Journal articles on the topic '2-Cells stage'
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1
Vanhecke, D., B. Verhasselt, V. Debacker, G. Leclercq, J. Plum, and B. Vandekerckhove. "Differentiation to T helper cells in the thymus. Gradual acquisition of T helper cell function by CD3+CD4+ cells." Journal of Immunology 155, no. 10 (November 15, 1995): 4711–18. http://dx.doi.org/10.4049/jimmunol.155.10.4711.
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Abstract We investigated at which point during thymocyte differentiation functions were acquired that are characteristic for mature Th cells. Differentiation from CD3+CD69-, CD4+CD8+ double-positive (DP) cells to terminally differentiated CD3+, CD4+CD8- single-positive (SP) cells was broken down into six discrete stages that were purified by four-color sorting: CD69-CD3+DP (stage 0), CD69+CD27-DP (stage 1), CD69+CD27-CD4+SP (stage 2), CD27+CD1+CD4+SP (stage 3), CD1-CD45RO+CD4+SP (stage 4), and CD1-CD45RO-CD4+SP cells (stage 5). Phenotypically, these stages seem to describe consecutive steps in differentiation from immature stage 0 to the terminally matured stage 5. Functionally, the capacity to proliferate on IL-2 after stimulation was absent in CD69- stage 0 cells, but was acquired gradually during stages 1 to 4. Clonal expandability and the capacity to respond to stimulation with the production of cytokines were acquired later and rather abruptly by CD1- stage 4 and 5 cells. Activation markers such as CD69 expression and in vivo IL-2 gene transcription came up simultaneously at the DP stage and peaked at stage 3 to 4. These data suggest that functional maturation of Th cells occurs over an extended period in differentiation, stages 1 to 4, and coincides with a gradual increase in activation markers. After completion of functional differentiation, at stage 5, in vivo IL-2 mRNA transcription and CD69 expression are down-regulated, and the cells become functionally resting naive T cells expressing CD45RA+.
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Mower, D. A., D. W. Peckham, V. A. Illera, J. K. Fishbaugh, L. L. Stunz, and R. F. Ashman. "Decreased membrane phospholipid packing and decreased cell size precede DNA cleavage in mature mouse B cell apoptosis." Journal of Immunology 152, no. 10 (May 15, 1994): 4832–42. http://dx.doi.org/10.4049/jimmunol.152.10.4832.
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Abstract Mature resting mouse spleen B cells progress stochastically into apoptosis at a uniform rate over the first 16 h in vitro in 3 stages. In stage 1, early apoptotic B cells decreased the normal phospholipid packing of their plasma membranes, detected as increased binding of the lipophilic dye merocyanine 540, and also decreased in volume, detected as decreased forward scatter. In stage 2 there was abrupt internucleosomal cleavage of DNA, quantitated as hypodiploid nuclei by flow cytometry. Some stage 2 cells entered stage 3, where the plasma membrane became permeable to propidium iodide. B cells in later stages of this sequence retained the characteristics of earlier stages, whereas nonapoptotic B cells remained in their original state. Cycloheximide increased the progression of B cells through these three stages, whereas dextran sulfate inhibited stage 1 more effectively than stages 2 or 3. Increased orthogonal scatter also occurred late in some of the cells that had passed through stage 1, but did not correlate well with propidium iodide permeability. Fresh small dense spleen B cells contained 5% to 7% stage 1 cells but only about 1% stage 2 cells. Macrophages have been reported to destroy preferentially apoptotic thymocytes by recognizing plasma membrane alterations deriving from loose packing of phospholipid head groups. The recognition of stage 1 rather than stage 2 B cells by macrophages may help to keep the proportion of apoptotic cells low in vivo.
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Bai, L., G. Meredith, and B. E. Tuch. "Glucagon-like peptide-1 enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells." Journal of Endocrinology 186, no. 2 (August 2005): 343–52. http://dx.doi.org/10.1677/joe.1.06078.
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Embryonic stem cells (ESCs) can be differentiated into insulin-producing cells by a five-stage procedure involving altering culture conditions and addition of nicotinamide. The amounts of insulin in these cells are lower than those found in pancreatic β cells. Glucagon-like peptide-1 (GLP-1) induces the differentiation of β cells from ductal progenitor cells. We examined the possibility of GLP-1, and its long-acting agonist exendin-4, enhancing the differentiation of insulin-producing cells from mouse ESCs (mESCs). A five-stage culturing strategy starting with embryoid bodies (EBs) was used in this study. mRNA for pancreatic duodenal homeobox gene 1 (PDX-1) and neurogenic differentiation (NeuroD) was detected from stage 1, hepatocyte nuclear factor 3 beta (HNF3β) and insulin 2 from stage 2, Ngn3 and glucose transporter 2 (GLUT2) from stage 3, and insulin 1 and other β-cell markers, at stages 4–5. Cells at stage 5 secreted C-peptide, being 0.68 ± 0.01 pmol/106 cells per 2 days, and had an immunoreactive insulin content of 13.5 ± 0.7 pmol/106 cells. Addition of GLP-1 (100 nM) and nicotinamide (10 mM) at stage 5 resulted in a 50% and 48% increase in insulin content and C-peptide secretion respectively compared with nicotinamide alone. Glucose-induced insulin secretion was enhanced 4-fold by addition of both growth factors. The GLP-1 receptor was present at all five stages of the culture. Addition of exendin-4 to cells at stage 2 resulted in a 4.9-fold increase in expression of the gene for insulin 1 and a 2-fold increase in insulin content compared with the effect of nicotinamide alone at stage 5. It is concluded that both GLP-1 and exendin-4 enhance the level of expression of insulin in glucose-responsive insulin-producing cells derived from the R1 mESC line.
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Ruggeri, Rosaria Maddalena, Salvatore Sciacchitano, Enrica Vitarelli, Francesco Trimarchi, Gaetano Barresi, and Maria Trovato. "Immunoexpression of Multidrug-Resistance Protein 2 and Cyclooxygenase 2 in Medullary Thyroid Carcinomas." Archives of Pathology & Laboratory Medicine 130, no. 7 (July 1, 2006): 1014–19. http://dx.doi.org/10.5858/2006-130-1014-iompac.
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Abstract Context.—Chemoresistance is due to the expression of multidrug-resistance proteins (MRPs). Cyclooxygenase 2 (COX2), a key enzyme in prostaglandins synthesis, upregulates MRP1. MRP1 is overexpressed in medullary thyroid carcinomas (MTCs), but it is not involved in resistance to doxorubicin and cisplatin, which are commonly used in MTC treatment. MRP2 is specifically involved in resistance to both chemotherapeutic agents, but no data exist on the expression of MRP2 and COX2 in MTC. Objective.—To evaluate MRP2 and COX2 expressions in MTC. Design.—We analyzed immunohistochemical expression of MRP2 and COX2 in 12 MTCs and in 6 lymph node metastases. Results were correlated with pTNM and clinical stage. Results.—MRP2 and COX2 expressions were observed only in tumor samples and metastases. Nine MTCs, all pTNM stage T4, were positive for MRP2, whereas 3 MTCs, pTNM stages T2 and T3, were unreactive for MRP2. Six metastatic MTCs at stage T4 showed higher proportion of MRP2+ cells, compared with primary tumors. All 12 MTCs were positive for COX2. Three MTCs, pTNM stage T2 and T3, showed COX2 positivity in all cells. The proportion of COX2+ cells decreased with increased pTNM stage. Four out of 6 metastatic MTCs, stage T4, showed a lower proportion of COX2+ cells, compared with primary tumors. The proportion of MRP2+ cells was inversely related to the proportion of COX2+ cells. Conclusions.—MRP2 and COX2 expression correlated with pTNM stage. High MRP2 and low COX2 expression may explain resistance to doxorubicin and cisplatin, which is observed in advanced stage MTC. Evaluation of the expression pattern of these 2 proteins may be useful to predict chemosensitivity of these types of tumors.
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Jones, E. A., and H. R. Woodland. "The development of animal cap cells in Xenopus: a measure of the start of animal cap competence to form mesoderm." Development 101, no. 3 (November 1, 1987): 557–63. http://dx.doi.org/10.1242/dev.101.3.557.
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Grafting, together with tissue identification by monoclonal antibodies, has been used to study the allocation of Xenopus animal cap cells to the ectodermal or mesodermal lineages. Animal cap cells become responsive at stage 61/2 and lose responsiveness to mesodermal induction at, or just after, stage 101/2 (depending on the batch of embryos). The ability of the vegetal yolky cells to induce mesoderm disappears between stages 101/2 and 11. It is present at stage 6–61/2 and may exist before this. The emergence of competence to respond at stage 61/2, coupled with the fact that the endoderm is already capable of induction at this stage, suggests that mesodermal induction begins at this point in the intact embryo.
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Rybicka, Krystyna. "Histogenesis of alveolar cell carcinoma." Proceedings, annual meeting, Electron Microscopy Society of America 45 (August 1987): 626–27. http://dx.doi.org/10.1017/s0424820100127566.
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Alveolar cell carcinoma (ACC) is a lung neoplasm characterized by the presence of lamellar bodies specific for normal type 2 alveolar cells. Tumor histogenesis is uncertain. The present study indicates that ACC originates from dedifferentiation of hyperplastic type 2 alveolar cells rather than migration of bronchial stem cells into alveoli as suggested earlier.An aliquot of human lung biopsy diagnosed as ACC was fixed in glutaraldehyde and osmium, treated with 1% aqueous uranyl acetate, and embedded in epoxy resin. Sections were stained for glycogen by periodic acid - thiosemicarbazide - silver proteinate, and post-stained by uranyl acetate and lead citrate.The tumor contained morphologically distinct cell clusters. Each cluster consisted of identical cells. Differences between clusters resulted from synchronous alterations in lamellar bodies, mitochondria, glycosomes, and ribosomes. These alterations revealed distinct stages in cell differentiation classified here as follows: stage 1-hyperplastic cells (Fig.1), stage 2-dedifferentiating cells (Fig.2), stage 3-undifferentiated cells (Fig.3), stage 4-differentiating cells (Fig.4).
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Loncarevic, Slobodan, Denis Brajkovic, Milka Gardasevic, Olivera Loncarevic, Nebojsa Ladjevic, Dejan Nesic, Dusica Stamenkovic, Ivana Likic-Ladjevic, Nikola Ladjevic, and Nemanja Rancic. "Expression of PCNA, CD-31 and HER-2 in Serbian patients with oral squamous cell carcinoma." Archives of Biological Sciences 71, no. 4 (2019): 703–10. http://dx.doi.org/10.2298/abs190705053l.
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Several studies have investigated the expression of tumor markers, including p53, HER-2, PCNA, EGFR, VEGFR CD-31 and Bcl-2 in patients with oral squamous carcinoma (OSC). This study aimed to determine the expression of proliferating cell nuclear antigen (PCNA), endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31) and human epidermal growth factor receptor-2 (HER-2) according to OSC stage. The prospective study included 62 patients diagnosed with OSC stages II and III. Surgical specimens were obtained from tumor and peritumoral tissues. We determined the pathohistological degree of tumor differentiation and the immunohistochemical expression of PCNA, CD-31 and HER-2 for each specimen. Immunohistochemical analysis of the expression of PCNA in tumor cells demonstrated poor staining of immunoreactive tumor cells in 23 patients (10 in stage II, 7 in stage IIIa and 6 in stage IIIb). Moderately expressed PCNA-immunoreactivity in the tumor cells in 17 patients (7 in stage II, 6 in stage IIIa and 4 in stage III), and extremely strong PCNA-immunoreactive staining in tumor cells of 10 patients with IIIb stage, was observed. These results suggest that PCNA expression combined with pathohistological findings could possess a prognostic value in determining the survival rates for patients with oral squamous cell carcinoma.
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Sepulveda-Rincon, L. P., D. Dube, P. Adenot, L. Laffont, S. Ruffini, L. Gall, W. E. Maalouf, V. Duranthon, and N. Beaujean. "78 NONINVASIVE CELL LINEAGE TRACING IN BOVINE EMBRYOS FROM 2-CELL STAGE UP TO BLASTOCYST STAGE." Reproduction, Fertility and Development 27, no. 1 (2015): 132. http://dx.doi.org/10.1071/rdv27n1ab78.
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The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.
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Hughes, Tiffany L., Michael Brian Becknell, Aharon Freud, Susan E. Schmidt, Jianhua Yu, Charlene Mao, Mikhail Gavrilin, Mark Wewers, and Michael A. Caligiuri. "The Stage-Specific Effect of Interleukin-1 Beta (IL-1β) during Human Natural Killer Cell Development." Blood 112, no. 11 (November 16, 2008): 3746. http://dx.doi.org/10.1182/blood.v112.11.3746.3746.
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Abstract Developmental intermediates of human natural killer (NK) cells are found within secondary lymphoid tissue (SLT), and five distinct stages of these intermediates have been identified. While it is well documented that developing NK cells are reliant on interleukin (IL)-15 as a survival factor, it is likely that additional cytokines and growth factors are required for complete NK cell differentiation. Microarray transcriptional profiling of purified stage 1–4 cells from human tonsil and stage 4 and 5 cells from peripheral blood (PB) identified a developmental window of interleukin-1 receptor 1 (IL-1R1) messenger RNA (mRNA) expression restricted to stages 2 and 3. We confirmed this finding by quantitative RT-PCR, and analysis of IL-1R1 surface protein expression revealed that, on average, 81% of stage 3 immature NK cells are IL-1R1(+), whereas the majority of cells from stages 1, 2, and 4 are IL-1R1(−). When cultured in vitro with IL-1β, a physiologic ligand for IL-1R1, cells from all four stages died within 48 hours, consistent with an absolute requirement for IL-15 as a survival factor. However, the combination of IL-1β and IL-15 led to a significant and reproducible 4.64±−0.68–fold increase in stage 3 cell number over that seen with IL-15 alone (p < 0.0005). This phenomenon was completely restricted to stage 3 immature NK cells, and is attributed to increased proliferation. The effects of IL-1β were abrogated by a molar excess of IL-1 receptor antagonist (IL-1RA), a physiologic competitor for IL-1R1 binding. Collectively, our data indicate that IL-1R1 expression fluctuates dramatically during NK cell development, and that unique responses of IL-1R1(+) stage 3 cells to IL-1β and IL-15 govern the expansion of these immature NK cells. Our findings support a model in which IL-1β promotes stage 3 proliferation and survival in vivo, driving stage 3 cells to be the most prevalent NK cell intermediates within SLT.
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Novikova, I. A., and O. I. Kit. "Expression of epithelial-mesenchymal transition markers E-cadherin and ZEB1 in colorectal cancer." Research and Practical Medicine Journal 8, no. 2 (June 23, 2021): 23–33. http://dx.doi.org/10.17709/2410-1893-2021-8-2-2.
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Purpose of the study. Evaluation of expression of the epithelial-mesenchymal transition markers E-cadherin and ZEB1 in patients with stage II-IV colorectal cancer (CRC).Materials and methods. The study included operational material obtained from 299 patients aged 42–86 years (mean age 64.2±1.7 years) with stage II-IV CRC treated at National Medical Research Centre for Oncology in 2013-2017. Stage II CRC (T3-4 N0 M0 ) was diagnosed in 110 patients, stage III (T1-4 N1-2 M0 ) – in 88 patients, stage IV (T1-4 N0-2 M1 ) – in 101 patients. Polyclonal rabbit antibodies to ZEB1 (Biorbyt Ltd., UK) and mouse monoclonal antibodies to E-cadherin (Diagnostic BioSystems, USA) were used for an IHC analysis. The intensity and degree of tumor cell staining, percentage of stained tumor cells in the sample and the number of patients with positive and negative marker expression were determined. Groups were compared using the Mann–Whitney U test and the Pearson's chi-square test.Results. Positive expression of E-cadherin was found in 64.5 % (193 of 299 patients), ZEB1 – in 80.6 % (241 of 299 patients). The number of patients with E-cadherin-positive tumors statistically significantly decreased (χ2 =15.888 at p<0.001) from stage II to stage IV, while for ZEB1, on the contrary, it statistically significantly increased (χ2 =43.912 at p><0.001) from stage II to stage IV. The mean values of expression in positively stained cells were: in stage II – E-cadherin 55.3±6.8 %, ZEB1 43.0±5.9 %; in stage III – E-cadherin 38.4±5.8 %, ZEB1 77.0±5.5 %; in stage IV – E-cadherin 14.7±4.7 %, ZEB1 76.9±3.5 %. Significant differences were observed between the mean values of ZEB1 expression in stages III and IV compared to stage II, as well as between the mean values of E-cadherin expression in stages II and III compared to stage IV (p><0.05). No significant differences were found in the mean values of ZEB1 and E-cadherin expression in stages III and IV, II and III respectively.Conclusions. The study demonstrated statistically significant relationship between tumor stages and expression of E-cadherin and ZEB1 in the epithelial-mesenchymal transition. The loss of the E-cadherin expression in tumor cells of patients from stage II to stage IV and increased expression of ZEB1 in the studied groups were statistically significant (p<0.05).
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Zou, J. P., J. Shimizu, K. Ikegame, N. Yamamoto, S. Ono, H. Fujiwara, and T. Hamaoka. "Tumor-bearing mice exhibit a progressive increase in tumor antigen-presenting cell function and a reciprocal decrease in tumor antigen-responsive CD4+ T cell activity." Journal of Immunology 148, no. 2 (January 15, 1992): 648–55. http://dx.doi.org/10.4049/jimmunol.148.2.648.
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Abstract Splenic CD4+ T cells from BALB/c mice bearing a syngeneic tumor (CSA1M) 2 to 3 wk after the inoculation with CSA1M cells produced IL-2 and macrophage-activating factor upon in vitro cultures. This lymphokine production was achieved without stimulation of these T cells with exogenous stimulating tumor Ag. However, elimination of APC from spleen cells resulted in almost complete abrogation of the capacity of CD4+ T cells to produce IL-2/macrophage-activating factor. The lymphokine production was regained when APC from CSA1M-bearing mice were added back to cultures. APC from normal or another syngeneic tumor (Meth A)-bearing mice failed to regain the lymphokine production. These observations demonstrated that the lymphokines were produced by CD4+ T cells from CSA1M-bearing hosts through their collaboration with APC binding CSA1M tumor Ag in the tumor-bearing state. The lymphokine-producing capacity of whole spleen cells from tumor-bearing mice reached the maximal level around 2 to 3 wk after tumor implantation but gradually decreased with the progress of tumor-bearing stages. Importantly, tumor-bearing stage-related changes were observed in a different fashion in the capacities of anti-CSA1M CD4+ T cells vs CSA1M tumor Ag-binding APC. The capacity of APC increased with the progress of tumor-bearing stages as demonstrated by the stimulation of CSA1M-immunized T cells with APC from different CSA1M-bearing stages. In contrast, the reactivity of anti-CSA1M T cells to APC from a given CSA1M-bearing stage decreased with the tumor-bearing stage. These results demonstrate a stage-related increase tumor Ag-binding APC function, as well as a reciprocal reduction in tumor Ag-responsive CD4+ T cell activity.
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Nakagawa, T., N. Nakagawa, H. Goldstein, D. J. Volkman, and A. S. Fauci. "Demonstration that human B cells respond differently to interleukin 2 and B cell differentiation factor based on their stages of maturation." Journal of Immunology 137, no. 10 (November 15, 1986): 3175–82. http://dx.doi.org/10.4049/jimmunol.137.10.3175.
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Abstract The mechanisms whereby interleukin 2 (IL 2), interferon-gamma (IFN-gamma), and B cell differentiation factor (BCDF) alone or in combination modulate human B cell differentiation are currently under intensive study. To dissect out the effects of individual lymphokines contained in mixed lymphocyte reaction-culture supernatants (MLR-CS) on B cell differentiation, we employed pure factors that possessed the same activity as factors contained in MLR-CS (IL 2: 50 U/ml, IFN-gamma: 7 U/ml, BCDF-Nal: 5 pM/ml, BCDF-YA2: 12.5% v/v) singly and in combination to human B cells. By activating purified human B cells with Staphylococcus aureus Cowan I (SAC) for 3 days, separating B blast cells by the Percoll centrifugation method, and then either using these B blast cells as B cells in the earlier stage after SAC-activation, or further culturing these B blast cells for 4 more days without any stimuli and using these B cells as B cells in the later stage after SAC-activation, we could define two different populations of cells. Disparity in the populations could be demonstrated by the observation that B cells in the earlier stage were 81.2% Tac-antigen+, 23.2% B2+, 68.9% transferrin receptor+, and 90.5% HLR-DR+, whereas B cells in the later stage were observed to be less positive for each surface antigen: 36.1% Tac-Ag+, 8.3% B2+, 45.3% transferrin receptor+, and 58.7% HLR-DR+. By adding each factor to both B cell fractions, we also demonstrated functional differences in the two populations. B cells in the earlier stage of activation only differentiated in response to IL 2 or IL 2 + IFN-gamma but not to BCDF, which was in contrast to B cells in the later stage that did not differentiate in response to IL 2 but did differentiate to BCDF. However, B cells in both stages proliferated in response to IL 2 but not to BCDF. Finally, we separated B cells in the later stage into two populations by the Percoll discontinuous gradient centrifugation. Lower density (larger) B cells were observed to proliferate but not to differentiate in response to IL 2, whereas higher density (smaller) B cells were observed to differentiate in response to BCDF. Therefore, we conclude that activated B cells initially become large and gain Tac-Ag and differentiate in response to IL 2 alone as well as the combination of IL 2 and IFN-gamma, whereas later in the more mature stage they become smaller again and differentiate into Ig-secreting cells only in response to BCDF.
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Smith, Roger K. W., and Martin H. Johnson. "DNA replication and compaction in the cleaving embryo of the mouse." Development 89, no. 1 (October 1, 1985): 133–48. http://dx.doi.org/10.1242/dev.89.1.133.
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The effects of aphidicolin, a reversible inhibitor of DNA polymerase alpha, both on replication and on development of the mouse embryo from the 2- and 4-cell stages to the compacted late 8-cell stage have been assessed. The continuous presence of aphidicolin from G1 of the 4-cell stage resulted in inhibition of DNA replication and prevention of division from 4 to 8 cells, but was without effect on the timing or incidence of cell flattening, surface polarization and cytoplasmic polarization. The continuous presence of aphidicolin from G1 of the 2-cell stage resulted in inhibition of DNA replication, division, and polarization. Some slight intercellular flattening in a few embryos did occur. If addition of aphidicolin was delayed by 10 h to early in G2 of the 2-cell stage, further rounds of replication were blocked and some embryos failed to cleave to 4-cells. Nevertheless, almost all embryos showed evidence of flattening and polarization regardless of cell number. In contrast, if aphidicolin was added in G1 of the 2-cell stage and removed after 10 h, the cells showed delayed DNA replication, little evidence of division, and no cell flattening or polarization. We conclude that DNA replication at the 2-cell stage may be essential for the components of compaction studied, but that DNA replication at the 4- and 8-cell stages is not.
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Boettiger, David, Francois Huber, Laura Lynch, and Scott Blystone. "Activation of αvβ3-Vitronectin Binding Is a Multistage Process in which Increases in Bond Strength Are Dependent on Y747 and Y759 in the Cytoplasmic Domain of β3." Molecular Biology of the Cell 12, no. 5 (May 2001): 1227–37. http://dx.doi.org/10.1091/mbc.12.5.1227.
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Integrin receptors serve as mechanical links between the cell and its structural environment. Using αvβ3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between αvβ3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the αvβ3-vitronectinbinding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the β3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of αvβ3 integrin.
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Borjigin, Uyunbilig, Rhonda Davey, Keryn Hutton, and Muren Herrid. "Expression of promyelocytic leukaemia zinc-finger in ovine testis and its application in evaluating the enrichment efficiency of differential plating." Reproduction, Fertility and Development 22, no. 5 (2010): 733. http://dx.doi.org/10.1071/rd09237.
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Identification and enrichment of spermatogonial stem cells (SSCs) are critical steps in testis germ cell transplantation. The present study shows that expression of the protein promyelocytic leukaemia zinc-finger (PLZF) does not occur in all cells, only in gonocytes in neonatal testis (Stage 1) and a subpopulation of Type A spermatogonia in peripubertal (Stage 2), prepubertal (Stage 3) and post-pubertal (Stage 4) ovine testes. Dolichos biflorus agglutinin (DBA) lectin binding does not occur at any stage of testis development. The numbers of putative undifferentiated spermatogonia, germ cells and Sertoli cells were assessed by PLZF, VASA and vimentin staining, respectively. In paraffin sections, the percentage of PLZF-positive cells per tubule in samples derived from Stage 2 testis (12.2 ± 2.8%) was twofold higher than that from Stage 1 testis (6.4 ± 0.4%), but the percentages decreased in Stage 3 and Stage 4 testes (4.6 ± 0.7% and 3.1 ± 0.6%, respectively). Single cell suspensions from Stage 1 and Stage 2 testis were generated by two-step enzymatic digestion. The spermatogonia were enriched by 2 h and 2 + 16 h (overnight) differential plating on 0.2% gelatin-coated coated flasks. For Stage 1 testes, a sixfold increase in PLZF-positive cells was observed in 2 h differential plating and an almost 10-fold increase was produced following 2 + 16 h enrichment. There was less than a twofold increase in PLZF-positive cells between the 2 h and 2 + 16 h differential plating. A similar level of enrichment efficiency was also obtained for Stage 2 testis, but the percentage of PLZF-positive cells in the final enrichment was approximately one-third of that Stage 1. The efficiency of isolation and/or enrichment of PLZF-positive cells appears to depend on the maturity of the testis and the neonatal testis is better suited for isolation of gonocytes and/or putative SSCs.
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Yi, Zuoan, Laura L. Stunz, and Gail A. Bishop. "TNF receptor associated factor 3 plays a key role in development and function of invariant natural killer T cells." Journal of Experimental Medicine 210, no. 6 (May 6, 2013): 1079–86. http://dx.doi.org/10.1084/jem.20122135.
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TCR signaling is a prerequisite for early stage development of invariant natural killer T (iNKT) cells, whereas IL-15 signaling is required for expansion and maturation at later stages. In this study, we show that TNF receptor associated factor 3 (TRAF3) plays a critical role in the transition between these two distinct signaling pathways and developmental stages. TRAF3-deficient iNKT cells in CD4CreTRAF3flox/flox (T-TRAF3−/−) mice exhibit defective up-regulation of T-bet and CD122, two critical molecules for IL-15 signaling, and as a consequence, IL-15–mediated iNKT cell proliferation and survival are impaired. Consistently, development of iNKT cells in T-TRAF3−/− mice shows a major defect at developmental stages 2 and 3, but not stages 0 and 1. We further demonstrated that defective T-bet up-regulation occurring during the stage 1 to stage 2 transition results from reduced TCR signaling in TRAF3−/− iNKT cells. In addition, mature TRAF3−/− iNKT cells displayed defective cytokine responses upon TCR stimulation. Collectively, our results reveal that by modulating the relative strength of TCR signaling, TRAF3 is an important regulator of iNKT cell development and functions.
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Pyaram, Kalyani, and Cheong-Hee Chang. "Wnt/β-catenin signaling regulates IL-15-mediated terminal maturation and interferon-gamma production of invariant NKT cells." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 121.14. http://dx.doi.org/10.4049/jimmunol.196.supp.121.14.
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Abstract The Wnt/β-catenin signaling pathway is important for various cellular functions including survival and proliferation. The indispensable role of this pathway in conventional T cell development is well established but little is known about its role in innate T cell development. In this study, we investigated the role of the Wnt/β-catenin signaling in the development of invariant natural killer T (NKT) cells using mice that express a constitutively active form of b-catenin (CAT-Tg) or deficient in β-catenin (CAT-KO). Thymic NKT cells were increased in CAT-Tg and decreased in CAT-KO mice compared to their wild type littermates. Thymic NKT cells progress from Stage 0 to Stage 3 as they mature. In the absence of Wnt signaling (CAT-KO), we observed accumulation of Stage 0 NKT cells. In contrast, in CAT-Tg mice, the final maturation was compromised - more Stage 2 but less Stage 3 NKT cells. Thus, stage-specific regulation of Wnt signaling may be critical for the thymic NKT development. Indeed, endogenous β-catenin expression was high in early stages but low in late stages, suggesting that its reduction mounts the stage-2 to -3 NKT transition. The CAT-Tg mice also had lower number of IFN-gamma expressing NKT cells, which, in these cells, correlated with reduced expression of transcription factor Tbet and CD122 (IL-2R & IL-15R subunit), requisites for final maturation and IFN-gamma expression of NKT cells. Further, although PLZF, a master transcription factor for NKT cell development is expressed at high levels in CAT-Tg NKT cells, Wnt signaling-mediated NKT cell maturation was found to be independent of PLZF. Together, our data shows that regulation of Wnt/β-catenin signaling is necessary for terminal maturation and IFN-gamma production of NKT cells.
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Sathananthan, Henry, Lynne Selwood, Isabel Douglas, and Kamani Nanayakkara. "Early cleavage to formation of the unilaminar blastocyst in the marsupial Antechinus stuartii: ultrastructure." Reproduction, Fertility and Development 9, no. 2 (1997): 201. http://dx.doi.org/10.1071/r96049.
Full textAbstract:
The development of Antechinus stuartiifrom the 2-cell stage to the blastocyst stage in vivo was examined by routine transmission electron microscopy. The 2–8-cell stages had a similar organization of organelles, whereas the 16- to 32-cell stages had pluriblast cells and trophoblast cells forming an epithelium closely apposed to the zona pellucida. Specialized cell–zona plugs were formed at the 8-cell stage, and primitive cell junctions appeared in later conceptuses. The cytoplasmic organelles included mitochondria, lysosomes, aggregates of smooth endoplasmic reticulum, lipid and protein yolk bodies and fibrillar arrays, possibly contractile in function. Nuclei had uniformly-dispersed dense chromatin. Nucleoli of 2–4-cell conceptuses were dense, compact and fibrillar, and those of 8-cell conceptuses and later conceptuses were finely granular and became progressively reticulated. The embryonic genome is probably not switched on before the 8-cell stage. Sperm tails were detected in cells in several early conceptuses. The yolk mass had the same organelles as cells. Centrioles were discovered for the first time in marsupial conceptuses. These were prominently situated at a spindle pole in a 32-cell blastomere and were associated with a nucleus and sperm tail at the 4-cell stage. It is very likely that the paternal centrosome is inherited at fertilization and perpetuated in Antechinus embryos during cleavage.
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Wu, Chia-Chao, Huey-Kang Sytwu, Kuo-Cheng Lu, and Yuh-Feng Lin. "Role of T Cells in Type 2 Diabetic Nephropathy." Experimental Diabetes Research 2011 (2011): 1–9. http://dx.doi.org/10.1155/2011/514738.
Full textAbstract:
Type 2 diabetic nephropathy (DN) is the most common cause of end-stage renal disease and is increasingly considered as an inflammatory disease characterized by leukocyte infiltration at every stage of renal involvement. Inflammation and activation of the immune system are closely involved in the pathogenesis of diabetes and its microvascular complications. Macrophage has been well recognized to play an important role in type 2 DN, leukocyte infiltration, and participated in process of DN, as was proposed recently. Th1, Th2, Th17, T reg, and cytotoxic T cells are involved in the development and progression of DN. The purpose of this review is to assemble current information concerning the role of T cells in the development and progression of type 2 DN. Specific emphasis is placed on the potential interaction and contribution of the T cells to renal damage. The therapeutic strategies involving T cells in the treatment of type 2 DN are also reviewed. Improving knowledge of the recognition of T cells as significant pathogenic mediators in DN reinforces the possibility of new potential therapeutic targets translated into future clinical treatments.
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Taylor, Tyl H., Darren K. Griffin, Seth L. Katz, Jack L. Crain, Lauren Johnson, and Susan Gitlin. "Technique to ‘Map' Chromosomal Mosaicism at the Blastocyst Stage." Cytogenetic and Genome Research 149, no. 4 (2016): 262–66. http://dx.doi.org/10.1159/000449051.
Full textAbstract:
The purpose of this study was to identify a technique that allows for comprehensive chromosome screening (CCS) of individual cells within human blastocysts along with the approximation of their location in the trophectoderm relative to the inner cell mass (ICM). This proof-of-concept study will allow for a greater understanding of chromosomal mosaicism at the blastocyst stage and the mechanisms by which mosaicism arises. One blastocyst was held by a holding pipette and the ICM was removed. While still being held, the blastocyst was further biopsied into quadrants. To separate the individual cells from the biopsied sections, the sections were placed in calcium/magnesium-free medium with serum for 20 min. A holding pipette was used to aspirate the sections until individual cells were isolated. Individual cells from each section were placed into PCR tubes and prepped for aCGH. A total of 18 cells were used for analysis, of which 15 (83.3%) amplified and provided a result and 3 (16.7%) did not. Fifteen cells were isolated from the trophectoderm; 13 (86.7%) provided an aCGH result, while 2 (13.3%) did not amplify. Twelve cells were euploid (46,XY), while 1 was complex abnormal (44,XY), presenting with monosomy 7, 10, 11, 13, and 19, and trisomy 14, 15, and 21. A total of 3 cells were isolated from the ICM; 2 were euploid (46,XY) and 1 did not amplify. Here, we expand on a previously published technique which disassociates biopsied sections of the blastocyst into individual cells. Since the blastocyst sections were biopsied in regard to the position of the ICM, it was possible to reconstruct a virtual image of the blastocyst while presenting each cell's individual CCS results.
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Mohd Yusof, Hazwani, Sharaniza Ab-Rahim, Wan Zurinah Wan Ngah, Sheila Nathan, A. Rahman A Jamal, and Musalmah Mazlan. "Extracellular Metabolites Profile of Different Stages Colorectal Cancer Cell Lines." Science Letters 15, no. 2 (June 15, 2021): 26–41. http://dx.doi.org/10.24191/sl.v15i2.13810.
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Metabolic footprinting involves the determination of metabolites excreted or secreted by the cells. This study aimed to identify the differential extracellular metabolites in colorectal cancer (CRC) cells for the determination of molecular changes that occur as CRC progresses. CRC cells at different stages ie; SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D) were grown in culture. The media in which the cells were grown are subjected to metabolomics profiling using Liquid Chromatography Mass Spectrometry-Quadrupole Time of Flight (LC/MS Q-TOF). Statistical and metabolic pathway analysis was performed using Metaboanalyst software and identification of metabolites was determined by the METLIN database. A total of 27 differential extracellular metabolites were identified in CRC cells of different stages compared to stage A cells. Data from the Partial least squares-discriminant analysis (PLS-DA) score plot shows a clear separation between CRC cells of different stages with a few overlaps between stage B and C. Further analysis using variable importance in projection (VIP) revealed 14 differential extracellular metabolites that were most significant in differentiating CRC cells of the advanced stages from stage A which are 5-hydroxy-L-tryptophan, indoleacetaldehyde, 4,5-dimethylthiazole, 8-oxodiacetoxyscirpenol, bisnorbiotin, 5-amino-6-(5'phosphoribosylamino) uracil, glyceryl 5-hydroxydecanoate, sphinganine, 8,8-diethoxy-2,6-dimethyl-2-octanol, l-cystine, thiamine acetic acid, phytosphingosine, PE (20:4(5Z,8Z,11Z,14Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)), N-(2R-hydroxypentacosano-yl)-2S-amino-1,3S,4R-octadecanetriol. The different expressions of metabolites may indicate altered metabolic pathways in the more advanced CRC cells compared to stage A. This study highlights the importance of conducting both metabolomics profiling of extracellular and intracellular to generate a more complete understanding on the molecular changes that occur as CRC progresses.
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Sawhney, V. K., and S. K. Bhadula. "Microsporogenesis in the normal and male-sterile stamenIess-2 mutant of tomato (Lycopersicon esculentum)." Canadian Journal of Botany 66, no. 10 (October 1, 1988): 2013–21. http://dx.doi.org/10.1139/b88-275.
Full textAbstract:
The development of microspores and the associated changes in the tapetum were examined in the normal (+/+) and male-sterile, stamenless-2 (sl-2/sl-2) mutant anthers of tomato (Lycopersicon esculentum). Anthers of eight comparable stages, from the microspore mother cell stage to anthesis, of both lines were processed for light microscopy. Until the formation of tetrads (stage ii), there were no differences in the sporogenous tissue, but the tapetal cells of the mutant were more enlarged than the normal and had, at places, divided to form a bilayer. Later, the tapetal cells in both lines became amoeboid and had sporopollenin-like deposits. At stage iv, whereas the tapetal cells of the normal had started to degenerate, those of the mutant were intact but had large vacuoles. Also at this stage, the deposition of exine was evident in normal microspores, but it was lacking in most mutant microspores, which enlarged considerably and eventually degenerated. From stage v onwards, the normal microspores progressed from the binucleate pollen to pollen containing many vacuoles to mature pollen. In the mutant, tapetum degeneration was delayed until stage v, and later, although some microspores closer to the tapetum appeared normal, most either were empty or had large vacuoles. It is suggested that the delay in tapetum degeneration coupled with the failure of exine deposition, presumably associated with low esterase activity, is responsible for pollen degeneration in the sl-2/sl-2 mutant.
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Gusev, Evgenii, Alexey Sarapultsev, Liliya Solomatina, and Valeriy Chereshnev. "SARS-CoV-2-Specific Immune Response and the Pathogenesis of COVID-19." International Journal of Molecular Sciences 23, no. 3 (February 2, 2022): 1716. http://dx.doi.org/10.3390/ijms23031716.
Full textAbstract:
The review aims to consolidate research findings on the molecular mechanisms and virulence and pathogenicity characteristics of coronavirus disease (COVID-19) causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and their relevance to four typical stages in the development of acute viral infection. These four stages are invasion; primary blockade of antiviral innate immunity; engagement of the virus’s protection mechanisms against the factors of adaptive immunity; and acute, long-term complications of COVID-19. The invasion stage entails the recognition of the spike protein (S) of SARS-CoV-2 target cell receptors, namely, the main receptor (angiotensin-converting enzyme 2, ACE2), its coreceptors, and potential alternative receptors. The presence of a diverse repertoire of receptors allows SARS-CoV-2 to infect various types of cells, including those not expressing ACE2. During the second stage, the majority of the polyfunctional structural, non-structural, and extra proteins SARS-CoV-2 synthesizes in infected cells are involved in the primary blockage of antiviral innate immunity. A high degree of redundancy and systemic action characterizing these pathogenic factors allows SARS-CoV-2 to overcome antiviral mechanisms at the initial stages of invasion. The third stage includes passive and active protection of the virus from factors of adaptive immunity, overcoming of the barrier function at the focus of inflammation, and generalization of SARS-CoV-2 in the body. The fourth stage is associated with the deployment of variants of acute and long-term complications of COVID-19. SARS-CoV-2’s ability to induce autoimmune and autoinflammatory pathways of tissue invasion and development of both immunosuppressive and hyperergic mechanisms of systemic inflammation is critical at this stage of infection.
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Asaoku, H., M. Kawano, K. Iwato, O. Tanabe, H. Tanaka, T. Hirano, T. Kishimoto, and A. Kuramoto. "Decrease in BSF-2/IL-6 response in advanced cases of multiple myeloma." Blood 72, no. 2 (August 1, 1988): 429–32. http://dx.doi.org/10.1182/blood.v72.2.429.429.
Full textAbstract:
Abstract Human myeloma cells freshly isolated from 40 patients with IgG multiple myeloma (MM, 10 in stage I and 30 in stage III), were cultured for 48 hours with recombinant B cell stimulatory factor 2 (rBSF-2)/interleukin- 6 (IL-6), which is considered a major growth factor for myeloma cells. Uptake of 3H-thymidine by these purified myeloma cells was measured, and BSF-2 response was evaluated by stimulation index and delta cpm induced by rBSF-2. Myeloma cells from cases in stage I responded to rBSF-2 better than those in stage III. Moreover rBSF-2 responders also showed better response to chemotherapy. Therefore, these results suggest that in vitro response of myeloma cells to BSF-2 correlates with disease progression and clinical response in patients of MM.
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Asaoku, H., M. Kawano, K. Iwato, O. Tanabe, H. Tanaka, T. Hirano, T. Kishimoto, and A. Kuramoto. "Decrease in BSF-2/IL-6 response in advanced cases of multiple myeloma." Blood 72, no. 2 (August 1, 1988): 429–32. http://dx.doi.org/10.1182/blood.v72.2.429.bloodjournal722429.
Full textAbstract:
Human myeloma cells freshly isolated from 40 patients with IgG multiple myeloma (MM, 10 in stage I and 30 in stage III), were cultured for 48 hours with recombinant B cell stimulatory factor 2 (rBSF-2)/interleukin- 6 (IL-6), which is considered a major growth factor for myeloma cells. Uptake of 3H-thymidine by these purified myeloma cells was measured, and BSF-2 response was evaluated by stimulation index and delta cpm induced by rBSF-2. Myeloma cells from cases in stage I responded to rBSF-2 better than those in stage III. Moreover rBSF-2 responders also showed better response to chemotherapy. Therefore, these results suggest that in vitro response of myeloma cells to BSF-2 correlates with disease progression and clinical response in patients of MM.
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Dominov, Janice A., Jonathan J. Dunn, and Jeffrey Boone Miller. "Bcl-2 Expression Identifies an Early Stage of Myogenesis and Promotes Clonal Expansion of Muscle Cells." Journal of Cell Biology 142, no. 2 (July 27, 1998): 537–44. http://dx.doi.org/10.1083/jcb.142.2.537.
Full textAbstract:
We show that Bcl-2 expression in skeletal muscle cells identifies an early stage of the myogenic pathway, inhibits apoptosis, and promotes clonal expansion. Bcl-2 expression was limited to a small proportion of the mononucleate cells in muscle cell cultures, ranging from ∼1–4% of neonatal and adult mouse muscle cells to ∼5–15% of the cells from the C2C12 muscle cell line. In rapidly growing cultures, some of the Bcl-2–positive cells coexpressed markers of early stages of myogenesis, including desmin, MyoD, and Myf-5. In contrast, Bcl-2 was not expressed in multinucleate myotubes or in those mononucleate myoblasts that expressed markers of middle or late stages of myogenesis, such as myogenin, muscle regulatory factor 4 (MRF4), and myosin. The small subset of Bcl-2–positive C2C12 cells appeared to resist staurosporine-induced apoptosis. Furthermore, though myogenic cells from genetically Bcl-2–null mice formed myotubes normally, the muscle colonies produced by cloned Bcl-2–null cells contained only about half as many cells as the colonies produced by cells from wild-type mice. This result suggests that, during clonal expansion from a muscle progenitor cell, the number of progeny obtained is greater when Bcl-2 is expressed.
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Heeger, David J. "Half-squaring in responses of cat striate cells." Visual Neuroscience 9, no. 5 (November 1992): 427–43. http://dx.doi.org/10.1017/s095252380001124x.
Full textAbstract:
AbstractSimple cells in striate cortex have been depicted as rectified linear operators, and complex cells have been depicted as energy mechanisms (constructed from the squared sums of linear operator outputs). This paper discusses two essential hypotheses of the linear/energy model: (1) that a cell's selectivity is due to an underlying (spatiotemporal and binocular) linear stage; and (2) that a cell's firing rate depends on the squared output of the underlying linear stage. This paper reviews physiological measurements of cat striate cell responses, and concludes that both of these hypotheses are supported by the data.
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Troilo, Arianna, Claudia Wehr, Iga Janowska, Justyna Rawluk, Jens Thiel, Nils Venhoff, Georg Herget, et al. "In vitro modeling of human B cell development can identify distinct physiopathological patterns in primary antibody deficiency (PAD)." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 53.18. http://dx.doi.org/10.4049/jimmunol.202.supp.53.18.
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Abstract Bone marrow (BM) analysis show that 20% of PAD patients harbor a block at an early stage of B cell development. The aim of this work is to study the mechanisms of this developmental block. We set up a feeder free cultivation system that in healthy donors leads to the development of lymphocyte progenitors until the stage of Immature B cells. BM derived CD34+ cells are expanded in cytokine cocktail for 2 weeks. From day 14 to day 49 cells are cultivated in cytokine-free medium and analyzed once a week by flow cytometry and RNA is collected for PCR assays. BM derived CD34+ cells from 15 PAD patients and 2 Btk-deficient patients were tested in vitro for their capability to develop into IgM+ Immature B cells. CD34+ cells from patients showing a normal B cell development in vivo (6/17) developed in vitro until the stage of Immature B cells. Among PAD patients presenting a block at early B cell stage, 5/11, including the Btk-deficient patients, could not reach the Immature B cell stage, while 6/11 could develop in vitro until the stage of IgM+ B cells. Among this last group, reaching Immature B cell stage in vitro but not in vivo, 2/6 patients showed a rapid in vitro development of Immature B cells at day 14, but presented a progressive exhaustion of the common lymphoid progenitors (CLP) compartment. In patients with a block in B cell development (in vivo and in vitro), PCR analysis revealed adelayed expression of transcription factors E2A, CD79a and PAX5, reflecting an impaired lineage commitment. Combining BM phenotyping and in vitro modeling we identified four patients’ groups characterized by (1) an intrinsic B cell defect, (2) a defect in the BM microenvironment impairing early stages of B cell development, (3) a defect in repopulation of CLP and (4) normal B cell development.
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Sauer, Karin, Anne K. Camper, Garth D. Ehrlich, J. William Costerton, and David G. Davies. "Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm." Journal of Bacteriology 184, no. 4 (February 15, 2002): 1140–54. http://dx.doi.org/10.1128/jb.184.4.1140-1154.2002.
Full textAbstract:
ABSTRACT Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.
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Feldherr, C., D. Akin, and M. S. Moore. "The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis." Journal of Cell Science 111, no. 13 (July 1, 1998): 1889–96. http://dx.doi.org/10.1242/jcs.111.13.1889.
Full textAbstract:
Previtellogenic, stage-1 Xenopus oocytes produce mainly 5S and tRNA, whereas vitellogenic oocytes, stages 2–6, synthesize predominantly 18S and 28S rRNA. Using nucleoplasmin-coated gold as a transport substrate, it was determined that the shift in synthesis from small to large RNAs during oogenesis is accompanied by an increase in both the rates of signal-mediated nuclear import and the functional size of nuclear pores. It was observed that, despite the reduction in transport capacity, gold still accumulated at the cytoplasmic surface of the pores in stage-1 oocytes. This suggested that transport in these cells is limited by translocation factors, rather than by cytoplasmic binding factors. Analysis of extracts prepared from stage-1 and vitellogenic oocytes revealed that the transport factor p10 is more abundant in stage-1 cells. Microinjection of purified p10 into stage-2 oocytes reduced the nuclear import of large gold particles to the level observed in stage-1 cells. It is concluded that p10 can modulate transport through the pores by regulating the functional size of the central transporter element.
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Foley, A. C., K. G. Storey, and C. D. Stern. "The prechordal region lacks neural inducing ability, but can confer anterior character to more posterior neuroepithelium." Development 124, no. 15 (August 1, 1997): 2983–96. http://dx.doi.org/10.1242/dev.124.15.2983.
Full textAbstract:
The avian equivalent of Spemann's organizer, Hensen's node, begins to lose its ability to induce a nervous system from area opaca epiblast cells at stage 4+, immediately after the full primitive streak stage. From this stage, the node is no longer able to induce regions of the nervous system anterior to the hindbrain. Stage 4+ is marked by the emergence from the node of a group of cells, the prechordal mesendoderm. Here we have investigated whether the prechordal region possesses the lost functions of the organizer, using quail-chick chimaeras to distinguish graft- and host-derived cells, together with several region-specific molecular markers. We find that the prechordal region does not have neural inducing ability, as it is unable to divert extraembryonic epiblast cells to a neural fate. However, it can confer more anterior character to prospective hindbrain cells of the host, making them acquire expression of the forebrain markers tailless and Otx-2. It can also rescue the expression of Krox-20 and Otx-2 from nervous system induced by an older (stage 5) node in extraembryonic epiblast. We show that these properties reflect a true change of fate of cells rather than recruitment from other regions. The competence of neuroectoderm to respond to anteriorizing signals declines by stages 7–9, but both posteriorizing signals and the ability of neuroectoderm to respond to them persist after this stage.
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Keerthivasan, Ganesan, Jing Yang, Piu Wong, John Doench, David E. Root, and Peng Ji. "Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis." Blood 120, no. 21 (November 16, 2012): 3199. http://dx.doi.org/10.1182/blood.v120.21.3199.3199.
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Abstract Abstract 3199 Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development. Disclosures: No relevant conflicts of interest to declare.
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Watanabe, M., M. Kinutani, M. Naito, O. Ochi, and Y. Takashima. "Distribution analysis of transferred donor cells in avian blastodermal chimeras." Development 114, no. 2 (February 1, 1992): 331–38. http://dx.doi.org/10.1242/dev.114.2.331.
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Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.
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Shimoni, Avichai, Hadar Marcus, Allon Canaan, David Ergas, Magda David, Alain Berrebi, and Yair Reisner. "A Model for Human B-Chronic Lymphocytic Leukemia in Human/Mouse Radiation Chimera: Evidence for Tumor-Mediated Suppression of Antibody Production in Low-Stage Disease." Blood 89, no. 6 (March 15, 1997): 2210–18. http://dx.doi.org/10.1182/blood.v89.6.2210.
Full textAbstract:
Abstract B-chronic lymphocytic leukemia (BCLL) is a lymphoproliferative disease that is characterized by clonal expansion of CD5+ B cells. BCLL is associated with secondary immunodeficiency and hypogammaglobulinemia. It has been suggested that T-cell dysregulation may play a role in the hypogammaglobulinemia and in the increased incidence of autoimmunity in BCLL patients. We attempted to transfer human peripheral blood mononuclear cells (PBMC) from BCLL patients in different stages of the disease into immunodeficient mice. PBMC from BCLL patients in stage 0, stages I to II, and stages III to IV were transplanted into the peritoneal cavity of lethally irradiated Balb/c or beige/nude/Xid (BNX) mice radioprotected with bone marrow (BM) from severe combined immunodeficiency (SCID) mice. Different engraftment profiles were found in the chimeric mice 2 weeks after transplantation of PBMC according to the disease stage of the BCLL donors. Infusion of PBMC from donors in stage 0 led to marked engraftment of human T cells, whereas the human tumor cells could hardly be detected. In contrast, chimeric mice receiving PBMC from patients in stage III to IV disease exhibited engraftment with a dominance of tumor cells, compared with a miniscule level of T cells. The ability of the engrafted cells to produce human Ig was also found to be correlated with the disease stage of the donor, although all donors had the same magnitude of hypogammaglobulinemia. Total human Ig production in the chimeric mice was normal in mice receiving PBMC from donors in stage 0, whereas in chimeric mice engrafted with PBMC from donors in stages III to IV almost no human Igs could be detected. This differential reconstitution of antibody production in the mouse model according to the stage of the patient's disease will allow further studies on possible cellular interactions between malignant and immune cells in BCLL.
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Vejlsted, M., H. Offenberg, and P. Maddox-Hyttel. "147 IDENTIFICATION OF PRIMORDIAL GERM CELLS IN PORCINE EMBRYOS FROM THE PRIMITIVE STREAK STAGE." Reproduction, Fertility and Development 18, no. 2 (2006): 181. http://dx.doi.org/10.1071/rdv18n2ab147.
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In embryonic stem cell research, Oct-4 is one of the most widely used markers of pluripotency. Moreover, at least in the mouse, this marker is restricted to primordial germ cells (PGCs) after gastrulation. Vimentin is often used as a marker of mesoderm/mesenchyme in embryonic tissues and appears to localize to the same embryonic cells as Oct-4, at least in the bovine epiblast. The expression of neither of these markers has been completely addressed in the pig. Therefore, the purpose of the present study was to examine the expression of Oct-4 and vimentin in the porcine epiblast during differentiation and establishment of the three germ layers, i.e. the process of gastrulation. A total of 410 porcine embryos were collected at 8 to 17 days post-insemination from 29 sows of the Danish Landrace breed. Embryos were categorized based on stereo-microscopic observations into the following stages: pre-streak stages 1 and 2, primitive streak stage, neural groove stage, and somite stage. Specimens were fixed at all stages, dehydrated and embedded in paraffin wax. Selected embryos at each stage (n = 28) were completely cut into serial sections for immunohistochemical evaluation of Oct-4 and vimentin. Pre-streak stage 1 embryos were defined by lack of polarization of the embryonic disk, whereas in pre-streak stage 2 embryos a crescent shaped thickening was seen at the posterior pole of the disk. This thickening, marking the first morphological anterior-posterior polarization of the embryo proper, was shown to be a site of incipient ingression of cells from the epiblast. Immunohistochemical analyses localized Oct-4 to nuclei and vimentin to cytoplasm of both founding and ingressing epiblast cells. During formation of mesoderm and endoderm, at the primitive streak stage, solitary Oct-4 positive cells, i.e. potential PGCs, were seen scattered in the endoderm. Cells of the epiblast displayed positive labeling for Oct-4 until specification for the ectoderm cell lineage at the subsequent neural groove stage. In mesoderm, Oct-4 likewise disappeared by the time of formation of the first somites, defining the following somite stage. Thus, at this stage the only cells labeled for Oct-4, i.e. potential PGCs, were seen solitarily scattered in the endoderm. By the 15-somite stage, such cells were no longer visible in the endoderm but were seen located in the mesoderm, spreading from the stalk of the yolk sac and allantois and extending through the mid- and hindgut areas into the incipient genital ridge. Vimentin localized to the mesenchyme and most other derivatives of neural crest and mesodermal origin. In conclusion, based on Oct-4 labeling and distribution pattern, we strongly believe that we have identified the porcine PGCs from the primitive streak stage.
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Fu, Bo, Hong Ma, and Di Liu. "2-Cell-like Cells: An Avenue for Improving SCNT Efficiency." Biomolecules 12, no. 11 (November 1, 2022): 1611. http://dx.doi.org/10.3390/biom12111611.
Full textAbstract:
After fertilization, the zygote genome undergoes dramatic structural reorganization to ensure the establishment of totipotency, and then the totipotent potential of the zygote or 2-cell-stage embryo progressively declines. However, cellular potency is not always a one-way street. Specifically, a small number of embryonic stem cells (ESCs) occasionally overcome epigenetic barriers and transiently convert to a totipotent status. Despite the significant potential of the somatic cell nuclear transfer (SCNT) technique, the establishment of totipotency is often deficient in cloned embryos. Because of this phenomenon, the question arises as to whether strategies attempting to induce 2-cell-like cells (2CLCs) can provide practical applications, such as reprogramming of somatic cell nuclei. Inspired by strategies that convert ESCs into 2CLCs, we hypothesized that there will be a similar pathway by which cloned embryos can establish totipotent status after SCNT. In this review, we provide a snapshot of the practical strategies utilized to induce 2CLCs during investigations of the development of cloned embryos. The 2CLCs have similar transcriptome and chromatin features to that of 2-cell-stage embryos, and we propose that 2CLCs, already a valuable in vitro model for dissecting totipotency, will provide new opportunities to improve SCNT efficiency.
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O'Reilly, L. A., A. W. Harris, D. M. Tarlinton, L. M. Corcoran, and A. Strasser. "Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo." Journal of Immunology 159, no. 5 (September 1, 1997): 2301–11. http://dx.doi.org/10.4049/jimmunol.159.5.2301.
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Abstract B lymphocyte differentiation proceeds through a series of alternating stages of proliferative expansion interspersed with noncycling stationary phases during which cells undergo either positive selection or apoptotic cell death. The molecular control of cell cycle progression and that of apoptosis appear to be interconnected. Overexpression of Bcl-2 in lymphocytes or fibroblasts antagonizes apoptosis and delays their transition from the quiescent state into the cell cycle. We have undertaken a systematic analysis of the impact of bcl-2 transgene expression on cell cycle distribution and turnover rate of developing B lymphocytes in normal mice and in mutant animals in which B cell differentiation is arrested at the pro-B/pre-BI or the pre-BII stage. These experiments revealed that overexpression of Bcl-2 reduces proliferation and slows turnover of B cells at all stages of development. This demonstrates that Bcl-2 can retard transition of B cells between the quiescent and the cycling state regardless of the mitogenic stimulus and the differentiation stage. The implications of these results for the normal control of B lymphopoiesis and for lymphomagenesis are discussed.
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haku, Sona, Hiromichi Wakui, Kengo Azushima, Kotaro Haruhara, Sho Kinguchi, Kohji Ohki, Kazushi Uneda, et al. "Early Enhanced Leucine-Rich α-2-Glycoprotein-1 Expression in Glomerular Endothelial Cells of Type 2 Diabetic Nephropathy Model Mice." BioMed Research International 2018 (November 1, 2018): 1–9. http://dx.doi.org/10.1155/2018/2817045.
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Abnormal angiogenesis plays a major role in the development of early stage diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a classical proangiogenic factor that regulates abnormal glomerular angiogenesis linked to glomerular hypertrophy in the early stage of diabetic nephropathy. Leucine-rich α-2-glycoprotein-1 (LRG1) was recently reported as a novel proangiogenic factor that is expressed in endothelial cells and promotes angiogenesis by modulating the transforming growth factor-β signaling pathway. However, the pathophysiology of LRG1 in diabetic nephropathy remains largely unknown. In the present study, we investigated intrarenal expression of the novel proangiogenic factor LRG1 in diabetic db/db mice by immunohistochemistry and a laser capture microdissection method during the development of diabetic nephropathy. We hypothesized that glomerular LRG1 expression is increased earlier than VEGF expression under conditions of pathological angiogenesis in the early stage of diabetic nephropathy. Thus, we compared glomerular expression of VEGF and LRG1 in diabetic db/db mice at 16 and 24 weeks of age. At 16 weeks, diabetic db/db mice exhibited glomerular hypertrophy with abnormal angiogenesis characterized by endothelial cell proliferation, which was concomitant with an increase in LRG1 expression of glomerular endothelial cells. However, glomerular VEGF expression was not increased at this early stage. At 24 weeks, the features of early diabetic nephropathy in db/db mice had developed further, along with further enhanced glomerular LRG1 expression. At this late stage, glomerular VEGF and fibrosis-related-gene expression was also significantly increased compared with nondiabetic db/m mice. These results suggest that LRG1 plays a pivotal role in the initial development of diabetic nephropathy by promoting abnormal angiogenesis, thereby suggesting that LRG1 is a potential preemptive therapeutic target of diabetic nephropathy.
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Hashem, M. A., D. P. Bhandari, S. K. Kang, and B. C. Lee. "40 TREATMENT OF DONOR CELLS AND ITS EFFECT ON INTERSPECIES NUCLEAR TRANSFER." Reproduction, Fertility and Development 19, no. 1 (2007): 138. http://dx.doi.org/10.1071/rdv19n1ab40.
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The present study was undertaken to examine the effect of donor cells, under a variety of treatment effects, on the development of goral porcine reconstructed embryos. Three experiments were performed, each with a one-way completely randomized design involving 3 to 4 replicates of all. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused on the effects of cycling, serum-starved (SS), and fully confluent stages of goral cells when reconstructed with porcine enucleated oocytes. In Experiment II, the effects of 2 antioxidants, β-mercaptoethanol (β-ME, 10 �M) and cysteine (2 mM), were examined after cells were fully confluent without serum starvation for 4 h. In Experiment III, the effect of different levels of dimethylsulfoxide (DMSO) at 0%, 0.5%, and 1.0% were tested, after 4 h of treatment, on the development rate after reconstruction with enucleated porcine oocytes. From the results, it appears that there were no significant (P > 0.05) differences from cleavage to morula among cyclic, SS, and fully confluent stages of the cell cycle. None of the treated group reached the blastocyst stage. There were no significant differences at the fused, 2- to 4-cell, and morula stages of embryo development after treatment of the donor cells with β-ME and cysteine before nuclear transfer. However, in the case of 8- to 16-cell stages, there were significant differences between β-ME and cysteine; the donor cells treated with β-ME had a better development rate than those treated with cysteine. No significant differences were observed in fusion, 2- to 4-cell, 8- to 16-cell, blastocyst, and hatching blastocyst stages at the 0.0, 0.5, and 1.0% levels of DMSO. However, there were statistically significant (P < 0.05) differences observed at the morula stage of embryo development. When donor cells were treated for 4 h with 0.5 and 1.0% levels of DMSO, goral-porcine reconstructed embryos reached the morula stage. From the results it can be concluded that goral somatic cells can be de-differentiated in porcine oocytes after treated with antioxidants and DMSO.
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Yu, Chen, Yanxia Liu, Zhenchuan Miao, Ming Yin, Wei Lu, Yaxin Lv, Mingxiao Ding, and Hongkui Deng. "Retinoic acid enhances the generation of hematopoietic progenitors from human embryonic stem cell–derived hemato-vascular precursors." Blood 116, no. 23 (December 2, 2010): 4786–94. http://dx.doi.org/10.1182/blood-2010-01-263335.
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Abstract Current induction schemes directing hematopoietic differentiation of human embryonic stem cells (hESCs) are not well defined to mimic the sequential stages of hematopoietic development in vivo. Here, we report a 3-stage method to direct differentiation of hESCs toward hematopoietic progenitors in chemically defined mediums. In the first 2 stages, we efficiently generated T-positive primitive streak/mesendoderm cells and kinase domain receptor–positive (KDR+) platelet-derived growth factor receptor α–negative (PDGFRα−) hemato-vascular precursors sequentially. In the third stage, we found that cells in a spontaneous differentiation condition mainly formed erythroid colonies. Addition of all-trans retinoic acid (RA) greatly enhanced generation of hematopoietic progenitors in this stage while suppressing erythroid development. The RA-treated cells highly expressed definitive hematopoietic genes, formed large numbers of multilineage and myeloid colonies, and gave rise to greater than 45% CD45+ hematopoietic cells. When hematopoietic progenitors were selected with CD34 and C-Kit, greater than 95% CD45+ hematopoietic cells could be generated. In addition, we found that endogenous RA signaling at the second stage was required for vascular endothelial growth factor/basic fibroblast growth factor–induced hemato-vascular specification, whereas exogenously applied RA efficiently induced KDR−PDGFRα+ paraxial mesoderm cells. Our study suggests that RA signaling plays diverse roles in human mesoderm and hematopoietic development.
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Palucka, Karolina A., Nicolas Taquet, Francoise Sanchez-Chapuis, and Jean Claude Gluckman. "Dendritic Cells as the Terminal Stage of Monocyte Differentiation." Journal of Immunology 160, no. 9 (May 1, 1998): 4587–95. http://dx.doi.org/10.4049/jimmunol.160.9.4587.
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Abstract Monocytes (MO) cultured for ≥5 days with either macrophage-CSF (M-CSF) or granulocyte macrophage (GM)-CSF and IL-4 differentiated without concomitant proliferation into CD14+ macrophages (Mφ) or CD1a+ dendritic cells (DC), respectively. When adherent and nonadherent CD14high Mφ from M-CSF cultures were separated and cultured further in cytokine-free medium or with GM-CSF/IL-4, most cells from both fractions that were exposed to GM-CSF/IL-4 acquired CD1a expression and DC morphology and function. Conversely, GM-CSF/IL-4 withdrawal at day 5 and additional culture of sorted CD1a+ DC for 2 to 7 days in cytokine-free medium led to cells rapidly becoming adherent CD1a−CD14+ Mφ. Replacing GM-CSF/IL-4 with M-CSF hastened the conversion of DC to Mφ without increasing cell numbers. CD1a+CD14−CD83+ mature DC were induced by a ≥2-day exposure to MO-conditioned medium, LPS, or TNF-α/IL-1β. Upon cytokine removal or culture with M-CSF, DC that had been pushed to maturation by conditioned medium or LPS remained stable or died in the new environment. TNF-α/IL-1β-driven DC displayed heterogeneous CD83 expression and could thus be sorted into CD83high and CD83low/− cells; in cytokine-free medium or in M-CSF, most CD83low/− cells converted to Mφ, whereas most CD83high cells remained nonadherent CD1a+CD14− or died and thus appeared truly terminally differentiated. Hence, MO are precursors of Mφ as well as of DC, with each cell type having the capability to convert into the other until late in the differentiation/maturation process. Accordingly, the cytokine environment and the presence of differentiation and/or other stimulatory signals may be the “final decision-making factors” determining whether these cells will acquire DC or Mφ characteristics and function.
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Miller, R. A., M. K. Rozans, A. A. Ythier, and T. B. Strom. "Stages of T cell activation: continued antigen dependence of IL 2-producing cells after IL 2 receptor expression." Journal of Immunology 136, no. 3 (February 1, 1986): 977–83. http://dx.doi.org/10.4049/jimmunol.136.3.977.
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Abstract Alloantigen stimulation leads, within 48 to 72 hr, to the expression of IL 2 receptors (IL 2R) on the surface of most of the helper T cells with specificity for the stimulating antigens. The IL 2R-bearing cells, separated by flow cytometry from 3-day human or mouse mixed lymphocyte cultures, were found by limiting dilution methods to be enriched 10- to 20-fold (compared to IL 2R- cells) for antigen-specific helper T cells detected by IL 2 production. Although these cells have been activated to an IL 2R+ stage, most of them are unable to produce detectable IL 2 unless they are cultured together with the original, activating antigen. Even when IL 2 is supplied, and lymphokine production measured by assay of a second factor, IL 3, the large majority of individual activated helper T cells remain dependent on further antigenic exposure for their continued maturation into lymphokine-secreting effectors. Helper T cells at this early stage of activation can therefore proliferate when given IL 2 alone, but lymphokine secretion involves a second antigen-dependent step.
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Jung, Minho, and Eun Young Choi. "TLR5 and TLR7 amplify different stage of myeloid cells." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 126.40. http://dx.doi.org/10.4049/jimmunol.202.supp.126.40.
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Abstract Balance between immune activation and immune suppression is important for immune homeostasis. Toll-like receptors (TLRs) play key roles in the innate immune system, initiating inflammatory responses against pathogen infections and internal danger signals. Different TLRs recognize different kinds of ligands, but not all the TLRs do have same effect: some TLRs are immune stimulatory, while the others are immune suppressive. For instance, TLR5 ligand has been known to be immune suppressive, while TLR7 ligand has been used for immune activation. To understand how these TLR ligands lead to opposite immune outcomes, we investigated the effects of the ligands on myeloid cell differentiation. Treatment of TLR5 ligand flagellin resulted in the increase of CD11b+ Ly6C+Ly6G+ myeloid-derived suppressor cells (MDSCs) on days 2 and 3 after culture initiation. However, TLR7 ligand gardiquimod led to enhancement of dendritic cell differentiation on day 1, and then increase of CD11c− MHCII− CD11b− Ly6C− Ly6G− progenitor population on day 2, which differentiate into CD11c− MHCIIow CD11b+ Ly6C+ cells with DC differentiation potential afterwards. Based on these results, we suggest that different immune outcomes of TLR ligands would depend on the stages of myeloid cells at which corresponding TLRs act, contributing to overall immune homeostasis.
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Haynes, Laura, Phyllis-Jean Linton, Sheri M. Eaton, Susan L. Tonkonogy, and Susan L. Swain. "Interleukin 2, but Not Other Common γ Chain–Binding Cytokines, Can Reverse the Defect in Generation of Cd4 Effector T Cells from Naive T Cells of Aged Mice." Journal of Experimental Medicine 190, no. 7 (October 4, 1999): 1013–24. http://dx.doi.org/10.1084/jem.190.7.1013.
Full textAbstract:
Development of effectors from naive CD4 cells occurs in two stages. The early stage involves activation and limited proliferation in response to T cell receptor (TCR) stimulation by antigen and costimulatory antigen presenting cells, whereas the later stage involves proliferation and differentiation in response to growth factors. Using a TCR-transgenic (Tg+) model, we have examined the effect of aging on effector generation and studied the ability of γc signaling cytokines to reverse this effect. Our results indicate that responding naive CD4 cells from aged mice, compared with cells from young mice, make less interleukin (IL)-2, expand poorly between days 3 to 5, and give rise to fewer effectors with a less activated phenotype and reduced ability to produce cytokines. When exogenous IL-2 or other γc signaling cytokines are added during effector generation, the Tg+ cells from both young and aged mice proliferate vigorously. However, IL-4, IL-7, and IL-15 all fail to restore efficient effector production. Only effectors from aged mice generated in the presence of IL-2 are able to produce IL-2 in amounts equivalent to those produced by effectors generated from young mice, suggesting that the effect of aging on IL-2 production is reversible only in the presence of exogenous IL-2.
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Vida, Carmen, Hikaru Kobayashi, Antonio Garrido, Irene Martínez de Toda, Eva Carro, José Molina, and Mónica De la Fuente. "Lymphoproliferation Impairment and Oxidative Stress in Blood Cells from Early Parkinson’s Disease Patients." International Journal of Molecular Sciences 20, no. 3 (February 12, 2019): 771. http://dx.doi.org/10.3390/ijms20030771.
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In Parkinson’s Disease (PD), the peripheral changes in the functional capacity and redox state of immune cells has been scarcely investigated, especially in the early PD stages. Aging is a risk factor for PD, and the age-related impairment of the immune system, based on a chronic-oxidative stress situation, is involved in the rate of aging. We analyzed several functions in isolated peripheral blood neutrophils and mononuclear cells from PD stage 2 patients, and compared the results to those in healthy elderly and adult controls. Several oxidative stress and damage parameters were studied in whole blood cells. The results showed an impairment of the lymphoproliferative response in stimulated conditions in the PD patients compared with age-matched controls, who also showed typical immunosenescence in comparison with adult individuals. Higher oxidative stress and damage were observed in whole blood cells from PD patients (lower glutathione peroxidase activity, and higher oxidized glutathione and malondialdehyde contents). Our results suggest an accelerated immunosenescence in PD stage 2, and that several of the parameters studied could be appropriate peripheral biomarkers in the early stages of PD.
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Sambrook, Joseph, and David W. Russell. "Exon Trapping and Amplification Stage 2: Electroporation of the Library into COS-7 Cells." Cold Spring Harbor Protocols 2006, no. 1 (June 2006): pdb.prot3733. http://dx.doi.org/10.1101/pdb.prot3733.
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Komanduri, Krishna V., Tae Kon Kim, Eric D. Wieder, and Lisa S. St. John. "Cytokine Production Signatures Are Intrinsically Associated with Human T Cell Maturation Stages." Blood 110, no. 11 (November 16, 2007): 1337. http://dx.doi.org/10.1182/blood.v110.11.1337.1337.
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Abstract Our recent published studies have suggested that impaired immune reconstitution after allogeneic stem cell transplantation is associated with a greater proportion of circulating late memory T cells, defined phenotypically. To characterize the relationship between immunophenotypic markers of T cell maturation and functional attributes of T cells, we optimized an 8-color, 10-parameter cytokine flow cytometry (CFC) approach and studied T cells from healthy donors. T cells were exposed to stimuli that both bypass (PMA:Ionomycin, P:I) and signal through the T cell receptor (Staph enterotoxin B, SEB; and CMV pp65 peptide pools) and stained with CD45RA and CD27 to demarcate naïve (N, CD45RA+CD27+), and three progressively mature memory subsets: M1 (CD45RA−CD27+), M2 (CD45RA−CD27−), M3 (CD45RA+CD27−) CD4+ and CD8+ T cells. We assessed the 15 possible combinations of cells producing IL-2, IFNγ, TNFα, and MIP1β alone or in combination within maturation subsets. When we initially studied the production of individual cytokines, we found that the bulk of IL-2 production was produced by activated N and M1 cells in both CD4 and CD8 lineages. In contrast, IFNγ and MIP1β were produced by later maturation stages (M2 and M3) of CD4+ and CD8+ T cells. In contrast to the polarized production of individual cytokines at the extremes of the maturation spectrum, early and middle memory cells (M1 and M2) cells produced heterogeneous combinations of cytokines (e.g, IL-2+IFNγ+ and TNFα+MIP-1β+ cells). We also found that IL-2/IFNγ co-producing cells, shown to be particularly important for the control of chronic viral pathogens, exist mainly in the M1 and M2 stages, and not the M3 stage. The above results were consistent with both P:I and SEB stimulation, and across several healthy subjects tested. Our cross-sectional results were confirmed by in vitro differentiation experiments, wherein we sorted naive (CD45RA+CD27+) CD4+ and CD8+ T cells and demonstrated that their function evolved as expected following stimulation with PHA and IL-2, which resulted in differentiation into M1 and M2 cells in culture. Finally, we stimulated PBMC from healthy CMV-seropositive donors with a CMV pp65 peptide mixtures and examined maturation and cytokine production. Consistent with prior observations, most CMV-specific T cells were M2 and M3 cells. Surprisingly, the most abundant functional subsets consisted of cells producing either MIP1β alone or MIP1β and other cytokines. Consistent with our results following polyclonal stimulation, we found that IL-2/IFNγ co-producing CMV specific T cells existed in M1 and M2, but not in the M3 stage. These results demonstrate that: Functional cytokine signature is strongly associated with T cell maturation stage; Nearly all IL-2 production occurs in N, M1 and M2 cells; M3 cells produce little IL-2, but substantial amounts of MIP1β; IL-2/IFNγ co-production is rare in M3 cells, but exist in M1 and M2 cells, perhaps suggesting why late stage skewing of memory T cells may lead to functional T cell impairment in vivo; and that MIP1β is the most abundant cytokine produced by CMV-specific T cells. Overall, our results demonstrate that phenotypically defined maturation stages in both CD4+ and CD8+ T cell lineages are strongly associated with functional signatures irrespective of stimulus type, and that multidimensional analyses of human T cells may be beneficial when assessing human T cells in the clinical setting.
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Rossi, Elisabetta, Angelica Zin, Antonella Facchinetti, Cristina Poggiana, Lucia Tombolan, Maria Carmen Affinita, Paolo Bonvini, et al. "Liquid Biopsy in Pediatric Renal Cancer: Stage I and Stage IV Cases Compared." Diagnostics 10, no. 10 (October 12, 2020): 810. http://dx.doi.org/10.3390/diagnostics10100810.
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Pediatric renal cancer is rare, and robust evidence for treatment recommendations is lacking. In the perspective of personalized medicine, clinicians need new biomarkers to improve risk stratification and patients’ follow-up. Herein, we analyzed some liquid biopsy tools, which have been never tested in pediatric renal cancer: namely, circulating tumor cells (CTCs); the expression of M30, an apoptosis marker, to test CTC metastatic potential; and c-MET expression in CTCs, because of its role in renal cancer progression and drug-resistance. Furthermore, we evaluated the Circulating Endothelial Cells (CECs), whose utility we previously demonstrated in adult metastatic renal cancer treated with anti-angiogenic therapy. We compared two renal cell carcinomas of clear-cell type, stage I and IV, which underwent surgery and surgery plus Sunitinib, respectively. Baseline CTC level and its changes during follow-up were consistent with patients’ outcome. In case 2, stage IV, the analysis of CECs performed during Sunitinib revealed a late response to treatment consistent with poor outcome, as the finding of M30-negative, viable cells. Noteworthily, few CTCs were MET-positive in both cases. Our study highlights the feasibility for a change in the prognostic approach and follow-up of childhood renal cancer, with a view to guide a better treatment design.
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Carrel, F., S. Dharmawardhane, A. M. Clark, J. A. Powell-Coffman, and R. A. Firtel. "Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation." Molecular Biology of the Cell 5, no. 1 (January 1994): 7–16. http://dx.doi.org/10.1091/mbc.5.1.7.
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Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.
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Fang, Jing, Madhu Menon, Diya Zhang, Bruce Torbett, Leif Oxburgh, Mario Tschan, Estelle Houde, and Don M. Wojchowski. "Attenuation of EPO-dependent erythroblast formation by death-associated protein kinase-2." Blood 112, no. 3 (August 1, 2008): 886–90. http://dx.doi.org/10.1182/blood-2008-02-138909.
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Abstract The adult erythron is maintained via dynamic modulation of erythroblast survival potentials. Toward identifying novel regulators of this process, murine splenic erythroblasts at 3 developmental stages were prepared, purified and profiled. Stage-to-stage modulated genes were then functionally categorized, with a focus on apoptotic factors. In parallel with BCL-X and NIX, death-associated protein kinase-2 (DAPK2) was substantially up-modulated during late erythropoiesis. Among hematopoietic lineages, DAPK2 was expressed predominantly in erythroid cells. In a Gata1-IE3.9int-DAPK2 transgenic mouse model, effects on steady-state reticulocyte and red blood cell (RBC) levels were limited. During hemolytic anemia, however, erythropoiesis was markedly deficient. Ex vivo ana-lyses revealed heightened apoptosis due to DAPK2 at a Kit−CD71highTer119− stage, together with a subsequent multifold defect in late-stage Kit−CD71highTer119+ cell formation. In UT7epo cells, siRNA knock-down of DAPK2 enhanced survival due to cytokine withdrawal, and DAPK2's phosphorylation and kinase activity also were erythropoietin (EPO)-modulated. DAPK2 therefore comprises a new candidate attenuator of stress erythropoiesis.