scholarly journals In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 248-256 ◽  
Author(s):  
G Migliaccio ◽  
AR Migliaccio ◽  
JW Adamson
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Colony Growth ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Concentration Dependent Manner ◽  
Gm Csf ◽  
Human Erythropoietin ◽  
Normal Humans

Abstract The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

scholarly journals In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum- deprived cultures

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 248-256
Author(s):  
G Migliaccio ◽  
AR Migliaccio ◽  
JW Adamson
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Colony Growth ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Concentration Dependent Manner ◽  
Gm Csf ◽  
Human Erythropoietin ◽  
Normal Humans

The effects of recombinant human erythropoietin (Ep), granulocyte/macrophage (GM) and granulocyte (G) colony-stimulating factors (CSF), and interleukin-3 (IL-3) on erythroid burst and GM colony growth have been studied in fetal bovine serum (FBS)- supplemented and FBS-deprived culture. Sources of progenitor cells were nonadherent or nonadherent T-lymphocyte-depleted marrow or peripheral blood cells from normal humans. G-CSF, in concentrations up to 2.3 X 10(-10) mol/L, induced only the formation of neutrophil colonies. In contrast, GM-CSF and IL-3 both induced GM colonies and sustained the formation of erythroid bursts in the presence of Ep. However, the activities of these growth factors were affected by the culture conditions. IL-3 induction of GM colonies depended on the presence of FBS, whereas the degree of GM-CSF induction of GM colonies in FBS- deprived cultures depended on the method by which adherent cells were removed. GM-CSF increased colony numbers in a concentration-dependent manner only if the cells had been prepared by overnight adherence. Both GM-CSF and IL-3 exhibited erythroid burst-promoting activity in FBS- deprived cultures. However, some lineage restriction was evident because GM-CSF was two- to threefold more active than IL-3 in inducing GM colonies but IL-3 was two- to threefold more active in promoting erythroid burst growth. Furthermore, in FBS-deprived cultures, the number of both erythroid bursts and GM colonies reached the maximum only when Ep, GM-CSF, and IL-3 or GM-CSF, IL-3, and G-CSF, respectively, were added together. These results suggest that the colonies induced by IL-3, GM-CSF, and G-CSF are derived from different progenitors.

Bovine in vitro oocyte maturation as a model for manipulation of the γ-glutamyl cycle and intraoocyte glutathione

2008 ◽  
Vol 20 (5) ◽  
pp. 579 ◽  
Author(s):  
E. C. Curnow ◽  
J. Ryan ◽  
D. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oocyte Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Maturation Medium

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.

Recombinant Human Erythropoietin Administration Decreases the Frequency of Excessive Apoptosis in Erythroid Progenitors in the Model Mouse of Anemia Due to Chronic Renal Failure.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3668-3668
Author(s):  
Tatsuo Oyake ◽  
Shigeki Ito ◽  
Shugo Kowata ◽  
Kazunori Murai ◽  
Yoji Ishida
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Recombinant Human Erythropoietin ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Color Flow ◽  
Human Erythropoietin ◽  
Model Mouse ◽  
Dose Dependent

Abstract Recombinant human erythropoietin (rh-EPO) administration results in a dramatic improvement of anemia due to chronic renal failure (ACRF). We reported that excessive apoptosis in erythroid progenitors was observed in ACRF patients before rh-EPO administration, and that we have established the model mouse of ACRF by administration of adenine-rich diets (ADRD) in ASH meetings 2001 and 2006. In this study, we evaluated the frequency of apoptosis in bone marrow erythroid cells of the model mouse before and after erythropoietin (EPO) administration, to confirm the hypothesis that excessive apoptosis in erythroid progenitors induced anemia via insufficient production of EPO. ADRD was administrated to C57B6 male mice for 8 weeks. Every day subcutaneous administration of EPO started at 6 weeks and continued for 2 weeks. Serum BUN and Crt levels began to elevate from 2 weeks after ADRD administration (Table1). Hb levels began to decrease from 5 weeks after ADRD administration and increased after EPO administration in a dose dependent manner (Table1, Figure1). The frequencies of apoptosis in erythroid progenitors and mature erythroblasts were evaluated by three color flow cytometric analysis using anti-CD34-antibody, anti-EPO-receptor (EPO-R)-antibody and AnnexinV. Erythroid progenitors were identified as CD34(+) and EPO-R(+) cells, and mature erythroblasts identified as CD34(−) and EPO-R(+) cells. Much higher frequency of apoptosis was observed in erythroid progenitors at 6 weeks after ADRD administration (76.4±5.3%, n=4), while, the frequency of apoptosis decreased dramatically after EPO administration in a dose dependent manner (0U/kg/day: 70.1±6.9% at 7 weeks, 73.8±8.7% at 8 weeks, 10U/kg/day: 48.1±5.4%* at 7 weeks, 46.9±4.2%* at 8 weeks, 100U/kg/day: 36.9±2.5%* at 7 weeks, 21.2±6.8%* at 8 weeks, 1000U/kg/day: 18.4±2.9%* at 7 weeks, 15.1±3.5%* at 8 weeks, each n=4, *p<0.01) (Figure2). The frequency of apoptosis was not increased in mature erythroblasts at each week after ADRD administration. Our findings suggested that excessive apoptosis occurred mainly in CD34(+) erythroid progenitors by insufficient production of EPO, and that EPO had the anti-apoptotic effect to erythroid progenitors. Insufficient production of EPO might be one of the important causes of ACRF. Table 1. BUN, Crt and Hb levels in C57B6 mice after ADRD administration. 0 week 2 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks BUN (mg/dl) 40.1±4.60 73.2±14.4 150.7±11.6 148.8±4.60 164.0±8.40 161.8±16.4 193.0±9.10 Crt (mg/dl) 0.06±0.04 0.30±0.03 0.51±0.07 0.52±0.09 0.68±0.09 0.65±0.04 0.80±0.17 Hb (g/dl) 13.6±0.4 13.3±0.3 13.2±0.2 11.1±0.1 10.2±0.5 9.6±0.1 8.3±0.5 Figure 1. Hb levels in C57B6 mice after ADRD and EPO administration. Figure 1. Hb levels in C57B6 mice after ADRD and EPO administration. Figure 2. The frequency of apoptotic cells positive for CD34 and EPO-R in C57B6 mice after ADRD and EPO administration. Figure 2. The frequency of apoptotic cells positive for CD34 and EPO-R in C57B6 mice after ADRD and EPO administration.

scholarly journals Interaction of the New Ketolide ABT-773 (Cethromycin) with Human Polymorphonuclear Neutrophils and the Phagocytic Cell Line PLB-985 In Vitro

2004 ◽  
Vol 48 (4) ◽  
pp. 1096-1104 ◽  
Author(s):  
Marie Thérèse Labro ◽  
Houria Abdelghaffar ◽  
Catherine Babin-Chevaye
Keyword(s):  
Cell Line ◽  
Active Transport ◽  
Transport System ◽  
Culture Conditions ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Erythromycin A ◽  
Active Transport System

ABSTRACT A classical velocity centrifugation technique was used to study the in vitro uptake of the new ketolide ABT-773 by human polymorphonuclear neutrophils (PMNs) and a myelomonoblastic cell line, PLB-985, which can be differentiated into PMNs under certain culture conditions, compared to that of HMR 3004. ABT-773 was rapidly taken up by PMNs (cellular concentration to extracellular concentration ratio [C/E], about 34 at 30 s and up to 207 at 5 min), and uptake plateaued from 30 to 180 min (C/E, about 300). ABT-773 was accumulated significantly better than HMR 3004 from 5 to 180 min. Nondifferentiated PLB-985 cells (ND-PLB) accumulated significantly less ABT-773 and HMR 3004 than PMNs and PLB-985 cells differentiated into PMNs (D-PLB). Whatever the cell type and in contrast to the results obtained with HMR 3004, ABT-773 was mainly located in the cytosol (about 75%) and was rapidly released from loaded cells (about 40% at 5 min), followed by a plateau, likely owing to avid reuptake. Verapamil and H89, an inhibitor of protein kinase A, increased drug efflux. Uptake was sensitive to external pH, and the activation energy was moderate (about 50 kJ/mol). The existence of an active transport system on the PMN membrane was suggested by the following findings: concentration-dependent and saturable uptake (V max, about 10 000 ng/2.5 × 106 PMNs/5 min; Km , about 60 μg/ml) the inhibitory effects of PMN activators or inhibitors (phorbol myristate acetate, verapamil, Ni2+) and the significantly decreased levels of accumulation by killed cells and cells treated at low temperatures. In addition, various macrolides impaired ABT-773 uptake, contrary to the findings for the quinolone levofloxacin. ND- and D-PLB also presented saturation kinetics that defined an active transport system (V max and Km values were similar to those obtained with PMNs), but the activation pathway of the carrier system did not seem to be fully functional in ND-PLB. As has been observed with other erythromycin A derivatives, ABT-773 impaired oxidant production by phagocytes in a time- and concentration-dependent manner. These data extend our previous results on the existence of an active transport system common to all macrolides and ketolides, at least in PMNs.

scholarly journals Cellular Fragments as Biomaterial for Rapid In Vitro Bone-Like Tissue Synthesis

2020 ◽  
Vol 21 (15) ◽  
pp. 5327
Author(s):  
Mst Nahid Akhter ◽  
Emilio Satoshi Hara ◽  
Koichi Kadoya ◽  
Masahiro Okada ◽  
Takuya Matsumoto
Keyword(s):  
Collagen Gel ◽  
Culture Conditions ◽  
Substrate Material ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Freeze Dried ◽  
Ionic Detergent ◽  
3D Collagen ◽  
Fetal Bovine

Current stem cell-based techniques for bone-like tissue synthesis require at least two to three weeks. Therefore, novel techniques to promote rapid 3D bone-like tissue synthesis in vitro are still required. In this study, we explored the concept of using cell nanofragments as a substrate material to promote rapid bone formation in vitro. The methods for cell nanofragment fabrication were ultrasonication (30 s and 3 min), non-ionic detergent (triton 0.1% and 1%), or freeze-dried powder. The results showed that ultrasonication for 3 min allowed the fabrication of homogeneous nanofragments of less than 150 nm in length, which mineralized surprisingly in just one day, faster than the fragments obtained from all other methods. Further optimization of culture conditions indicated that a concentration of 10 mM or 100 mM of β-glycerophosphate enhanced, whereas fetal bovine serum (FBS) inhibited in a concentration-dependent manner, the mineralization of the cell nanofragments. Finally, a 3D collagen-cell nanofragment-mineral complex mimicking a bone-like structure was generated in just two days by combining the cell nanofragments in collagen gel. In conclusion, sonication for three min could be applied as a novel method to fabricate cell nanofragments of less than 150 nm in length, which can be used as a material for in vitro bone tissue engineering.

Recombinant Erythropoietin in Anemia of Prematurity: Five Years Later

PEDIATRICS ◽  
1993 ◽  
Vol 92 (4) ◽  
pp. 614-617
Author(s):  
KEVIN SHANNON
Keyword(s):  
Recombinant Human Erythropoietin ◽  
Recombinant Erythropoietin ◽  
Red Cell ◽  
Erythroid Progenitors ◽  
Human Erythropoietin ◽  
Therapeutic Trials ◽  
Premature Babies ◽  
Large Numbers ◽  
Anemia Of Prematurity

In 1988, Dr James Stockman discussed the anemia of prematurity in a fascinating editorial that anticipated much of what has unfolded as therapeutic trials using recombinant human erythropoietin (r-HuEPO) were initiated. Dr Stockman began by addressing the implications of then-recent results from my laboratory and from that of Robert Christensen which demonstrated large numbers of committed erythroid progenitors in the blood and bone marrow of small premature babies that responded normally to r-HuEPO in vitro. These data effectively closed the pathophysiologic loop in anemia of prematurity and strongly implicated inadequate production of erythropoietin (EPO) as the underlying reason erythropoiesis is quantitatively insufficient in this clinical setting (see reference 4 for a review). By then, early studies had shown that treatment with r-HuEPO corrects the anemia associated with chronic renal failure, a disorder that resembles anemia of prematurity in that it is characterized by low EPO levels and inadequate red cell production.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

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