Use of Flow Cytometry to Assess Basophil Activation in Patients Allergic to Betalactam Antibiotics: Correlation between Flow-Cytometric Allergen Stimulation Test and Other in vivo and in vitro Tests

2001 ◽  
Vol 124 (1-3) ◽  
pp. 307-308
Author(s):  
M.L. Sanz ◽  
A.L. de Weck ◽  
C. Uasuf ◽  
P.M. Gamboa ◽  
M. Chazot
Keyword(s):  
Flow Cytometry ◽  
Basophil Activation ◽  
Stimulation Test ◽  
In Vitro Tests ◽  
Flow Cytometric

Flow cytometry as an auxiliary tool for the selection of probiotic bacteria

2015 ◽  
Vol 6 (5) ◽  
pp. 727-734 ◽  
Author(s):  
D. Mudroňová
Keyword(s):  
Flow Cytometry ◽  
Antimicrobial Agents ◽  
Probiotic Bacteria ◽  
In Vitro Tests ◽  
Good Growth ◽  
Bacterial Strains ◽  
Functional Characterisation ◽  
Selection Of

Selection of appropriate bacterial strains is crucial for development of new probiotic preparations. The fundamental prerequisite for potential efficacy of a probiotic preparation for oral application is the selection of appropriate bacterial strains with good gastrointestinal colonisation abilities, antimicrobial activity, and tolerance of conditions in the gastrointestinal tract, resistance to different antimicrobial agents, survival during processing and storage. The strain should be genetically stable, it should have good growth properties, to maintain its high viability at processing and when in storage. Mostly, the properties of promising strains are tested in the first phase in vitro, and only the best ones undergo subsequent in vivo testing. in vitro tests are often performed by classical microbiological cultivation methods which are material and time consuming, and they are not able to distinguish between ‘viable but nonculturable’ and dead bacteria. Flow cytometry is usually used for counting, phenotyping or functional characterisation of immune cells. Nowadays, flow cytometry is increasingly used in microbiology for counting bacteria, determining their viability and metabolic activity, detecting specific strains or testing their adherence abilities. The utilisation of flow cytometry in combination with an appropriate fluorescent labelling represents an effective and rapid method for the selection of probiotic bacteria.

scholarly journals Longer in vivo survival of CD59- and decay-accelerating factor-almost normal positive and partly positive erythrocytes in paroxysmal nocturnal hemoglobinuria as compared with negative erythrocytes: a demonstration by differential centrifugation and flow cytometry

Blood ◽  
1992 ◽  
Vol 79 (7) ◽  
pp. 1842-1845
Author(s):  
S Fujioka ◽  
T Yamada
Keyword(s):  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Flow Cytometric Analysis ◽  
Differential Centrifugation ◽  
Decay Accelerating Factor ◽  
Complement Regulatory Proteins ◽  
Flow Cytometric ◽  
Complement Lysis

Three populations of erythrocytes have been shown by flow cytometric analysis on complement regulatory proteins: CD59 and decay-accelerating factor (DAF) on erythrocytes in paroxysmal nocturnal hemoglobinuria (PNH). CD59 and DAF in PNH may be completely deficient in CD59- and DAF- negative erythrocytes, they may be decreased varyingly in partly positive erythrocytes, and they may be approximately normal in almost normal positive erythrocytes. Control erythrocytes are always CD59- and DAF-normal positive. CD59- and DAF-negative erythrocytes have been shown to be most sensitive to complement lysis in vitro. However, it has not yet been elucidated whether CD59- and DAF-almost normal positive and partly positive erythrocytes in a patient have a longer in vivo survival than negative erythrocytes. Blood from controls and PNH patients was separated in five fractions by differential centrifugation. CD59 and DAF on the fractionated erythrocytes were determined by flow cytometry using specific antibodies. Ratios of CD59- and DAF-almost normal positive and partly positive cells to negative erythrocytes were increased progressively from the top fraction to the bottom. The erythrocytes in the top fraction are younger and reticulocyte-rich, while those in the bottom are older and reticulocyte- poor. Hence, the present results indicate that CD59- and DAF-partly positive erythrocytes as well as almost normal positive erythrocytes in patients may have a longer in vivo survival than negative erythrocytes.

scholarly journals Longer in vivo survival of CD59- and decay-accelerating factor-almost normal positive and partly positive erythrocytes in paroxysmal nocturnal hemoglobinuria as compared with negative erythrocytes: a demonstration by differential centrifugation and flow cytometry

Blood ◽  
1992 ◽  
Vol 79 (7) ◽  
pp. 1842-1845 ◽  
Author(s):  
S Fujioka ◽  
T Yamada
Keyword(s):  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Flow Cytometric Analysis ◽  
Differential Centrifugation ◽  
Decay Accelerating Factor ◽  
Complement Regulatory Proteins ◽  
Flow Cytometric ◽  
Complement Lysis

Abstract Three populations of erythrocytes have been shown by flow cytometric analysis on complement regulatory proteins: CD59 and decay-accelerating factor (DAF) on erythrocytes in paroxysmal nocturnal hemoglobinuria (PNH). CD59 and DAF in PNH may be completely deficient in CD59- and DAF- negative erythrocytes, they may be decreased varyingly in partly positive erythrocytes, and they may be approximately normal in almost normal positive erythrocytes. Control erythrocytes are always CD59- and DAF-normal positive. CD59- and DAF-negative erythrocytes have been shown to be most sensitive to complement lysis in vitro. However, it has not yet been elucidated whether CD59- and DAF-almost normal positive and partly positive erythrocytes in a patient have a longer in vivo survival than negative erythrocytes. Blood from controls and PNH patients was separated in five fractions by differential centrifugation. CD59 and DAF on the fractionated erythrocytes were determined by flow cytometry using specific antibodies. Ratios of CD59- and DAF-almost normal positive and partly positive cells to negative erythrocytes were increased progressively from the top fraction to the bottom. The erythrocytes in the top fraction are younger and reticulocyte-rich, while those in the bottom are older and reticulocyte- poor. Hence, the present results indicate that CD59- and DAF-partly positive erythrocytes as well as almost normal positive erythrocytes in patients may have a longer in vivo survival than negative erythrocytes.

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.

A Novel Camptothecin Derivative 3j Inhibits Nsclc Proliferation Via Induction of Cell Cycle Arrest By Topo I-Mediated DNA Damage

2019 ◽  
Vol 19 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Yang Liu ◽  
Jingyin Zhang ◽  
Shuyun Feng ◽  
Tingli Zhao ◽  
Zhengzheng Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Cyclin D ◽  
Dna Relaxation ◽  
Cycle Arrest ◽  
H460 Cell ◽  
H1975 Cell

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 µM lower than irinotecan with an IC50 of 13.86±0.80 µM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 µM lower than irinotecan (IC50±SD, 5.35±0.38 µM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 µM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10µM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

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 A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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