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Menotti, J., A. Alanio, A. Sturny-Leclere, S. Vitry, F. Sauvage, F. Dromer, G. Barratt, and S. Bretagne. "Comparaison de la toxicité de l’amphotéricine B liposomale et désoxycholate sur des cellules épithéliales alvéolaires par mesures de l’impédance cellulaire en temps réel et du niveau d’expression de gènes de cytokines pro-inflammatoires." Journal de Mycologie Médicale 26, no. 2 (June 2016): e12-e13. http://dx.doi.org/10.1016/j.mycmed.2016.04.030.
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Lee, Da Hee, Na Eun Kwon, Won-Ji Lee, Moo-Seung Lee, Doo-Jin Kim, Ji Hyung Kim, and Sung-Kyun Park. "Increased O-GlcNAcylation of c-Myc Promotes Pre-B Cell Proliferation." Cells 9, no. 1 (January 8, 2020): 158. http://dx.doi.org/10.3390/cells9010158.
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O-linked β-N-acetylglucosamine (O-GlcNAc) modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes, such as gene expression, signaling, and cell growth; however, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation remains largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affected the growth of pre-B cells, which rapidly proliferate to allow expansion of functional clones that express successfully rearranged heavy chains at the pro-B stage during early B cell development. In our study, the overall O-GlcNAc levels in these proliferative pre-B cells, which are linked to the glucose uptake rate, were highly induced when compared with those in pro-B cells. Thus, pharmacologically, genetically, or nutritionally, inhibition of O-GlcNAcylation in pre-B cells markedly downregulated c-Myc expression, resulting in cell cycle arrest via blockade of cyclin expression. Importantly, the population of B cells after the pro-B cell stage in mouse bone marrow was severely impaired by the administration of an O-GlcNAc inhibitor. These results strongly suggest that O-GlcNAcylation-dependent expression of c-Myc represents a new regulatory component of pre-B cell proliferation, as well as a potential therapeutic target for the treatment of pre-B cell-derived leukemia.
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Urbanczyk, Sophia, Merle Stein, Wolfgang Schuh, Hans-Martin Jäck, Dimitrios Mougiakakos, and Dirk Mielenz. "Regulation of Energy Metabolism during Early B Lymphocyte Development." International Journal of Molecular Sciences 19, no. 8 (July 27, 2018): 2192. http://dx.doi.org/10.3390/ijms19082192.
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The most important feature of humoral immunity is the adaptation of the diversity of newly generated B cell receptors, that is, the antigen receptor repertoire, to the body’s own and foreign structures. This includes the transient propagation of B progenitor cells and B cells, which possess receptors that are positively selected via anabolic signalling pathways under highly competitive conditions. The metabolic regulation of early B-cell development thus has important consequences for the expansion of normal or malignant pre-B cell clones. In addition, cellular senescence programs based on the expression of B cell identity factors, such as Pax5, act to prevent excessive proliferation and cellular deviation. Here, we review the basic mechanisms underlying the regulation of glycolysis and oxidative phosphorylation during early B cell development in bone marrow. We focus on the regulation of glycolysis and mitochondrial oxidative phosphorylation at the transition from non-transformed pro- to pre-B cells and discuss some ongoing issues. We introduce Swiprosin-2/EFhd1 as a potential regulator of glycolysis in pro-B cells that has also been linked to Ca2+-mediated mitoflashes. Mitoflashes are bioenergetic mitochondrial events that control mitochondrial metabolism and signalling in both healthy and disease states. We discuss how Ca2+ fluctuations in pro- and pre-B cells may translate into mitoflashes in early B cells and speculate about the consequences of these changes.
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Mourcin, Frédéric, Caroline Breton, Julie Tellier, Priyanka Narang, Lionel Chasson, Audrey Jorquera, Mark Coles, Claudine Schiff, and Stéphane J. C. Mancini. "Galectin-1–expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow." Blood 117, no. 24 (June 16, 2011): 6552–61. http://dx.doi.org/10.1182/blood-2010-12-323113.
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Abstract In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7–producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1+ reticular cells. We also determined that IL-7+ and GAL1+ cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7+ to GAL1+ supportive BM niches during their development.
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KÖhrer, Stefan, Greg Coffey, Ekaterina Kim, Nathalie Y. Rosin, Uma Sinha, Anjali Pandey, Medhat Shehata, et al. "Efficacy of PRT060318, a Novel Highly Specific SYK Inhibitor, in Acute Lymphoblastic Leukemia (ALL)." Blood 120, no. 21 (November 16, 2012): 3532. http://dx.doi.org/10.1182/blood.v120.21.3532.3532.
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Abstract Abstract 3532 B cell acute lymphoblastic leukemia (B-ALL) arises by transformation of progenitor (pre-B) cells. Cure rates in adults remain low and treatment is complicated by microenvironment-mediated resistance to cytotoxic drugs, indicating an urgent need for development of new, more targeted treatment approaches. Spleen tyrosine kinase (Syk), a B cell receptor (BCR)-associated tyrosine kinase, recently was identified as a novel therapeutic target in mature B cell malignancies, such as chronic lymphocytic leukemia (CLL). Besides its role in BCR signaling in mature B cells, Syk also plays an important role in maintenance and expansion of immature B cells. Syk-deficient mice display a severe defect of B lymphopoiesis, with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-BCR signaling. We therefore hypothesized that pre-B ALL cells which express pre-BCRs might be more sensitive to Syk inhibition than other ALL subtypes, which either do not have functional pre-BCRs (pro-B ALL) or display modifying genetic lesions (Ph+/BCR-ABL+ B-ALL). PRT060318, a highly specific, ATP-competitive, small molecular inhibitor of Syk, has preclinical activity in CLL and DLBCL models (Hoellenriegel J et al., Leukemia 26:1576–83, 2012). Syk specificity was confirmed by cellular and non-cellular kinase inhibition assays. We performed metabolic assays using the tetrazolium dye XTT to screen B-ALL cell lines (RS4;11, REH, TOM-1, Nalm-20, Nalm-21, Nalm-6, 697, Kasumi-2, KOPN-8, RCH-ACV, SMS-SB) for responses to PRT060318. ALL cells were incubated with increasing concentrations of PRT060318 (100 nM – 10 μM) for 72 hours. Based on the XTT assays, we determined half maximal inhibitory concentration (IC50), and separated B-ALL cells into responsive (IC50 < 4.5 μM) and non-responsive (IC50 > 4.5 μM) groups. Interestingly, the responsive group consisted only of pre-B ALL cells (CD10+, TdT+, cytoIgμ+), whereas the resistant group comprised only ALL cell lines with a pro-B cell phenotype (CD10+/−, TdT+, cytoIgμ-). All BCR-ABL positive cell lines tested exhibit the pro-B cell phenotype and, similar to their BCR-ABL negative counterparts, were resistant to PRT060318. The Figure depicts XTT assay results for two PRT060318-sensitive pre-B (grey lines) and two resistant pro-B (black lines) B-ALL cell lines. Western Blot analysis demonstrated SYK protein expression in all B-ALL cell lines, excluding lack of target protein expression as possible cause for different response rates between pre-B and pro-B ALL cells. Cell proliferation assays revealed a significant, dose-dependent inhibition of pre-B ALL cell proliferation by PRT060318. Concordantly, dose depended S phase reduction was detected in all PRT060318-sensitive cell lines. In apoptosis assays, in which sensitive pre-B ALL lines were incubated with increasing concentrations of PRT060318 (up to 5 μM) we did not find any significant induction of apoptosis, suggesting anti-proliferative, rather than cytotoxic effects as a main mechanism of action of PRT060318. Preliminary data of primary ALL patient samples, cultured with KUSA-H1 marrow stroma cells in the presence or absence of PRT060318 demonstrate induction of apoptosis in 3 out of 10 samples, other assays are ongoing. In conclusion, we provide evidence that the Syk inhibitor PRT060318 thwarts pre-B ALL cell proliferation, providing a first rationale for clinical testing of PRT060318 in selected patients with B cell ALL. Disclosures: Coffey: Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment.
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Wang, Yui-Hsi, Zhixin Zhang, Peter D. Burrows, Hiromi Kubagawa, S. Louis Bridges, Harry W. Findley, and Max D. Cooper. "V(D)J recombinatorial repertoire diversification during intraclonal pro-B to B-cell differentiation." Blood 101, no. 3 (February 1, 2003): 1030–37. http://dx.doi.org/10.1182/blood-2002-06-1828.
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Abstract The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple VH and VL gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.
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Fathi, Afshin, Mehrdad Mirzarahimi, and Homa Farajkhah. "Réponse à un schéma chimiothérapeutique administré à des enfants atteints de LAL à cellules pré-B à risque élevé selon le protocole COG." Canadian Oncology Nursing Journal 31, no. 3 (July 22, 2021): 334–38. http://dx.doi.org/10.5737/23688076313334338.
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Objectif : La présente étude a pour but d’examiner la réponse à un schéma chimiothérapeutique administré à des enfants atteints de LAL à cellules pré-B à risque élevé selon le protocole COG. Méthode : L’étude transversale porte sur 55 enfants traités selon le protocole du groupe d’oncologie pédiatrique (mieux connu sous le nom de Children’s Oncology Group ou COG), de septembre 2010 à février 2015, et évalue les résultats du schéma chimiothérapeutique. Résultats : Durant la première semaine suivant le traitement, le taux de rétablissement complet a été de 76,4 %. Les taux de survie après trois ans et cinq ans étaient respectivement de 85,5 % et de 81 %. Le taux de rechute après le premier épisode de rémission a été de 20 % et le taux de mortalité consécutif à cette rechute a été de 50 %. Trente pour cent de l’ensemble des décès ont eu lieu durant la période d’induction. Dans tous les cas, une septicémie en est la cause. Conclusion : Les résultats indiquent que le taux de survie a augmenté. Il est donc possible d’améliorer le taux de survie en optant pour le protocole COG et en contrôlant les infections chez les patients, et ce, sans égard au groupe de risque.
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Khoor, A., M. T. Stahlman, M. E. Gray, and J. A. Whitsett. "Temporal-spatial distribution of SP-B and SP-C proteins and mRNAs in developing respiratory epithelium of human lung." Journal of Histochemistry & Cytochemistry 42, no. 9 (September 1994): 1187–99. http://dx.doi.org/10.1177/42.9.8064126.
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We determined the temporal and spatial distribution of surfactant protein B (pro-SP-B) and C (pro-SP-C) mRNAs and proteins by immunohistochemistry and in situ hybridization in fetal, neonatal, and adult human lung. Pro-SP-B and SP-B mRNA were detected in bronchi and bronchioles by 15 weeks' gestation. After 25 weeks, pro-SP-B, active SP-B peptide, and SP-B mRNA were co-localized in bronchiolo-alveolar portal cells and in Type II epithelial cells. In adult lung, pro-SP-B and SP-B mRNA were detected primarily in non-ciliated bronchiolar epithelial cells and in Type II cells in the alveolus. Pro-SP-C and SP-C mRNA were detected in cells lining terminal airways from 15 weeks' gestation and thereafter. After 25 weeks, SP-C mRNA and precursor protein were detected in epithelial cells of the bronchiolo-alveolar portals and in Type II cells, where expression increased with advancing gestational age. Distinct cellular patterns of staining for pro-SP-B compared with SP-B active peptide support the concept that its proteolytic processing or cellular routing may be influenced by cell type and/or cell differentiation. SP-B and SP-C are expressed primarily in distal conducting and terminal airway epithelium of human fetal lung well in advance of surfactant lipid synthesis or physiologic requirements to produce pulmonary surfactant at the time of birth.
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Zhu, Jiang, Russell Garrett, Younghun Jung, Yi Zhang, Nacksung Kim, Jingcheng Wang, Gerard J. Joe, et al. "Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells." Blood 109, no. 9 (January 16, 2007): 3706–12. http://dx.doi.org/10.1182/blood-2006-08-041384.
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Abstract Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin−Sca-1+Rag-2− BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Δ-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.
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Simmons, Szandor, Marko Knoll, Christopher Drewell, Ingrid Wolf, Hans-Joachim Mollenkopf, Corinne Bouquet, and Fritz Melchers. "Biphenotypic B-lymphoid/myeloid cells expressing low levels of Pax5: potential targets of BAL development." Blood 120, no. 18 (November 1, 2012): 3688–98. http://dx.doi.org/10.1182/blood-2012-03-414821.
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Abstract The expression of Pax5 commits common lymphoid progenitor cells to B-lymphoid lineage differentiation. Little is known of possible variations in the levels of Pax5 expression and their influences on hematopoietic development. We have developed a retroviral transduction system that allows for the study of possible intermediate stages of this commitment by controlling the levels of Pax5 expressed in Pax5-deficient progenitors in vitro and in vivo. Retroviral transduction of Pax5-deficient pro-/pre-B cell lines with a doxycycline-inducible (TetON) form of the human Pax5 (huPax5) gene yielded cell clones that could be induced to different levels of huPax5 expression. Clones inducible to high levels developed B220+/CD19+/IgM+ B cells, while clones with low levels differentiated to B220+/CD19−/CD11b+/Gr-1− B-lymphoid/myeloid biphenotypic cells in vitro and in vivo. Microarray analyses of genes expressed at these lower levels of huPax5 identified C/ebpα, C/ebpδ, Pu.1, Csf1r, Csf2r, and Gata-3 as myeloid-related genes selectively expressed in the pro-/pre-B cells that can develop under myeloid/lymphoid conditions to biphenotypic cells. Therefore, reduced expression of huPax5 during the induction of early lymphoid progenitors to B-lineage–committed cells can fix this cellular development at a stage that has previously been seen during embryonic development and in acute lymphoblastic lymphoma–like biphenotypic acute leukemias.
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Xiao, Gang, Zhengshan Chen, Brass Daniel, Lai N. Chan, Huimin Geng, Xiaoyan Jiang, and Markus Müschen. "PP2A Balances Glucose Metabolism and Foxo Activation to Maintain Cellular Redox Homeostasis in Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 1056. http://dx.doi.org/10.1182/blood.v128.22.1056.1056.
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Abstract Introduction : Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase negatively regulates a diverse set of signaling pathways promoting tumor growth. While PP2A functions as tumor suppressor in multiple types of cancer, here, we demonstrate an unexpected pro-survival role of PP2A in pre-B acute lymphoblastic leukemia (ALL) cells. By calibrating PI3K-AKT-mTOR signaling strength, PP2A regulates glycolysis rate and thereby balances energy demands against anti-oxidant protection of pre-B ALL cells. In addition, PP2A reinstates activity of FOXO factors by dephosphorylation and thereby enables the anti-oxidant function of FOXO1 and FOXO3. Consistent with previous studies, we find PP2A is dispensable for the survival of myeloid leukemia cells which indicates a lineage-specific role of PP2A. Combined with genetic study and small molecule inhibitor, we verify that regulation in patient-derived xenografts and highlight PP2A as a therapeutic target in pre-B ALL. Results: Consistent with a divergent role of PP2A in pre-B ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. Consistent with these findings, mutations in PP2A subunits are extremely rare in B cell malignancies but relatively common in solid tumors and myeloid malignancies (COSMIC). We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A and the catalytic subunit C, which leads to near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of FoxO1, FoxO3a, p70S6K and S6 ribosomal protein, which indicated elevated PI3K-Akt-mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. PI3K and mTOR inhibitors also have rescue effect on PP2A deficient ALL cells in growth-competition assay. Luciferase-labeled PP2A-deleted ALL cells showed reduced cell growth and leukemia progression after being transplanted into recipient mice. However, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. This lineage-specific role of PP2A was verified by inducible CEBPα expression to reprogram B cell lineage ALL cells into myeloid cells. Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in Ph+ ALL but not in CML cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. By employing glucose flux metabolic profiling assay using [1,2-13C2]-D-glucose tracer, we found elevated glycolysis and repressed pentose phosphate pathway (PPP) flux in PP2A-deleted pre-B ALL cells. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted pre-B ALL cells, together with decreased anti-oxidant gene expression, increased DNA damage, including H2AX phosphorylation and p53 expression. The mechanistic role of ROS downstream of PP2A was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation. Conclusion: Here we revealed an unexpected role of PP2A in maintaining redox homeostasis in pre-B ALL cells. By regulating AKT-mTOR signaling, PP2A keeps the balance of glycolysis and PPP to meet the energy demands of pre-B ALL cells and avoids extreme levels of oxidative stress. We confirmed this pro-survival role of PP2A in both genetic mouse ALL model and human Ph+ ALL-patients derived leukemia cells. These findings highlight PP2A as a therapeutic target and suggest that agents like the PP2A inhibitor LB-100 may be of interest for pre-clinical development and testing. Disclosures No relevant conflicts of interest to declare.
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Chappel, M. S., M. R. Hough, A. Mittel, F. Takei, R. Kay, and R. K. Humphries. "Cross-linking the murine heat-stable antigen induces apoptosis in B cell precursors and suppresses the anti-CD40-induced proliferation of mature resting B lymphocytes." Journal of Experimental Medicine 184, no. 5 (November 1, 1996): 1639–49. http://dx.doi.org/10.1084/jem.184.5.1639.
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The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.
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Patrick, C. W., T. W. Smith, L. V. McIntire, and H. S. Juneja. "Cellular Interactions Among Marrow Stromal and Normal/Neoplastic Pre-B- and B-Lymphoblastic Cells." Leukemia & Lymphoma 22, no. 3-4 (January 1996): 205–19. http://dx.doi.org/10.3109/10428199609051751.
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Greenberg, Zev J., Darlene A. Monlish, Rachel L. Bartnett, Jeffrey J. Bednarski, and Laura G. Schuettpelz. "The Tetraspanin CD53 Regulates Early B Cell Development By Promoting IL-7R Signaling." Blood 134, Supplement_1 (November 13, 2019): 79. http://dx.doi.org/10.1182/blood-2019-131641.
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The tetraspanin CD53 has been implicated in B cell development and function. Tetraspanins are a family of transmembrane proteins important for organization of the plasma membrane and regulation of cellular migration, adhesion, and activation. CD53 has been shown to be a transcriptional target of EBF1, a critical transcription factor for early B cell development. Additional signaling for early B cell development occurs through the IL-7 receptor (IL-7R), where ligation promotes continued B cell differentiation and pro-survival/anti-apoptotic gene expression. Human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulins. While prior studies have implicated a role for CD53 in regulating mature B cells, its role in early B cell development is not well understood. Herein, we show that CD53 expression rapidly increases throughout B cell development, beginning at the pre-pro-B cell stage. With a CRISPR-generated knockout mouse, we show that Cd53-/- mice have significantly reduced bone marrow (25% fewer, p<0.005), splenic (35% fewer, p<0.05), lymphatic (65% fewer, p<0.0001), and peripheral (30% fewer, p<0.005) B cells compared to wild-type (WT) littermate controls. Mirroring the human phenotype, Cd53-/- mice have significantly reduced serum IgG and IgM (40% reduced, p<0.01). In addition, hematopoietic stem cells isolated from Cd53-/- mice give rise to 30% fewer B cells compared to controls in vitro (p=0.005). Analysis of bone marrow B cell development demonstrates that this loss of B cells originates with early B cell progenitors, which express nearly 50% less IL-7Ra than WT and reduced IL-7 signaling. Using mass cytometry, we identified differential signaling pathways downstream of IL-7R in B cell progenitors. Specifically, we observe impaired PI3K and STAT5 activation in pre-pro- and pro-B cells in the absence of CD53, with a consequent increase in apoptosis in these populations (p<0.01). Decreased STAT5 phosphorylation was confirmed by western blot. Finally, co-immunoprecipitation studies demonstrate a physical interaction between CD53 and IL-7Ra, suggesting that these proteins associate at the cell surface. Together, these data suggest a novel role for CD53 during IL-7 signaling to promote early B cell development. Ongoing studies are focused on determining the CD53 residues required for interaction with IL-7R. Disclosures No relevant conflicts of interest to declare.
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Xu, Shengli, and Kong-Peng Lam. "Delayed Cellular Maturation and Decreased Immunoglobulin κ Light Chain Production In Immature B Lymphocytes Lacking B Cell Linker Protein." Journal of Experimental Medicine 196, no. 2 (July 8, 2002): 197–206. http://dx.doi.org/10.1084/jem.20020172.
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B cell linker (BLNK) protein is a component of the B cell receptor (BCR) signaling pathway and BLNK−/− mice have a block in B lymphopoiesis at the pro-B/pre-B cell stage. To study the effect of BLNK mutation at later stages of B cell development, we introduce an innocuous transgenic BCR into BLNK−/− mice and show that two populations of immature B cells distinguishable by their IgMlow (lo) and IgMhigh (hi) phenotypes are found in the bone marrow of these mice in contrast to a single population of IgMhi cells found in control BCR-transgenic BLNK+/+ mice. The mutant IgMlo and IgMhi cells are at an earlier developmental stage compared with the control IgMhi cells as indicated by their differential expression of CD43, B220, and major histocompatibility complex class II antigens and their timing of generation in culture. Thus, in the absence of BLNK the differentiation of immature B cells is delayed. Furthermore, mutant IgMlo cells produce equivalent level of immunoglobulin (Ig) μ but less Ig κ proteins than control and mutant IgMhi cells and this defect is attributed to a decrease in the amount of κ transcripts being generated. Finally, splenic B cells in BCR-transgenic BLNK−/− mice are predominantly of the transitional B cell phenotype and are rapidly lost from the peripheral B cell pool. Taken together, the data suggest a role for BLNK and perhaps BCR signaling, in the regulation of κ light chain expression and continued immature B cell differentiation.
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Maki, Kazushige, Kisaburo Nagata, Fujiko Kitamura, Toshitada Takemori, and Hajime Karasuyama. "Immunoglobulin β Signaling Regulates Locus Accessibility for Ordered Immunoglobulin Gene Rearrangements." Journal of Experimental Medicine 191, no. 8 (April 17, 2000): 1333–40. http://dx.doi.org/10.1084/jem.191.8.1333.
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The antigen receptor gene rearrangement at a given locus is tightly regulated with respect to cell lineage and developmental stage by an ill-defined mechanism. To study the possible role of precursor B cell antigen receptor (pre-BCR) signaling in the regulation of the ordered immunoglobulin (Ig) gene rearrangement during B cell differentiation, a newly developed system using μ heavy (H) chain membrane exon (μm)-deficient mice was employed. In this system, the antibody-mediated cross-linking of Igβ on developmentally arrested progenitor B (pro-B) cells mimicked pre-BCR signaling to induce early B cell differentiation in vivo. Analyses with ligation-mediated polymerase chain reaction revealed that the Igβ cross-linking induced the redirection of Ig gene rearrangements, namely, the suppression of ongoing rearrangements at the H chain locus and the activation of rearrangements at the light (L) chain locus. Upon the cross-linking, the κL chain germline transcription was found to be upregulated whereas the VH germline transcription was promptly downregulated. Notably, this alteration of the accessibility at the H and L chain loci was detected even before the induction of cellular differentiation became detectable by the change of surface phenotype. Thus, the pre-BCR signaling through Igβ appears to regulate the ordered Ig gene rearrangement by altering the Ig locus accessibility.
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Liu, Li, Marilyn Sanchez-Bonilla, Matthew Crouthamel, Cecilia Giachelli, and Sioban B. Keel. "Murine Terminal Erythroid Differentiation and Early B Development Require the Sodium-Dependent Phosphate Import Protein, PiT1." Blood 120, no. 21 (November 16, 2012): 2307. http://dx.doi.org/10.1182/blood.v120.21.2307.2307.
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Abstract Abstract 2307 Phosphate is critical in multiple biological processes, including phosphorylation reactions, ATP production, and DNA structure and synthesis, and is likely an important determinant of cell growth. It remains unclear how individual cells initially sense changes in extracellular phosphate concentration and the cellular consequences of these changes. PiT1 is a constitutively expressed, high affinity sodium-dependent phosphate import protein and studies in nonhematopoietic cells suggest it plays a role in governing cellular proliferation. Recently, we reported that conditional deletion of Pit1 in neonatal mice causes a profound macrocytic anemia, characterized by a block in terminal erythroid differentiation, dyserythropoiesis, and increased apoptosis. The animals also have marked thrombocytosis and mild neutropenia. Importantly, the phenotype is intrinsic to the hematopoietic system (ASH Annual Meeting Abstracts, November 2011;118:681). Further characterization of their hematopoietic phenotype reveals equivalent numbers of marrow-derived hematopoietic progenitor cells (common myeloid, megakaryocyte-erythroid, and granulocyte-macrophage progenitors) compared to controls. Deleted mice demonstrate a relative expansion in Lin−c-KithighSca-1−CD16/CD32highCD34− cells, which is absent in control animals; morphology and additional flow cytometric characterization (high endoglin and CD150 expression) of this population suggest it includes early erythroid precursors (Pre CFU-E) with aberrant expression of the myeloid antigen CD16. Thus, erythroid differentiation is impacted by a lack of PiT1 from the early CFU-E through basophilic erythroblast stages. Additionally, we discovered that the animals have a marked B cell lymphocytopenia (0.4 K/uL ± 0.1 vs. 2.6 ± 0.5, p<1.0E−4, deleted n=11, control n=4, mean±SEM, Student's t-test) due to a defect in B cell development prior to the pre-pro B cell stage and an additional defect in B cell development unique to late, early pro-B cell development. We confirmed that the defect in B cell development in Pit1-deleted mice is intrinsic to the hematopoietic system by demonstrating B cell lymphocytopenia in lethally irradiated mice transplanted with Pit-1flox/flox;Mx-cre marrow and then treated with poly(I)poly(C) to delete Pit-1 specifically in engrafted cells (1.1 K/uL ± 0.3 vs. 4.9 ± 0.4, p<1.0E−3, deleted n=4, control n=3, mean±SEM, Student's t-test). Cell cycle profiles and BrdU studies show that erythroid cells and B cells lacking PiT1 have impaired cell cycle progression akin to that seen in siRNA knockdown studies of PiT1 in nonhematopoietic cells. Total and sodium-dependent phosphate uptake in flow-cytometrically sorted basophilic erythroblasts/proerythroblasts (CD71highCD44highFSChighB220−Gr1−Mac1−) and whole bone marrow cells are equivalent in deleted and control populations, proving that the phenotype is independent of phosphate uptake (p>0.5). We hypothesize that the profound anemia in mice lacking PiT1 reflects a unique vulnerability of proerythroblasts/basophilic erythroblasts to defects in cell cycle progression due to their high proliferative requirements and the unique coordinate control of cell cycle exit with terminal erythroid differentiation. Late, early pro-B cells may also be particularly vulnerable to perturbations in the cell cycle since they undergo a proliferative burst, likely dependent on the assembly and signaling of the pre-B cell receptor. Ongoing genomic and proteomic studies of flow-cytometrically sorted proerythroblasts/basophilic erythroblasts from Pit1-deleted and control mice are aimed at defining activated signaling networks to account for the anemia in mice lacking PiT1. Our work may offer further insight into how erythroid differentiation is intimately coupled with cellular proliferation, one possible mechanism of ineffective erythropoiesis in low grade acquired myelodysplastic syndromes, and the proliferative stresses shared between terminal erythroid differentiation and early B cell development. Disclosures: No relevant conflicts of interest to declare.
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Rosin, Nathalie Y., Stefan Koehrer, Ekaterina Kim, Susan O'Brien, William G. Wierda, Deborah A. Thomas, Zeev Estrov, Hagop M. Kantarjian, Brian J. Lannutti, and Jan A. Burger. "In Vitro Effects of PI3Kδ Inhibitor GS-1101 (Cal-101) in Acute Lymphoblastic Leukemia (ALL)." Blood 120, no. 21 (November 16, 2012): 3534. http://dx.doi.org/10.1182/blood.v120.21.3534.3534.
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Abstract Abstract 3534 Acute lymphoblastic leukemia (ALL) is a highly heterogeneous disease. B-cell acute lymphoblastic leukemia (B-ALL) is characterized by uncontrolled proliferation of immature lymphoid blasts with suppression of normal hematopoiesis. Phosphoinositide 3-kinases (PI3K) transmit activation signals from diverse transmembrane receptors, leading to generation of phosphatidylinositol- 3,4,5-trisphosphate (PIP3) which promotes proliferation, differentiation, migration, and survival in lymphocytes and various other cell types. A knockout mouse model of the PI3K isoform p110δ demonstrates a unique role of p110δ (PI3Kδ) in B cell receptor (BCR) signaling. This is corroborated by clinical efficacy of the PI3Kδ inhibitor GS-1101 in mature B cell malignancies, especially in chronic lymphocytic leukemia (CLL). In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. We therefore analyzed PI3Kδ expression and effects of the PI3Kδ inhibitor GS-1101 in B-ALL. To screen efficacy of GS-1101 in B-ALL subsets, we performed viability and proliferation assays, using a panel of B-ALL cell lines, derived from different B-cell development stages (Pro-B: REH, RS4;11, Nalm-20, Nalm-21, TOM-1; Pre-B: Nalm-6, Kasumi-2, KOPN-8, SMS-SB, RCH-ACV, 697; Mature: Tanoue, Ball-1 unknown: CCRF-SB). A key downstream effector of PI3K is the serine/threonine kinase Akt, whose phosphorylation is used as a common readout of PI3K activation status. Western Blot analysis of the 15 cell lines showed almost identical levels of phospho-Akt (Ser473) in all tested cell lines, suggesting constitutive PI3K activity. To investigate the ability of GS-1101 to inhibit B-ALL cell proliferation, we performed cell growth experiments. Among the pre-B cell lines 4 of 6 showed a marked decrease in proliferation, 2 other pre-B cell lines showed a minor decrease. In contrast, none of the pro-B or mature B-ALL cell lines were affected by GS-1101. To explore the effects of GS-1101 on cell cycle of B-ALL cells, cell lines were treated with GS-1101 at concentrations ranging from 0.5μM to 5μM. In accordance with the cell growth experiments, G1 phase arrest and reduced numbers of S phase cells were detected in pre-B cell lines after GS-1101 treatment, but not in the pro-B or mature B cell lines. Next, we examined GS-1101 effects on metabolism of B-ALL cells via XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) staining. Cell lines were treated with GS-1101 concentrations between 0.1μM and 5μM for 3 days prior to XTT measurement. Pre-B cells showed a significant (p-value <0.0001) decrease in normalized absorbance compared to the control (without treatment) indicating a decrease in cellular viability. Finally, preliminary co-culture experiments of primary B-ALL samples and KUSA-H1 bone marrow stromal cells revealed significantly reduced B-ALL cell viability after GS-1101 treatment, signifying that GS-1101 can overcome microenviromental-mediated B-ALL cell protection; this is similar to that in other B cell malignances. In summary, these experiments demonstrate that GS-1101 inhibits growth, cell cycle progression and metabolic activity of pre-B ALL cells. Validation of these data with primary patient samples is ongoing. Disclosures: Lannutti: Gilead Sciences Inc: Employment.
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Nahar, Rahul, Parham Ramezani-Rad, Maximilian Mossner, Cihangir Duy, Leandro Cerchietti, Huimin Geng, Sinisa Dovat, et al. "Pre-B cell receptor–mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC." Blood 118, no. 15 (October 13, 2011): 4174–78. http://dx.doi.org/10.1182/blood-2011-01-331181.
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Abstract Initial cell surface expression of the pre-B cell receptor induces proliferation. After 2 to 5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). The mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. The checkpoint at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling induces cell-cycle exit through up-regulation of the transcriptional repressor BCL6. Inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Hence, pre-B cell receptor-mediated activation of BCL6 limits pre-B cell proliferation and induces cellular quiescence at the small pre-BII (Fraction D) stage.
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Duy, Cihangir, J. Jessica Yu, Srividya Swaminathan, Rahul R. Nahar, Soo-mi Kweon, Jose M. Polo, Ester Valls, et al. "BCL6 Is Critical for the Development of a Diverse Primary B Cell Repertoire." Blood 114, no. 22 (November 20, 2009): 91. http://dx.doi.org/10.1182/blood.v114.22.91.91.
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Abstract Abstract 91 Through DNA strand breaks resulting from somatic hypermutation and class-switch recombination, germinal center (GC) B cells are exposed to a high level of DNA damage stress. At the GC stage of development, B cells are protected against apoptosis by specific expression of BCL6, which functions as transcriptional repressor of genes in the DNA damage response pathway. In the absence of BCL6, GC formation is abrogated. During normal B cell development, BCL6 expression was only found in GCs, where the secondary B cell repertoire is shaped. Extensive DNA damage, however, also occurs during the development of the primary B cell repertoire in the bone marrow. B cell precursors in the bone marrow sustain DNA damage during V(D)J recombination at immunoglobulin heavy and light chain loci. It is currently unclear, through which mechanisms early B cell precursors are protected against extensive DNA damage stress caused by V(D)J recombination. Here we report that BCL6 plays a critical role during early B cell development by protecting pre-B cells from DNA damage-induced apoptosis during V(D)J recombination. At the transition from IL7-dependent to IL7-independent stages of B cell development, BCL6 is activated, reaches similar expression levels as in GC B cells. Compared to IL7-dependent pro-B cells and large cycling pre-B cells, BCL6 mRNA and protein levels in IL7-independent small resting pre-B cells were increased by 60- to 90-fold, respectively. We identified STAT5 as a critical negative regulator of BCL6 downstream of IL7 receptor signaling in pre-B cells. Expression of a constitutively active STAT5 mutant prevented BCL6 upregulation in differentiating pre-B cells at the transition from IL7-dependent to IL7-independent stages of B cell development. BCL6 function was then tested in bone marrow precursor cells from BCL6−/− and BCL6+/+ mice: Comparing the gene expression pattern of BCL6−/− and BCL6+/+ pre-B cells, we found that BCL6 is required for transcriptional repression of the ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 genes, which all contribute to cellular senescence and cell cycle arrest. In agreement with gene expression analyses, ChIP-chip and single-locus q-ChIP studies identified ARF (Cdkn2a), p21 (Cdkn1a), Gadd45a and p53 as transcriptional targets of BCL6 in pre-B cells. BCL6-dependent transcriptional repression of these genes in pre-B cells is critical because BCL6+/+ but not BCL6−/− pre-B cells were capable to proliferate in vitro and to form pre-B cell colonies in semisolid agar. Of note, peptide-inhibition of BCL6 suppressed growth and colony formation in ARF+/+ but not ARF−/− pre-B cells, suggesting that ARF-deficiency rescues lack of BCL6 function. We conclude that BCL6-mediated transcriptional repression of ARF is critical for pre-B cell self-renewal. To determine whether BCL6 function is also required for normal early B cell differentiation in vivo, we performed a comprehensive analysis of B cell differentiation stages in bone marrow from BCL6−/− and BCL6+/+ mice. In agreement with previous studies, the overall number of B cell precursors in the bone marrow was only slightly reduced and pro-B cell and large pre-B cell populations were normal. However, the pools of small-resting pre-B cells and new emigrant B cells were reduced in BCL6−/− mice by 3- and 7-fold, respectively. While the overall numbers of mature B cells in BCL6−/− mice were normal, we found that their clonal repertoire was extremely restricted. Using spectratype analysis, we found a broad polyclonal primary B cell repertoire in BCL6+/+ mice, whereas the B cell repertoire in their BCL6−/− counterparts was strictly oligoclonal. We conclude that pre-B cell self-renewal and polyclonal B cell production critically depends on BCL6. While the self-renewal defect of BCL6-deficient pre-B cells can be numerically compensated by increased proliferation at later stages of development, the diversity of the B cell repertoire in BCL6−/− mice is permanently restricted. We conclude that BCL6 is required for pre-B cell self-renewal and the formation of a normal polyclonal B cell repertoire. Disclosures: No relevant conflicts of interest to declare.
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Wang, Lili, Rutendo Gambe, Jean Fan, Youzhong Wan, Angela N. Brooks, Jing Sun, Esther A. Obeng, et al. "Expressionof Sf3b1- K700Ein Murine B Cells Causes Pre-mRNA Splicing and Altered B Cell Differentiation and Function." Blood 126, no. 23 (December 3, 2015): 366. http://dx.doi.org/10.1182/blood.v126.23.366.366.
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Abstract Mutations in the RNA splicing factor SF3B1 have been identified by large-scale sequencing as putative drivers in chronic lymphocytic leukemia (CLL), but their precise roles in the pathogenesis of CLL remains unknown. Although prior transcriptomic studies using primary CLL samples have led to the appreciation of altered RNA splicing in association with these mutations, understanding of their impact on cellular function has been complicated by their variable mutant allele frequency across samples as well as their common co-occurrence with other heterogeneous gene mutations. We therefore generated a mouse line that conditionally expresses the commonly occurring Sf3b1-K700E mutation at its endogenous murine locus. We obtained B-cell lineage specific expression of the mutant allele by crossing heterozygous floxed Sf3b1-K700E mice with homozygous CD19-Cre knockin mice. We confirmed that expression of the mutant allele was uniquely present as a heterozygous mutation in B cells, but not in other cell lineages. We sought to characterize the impact of Sf3b1-K700E on RNA splicing, B cell function, and CLL in this in vivo model. By unbiased RNA-sequencing of splenic B cells from wildtype and mutant mice (n=3), we profiled the splice isoform changes that were associated with Sf3b1-K700E. Using the tool JuncBASE, we detected, classified and quantified 54 differentially spliced transcripts (P<0.05, absolute delta percent spliced in >10%). Consistent with the altered splicing pattern reported in human CLL samples, the splice variants in our mouse model were highly enriched with altered selection of 3' splice sites (49 of 54 events, P<0.001). Of these, we validated 3 selected splice variants in independent samples by qPCR. Our murine model of Sf3b1-K700E mutation thus recapitulates altered RNA splicing, as per in human disease. We therefore next investigated whether Sf3b1-K700E affects B cell development and function. Splenic B cell numbers were significantly lower in the mutant (n=37) compared to control (n=33, P=0.0027) mice while T cell numbers were equivalent. Flow cytometric analysis of various B cell subpopulations from bone marrow and spleen revealed a significant increase in marginal zone B cells in the mutant mice (n=6, P<0.01). Consistent with this finding, we observed evidence of enlarged marginal zone areas on sections of mutant mouse spleens by visual inspection, with a mean reduction by 25% of proliferating germinal centers (per Ki67+ staining, n=6, average, P<0.05). On average, in vitro culture of splenic B cells with LPS and IL4 revealed mutant B cells to undergo 10-15% less proliferation, with reduced survival upon stimulation. Moreover, serum analysis revealed a 30-45% reduction in production of IgG1 and IgG3 from mutant mice (n=8, P<0.05). Altogether, these results suggest that mutant Sf3b1 induces an intrinsic B cell defect leading preferentially to impaired cellular proliferation. Cellular senescence has been commonly detected in pre-malignant lesions and is related to impaired cell proliferation. Indeed, by quantitative PCR array of 84 genes associated with cellular senescence, we found comparable levels of expression of all genes in T cells from wildtype and mutant splenocytes, but an overall trend of upregulation (range: by 1.5- to 21-fold) in the entire set of 84 genes in mutant B splenocytes, with significant upregulation in 20 genes (n=5, P<0.05). These genes included the critical senescence regulators Cdkn2a (p16) and Cdkn1a (p21), whose elevated expression in Sf3b1 mutated B cells we confirmed at the protein level. Furthermore, we detected increased levels of the cellular senescence mediators Igfbp6 and Igfbp7 in the sera from mutant mice (n=31, P<0.05). Collectively, the data demonstrate that expression of Sf3b1-K700E in B cells leads to the cellular senescent phenotype. While we observed that expression of Sf3b1-K700E results in RNA splicing changes, B cell developmental dysregulation and cellular senescence, expression of this mutation alone did not lead to expansion of CD5+CD19+ cells in vivo over time, despite observations up to 18-months (n=50). Our ongoing studies are now focused on the combined effects of Sf3b1-K700E and other recurrent CLL mutations on evasion of senescence and CLL disease progression. Disclosures No relevant conflicts of interest to declare.
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Kim, Yong-Soo, Dong-Mi Shin, Hongsheng Wang, and Herbert Morse. "MAZ Is a Substrate for the Deubiquitinating Enzyme DUB1." Blood 116, no. 21 (November 19, 2010): 3913. http://dx.doi.org/10.1182/blood.v116.21.3913.3913.
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Abstract Abstract 3913 Ubiquitination is a fundamental mechanism of signal transduction that regulates immune responses and many other biological processes. Similar to phosphorylation, ubiquitination is a reversible process that is counter-regulated by ubiquitinating enzymes and deubiquitinating enzymes (DUBs). DUB1 has been identified as a member of a subfamily of deubiquitinating enzymes that are induced by Interleukin-3 (IL-3) in Ba/F3 cells. Recently, we've known that DUB1 is expressed in some pro-B and pre-B cell lines and is differentially regulated during normal B cell differentiation with highest expression in small pre-B cells. To understand the functional role of DUB1 in early B cell development, we identified a transcription factor, MAZ, as an interacting partner or substrate of DUB1 in the Abelson-transformed 220-8 pro-B cell line by using a retrovirus-based protein-fragment complementation assay (RePCA) screen. MAZ has been identified as a critical regulator of p21 gene induction, which controls cell cycle progression in synovial fibroblast cells. Immunoprecipitation and immunoblot analysis confirmed that MAZ and DUB1 interact with each other in cells. DUB1 expression significantly increased the steady-state cellular levels of MAZ. Notably, the half-life of MAZ in the pEGFP-DUB1-transfected cells was significantly increased by DUB1 expression, whereas the half-life of MAZ in the mock-transfected cells was less than 1 hr. These data therefore demonstrate that DUB1 specifically stabilizes MAZ in vivo. We found that the overexpressed MAZ was polyubiquitinated and that the polyubiquitinated MAZ was the substrate for proteasome inhibitor MG132 caused a robust increase of MAZ polyubiquitination. Overexpression of DUB1 in 293T cells caused a decrease of MAZ polyubiquitination in a DUB1 dose-dependent manner. Taken together, these results indicate that DUB1 mediate the ubiquitination-dependent degradation of MAZ. Other study shows that the DUB1 targets both K-48 and K-63 linked ubiquitination suggesting that it may be involved in both protein degradative and non-degradative functions during early B cell differentiation. Disclosures: No relevant conflicts of interest to declare.
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Palmi, Chiara, Grazia Fazio, Ilaria Brunati, Valeria Cazzaniga, Valentina André, Silvano Sozzani, Antonello Villa, et al. "TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia." Blood 116, no. 21 (November 19, 2010): 3624. http://dx.doi.org/10.1182/blood.v116.21.3624.3624.
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Abstract Abstract 3624 Introduction: The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aim of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods: the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results: The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions: in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures: No relevant conflicts of interest to declare.
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Maemunah, Siti, Theo A. Priatna, and Achmad Ali Sjamsuruputra. "Aplikasi enzim selulase dari trichoderma reesei qm 9414 untuk peningkatan produksi etanol dari singkong melalui proses sakarifikasi fermentasi simultan." Jurnal Teknik Kimia Indonesia 4, no. 2 (October 2, 2018): 219. http://dx.doi.org/10.5614/jtki.2005.4.2.5.
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Cellulase enzyme is a complex enzyme which catalyzes the degradation of cellulose. The use of cellulase enzyme on the pretreatment of cassava powder to produce ethanol before saccharified and fermented simultaneously have done in order to destroy most of the cell wall which comprise mostly of cellulose so more starch grain were availabe from the cell. The simultaneous saccharification and fermentation process were carried out at 30°C and at pH of5 with 0,632 unit/g of rice brand koji as glucoamylase and 2,6x107cell/gsubstrate of Saccharomyces cerevisiae yeast. The result showed that, the SSF process gave higher ethanol conversion from starch in cassava powder when the substrate had been pre-treated with cellulase enzyme. Ethanol was obtained with the conversion of 48,6% from starch in cassava powder with 9,88%(wt) in concentration bypreviously soaking the cassava powder in 5% v/v cellulase enzymesfor 7 days before SSF process carried out. Ethanol was obtained with the conversion of 42,07% from raw cassava starch and 7,8%(wt) in concentration by carried out SSF process with 75% v/v cellulase enzymes directly. While the SSF process without ellulase enzvmes only gave 5,6%(wt) of ethanol with 25% conversion.Keywords: Cellulase, SFS, Cassava Starch, Conversion, Ethanol Abstrak Enzim selulase merupakan kompleks enzim yang dapat mengkatalisis penguraian selulosa. Penggunaan enzim selulase pada tepung singkong melalui proses sakarifikasi fermentasi secara simultan diharapkan dapat merusak sebagian besar dinding sel singkong yang masih utuh yang tersusun dari selulosa sehingga membebaskan lebih banyak pati dari dalam sel. Usaha ini dilakukan untuk meningkatkan konversi etanol dari tepung singkong melalui proses sakarifikasi dan fermentasi simultan. Proses produksi etanol melalui proses sakarifikasi dan fermentasi simultan (SFS) dilakukan dengan komposisi tepung singkong 20%(b/b), kadar air medium SFS 80%, pH 5 dan temperatur 30°C. Aktifitas glukoamilase yang digunakan adalah 0,632 unit/gTSK dan jumlah ragi Saccharomyces cerevisiae 2,6x107 sel/gTSK. Hasil percobaan menunjukkan produksi etanol pada SFS dengan perendaman selulase sebesar 5%(v/v) (0,63 UA/gTSK) terlebih dulu selama 7 hari, akan meningkatkan konversi etanol dari tepung singkong hingga 48,6% dengan kadar etanol 9,88%(blb). Konversi etanol dari tepung singkong pada SFS dengan penambahan langsung enzim selulase sebesar 75%(v/v) (8,37 UA/gTSK) tanpa direndam terlebih dulu, adalah 42,07% dengan kadar etanol 7,8%(b/b). Sedangkan pad a SFS tanpa penambahan selulase sam a sekali, hanya diperoleh konversi etanol dari tepung singkong sebesar 25% dengan kadar etanol 5,6%(b/b) .Kata Kunci: Selulase, SFS, Tepung Singkong, Konversi, Etanol
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Agosti, Valter, Selim Corbacioglu, Imke Ehlers, Claudia Waskow, Gunhild Sommer, Georgina Berrozpe, Holger Kissel, et al. "Critical Role for Kit-mediated Src Kinase But Not PI 3-Kinase Signaling in Pro T and Pro B Cell Development." Journal of Experimental Medicine 199, no. 6 (March 15, 2004): 867–78. http://dx.doi.org/10.1084/jem.20031983.
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The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec®) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.
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Mentrup, Torben, Kosta Theodorou, Florencia Cabrera-Cabrera, Andreas O. Helbig, Kathrin Happ, Marion Gijbels, Ann-Christine Gradtke, et al. "Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis." Journal of Experimental Medicine 216, no. 4 (February 28, 2019): 807–30. http://dx.doi.org/10.1084/jem.20171438.
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The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1–mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase–like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling.
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Goetze, Jens Peter. "Biochemistry of Pro-B-Type Natriuretic Peptide-Derived Peptides: The Endocrine Heart Revisited." Clinical Chemistry 50, no. 9 (September 1, 2004): 1503–10. http://dx.doi.org/10.1373/clinchem.2004.034272.
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Abstract Background: Since the discovery of cardiac hormones almost 25 years ago, a vast amount of clinical research has identified the cardiac natriuretic peptides and their precursors as markers of heart failure. It even seems likely that the pro-B-type natriuretic peptide (proBNP)-derived peptides in plasma may become the most frequently measured peptides in the daily diagnosis and control of therapy. In contrast, the biochemistry of the peptides has received less attention. Methods: Published data available on the National Library of Medicine (NLM) were used as the basis for the review. Outcome: This review shows that the present understanding of the biochemistry of peptides is far from complete. In particular, cellular synthesis, including posttranslational precursor maturation, is poorly understood. Moreover, elimination of the precursor fragments is unknown. Elucidation of the molecular heterogeneity of proBNP products will therefore contribute to the understanding of the endocrine heart and may also have important diagnostic consequences. Above all, the different proBNP-derived peptides may not always be equal markers of the same pathophysiologic processes. A different metabolism and peripheral elimination may also impose new and peptide-specific limitations for diagnostic use. Conclusions: It is necessary to focus more on the biology of the proBNP-derived peptides. In turn, new insight into the biochemistry could pave the way for more sensitive and disease-specific assays in the clinical setting.
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Lin, Shan, Roger T. Luo, Mark Wunderlich, Joseph J. Kaberlein, Ahmad Rayes, John Anastasi, Maureen M. O'Brien, James C. Mulloy, and Michael J. Thirman. "Lymphoid Lineage Preference of MLL-AF4 Is Revealed in a Species-Specific Model." Blood 126, no. 23 (December 3, 2015): 2454. http://dx.doi.org/10.1182/blood.v126.23.2454.2454.
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Abstract The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes that drive both acute myeloid and acute lymphoid leukemia (AML and ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (also referred to as AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently identified in AML, for example MLL-AF9 caused by t(9;11)(p22;q23), MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It is the most frequent MLL fusion in ALL and accounts for 10-15% of ALL cases. Patients with t(4;11) have a poor prognosis compared with other cytogenetically defined subsets. Although many MLL-fusion leukemia models have successfully been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful human cell based model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Transduced with a modified MLL-AF4 fusion gene, human hematopoietic CD34+ cells successfully initiate ALL in a xenograft system with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells with MLL-AF4 results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNAseq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusions in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to reprogramming in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Strikingly, this decreased BMI1 expression is evident in primary B-ALL patient samples as well, with MLL-AF9 B-ALL samples demonstrating increased BMI1 relative to MLL-AF4. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11) oncogene and highlight the limitations of using murine cells in human disease modeling. The model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. Disclosures Thirman: AbbVie: Research Funding; Pharmacyclics: Research Funding; Gilead: Research Funding.
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Mead, Adam J., Shabnam Kharazi, Iain C. Macaulay, Debbie Atkinson, Stephen Loughran, Michael Lutteropp, Petter Woll, et al. "FLT3-ITDs Introduce a Myeloid Differentiation and Transformation Bias to Multipotent Lympho-Myeloid Progenitors." Blood 118, no. 21 (November 18, 2011): 1380. http://dx.doi.org/10.1182/blood.v118.21.1380.1380.
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Abstract Abstract 1380 Although the growth factor receptor (GFR) FLT3 has a crucial role in normal early B- and T-lymphoid development, constitutively activating internal tandem duplications (ITDs) of FLT3 are almost entirely restricted to patients with adverse-risk acute myeloid leukemia. We used a murine knock-in model of FLT3-ITD myeloproliferative disease (MPD) to gain a better understanding of the cellular and molecular basis for the myeloid-bias of FLT3-ITD-induced hematological malignancies. As Flt3 cell surface expression is lacking in homozygous Flt3-itd mice, we used CD48 and CD150 expression to investigate the distribution of multipotent progenitors (MPPs) and hematopoietic stem cells (HSCs) within the primitive Lin-Sca1+Kit+ (LSK) compartment. Notably, phenotypic (LSKCD150+CD48-) and functional HSCs were markedly reduced in adult Flt3-itd mice. Competitive transplantation experiments using fetal liver confirmed that HSC numbers were reduced (20-fold reduction) by Flt3-ITDs, in a cell-extrinsic manner. Rather, LSKCD48+150- cells (MPPs) were expanded 2.7-fold in Flt3-itd mice comprising >90% of LSK cells. Similarly to Flt3high wild type (WT) lymphoid-primed multipotent progenitors (LMPPs), nanofluidic gene-expression analysis demonstrated that WT MPPs and Flt3-itd MPPs were myeloid-primed (Csf1r, Csf2r, Cebpa, Mpo) with loss of megakaryocyte and erythroid (MkE) priming (Eklf, Epor, Vwf, Gata1). In contrast, the lymphoid (Il7r, Rag1, sIgH) transcriptional priming of WT MPPs was downregulated in Flt3-itd MPPs. In agreement with this, Flt3-itd MPPs sustained extensive GM potential in vitro, with no MkE potential and, unlike WT MPPs, considerably reduced lymphoid potential. Furthermore, microarray analysis demonstrated global upregulation of the myeloid program in Flt3-itd MPPs. These findings demonstrate that primitive lympho-myeloid MPPs, are expanded and biased towards myeloid development by Flt3-ITDs. In agreement with reduced lymphoid-priming of Flt3-itd MPPs, analysis of early thymic development demonstrated a 10-fold reduction of early thymic progenitors (DN1 Kit+) in Flt3-itd mice. Subsequent stages of thymic development were also reduced, as was overall thymic cellularity. Interestingly, expression of the chemokine receptor CCR9 was 5.5-fold reduced in Flt3-itd MPPs suggesting that thymic seeding progenitors in the bone marrow are suppressed by FLT3-ITDs. Previous studies have suggested that the earliest stage of B-cell development, pre-pro-B cells, retain both B-cell and myeloid potential. Lin-CD19-CD24-AA4.1+CD43+B220+ pre-pro-B cells were expanded 13.7-fold in Flt3-itd mice, whereas subsequent stages of CD19+ B-lymphopoiesis were all reduced. The expanded pre-pro-B cells in Flt3-itd mice were myeloid biased at the transcriptional level with markedly reduced expression of lymphoid genes. Pu1 is a master-regulator of myeloid commitment in early hematopoiesis and a STAT3 target gene. As FLT3-ITDs are known to activate STAT3, unlike WT FLT3, we therefore investigated Pu1 expression in Flt3-itd mice using a Pu1-YFP reporter. Expression of Pu1 was significantly increased in LSK cells (1.4 fold) and in pre-pro-B cells (2.6 fold) in Flt3-itd mice. Furthermore, other STAT3 target genes (Cish, Id1, Pim1, Socs1, Junb) were also upregulated in these cell populations in Flt3-itd mice. Moreover, gene-set enrichment analysis in MPPs demonstrated upregulation of Pu1 target genes in Flt3-itd mice, thus providing a link between aberrant ITD signaling and the observed myeloid bias. In order to determine the functional relevance of this myeloid-bias of Flt3-itd MPPs for disease transformation, we targeted a conditional Aml1-ETO fusion-gene to the earliest B-cell progenitors in Flt3-itd mice using Mb1-Cre. Expression of AML1-ETO in WT mice did not induce any phenotype. However, Mb1-Cre induced AML1-ETO expression in Flt3-itd mice led to a high-penetrance, short latency acute leukaemia. All leukaemias expressed myeloid markers (Mac1 and Gr1) but lacked CD19 and B220 expression. These data demonstrate that Flt3-ITDs expand primitive MPPs with a myeloid lineage bias at the molecular and cellular level, at the expense of HSCs and early lymphoid development. This provides insight into the mechanisms by which mutations resulting in activation of a GFR introduce a lineage bias of resulting hematological malignancies. Disclosures: No relevant conflicts of interest to declare.
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Arnberg, Niklas, Karin Edlund, Alistair H. Kidd, and Göran Wadell. "Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor." Journal of Virology 74, no. 1 (January 1, 2000): 42–48. http://dx.doi.org/10.1128/jvi.74.1.42-48.2000.
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ABSTRACT Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2.
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Pal, Saumen, Jing Wu, Justin K. Murray, Samuel H. Gellman, Michele A. Wozniak, Patricia J. Keely, Meghan E. Boyer, et al. "An antiangiogenic neurokinin-B/thromboxane A2 regulatory axis." Journal of Cell Biology 174, no. 7 (September 21, 2006): 1047–58. http://dx.doi.org/10.1083/jcb.200603152.
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Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However, the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a “neuropeptide,” neurokinin-B (NK-B), reversibly inhibits endothelial cell vascular network assembly and opposes angiogenesis in the chicken chorioallantoic membrane. Disruption of endogenous NK-B signaling promoted angiogenesis. Mechanistic analyses defined a multicomponent pathway in which NK-B signaling converges upon cellular processes essential for angiogenesis. NK-B−mediated ablation of Ca2+ oscillations and elevation of 3′–5′ cyclic adenosine monophosphate (cAMP) reduced cellular proliferation, migration, and vascular endothelial growth factor receptor expression and induced the antiangiogenic protein calreticulin. Whereas NK-B initiated certain responses, other activities required additional stimuli that increase cAMP. Although NK-B is a neurotransmitter/ neuromodulator and NK-B overexpression characterizes the pregnancy-associated disorder preeclampsia, NK-B had not been linked to vascular remodeling. These results establish a conserved mechanism in which NK-B instigates multiple activities that collectively oppose vascular remodeling.
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Nahar, Rahul, Parham Ramezani-Rad, Maximilian Mossner, Cihangir Duy, Leandro Cerchietti, Huimin Geng, Hassan Jumaa, B. Hilda Ye, Ari Melnick, and Markus Muschen. "Pre-B Cell Receptor-Mediated Activation of BCL6 Induces Pre-B Cell Quiescence Through Transcriptional Repression of MYC." Blood 118, no. 21 (November 18, 2011): 1406. http://dx.doi.org/10.1182/blood.v118.21.1406.1406.
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Abstract Abstract 1406 Background: Pre-B cell receptor signaling is critical to induce initial expansion of the pool of differentiating B cell precursors but also mediates subsequent cell cycle exit and quiescence. Initial cell surface expression of the pre-B cell receptor induces a series of tyrosine phosphorylation events resulting in MYC and CCND2-mediated proliferation. After 2–5 divisions, however, large pre-BII (Fraction C') cells exit cell cycle to become resting, small pre-BII cells (Fraction D). While the signaling pathway resulting in pre-B cell receptor-induced proliferation is well understood, the mechanism by which pre-BII cells exit cell cycle, however, is currently unclear. Results: This checkpoint and cell cycle exit at the Fraction C'-D transition is critical for immunoglobulin light chain gene recombination and to prevent malignant transformation into acute lymphoblastic leukemia. Here we demonstrate that inducible activation of pre-B cell receptor signaling recapitulates the initial proliferative burst followed by cell cycle exit, which is characterized by strong upregulation of BCL6 and subsequent loss of MYC and CCND2 expression. ChIP-on-chip analysis revealed that the transcriptional repressor BCL6 is directly recruited to the MYC promoter. Inducible activation of pre-B cell receptor signaling in pre-B cells from BCL6-deficient mice failed to induce cell cycle exit and quiescence. We conclude that activation of BCL6 downstream of the pre-B cell receptor is crucial for the induction of quiescence at the Fraction C'-D checkpoint. As expected, inducible activation of BCL6 downstream of the pre-B cell receptor results in transcriptional repression of MYC and CCND2. Overexpression of MYC prevented pre-B cell receptor/BCL6-induced cell cycle exit. Conclusion: Hence, pre-B cell receptor signaling induces cellular quiescence of Fraction C' pre-B cells through activation of BCL6 and BCL6-mediated transcriptional repression of MYC and CCND2. Disclosures: No relevant conflicts of interest to declare.
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Nybo, Mads, Lars Bo Nielsen, Søren Junge Nielsen, Marie Lindegaard, Peter Damm, Jens F. Rehfeld, and Jens Peter Goetze. "Discordant expression of pro-B-type and pro-C-type natriuretic peptide in newborn infants of mothers with type 1 diabetes." Regulatory Peptides 141, no. 1-3 (June 2007): 135–39. http://dx.doi.org/10.1016/j.regpep.2006.12.026.
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Nesslein, Lori L., Kristin R. Melton, Machiko Ikegami, Cheng-Lun Na, Susan E. Wert, Ward R. Rice, Jeffrey A. Whitsett, and Timothy E. Weaver. "Partial SP-B deficiency perturbs lung function and causes air space abnormalities." American Journal of Physiology-Lung Cellular and Molecular Physiology 288, no. 6 (June 2005): L1154—L1161. http://dx.doi.org/10.1152/ajplung.00392.2004.
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Surfactant protein B (SP-B) is required for function of newborn and adult lung, and partial deficiency has been associated with susceptibility to lung injury. In the present study, transgenic mice were produced in which expression of SP-B in type II epithelial cells was conditionally regulated. Concentrations of SP-B were maintained at 60–70% of that normally present in control. Immunostaining for SP-B demonstrated cellular heterogeneity in expression of the protein. In subsets of type II cells in which SP-B staining was decreased, immunostaining for pro-SP-C was increased and lamellar body ultrastructure was disrupted, consistent with focal SP-B deficiency. Fluorescence-activated cell sorting analyses of freshly isolated type II cells identified a population of cells with low SP-B content and a smaller population with increased SP-B content, confirming nonuniform expression of the SP-B transgene. Focal air space enlargement, without cellular infiltration or inflammation, was observed. Pressure-volume curves indicated that maximal tidal volume was unchanged; however, hysteresis was modestly altered and residual volumes were significantly decreased in the SP-B-deficient mice. Chronic, nonuniform SP-B deficiency perturbed pulmonary function and caused air space enlargement.
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Scott, Gina B., Paul A. Bowles, Erica B. Wilson, Josephine L. Meade, Boon Chuan Low, Adam Davison, G. Eric Blair, and Graham P. Cook. "Identification of the BCL2/adenovirus E1B-19K protein-interacting protein 2 (BNIP-2) as a granzyme B target during human natural killer cell-mediated killing." Biochemical Journal 431, no. 3 (October 11, 2010): 423–31. http://dx.doi.org/10.1042/bj20091073.
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Cytotoxic lymphocytes eliminate infected cells and tumours via the perforin-mediated delivery of pro-apoptotic serine proteases known as granzymes. Granzyme B triggers apoptosis via the cleavage of a repertoire of cellular proteins, leading to caspase activation and mitochondrial depolarization. A simple bioinformatics strategy identified a candidate granzyme B cleavage site in the widely expressed BNIP-2 (BCL2/adenovirus E1B-19K protein-interacting protein 2). Granzyme B cleaved recombinant BNIP-2 in vitro and endogenous BNIP-2 was cleaved during the NK (natural killer) cell-mediated killing of tumour cells. Cleavage required the site identified in the bioinformatics screen and was caspase-independent. Expression of either full-length BNIP-2 or a truncated molecule mimicking the granzyme B cleaved form was pro-apoptotic and led to the caspase-dependent cleavage of BNIP-2 at a site distinct from granzyme B cleavage. Inhibition of BNIP-2 expression did not affect the susceptibility to NK cell-mediated killing. Furthermore, target cells in which BID (BH3-interacting domain death agonist) expression was inhibited also remained highly susceptible to NK cell-mediated killing, revealing redundancy in the pro-apoptotic response to human cytotoxic lymphocytes. Such redundancy reduces the opportunity for escape from apoptosis induction and maximizes the chances of immune-mediated clearance of infected cells or tumour cells.
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Leguit, Roos J., Reinier A. P. Raymakers, Konnie M. Hebeda, and Roel Goldschmeding. "CCN2 (Cellular Communication Network factor 2) in the bone marrow microenvironment, normal and malignant hematopoiesis." Journal of Cell Communication and Signaling 15, no. 1 (January 11, 2021): 25–56. http://dx.doi.org/10.1007/s12079-020-00602-2.
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AbstractCCN2, formerly termed Connective Tissue Growth Factor, is a protein belonging to the Cellular Communication Network (CCN)-family of secreted extracellular matrix-associated proteins. As a matricellular protein it is mainly considered to be active as a modifier of signaling activity of several different signaling pathways and as an orchestrator of their cross-talk. Furthermore, CCN2 and its fragments have been implicated in the regulation of a multitude of biological processes, including cell proliferation, differentiation, adhesion, migration, cell survival, apoptosis and the production of extracellular matrix products, as well as in more complex processes such as embryonic development, angiogenesis, chondrogenesis, osteogenesis, fibrosis, mechanotransduction and inflammation. Its function is complex and context dependent, depending on cell type, state of differentiation and microenvironmental context. CCN2 plays a role in many diseases, especially those associated with fibrosis, but has also been implicated in many different forms of cancer. In the bone marrow (BM), CCN2 is highly expressed in mesenchymal stem/stromal cells (MSCs). CCN2 is important for MSC function, supporting its proliferation, migration and differentiation. In addition, stromal CCN2 supports the maintenance and longtime survival of hematopoietic stem cells, and in the presence of interleukin 7, stimulates the differentiation of pro-B lymphocytes into pre-B lymphocytes. Overexpression of CCN2 is seen in the majority of B-acute lymphoblastic leukemias, especially in certain cytogenetic subgroups associated with poor outcome. In acute myeloid leukemia, CCN2 expression is increased in MSCs, which has been associated with leukemic engraftment in vivo. In this review, the complex function of CCN2 in the BM microenvironment and in normal as well as malignant hematopoiesis is discussed. In addition, an overview is given of data on the remaining CCN family members regarding normal and malignant hematopoiesis, having many similarities and some differences in their function.
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Sesler, Cheryl L., and Majd Zayzafoon. "NFAT Signaling in Osteoblasts Regulates the Hematopoietic Niche in the Bone Microenvironment." Clinical and Developmental Immunology 2013 (2013): 1–11. http://dx.doi.org/10.1155/2013/107321.
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Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT) negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1), a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic developmentin vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFATOB). Bone histomorphometry showed that dnNFATOBmice have significant increases in bone volume (44%) and mineral apposition rate (131%) and decreased trabecular thickness (18%). In the bone microenvironment, dnNFATOBmice displayed a significant increase (87%) in Lineage−cKit+Sca-1+(LSK) cells and significant decreases in B220+CD19−IgM−pre-pro-B cells (41%) and B220+CD19+IgM+immature B cells (40%). Concurrent with these findings, LSK cell differentiation into B220+cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFATOBmice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFATOBmice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts.
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Nie, Pingping, Chen Chen, Qian Yin, Chunhao Jiang, Jianhua Guo, Hongwei Zhao, and Dongdong Niu. "Function of miR825 and miR825* as Negative Regulators in Bacillus cereus AR156-elicited Systemic Resistance to Botrytis cinerea in Arabidopsis thaliana." International Journal of Molecular Sciences 20, no. 20 (October 11, 2019): 5032. http://dx.doi.org/10.3390/ijms20205032.
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Small RNAs function to regulate plant defense responses to pathogens. We previously showed that miR825 and miR825* downregulate Bacillus cereus AR156 (AR156)-triggered systemic resistance to Pseudomonassyringae pv. tomato DC3000 in Arabidopsis thaliana (Arabidopsis). Here, Northern blotting revealed that miR825 and miR825* were more strongly downregulated in wild type Arabidopsis Col-0 (Col-0) plants pretreated with AR156 than in nontreated plants upon Botrytis cinerea (B. cinerea) B1301 infection. Furthermore, compared with Col-0, transgenic plants with attenuated miR825 and miR825* expression were more resistant to B. cinerea B1301, yet miR825- and miR825*-overexpressing (OE) plants were more susceptible to the pathogen. With AR156 pretreatment, the transcription of four defense-related genes (PR1, PR2, PR5, and PDF1.2) and cellular defense responses (hydrogen peroxide production and callose deposition) were faster and stronger in miR825 and miR825* knockdown lines but weaker in their OE plants than in Col-0 plants upon pathogen attack. Also, AR156 pretreatment caused stronger phosphorylation of MPK3 and MPK6 and expression of FRK1 and WRKY53 genes upon B. cinerea B1301 inoculation in miR825 and miR825* knockdown plants than in Col-0 plants. Additionally, the assay of agrobacterium-mediated transient co-expression in Nicotiana benthamiana confirmed that AT5G40910, AT5G38850, AT3G04220, and AT5G44940 are target genes of miR825 or miR825*. Compared with Col-0, the target mutant lines showed higher susceptibility to B. cinerea B1301, while still expressing AR156-triggered induced systemic resistance (ISR). The two-way analysis of variance (ANOVA) revealed a significant (P < 0.01) interactive effect of treatment and genotype on the defense responses. Hence, miR825 and miR825*act as negative regulators of AR156-mediated systemic resistance to B. cinerea B1301 in Arabidopsis.
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Urban, Stephan, Klaus M. Breiner, Frank Fehler, Ursula Klingmüller, and Heinz Schaller. "Avian Hepatitis B Virus Infection Is Initiated by the Interaction of a Distinct Pre-S Subdomain with the Cellular Receptor gp180." Journal of Virology 72, no. 10 (October 1, 1998): 8089–97. http://dx.doi.org/10.1128/jvi.72.10.8089-8097.1998.
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ABSTRACT Functionally relevant hepadnavirus-cell surface interactions were investigated with the duck hepatitis B virus (DHBV) animal model by using an in vitro infection competition assay. Recombinant DHBV pre-S polypeptides, produced in Escherichia coli, were shown to inhibit DHBV infection in a dose-dependent manner, indicating that monomeric pre-S chains were capable of interfering with virus-receptor interaction. Particle-associated pre-S was, however, 30-fold more active, suggesting that cooperative interactions enhance particle binding. An 85-amino-acid pre-S sequence, spanning about half of the DHBV pre-S chain, was characterized by deletion analysis as essential for maximal inhibition. Pre-S polypeptides from heron hepatitis B virus (HHBV) competed DHBV infection equally well despite a 50% difference in amino acid sequence and a much-reduced infectivity of HHBV for duck hepatocytes. These observations are taken to indicate (i) that the functionality of the DHBV pre-S subdomain, which interacts with the cellular receptor, is determined predominantly by a defined three-dimensional structure rather than by primary sequence elements; (ii) that cellular uptake of hepadnaviruses is a multistep process involving more than a single cellular receptor component; and (iii) that gp180, a cellular receptor candidate unable to discriminate between DHBV and HHBV, is a common component of the cellular receptor complex for avian hepadnaviruses.
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Ott, German. "Impact of MYC on malignant behavior." Hematology 2014, no. 1 (December 5, 2014): 100–106. http://dx.doi.org/10.1182/asheducation-2014.1.100.
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Abstract MYC, a member of the helix-loop-helix leucine zipper family of nuclear transcription factors, is a potent proto-oncogene primarily identified as the target of the t(8;14)(q24;q32) chromosome translocation in Burkitt lymphoma. Activation of the MYC gene in normal cells both results in enhanced cellular proliferation and up-regulation of pro-apoptotic pathways, reflecting the tight regulation of the molecule in the normal cellular system. In the process of transformation, these secondary inhibitory functions of the MYC molecule have to be overcome through secondary mutations of the MYC gene itself and/or by abrogating the inhibitory effects of physiological regulators and/or repressors of proliferation such as BCL2, BCL6, BLIMP1, or others. Most aggressive lymphomas, therefore, harbor additional oncogenic alterations that cooperate with MYC deregulation, with different alterations identified in human solid or hematological tumors. These alterations are likely to counteract the pro-apoptotic function of MYC. MYC gene alterations in diffuse large B-cell lymphomas and in B-cell lymphomas, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma are frequently associated with BCL2 or/and BCL6 translocations conferring a very aggressive behavior. This review summarizes inherent factors of the biology and function of MYC important in the process of transformation, especially taking account the interdependence of MYC on various cellular networks that have to be co-deregulated to achieve the full malignant phenotype.
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Jeong, Kang-Hyun, Hyun Jeong Lee, Tae-Sik Park, and Soon-Mi Shim. "Catechins Controlled Bioavailability of Benzo[a]pyrene (B[α]P) from the Gastrointestinal Tract to the Brain towards Reducing Brain Toxicity Using the In Vitro Bio-Mimic System Coupled with Sequential Co-Cultures." Molecules 24, no. 11 (June 10, 2019): 2175. http://dx.doi.org/10.3390/molecules24112175.
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The aim of the current study was to examine the preventive effect of green tea catechins on the transport of Benzo[a]pyrene (B[α]P) into the brain using an in vitro bio-mimic system coupled with sequential co-cultures. When 72 μM of catechins was pre-treated, cellular cytotoxicity induced by IC50 of B[α]P in human liver hepatocellular carcinoma (HepG2) and human brain microvascular endothelial cells (HBMECs) was reduced by 27% and 26%, respectively. The cellular integrity measured in HBMECs, which was exposed to IC50 of B[α]P, slowly decreased. However, the pre-treatment of catechins retained cellular integrity that was 1.14 times higher than with the absence of catechins. Co-consumption of catechins reduced not only the bio-accessibility of B[α]P in digestive fluid, but it also decreased absorption of B[α]P in human intestinal epithelial cells (Caco-2) with a HepG2 co-culture system. It was found that approximately a two times lower amount of B[α]P was transported via the blood-brain barrier (BBB) compared to only the B[α]P intake. These results are taken in conjunction with each other support that catechins could be able to prevent brain toxicity induced by B[α]P in the human body by limiting the bio-availability of B[α]P.
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Stenger, Rachel M., Martien C. M. Poelen, Ed E. Moret, Betsy Kuipers, Sven C. M. Bruijns, Peter Hoogerhout, Marcel Hijnen, et al. "Immunodominance in Mouse and Human CD4+ T-Cell Responses Specific for the Bordetella pertussis Virulence Factor P.69 Pertactin." Infection and Immunity 77, no. 2 (November 17, 2008): 896–903. http://dx.doi.org/10.1128/iai.00769-08.
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ABSTRACT P.69 pertactin (P.69 Prn), an adhesion molecule from the causative agent of pertussis, Bordetella pertussis, is present in cellular and most acellular vaccines that are currently used worldwide. Although both humoral immunity and cellular immunity directed against P.69 Prn have been implicated in protective immune mechanisms, the identities of CD4+ T-cell epitopes on the P.69 Prn protein remain unknown. Here, a single I-Ad-restricted B. pertussis conserved CD4+ T-cell epitope at the N terminus of P.69 Prn was identified by using a BALB/c T-cell hybridoma. The epitope appeared immunodominant among four other minor strain-conserved P.69 Prn epitopes recognized after vaccination and B. pertussis infection, and it was capable of evoking a Th1/Th17-type cytokine response. B. pertussis P.69 Prn immune splenocytes did not cross-react with natural variants of the epitope as present in Bordetella parapertussis and Bordetella bronchiseptica. Finally, it was found that the immunodominant P.69 Prn epitope is broadly recognized in the human population by CD4+ T cells in an HLA-DQ-restricted manner. During B. pertussis infection, the epitope was associated with a Th1-type CD4+ T-cell response. Hence, this novel P.69 Prn epitope is involved in CD4+ T-cell immunity after B. pertussis vaccination and infection in mice and, more importantly, in humans. Thus, it may provide a useful tool for the evaluation of the type, magnitude, and maintenance of B. pertussis-specific CD4+ T-cell mechanisms in preclinical and clinical vaccine studies.
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Lapalombella, Rosa, Xiaobin B. Zhao, Peter R. Baum, Jeffrey A. Ledbetter, Natarajan Muthusamy, and John C. Byrd. "Role of CD37SMIP a Novel Engineered Small Modular Immunopharmaceutical in the Treatment of CLL." Blood 110, no. 11 (November 16, 2007): 801. http://dx.doi.org/10.1182/blood.v110.11.801.801.
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Abstract CD37 is a lineage-specific B-cell antigen highly expressed on both normal and transformed B-cells. The significant B-cell selective CD37 expression makes it a valuable target for therapy against B cell malignancies including chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), and non-Hodgkins lymphoma (B-NHL). A novel CD37-specific small modular immunopharmaceutical (CD37SMIP) was engineered to contain a single chain variable region (scFv) linked to a human IgG1 hinge, CH2 and CH3 domains. CD37SMIP induces potent apoptosis in the presence of a crosslinker and antibody dependent cellular cytotoxicity (ADCC) against primary CLL cells. Significant in vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model that is dependent upon NK cell function. The induced cytotoxicity in primary CLL cells was independent of activation of caspase cascade, and consistent with this, the pan-caspase inhibitor z-VAD-fmk failed to rescue CD37 SMIP drug induced cytotoxicity. In an attempt to define the CD37 mediated early signaling events and their role in cytotoxicity, we investigated protein tyrosine phosphorylation as a potential early activation event responsible for CD37 SMIP drug mediated cytotoxicity. Primary B CLL cells were stimulated with increasing concentration of CD37 SMIP with or without crosslinker. Western blot analysis of cellular lysates with anti-phosphotyrosine antibody revealed several tyrosine phosphorylated proteins including a predominant ∼65KDa protein in response to the cross-linking of CD37 SMIP within 10 minutes. Further, pre-treatment with the tyrosine kinase inhibitor herbimycin prevented tyrosine phosphorylation of the predominant 65KDa protein and inhibited CD37 SMIP induced apoptosis in a dose dependent manner. Attempts to identify the tyrosine phosphorylated proteins, using MALDI-TOF mass spectrometry, and to define the potential role of these target proteins in the apoptotic pathway are ongoing. To further examine the molecular mechanism whereby CD37 SMIP triggers cell death, we investigated the involvement of mitochondrial pathways including changes in mitochondrial potential (Δψm) and generation of reactive oxygen species. Treatment with CD37 SMIP induces a time dependent mitochondrial membrane depolarization and increased production of ROS. Further, primary B CLL cells cultured with CD37SMIP induced cleavage and mitochondrial translocation of Bax and upregulation of Bim in a time dependent manner indicating a potential role for Bim and Bax pro apoptotic proteins in CD37 induced apoptosis. These findings provide strong justification for CD37 as a therapeutic target aimed at modulation of pro apoptotic proteins and introduce small modular immunopharmaceuticals as a novel class of targeted therapies for B-cell malignancies. (SMIP trademark is owned by Trubion Pharmaceuticals).
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Bueno, C., R. Montes, P. Catalina, R. Rodríguez, and P. Menendez. "Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement." Leukemia 25, no. 3 (December 7, 2010): 400–410. http://dx.doi.org/10.1038/leu.2010.284.
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Ungerbäck, Jonas, Josefine Åhsberg, Tobias Strid, Rajesh Somasundaram, and Mikael Sigvardsson. "Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors." Journal of Experimental Medicine 212, no. 7 (June 8, 2015): 1109–23. http://dx.doi.org/10.1084/jem.20132100.
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To investigate how transcription factor levels impact B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19+ progenitors, these cells displayed an increased T cell potential in vivo and in vitro. The alteration in lineage fate depended on a Notch1-mediated conversion process, whereas no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5+/−Ebf1+/− pro–B cells were reflected in the transcriptional response. Both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, but only the Pax5+/−Ebf1+/− pro–B cells down-regulated genes central for the preservation of stable B cell identity. This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling. This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.
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Firouzbakht, Hossein, Arash Zibaee, Hassan Hoda, and Mohammad Mehdi Sohani. "Purification and characterization of the cuticle-degrading proteases produced by an isolate of Beauveria bassiana using the cuticle of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae)." Journal of Plant Protection Research 55, no. 2 (April 1, 2015): 179–86. http://dx.doi.org/10.1515/jppr-2015-0024.
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Abstract The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect to reach the hemocoel. Since proteins are the molecules responsible for integument strength in insects, the proteins must synthesise the cuticle degrading proteases which will then enable the proteases to penetrate. It is important to determine the biochemical properties of these proteases so that fungal virulence can be better understood. In the current study, a recently collected isolate of B. bassiana, namely AM-118, was inoculated in liquid media containing 0.5% of Andrallus spinidens Fabricus cuticle to obtain specific proteases. The crude samples were purified via a three step process using ammonium sulfate, Sepharyl G-100, and DEAE-Cellulose Fast Flow. The results revealed two proteases known as subtilisin-like (Pr1), and trypsin-like (Pr2), with the molecular weights of 105 and 103 kDa. The optimal pH and temperature values were found to be 8 and 35°C for Pr1 and 8 and 40°C for Pr2, respectively. Inhibitors like AEBSF, EDTA, TPCK, and phenanthroline significantly affected proteolytic activities. Here, we reported two fungal proteases by high molecular weight from an Iranian isolate of B. bassiana. These findings will help us to better understand fungal virulence against insects.
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Sanchez-Aguilera, Abel, Jose Cancelas, and David A. Williams. "RhoH-Deficient Mice Show Altered B Cell Populations In Vivo." Blood 110, no. 11 (November 16, 2007): 2307. http://dx.doi.org/10.1182/blood.v110.11.2307.2307.
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Abstract RhoH is a GTPase-deficient, hematopoietic-specific member of the family of Rho GTPases (Li et al, 2002). RhoH has been described as regulating proliferation and engraftment of hematopoietic progenitor cells (Gu et al, 2005) and integrin-mediated adhesion in T cells (Cherry et al, 2004). Additionally, RhoH plays a critical role in T-cell development and T-cell receptor signaling (Gu et al, 2006; Dorn et al, 2007). However, the potential role of RhoH in the differentiation and biological functions of B cells are unknown. To answer these questions, we analyzed the B-cell phenotype of RhoH−/− mice and the in vitro properties of RhoH-deficient splenic B cells compared to their wild-type counterparts. RhoH−/− mice showed increased B-cell numbers in the bone marrow, mainly due to an increase in the number of pro-B, pre-B and immature B cells. In the spleen, lymph nodes and peripheral blood, RhoH−/− mice showed a significant decrease in the number of follicular (B-2) cells (B220+ CD93– IgDhigh CD21low). The number of splenic marginal zone B cells (B220+ CD93– IgDlow CD21high), plasma cells (CD93– CD38+ CD138+) in bone marrow and spleen, and B-1 cells (IgM+ CD5+) in peritoneal cavity were not significantly different from those in wild-type animals. These alterations have functional significance, since the serum concentrations of IgM and IgG1 were significantly lower in RhoH−/− mice. However, splenic B cells isolated from RhoH−/− mice did not show any significant differences in their in vitro activation by anti-IgM, CD40 ligation or IL-4 stimulation, nor did they differ in their proliferative response to lipopolysaccharide. In vitro migration of RhoH-deficient B cells in response to CXCL12 or CXCL13 was similar to that of wild-type B cells. Given the important role of RhoH in signal transduction downstream the T cell receptor, we investigated the possible role of RhoH in B cell receptor signaling. Although total splenic B cells from RhoH−/− mice showed markedly increased phosphorylation of SYK and ERK after anti-IgM stimulation compared to wild-type B cells, sorted populations of splenic B-2 and marginal zone B cells from RhoH−/− and wild-type animals did not differ in the activation of these kinases, suggesting that the observed difference can be attributed to the different cellular composition of the B cell compartment (i.e. B-2 vs marginal zone B cells) in RhoH−/− mice. These data imply that the phenotype observed in RhoH−/− mice may not reflect an intrinsic defect in B cells but may be attributed to crosstalk between B cells and other hematopoietic cell populations. Composition of B cell subsets in wild-type and RhoH−/− mice (total cell number ×106, ± standard deviation, N=9) Bone marrow Spleen (*) indicates p<0.05; (**), p<0.01; (***), p<0.005 RhoH+/+ RhoH−/− RhoH+/+ RhoH−/− total B cells 7.8±1.8 11.0±2.4 (**) total B cells 31.7±10.1 25.4±8.8 pro-B 0.12±0.03 0.15±0.04 (*) transitional 8.7±1.2 8.6±2.8 pre-B 2.6±0.6 3.8±0.8 (***) B-2 11.6±4.1 7.6±2.5 (*) immature 1.5±0.4 2.1±0.5 (*) marginal 3.2±1.1 3.9±1.6 mature 1.4±0.7 1.7±0.9
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Camandola, Simonetta, and Mark P. Mattson. "Pro-apoptotic action of PAR-4 involves inhibition of NF-?B activity and suppression of BCL-2 expression." Journal of Neuroscience Research 61, no. 2 (2000): 134–39. http://dx.doi.org/10.1002/1097-4547(20000715)61:2<134::aid-jnr3>3.0.co;2-p.
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SEIDAH, Nabil G., Suzanne BENJANNET, Sangeeta PAREEK, Diane SAVARIA, Josée HAMELIN, Brigitte GOULET, Jacynthe LALIBERTÉ, Claude LAZURE, Michel CHRÉTIEN, and Richard A. MURPHY. "Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases." Biochemical Journal 314, no. 3 (March 15, 1996): 951–60. http://dx.doi.org/10.1042/bj3140951.
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In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PC2, PACE4, PC5 and the PC5 isoform PC5/6-B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C-terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Co-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro-segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans-Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment.
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Carlson, Christopher S., Michael A. Pulsipher, Bryan Langholz, Anna Sherwood, Cindy Desmarais, Harlan Robins, Michael Kalos, and Stephan A. Grupp. "Immune Profiling Suggests an IGH Signaling-Dependent Subtype of Aggressive B-ALL." Blood 120, no. 21 (November 16, 2012): 1428. http://dx.doi.org/10.1182/blood.v120.21.1428.1428.
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Abstract Abstract 1428 The majority of B-cell acute lymphocytic leukemias (ALL) in childhood are derived from precursor B cells. In order to more precisely define the stage in cellular differentiation of the B-cell that gave rise to the tumor, we applied high-throughput sequencing techniques to profile the IGH repertoire in marrow at the time of diagnosis in a set of nine B-ALL patients who ultimately received a bone marrow transplant. These pediatric patients were enrolled on the Children's Oncology Group ASCT0431 trial, a phase 3, randomized trial stem cell transplantation for high risk ALL. In eight of these patients we identified a dominant IGH VDJ clone accounting for 45%-99% of all sequences, consistent with the oncogenic transformation event occurring in a cell no longer capable of further IGH rearrangement. In the ninth patient, no single VDJ clone dominated, but we observed a highly recurrent rearrangement of a specific pair of IGH diversity-joining segments (D7–27 and J4) which also conserved a four base pair non-templated sequence at the N2 junction between D and J. We observed at least 143 rearrangements sharing this D-N2-J sequence, accounting for 22% of total sequences from the patient. These sequences recombined a variety of V-N1 sequences with the common D-N2-J sequence, consistent with a primary tumor that had initially rearranged the D-J segments but continued to express the RAG recombinases, allowing for full for V-DJ recombination. Of note, 140 out of 143 of these sequences were in frame, significantly more frequently than the one in three expected. This suggests that in this specific patient, the primary tumor bearing the common D-J rearrangement was relatively indolent, and signaling through a subsequently, successfully rearranged and expressed IGH protein was an important event contributing to to the malignancy. The rearranged IGH sequences show little evidence of somatic hypermutation, so the expansion signal being received through the IGH protein appears to be coinsistent with normal positive selection during pro-B cell development, rather than an antigen-specific signal. This is consistent with the signal delivered by the pre-B receptor at the pro to pre-B stage of B cell development. We are investigating additional samples to assess the frequency of this subtype of B-ALL (a single clonal rearrangement of D-J, followed by many subclones expressing different, in frame full V-DJ rearrangements), by sequencing patients at diagnosis, relapse pre-transplant, and relapse post-transplant. Our ongoing analysis of patients enrolled on this trial at various stages of relapse will allow us to determine the prevalence of this subtype among high-risk cases, and whether differential outcomes are observed in similar cases. Disclosures: Carlson: Adaptive Biotechnologies: Consultancy, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership. Kalos:University of Pennsylvania: Employment, Patents & Royalties.