An in vitro testing of chemoresistance in leukaemic patients

Biologia ◽  
2009 ◽  
Vol 64 (1) ◽  
Author(s):  
Jozef Hatok ◽  
Tatiana Matáková ◽  
Juraj Chudej ◽  
Ján Staško ◽  
Miroslava Dobrotová ◽  
...  
Keyword(s):  
Mtt Assay ◽  
Study Drug ◽  
Cellular Level ◽  
In Vitro Testing ◽  
In Vitro Test ◽  
Vitro Assay ◽  
Drug Concentrations ◽  
Leukaemic Cells ◽  
Relapsed Disease

AbstractThe fact that leukaemic cells are primarily or secondarily resistant to cytostatics is a serious phenomenon, which leads to the failure of chemotherapy of malignant diseases in clinical practise. Some detoxification and transporting systems are responsible for the generation of chemoresistance on the cellular level and the decrease of effectiveness in treatment. In vitro testing of chemoresistance of leukaemic cells is presently an inseparable component of “tailoring” therapy in the developing field of predictive oncology. The aim of this work was to estimate profiles of drug resistance, based on the predictive in vitro test, and to help in choosing the most effective cytostatic. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoline (MTT) assay was used, based on the direct effect of cytostatics on the viability of leukaemic cells in vitro. The number of living leukaemic cells was evaluated by a computer program, where LC50 (concentration of cytostatics lethal to 50% of leukaemic cells) was established from the achieved dose-relation curves. Seventy-one samples of leukaemic cells isolated from the patients’ peripheral blood or bone marrow were examined. All samples were tested to 3 cytostatics minimally. It was found by the in vitro assay, that resistance to dexamethasone, prednisolone, etoposide and vincristine is increased in patients with acute myeloid leukaemia disease, compared to the acute lymphoblastic leukaemia patients. In patients with a relapsed disease population, leukaemic cells are highly heterogeneous in the MTT assay. It was concluded that the MTT assay can be used to study drug interactions in vitro in leukaemia samples. The type of interaction was highly different between patients, and depended on drug concentrations.

Synthesis and Biological Evaluation of Amoxicillin Loaded Hybrid Material Composite Spheres Against Methicillin-Resistant Staphylococcus aureus

2020 ◽  
Vol 21 ◽  
Author(s):  
Muhammad Arfat Yameen ◽  
Amir Zeb ◽  
Raza E Mustafa ◽  
Sana Mushtaq ◽  
Nargis Aman ◽  
...  
Keyword(s):  
Mtt Assay ◽  
Hybrid Material ◽  
Ag Nanoparticles ◽  
Hybrid Composite ◽  
Synthesis Method ◽  
Biological Evaluation ◽  
Vitro Assay ◽  
Cytotoxicity Studies ◽  
Material Composite

Background: Incoherent use of antibiotics has led toward resistance in MRSA, which is becoming multidrugresistant with high rate of virulence in the community and hospital settings. Objective: Synergistic anti-MRSA activity was investigated in this study for hybrid material composite spheres of amoxicillin, Ag nanoparticles and chitosan which were prepared by one-step synthesis method and various characterizations were performed. Methods: Antimicrobial-susceptibility assay on MRSA was achieved by disc diffusion and agar dilution techniques while agar well diffusion was used for hybrid composite spheres. The in vitro and cytotoxicity studies was done by skin abrasion mouse model and MTT assay on RD cell respectively. Results: All isolates were resistant with the tested antibiotics except vancomycin. MIC against MRSA showed high resistance with amoxicillin from 4 to 128 mg L-1. The mean diameter of chitosan spheres and Ag nanoparticles was 02 mm and 277 nm respectively. Morphology of spheres was uneven, varied, porous and irregular in SEM and Ag nanoparticles presence and formation was also seen in micrograph. No substantial interface among drug, nanoparticles and polymer was found in XRD and IR showed characteristic peaks of all compound in the formulation. The in vitro assay showed augmented anti-MRSA activity with amoxicillin loaded hybrid composite spheres (22-29 mm). A significant reduction in microbial burden (~6.5 log10 CFU ml-1) was seen in vivo with loaded hybrid composite spheres formulation. The MTT assay indicated no potential cytotoxicity with hybrid composite spheres. Conclusion: Synergistic effect, amoxicillin, new hybrid formulation, anti-MRSA activity, composite spheres. nanoparticles.

Development and Application of a Transcriptomic Signature of Bioactivation in an Advanced In Vitro Liver Model to Reduce Drug-induced Liver Injury Risk Early in the Pharmaceutical Pipeline

2020 ◽  
Vol 177 (1) ◽  
pp. 121-139 ◽  
Author(s):  
Wen Kang ◽  
Alexei A Podtelezhnikov ◽  
Keith Q Tanis ◽  
Stephen Pacchione ◽  
Ming Su ◽  
...  
Keyword(s):  
Liver Injury ◽  
Human Model ◽  
Reactive Metabolites ◽  
In Vitro Test ◽  
Drug Induced ◽  
Vitro Assay ◽  
Drug Induced Liver Injury ◽  
Drug Candidates ◽  
Transcriptomic Signature

Abstract Early risk assessment of drug-induced liver injury (DILI) potential for drug candidates remains a major challenge for pharmaceutical development. We have previously developed a set of rat liver transcriptional biomarkers in short-term toxicity studies to inform the potential of drug candidates to generate a high burden of chemically reactive metabolites that presents higher risk for human DILI. Here, we describe translation of those NRF1-/NRF2-mediated liver tissue biomarkers to an in vitro assay using an advanced micropatterned coculture system (HEPATOPAC) with primary hepatocytes from male Wistar Han rats. A 9-day, resource-sparing and higher throughput approach designed to identify new chemical entities with lower reactive metabolite-forming potential was qualified for internal decision making using 93 DILI-positive and -negative drugs. This assay provides 81% sensitivity and 90% specificity in detecting hepatotoxicants when a positive test outcome is defined as the bioactivation signature score of a test drug exceeding the threshold value at an in vitro test concentration that falls within 3-fold of the estimated maximum drug concentration at the human liver inlet following highest recommended clinical dose administrations. Using paired examples of compounds from distinct chemical series and close structural analogs, we demonstrate that this assay can differentiate drugs with lower DILI risk. The utility of this in vitro transcriptomic approach was also examined using human HEPATOPAC from a single donor, yielding 68% sensitivity and 86% specificity when the aforementioned criteria are applied to the same 93-drug test set. Routine use of the rat model has been adopted with deployment of the human model as warranted on a case-by-case basis. This in vitro transcriptomic signature-based strategy can be used early in drug discovery to derisk DILI potential from chemically reactive metabolites by guiding structure-activity relationship hypotheses and candidate selection.

scholarly journals Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

2004 ◽  
Vol 11 (1) ◽  
pp. 21-28 ◽  
Author(s):  
James J. McSharry ◽  
Ann C. McDonough ◽  
Betty A. Olson ◽  
George L. Drusano
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Virus Yield ◽  
Drug Resistant ◽  
Wild Type ◽  
Vitro Assay ◽  
Drug Concentrations ◽  
Clinical Resistance ◽  
Na Gene

ABSTRACT A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.

scholarly journals Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level.

1996 ◽  
Vol 132 (3) ◽  
pp. 345-357 ◽  
Author(s):  
X M Wang ◽  
J G Peloquin ◽  
Y Zhai ◽  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Posttranslational Modifications ◽  
Glutathione Transferase ◽  
Cultured Cells ◽  
Human Fibroblasts ◽  
Cellular Level ◽  
Vitro Assay ◽  
Normal Functions ◽  
Projection Domain

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT-dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.

An in Silico, Biomarker-Based Method for the Evaluation of Virtual Neuropsychiatric Drug Effects

2017 ◽  
Vol 29 (4) ◽  
pp. 1021-1052 ◽  
Author(s):  
Peter J. Siekmeier
Keyword(s):  
In Silico ◽  
Fragile X ◽  
Drug Effects ◽  
Cellular Level ◽  
Model Parameters ◽  
Virtual Agents ◽  
In Vitro Test ◽  
Pharmacologic Treatments

The recent explosion in neuroscience research has markedly increased our understanding of the neurobiological correlates of many psychiatric illnesses, but this has unfortunately not translated into more effective pharmacologic treatments for these conditions. At the same time, researchers have increasingly sought out biological markers, or biomarkers, as a way to categorize psychiatric illness, as these are felt to be closer to underlying genetic and neurobiological vulnerabilities. While biomarker-based drug discovery approaches have tended to employ in vivo (e.g., rodent) or in vitro test systems, relatively little attention has been paid to the potential of computational, or in silico, methodologies. Here we describe such a methodology, using as an example a biophysically detailed computational model of hippocampus that is made to generate putative schizophrenia biomarkers by the inclusion of a number of neuropathological changes that have been associated with the illness (NMDA system deficit, decreased neural connectivity, hyperdopaminergia). We use the specific inability to attune to gamma band (40 Hz) auditory stimulus as our illness biomarker. We expose this system to a large number of virtual medications, defined by systematic variation of model parameters corresponding to five cellular-level effects. The potential efficacy of virtual medications is determined by a wellness metric (WM) that we have developed. We identify a number of virtual agents that consist of combinations of mechanisms, which are not simply reversals of the causative lesions. The manner in which this methodology could be extended to other neuropsychiatric conditions, such as Alzheimer’s disease, autism, and fragile X syndrome, is discussed.

scholarly journals Expression and Purification of an Active Form of the Mycobacterium leprae DNA Gyrase and Its Inhibition by Quinolones

2007 ◽  
Vol 51 (5) ◽  
pp. 1643-1648 ◽  
Author(s):  
Stéphanie Matrat ◽  
Stéphanie Petrella ◽  
Emmanuelle Cambau ◽  
Wladimir Sougakoff ◽  
Vincent Jarlier ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Active Form ◽  
Mycobacterium Leprae ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Drug Induced ◽  
Vitro Assay ◽  
Drug Concentrations

ABSTRACT Mycobacterium leprae, the causative agent of leprosy, is noncultivable in vitro; therefore, evaluation of antibiotic activity against M. leprae relies mainly upon the mouse footpad system, which requires at least 12 months before the results become available. We have developed an in vitro assay for studying the activities of quinolones against the DNA gyrase of M. leprae. We overexpressed in Escherichia coli the M. leprae GyrA and GyrB subunits separately as His-tagged proteins by using a pET plasmid carrying the gyrA and gyrB genes. The soluble 97.5-kDa GyrA and 74.5-kDa GyrB subunits were purified by nickel chelate chromatography and were reconstituted as an enzyme with DNA supercoiling activity. Based on the drug concentrations that inhibited DNA supercoiling by 50% or that induced DNA cleavage by 25%, the 13 quinolones tested clustered into three groups. Analysis of the quinolone structure-activity relationship demonstrates that the most active quinolones against M. leprae DNA gyrase share the following structural features: a substituted carbon at position 8, a cyclopropyl substituent at N-1, a fluorine at C-6, and a substituent ring at C-7. We conclude that the assays based on DNA supercoiling inhibition and drug-induced DNA cleavage on purified M. leprae DNA gyrase are rapid, efficient, and safe methods for the screening of quinolone derivatives with potential in vivo activities against M. leprae.

scholarly journals Comparison of adsorption of selected antibiotics on the filters in continuous renal replacement therapy circuits: in vitro studies

2019 ◽  
Vol 23 (2) ◽  
pp. 163-170 ◽  
Author(s):  
Dariusz Onichimowski ◽  
Hubert Ziółkowski ◽  
Krzysztof Nosek ◽  
Jerzy Jaroszewski ◽  
Elżbieta Rypulak ◽  
...  
Keyword(s):  
Renal Replacement Therapy ◽  
Continuous Renal Replacement Therapy ◽  
Replacement Therapy ◽  
Drug Concentration ◽  
Initial Level ◽  
Clinical Importance ◽  
Study Drug ◽  
Crystalloid Fluid ◽  
Drug Concentrations

Abstract The aim of this study was to assess the adsorption of selected antibiotics: vancomycin, gentamicin, ciprofloxacine and tigecycline in an experimental continuous veno-venous hemofiltration circuit with the use of both polyethyleneimine-treated polyacrylonitrile (PAN) and the polysulfone (PS) filter membranes. The crystalloid fluid dosed with one of antibiotic was pumped from a reservoir through a hemofiltration circuit (with PAN or PS membrane) and back to reservoir. All ultrafiltrate was also returned to the reservoir. During the procedures samples were collected from the post-hemofilter port at 5, 15, 30, 45, 60, 90, and 120 min. To determine spontaneous degradation of the antimicrobials, an additional bag with each study drug was prepared, which was not attached to the hemofiltration circuit. The samples from these bags were used as controls. In the case of vancomycin, gentamycin and tigecycline there was a statistically significant decrease in the drug concentration in the hemofiltration circuit in comparison to the control for PAN membrane (P < 0.05, P < 0.001, P < 0.001, respectively). In the case of ciprofloxacine adsorption was reversible and the drug concentrations increase to achieve the initial level for both membranes. Our studies indicated that a large portion of the administered dose of antibiotics may be adsorbed on a PAN membrane. In the case of gentamicin and tigecycline this amount is sufficiently big (over 90% of the administered dose) to be of clinical importance. In turn, adsorption on PS membranes is clearly lower (up to 10%) and may be clinically unimportant.

The Application of in Vitro Models to Research on Demineralization and Remineralization of the Teeth

1995 ◽  
Vol 9 (3) ◽  
pp. 175-193 ◽  
Author(s):  
D.J. White
Keyword(s):  
Analytical Techniques ◽  
Test Methods ◽  
In Vitro Models ◽  
In Vitro Tests ◽  
In Vitro Testing ◽  
In Vitro Test ◽  
Dental Tissues ◽  
Testing Protocols

Progress in in vivo and in situ experimentation has led many researchers to speculate as to the relevance and importance of in vitro testing protocols in caries research. A Medline/Biosis search for the present review revealed well over 300 citations (since 1989) documenting in vitro tests associated with caries research on mineralization and fluoride reactivity. The present survey documents these recent applications of in vitro test methods in both mechanistic and 'profile'* caries research. In mechanistic studies, in vitro protocols over the past five years have made possible detailed studies of dynamics occurring in mineral loss and gain from dental tissues and the reaction dynamics associated with fluoride anticaries activity. Similarly, in profile applications, in vitro protocols make possible the inexpensive and rapid-yet sensitive-assessment of F anticaries efficacy within fluoride-active systems, and these tests represent a key component of product activity confirmation. The ability to carry out single variable experiments under highly controlled conditions remains a key advantage in in vitro experimentation, and will likely drive even further utilization, as advances continue in physical-chemical and analytical techniques for substrate analysis in these protocols. Despite their advantages, in vitro testing protocols have significant limitations, most particularly related to their inability to simulate the complex biological processes involved in caries.

Development and Evaluation of a Novel Artificial Catheter-Deliverable Prosthetic Heart Valve and Method for in Vitro Testing

2009 ◽  
Vol 32 (5) ◽  
pp. 262-271 ◽  
Author(s):  
Thomase Claiborne ◽  
Danny Bluestein ◽  
Richard T. Schoephoerster
Keyword(s):  
Heart Valve ◽  
State Of The Art ◽  
Prosthetic Heart Valve ◽  
Test Methods ◽  
In Vitro Testing ◽  
In Vitro Test ◽  
Vitro Test ◽  
Percutaneous Heart Valve

Background This work presents a novel artificial prosthetic heart valve designed to be catheter or percutaneously deliverable, and a method for in vitro testing of the device. The device is intended to create superior characteristics in comparison to tissue-based percutaneous valves. Methods The percutaneous heart valve (PHV) was constructed from state-of-the-art polymers, metals and fabrics. It was tested hydrodynamically using a modified left heart simulator (LHS) and statically using a tensile testing device. Results The PHV exhibited a mean transvalvular pressure gradient of less than 15 mmHg and a mean regurgitant fraction of less than 5 percent. It also demonstrated a resistance to migration of up to 6 N and a resistance to crushing of up to 25 N at a diameter of 19 mm. The PHV was crimpable to less than 24 F and was delivered into the operating LHS via a 24 F catheter. Conclusion An artificial PHV was designed and optimized, and an in vitro methodology was developed for testing the valve. The artificial PHV compared favorably to existing tissue-based PHVs. The in vitro test methods proved to be reliable and reproducible. The PHV design proved the feasibility of an artificial alternative to tissue based PHVs, which in their traditional open-heart implantable form are known to have limited in vivo durability.

scholarly journals Evaluation of the effects of acepromazine and xylazineon viability in an equine dermal cell line

2020 ◽  
Vol 14 (4) ◽  
pp. 259-264
Author(s):  
Jéssica Grace da Silveira ◽  
Bruno Egídio Cappelari ◽  
Ana Paula Muterle Varela ◽  
Thais Fumaco Teixeira ◽  
Giovana Dantas de Araujo
Keyword(s):  
Veterinary Medicine ◽  
Cell Viability ◽  
Cell Line ◽  
Mtt Assay ◽  
Tissue Damage ◽  
The Other ◽  
Drug Concentrations ◽  
Dermal Cell

Xylazine and acepromazine are drugs used exclusively in veterinary medicine. Xylazineis used as a sedative, analgesic, and tranquilizer while acepromazine is used as a sedative, pre-anesthetic, and anesthetic adjuvant. In vitrodrug toxicity experimentation is essential to predict possible damage associated with treatment. This study was carried out to evaluate and compare the in vitroeffects of acepromazine and xylazine on cell viability. Equine Dermis cells lines were used to examine different drug concentrations (0.02 mg/mL, 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL). An MTT assay was carried out to reveal cell viability. Both tested drugs reduced the viability of ED cells at 0.02 and 0.01 mg/mL. At 0.005 mg/mL, only acepromazine presented an effect. These results corroborate previous studies with xylazine. On the other hand, this is thefirst report about acepromazine and cell viability. Previous studies suggest that the mechanisms involved in reducing cell viability are apoptosis for xylazine and the activation of the autophagic pathway for acepromazine. Both mechanisms have been seen in other drugs of the same classes. These findings reveal that both acepromazine and xylazine cause concentration-dependent cytotoxicity in vitro. Future experiments could further elucidate the mechanisms by which this effect happens and thus circumvent therisk of potential tissue damage in vivo.

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