C.E.R.A. Acts Differently at the Erythropoietin (EPO) Receptor Compared with Epoetin Beta: UT-7 and CD34+ Cell Stimulation Assays.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4206-4206 ◽  
Author(s):  
M. Jarsch ◽  
M. Brandt ◽  
M. Kubbies ◽  
A. Haselbeck
Keyword(s):  
Blood Cell ◽  
Cell Line ◽  
Glycophorin A ◽  
Epo Receptor ◽  
Epoetin Beta ◽  
Human Cd34 ◽  
Maximal Stimulation ◽  
Cd34 Cells ◽  
Stimulation Of

Abstract Continuous Erythropoietin Receptor Activator (C.E.R.A.), an innovative erythropoietic agent with unique receptor activity, is currently in development to provide correction of anemia and stable maintenance of hemoglobin (Hb) levels at extended administration intervals up to once monthly in patients with all stages of chronic kidney disease (CKD), and is also in development for the treatment of chemotherapy-induced anemia. In vitro studies show that C.E.R.A. has a 45-fold lower affinity for the EPO receptor than epoetin beta, due mainly to a reduced association rate. To further investigate the in vitro activity of C.E.R.A., two cell stimulation studies were undertaken. Study 1 evaluated an assay for the analysis of the molecular mechanism of C.E.R.A.- and epoetin beta-mediated cell activation. UT-7 cells were activated with C.E.R.A. or epoetin beta for 72 h or 96 h, followed by WST (tetrazolium salt) staining and spectrophotometric detection. UT-7 is a human myeloid leukemia cell line expressing the EPO receptor, and has growth dependency on EPO if no other growth factors are present. Results showed that the EC50 (concentration giving half maximal stimulation) value was approximately 10-fold higher for C.E.R.A. (range 300–400 pM) than for epoetin beta (30–60 pM). Maximal activation of UT-7 cells was achieved at C.E.R.A. 1000–2000 pM and epoetin beta 100–200 pM, but the maximal stimulation of cells was similar for both agents. Study 2 investigated the effects of C.E.R.A. and epoetin beta on stimulation of the proliferation and differentiation of human CD34+ cells. Human CD34+ stem cells from cord blood and bone marrow were cultivated with C.E.R.A. or epoetin beta for 8–14 days. After labeling, using fluorescence-tagged antibodies to proteins specific for erythroid cells (glycophorin A) and other blood cell types (CD13, CD14, CD16, CD41, CD42b, and CD61), cells were analyzed using three-color flow cytometry with a FACScan instrument (Becton Dickinson, CA). The maximal number of glycophorin A positive cells at plateau phase was used for EC50 calculation. Following stimulation of CD34+ cells, glycophorin A+ cells increased to a similar level with C.E.R.A. and epoetin beta. This stimulation was specific for erythroid precursors since the differentiation of white blood cells and megakaryocytes was not affected by C.E.R.A. or epoetin beta. Notably, mean EC50 values were 43.4-fold higher with C.E.R.A. (2.807 nM) than with epoetin beta (0.076 nM). In conclusion, C.E.R.A. and epoetin beta activate UT-7 cells and induce differentiation and expansion of CD34+ cells. These studies provide further evidence that C.E.R.A. has different EPO receptor binding properties compared with epoetin beta and demonstrate its specificity for the red blood cell line. Preclinical studies have shown that these properties translate into more continuous stimulation of erythropoiesis in vivo compared with epoetin beta and Phase III data indicate that C.E.R.A. achieved and maintained stable Hb levels in all patients with CKD.

scholarly journals Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.

1996 ◽  
Vol 183 (3) ◽  
pp. 837-845 ◽  
Author(s):  
X Sui ◽  
K Tsuji ◽  
S Tajima ◽  
R Tanaka ◽  
K Muraoka ◽  
...  
Keyword(s):  
Stem Cell Factor ◽  
Current Data ◽  
Erythroid Cells ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Human Sera ◽  
Stimulation Of

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

scholarly journals Preclinical Evaluation of ALS20, a New and Improved Lentiviral Vector for Beta-Globinopathies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2242-2242
Author(s):  
Laura Breda ◽  
Valentina Ghiaccio ◽  
Silvia Pires Lourenco ◽  
Danuta Jadwiga Jarocha ◽  
Yasuhiro Ikawa ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Line ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Globin Gene ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Primary Mouse

The ongoing clinical trials, based on the use of lenti-globin vectors for beta-globinopathies, indicate that current vectors require high number of integrations (~3-4 copies per genome) in a pancellular fashion to make a patient transfusion independent, regardless of the genotype. This increased VCN requirement could increase the risk of genome toxicity, limiting the application of these vectors and preventing their use in a reduced myeloablative regimen. To overcome this limitation we designed new vectors, using modification or inclusion of a variety of regulatory genomic elements aiming to increase expression of the beta-globin gene. We performed in vitro and in vivo studies to compare the ability of these constructs to express the therapeutic gene with a low number of integrations and reduced chimerism. We screened new constructs using a CRISPR-Cas9 modified clonal cell line, HUDEPM#13, which derives from the previously described erythroid HUDEP-2 cell line (Kurita, 2013). Upon differentiation, HUDEPM#13 cells produce a hemoglobin variant (HbMut) that can be discriminated by liquid chromatography from the adult hemoglobin (HbA) produced by the transgene in the lentiviruses. Among our candidates we identified a construct, indicated as ALS20, that synthesizes HbA at high level at a single integration (VCN). Moreover, ALS20 produced significantly more HbA per copy than constructs currently utilized in clinical trials, which were reproduced based on the literature (Negre, 2015; Miccio, 2008; and Boulad, 2014) and designated CV-1, CV-2, and CV-3, respectively. In erythroblasts differentiated in vitro from patients with SCD ALS20 produces, on average, 21% HbA at VCN=1(P<0.001). CV1, which proved to be the most powerful among the clinical vectors tested in HUDEP#M13, produces, on average, 14.9% HbA at VCN=1 (P<0.001). Overall, ALS20 expresses 40% more HbA per VCN in patients' cells, confirming results observed in the mutant HUDEP cell line. In a parallel potency assay, we tested ALS20 using beta0/beta0 thalassemic specimens, which represent the most severe phenotype to correct, due to complete absence of HbA production. Statistical analyses indicate that ALS20 produces, on average, 32.5% HbA at VCN=1 (P<0.001). All results were obtained targeting a range between 0 and 3 integrations, using dilutions of the viral product. Our ongoing bone marrow transplantation studies, using a semi-myeloablative conditioning based on busulfan administration on the beta thalassemia Hbbth3/+mouse model, indicate that ALS20 is curative at VCN lower than 1. In fact, mice with an average of 0.8 copies per genome and 65% transgenic chimerism, present Hb levels of 13.6g/dL, reiterating the potential of this new lentivirus in vivo. In immunocompromised NSG mice, injection of human CD34+cells transduced with ALS20 did not trigger tumor formation or meaningful pathological changes. The safety of our vector has been further assessed using an in vitro immortalization assay on primary mouse BM cells, in which no clonal survival has been observed. Finally, genome integration analyses on human CD34+cells infected with ALS20 showed that none of the examined samples had clones that exceeded the 20% abundance threshold, in accordance with the expected level of safety for gene therapy-based approaches. In summary, we have identified a powerful new lentiviral vector with an enhanced ability to synthesize hemoglobin with a low number of integrations. ALS20's performance has been demonstrated in specimens from patients with hemoglobinopathies as well as in mice affected by thalassemia. Treatment of thalassemia and SCD with ALS20 could reduce the risk of cytotoxic events due to high levels of integration, and also lessen the intensity of the myeloablative regimen to correct anemia in patients. This, in addition with its confirmed safety features, makes this construct an outstanding candidate for clinical trial. Disclosures Kwiatkowski: Imara: Consultancy; Apopharma: Research Funding; Terumo: Research Funding; Novartis: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Celgene: Consultancy. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

Expression and Functional Differences of CBFA2T (ETO, MTG) Gene Family Members in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4208-4208
Author(s):  
Fariborz Mortazavi ◽  
ShriHari Kadkol ◽  
Annette Bruno ◽  
Kristine Baraoidan ◽  
Steven Ackerman ◽  
...  
Keyword(s):  
Gene Family ◽  
Cell Line ◽  
Cell Lines ◽  
Family Members ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Macrophage Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract The CBFA2T (ETO, MTG) family has three similar family members - CBFA2T1-T3. CBFA2T1 (ETO, MTG8) and CBFA2T3 (ETO2, MTG16) are targeted by chromosomal translocations in acute myeloid leukemia. To better understand the usual hematopoietic function of this gene family, we examined the expression of CBFA2T RNA using RQ-PCR in cell-lines and human CD34+ hematopoietic cells during macrophage and erythroid differentiation. RQ-PCR on extracted RNA was performed with an icyclerQ instrument (Bio-Rad) using the Quantitect SYBR Green RT-PCR kit (Qiagen) and in vitro transcribed RNA to construct standard curves. CBFA2T3 was the most highly expressed family member in human CD34+ cells, the erythro-leukemia line K562, the myeloid line MPD, the T cell line Jurkatt and the B-cell line LCL-11. However, CBFA2T3 expression decreased by &gt;50% during both macrophage and erythroid differentiation of human CD34+ cells. In contrast, CBFA2T1 expression was almost undetectable in human CD34+ cells and all cell lines except K562 but increased more than 20 fold during erythroid (but not macrophage) differentiation of human CD34+ cells. Extrinsic over-expression of CBFA2T1, but not CBFA2T2, significantly increased glycophorin-A and hemoglobin A expression in K562 cells, consistent with a regulatory role for CBFA2T1 in erythroid differentiation. CBFA2T2 (MTGR1) was moderately expressed in human CD34+ cells and all the cell lines and demonstrated a 2.5 fold increase in expression with macrophage differentiation but essentially no change with erythroid differentiation of human CD34+ cells. These findings suggest that despite their similarity, the CBFA2T family members have distinctive regulatory roles in hematopoietic differentiation.

EFFECT OF VARIOUS PREPARATIONS OF PITUITARY AND DIENCEPHALON ON THE IN VITRO SECRETION OF ALDOSTERONE AND CORTICOSTERONE BY THE RAT ADRENAL GLAND

1961 ◽  
Vol 39 (5) ◽  
pp. 901-913 ◽  
Author(s):  
O. J. Lucis ◽  
I. Dyrenfurth ◽  
E. H. Venning
Keyword(s):  
Adrenal Gland ◽  
Linear Relationship ◽  
Dose Response ◽  
Response Curve ◽  
Maximum Effect ◽  
Percentage Increase ◽  
Aldosterone Secretion ◽  
Maximal Stimulation ◽  
Stimulation Of

Purified corticotropin and ACTH peptides increased the secretion of aldosterone, corticosterone, and an unidentified compound RT4in incubated rat adrenal tissue. When the response was expressed as a percentage increase above that of the control tissue, the increases in corticosterone and compound RT4followed a sigmoid log dose – response curve. The maximum effect on aldosterone was obtained at a time when the response curve for corticosterone assumed a linear relationship between the response and the logarithm of the dose of ACTH. This dose level was considerably less than that required for maximal stimulation of corticosterone.The capacity of the ACTH peptides α1+α2and δ′ for stimulating aldosterone secretion could be greatly diminished by allowing solutions of these fractions to stand at 5 °C for 1 week. These solutions still retained their ability to stimulate corticosterone secretion.Saline suspensions and extracts of fresh hog diencephalon contained a factor which selectively stimulated aldosterone secretion.

scholarly journals Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2063-2063
Author(s):  
Naoya Uchida ◽  
Claire Drysdale ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Rhesus Macaques ◽  
Fetal Hemoglobin ◽  
Selective Advantage ◽  
Erythroid Cells ◽  
Post Transplant ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Abstract P019: Exosomes from Human CD34+ Cells: Critical Mediators of Proangiogenic Paracrine Effect of EPCs

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Susmita Sahoo ◽  
Sol Misener ◽  
Tina Thorne ◽  
Meredith Millay ◽  
Kathryn M Schultz ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Human Peripheral Blood ◽  
Limb Ischemia ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Paracrine Effect ◽  
Human Cd34 ◽  
Cd34 Cells

Local transplantation of human CD34+ hematopoietic stem cells has been shown to promote neovascularization in pre-clinical studies in models of myocardial and limb ischemia. In early phase clinical trials, transplantation of CD34+ cells has been associated with reduced angina, improved exercise time and reduced amputation rates. Several studies have suggested that paracrine effects by these pro-angiogenic cells mediate the effects induced by cell transplantation. We hypothesized that CD34+ cells secrete exosomes (Exo), which mediate at least a part of the therapeutic function of the cells. Methods and Results: We isolated Exo from the conditioned media of adult human peripheral blood (PB) CD34+ cells. The angiogenic and therapeutic potency of CD34+ Exo was compared with the intact CD34+ cells and also with PB mononuclear cell (MNC) Exo. Exo from both CD34+ cells and MNC are 50–90nm in size, have cup shaped morphology, and carry known Exo-marker proteins such as CD63, TSG101 and Annexin V as shown by electron microscopy, Western blot and flow cytometry. Compared to CD34+ cells or MNC Exo, CD34+ Exo significantly induces in vitro angiogenic activities such as viability, proliferation and tube formation of HUVECs on matrigel- in a dose dependent manner. In vivo, CD34+ Exo stimulated significant neovascularization in mouse corneal angiogenesis assay (14±4 mm v MNC Exo, 4±1 mm, p<0.01) and incorporation of endothelial (CD31+) cells in mouse matrigel-plug assay (6±1.7% v CD34+ cells, 2±0.8%, p<0.01). Finally, in a mouse model of hind limb ischemia (HLI), CD34+ Exo significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P<0.05), increased capillary density (1.8±0.3/HPF v 0.9±0.1/HPF, p<0.001) and prevented ischemic leg amputation (16% v 100%), as compared with MNC Exo. Conclusions: These data demonstrate that CD34+ Exo induce angiogenic activity and ischemic tissue repair in the absence of CD34+ cells, and suggest that Exo represent important mediators of the therapeutic effects associated with CD34+ cell therapy. We speculate that Exo derived from CD34+ cells may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Soluble Interleukin-6 (IL-6) Receptor With IL-6 Stimulates Megakaryopoiesis From Human CD34+ Cells Through Glycoprotein (gp)130 Signaling

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2525-2532 ◽  
Author(s):  
Xingwei Sui ◽  
Kohichiro Tsuji ◽  
Yasuhiro Ebihara ◽  
Ryuhei Tanaka ◽  
Kenji Muraoka ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Ex Vivo ◽  
Serum Free ◽  
Human Cd34 ◽  
Receptor Component ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Clonal Cultures

Abstract We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34+ cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34+ cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34+ cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34+ cells were subfractionated into two populations of IL-6R–negative (CD34+ IL-6R−) and IL-6R–positive (CD34+ IL-6R+) cells by fluorescence-activated cell sorting. The CD34+IL-6R− cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34+ IL-6R+cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

Insulin sensitivity of rates of glycolysis and glycogen synthesis in soleus, stripped soleus, epitrochlearis, and hemi-diaphragm muscles isolated from sedentary rats

1983 ◽  
Vol 3 (7) ◽  
pp. 675-679 ◽  
Author(s):  
R. A. J. Challiss ◽  
J. Espinal ◽  
E. A. Newsholme
Keyword(s):  
Fatty Acid ◽  
Glucose Utilization ◽  
Glycogen Synthesis ◽  
Maximal Inhibition ◽  
Maximal Stimulation ◽  
Glucose Fatty Acid Cycle ◽  
Isolated Adipocytes ◽  
Stimulation Of

The effect of insulin concentrations on the rates of glycolysis and glycogen synthesis in four different in vitro rat muscle preparations (intact soleus, stripped soleus, epitrochlearis, and hemi-diaphragm) were investigated: the concentrations of insulin that produced half-maximal stimulation of the rates of these two processes in the four muscle preparations were similar – about 100 μunits/ml. This is at least 10-fold greater than the concentration that produced half-maximal inhibition of lipolysis in isolated adipocytes. Since 100 μunits/ml insulin is outside the normal physiological range in the rat, it is suggested that, in vivo, insulin influences glucose utilization in muscle mainly indirectly, via changes in the plasma fatty acid levels and the ‘glucose/fatty acid cycle’. Consequently the view that insulin stimulates glucose utilization in muscle mainly by a direct effect on membrane transport must be treated with caution.

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