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1
Liu, Jing, Phoebe Tzou, Russell J. Hill und Paul W. Sternberg. „Structural Requirements for the Tissue-Specific and Tissue-General Functions of the Caenorhabditis elegans Epidermal Growth Factor LIN-3“. Genetics 153, Nr. 3 (01.11.1999): 1257–69. http://dx.doi.org/10.1093/genetics/153.3.1257.
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Abstract Caenorhabditis elegans lin-3 encodes a homolog of the epidermal growth factor (EGF) family of growth factors. LIN-3 is the inductive signal for hermaphrodite vulval differentiation, and it is required for animal viability, hermaphrodite fertility, and the specification of anterior cell fates in the male B cell lineage. We describe the cloning of a lin-3 homolog from C. briggsae, sequence comparison of C. elegans lin-3 with C. briggsae lin-3, and the determination of molecular lesions in alleles of C. elegans lin-3, including three new alleles. We also analyzed the severity of phenotypes caused by the new and existing alleles of lin-3. Correlation of mutant phenotypes and their molecular lesions, as well as sequence comparison between two species, reveal that the EGF motif and the N-terminal portion of the cytoplasmic domain are important for the functions of LIN-3 in all tissues, while the C-terminal portion of the cytoplasmic domain is involved in the tissue-specific functions of lin-3. We discuss how the structure of lin-3 contributes to its functions in multiple developmental processes.
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Jiang, L. I., und P. W. Sternberg. „Interactions of EGF, Wnt and HOM-C genes specify the P12 neuroectoblast fate in C. elegans“. Development 125, Nr. 12 (15.06.1998): 2337–47. http://dx.doi.org/10.1242/dev.125.12.2337.
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We investigate how temporal and spatial interactions between multiple intercellular and intracellular factors specify the fate of a single cell in Caenorhabditis elegans. P12, which is a ventral cord neuroectoblast, divides postembryonically to generate neurons and a unique epidermal cell. Three classes of proteins are involved in the specification of P12 fate: the LIN-3/LET-23 epidermal growth factor signaling pathway, a Wnt protein LIN-44 and its candidate receptor LIN-17, and a homeotic gene product EGL-5. We show that LIN-3 is an inductive signal sufficient to promote the P12 fate, and the conserved EGF signaling pathway is utilized for P12 fate specification; egl-5 is a downstream target of the lin-3/let-23 pathway in specifying P12 fate; and LIN-44 and LIN-17 act synergistically with lin-3 in the specification of the P12 fate. The Wnt pathway may function early in development to regulate the competence of the cells to respond to the LIN-3 inductive signal.
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Keller, JR, JM Gooya und FW Ruscetti. „Direct synergistic effects of leukemia inhibitory factor on hematopoietic progenitor cell growth: comparison with other hematopoietins that use the gp130 receptor subunit“. Blood 88, Nr. 3 (01.08.1996): 863–69. http://dx.doi.org/10.1182/blood.v88.3.863.863.
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Abstract Because leukemia inhibitory factor (LIF) has little or no effect on murine hematopoietic progenitor cell growth yet enhances hematopoiesis in vivo, we sought to determine whether the effects of LIF were directly or indirectly mediated, or a combination of both. Although LIF alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) has no effect on colony formation of unfractionated bone marrow cells (BMCs), it enhances M-CSF- induced colony formation. In comparison, LIF synergizes with IL-3, GM- CSF, M-CSF, and Steel Factor (SLF) to promote the colony formation of partially purified lineage-negative (Lin-) BM progenitors without altering their differentiation. These effects were directly mediated since identical results were observed in single-cell assays. Comparing the effect of LIF with other members of this subclass of hematopoietins (IL-6, oncostatin M [OSM], and ciliary neurotrophic factor [CNTF]), we found that while LIF and IL-6 equally synergize with M-CSF and SLF to promote the colony formation of Lin- BMCs, OSM, and CNTF have no effect. In agreement with OSMs ability to directly bind gp130, preincubation of BMCs with OSM inhibits progenitor cell growth stimulated by the combination of LIF or IL-6 plus SLF. LIF can also directly enhance the growth of further purified more primitive Lin- c- kit+ progenitor cells in the presence of IL-3, GM-CSF, or SLF. Thus, LIF can directly synergize with growth factors to promote the proliferation of purified hematopoietic progenitors, suggesting that the direct effects of LIF on hematopoietic cell growth can, in part, explain the observed hematopoietic effects in vivo. This is a US government work. There are no restrictions on its use.
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Keller, JR, JM Gooya und FW Ruscetti. „Direct synergistic effects of leukemia inhibitory factor on hematopoietic progenitor cell growth: comparison with other hematopoietins that use the gp130 receptor subunit“. Blood 88, Nr. 3 (01.08.1996): 863–69. http://dx.doi.org/10.1182/blood.v88.3.863.bloodjournal883863.
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Because leukemia inhibitory factor (LIF) has little or no effect on murine hematopoietic progenitor cell growth yet enhances hematopoiesis in vivo, we sought to determine whether the effects of LIF were directly or indirectly mediated, or a combination of both. Although LIF alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) has no effect on colony formation of unfractionated bone marrow cells (BMCs), it enhances M-CSF- induced colony formation. In comparison, LIF synergizes with IL-3, GM- CSF, M-CSF, and Steel Factor (SLF) to promote the colony formation of partially purified lineage-negative (Lin-) BM progenitors without altering their differentiation. These effects were directly mediated since identical results were observed in single-cell assays. Comparing the effect of LIF with other members of this subclass of hematopoietins (IL-6, oncostatin M [OSM], and ciliary neurotrophic factor [CNTF]), we found that while LIF and IL-6 equally synergize with M-CSF and SLF to promote the colony formation of Lin- BMCs, OSM, and CNTF have no effect. In agreement with OSMs ability to directly bind gp130, preincubation of BMCs with OSM inhibits progenitor cell growth stimulated by the combination of LIF or IL-6 plus SLF. LIF can also directly enhance the growth of further purified more primitive Lin- c- kit+ progenitor cells in the presence of IL-3, GM-CSF, or SLF. Thus, LIF can directly synergize with growth factors to promote the proliferation of purified hematopoietic progenitors, suggesting that the direct effects of LIF on hematopoietic cell growth can, in part, explain the observed hematopoietic effects in vivo. This is a US government work. There are no restrictions on its use.
5
Chamberlin, H. M., und P. W. Sternberg. „The lin-3/let-23 pathway mediates inductive signalling during male spicule development in Caenorhabditis elegans“. Development 120, Nr. 10 (01.10.1994): 2713–21. http://dx.doi.org/10.1242/dev.120.10.2713.
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During Caenorhabditis elegans male spicule development, four pairs of precursor cells respond to multiple positional cues and establish a pattern of fates that correlates with relative anterior-posterior cell position. One of the extracellular cues is provided by the F and U cells, which promote anterior fates. We show that the genes in the lin-3/let-23 signalling pathway required for hermaphrodite vulval induction also mediate this F/U signal. Reduction-of-function mutations in lin-3, let-23, sem-5, let-60 or lin-45 disrupt the fate of anterior cells. Likewise, activation of the pathway with ubiquitously produced signal results in posterior cells inappropriately adopting the anterior fates even in the absence of F and U. We have further used this genetic pathway to begin to understand how multiple positional cues are integrated to specify cell fate. We demonstrate that lin-15 acts in spicule development as it does in vulval induction, as a negative regulator of let-23 receptor activity. A second extracellular cue, from Y.p, also acts antagonistically to the lin-3/let-23 pathway. However, this signal is apparently integrated into the lin-3/let-23 pathway at some step after lin-45 raf and is thus functionally distinct from lin-15. We have also investigated the role of lin-12 in forming the anterior/posterior pattern of fates. A lin-12 gain-of-function defect is masked by redundant positional information from F and U.
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Ruvkun, Gary, Bruce Wightman, Thomas Bürglin und Prema Arasu. „Dominant gain-of-function mutations that lead to misregulation of the C. elegans heterochronic gene lin-14, and the evolutionary implications of dominant mutations in pattern-formation genes“. Development 113, Supplement_1 (01.01.1991): 47–54. http://dx.doi.org/10.1242/dev.113.supplement_1.47.
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The heterochronic gene lin-14 controls the temporal sequence of developmental events in the C. elegans postembryonic cell lineage. It encodes a nuclear protein that is normally present in most somatic cells of late embryos and LI larvae but not in later larval stages or adults. Two lin-14 gain-of-function mutations cause an inappropriately high level of the lin-14 nuclear protein late in development. These mutations delete 3′ untranslated sequences from the lin-14 mRNAs and identify a negative regulatory element that controls the formation of the lin-14 protein temporal gradient. The 21 kb lin-14 gene contains 13 exons that are differentially spliced to generate two lin-14 protein products with variable N-terminal regions and a constant C-terminal region. No protein sequence similarity to any proteins in various databases was found. The temporal and cellular expression patterns of lin-14 protein accumulation is altered by mutations in the heterochronic genes lin-4 and lin-28. The lin-4 gene is required to down-regulate lin-14 protein levels during the mid-Ll stage. The lin-4 gene product could be the trans-acting factor that binds to the negative regulatory element in the lin-14 3′ untranslated region. In contrast, the lin-28 gene activity positively regulates lin-14 protein levels during early LI. Thus, these genes act antagonistically to regulate the lin-14 temporal switch. The normal down-regulation of lin-14 within 10 h of hatching is not determined by the passage of time per se, but rather is triggered when feeding induces postembryonic development. Loss of lin-28 gene activity causes precocious down-regulation of lin-14 protein levels before feeding, whereas loss of lin-4 gene activity does not affect the level of lin-14 protein before feeding. These data suggest that to trigger the lin-14 temporal switch, the lin-4 gene is up-regulated after feeding which in turn down-regulates lin-14 via its 3′ untranslated region. We speculate on the evolutionary implications of dominant mutations in pattern-formation genes.
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Sommer, R. J., A. Eizinger, K. Z. Lee, B. Jungblut, A. Bubeck und I. Schlak. „The Pristionchus HOX gene Ppa-lin-39 inhibits programmed cell death to specify the vulva equivalence group and is not required during vulval induction“. Development 125, Nr. 19 (01.10.1998): 3865–73. http://dx.doi.org/10.1242/dev.125.19.3865.
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In the two nematode species Caenorhabditis elegans and Pristionchus pacificus the vulva equivalence group in the central body region is specified by the Hox gene lin-39. C. elegans lin-39 mutants are vulvaless and the vulval precursor cells fuse with the surrounding hypodermis, whereas in P. pacificus lin-39 mutants the vulval precursor cells die by apoptosis. Mechanistically, LIN-39 might inhibit non-vulval fate (cell fusion in C. elegans, apoptosis in P. pacificus), promote vulval fate or do both. To study the mechanism of lin-39 function, we isolated P. pacificus cell death mutants and identified mutations in ced-3. Surprisingly, P. pacificus ced-3; lin-39 double mutants form a functional vulva in the absence of LIN-39 activity. Thus, in P. pacificus lin-39 specifies the vulva equivalence group by inhibiting programmed cell death. Furthermore, these data reveal an important difference in a later function of lin-39 between the two species. In C. elegans, LIN-39 specifies vulval cell fates in response to inductive RAS signaling, and in P. pacificus LIN-39 is not required for vulval induction. Thus, the comparative analysis indicates that lin-39 has distinct functions in both species although the gene is acting in a homologous developmental system.
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Saito, Morihiro, Shinya Yamada, Taro Ishikawa, Hiromi Otsuka, Kimihiko Ito und Yoshimi Kubo. „Factors influencing fast ion transport in glyme-based electrolytes for rechargeable lithium–air batteries“. RSC Adv. 7, Nr. 77 (2017): 49031–40. http://dx.doi.org/10.1039/c7ra07501d.
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To elucidate the factors affecting Li-ion transport in glyme-based electrolytes, six kinds of 1.0 M tetraglyme (G4) electrolytes were prepared containing a Li salt (LiSO3CF3, LiN(SO2CF3)2, or LiN(SO2F)2) or different concentrations (0.5, 2.0, or 2.7 M) of LiN(SO2CF3)2.
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Fujisaki, Tomoaki, Marc G. Berger, Stefan Rose-John und Connie J. Eaves. „Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro“. Blood 94, Nr. 6 (15.09.1999): 1926–32. http://dx.doi.org/10.1182/blood.v94.6.1926.
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Abstract Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.
10
Fujisaki, Tomoaki, Marc G. Berger, Stefan Rose-John und Connie J. Eaves. „Rapid Differentiation of a Rare Subset of Adult Human Lin−CD34−CD38− Cells Stimulated by Multiple Growth Factors In Vitro“. Blood 94, Nr. 6 (15.09.1999): 1926–32. http://dx.doi.org/10.1182/blood.v94.6.1926.418k14_1926_1932.
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Recently, several reports of lineage-negative (lin−) CD34− cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin−CD34−CD38− cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)–mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin−CD34−CD38− cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin−CD34−CD38− cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper–IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 ± 7 CFC per lin−CD34−CD38− pre-CFC). In contrast to lin−CD34+CD38−cells, this response of lin−CD34−CD38− cells required exposure to all of the 5 growth factors used. Up to 1.7 × 105 lin−CD34− adult marrow cells failed to engraft sublethally irradiated NOD/SCID-β2M−/− mice. These studies demonstrate unique properties of a rare subset of lin−CD34−CD38− cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.
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Bernstein, ID, RG Andrews und KM Zsebo. „Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor“. Blood 77, Nr. 11 (01.06.1991): 2316–21. http://dx.doi.org/10.1182/blood.v77.11.2316.2316.
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Abstract We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
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Bernstein, ID, RG Andrews und KM Zsebo. „Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor“. Blood 77, Nr. 11 (01.06.1991): 2316–21. http://dx.doi.org/10.1182/blood.v77.11.2316.bloodjournal77112316.
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We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
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Hess, David A., Phillip E. Herrbrich, Louisa Wirthlin, Timothy P. Craft und Jan A. Nolta. „Isolation of Human CD34- Cells with High Aldehyde Dehydrogenase Activity Reveals a Novel Population with Hematopoietic Repopulating Potential.“ Blood 104, Nr. 11 (16.11.2004): 3214. http://dx.doi.org/10.1182/blood.v104.11.3214.3214.
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Abstract The successful development of stem cell-based therapies requires a thorough understanding of human hematopoietic stem cell (HSC) populations. Human CD34− cells engraft NOD/SCID mice with low efficiency by intravenous (IV) transplant. However, intra-femoral injection into immune deficient mice has identified potent human repopulating cells from CD34+ and CD34− subfractions. We recently described a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin−) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. Hematopoietic progenitor function and in vivo reconstituting ability were exclusively maintained within the ALDHhiLin− population, which demonstrated variable expression of CD34. Here, we compared the repopulating ability of purified CD34+ALDHhiLin− and CD34−ALDHhiLin− populations to traditionally isolated CD34+Lin− and CD34−Lin− cells. Sorting of Lin− cells from human UCB isolated CD34−ALDHhi and CD34+ALDHhi cells (>96% purity) at an overall frequency of 4.4±1.3% or 29.1±3.5%, respectively. In contrast to CD34−Lin− cells, ALDHhiCD34−Lin− cells demonstrated robust clonogenic progenitor function in vitro (1 CFU in 9 cells, n=3), and total colony production was further increased in ALDHhiCD34+Lin− cells (1 CFU in 4.5 cells, n=4) (p<0.05). Human hematopoietic repopulation was consistently observed in the bone marrow (17.2±4.2%), spleen (0.8±0.2%), and peripheral blood (0.7±0.3%) of NOD/SCID β2M null mice 6–8 weeks after IV transplant with 103–104 purified ALDHhiCD34+Lin− cells (n=14). Similarly, intra-femoral injection (IF) of ALDHhiCD34+Lin− cells resulted in robust human repopulation (n=5). IV injection of equivalent doses of either ALDHhiCD34+Lin− or CD34+Lin− cells showed similar levels and frequencies of human hematopoietic engraftment. Repopulating ALDHhiCD34+Lin− cells also differentiated into cells expressing markers for mature myeloid (CD33, CD14), B-lymphoid (CD19, CD20) cells and primitive repopulating cells (CD34+CD38−) at similar frequencies as CD34+Lin− cells (n=5). IV injection of 2x104–1x105 ALDHhiCD34−Lin− cells engrafted at 0.2–0.3% in the BM of 3 of 4 NOD/SCID β2M null mice, whereas IV injection of up to 4x105 CD34−Lin− cells produced no detectable human engraftment (n=6). IF-injected ALDHhiCD34−Lin− cells engrafted the injected bone in 2 of 3 NOD/SCID mice at low levels and did not efficiently migrate to the non-injected femur or tibiae. In summary, the human UCB ALDHhiLin− population includes both CD34+ and CD34− cells capable of bone marrow homing and hematopoietic reconstitution. Therefore, isolation of CD34− cells based on high ALDH activity may reveal a novel population of hematopoietic stem and progenitor cells.
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Huang, L. S., P. Tzou und P. W. Sternberg. „The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development.“ Molecular Biology of the Cell 5, Nr. 4 (April 1994): 395–411. http://dx.doi.org/10.1091/mbc.5.4.395.
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During Caenorhabditis elegans vulval development, an inductive signal from the anchor cell stimulates three of the six vulval precursor cells (VPCs) to adopt vulval rather than nonvulval epidermal fates. Genes necessary for this induction include the lin-3 growth factor, the let-23 receptor tyrosine kinase, and let-60 ras. lin-15 is a negative regulator of this inductive pathway. In lin-15 mutant animals, all six VPCs adopt vulval fates, even in the absence of inductive signal. Previous genetic studies suggested that lin-15 is a complex locus with two independently mutable activities, A and B. We have cloned the lin-15 locus by germline transformation and find that it encodes two nonoverlapping transcripts that are transcribed in the same direction. The downstream transcript encodes the lin-15A function; the upstream transcript encodes the lin-15B function. The predicted lin-15A and lin-15B proteins are novel and hydrophilic. We have identified a molecular null allele of lin-15 and have used it to analyze the role of lin-15 in the signaling pathway. We find that lin-15 acts upstream of let-23 and in parallel to the inductive signal.
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Li, Lei, Haowen Liu, Kang-Ying Qian, Stephen Nurrish, Xian-Ting Zeng, Wan-Xin Zeng, Jiafan Wang, Joshua M. Kaplan, Xia-Jing Tong und Zhitao Hu. „CASK and FARP localize two classes of post-synaptic ACh receptors thereby promoting cholinergic transmission“. PLOS Genetics 18, Nr. 10 (24.10.2022): e1010211. http://dx.doi.org/10.1371/journal.pgen.1010211.
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Changes in neurotransmitter receptor abundance at post-synaptic elements play a pivotal role in regulating synaptic strength. For this reason, there is significant interest in identifying and characterizing the scaffolds required for receptor localization at different synapses. Here we analyze the role of two C. elegans post-synaptic scaffolding proteins (LIN-2/CASK and FRM-3/FARP) at cholinergic neuromuscular junctions. Constitutive knockouts or muscle specific inactivation of lin-2 and frm-3 dramatically reduced spontaneous and evoked post-synaptic currents. These synaptic defects resulted from the decreased abundance of two classes of post-synaptic ionotropic acetylcholine receptors (ACR-16/CHRNA7 and levamisole-activated AChRs). LIN-2’s AChR scaffolding function is mediated by its SH3 and PDZ domains, which interact with AChRs and FRM-3/FARP, respectively. Thus, our findings show that post-synaptic LIN-2/FRM-3 complexes promote cholinergic synaptic transmission by recruiting AChRs to post-synaptic elements.
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Oner, Z., E. Altınoz, H. Elbe und N. Ekinci. „The protective and therapeutic effects of linalool against doxorubicin-induced cardiotoxicity in Wistar albino rats“. Human & Experimental Toxicology 38, Nr. 7 (12.04.2019): 803–13. http://dx.doi.org/10.1177/0960327119842634.
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The aim of the present study was to determine the protective and therapeutic effects of linalool (LIN) against doxorubicin (DOX)-induced cardiotoxicity in rats histologically and biochemically. In experiments, 64 male Wistar albino rats were randomly divided into eight groups ( n = 8). These groups were control (C) (0.9% saline solution), DOX (20 mg/kg DOX), LIN50 (50 mg/kg LIN), LIN100 (100 mg/kg LIN), DOX + LIN50 (20 mg/kg DOX and 50 mg/kg LIN), DOX + LIN100 (20 mg/kg DOX and 100 mg/kg LIN), LIN50 + DOX (50 mg/kg LIN and 20 mg/kg DOX), and LIN100 + DOX (100 mg/kg LIN and 20 mg/kg DOX). It was determined that necrosis and extensive inflammatory cell infiltration were observed in the DOX group. It was determined that histopathological changes significantly decreased in groups treated with LIN after DOX administration. While the caspase-3 immunostaining was highly evident in DOX group apoptotic cells ( p < 0.001, for all), the intensity of caspase-3 immunostaining in the treatment groups decreased ( p < 0.05). While DOX administration resulted in a significant increase in malondialdehyde (MDA) levels and plasma Creatine kinase (CK) and lactate dehydrogenase (LDH) levels in cardiac tissue when compared to the C groups, it was observed that DOX + LIN administration led to a significant decrease in MDA, plasma CK and LDH levels and a significant increase in glutathione (GSH), superoxide dismutase, and catalase enzyme levels. Finally, it was concluded that DOX led to heavy cardiotoxicity and DOX + LIN administration could remove cardiomyopathy symptoms.
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Mori, Takehiko, Kiyoshi Ando, Kazuo Tanaka, Yasuo Ikeda und Yasuhiro Koga. „Fas-Mediated Apoptosis of the Hematopoietic Progenitor Cells in Mice Infected With Murine Cytomegalovirus“. Blood 89, Nr. 10 (15.05.1997): 3565–73. http://dx.doi.org/10.1182/blood.v89.10.3565.
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Abstract The effects of cytomegalovirus (CMV) infection on hematopoietic progenitor cells in vivo were investigated to elucidate the pathogenesis of CMV-induced myelosuppression. BALB/c mice were inoculated with 0.2LD50 of murine CMV (MCMV). Lineage marker negative, c-kit positive (Lin−c-kit+) and Lin−CD34+ cells, which are both phenotypically defined as hematopoietic progenitor cells, showed a significant reduction in number on day 3 postinfection (pi). Moreover, the reduction in the number of day-14 colony-forming units-spleen (CFU-S), another indicator to identify hematopoietic progenitor cells, was noted on day 3 pi. To clarify the mechanism of such depletion, we examined the cells undergoing apoptosis in the Lin− populations and found a 15-fold increase in the apoptosis-induction of these cells. Furthermore, an increase in the expression level of Fas, which mediates apoptosis, was observed in such Lin−c-kit+ and Lin−Sca-1+ cells on day 3 pi. In vitro treatment with the anti-Fas antibody accelerated the apoptosis in Lin− cells, but not in the uninfected control cells, thus indicating that the upregulated Fas on Lin− cells is directly related to the acceleration of apoptosis found in these cells in vivo. These results suggest that MCMV infection reduces the number of hematopoietic progenitor cells in bone marrow at least in part due to Fas-mediated apoptosis, and this phenomenon is thus considered to contribute to CMV-induced myelosuppression.
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Mori, Takehiko, Kiyoshi Ando, Kazuo Tanaka, Yasuo Ikeda und Yasuhiro Koga. „Fas-Mediated Apoptosis of the Hematopoietic Progenitor Cells in Mice Infected With Murine Cytomegalovirus“. Blood 89, Nr. 10 (15.05.1997): 3565–73. http://dx.doi.org/10.1182/blood.v89.10.3565.3565_3565_3573.
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The effects of cytomegalovirus (CMV) infection on hematopoietic progenitor cells in vivo were investigated to elucidate the pathogenesis of CMV-induced myelosuppression. BALB/c mice were inoculated with 0.2LD50 of murine CMV (MCMV). Lineage marker negative, c-kit positive (Lin−c-kit+) and Lin−CD34+ cells, which are both phenotypically defined as hematopoietic progenitor cells, showed a significant reduction in number on day 3 postinfection (pi). Moreover, the reduction in the number of day-14 colony-forming units-spleen (CFU-S), another indicator to identify hematopoietic progenitor cells, was noted on day 3 pi. To clarify the mechanism of such depletion, we examined the cells undergoing apoptosis in the Lin− populations and found a 15-fold increase in the apoptosis-induction of these cells. Furthermore, an increase in the expression level of Fas, which mediates apoptosis, was observed in such Lin−c-kit+ and Lin−Sca-1+ cells on day 3 pi. In vitro treatment with the anti-Fas antibody accelerated the apoptosis in Lin− cells, but not in the uninfected control cells, thus indicating that the upregulated Fas on Lin− cells is directly related to the acceleration of apoptosis found in these cells in vivo. These results suggest that MCMV infection reduces the number of hematopoietic progenitor cells in bone marrow at least in part due to Fas-mediated apoptosis, and this phenomenon is thus considered to contribute to CMV-induced myelosuppression.
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Hsu, Virginia, Cheri L. Zobel, Eric J. Lambie, Tim Schedl und Kerry Kornfeld. „Caenorhabditis elegans lin-45 raf Is Essential for Larval Viability, Fertility and the Induction of Vulval Cell Fates“. Genetics 160, Nr. 2 (01.02.2002): 481–92. http://dx.doi.org/10.1093/genetics/160.2.481.
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Abstract The protein kinase Raf is an important signaling protein. Raf activation is initiated by an interaction with GTP-bound Ras, and Raf functions in signal transmission by phosphorylating and activating a mitogen-activated protein (MAP) kinase kinase named MEK. We identified 13 mutations in the Caenorhabditis elegans lin-45 raf gene by screening for hermaphrodites with abnormal vulval formation or germline function. Weak, intermediate, and strong loss-of-function or null mutations were isolated. The phenotype caused by the most severe mutations demonstrates that lin-45 is essential for larval viability, fertility, and the induction of vulval cell fates. The lin-45(null) phenotype is similar to the mek-2(null) and mpk-1(null) phenotypes, indicating that LIN-45, MEK-2, and MPK-1 ERK MAP kinase function in a predominantly linear signaling pathway. The lin-45 alleles include three missense mutations that affect the Ras-binding domain, three missense mutations that affect the protein kinase domain, two missense mutations that affect the C-terminal 14-3-3 binding domain, three nonsense mutations, and one small deletion. The analysis of the missense mutations indicates that Ras binding, 14-3-3-binding, and protein kinase activity are necessary for full Raf function and suggests that a 14-3-3 protein positively regulates Raf-mediated signaling during C. elegans development.
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Wu, Ligang, und Joel G. Belasco. „Micro-RNA Regulation of the Mammalian lin-28 Gene during Neuronal Differentiation of Embryonal Carcinoma Cells“. Molecular and Cellular Biology 25, Nr. 21 (01.11.2005): 9198–208. http://dx.doi.org/10.1128/mcb.25.21.9198-9208.2005.
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ABSTRACT Vertebrate genomes each encode hundreds of micro-RNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3′ untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3′ UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3′ UTR that respond to miR-125. At the 3′ end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5′-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation.
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BRUNSCHWIG, P., C. HURTAUD, Y. CHILLIARD und F. GLASSER. „L’apport de lin dans la ration des vaches laitières : Effets sur la production, la composition du lait et des produits laitiers, les émissions de méthane et les performances de reproduction“. INRAE Productions Animales 23, Nr. 4 (14.11.2010): 307–18. http://dx.doi.org/10.20870/productions-animales.2010.23.4.3310.
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La supplémentation en lin des rations des vaches laitières est une pratique qui se développe, avec pour objectifs l’amélioration de la qualité nutritionnelle du lait et la diminution des rejets de méthane. En effet, la recherche de laits moins riches en Acides Gras (AG) saturés et avec un rapport plus faible entre acide linoléique et acide linolénique, incite à utiliser des aliments riches en acides gras polyinsaturés (et en particulier en acide alpha-linolénique C18:3 n-3) pour corriger des rations insuffisamment riches en cet AGPI. Parmi les aliments des vaches laitières, le lin est un aliment particulièrement riche en C18:3 n-3. La diminution des rejets de gaz à effet de serre (dont le méthane) est également une préoccupation actuelle des filières animales. De nombreux essais de supplémentation en lin, sous différentes formes, ont été publiés ces dernières années, et les données disponibles permettent de tirer des conclusions sur ces effets attendus. Le présent article fait le point sur les disponibilités en lin et sur les différentes formes d’apport dans les rations. Les effets du lin sur la production laitière, sur la composition du lait et des produits laitiers, la production de méthane et la reproduction sont passés en revue. L’analyse des effets sur le lait s’appuie sur 41 essais zootechniques publiés. La culture de lin oléagineux est peu importante en France. L’approvisionnement est fait dans des pays européens et au Canada. Les variétés présentent des teneurs variables en acide alpha-linolénique. L’introduction de lin dans la ration diminue un peu la quantité de MS ingérée mais ne modifie en général pas la production laitière (volumes et taux). La teneur du lait en AG saturés diminue et le pourcentage en C18:1-trans est augmenté, et ce d’autant plus que l’apport de lipides se fait sous forme non protégée (graines extrudées, huile) et avec des rations riches en amidon (pour les AG trans). La teneur en C18:2 n-6 n’est en moyenne pas modifiée, sauf par l’apport d’huile. La proportion en C18:3 n-3 du lait est multipliée en moyenne par 2 ou 3 pour les formes pratiques les plus efficaces (graines aplaties, farine), et peut atteindre jusqu’à 1,4% des AG du lait avec ce type de supplémentations. Il n’apparaît pas d’effet dose de lipides apportée pour le C18:2 n-6 et le C18:3 n-3, alors qu’il en existe un pour les C18:1-trans. Le beurre et les fromages ont la même composition en AG que le lait dont ils proviennent. Les qualités organoleptiques de beurres et fromages ne sont pas modifiées par l’addition de lin dans la ration. Différents effets sont cités dans la bibliographie pour expliquer une augmentation potentielle de la fertilité, qui reste à confirmer. La production ruminale de méthane est diminuée par l’ajout de lin dans la ration. En conclusion, l’ajout de lin à la ration des vaches laitières a des effets analogues à ceux d’introduction d’herbe dans le régime fourrager, à l’exception d’une teneur en AG trans supérieure.
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Cainap, Calin, Shukui Qin, Wen-Tsung Huang, Ik-Joo Chung, Hongming Pan, Ying Cheng, Masatoshi Kudo et al. „Phase III trial of linifanib versus sorafenib in patients with advanced hepatocellular carcinoma (HCC).“ Journal of Clinical Oncology 31, Nr. 4_suppl (01.02.2013): 249. http://dx.doi.org/10.1200/jco.2013.31.4_suppl.249.
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249 Background: Linifanib (ABT-869; Lin) is a potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinase families. In a phase II trial in patients (pts) with advanced HCC, Lin showed clinical activity (objective response rate [ORR] 10.5% in Child-Pugh A [CPA] pts). This open-label, global phase 3 trial evaluated Lin versus sorafenib (Sor) as first-line therapy in pts with advanced CPA HCC (NCT01009593). Methods: Pts were randomized 1:1 to Lin 17.5 mg QD or Sor 400 mg BID and stratified by region (non-Asia/Japan/rest of Asia), ECOG performance status (0/1), vascular invasion or extrahepatic spread (yes/no) and HBV infection (yes/no). The primary efficacy endpoint was overall survival (OS); both non-inferiority (margin 1.0491) and superiority hypotheses were to be tested. Secondary efficacy endpoints included time to progression (TTP) and ORR, using RECIST v1.1. AE severity was graded using NCI-CTCAE v4.0. Results: 1035 pts (median age 60 y, 68% Asian, 65% ECOG 0, 49% HBV, 70% vascular invasion or extrahepatic spread) were randomized at 149 sites in 26 countries. Hazard ratio (HR) for OS was 1.046 (95% CI: 0.896, 1.221). Median OS (95% CI) was 9.1 months (m) (8.1, 10.2) on Lin and 9.8 m (8.3, 11.0) on Sor. For all pre-specifed subgroup analyses, OS HRs ranged from 0.793-1.119, and the 95% CI contained 1.0. TTP HR was 0.759 (95% CI: 0.643, 0.895; p=0.001) favoring Lin. Median TTP (95% CI) was 5.4 m (4.2, 5.6) on Lin and 4.0 m (2.8. 4.2) on Sor. ORR was 13.0% on Lin and 6.9% on Sor. Grade 3/4 AEs, serious AEs and AEs leading to discontinuations, dose interruptions and reductions were more frequent on Lin versus Sor (all p<0.001). Conclusions: Lin and Sor resulted in similar OS in advanced HCC. Predefined superiority and non-inferiority OS boundaries were not met for Lin. Secondary endpoints (TTP and ORR) favored Lin while safety results favored Sor. Clinical trial information: NCT01009593.
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Poureslami, R., K. Raes, G. M. Turchini, G. Huyghebaert und S. De Smet. „Effect of diet, sex and age on fatty acid metabolism in broiler chickens:n-3 andn-6 PUFA“. British Journal of Nutrition 104, Nr. 2 (01.03.2010): 189–97. http://dx.doi.org/10.1017/s0007114510000395.
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The PUFA metabolism in broiler chicken was studied through the whole body fatty acid balance method. Four dietary lipid sources (palm fat, Palm; soyabean oil, Soya; linseed oil, Lin; fish oil, Fish) were added at 3 % to a basal diet containing 5 % palm fat. Diets were fed to female and male birds from day 1 to either day 21 or day 42 of age. Birds fed the Lin diet showed a significantly higher 18 : 2n-6 accumulation compared with the other diets (85·2v.73·6 % of net intake), whereas diet did not affect 18 : 3n-3 accumulation (mean 63 % of net intake). Bioconversion of 18 : 2n-6 significantly decreased in the order Palm>Lin>Soya>Fish (4·7, 3·9, 3·4 and 1 % of net intake, respectively). The 18 : 3n-3 bioconversion on the Palm and Soya diets was similar and significantly higher than in broilers on the Lin diet (9·1v.5·8 % of net intake). The β-oxidation of 18 : 2n-6 was significantly lower on the Lin diet than on the other diets (10·8v.23·3 % of net intake), whereas β-oxidation of 18 : 3n-3 was significantly higher on the Fish diet than on the other diets (41·5v.27·3 % of net intake). Feeding fish oil suppressed apparent elongase and desaturase activity, whereas a higher dietary supply of 18 : 3n-3 and 18 : 2n-6 enhanced apparent elongation and desaturation activity on the PUFA involved in then-3 andn-6 pathway, respectively. Accumulation of 18 : 2n-6 and 18 : 3n-3 increased and β-oxidation decreased with age. Sex had a marginal effect on the PUFA metabolism.
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de la Cova, Claire C., Robert Townley und Iva Greenwald. „Negative feedback by conserved kinases patterns the degradation of Caenorhabditiselegans Raf in vulval fate patterning“. Development 147, Nr. 24 (03.11.2020): dev195941. http://dx.doi.org/10.1242/dev.195941.
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ABSTRACTActivation of a canonical EGFR-Ras-Raf-ERK cascade initiates patterning of multipotent vulval precursor cells (VPCs) of Caenorhabditis elegans. We have previously shown that this pathway includes a negative-feedback component in which MPK-1/ERK activity targets the upstream kinase LIN-45/Raf for degradation by the SEL-10/FBXW7 E3 ubiquitin ligase. This regulation requires a Cdc4 phosphodegron (CPD) in LIN-45 that is conserved in BRAF. Here, we identify and characterize the minimal degron that encompasses the CPD and is sufficient for SEL-10-mediated, MPK-1-dependent protein degradation. A targeted screen of conserved protein kinase-encoding genes yielded gsk-3 (an ortholog of human GSK3B) and cdk-2 (a CDK2-related kinase) as required for LIN-45 degron-mediated turnover. Genetic analysis revealed that LIN-45 degradation is blocked at the second larval stage due to cell cycle quiescence, and that relief of this block during the third larval stage relies on activation of CDKs. Additionally, activation of MPK-1 provides spatial pattern to LIN-45 degradation but does not bypass the requirement for gsk-3 and cdk-2. This analysis supports a model whereby MPK-1/ERK, GSK-3/GSK3 and CDK-2/CDK2, along with SEL-10/FBXW7, constitute a regulatory network that exerts spatial and temporal control of LIN-45/Raf degradation during VPC patterning.
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Shibuya, A., K. Nagayoshi, K. Nakamura und H. Nakauchi. „Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells“. Blood 85, Nr. 12 (15.06.1995): 3538–46. http://dx.doi.org/10.1182/blood.v85.12.3538.bloodjournal85123538.
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We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)-cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.
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Rowley, SD, C. Brashem-Stein, R. Andrews und ID Bernstein. „Hematopoietic precursors resistant to treatment with 4- hydroperoxycyclophosphamide: requirement for an interaction with marrow stroma in addition to hematopoietic growth factors for maximal generation of colony-forming activity“. Blood 82, Nr. 1 (01.07.1993): 60–65. http://dx.doi.org/10.1182/blood.v82.1.60.bloodjournal82160.
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We tested the ability of CD34+lin- precursor cells isolated from marrow after treatment with 4-hydroperoxycyclophosphamide (4HC) to generate colony-forming cells (CFC). In liquid cultures, recombinant human stem cell factor (SCF), in combination with interleukin-1 (IL-1), IL-3, IL- 6, granulocyte-macrophage colony-stimulating factor, or granulocyte colony-stimulating factor caused untreated, but not 4HC-treated, CD34+lin- cells to form CFC. However, generation of CFC from CD34+lin- cells treated with 60 micrograms/mL of 4HC was possible in the presence of an irradiated allogeneic stromal cell layer. This generation was increased when combinations of hematopoietic growth factors including SCF and IL-3 were added. Maximal generation of CFC was seen after 11 to 21 days of culture. At that time, generation of CFC from CD34+lin- 4HC- treated cells equalled that from untreated cells. The phenotype of these 4HC-resistant CD34+lin- precursors was also further defined as CD38-. These studies show that the generation of CFC from the 4HC- resistant, highly immature population of CD34+lin- cells requires an as yet undefined interaction with marrow stroma in addition to known hematopoietic growth factors.
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Jiang, Xiaoyan, Yun Zhao, Wing Yiu Chan, Emily Pang, Allen Eaves und Connie Eaves. „Leukemic Stem Cells of Chronic Phase CML Patients Consistently Display Very High BCR-ABL Transcript Levels and Reduced Responsiveness to Imatinib Mesylate in Addition to Generating a Rare Subset That Produce Imatinib Mesylate-Resistant Differentiated Progeny.“ Blood 104, Nr. 11 (16.11.2004): 711. http://dx.doi.org/10.1182/blood.v104.11.711.711.
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Abstract Imatinib mesylate (IM) is an inhibitor of the BCR-ABL oncoprotein associated with human chronic myeloid leukemia (CML). IM therapy has shown remarkable effects in initial clinical trials, but both clinical and laboratory studies increasingly suggest that, on its own, IM may have limited curative potential, due to a reduced IM sensitivity of the more primitive, slowly proliferating CD34+ CML cells thought to be responsible for sustaining the disease in vivo. To investigate the basis of this unresponsiveness, we compared the IM sensitivity and BCR-ABL expression of FACS-purified subsets of lin−CD34+ cells from 4 CML chronic phase patients. None of these had been treated with IM and their cells at all stages of differentiation were exclusively leukemic; i.e., >95% of the lin−CD34+CD38−, lin−CD34+CD38+ and lin+CD34− cells were BCR-ABL+ (by direct FISH) and all longterm culture-initiating cell (LTC-IC) -derived CFCs were Ph+. In the absence of IM, suspension cultures initiated with these lin−CD34+CD38− CML cells (0.5–5% of the lin−CD34+ cells) showed a net expansion of viable cells after 3 weeks; 100x with and 10x without added growth factors (GFs). Addition of 0.1–10 μM/ml IM reduced the yield of viable cells in a dose-dependent fashion, particularly when GFs were not added (100-fold decrease with 10 μM/ml IM). Parallel cultures of the corresponding lin−CD34+CD38+ CML cells showed these did not expanded as much (~8x +GFs, 2x -GFs) and were more sensitive to IM (1000-fold decrease after 3 weeks in 10 μM/ml IM -GFs). Quantitative real-time RT-PCR analysis revealed BCR-ABL transcripts to be present in the most primitive, freshly isolated lin−CD34+CD38− cells (n=12) at >300-fold higher levels than in the terminally differentiating lin+CD34− CML cells (n=21), at >10-fold higher levels than the normal BCR transcripts in the same lin−CD34+CD38− cells, and at 40-fold higher levels than in the less primitive lin−CD34+CD38+ cells (n=12), indicating a correlation between decreasing BCR-ABL transcripts and increasing IM sensitivity during CML stem cell differentiation in vivo. Interestingly, maintenance of the lin−CD34+CD38− CML cells for 3 weeks in vitro with 10 μM/ml IM (±GFs) consistently selected for a subset of leukemic cells (80–100% BCR-ABL+ by FISH) that showed complete resistance to 5 μM/ml IM in CFC assays, in marked contrast to the CFCs in the starting lin−CD34+CD38− cells that were inhibited 5–10-fold by 5 μM/ml IM. Moreover, although the Ph was the sole abnormality present in all direct metaphases, initial CFCs and LTC-IC-derived CFCs from all samples, a 17p+ abnormality was seen in 4/4 metaphases obtained from one colony generated from the cells present in one of the 3-week IM-containing cultures, suggesting the selective survival of differentiating progeny of rare, pre-existing, IM-resistant stem cells. Consistent with this possibility was the finding that BCR-ABL transcript levels in the cells present in the 3 week cultures were reduced 50-fold relative to the input lin−CD34+CD38− cells. Taken together, these findings suggest a previously undescribed epigenetic mechanism of IM unresponsiveness characteristic of chronic phase CML stem cells, in addition to the silent accumulation of genetically-determined IM-resistant members as the CML stem cell population expands during the development of the chronic phase of the disease.
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Muench, MO, MG Roncarolo, S. Menon, Y. Xu, R. Kastelein, S. Zurawski, CH Hannum, J. Culpepper, F. Lee und R. Namikawa. „FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver“. Blood 85, Nr. 4 (15.02.1995): 963–72. http://dx.doi.org/10.1182/blood.v85.4.963.bloodjournal854963.
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The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL- cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.
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Hoskins, R., A. F. Hajnal, S. A. Harp und S. K. Kim. „The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins“. Development 122, Nr. 1 (01.01.1996): 97–111. http://dx.doi.org/10.1242/dev.122.1.97.
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The lin-2 gene is required for the induction of the Caenorhabditis elegans vulva. Vulval development is initiated by a signal from the anchor cell that is transduced by a receptor tyrosine kinase/Ras pathway. We show that lin-2 acts in the vulval precursor cell P6.p, downstream of lin-3 EGF and upstream of let-60 ras, to allow expression of the 1 degrees cell fate. lin-2 encodes a protein of relative molecular mass 109,000 (LIN-2A) with regions of similarity to CaM kinase II and membrane-associated guanylate kinases. Mutant lin-2 transgenes designed to lack either protein kinase or guanylate kinase activity are functional, indicating that LIN-2A has a structural rather than an enzymatic role in vulval induction. Most or all identified membrane-associated guanylate kinases are components of cell junctions, including vertebrate tight junctions and arthropod septate junctions in epithelia. Thus, LIN-2A may be a component of the cell junctions of the epithelial vulval precursor cells that is required for signaling by the receptor tyrosine kinase LET-23. We propose that LIN-2A is required for the localization of one or more signal transduction proteins (such as LET-23) to either the basal membrane domain or the cell junctions, and that mislocalization of signal transduction proteins in lin-2 mutants interferes with vulval induction.
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Keller, JR, SH Bartelmez, E. Sitnicka, FW Ruscetti, M. Ortiz, JM Gooya und SE Jacobsen. „Distinct and overlapping direct effects of macrophage inflammatory protein-1 alpha and transforming growth factor beta on hematopoietic progenitor/stem cell growth“. Blood 84, Nr. 7 (01.10.1994): 2175–81. http://dx.doi.org/10.1182/blood.v84.7.2175.2175.
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Abstract Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL- 3)/and granulocyte-macrophage colony-stimulating factor (GM- CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G- CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM- CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Hoechst 33342/Rhodamine 123 [Ho/Rh123] and Lin- Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP- 1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP- CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.
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Keller, JR, SH Bartelmez, E. Sitnicka, FW Ruscetti, M. Ortiz, JM Gooya und SE Jacobsen. „Distinct and overlapping direct effects of macrophage inflammatory protein-1 alpha and transforming growth factor beta on hematopoietic progenitor/stem cell growth“. Blood 84, Nr. 7 (01.10.1994): 2175–81. http://dx.doi.org/10.1182/blood.v84.7.2175.bloodjournal8472175.
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Both transforming growth factor beta (TGF beta) and macrophage inflammatory protein 1 alpha (MIP-1 alpha) have been shown to be multifunctional regulators of hematopoiesis that can either inhibit or enhance the growth of hematopoietic progenitor cells (HPC). We report here the spectrum of activities of these two cytokines on different hematopoietic progenitor and stem cell populations, and whether these effects are direct or indirect. MIP-1 alpha enhances interleukin-3 (IL- 3)/and granulocyte-macrophage colony-stimulating factor (GM- CSF)/induced colony formation of normal bone marrow progenitor cells (BMC) and lineage-negative (Lin-) progenitors, but has no effect on G- CSF or CSF-1/induced colony formation. Similarly, TGF beta enhances GM- CSF/induced colony formation of normal BMC and Lin- progenitors. In contrast, TGF beta inhibits IL-3/ and CSF-1/induced colony formation of Lin- progenitors. The effects of MIP-1 alpha and TGF beta on the growth of Lin- progenitors were direct and correlate with colony formation in soft agar. Separation of the Lin- cells into Thy-1 and Thy-1lo subsets showed that the growth of Thy-1lo Lin- cells is directly inhibited by MIP-1 alpha and TGF beta regardless of the cytokine used to stimulate growth (IL-3), GM-CSF, or CSF-1). In contrast, two other stem cell populations (0% to 15% Hoechst 33342/Rhodamine 123 [Ho/Rh123] and Lin- Sca-1+ cells) were markedly inhibited by TGF beta and unaffected by MIP- 1 alpha. Furthermore, MIP-1 alpha has no effect on high proliferative potential colony-forming cells 1 or 2 (HPP-CFC/1 or /2) colony formation in vitro, whereas TGF beta inhibits both HPP-CFC/1 and HPP- CFC/2. Thus, MIP-1 alpha and TGF beta are direct bidirectional regulators of HPC growth, whose effects are dependent on other growth factors present as well as the maturational state of the HPC assayed. The spectrum of their inhibitory and enhancing activities shows overlapping yet distinct effects.
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Horne, Gillian A., Chinmay Rajiv Munje, Ross Kinstrie, Eduardo Gómez-Castañeda, Helen Wheadon und Mhairi Copland. „Gene Expression Profiling of CD93-Selected CP-CML Stem Cells Confirms Their Quiescent Character and Biomarker Potential“. Blood 128, Nr. 22 (02.12.2016): 4231. http://dx.doi.org/10.1182/blood.v128.22.4231.4231.
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Abstract The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". Disease persistence suggests, that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of a small population of cells, termed leukemic stem cells (LSC). We previously identified CD93 expression as a promising biomarker of LSC in chronic phase (CP)-CML. Our group has described the long term self-renewal potential of Lin-CD34+93+ CP-CML cells compared to their Lin-CD34+93- counterparts through LTCIC assays (n=3, p<0.0001) and NSG engraftment models (3.5-30-fold increased in engraftment with Lin-CD34+93+ cells, p<0.03). We hypothesized that CD93+-selected cells would represent a more immature functional phenotype compared to CD93- selected cells. The aim of this study was to characterize differences in the gene expression profile between CD93+ and CD93- CML LSC populations and determine heterogeneity of each population at a single cell level. To interrogate this, we initially identified CP-CML subpopulations with the greatest functional capability compared to normal. Normal and CP-CML samples were FACS-sorted into HSC/LSC, CMP, GMP, and MEP sub-populations. Results suggest a significant change in functional status between normal and CP-CML subpopulations within the HSC/LSC compartment (lin-CD34+CD38-CD45RA-CD90+), where CML LSC demonstrated significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture in physiological growth factors. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively (p<0.05)). Furthermore, fluorescence in situ hybridization demonstrated that >90% of lin-CD34+CD38-CD45RA-CD90+ CML LSC from all patient samples were BCR-ABL positive. Subsequent experiments were confined to the LSC population. We hypothesized that lin-CD34+CD38-CD90+CD93- CML cells would have a more mature gene expression profile compared to lin-CD34+CD38-CD90+CD93+ cells. CP-CML cells were sorted into (1) lin-CD34+, (2) lin-CD34+CD38-CD90+CD93- and (3) lin-CD34+CD38-CD90+CD93+ populations. RNA was harvested at baseline from bulk populations (1) to (3) and cDNA was generated from single cells using the Fluidigm C1 autoprep system. Using Fluidigm technology, quantitative PCR of 90 lineage-specific and cell survival genes was performed within all populations of cells (1) to (3) in 'bulk' samples (n=3), and at single cell level (n=123 CD93+, n=120 CD93-single cells; n=3 samples in total). Bulk sample analysis demonstrated a significant increase in expression of lineage commitment genes within the lin-CD34+CD38-CD90+CD93- population, as shown by increased expression of GATA1 (p=0.0007), and CBX8 (p=0.0002). The lin-CD34+CD38-CD90+CD93+ population displayed a less lineage-restricted profile with increased expression of CDK6 (p=0.05), HOXA6 (ns), CDKN1C (ns) and CKIT (p=0.0014), compared to the lin-CD34+CD38-CD90+CD93- population. Furthermore, the two populations could be segregated by differential gene expression through gene clustering. At a single cell level, differences were noted in the frequency of expression between lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations, particularly in GATA1, TPOR, and VWF. Although a statistically significant change was demonstrated in gene expression between the lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations in a number of genes, we were not able to segregate the populations by differential expression using gene clustering. This highlights the heterogeneous nature of the cell populations and the inability to distinctly characterize between the two populations at a single cell level. Our results validate CD93 as a potential biomarker to separate the primitive CP-CML LSC population and highlight key lineage and cell survival pathways that are altered in CML LSC. The results demonstrate the heterogeneity seen within gene expression at the single cell level, which may allow for further insight into the CML LSC compartment with further analyses. Disclosures Wheadon: GlaxoSmithKline: Research Funding. Copland:Shire: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.
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Calado, Rodrigo T., Neal S. Young und Jichun Chen. „Lin−CD117+ Hematopoietic Cells Preferentially Home to Spleen and Their Migration Is Affected by Selectins.“ Blood 106, Nr. 11 (16.11.2005): 1400. http://dx.doi.org/10.1182/blood.v106.11.1400.1400.
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Abstract In hematopoietic stem and progenitor cell transplantation, infused donor cells home to recipient bone marrow (BM) where they become residents and sustain normal hematopoiesis. Understanding the molecular mechanisms that regulate hematopoietic cell homing might help to increase homing efficiency and to optimize cell engraftment in patients. When BM cells from enhanced green fluorescence protein (GFP) transgenic mice were transplanted into normal C57BL/6 recipients, GFP+Lin− cell recovery in BM peaked to 17 ± 2 %, 21 ± 8 %, and 22 ± 14 % at 18, 48 and 72 hours after cell infusion; likewise, GFP+Lin− cell recovery in the spleen peaked to 7 ± 3 %, 10 ± 0 %, and 14 ± 3 % at these time points. Conversely, GFP+Lin− cells were not detected in recipient thymus at any time. Of the recovered GFP+Lin− cells in the BM and spleen, GFP+Lin−CD117+ cells preferentially homed to the spleen (7.0 ± 0.5 % recovery in spleen vs. 0.3 ± 0.5 % recovery in BM; P<0.0001) since only 0.5 ± 1.1 % of recovered GFP+Lin− cells in the BM were CD117+, compared to a 25 ± 1.1% CD117+ recovery rate in the spleen (P<0.0001). Total body irradiation did not increase but instead reduced GFP+Lin− cell homing in both BM (P<0.002) and spleen (P<0.005). Also, the proportion of CD117+ cells among recovered GFP+Lin− cells was much lower (P<0.05) in irradiated than in unirradiated recipients. When sorted GFP+Lin−CD117+ and GFP+Lin−CD117− cells were transplanted into sublethally-irradiated recipients at 20–180 ×103 cells per recipient, no GFP+ cells reached detectable level in either the spleen or BM. When the same number of GFP+Lin−CD117+ and GFP+Lin−CD117− cells were co-transplanted with 20 ×106 normal B6 BM cells, we detected 0.15% to 0.86% cell recovery in recipient BM and spleen, indicating that total number of injected cells affects cell homing and recovery. All recovered cells carried the same GFP+Lin−CD117+ or GFP+Lin−CD117− phenotype as they were injected, indicating that there was no phenotype switch during the first 18 hours of homing. In a short-term engraftment assay, 81 ×103 GFP+Lin−CD117+ cells transplanted along with 20 ×106 normal BM cells resulted in 1.7–3.8%, 3.2–5.2% and 11–17% engraftment in recipient spleen, BM and peripheral blood respectively at 10 days after transplantation, while 96 ×103 GFP+Lin−CD117− cells transplanted along with 20 ×106 normal BM cells produced in no detectable engraftment. Similarly, in a long-term engraftment assay, donor engraftment was detected from GFP+Lin−CD117+ but not from GFP+Lin−CD117− cells when recipients were monitored at 15, 30, 60 and 90 days after transplantation. To test the role of selectin proteins in hematopoietic cell homing, we transplanted 20 ×106 GFP+ BM cells to B6 recipients that carry targeted deletions for E-, L- and P-selectins. At 18 hours, GFP+Lin- cell recovery was at 5.0 ± 0.4 % and 9.8 ± 1.8 % in the spleen and 19 ± 1.4 % and 11 ± 1.9 % in the BM for normal and selectin-deficient recipients. In conclusion, GFP+Lin− cells homed to BM and spleen but not to thymus, indicating that an appropriate microenvironment is necessary for adequate homing. Irradiation did not attract Lin− cells to the thymus but reduced Lin− cell homing to BM and spleen. While only a very small fraction of GFP+Lin−CD117+ cells homed to BM, they are likely the cells responsible for short- and long-term hematopoietic engraftment in recipients. Deletion of selectin genes may alter the microenvironment and shift Lin- cell homing to spleen, thus reducing hematopoietic cell homing to BM.
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Paczkowska, Edyta, Karolina Łuczkowska, Katarzyna Piecyk, Dorota Rogińska, Ewa Pius-Sadowska, Przemysław Ustianowski, Elżbieta Cecerska, Barbara Dołęgowska, Zbigniew Celewicz und Bogusław Machaliński. „The influence of BDNF on human umbilical cord blood stem/progenitor cells: Implications for stem cell-based therapy of neurodegenerative disorders“. Acta Neurobiologiae Experimentalis 75, Nr. 2 (30.06.2015): 172–91. http://dx.doi.org/10.55782/ane-2015-2026.
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Umbilical cord blood (UCB)-derived stem/progenitor cells (SPCs) have demonstrated the potential to improve neurologic function in different experimental models. SPCs can survive after transplantation in the neural microenvironment and indu ce neuroprotection, endogenous neurogenesis by secreting a broad repertoire of trophic and immunomodulatory cytokines. In this study, the influence of brain-derived neurotrophic factor (BDNF) pre-treatment was comprehensively evaluated in a UCB-derived lineage-negative (Lin−) SPC population. UCB-derived Lin− cells were evaluated with respect to the expression of (i) neuronal markers using immunofluorescence staining and (ii) specific (TrkB) receptors for BDNF using flow cytometry. Next, after BDNF pre-treatment, Lin− cells were extensively assessed with respect to apoptosis using Western blotting and proliferation via BrdU incorporation. Furthermore, NT-3 expression levels in Lin− cells using RQ PCR and antioxidative enzyme activities were assessed. We demonstrated neuronal markers as well as TrkB expression in Lin− cells and the activation of the TrkB receptor by BDNF. BDNF pre-treatment diminished apoptosis in Lin− cells and influenced the proliferation of these cells. We observed significant changes in antioxidants as well as in the increased expression of NT-3 in Lin− cells following BDNF exposure. Complex global miRNA and mRNA profiling analyses using microarray technology and GSEA revealed the differential regulation of genes involved in the proliferation, gene expression, biosynthetic processes, translation, and protein targeting. Our results support the hypothesis that pre-treatment of stem/progenitor cells could be beneficial and may be used as an auxiliary strategy for improving the properties of SPCs.
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Trojani, Alessandra, Milena Lodola, Barbara Di Camillo, Giuseppe Rossi, Adele Capucci, Alessandra Perego, Enrico Maria Pogliani et al. „Microarray of Bone Marrow CD34+/Lin- Cells from Patients with Chronic Myeloid Leukemia (CML)“. Blood 124, Nr. 21 (06.12.2014): 5178. http://dx.doi.org/10.1182/blood.v124.21.5178.5178.
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Abstract This study aimed to determine the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of CML patients at diagnosis and after nilotinib treatment.Microarray was performed on 30 CML patients at diagnosis as well as 8 patients after 12 months of nilotinib treatment using the latest generation Affymetrix GeneChip HTA 2.0 to further investigate genomic complexity. Bone marrow blood (in a range of 5-20 ml) samples were collected from patients at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation at 800 rpm for 20 min and soon after CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin-cells was about 97% as determined by flow citometry and the cells were counted at diagnosis and after the treatment with nilotinib (at 3, 6, and 12 months). BM CD34+/lin- cells of 30 patients at diagnosis and after 3 and 6 months of nilotinib were resuspended in RNAlater (Ambion, Life Technologies) and stored at -20°C until RNA extraction was performed. Total RNA was extracted from BM CD34+/lin- cells using MagMAX 96 Total RNA Isolation Kit (Ambion). BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis. cRNA was prepared according to OvationPico WTA System V2 kit followed by Encore Biotin Module Kit (NuGEN). Ultimately, cRNA was hybridized to HTA 2.0 and signals were scanned by Affymetrix GeneChip Scanner 3000 following the manufacturer’s instructions. We observed that the number of CD34+/lin- cells dramatically decreased at 3 months (1,000-600,000), after 6 months (1,000-260,000) and after 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 after 12 months of nilotinib. If the number of CD34+/lin- cells after 12 months of nilotinib was less than 1000, we directly resuspended the cells in Prelude direct Lysis module (NuGEN) and microarray experiments were performed without RNA extraction. In this case, cRNA was prepared using Ovation One Direct System kit followed by Encore Biotin Module Kit (Nugen) and finally hybridized to the HTA 2.0. Microarray experiments were performed on CD34+/lin- cells of 30 CML patients at diagnosis with HTA 2.0. Moreover, we performed microarray experiments on CD34+/lin- cells of 8 CML patients at diagnosis vs. 8 CML patients after 12 months of nilotinib using HTA 2.0. Data was preprocessed and normalized using Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression in CML patients at diagnosis and at 12 months of treatment. P-values were corrected for multiple testing using false discovery rate. In conclusion, as far as we know, this is the first study which has isolated BM CD34+/lin-cells from CML patients at diagnosis and after nilotinib treatment. We observed a wide variety of the number of CD34+/lin- cells in 30 CML patients at diagnosis as well as after 3, 6 and 12 months of nilotinib. In particular, CD34+/lin- cells of CML patients at diagnosis were over 100.000. RNA was extracted and cRNA was prepared with OvationPico WTA System V2 kit followed by Encore Biotin Module Kit (NuGEN) and finally hybridized to HTA 2.0. On the other hand, the number of CD34+/lin- cells of CML patients after 12 months of nilotinib markedly decreased as low as 100 cells. In this case, we decided to avoid RNA extraction from the cells and we directly prepared cRNA using Ovation One Direct System kit and Encore Biotin Module Kit (Nugen) followed by the hybridization to the HTA 2.0 Array. To increase the statistical power in biomarker identification, we combined samples pre-processed with different experimental protocols (NuGEN) using ComBat, an empirical Bayes framework that adjusts data for batch effects and is robust to outliers in small datasets. Principal component analysis applied to the expression data before and after ComBat adjustment shows that the adopted normalization procedure effectively removes the systematic bias introduced by different protocols. Disclosures No relevant conflicts of interest to declare.
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Grundt, Inger K., und Harald Nyland. „Effects of Polyunsaturated Fatty Acids on Glial Cell Activation. A Study Using Primary Cultures“. Alternatives to Laboratory Animals 22, Nr. 3 (Mai 1994): 201–6. http://dx.doi.org/10.1177/026119299402200311.
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The aim of this study was to examine the effects of essential fatty acids (gammalinolenic acid [18:3 n-6; GLA] and alpha-linolenic acid [18:3 n-3; Lin]) on the activation of glial cells, using lipopolysaccharides as the activating agent. Primary cultures of mixed glial cells from rat brain were used as the model. The morphological activation of microglia was the most significant response to the exposure. This activation was followed by an increase in 5’-nucleotidase (5’-NT) activity. The 5’-NT activity was increased by GLA or Lin alone to 250–350% of the control value and further increased by co-incubation with lipoteichoic acid (a lipopolysaccharide) to 500–600% of the control value. The lipopolysaccharide-induced activation of glial cells was also followed by an augmented release of prostaglandin E2. GLA increased the release of prostaglandin E2 in a dose-dependent manner, whereas Lin had no effect on its release. The results show that this model system is useful for studies on factors affecting the activation of glial cells. GLA and Lin did not reverse glial activation induced by lipopolysaccharides under these experimental conditions.
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Guelzim, Najoua, Jean-François Huneau, Véronique Mathé, Annie Quignard-Boulangé, Pascal G. Martin, Daniel Tomé und Dominique Hermier. „Consequences of PPARαInvalidation on Glutathione Synthesis: Interactions with Dietary Fatty Acids“. PPAR Research 2011 (2011): 1–10. http://dx.doi.org/10.1155/2011/256186.
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Glutathione (GSH) derives from cysteine and plays a key role in redox status. GSH synthesis is determined mainly by cysteine availability andγ-glutamate cysteine ligase (γGCL) activity. Because PPARαactivation is known to control the metabolism of certain amino acids, GSH synthesis from cysteine and related metabolisms were explored in wild-type (WT) and PPARα-null (KO) mice, fed diets containing either saturated (COCO diet) or 18 : 3 n-3, LIN diet. In mice fed the COCO diet, but not in those fed the LIN diet, PPARαdeficiency enhanced hepatic GSH content andγGCL activity, superoxide dismutase 2 mRNA levels, and plasma uric acid concentration, suggesting an oxidative stress. In addition, in WT mice, the LIN diet increased the hepatic GSH pool, without effect onγGCL activity, or change in target gene expression, which rules out a direct effect of PPARα. This suggests that dietary 18 : 3 n-3 may regulate GSH metabolism and thus mitigate the deleterious effects of PPARαdeficiency on redox status, without direct PPARαactivation.
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Galy, AH, D. Cen, M. Travis, S. Chen und BP Chen. „Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow“. Blood 85, Nr. 10 (15.05.1995): 2770–78. http://dx.doi.org/10.1182/blood.v85.10.2770.bloodjournal85102770.
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T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin-ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA-cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.
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Glodowski, Doreen R., Tricia Wright, Keri Martinowich, Howard Chia-Hao Chang, Douglas Beach und Christopher Rongo. „Distinct LIN-10 Domains Are Required for Its Neuronal Function, Its Epithelial Function, and Its Synaptic Localization“. Molecular Biology of the Cell 16, Nr. 3 (März 2005): 1417–26. http://dx.doi.org/10.1091/mbc.e04-10-0885.
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α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) mediate excitatory neurotransmission at neuronal synapses, and their regulated localization plays a role in synaptic plasticity. In Caenorhabditis elegans, the PDZ and PTB domain-containing protein LIN-10 is required both for the synaptic localization of the AMPAR subunit GLR-1 and for vulval fate induction in epithelia. Here, we examine the role that different LIN-10 domains play in GLR-1 localization. We find that an amino-terminal region of LIN-10 directs LIN-10 protein localization to the Golgi and to synaptic clusters. In addition, mutations in the carboxyl-terminal PDZ domains prevent LIN-10 from regulating GLR-1 localization in neurons but do not prevent LIN-10 from functioning in the vulval epithelia. A mutation in the amino terminus prevents the protein from functioning in the vulval epithelia but does not prevent it from functioning to regulate GLR-1 localization in neurons. Finally, we show that human Mint2 can substitute for LIN-10 to facilitate GLR-1 localization in neurons and that the Mint2 amino terminus is critical for this function. Together, our data suggest that LIN-10 uses distinct modular domains for its functions in neurons and epithelial cells and that during evolution its vertebrate ortholog Mint2 has retained the ability to direct AMPAR localization in neurons.
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Nezir, Veysel, und Nizami Mustafa. „c0 can be renormed to have the fixed point property for affine nonexpansive mappings“. Filomat 32, Nr. 16 (2018): 5645–63. http://dx.doi.org/10.2298/fil1816645n.
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P.K. Lin gave the first example of a non-reflexive Banach space (X,||?||) with the fixed point property (FPP) for nonexpansive mappings and showed this fact for (l1,||?||1) with the equivalent norm ||?|| given by ||x|| = sup k?N 8k/1+8k ?1,n=k |xn|, for all x = (xn)n?N ? l1. We wonder (c0, ||?||1) analogue of P.K. Lin?s work and we give positive answer if functions are affine nonexpansive. In our work, for x = (?k)k ? c0, we define |||x||| := lim p?? sup ?k?N ?k (?1,j=k |?j|p/2j)1/p where ?k ?k 3, k is strictly increasing with ?k > 2, ?k ? N, then we prove that (c0,|||?|||) has the fixed point property for affine |||?|||-nonexpansive self-mappings. Next, we generalize this result and show that if ?(?) is an equivalent norm to the usual norm on c0 such that lim sup n ?(1/n ?n,m=1 xm + x) = lim sup n ?(1/n ?n,m=1 xm) + ?(x) for every weakly null sequence (xn)n and for all x ? c0, then for every ? > 0, c0 with the norm ||?||? = ?(?)+?|||?||| has the FPP for affine ||?||?-nonexpansive self-mappings.
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Moskovszky, Linda, Barbara Berger, Achim Fleischmann, Thomas Friedrich, Birgit Helmchen, Meike Körner, Tilman T. Rau und Zsuzsanna Varga. „Inter-observer reproducibility of classical lobular neoplasia (B3 lesions) in preoperative breast biopsies: a study of the Swiss Working Group of breast and gynecopathologists“. Journal of Cancer Research and Clinical Oncology 146, Nr. 6 (30.03.2020): 1473–78. http://dx.doi.org/10.1007/s00432-020-03195-w.
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Abstract Purpose Classical type of lobular neoplasia (LN) spans a spectrum of disease, including atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS), classical lobular neoplasia (LN), and the three-tiered classification of lobular intraepithelial neoplasia (LIN-1, -2, -3). This study addressed inter-observer variability of classical lobular neoplasias (LN) (B3 lesions) in preoperative breast biopsies among breast and gynecopathologists Methods A retrospective, observational, cross-sectional study was conducted. 40 preoperative digital images of breast core/vacuum biopsies were analyzed by eight experienced breast- and gynecopathologists. Evaluation criteria were ALH, LCIS, LN classic, LIN-1, LIN-2, LIN-3, focal B3 (one focus), extensive B3 (> one focus). Kappa-index and Chi-square tests were used for statistics. Digital scanned slides were provided to each participant. Agreement between the categories was defined as at least six of eight (cut-off 75%) concordant diagnoses. Results The highest agreement between eight pathologists was reached using the category lobular neoplasia (LN, classical), 26/40 (65%) cases were diagnosed as such. Agreements in other categories was low or poor: 12/40 (30%) (ALH), 9/40 (22%) (LCIS), 8/40 (20%) (LIN-1), 8/40 (20%) (focal B3), 3/40 (7.5%) (LIN-2), and 2/40 (5%) (extensive B3). Chi-square-test (classical LN versus the other nomenclatures) was significant (p = 0.001137). Conclusion Our data suggest that among Swiss breast pathologists, the most reproducible diagnosis for B3 lobular lesions is the category of classical LN. These data further support lack of consistent data in retrospective studies using different terminologies. Validation of reproducible nomenclature is warranted in further studies. This information is useful especially in view of retro- and prospective data analysis with different diagnostic categories.
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Pungolino, Ester, Giuseppe Rossi, Maria Angela Mura, Alessandra Perego, Ester Maria Orlandi, Mauro Turrini, Lorenza Borin et al. „REL-Protocol PhilosoPhi34: An Open Label, Single Arm, Phase II Study of Nilotinib 300 Mg BID in Newly Diagnosed Chronic Phase Chronic Myeloid Leukaemia Patients, to Study the Disappearance of CD34+/Lin-Ph+ Cells from Bone Marrow during Treatment. Preliminary Data“. Blood 126, Nr. 23 (03.12.2015): 1597. http://dx.doi.org/10.1182/blood.v126.23.1597.1597.
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Abstract Background. Chronic Myeloid Leukaemia (CML) can be effectively treated with the first generation Tyrosine Kinase Inhibitor (TKI) Imatinib, and more effectively with the second generation TKI, like Nilotinib. However, despite the deeper and faster responses induced by nilotinib in a large proportion of patients, the possible eradication of the pathological stem cells is not yet clearly elucidated. In fact, in vitro data suggest that quiescent stem cells are not sensitive to Bcr/Abl inhibition (Corbin AS, et al 2011; Hamilton A, et al 2012). A preliminary in-vivo study (Defina M, et al 2012) shows that in patients in CCyR even after short-term of nilotinib therapy, residual leukemic progenitors are rarely detected. Methods. On behalf of Rete Ematologica Lombarda (REL), Italy, we designed a single arm prospectic study, PhilosoPhi34 (EudraCT: 2012-005062-34), with the aim to investigate the efficacy of nilotinib 300 mg BID in obtaining the disappearance of Bone Marrow (BM) leukemic stem cells (CD34+/lin-Ph+) in newly diagnosed CP-CML. Primary objective of the study: to enumerate the BM CD34+/lin-Ph+ cells at the end of 6 months of treatment. Secondary objectives: to enumerate the BM CD34+/lin-Ph+ cells at 3 and 12 months; to assess the percentage of patients showing MR ≤10% IS at 3 months and MR ≤1% IS at 6 months and the MMR IS and MR4.5 IS by 3, 6 and 12 months of treatment. BM blood samples (range of 5-20 ml) were collected at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation and then CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin- cells was about 97% as determined by flow cytometry. BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis. After the treatment we observed that the number of CD34+/lin- cells dramatically decreased after 3 (1,000-600,000), 6 (1,000-260,000) and 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 at 12 month of treatment. In order to verify the disappearance of leukemic stem cells, isolated CD34+/lin- cells were tested by standard FISH (i.e. to categorize a sample as negative at least 200 nucleus were examined). From April 2013 and June 2015 we enrolled 87 pts, as for protocol. We report here the preliminary results. Results. Of 56 patients in CCyR after 6 months of treatment, FISH performed on BM CD34+/lin- cells nuclei was evaluable in 51 cases (5 negative cases were excluded because of less than 200 nucleus were analysed). In 4 out of 51 patients (7.8%), Ph+ nuclei were detected. The Sokal score of these 4 patients was 1 low, 2 intermediate and 1 high risk with a ratio (positive nuclei/total nuclei) of 295/300, 1/200, 2/92, 3/300, respectively. Among 58 patients tested at 3 months and 44 tested at 12 months of treatment, the number of evaluable patients was 48 and 37, respectively; 8/48 (16.6%) and 0/37 (0%) patients showed Ph+ nuclei. Only 2 out of 8 positive patients had a high Sokal score. Regarding efficacy of treatment, Table 1 summarizes the MRs IS observed after 3, 6 and 12 months of treatment in 71, 57 and 41 patients, respectively. Conclusion. Data of this prospective study confirms that nilotinib 300 mg BID, rapidly and progressively induces the clearance of BM CD34+/lin-Ph+ cells in CP-CML patients. In particular, on CD34+/lin- cells, after 6 months of treatment, only 7.8% of patients showed positive nuclei. On 37 patients after 12 months of treatment, no positive nuclei were detected. So far, the kinetic of reduction of such cells seems not influenced by Sokal score. According to international studies, PhilosoPhi34 shows a very high efficacy of Nilotinib to induce MRs in CP-CML patients, at the standard time points. Table 1. Molecular Response (MR) in CP-CML patients treated with Nilotinib 300mg BID from diagnosis. MR IS 3 months 6 months 12 months ≤10% 67/71 94% 57/57 100% 40/41 97.50% ≤1% 57/71 80% 55/57 96.50% 40/41 97.50% ≤0.1% 17/71 24% 41/57 72% 35/41 85% ≤0.01% 3/71 4% 19/57 33% 20/41 48.70% MR4.5(UD) 2/71 2.80% 10 (6)/57 17.5% (10.5%) 15 (9)/41 36.5% (22%) UD: undetectable We acknowledge all REL Colleagues for their collaboration and Novartis SpA for the partial financial support to the study. Disclosures No relevant conflicts of interest to declare.
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Crittenden, N., D. McGeeney, Y. Assouline-Dayan, K. Boutros, M. Syrzycka, W. Bartolini, R. Boinpally und C. N. Andrews. „A236 LINACLOTIDE IS NOT DETECTABLE IN BREAST MILK OF LACTATING WOMEN: AN OPEN-LABEL, PHASE 1 STUDY“. Journal of the Canadian Association of Gastroenterology 4, Supplement_1 (01.03.2021): 285–87. http://dx.doi.org/10.1093/jcag/gwab002.234.
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Abstract Background Linaclotide (LIN) is a guanylate cyclase-C agonist approved to treat adults with irritable bowel syndrome with constipation (IBS-C) and chronic idiopathic constipation (CIC). As LIN is a 14-amino acid peptide, no absorption from the gastrointestinal tract is expected following oral administration. However, LIN levels in human breast milk have not been determined. Aims To determine the levels of LIN and its active metabolite, MM-419447, excreted in breast milk after multiple once-daily doses of LIN (72 µg, 145 μg, or 290 μg) in lactating women. Methods This was a multicenter, open-label, multidose, Phase 1, milk-only lactation study (NCT02220348) in lactating women aged 18–45 years who were actively breastfeeding or pumping for ≥4 weeks and were already taking LIN 72 μg, 145 μg, or 290 μg therapeutically for IBS-C or CIC. Participants continued their LIN dose once daily for the 3 study days, with breast milk extractions at −1 to 0 hours (pre-dose) on Day 1 and −1 to 0 hours and 0–2, 2–4, 4–8, 8–12, 12–16, and 16–24 hours after dosing on Day 3. The pharmacokinetic endpoints were the cumulative amount of LIN and MM-419447 and the percentage dose of LIN excreted into the breast milk over the dosing interval. Adverse events (AEs) were monitored. The study has institutional review board approval. Results Seven women were enrolled in the study (IBS-C, n=1; CIC, n=6) and received LIN (72 μg, n=5; 145 μg, n=1; and 290 μg, n=1). Mean age was 28 (range: 19–35), mean weight was 70 kg (standard deviation [SD]: 12 kg), and mean body mass index was 26 kg/m2 (SD: 4 kg/m2); the majority were white (n=6). Concentrations of LIN and MM-419447 in breast milk samples were below the quantitation limits (<0.25 ng/mL and <1.00 ng/mL, respectively) at all time points for all participants. Cumulative exposure for each dose was 216 μg (72 μg dose), 435 μg (145 μg dose), and 870 μg (290 μg dose). One treatment-emergent AE was reported (mild rash). Conclusions The data collected in this study provide no evidence that breastfeeding infants receive LIN or MM-419447 through breast milk as their concentrations were below the quantitation limit following multiple once-daily doses of LIN 72 μg, 145 μg, or 290 μg. Funding Agencies This study was sponsored by Allergan plc, Dublin, Ireland (prior to acquisition by AbbVie Inc.). Writing and editorial assistance were provided to the authors by Stephanie J. Rippon, MBio, Jane Beck, MA, and Rebecca Fletcher, BA(Hons), of Complete HealthVizion, Inc., Chicago, IL, USA and funded by Allergan plc (prior to acquisition by AbbVie Inc.).
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Ambros, V. „Cell cycle-dependent sequencing of cell fate decisions in Caenorhabditis elegans vulva precursor cells“. Development 126, Nr. 9 (01.05.1999): 1947–56. http://dx.doi.org/10.1242/dev.126.9.1947.
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In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1 degrees) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1 degrees cell each express secondary (2 degrees) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non-vulval tertiary (3 degrees) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two developmental decisions separable by cell cycle phase: lin-12 must act prior to the end of VPC S phase to influence a 1 degrees versus 2 degrees cell fate choice, but must act after VPC S phase to influence a 3 degrees versus 2 degrees cell fate choice. Coupling developmental decisions to cell cycle transitions may provide a mechanism for prioritizing or ordering choices of cell fates for multipotential cells.
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Lee, Youngae, Yutaro Kumagai, Shizuo Akira und Myoung Ho Jang. „Intestinal mast cell progenitors function as innate lymphoid cells (P497) (71.3)“. Journal of Immunology 188, Nr. 1_Supplement (01.05.2012): 71.3. http://dx.doi.org/10.4049/jimmunol.188.supp.71.3.
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Abstract Small intestinal innate lymphoid cells (mainly NKp46+ILCs) are known to have a protective role from infection of pathogenic bacteria through the production of IL-22. The proportions of ILCs localized intestinal lamina propria are higher than those in the others lymphoid tissues, such as mesenteric lymph node, spleen, liver, and bone marrow. In this study, we did a microarray experiment to study whether small intestinal ILCs (Lin-c-Kit+Sca-1- cells) highly express which genes as compared with non-ILCs (Lin-c-Kit-Sca-1- cells). We selected 251 up-regulated genes and 219 down-regulated genes in Lin-c-Kit+Sca-1- cells. Especially, Lin-c-Kit+Sca-1- cells highly expressed genes coding to Il22, Csf2rb2, mast cell proteases (Mcpts), and Rorc. To address whether which ILC populations are main source of IL-22, we more detailed divided Lin-c-Kit+Sca-1- cells into the two groups [Lin-c-Kit+NKp46+ (NKp46+ILCs) and Lin-c-Kit+NKp46-CD4-]. Only Lin-c-Kit+NKp46-CD4- cells expressed Csf2rb2 and Mcpts transcripts. These cells expressed higher level of Il22 mRNA, IL-22 protein, and IL-17F protein in physiological condition and in IL-23 stimulated condition compared with NKp46+ILCs. Moreover, these cells could differentiate into mast cells in the presence of mIL-3 and mSCF. Taken together, Lin-c-Kit+NKp46-CD4- cells as mast cell progenitors may regulate small intestinal homeostasis through the production of Mcpts, IL-17F, and IL-22 in physiological condition and in pathophysiological condition.
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Arinobu, Yojiro, Hiromi Iwasaki, Michael F. Gurish, Shi-ichi Mizuno, Hirokazu Shigematsu und Koichi Akashi. „Identification of Progenitors Committed to Basophil and/or Mast Cell Lineages.“ Blood 104, Nr. 11 (16.11.2004): 779. http://dx.doi.org/10.1182/blood.v104.11.779.779.
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Abstract Eosinophils, basophils and mast cells are multifunctional hematopoietic effectors that co-operate to mount a variety of allergic and innate immune responses. Their origin and developmental relationships, however, have not yet been resolved, and remain as one of the major issues in the biology of hematopoiesis. Here we report that progenitors bipotent for basophils and mast cells (basophil/mast cell progenitors: BMCPs) are prospectively isolatable downstream of granulocyte/monocyte progenitors (GMPs). Since both basophils and mast cells express the αβγ2 form of FcεRI on their surface, we hypothesized that early progenitors restricted to these lineages may have already upregulated these molecules. Thus, FcεRIα-expressing cells were searched within the Lin−CD34+ bone marrow and spleen cells. Lin−CD34+ bone marrow cells contained a small fraction of cells expressing a high level of FcεRIα that were all c-Kit−. Purified Lin−CD34+FcεRIαhic-Kit− cells were cultured, and they gave rise exclusively to pure basophil colonies, which were named as basophil progenitors (BaPs). In contrast, the spleen had a small fraction of Lin−CD34+ cells expressing a low level of FcεRIα and a high level of c-Kit. Strikingly, single Lin−CD34+FcεRIαloc-Kithi cells formed colonies containing both basophils and mast cells as well as pure mast cell or basophil colonies. This indicates that at least a fraction of Lin−CD34+FcεRIαloc-Kithi cells are bipotent for basophils and mast cells. We thus named the Lin−CD34+FcεRIαloc-Kithi cells as BMCPs. Identification of BMCPs formally proves for the first time that basophils and mast cells share a common progenitor stage. After 3-day culture, BMCPs gave rise to Lin−CD34+FcεRIαhic-Kit−BaPs and Lin−CD34+FcεRIαhic-Kit+ cells which exclusively formed pure mast cell colonies. Lin−CD34+FcεRIαhic-Kit+ cells were named as mast cell progenitors (MCPs). All of these progenitors are located downstream of GMPs since GMPs gave rise to BMCPs, BaPs and MCPs in vitro after 3-day culture with SCF, IL-3, and IL-9. The intestine is known to collect mast cell colony-forming activity. Since MCPs were not isolatable as a distinct population in the bone marrow or the spleen, we searched for MCPs in the intestine by using similar markers. We newly identified Lin−CD34+FcεRIαloc-Kitlo cells in the intestine, and these cells exclusively formed pure mast cell colonies. In mice sensitized with OVA to induce allergic reaction, BMCPs, BaPs and intestinal MCPs expanded by1.5- to 5-fold in number after OVA administration. This strongly suggests that these populations constitute critical stages in physiological pathways for each lineage development. Taken together, it is likely that the initial commitment into basophil/mast cell lineages occurs in the spleen, and that spleen BMCPs may migrate into the bone marrow to become BaPs or into the intestine to become MCPs. These progenitor populations should be useful to analyze the mechanism of commitment into each of these lineages, and could also be therapeutic targets for a variety of allergic and autoimmune disorders.
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Okada, S., H. Nakauchi, K. Nagayoshi, S. Nishikawa, Y. Miura und T. Suda. „In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells“. Blood 80, Nr. 12 (15.12.1992): 3044–50. http://dx.doi.org/10.1182/blood.v80.12.3044.3044.
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Abstract c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.
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Okada, S., H. Nakauchi, K. Nagayoshi, S. Nishikawa, Y. Miura und T. Suda. „In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells“. Blood 80, Nr. 12 (15.12.1992): 3044–50. http://dx.doi.org/10.1182/blood.v80.12.3044.bloodjournal80123044.
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c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.
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Koga, Makoto, und Yasumi Ohshima. „Drosophila MAP kinase kinase suppresses the vulvaless phenotype of lin-3, let-23 and lin-45 mutations in Caenorhabditis elegans“. Mechanisms of Development 53, Nr. 1 (September 1995): 15–22. http://dx.doi.org/10.1016/0925-4773(95)00417-3.
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Miller, Leilani M., Heather A. Hess, David B. Doroquez und Noelle M. Andrews. „Null Mutations in thelin-31Gene Indicate Two Functions DuringCaenorhabditis elegansVulval Development“. Genetics 156, Nr. 4 (01.12.2000): 1595–602. http://dx.doi.org/10.1093/genetics/156.4.1595.
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AbstractThe lin-31 gene is required for the proper specification of vulval cell fates in the nematode Caenorhabditis elegans and encodes a member of the winged-helix family of transcription factors. Members of this important family have been identified in many organisms and are known to bind specific DNA targets involved in a variety of developmental processes. DNA sequencing of 13 lin-31 alleles revealed six nonsense mutations and two missense mutations within the DNA-binding domain, plus three deletions, one transposon insertion, and one frameshift mutation that all cause large-scale disruptions in the gene. The missense mutations are amino acid substitutions in the DNA-binding domain and probably disrupt interactions of the LIN-31 transcription factor with its DNA target. In addition, detailed phenotypic analysis of all 19 alleles showed similar penetrances for several characteristics examined. From our analysis we conclude: (1) the null phenotype of lin-31 is the phenotype displayed by almost all of the existing alleles, (2) the DNA-binding domain plays a critical role in LIN-31 function, and (3) direct screens for multivulva and vulvaless mutants will probably yield only null (or strong) alleles of lin-31.