PLATELET-ACTIVATING FACTOR INDUCES DE NOVO SYNTHESIS OF MTOR IN TERMINALLY DIFFERENTIATED POLYMORPHONUCLEAR LEUKOCYTES.
Frederick J. RubnerMark J. CodyG. A. ZimmermanAndrew S. WeyrichHeather R. HillTimothy R LapineC.C. Yost
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Background The mammalian target of rapamycin (mTOR) is known to play a pivotal role in the differentiation of hematopoietic stem cells (HSCs) via control of cell cycling. In addition to this function, mTOR is known to mediate translation of constitutively expressed mRNAs. Specifically, we have demonstrated the role of mTOR in translational regulation of key inflammatory mediators. In this investigation we hypothesized that an inflammatory agonist would increase synthesis of mTOR in a pool of terminally differentiated polymorphonuclear leukocytes (PMN). Methods Human HSCs were isolated from umbilical cord blood obtained from full-term neonates. This population of HSCs underwent proliferation and differentiation into mature PMNs. These cells were purified by magnetically selecting cells expressing the CD16+ cell surface antigen. The CD16+ cells were exposed to platelet-activating factor (PAF) 10 nM or HBSS for 0, 60, or 120 minutes. The cells were subsequently fixed and examined by immunocytochemistry for their expression of mTOR. Results The population of terminally differentiated PMNs demonstrated increased expression of mTOR following stimulation with PAF compared with control. Further, this population of cells demonstrated a time-dependent increase in production of mTOR consistent with the expected rapid response of primary host defense cells. Conclusions HSC-derived, terminally differentiated PMNs demonstrate functional integrity of inflammatory mediator-induced mTOR expression. This capacity likely enables the cell to control the rapid synthesis of other mediators of inflammation. This finding will be key for further investigation using this model to perform loss of mTOR function experiments on downstream inflammatory mediators and the concomitant alteration in inflammatory processes.RPTOR
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<sup>111</sup>In-labelled human platelets were aggregated with ADP and subjected to ultra-structural morphometric analysis. In addition, the hemostatic function in vivo and the ultra-structural morphology ex vivo of rabbit platelets were examined. The platelet isolation and labelling procedures exerted no certain influence on the aggregation response, and platelet surface/volume calculations did not indicate that platelet activation had taken place. Bleeding time experiments in rabbits indicated that the hemostatic effectiveness of the labelled platelets was unimpaired. Transfused <sup>111</sup>In-labelled platelets isolated from the recipient rabbits exhibited fewer electronmicroscopic signs of platelet activation than the same platelets prior to transfusion. Our results indicate that the described procedure for isolation and <sup>111</sup>In-labelling of platelets induces only insignificant damage to the platelets.
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Objective Human platelets vary in size, function, and age. Large platelets are often considered to be young platelets. Two situations have to be distinguished, normal steady state platelet production and increased platelet turnover. Here we focused on large and small platelets in humans during increased platelet turnover. To avoid artefacts by interfering factors (medication, comorbidities), we established a platelet apheresis model to deplete platelets from healthy volunteers with subsequent increased platelet production.
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SUMMARY. Average platelet size, platelet count, and 35 S‐incorporation into platelets were compared as methods for the measurement of thrombopoietin‐stimulated thrombopoiesis. In mice injected with rabbit anti‐mouse platelet serum (RAMPS) average platelet size was shown to be increased as mice were recovering from thrombocytopenia. Also, 35 S‐measurements on platelets of these mice showed significant increases in cpm/average platelet 2–4 days after RAMPS treatment. Significant increases in 35 S‐incorporation into the total circulating mass of platelets were found on days 3–4. In normal mice or mice in rebound‐thrombocytosis injected with thrombopoietin, platelet size remained unchanged, whereas the platelet count and 35 S‐incorporation into platelets were shown to be significantly increased. Moreover, a dose‐response experiment in mice pretreated with RAMPS showed a slight increase in platelet count as the dose of TSF was increased, but platelet sizes were unaltered. The % 35 S‐incorporation into platelets showed a significant linear dose‐response, i.e. as the dose of thrombopoietin was increased, an increase in % 35 S‐incorporation into platelets was observed. These data indicated that of the three indirect measurements of thrombopoietin, the % 35 S‐incorporation into mouse platelets was the most sensitive, followed by platelet counting; the least sensitive measurement of thrombopoiesis was change in platelet size.
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The function of 111 In‐labelled platelets has been assessed by collagen‐induced aggregation of platelets in samples of whole blood. The blood samples were drawn after injection of autologous 111 In‐labelled platelets in 19 subjects undergoing platelet kinetic studies. It was thus possible to measure the aggregability of labelled and unmanipulated platelets simultaneously. 111 In‐labelled platelets aggregated to the same extent as unmanipulated platelets when tested from 10 min to 24 h after injection of the labelled platelets. The results confirm the assumption that minimal damage is inflicted on the platelets during the isolation and labelling procedures, and support the concept that platelets manipulated in vitro may recover in vivo within a few minutes after reinjection.
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The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway regulates numerous cellular processes such as growth, proliferation, cell cycle progression, motility, adhesion, and angio-genesis, and appears to be constitutively active in a majority of renal cell carcinoma (RCC). The integrity of the pathway is clearly vital to the survival and growth of RCC as pharmacologic inhibition of PI3K or Akt induces apoptosis in RCC tumor cells and tumor regression in vivo. These observations suggest that the PI3K/Akt/ mTOR pathway may be an attractive target for drug development in the treatment of RCC. The recently demonstrated clinical efficacy of inhibitors of mTOR supports this hypothesis and demonstrates the relevance of this pathway in RCC. As Akt activates numerous kinases, transcription factors and other proteins associated with cell growth and survival in addition to mTOR, it is possible even greater clinical responses may be achieved with agents that disrupt the PI3K/Akt/mTOR pathway upstream of mTOR. Concurrent with the development of inhibitors of PI3K or Akt for clinical application are efforts to identify predictive biomarkers of response to agents targeting elements of the PI3K/Akt/mTOR pathway so as to develop more individualized patient selection strategies. In this chapter, we will review the molecular biology of the PI3K/Akt/mTOR pathway, its relevance to RCC, and its potential as a therapeutic target in RCC.
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In the previous communication, suggestive evidence was presented for large-heavy platelets being "young" platelets and light-small platelets being "old" platelets. Large-heavy, light-small, and total human platelet populations were compared with respect to their platelet function. After addition of adenosine diphosphate (ADP), thrombin, or epinephrine, platelet aggregation time was 3.0-, 4.5-, and 3.3-fold shorter with large-heavy platelets compared with light-small platelets, and large-heavy platelets released 3.7-, 7.6-, and 8.1-fold greater adenosine triphosphate (ATP) into the medium, respectively, than did light-small platelets. After platelet aggregation by thrombin or epinephrine, large-heavy platelets released 6.0- and 3.8-fold more ADP into the medium than did light-small platelets. After platelet aggregation by ADP, light-small platelets consumed 5.9-fold greater added extracellular ADP than did large-heavy platelets.Large-heavy platelets aggregated by ADP, thrombin, or epinephrine released 9.1-, 8.5-, and 12.7-fold greater platelet factor 4 than light-small platelets similarly treated.
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