A multipurpose instrument for quantitative intravital microscopy

1992 ◽  
Vol 73 (1) ◽  
pp. 296-306 ◽  
Author(s):  
A. Toth ◽  
M. E. Tischler ◽  
M. Pal ◽  
A. Koller ◽  
P. C. Johnson
Keyword(s):  
Parenchymal Cell ◽  
Fluorescence Emission ◽  
Repeated Measurements ◽  
Open Architecture ◽  
In Vitro Tests ◽  
Fluorescence Measurements ◽  
Tissue Area ◽  
Microcirculatory Function

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18–30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.

Abstract 342: Ultrasound-Enhanced Release of Bevacizumab from Echogenic Liposomes for Therapeutic Inhibition of Atherogenesis

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Melvin E Klegerman ◽  
Yuejiao Zou ◽  
George L Britton ◽  
Ali K Naji ◽  
Shao-Ling Huang ◽  
...  
Keyword(s):  
Doppler Ultrasound ◽  
Color Doppler ◽  
Fluorescence Emission ◽  
Color Doppler Ultrasound ◽  
Binding Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fluorescence Measurements ◽  
Echogenic Liposomes

Introduction. Bevacizumab (BEV) is a humanized monoclonal antibody to vascular endothelial growth factor (VEGF) that can ameliorate atheroma progression, the mechanism of which is believed to be through inhibition of neovascularization. Systemically, BEV has multiple adverse effects, which may be diminished in a controlled release delivery vehicle. Hypothesis. The purpose of this study was to test the hypothesis that BEV release from echogenic liposomes (BEV-ELIP) could be enhanced by color Doppler ultrasound (US). Methods. BEV-ELIP were prepared by a rehydration/loading-freeze/thaw-centrifugation-lyophilization method. BEV-ELIP samples were resuspended in 50ml of porcine plasma and divided into two portions, one of which was subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 minutes. After various time intervals, aliquots were fractionated by centrifugation and assayed for immunoreactive BEV with a direct enzyme-linked immunosorbent assay (ELISA). We considered immunoreactive BEV in washed pellet suspensions to represent ELIP surface-exposed VEGF-binding sites. To assure that BEV was accurately measured in plasma, we repeated the release protocol with fluoresceinated BEV (FITC-BEV) loaded into ELIP by the same method; BEV was measured by quantitation of fluorescence emission using a SpectraMax M5 microplate reader. Results. BEV-ELIP encapsulation efficiency was 43.2 ± 9.7% (SD, n = 5); 34.1 ± 29.3% of the encapsulated BEV was spontaneously released within the first 30 minutes. US caused an additional 4.1% of BEV to be released from the surface exposed compartment. Fluorescence measurements indicated that 43.2 ± 6.2% (n = 3) of encapsulated FITC-BEV-ELIP was spontaneously released in 60 minutes and that US caused an additional 5.7% to be released in that period, but, overall, US caused an additional 100 μg of BEV to be released or exposed per aliquot. Conclusions. We have demonstrated that BEV-ELIP retains its VEGF-binding activity in a liposomal formulation and that clinical Doppler US can significantly increase that activity, both by releasing free BEV and by enhancing the surface exposure of the immunoreactive antibody. These results warrant investigation of the formulation’s anti-angiogenic efficacy in vitro and in vivo.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Inhibition of Fibrinolysis and Fibrinogenolysis in Man: Comparison of ε-Aminocaproic Acid and Kallikrein Inhibitor

1963 ◽  
Vol 10 (01) ◽  
pp. 106-119 ◽  
Author(s):  
E Beck ◽  
R Schmutzler ◽  
F Duckert ◽  
Keyword(s):  
Aminocaproic Acid ◽  
Side Reactions ◽  
In Vitro Tests ◽  
Factor V ◽  
Clinical Use ◽  
Strong Inhibitor ◽  
Kallikrein Inhibitor ◽  
Therapeutic Possibilities

SummaryInhibitor of kallikrein and trypsin (KI) extracted from bovine parotis was compared with ε-aminocaproic acid (EACA): both substances inhibit fibrinolysis induced with streptokinase. EACA is a strong inhibitor of fibrinolysis in concentrations higher than 0, 1 mg per ml plasma. The same amount and higher concentrations are not able to inhibit completely the proteolytic-side reactions of fibrinolysis (fibrinogenolysis, diminution of factor V, rise of fibrin-polymerization-inhibitors). KI inhibits well proteolysis of plasma components in concentrations higher than 2,5 units per ml plasma. Much higher amounts of KI are needed to inhibit fibrinolysis as demonstrated by our in vivo and in vitro tests.Combination of the two substances for clinical use is suggested. Therapeutic possibilities are discussed.

Role of in vivo and in vitro Tests in the Diagnosis of Severe Cutaneous Adverse Reactions (SCAR) to Drug

2019 ◽  
Vol 25 (36) ◽  
pp. 3872-3880 ◽  
Author(s):  
Marcel M. Bergmann ◽  
Jean-Christoph Caubet
Keyword(s):  
Adverse Reactions ◽  
Lymphocyte Transformation ◽  
Stevens Johnson Syndrome ◽  
In Vitro Tests ◽  
High Morbidity ◽  
Patch Tests ◽  
Severe Cutaneous Adverse Reactions ◽  
Cutaneous Adverse Reactions

Severe cutaneous adverse reactions (SCAR) are life-threatening conditions including acute generalized exanthematous pustulosis (AGEP), Stevens-Johnson Syndrome (SJS), toxic epidermal necrolysis (TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Diagnosis of causative underlying drug hypersensitivity (DH) is mandatory due to the high morbidity and mortality upon re-exposure with the incriminated drug. If an underlying DH is suspected, in vivo test, including patch tests (PTs), delayed-reading intradermal tests (IDTs) and in vitro tests can be performed in selected patients for which the suspected culprit drug is mandatory, or in order to find a safe alternative treatment. Positivity of in vivo and in vitro tests in SCAR to drug varies depending on the type of reaction and the incriminated drugs. Due to the severe nature of these reactions, drug provocation test (DPT) is highly contraindicated in patients who experienced SCAR. Thus, sensitivity is based on positive test results in patients with a suggestive clinical history. Patch tests still remain the first-line diagnostic tests in the majority of patients with SCAR, followed, in case of negative results, by delayed-reading IDTs, with the exception of patients with bullous diseases where IDTs are still contra-indicated. In vitro tests have shown promising results in the diagnosis of SCAR to drug. Positivity is particularly high when the lymphocyte transformation test (LTT) is combined with cytokines and cytotoxic markers measurement (cyto-LTT), but this still has to be confirmed with larger studies. Due to the rarity of SCAR, large multi-center collaborative studies are needed to better study the sensitivity and specificity of in vivo and in vitro tests.

Hemostatic wound dressings: Predicting their effects by in vitro tests

2019 ◽  
Vol 33 (9) ◽  
pp. 1285-1297 ◽  
Author(s):  
Cornelia Wiegand ◽  
Martin Abel ◽  
Uta-Christina Hipler ◽  
Peter Elsner ◽  
Michael Zieger ◽  
...  
Keyword(s):  
Blood Clot ◽  
Wound Dressings ◽  
In Vitro Tests ◽  
Regenerated Cellulose ◽  
Oxidized Regenerated Cellulose ◽  
Inflammatory Reactions ◽  
In Vitro Techniques ◽  
Cotton Gauze

Background Application of controlled in vitro techniques can be used as a screening tool for the development of new hemostatic agents allowing quantitative assessment of overall hemostatic potential. Materials and methods Several tests were selected to evaluate the efficacy of cotton gauze, collagen, and oxidized regenerated cellulose for enhancing blood clotting, coagulation, and platelet activation. Results Visual inspection of dressings after blood contact proved the formation of blood clots. Scanning electron microscopy demonstrated the adsorption of blood cells and plasma proteins. Significantly enhanced blood clot formation was observed for collagen together with β-thromboglobulin increase and platelet count reduction. Oxidized regenerated cellulose demonstrated slower clotting rates not yielding any thrombin generation; yet, led to significantly increased thrombin-anti-thrombin-III complex levels compared to the other dressings. As hemostyptica ought to function without triggering any adverse events, induction of hemolysis, instigation of inflammatory reactions, and initiation of the innate complement system were also tested. Here, cotton gauze provoked high PMN elastase and elevated SC5b-9 concentrations. Conclusions A range of tests for desired and undesired effects of materials need to be combined to gain some degree of predictability of the in vivo situation. Collagen-based dressings demonstrated the highest hemostyptic properties with lowest adverse reactions whereas gauze did not induce high coagulation activation but rather activated leukocytes and complement.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

In Vitro Methods as a Tool in Neurotoxicity Studies

1995 ◽  
Vol 23 (4) ◽  
pp. 491-496
Author(s):  
Hanna Tähti ◽  
Leila Vaalavirta ◽  
Tarja Toimela
Keyword(s):  
Risk Evaluation ◽  
Toxicity Testing ◽  
In Vitro Models ◽  
In Vitro Tests ◽  
Industrial Chemicals ◽  
Mercury Compounds ◽  
Toxicological Data ◽  
In Vivo Tests

— There are several hundred industrial chemicals with neurotoxic potential. The neurotoxic risks of most of these chemicals are unknown. Additional methods are needed to assess the risks more effectively and to elucidate the mechanisms of neurotoxicity more accurately than is possible with the conventional methods. This paper deals with general tasks concerning the use of in vitro models in the evaluation of neurotoxic risks. It is based on our previous studies with various in vitro models and on recent literature. The induction of glial fibrillary acidic protein in astrocyte cultures after treatment with known neurotoxicants (mercury compounds and aluminium) is discussed in more detail as an important response which can be detected in vitro. When used appropriately with in vivo tests and with previous toxicological data, in vitro neurotoxicity testing considerably improves risk assessment. The incorporation of in vitro tests into the early stages of risk evaluation can reduce the number of animals used in routine toxicity testing, by identifying chemicals with high neurotoxic potential.

A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters

2006 ◽  
Vol 77 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Claudia Pellacani ◽  
Annamaria Buschini ◽  
Mariangela Furlini ◽  
Paola Poli ◽  
Carlo Rossi
Keyword(s):  
Surface Waters ◽  
In Vitro Tests ◽  
Pollutant Detection

scholarly journals Comparative Characteristics of Determination of Antioxidant Activity of Sea Buckthorn Fruits by Various Methods

2019 ◽  
Vol 8 (4) ◽  
pp. 48-52
Author(s):  
O. V. Trineeva
Keyword(s):  
Antioxidant Activity ◽  
Medicinal Plant ◽  
Plant Material ◽  
Natural Antioxidants ◽  
Sea Buckthorn ◽  
In Vitro Tests ◽  
Medicinal Plant Material

Introduction. Recently, much attention has been paid to the primary assessment of the pharmacological effect of various drugs using in vivo and in vitro tests. It is known that such a medicinal plant as sea buckthorn, in its phytochemical composition is rich in natural antioxidants: carotenoids, tocopherols, flavonoids, ascorbic acid, etc. In some publications there is information about the antioxidant activity of sea buckthorn and fatty oil based on them. However, information on the comparative characteristics of the use of various methods for determining the antioxidant activity of this type of medicinal plant material and the results obtained are not found in the scientific literature.Aim. The aim of this work was a comparative determination of the antioxidant activity of medicinal plant material of buckthorn fruits of various species of buckthorn.Materials and methods. The total antioxidant activity of water and water-alcohol extracts from the fruits of sea buckthorn fruits was determined using various techniques recommended in the literature. The antioxidant activity of the extracts was determined by permanganometric titration, in vitro inhibition of adrenaline autooxidation, and also in a biological model, Parametium caudatum cell culture.Results and discussion. The effect of the extractant polarity on the value of antioxidant activity was studied. It was found that the highest content of antioxidants in the extraction is observed when using 96 % ethanol as an extractant.Conclusion. Using three methods, the prospects of using sea buckthorn fruits and preparations based on them as a source of antioxidants are shown. 

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