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.

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.

Cholinergic responsiveness of respiratory and vascular tissues in two different rat strains

1988 ◽  
Vol 64 (1) ◽  
pp. 323-328 ◽  
Author(s):  
M. Badier ◽  
M. Soler ◽  
M. Mallea ◽  
S. Delpierre ◽  
J. Orehek
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Dose Response ◽  
Response Curve ◽  
Dose Response Curve ◽  
Maximal Response ◽  
Arterial Smooth Muscle ◽  
Cholinergic Agents

The airway and systemic arterial smooth muscle responsiveness to cholinergic agents of two strains of rats, Rat Albino (RA) and Brown Norway (BN), was compared in vivo and in vitro. In vivo, we measured the doses of carbachol that induced a 100% increase in lung resistance (PD100 RL), a 50% decrease in dynamic lung compliance (PD50 Cdyn), and the value of systolic blood pressure at the carbachol dose of 10 micrograms (Pa 10 micrograms). In vitro airway smooth muscle and systemic arterial smooth muscle responsiveness was assessed by measuring the maximal response to acetylcholine, the slope of the linear portion of the dose-response curve, and the negative logarithm of the molar concentration of acetylcholine producing 50% of the maximal response (pD2). PD100 and PD50 were about four times greater in BN rats than in RA rats. In contrast, Pa 10 micrograms was 1.5 lower in the BN rats. These differences persisted after bivagotomy. Tracheal pD2 was 25% greater in the RA than in the BN strain. The mean dose-response curve of parenchymal strips of RA rats was situated upward and to the left of the BN curve, but the reverse was observed for aortic smooth muscle dose-response curves. Thus 1) airway smooth muscle responsiveness to cholinergic agents is greater in RA strain than in BN, but the reverse is true for systemic arterial smooth muscle responsiveness; and 2) these differences are not due to factors extrinsic to the smooth muscle, since they occurred in vitro and may depend on different densities of muscarinic receptors.

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.

Study of dosimetric characteristics of a commercial optically stimulated luminescence system

2017 ◽  
Vol 16 (4) ◽  
pp. 461-475 ◽  
Author(s):  
Gourav K. Jain ◽  
Arun Chougule ◽  
Ananth Kaliyamoorthy ◽  
Suresh K. Akula
Keyword(s):  
Dose Response ◽  
Response Curve ◽  
Light Exposure ◽  
Dose Range ◽  
Optically Stimulated Luminescence ◽  
Dose Response Curve ◽  
Field Size ◽  
In Vivo Dosimetry ◽  
Detector Response

AbstractBackgroundOptically stimulated luminescence dosimeters (OSLDs) have a number of advantages in radiation dosimetry making them an excellent dosimeter for in vivo dosimetry. The study aimed to study the dosimetric characteristics of a commercial optically stimulated luminescence (OSL) system by Landauer Inc., before using it for routine clinical practice for in vivo dosimetry in radiotherapy. Further, this study also aimed to investigate the cause of variability found in the literature in a few dosimetric parameters of carbon-doped aluminium oxide (Al2O3:C).Materials and methodsThe commercial OSLD system uses Al2O3:C nanoDotTM as an active radiation detector and InLightTM microStar® as a readout assembly. Inter-detector response, energy, dose rate, field size and depth dependency of the detector response were evaluated for all available clinical range of photon beam energies in radiotherapy.ResultsInter-detector variation in OSLD response was found within 3·44%. After single light exposure for the OSL readout, detector reading decreased by 0·29% per reading. The dose linearity was investigated between dose range 50–400 cGy. The dose response curve was found to be linear until 250 cGy, after this dose, the dose response curve was found to be supra-linear in nature. OSLD response was found to be energy independent for Co60 to 10 MV photon energies.ConclusionsThe cause of variability found in the literature for some dosimetric characteristics of Al2O3:C is due to the difference in general geometry, construction of dosimeter, geometric condition of irradiation, phantom material and geometry, beam energy. In addition, the irradiation history of detector used and difference in readout methodologies had varying degree of uncertainties in measurements. However, the large surface area of the detector placed in the phantom with sufficient build-up and backscatter irradiated perpendicularly to incident radiation in Co60 beam is a good method of choice for the calibration of a dosimeter. Understanding the OSLD response with all dosimetric parameters may help us in estimation of accurate dose delivered to patient during radiotherapy treatment.

Mechanisms of histamine-induced contraction of canine airway smooth muscle

1983 ◽  
Vol 55 (1) ◽  
pp. 22-26 ◽  
Author(s):  
S. Shore ◽  
C. G. Irvin ◽  
T. Shenkier ◽  
J. G. Martin
Keyword(s):  
Smooth Muscle ◽  
Dose Response ◽  
Response Curve ◽  
Line Tension ◽  
Isometric Tension ◽  
Dose Response Curve ◽  
Base Line ◽  
Histamine Concentration ◽  
Release Of Acetylcholine

We studied the effects of atropine (10(-10) to 10(-6) M), tetrodotoxin (TTX) (10(-6) g/ml), and neostigmine (10(-7) M) on the histamine dose-response curve of canine tracheal smooth muscle (TSM) in vitro. Pretreatment with atropine or TTX reduced base-line tension in some TSM samples, whereas neostigmine invariably caused contraction of TSM. All concentrations of atropine reduced the maximum isometric tension produced by histamine (Tmax). With 10(-6), 10(-8), and 10(-10) M atropine, Tmax was 57, 74, and 88%, respectively, of its value in paired control samples. Atropine, 10(-9) to 10(-6) M, increased the concentration of histamine which produced 20% of Tmax, whereas 10(-6) M also increased the concentration required to produce 50% of Tmax. TTX reduced tension produced by low concentrations of histamine but had no effect at higher concentrations. Neostigmine shifted the histamine dose-response curve and caused greater tension for any given histamine concentration; Tmax increased by 30% (P less than 0.05). Our data are consistent with spontaneous release of acetylcholine from cholinergic nerves in the airway tissue and suggest that histamine either accelerates this release or interacts supra-additively with the acetylcholine at the smooth muscle.

Comparison of Activated Partial Thromboplastin Time Ratios with Ecarin Clotting Time Ratios for Monitoring Direct Thrombin Inhibitor Therapy: In-Vitro Study and Case Study of Lepirudin Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4149-4149
Author(s):  
Harry L. Messmore ◽  
Nancy J. Fabbrini ◽  
Ketty Badrinath ◽  
Richard Harriman ◽  
Omar Iqbal ◽  
...  
Keyword(s):  
Dose Response ◽  
Partial Thromboplastin Time ◽  
Response Curve ◽  
Activated Partial Thromboplastin Time ◽  
Dose Response Curve ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Blood Levels ◽  
Clotting Time

Abstract The direct thrombin inhibitors lepirudin and argatroban are widely used to treat heparin induced thrombocytopenia (HIT). It has been suggested that the Ecarin™ (Echis carinatus venom) clotting time may be superior to the activated partial thromboplastin time (APTT) for monioring purposes. We have prepared standard curves for lepirudin (Refludan™) and Argatroban covering therapeutic drug levels and corresponding APTT ratios (clotting time/control clotting time). Ecarin™ clotting time ratios were performed to demonstrate the practical application of these curves in the clinical care of patients. We report the case of an 80 year old man with HIT/HITT syndrome that occurred during therapy for deep vein thrombosis (DVT) with a low molecular weight heparin (LMWH). His initial coagulation studies were abnormal due to warfarin and LMWH therapy. The patient had a moderate impairment of renal function. Lepirudin therapy in a bolus dose of 14 mg (patient weight: 103.0 kg) resulted in supratherapeutic blood levels of drug and hematuria (Platelet count: 〉200 x 103). Dosage adjustment to maintain an APTT ratio of 1.5 for five days caused no hematuria, but thromboembolic complications occurred at that ratio. The in-vitro dose response curve for Lepirudin was compared with the Ecarin™ clotting time (ECT) ratio at those same concentration ranges in the same plasma. For comparison, Argatroban dose response curves in-vitro were made as well. ECT ratios were very similar to the APTT ratios in the patient’s samples. Representative ratios after the initial bolus, during the infusion period of five days and at the termination of that period are shown in the following table: Comparison of APTT and ECT Ratios APTT Ratio ECT Ratio 1.43 1.07 3.98 3.08 2.91 1.95 2.74 1.95 2.79 1.90 2.58 1.78 2.44 2.42 3.83 4.78 Conclusion: The ECT ratios reflect a steeper dose response curve than that observed with the APTT ratios. This may permit more accurate measurement of blood levels using ECT ratios.

scholarly journals Interim In vitro Dose-Response Curve for the Dicentric Biodosimeter Assay from a Philippine Radiotherapy Facility using a Linear Accelerator

2021 ◽  
Vol 55 (1) ◽  
Author(s):  
Antonio Carlo D. De Guzman ◽  
Carmencita D. Padilla ◽  
Henri Cartier S. Co ◽  
Elrick T. Inocencio ◽  
Edsel Allan G. Salonga
Keyword(s):  
Radiation Exposure ◽  
Dose Response ◽  
Linear Accelerator ◽  
Response Curve ◽  
Dose Response Curve ◽  
Quadratic Model ◽  
Linear Quadratic ◽  
Response Curves ◽  
Energy Agency

Background. Accidental radiation exposure can occur anytime. Biodosimeters help in quantifying the absorbed dose of individuals who are not equipped with personal dosimeters during radiation exposure. The dicentric assay can quantify radiation damage by correlating radiation dose exposure with the frequency of dicentric chromosomes in the peripheral lymphocytes extracted from exposed individuals. Objective. The study aims to present the interim results of the reference dose-response curve for a Philippine radiotherapy facility constructed using a 6MV linear accelerator (ClinacX, Varian). Methods. Samples of peripheral blood from healthy volunteers were irradiated in a customized water phantom of doses 0.10 to 5.0 Gray using a linear accelerator. The irradiated samples were cultured and analyzed following the International Atomic Energy Agency Cytogenetic Dosimetry Protocol (2011) with modifications. Linear-quadratic model curve fitting and further statistical analysis were done using CABAS (Chromosome Aberration Calculation Software Version 2.0) and Dose Estimate (Version 5.2). Interim results of the samples were used to generate these curves. Results. The dose-response curve generated from the preliminary results were comparable to published dose response curves from international cytogenetic laboratories. Conclusion. The generated dose-response calibration curve will be useful for medical triage of the public and radiologic staff accidentally exposed to radiation during medical procedures or in the event of nuclear accidents.

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