ETUDE DE L'ACTION DU FACTEUR HYPOTHALAMIQUE LRF (LH-RELEASING FACTOR) CHEZ LE RAT IN VIVO & IN VITRO

1967 ◽  
Vol 55 (3) ◽  
pp. 481-496 ◽  
Author(s):  
Marian Jutisz ◽  
Annette Bérault ◽  
Marie-Anne Novella ◽  
Geneviève Ribot
Keyword(s):  
Dose Response ◽  
Crude Extract ◽  
Response Curve ◽  
Assay Method ◽  
Hypothalamic Extract ◽  
Rat Pituitary ◽  
Pituitary Glands ◽  
Releasing Effect

ABSTRACT A highly purified ovine LH-releasing factor (LRF) was obtained by a modification of the method previously described. After the fractionation of a crude hypothalamic extract on a Sephadex G-25 column, the LRF fraction was desalted and partially purified by chromatography on a Dowex 50 × 12 column and on an Amberlite CG 4B column. The last step of this method, chromatography on a CMC column, gave a purification of about 1600 times with respect to the crude extract. The action of this highly purified LRF preparation was studied on rat pituitary glands in vivo and in vitro. The method used in vivo was the evaluation of the LH-releasing effect of LRF in chronically ovariectomized, steroid-blocked rats (Ramirez & McCann 1963 b). A procedure was developed which allows a 4-fold concentration of the plasma LH from these rats, so that it can be assayed by a 4-point assay method. In the in vitro method, the pituitary glands of ovariectomized steroidblocked rats (Schally & Bowers 1964 a) were incubated in a Krebs-Ringer buffer with or without LRF, and the LH released into the medium was assayed using the O.A. A.D. method of Parlow. A dose-response curve was established between the log doses of LRF and the amount of LH released. This method can be used as a sensitive and specific assay for LRF. It was shown that a dose of 1.22 μg of LRF releases approximately 5 μg of LH per mg of pituitary tissue. This is about double of the amount of this hormone originally present in the pituitary glands of these rats (2.7 μg/mg). This leads us to the conclusion that the excess of this hormone was probably synthetized during the process of incubation. The amount of steroids injected as a blocking agents, appears to be very important for both in vivo and in vitro tests.

scholarly journals Predicting the in vivo developmental toxicity of benzo[a]pyrene (BaP) in rats by an in vitro–in silico approach

2021 ◽  
Author(s):  
Danlei Wang ◽  
Maartje H. Rietdijk ◽  
Lenny Kamelia ◽  
Peter J. Boogaard ◽  
Ivonne M. C. M. Rietjens
Keyword(s):  
Dose Response ◽  
In Silico ◽  
Response Curve ◽  
Developmental Toxicity ◽  
Toxicity Testing ◽  
Maximal Effect ◽  
Blood Concentrations ◽  
Reverse Dosimetry

AbstractDevelopmental toxicity testing is an animal-intensive endpoints in toxicity testing and calls for animal-free alternatives. Previous studies showed the applicability of an in vitro–in silico approach for predicting developmental toxicity of a range of compounds, based on data from the mouse embryonic stem cell test (EST) combined with physiologically based kinetic (PBK) modelling facilitated reverse dosimetry. In the current study, the use of this approach for predicting developmental toxicity of polycyclic aromatic hydrocarbons (PAHs) was evaluated, using benzo[a]pyrene (BaP) as a model compound. A rat PBK model of BaP was developed to simulate the kinetics of its main metabolite 3-hydroxybenzo[a]pyrene (3-OHBaP), shown previously to be responsible for the developmental toxicity of BaP. Comparison to in vivo kinetic data showed that the model adequately predicted BaP and 3-OHBaP blood concentrations in the rat. Using this PBK model and reverse dosimetry, a concentration–response curve for 3-OHBaP obtained in the EST was translated into an in vivo dose–response curve for developmental toxicity of BaP in rats upon single or repeated dose exposure. The predicted half maximal effect doses (ED50) amounted to 67 and 45 mg/kg bw being comparable to the ED50 derived from the in vivo dose–response data reported for BaP in the literature, of 29 mg/kg bw. The present study provides a proof of principle of applying this in vitro–in silico approach for evaluating developmental toxicity of BaP and may provide a promising strategy for predicting the developmental toxicity of related PAHs, without the need for extensive animal testing.

scholarly journals Cd2 Sets Quantitative Thresholds in T Cell Activation

1999 ◽  
Vol 190 (10) ◽  
pp. 1383-1392 ◽  
Author(s):  
Martin F. Bachmann ◽  
Marijke Barner ◽  
Manfred Kopf
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Dose Response ◽  
Response Curve ◽  
Cell Activation ◽  
Dose Response Curve ◽  
Lymphocyte Function

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell–antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose–response curve in vitro by a factor of 3–10. In comparison, stimulation of T cells in the absence of lymphocyte function–associated antigen (LFA)-1–intercellular adhesion molecule (ICAM)-1 interaction shifted the dose–response curve by a factor of 10, whereas absence of both CD2–CD48 and LFA-1–ICAM-1 interactions shifted the response by a factor of ∼100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca2+ fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.

Adenosine preconditions against endothelin-induced constriction of coronary arterioles

2000 ◽  
Vol 279 (6) ◽  
pp. H2593-H2597 ◽  
Author(s):  
Daphne Merkus ◽  
David W. Stepp ◽  
Deron W. Jones ◽  
Yasuhiro Nishikawa ◽  
William M. Chilian
Keyword(s):  
Ischemic Preconditioning ◽  
Dose Response ◽  
Response Curve ◽  
Protective Function ◽  
Vasodilatory Effect ◽  
Arteriolar Diameter ◽  
Myocardial Hypoperfusion ◽  
Coronary Arterioles

Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng · kg−1 · min−1administered intracoronary, n = 10) induced progressive constriction over 45 min [25 ± 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 μg · kg−1 · min−1administered intracoronary) for 20 min and 10 min of washout ( n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 ± 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50–150 μm) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM–5 nM) was rightward shifted after preconditioning with adenosine (1 μM) for 20 min and 10 min of washout ( n = 11). Blockade of A2 receptors [8-(3-chlorostyryl)caffeine, 1 μM, n = 9] but not A1 receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A2 receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.

RELEASE OF THYROTROPHIN IN VIVO AND IN VITRO BY SYNTHETIC NEUROHYPOPHYSIAL HORMONES

1964 ◽  
Vol 42 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Frank S. LaBella
Keyword(s):  
Dose Response ◽  
In Vitro Release ◽  
Optimal Level ◽  
Response Curves ◽  
Low Concentrations ◽  
Pituitary Glands ◽  
In Vivo Effects ◽  
Synthetic Oxytocin

The influence of synthetic oxytocin and synthetic lysine-8-vasopressin on the release of thyrotrophin (TSH) from slices of the "basophilic" zone of bovine anterior pituitary glands was determined. Up to 10-fold stimulation of TSH release occurred in the presence of the peptide hormones at low concentrations (approximately 10−11 to 10−9 M). Concentrations greater than 10−9 M were less stimulatory, ineffective, or inhibitory. In general, vasopressin stimulated at lower concentrations than did oxytocin. The dose–response curve of oxytocin began to descend at lower concentrations than did that of vasopressin.Stimulation of I131 discharge from the thyroids of propylthiouracil (PTU)-treated, day-old chicks was produced by the intraperitoneal injection of as little as 4 ng vasopressin or 25 ng oxytocin. As the injected dose of either peptide was increased beyond an optimal level, there was less enhancement of I131 discharge, and, with further increases, inhibition. The decreasing response began with lower doses of oxytocin than of vasopressin. The similarities of the dose–response curves of thyroid I131 discharge and of in vitro release of TSH indicate that the in vivo effects of injected neurohypophysial peptides are mediated through the release of endogenous TSH.

scholarly journals Correspondence of In Vitro and In Vivo Fluconazole Dose-Response Curves for Cryptococcus neoformans

2005 ◽  
Vol 49 (8) ◽  
pp. 3297-3301 ◽  
Author(s):  
Robert A. Larsen ◽  
Madeline Bauer ◽  
Ann M. Thomas ◽  
Alejandro Sanchez ◽  
Diane Citron ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dose Response ◽  
Response Curve ◽  
Drug Susceptibility Testing ◽  
Response Curves ◽  
In Vitro Susceptibility ◽  
Drug Concentrations ◽  
Wide Range

ABSTRACT We conducted in vitro experiments to evaluate the susceptibility of a clinical isolate of Cryptococcus neoformans to a wide range of concentrations of fluconazole. In vitro susceptibility was tested using broth macrodilution methods modified to provide a numeric count of viable organisms. The association between the quantitative in vitro response and fluconazole drug concentrations was estimated using local nonparametric regression. Regression analysis was used to assess the correspondence between the in vitro fluconazole concentration-response curve and the murine dose-response curve observed in our previously reported murine model. The regression model was then used to predict the murine response. There was a strong correspondence between in vitro measures of response to fluconazole alone and the previously reported biologic effects seen in the mouse. In vitro antifungal drug susceptibility testing can reliably predict the murine response to fluconazole.

Commercially available analogues of GnRH and LH secretion

1988 ◽  
Vol 119 (3) ◽  
pp. 345-352 ◽  
Author(s):  
T. R. Koiter ◽  
H. Moes ◽  
G. A. Schuiling
Keyword(s):  
Clinical Trials ◽  
Test System ◽  
In Vitro Test ◽  
Rat Pituitary ◽  
Pituitary Glands ◽  
Vitro Test ◽  
Lh Secretion ◽  
Derivatives Of

Abstract. Six agonistic derivatives of GnRH, four of which have already been evaluated in clinical trials, were compared with GnRH itself in an in vitro test system (incubation of rat pituitary glands). It was investigated 1) how the release of LH was affected when the pituitary glands were incubated in the presence of these analogues or GnRH, and 2) how the release of LH continued after removal of the analogues or GnRH from the medium. It was also investigated how an in vivo pretreatment for 6 days with several doses of 5 of these analogues or GnRH affects 3) the plasma concentration of LH, 4) the pituitary content of LH, and, in vitro, 5) the autonomous and 6) agonist-stimulated secretion of LH. Each of the analogues showed for each of the six investigated parameters a 10- to 100-fold higher potency than GnRH itself. Between the six analogues there were only minor differences. It is discussed how the six investigated parameters may be the expression of one single property of all these analogues, namely a long retention in the pituitary gland with a strong binding to the GnRH receptor.

scholarly journals 856 INBRX-106: a novel hexavalent anti-OX40 agonist for the treatment of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A897-A897
Author(s):  
Emily Rowell ◽  
Heather Kinkead ◽  
Elisabeth Torretti ◽  
Bryan Becklund ◽  
Florian Sulzmaier ◽  
...  
Keyword(s):  
T Cell ◽  
Dose Response ◽  
Response Curve ◽  
Single Agent ◽  
Cell Assays ◽  
Human Igg1 ◽  
Anti Tumor Activity ◽  
T Cell Assays

BackgroundOX40 is a co-stimulatory receptor enriched on immune cells in the tumor microenvironment. OX40 agonism promotes anti-tumor responses, both singly and in combination with checkpoint inhibitors. The cognate OX40 ligand, OX40L, is a trimeric protein that activates robust signaling through clustering. INBRX-106 is a novel hexavalent OX40 agonist that has been rationally designed to optimize target clustering and provide superior agonism to previously explored bivalent entities, leading to more potent anti-tumor activity.MethodsINBRX-106 is a homodimer, each half comprising three identical humanized, camelid single-domain antibody binding domains targeting OX40 linked end-to-end, and fused to an effector-enabled human IgG1 constant domain (Fc). Due to lack of rodent cross-reactivity, a valency, affinity and activity-matched murine surrogate, Hex-C04, was generated for the purpose of preclinical modeling. Hex-C04 contains an mIgG2a effector enabled Fc, the mouse isotype most analogous to the activity of human IgG1. The activity and potency of INBRX-106 and Hex-C04 were evaluated in functional in vitro T-cell assays, and the anti-tumor efficacy of Hex-C04 was evaluated alone or in combination with PD-1 blockade across a number of syngeneic tumor models.ResultsINBRX-106 binds specifically to OX40 with a sub nanomolar apparent affinity, without blocking the binding of its ligand OX40L. In vitro, cross-linking by INBRX-106 rapidly induces loss of OX40 surface expression in addition to driving receptor signaling. In primary T-cell assays, INBRX-106 is more potent than a bivalent comparator antibody, inducing greater upregulation of activation markers, cytokine production and proliferation. This costimulatory activity exhibits a bell-shaped dose-response curve, with maximal activity occurring at receptor occupancies of 30–100%. In vivo, tumor growth control by Hex-C04 also follows a bell-shaped dose response curve. Rapid loss of OX40 is observed in vivo as well, with both the degree and duration of OX40 loss dependent on Cmax and exposure. Hex-C04 demonstrated strong single-agent activity across a variety of preclinical tumor models including models that do not respond to a PD-1/PD-L1 checkpoint inhibitor, and this activity was improved in combination with a PD-1 blocking antibody.ConclusionsPreclinically, INBRX-106 significantly outperforms bivalent antibodies in co-stimulatory capacity and anti-tumor activity. On the weight of this data, Inhibrx Inc. has initiated a first-in-human Phase 1 trial of INBRX-106 as a single agent or in combination with Keytruda® (pembrolizumab). The complex relationship between dose, OX40 target modulation and activity indicate the importance of integrating preclinical data sets with emerging clinical data to make informed decisions regarding INBRX-106 dose and schedule.Trial RegistrationNCT04198766Ethics ApprovalThe care and use of all animals were reviewed and approved by the IACUC committees of Explora BioLabs and Molecular Diagnostic Services and conducted in accordance with AAALAC regulations.

226 EFFECTS OF MICROVESICLES SECRETED FROM EQUINE AMNIOTIC-DERIVED PROGENITOR CELLS ON IN VITRO LIPOPOLYSACCHARIDE-TREATED TENDON AND ENDOMETRIAL CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 244 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
M. C. Deregibus ◽  
G. Camussi ◽  
...  
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Calf Serum ◽  
Inverse Correlation ◽  
Dose Response Curve ◽  
Inflammatory Genes ◽  
Endometrial Cells ◽  
Tendon Cells

Administration of horse amniotic mesenchymal cell conditioned medium (AMC-CM) improves the in vivo recovery of spontaneous equine tendon lesions. This effect may involve paracrine mechanisms whose nature remains unknown. It has recently been demonstrated that microvesicles (MV) released from cells are an integral component of cell-to-cell communication during tissue regeneration. Aims of this study were to investigate the presence and type of MV secreted by AMC using Nanosight instrument (Malvern Instruments, Malvern, UK) and transmission electron microscopy (TEM) and the incorporation of MV in equine tendon and endometrial cells by fluorescence semiquantitative analysis. Tendon cells were used to understand the in vitro role of MV on stressed cells compared with the in vivo results previously obtained, while the endometrial cells were investigated in view of the prospective use of AMC-CM or MV in in vivo inflammatory endometrial diseases. Moreover, the ability of MV to counteract in vitro inflammation of tendon and endometrial cells induced by lipopolysaccharide (LPS) was also evaluated. The MV were obtained by ultracentrifugation at 100 000 × g for 1 h at 4°C of the media obtained by culturing AMC isolated from 3 different placentas. Tendon and endometrial cells were obtained from collagenase digestion for 17 and 3 h, respectively and cultured in HG-DMEM with 10% fetal calf serum. To study the ability of tendon and endometrial cells to incorporate MV, a dose-response curve was performed adding 10, 20, 30, 40, and 50 × 106 MV mL–1 labelled with PKH-26 for 24, 48, and 72 h. The uptake of MV was evaluated by an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with software for image acquisition. A dose-response curve of LPS was also investigated by apoptotic and MTT tests showing that 100 ng mL–1 at 48 h on tendon cells and 10 ng at 24 h on endometrial cells were the doses and times most effective in inducing cellular stress. RT-qPCR expression of pro-inflammatory genes such as metallopeptidase (MMP) 1 and 13 was evaluated in the in vitro LPS stress by Mann-Whitney U-test. Results by Nanosight Instrument showed that AMC secrete MV in the range of 100 to 200 nm; TEM showed budding of the AMC membrane, proving that these MV fall within the shedding vesicles category. The same semiquantitative fluorescence uptake signal was obtained when 50 × 106 MV were incorporated at 24 h, or 40 × 106 MV at 48 h, and 30 × 106 MV at 72 h, suggesting that an inverse correlation between concentration and time was found in MV uptake equally by tendon and endometrial cells. The MV induced a significant (P < 0.05) down-regulation of MMP1 and MMP13 expression in both cell lines after in vitro LPS stress. Our data suggest that these MV can be incorporated in tendon and endometrial cells and have a role in modulating inflammatory genes in vitro.

Synergism between Tissue-Type Plasminogen Activator and a Genetically Engineered Variant Lacking the Finger Domain, the Growth Factor Domain and the First Kringle Domain

1991 ◽  
Vol 65 (03) ◽  
pp. 286-290 ◽  
Author(s):  
C Mattson ◽  
K Wikström ◽  
C Sterky ◽  
G Pohl
Keyword(s):  
Synergistic Effect ◽  
Dose Response ◽  
Response Curve ◽  
Tissue Type ◽  
Lag Phase ◽  
Individual Dose ◽  
Response Curves ◽  
Type Plasminogen Activator

SummaryA modified variant of human tissue-type plasminogen activator (t-PA) lacking the finger domain (F), the growth factor domain (G) and the first kringle domain (K1), has an extended plasma half-life in vivo, compared to that of t-PA. When the variant (denoted K2P) was tested in vitro for its ability to lyse human plasma clots we found that the activity was characterized by a time lag phase and a sigmoidal dose-response curve. However, an attenuation of the lag phase in vitro was observed both when K2P was mixed with t-PA in a w/w ratio of 4 : 1 and when K2P was allowed to lyse a clot that had been pre-exposed to t-PA i.e. submitted to a limited plasmic digestion. Dosis that in vitro caused 50% lysis within 6 h were calculated from individual dose-response curves and were for K2P, t-PA and K2P/t-PA (4 : 1 w/w) 540 ng/ml, 360 ng/ml and 310 ng/ml, respectively. These results indicated a synergistic effect between K2P and t-PA. However, the data from individual dose-response curves showed that the effect of the K2P/t-PA mixture never was better than that of t-PA alone, and the synergistic effect in vitro is therefore considered to be of limited use. The thrombolytic activity in vivo was evaluated in a rabbit jugular vein thrombus model. Despite the lag phase observed in vitro, K2P was approximately 3 times as effective as t-PA in vivo (bolus injection). The thrombolytic effect of K2P was further potentiated when it was administred together with a small amount of t-PA (4 : 1 w/w). This potentiation in vivo was, in contrast to the effect in vitro, a useful synergistic effect as the dose-response curve for the K2P/t-PA mixture was steeper than that of t-PA and K2P alone. Doses that caused 50% lysis within 3 h were for t-PA, K2P and K2P/t-PA 1.28 mg/kg, 0.56 mg/kg and 0.35 mg/kg, respectively.

IN VITRO STUDIES OF THE RELEASE MECHANISM FOR VASOPRESSIN IN RATS

1966 ◽  
Vol 53 (4) ◽  
pp. 644-654 ◽  
Author(s):  
N. A. Thorn
Keyword(s):  
Release Mechanism ◽  
Posterior Pituitary ◽  
High Concentration ◽  
Vasopressin Release ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
Stimulation Period ◽  
Releasing Effect

ABSTRACT A study was made of the release of vasopressin activity from groups of isolated posterior pituitary hemilobes of rats, incubated in media with different ionic compositions and with different drugs added to the medium. Nicotine, amyl nitrite and ATP, which have been reported to cause a large in vivo release of hormone had no significant releasing effect in vitro. Caffeine too did not cause any release of hormone activity. About 5% of the vasopressin activity extractable from the isolated posterior pituitary hemilobes was released during stimulation with a high concentration of potassium in the medium. No more than this percentage could be mobilized during such a stimulation, even after further subdivision of the posterior pituitary glands, prolongation of the stimulation period or after increasing the calcium concentration in the medium five-fold. No release into the medium of vasopressin binding protein could be demonstrated during stimulation of vasopressin release. The results seem to be in agreement with the hypothesis that the release of vasopressin is intimately associated with arrival of impulses to the nerve endings in the posterior pituitary gland and that it takes place from a small pool of readily available hormone, presumably by dissociation of the hormone from the carrier protein.

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