THE INFLUENCE OF SERUM ON THE UPTAKE, CONVERSION AND ACTION OF DIHYDROTESTOSTERONE IN RAT PROSTATE GLANDS IN ORGAN CULTURE

1975 ◽  
Vol 64 (2) ◽  
pp. 289-297 ◽  
Author(s):  
ILSE LASNITZKI ◽  
HILARY R. FRANKLIN
Keyword(s):  
Organ Culture ◽  
Horse Serum ◽  
Target Tissue ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Human Pregnancy ◽  
Human Male ◽  
Columnar Cells

SUMMARY The influence of serum on the uptake, conversion and action of dihydrotestosterone in relation to the sex steroid binding protein, TeBG, has been investigated in rat ventral prostates in organ culture. The organs were incubated with [1,2-3H]dihydrotestosterone in: (1) serum-free medium, (2) horse serum, foetal and newborn bovine serum or (3) human male and human pregnancy serum. With all sera the uptake of dihydrotestosterone fell with rising serum concentration, at first steeply and then more gradually. At the same concentration, the uptake was significantly lower in explants incubated with human pregnancy serum than in those kept with human male serum. The conversion of dihydrotestosterone to androstanediol followed the same pattern and less androstanediol was formed in the presence of pregnancy serum. Since pregnancy serum contains higher amounts of TeBG than male serum, the lowered uptake suggests that only the free hormone was available to the target organ. Addition of unlabelled dihydrotestosterone resulted in a higher uptake than that measured in explants incubated with the labelled steroid only. The effect of the human sera on uptake and conversion was correlated with the androgenic activity of dihydrotestosterone applied at physiological concentrations and expressed as the percentage of secretory columnar cells present. The degree of maintenance closely corresponded to the uptake of the hormone. In serum-free medium, the number of columnar cells approached the values found in vivo, with male serum their number, though reduced, was still substantial, with pregnancy serum it was extremely low. It is concluded that the amounts of TeBG present in serum regulate the supply of the hormone to the target tissue and thus control its biological action.

Developmental pattern of neurotensin content in rat hypothalamic neurons cultured in serum-free medium: comparison with in vivo data

1994 ◽  
Vol 81 (1) ◽  
pp. 128-130 ◽  
Author(s):  
V. Scarcériaux ◽  
D. Pélaprat ◽  
A.M. Lhiaubet ◽  
R.M. Schimpff ◽  
G. Tramu ◽  
...  
Keyword(s):  
Developmental Pattern ◽  
Serum Free Medium ◽  
Free Medium ◽  
Hypothalamic Neurons ◽  
Serum Free

Insulin/IGF1 Pathway Inhibition in Combination with All-Trans Retinoic Acid in Acute Promyelocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1347-1347
Author(s):  
Tiphanie Durfort ◽  
Luca Mazzarella ◽  
Pier Giuseppe Pelicci
Keyword(s):  
Insulin Receptor ◽  
Drug Target ◽  
Promyelocytic Leukemia ◽  
Serum Free Medium ◽  
Free Medium ◽  
Atra Treatment ◽  
Serum Free ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract Abstract 1347 Chemotherapy-free regimens are now a realistic goal in Acute Promyelocytic Leukemia (APL), as demonstrated by recent studies combining All-Trans Retinoic Acid (ATRA) with Arsenic Trioxide and Gemtuzumab Ozogamicin. Combinations of ATRA with kinase inhibitors, which have shown tremendous efficacy in several malignancies, are interesting alternatives but remain poorly characterized in APL. The Insulin-like growth factor 1 (IGF1) pathway is an attractive drug target because of its pervasive involvement in cell proliferation and metabolism of cancer cells. A variety of strategies to target the Insulin/IGF1 axis have been developed, but trials have struggled to demonstrate real efficacy over standard treatments, probably due to our still incomplete understanding of the intricacies of the downstream signalling cascade. Importantly, recent evidence suggests that inhibition of the sole IGF1 receptor may be inadequate because the structurally similar insulin receptor (IR) or IGF1R/IR heterodimeric receptors may be sufficient to support tumour growth. We dissected the IGF1R/IR pathway in the well established APL cell line NB4. IGF1 and insulin, but not the structurally similar IGF1R ligand IGF2, significantly increased NB4 growth in serum-free medium. ATRA treatment resulted in the downregulation, at both transcript and protein level, of several components of the proximal IR/IGF1R pathway: IGF1R (down by 64,9 ±1,7%), IR (down by 82,5 ±4,9%), the transducer proteins insulin receptor substrate 1 (IRS1) (down by 85,7 %) and IRS2 insulin receptor IRS2) by 98,1% (+/− 0,8). However, the downstream cascade remained ligand-responsive: stimulation of serum-starved cells with IGF1 induced a dose-dependent phosphorylation of AKT, FOXO, the mTOR target ribosomal protein S6 and ERK1-2. In addition, baseline levels of phospho-ERK were elevated after ATRA treatment. This suggested that ATRA-treated cells may increase their dependence on IGF1R for their survival. In agreement with this model, NB4 cell growth was completely inhibited by the anti-IGF1R antibody aIR3, but concomitant treatment with ATRA halved the half-maximal dose (IC50) of aIR3 to 1 ug/ml and promoted apoptosis (as assessed by flow cytometry). To identify IGF1R-targeting compounds with higher efficacy we screened novel small molecule inhibitors and found that the imidazopyrazine-derivative OSI-906 stopped NB4 cell growth with an IC50 of 1.5 μM in IGF1-supplemented serum-free medium. This compound is an orally available dual IR/IGF1R inhibitor, currently on trial for several solid tumours. In conclusion, we show that the Insulin/IGF1 signalling pathway is modulated at different levels by ATRA in APL cell lines and that this pathway represents a suitable and attractive drug target in combination with ATRA treatment. On the basis of data presented here we are currently testing the in vivo efficacy of ATRA/OSI906 combinations in in vivo models of APL. Disclosures: Off Label Use: Linsitinib (OSI-906) is a potent, selective orally active inhibitor of the insulin-like growth factor-1 receptor (IGF-1R).

Expansion Culture Of Hematopoietic Stem & Progenitor Cells From Frozen-Thawed, Non-Enriched Human Umbilical Cord Blood In Animal Component– & Serum–Free Medium Enhances Engraftment & Reduces Graft-Versus-Host-Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4460-4460
Author(s):  
Sudipto Bari ◽  
Pat PY Chu ◽  
Andrea Lim ◽  
Xiubo Fan ◽  
Gigi NC Chiu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Serum Free Medium ◽  
Free Medium ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Graft Versus Host ◽  
Nsg Mice ◽  
Animal Component

Hematopoietic stem cell transplantation in adults using umbilical cord blood (UCB) is limited by low cell dosage & post-thaw viability. In several clinical trials cytokine supplementation & stromal cell support have been shown to enhance total nucleated cells (TNC). However, clinical safety is compromised due to source inconsistency & population heterogeneity of stromal cells along with animal components of the conventional growth media. In this study, we demonstrate effective use of an animal component– & serum–free growth medium to enhance the viability & ex vivo expansion of SCID repopulating cells (SRC) from frozen-thawed, non-enriched UCB–mononucleated cells (UCB-MNC). UCB-MNC were cultured in a commercially available animal component– & serum–free medium, StemSpanTM–ACF (ACF), while StemSpanTM–SFEM (SFEM), a conventional serum–free medium with human and bovine components served as control. Both media (from STEMCELL Technologies INC. Vancouver, Canada) were supplemented with clinical grade SCF, Flt-3 ligand, TPO, & IGFBP2. The expansion effects were characterized based on cell viability, phenotypic stem & progenitor cells & functional in vitro & in vivo assays. After 3-days of culturing, viability of CD45+ UCB-MNC was maintained at a significantly higher level in ACF (90.7±0.2%) compared to SFEM (75.4±0.1%) (p<0.0001; n=3). Culturing for 11-days significantly (p<0.0001; n=6) increased CD45+CD34+CD38– hematopoietic progenitors in ACF (90.6±13.5 fold) compared to control (4.8±0.4 fold). Further phenotypic study of ACF expanded cells showed significant increases of 4.1-fold for CD45+CD34+C38–CD90+ stem cells (p<0.0001), 2.1-fold for CD45+CD34+CD13+CD33+ myeloid progenitors (p<0.01) and 2.3-fold for CD45+CD34+C38–CD7+(p<0.01) lymphoid progenitors compared to SFEM (n=6). Viable TNC expansions were 4.3±0.2 fold and 5.9±0.7 fold in ACF and SFEM respectively (n=6; p<0.05). Colony forming unit (CFU) assay showed that ACF supported significantly higher expansion of GM progenitors than SFEM (60.1±7.9 vs. 14.6±2.1 fold; p<0.00001; n=16). The numbers of multi-potent progenitors, CFU-GEMM, were maintained in ACF but decreased in SFEM (0.83±0.21 vs. 0.09±0.04 fold relative to non-expanded UCB; p<0.01; n=16). UCB-MNC cultured for 11 days reconstituted the bone marrow (BM) of sub-lethally irradiated NOD/SCID gamma (NSG) mice with human CD45+/71+ cells as measured 16 weeks after transplantation at a dosage of 1x108 cells/kg. The frequency of human cells was higher for UCB expanded in ACF (38.1±15.4%; n=5) than for UCB expanded in SFEM (3.4±2.1; n=14; p<0.01). Human CD34+ progenitors were also detected in BM of the engrafted mice at frequencies of 2.4±1.4% and 0.2±0.1% for ACF and SFEM expanded cells respectively (p<0.05). Human hematopoiesis was multi-lineage with significantly higher numbers of CD45+/71+ & CD15+/66b+ granulopoietic cells (71.4-fold; p<0.001) and CD19+/20+ B-lineage cells (23.1-fold; p<0.001) in mice transplanted with cells expanded in ACF (n=5) as compared to SFEM (n=14). At a transplantation dosage of 2.5x107 cells/kg, non-expanded grafts (n=10) had similar engraftment of CD45+/71+cells compared to ACF expanded grafts (n=5; p=0.14), while engraftment was lower for SFEM expanded grafts (n=12; p<0.01). Limiting dilution analysis revealed that SRC frequencies were increased, on average, 7.9– and 1.2–fold in ACF relative to SFEM expanded & non-expanded grafts respectively. NSG mice transplanted with non-expanded grafts had a significantly lower (p<0.001) survival rate (40.4%, n=47) compared to those transplanted with grafts expanded in ACF (90.9%, n=11) or SFEM (92.3% n=26), or injected with saline only (100%, n=7). The high mortality rate in recipients of non-expanded grafts was due to higher incidence of graft-versus-host-disease (GVHD) associated with significantly (p<0.01; n=6) higher CD45+CD7+T cells in comparison to expanded grafts. In conclusion, expansion of freeze-thawed, non-enriched UCB-MNC in animal component– & serum–free medium improves in vivo repopulation and reduces mortality due to GVHD in a xenotransplantation model. These findings could set the platform for developing safer, cheaper & time efficient clinical transplantation, since no animal components, in the form of serum albumin or stromal cells, are required to achieve desired ex vivo expansion of hematopoietic stem & progenitor cells & pre-clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IGF-I-induced enhancement of contractile response in organ-cultured aortae from diabetic rats is mediated by sustained thromboxane A2 release from endothelial cells

2005 ◽  
Vol 186 (2) ◽  
pp. 367-376 ◽  
Author(s):  
Tsuneo Kobayashi ◽  
Takayuki Matsumoto ◽  
Katsuo Kamata
Keyword(s):  
Endothelial Cells ◽  
Organ Culture ◽  
Contractile Response ◽  
Diabetic Rats ◽  
Receptor Expression ◽  
Diabetic Rat ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Igf I

We have investigated the mechanisms underlying the changes in vascular contractile responsiveness induced by insulin and IGF-I in established streptozotocin-induced diabetic rats. The contractile response to noradrenaline (NA) in organ-cultured diabetic rat aortae cultured with insulin or IGF-I was significantly greater than the corresponding responses in (a) diabetic rat aortae cultured in serum-free medium and (b) control rat aortae cultured with insulin or IGF-I. In aortae from which the endothelium was removed after organ culture the contractile response to NA was greater in those cultured with insulin or IGF-I than in those cultured in serum-free medium. This was not true of aortae endothelium denuded before organ culture. The IGF-I-induced enhancement was prevented by treatment with indomethacin (cyclo-oxygenase inhibitor), SQ29548 (thromboxane (TX) A2 receptor antagonist) or fregrelate (TXA2 synthase inhibitor). IGF-I-induced production of TXB2, a metabolite of TXA2, was greater in diabetic than in control aortae and was attenuated by endothelium denudation, indomethacin or AG1024 (IGF-I receptor inhibitor). The expression of the protein and mRNA for the IGF-I receptor (as assessed by RT-PCR and immunohistochemistry) was markedly increased within endothelial cells in diabetic aortae but only slightly increased within smooth muscle cells (versus control rat aortae). Thus, the NA-induced contractile response in aortae from diabetic rats was enhanced by both insulin and IGF-I and this enhancement may be mediated by sustained cyclo-oxygenase-dependent TXA2 production from endothelial cells. The observed enhancement of IGF-I receptor expression within endothelial cells may be causally related to the potentiation of vascular contractility and the increase in TXA2 production.

scholarly journals The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 167-178 ◽  
Author(s):  
JM Harlan ◽  
PD Killen ◽  
FM Senecal ◽  
BR Schwartz ◽  
EK Yee ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Membrane Glycoprotein ◽  
Ionophore A23187 ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Endothelial Monolayers ◽  
Neutrophil Adherence

Abstract We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of insulin and dexamethasone on lipoprotein lipase in human adipose tissue

1992 ◽  
Vol 262 (5) ◽  
pp. E695-E699 ◽  
Author(s):  
B. Appel ◽  
S. K. Fried
Keyword(s):  
Adipose Tissue ◽  
Organ Culture ◽  
Lipoprotein Lipase ◽  
Human Adipose Tissue ◽  
Maximal Dose ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Synthesis And Degradation ◽  
Synthetic Glucocorticoid

The mechanisms by which insulin and glucocorticoids modulate lipoprotein lipase (LPL) synthesis and degradation were examined in human adipose tissue fragments maintained in organ culture. Tissue fragments were cultured for 7 days in serum-free medium supplemented with or without insulin (7 nM) and with or without dexamethasone (30 nM), a synthetic glucocorticoid. Responses of LPL activity to both insulin and dexamethasone were obtained at doses within the physiological range. At a maximal dose, insulin increased heparin-releasable and total LPL activity (approximately 7-fold) by specifically increasing the rate of LPL synthesis (approximately 5-fold) determined by pulse labeling with [35S]methionine and [35S]cysteine and immunoprecipitation. Dexamethasone added in the presence of insulin increased heparin-releasable and total LPL activity approximately 8-fold but did not alter rates of LPL synthesis compared with insulin alone. Pulse-chase studies showed that the rate of LPL degradation was markedly slowed in the presence of dexamethasone plus insulin compared with insulin alone. These data suggest that, in human adipose tissue, insulin is essential for maintaining rates of LPL synthesis and that cortisol may play a key role in regulating human adipose tissue LPL at the posttranslational level by inhibiting the degradation of newly synthesized LPL.

scholarly journals Improved Expansion and In Vivo Function of Patient T Cells by a Serum-free Medium

2018 ◽  
Vol 8 ◽  
pp. 65-74 ◽  
Author(s):  
Andrew R. Medvec ◽  
Christopher Ecker ◽  
Hong Kong ◽  
Emily A. Winters ◽  
Joshua Glover ◽  
...  
Keyword(s):  
T Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free
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