Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

PubMed

García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

2015-09-01

Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the blastocyst stage in the BMP5 group. Moreover, reverse transcription quantitative real-time polymerase chain reaction analysis showed a significant increase in the relative abundance of SOX2 in two-cell stage embryos, ID2 and OCT4 in eight-cell stage embryos, and NANOG and OCT4 in blastocysts derived from BMP5-treated embryos. In conclusion, our results report that early addition of BMP5 to the embryo culture medium had a positive effect on the blastocyst rate and affected the relative expression of BMP target and pluripotency genes, suggesting that BMP5 could play an important role in the preimplantation development of bovine embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

B cell-intrinsic TLR7 signaling is required for optimal B cell responses during chronic viral infection

PubMed Central

Clingan, Jonathan M.; Matloubian, Mehrdad

2013-01-01

The importance for activation of innate immunity by pattern recognition receptors in forming an effective adaptive immune response is well known. Toll-like receptors (TLRs) have been demonstrated to be critical for antibody responses to a variety of immunizations. In particular, recent evidence suggests that B cell-intrinsic TLR signaling is required for optimal responses to virus-like antigens, but mechanisms by which TLR signaling impacts antibody responses during infection in vivo is unclear. In the present study, we demonstrate that deficiency of TLR7 in B cells alone is sufficient to significantly impact antibody responses in mice during chronic viral infection. This effect was independent of T follicular helper cells, and resulted in a loss of plasma cells generated later, but not early, in the response. The defect in plasma cell formation appeared to be secondary to a qualitative effect of TLR signaling on the germinal center (GC) B cell response. GC B cells in TLR7-deficient mice proliferated to a lesser extent and had a greater proportion of cells with phenotypic characteristics of light zone, relative to dark zone GC B cells. These results suggest that B cell-intrinsic TLR signaling in vivo likely affects plasma cell output by altered selection of antigen-specific B cells in the germinal center. PMID:23761632

The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo

PubMed Central

Ponader, Sabine; Chen, Shih-Shih; Buggy, Joseph J.; Balakrishnan, Kumudha; Gandhi, Varsha; Wierda, William G.; Keating, Michael J.; O'Brien, Susan; Chiorazzi, Nicholas

2012-01-01

B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCR-associated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in CLL, using in vitro and in vivo models, and performed correlative studies on specimens from patients receiving therapy with PCI-32765. PCI-32765 significantly inhibited CLL cell survival, DNA synthesis, and migration in response to tissue homing chemokines (CXCL12, CXCL13). PCI-32765 also down-regulated secretion of BCR-dependent chemokines (CCL3, CCL4) by the CLL cells, both in vitro and in vivo. In an adoptive transfer TCL1 mouse model of CLL, PCI-32765 affected disease progression. In this model, PCI-32765 caused a transient early lymphocytosis, and profoundly inhibited CLL progression, as assessed by weight, development, and extent of hepatospenomegaly, and survival. Our data demonstrate that PCI-32765 effectively inhibits CLL cell migration and survival, possibly explaining some of the characteristic clinical activity of this new targeted agent. PMID:22180443

Generation of Rat Monoclonal Antibodies Against Human Pancreatic Ductal Adenocarcinoma Cells.

PubMed

Higashi, Kiyoshi; Fujii, Nobuaki; Kushida, Masahiko; Yamada, Keita; Suzuki, Noriyuki; Saito, Koichi; Tachibana, Taro

2016-06-01

Pancreatic ductal adenocarcinoma is an aggressive tumor with a poor prognosis. Biomarkers that can detect the tumor in its early stages when it may be amenable to curative resection might improve prognosis. To discover novel markers expressed in primary pancreatic cancer, we generated a panel of monoclonal antibodies against pancreatic ductal adenocarcinoma cell line BxPC3 using a rat medial iliac lymph node method. The antigen recognized by 1B5A5 was expressed on the cell surface and secreted into the conditioned medium of BxPC3 cells, and characterized as glycoproteins with molecular mass between 60 and 90 kDa. A wide range of molecular weights of 1B5A5 antigen in several pancreatic cancer cell lines were observed. Immunohistochemistry using a human multiple organ tumor tissue array showed an enhanced expression of 1B5A5 antigen in pancreas, lung, stomach, breast, urinary bladder, colon, and cervix uteri cancers. Immunoprecipitation followed by proteomic analyses identified CEACAM6 as a 1B5A5 antigen. In addition, western blot analysis results indicated that the 1B5A5 epitope is located within an amino-terminal domain of CEACAM6. These results raised the possibility that our approach could lead to discovery of novel biomarkers for the early stage of cancers in a relatively short period of time.

Mechanisms underlying the organizer formation in Bufo arenarum embryos.

PubMed

Manes, M E; Nieto, O L

1989-06-01

In the early gastrula of Bufo arenarum the prospective mesoderm was previously identified as a marginal belt of grey cells. To analyze their differentiation capacity explants of these cells were cultured within ectodermal vesicles, in isolation and in combination with vegetal components. When cultured in isolation, dorsal and ventral fragments from the deep marginal zone behaved differently. Whilst ventral explants produced blood cells, dorsal explants failed to differentiate, remaining as masses of yolk-laden cells. On the other hand, both cultures were drastically modified when associated with superficial cells from the blastoporal zone, which caused the following effects: a) Promotion of differentiation in dorsal marginal explants, able now to produce notochordal and somitic structures, in addition to mesenchymatic cells. b) Promotion of dorsalization in ventral marginal explants, which changed their expected destiny developing axial components, similar to those furnished by "activated" dorso marginal explants. On the contrary, combined cultures of animal and vegetal pieces were unable to generate mesodermal structures. These studies suggest that the axial mesoderm, identified as the "organizer", develops from a marginal substrate of genuine mesodermal cells through a dorsalizing inductive stimulus originated in superficial periblastoporal cells.

Prenatal exposure to non-ionizing radiation: effects of WiFi signals on pregnancy outcome, peripheral B-cell compartment and antibody production.

PubMed

Sambucci, Manolo; Laudisi, Federica; Nasta, Francesca; Pinto, Rosanna; Lodato, Rossella; Altavista, Pierluigi; Lovisolo, Giorgio Alfonso; Marino, Carmela; Pioli, Claudio

2010-12-01

During embryogenesis, the development of tissues, organs and systems, including the immune system, is particularly susceptible to the effects of noxious agents. We examined the effects of prenatal (in utero) exposure to WiFi signals on pregnancy outcome and the immune B-cell compartment, including antibody production. Sixteen mated (plug-positive) female mice were assigned to each of the following groups: cage control, sham-exposed and microwave-exposed (WiFi signals at 2.45 GHz, whole body, SAR 4 W/kg, 2 h/day, 14 consecutive days starting 5 days after mating). No effects due to exposure to WiFi signals during pregnancy on mating success, number of newborns/mother and body weight at birth were found. Newborn mice were left to grow until 5 or 26 weeks of age, when immunological analyses were performed. No differences due to exposure were found in spleen cell number, B-cell frequency or antibody serum levels. When challenged in vitro with LPS, B cells from all groups produced comparable amounts of IgM and IgG, and proliferated at a similar level. All these findings were consistently observed in the female and male offspring at both juvenile (5 weeks) and adult (26 weeks) ages. Stress-associated effects as well as age- and/or sex-related differences were observed for several parameters. In conclusion, our results do not show any effect on pregnancy outcome or any early or late effects on B-cell differentiation and function due to prenatal exposure to WiFi signals.

Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.

PubMed

Parisi, C; Mastoraki, S; Markou, A; Strati, A; Chimonidou, M; Georgoulias, V; Lianidou, E S

2016-10-01

Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer. Copyright © 2016. Published by Elsevier B.V.

Deficient EBV-specific B- and T-cell response in patients with chronic fatigue syndrome.

PubMed

Loebel, Madlen; Strohschein, Kristin; Giannini, Carolin; Koelsch, Uwe; Bauer, Sandra; Doebis, Cornelia; Thomas, Sybill; Unterwalder, Nadine; von Baehr, Volker; Reinke, Petra; Knops, Michael; Hanitsch, Leif G; Meisel, Christian; Volk, Hans-Dieter; Scheibenbogen, Carmen

2014-01-01

Epstein-Barr virus (EBV) has long been discussed as a possible cause or trigger of Chronic Fatigue Syndrome (CFS). In a subset of patients the disease starts with infectious mononucleosis and both enhanced and diminished EBV-specific antibody titers have been reported. In this study, we comprehensively analyzed the EBV-specific memory B- and T-cell response in patients with CFS. While we observed no difference in viral capsid antigen (VCA)-IgG antibodies, EBV nuclear antigen (EBNA)-IgG titers were low or absent in 10% of CFS patients. Remarkably, when analyzing the EBV-specific memory B-cell reservoir in vitro a diminished or absent number of EBNA-1- and VCA-antibody secreting cells was found in up to 76% of patients. Moreover, the ex vivo EBV-induced secretion of TNF-α and IFN-γ was significantly lower in patients. Multicolor flow cytometry revealed that the frequencies of EBNA-1-specific triple TNF-α/IFN-γ/IL-2 producing CD4(+) and CD8(+) T-cell subsets were significantly diminished whereas no difference could be detected for HCMV-specific T-cell responses. When comparing EBV load in blood immune cells, we found more frequently EBER-DNA but not BZLF-1 RNA in CFS patients compared to healthy controls suggesting more frequent latent replication. Taken together, our findings give evidence for a deficient EBV-specific B- and T-cell memory response in CFS patients and suggest an impaired ability to control early steps of EBV reactivation. In addition the diminished EBV response might be suitable to develop diagnostic marker in CFS.