Cytotoxicity of quahog parasite unknown (QPX) toward hard clam (Mercenaria mercenaria) haemocytes and interactions between different pathogen isolates and host strains

Parasitology ◽  
2009 ◽  
Vol 136 (11) ◽  
pp. 1281-1289 ◽  
Author(s):  
MICKAEL PERRIGAULT ◽  
BASSEM ALLAM
Keyword(s):  
Mercenaria Mercenaria ◽  
Neutral Red ◽  
Hard Clam ◽  
Field Investigations ◽  
Uptake Assay ◽  
Successful Establishment ◽  
Quahog Parasite Unknown ◽  
Transmission Studies

SUMMARYThe ability of pathogens to neutralize host defence mechanisms represents a fundamental requisite in the successful establishment of an infection. Host-pathogen interactions between quahog parasite unknown (QPX) and its hard clam host are poorly understood. Our prior in vivo investigations have shown that different QPX isolates display varying levels of pathogenicity toward clams. Similarly, field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. An in vitro approach was developed in this study to evaluate the toxicity of QPX cells and extracellular products toward haemocytes using a neutral red uptake assay. Results demonstrated that QPX produces virulence factors that are cytotoxic to M. mercenaria haemocytes. This cytotoxicity appears to be induced by clam factors, suggesting that it may play an important role in supporting QPX infection and proliferation within the host. Moreover, application of this technique to different QPX isolates and clam broodstocks indicates variations of QPX cytotoxicity in agreement with previous in vivo experiments, strengthening the existence of different QPX strains.

scholarly journals Mainstream Smoke Chemistry and in Vitro and In Vivo Toxicity of the Reference Cigarettes 3R4F and 2R4F

2012 ◽  
Vol 25 (1) ◽  
pp. 316-335 ◽  
Author(s):  
E Roemer ◽  
H Schramke ◽  
H Weiler ◽  
A Buettner ◽  
S Kausche ◽  
...  
Keyword(s):  
Neutral Red ◽  
Kinase Assay ◽  
Mainstream Smoke ◽  
Total Particulate Matter ◽  
Inhalation Study ◽  
Uptake Assay ◽  
Toxicological Activity ◽  
Health Canada

AbstractA new reference cigarette, the 3R4F, has been developed to replace the depleting supply of the 2R4F cigarette. The present study was designed to compare mainstream smoke chemistry and toxicity of the two reference cigarettes under the International Organization for Standardization (ISO) machine smoking conditions, and to further compare mainstream smoke chemistry and toxicological activity of the 3R4F cigarette by two different smoking regimens, i.e., the machine smoking conditions specified by ISO and the Health Canada intensive (HCI) smoking conditions.The in vitro cytotoxicity and mutagenicity was determined in the neutral red uptake assay, the Salmonella reverse mutation assay, and the mouse lymphoma thymidine kinase assay. Additionally, a 90-day nose-only inhalation study in rats was conducted to assess the in vivo toxicity. The comparison of smoke chemistry between the two reference cigarettes found practically the same yields of total particulate matter (TPM), ‘tar’, nicotine, carbon monoxide, and most other smoke constituents. For both cigarettes, the in vitro cytotoxicity, mutagenicity, and in vivo toxicity showed the expected smoke-related effects compared to controls without smoke exposure. There were no meaningful differences between the 2R4F and 3R4F regarding these toxicological endpoints. The assessments for the 3R4F cigarette by smoking regimen found as a trivial effect, due to the higher amount of smoke generated per cigarette under HCI conditions, an increased yield of toxicant and higher toxicological activity per cigarette. However, per mg TPM, ‘tar’, or nicotine, the amounts of toxicants and the in vitro toxicity were generally lower under HCI conditions, but the in vivo activity was not different between the two machine smoking conditions. Overall, as the main result, the present study suggests equivalent smoke chemistry and in vitro and in vivo toxicity for the 2R4F and 3R4F reference cigarettes.

The Neutral Red Uptake Assay: Comments on the Results of the Istituto Superiore di Sanità in the EC/HO Validation Study

1998 ◽  
Vol 26 (1) ◽  
pp. 61-68
Author(s):  
Annalaura Stammati ◽  
Franco Zampaglioni ◽  
Cristiana Zanetti
Keyword(s):  
Validation Study ◽  
Rank Correlation ◽  
Neutral Red ◽  
Home Office ◽  
Uptake Assay ◽  
Neutral Red Uptake ◽  
British Home ◽  
Chemical Groups

The neutral red uptake (NRU) assay was included, among others, in a validation study sponsored by the European Commission/British Home Office (EC/HO) study, for its reliability as an in vitro alternative to the Draize eye irritancy test. The test was performed in parallel by four laboratories (Istituto Superiore di Sanità [ISS], Microbiological Associates, Hatano Research Institute and Kurabo Industries) on 60 selected chemicals. The results obtained by the ISS are reported in this paper. A poor rank correlation was obtained between the in vivo endpoint and the ISS in vitro results for the full set of chemicals and for the subsets, with the exception of surfactants, by an independent statistics group. The same unsatisfactory results were obtained by the ISS group when the rank correlation was calculated for compounds divided into chemical groups. The performance of the NRU assay, as an alternative to the Draize eye irritancy test, is discussed.

A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test

1998 ◽  
Vol 26 (5) ◽  
pp. 679-708 ◽  
Author(s):  
Horst Spielmann ◽  
Michael Balls ◽  
Jack Dupuis ◽  
Wolfgang J. W. Pape ◽  
Odile de Silva ◽  
...  
Keyword(s):  
Neutral Red ◽  
Optimum Concentration ◽  
Alternative Methods ◽  
Uv Filter ◽  
Eu Directive ◽  
Optimum Concentration Range ◽  
The Impact ◽  
Positive Results

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1μg/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100μg/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1μg/ml and 10μg/ml, and that false positive results can be obtained at concentrations greater than 100μg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100μg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

scholarly journals Biocompatibility Evaluation of Electrospun Scaffolds of Poly (L-Lactide) with Pure and Grafted Hydroxyapatite

2017 ◽  
Vol 58 (4) ◽  
Author(s):  
José Manuel Cornejo-Bravo ◽  
Luis Jesús Villarreal-Gómez ◽  
Ricardo Vera-Graziano ◽  
María Raquel Vega-Ríos ◽  
José Luis Pineda-Camacho ◽  
...  
Keyword(s):  
Bacterial Adhesion ◽  
Neutral Red ◽  
C2c12 Cells ◽  
Surrounding Tissue ◽  
Neutral Red Assay ◽  
Electrospun Scaffolds ◽  
Muscle Tissues ◽  
Hematoxylin Eosin

<p>The objective of this work was to evaluate the biocompatibility of scaffolds of poly(<em>L</em>-lactide) with pure and grafted hydroxyapatite, at various concentrations of reinforcement. The biocompatibility tests were carried out <em>in vivo </em>in Wistar rats by implanting the material into the subcutaneous and muscle tissues from 1 to 14 weeks and evaluating the surrounding tissue stained with hematoxylin-eosin. For <em>in vitro </em>assays, MTT and neutral red assay were used to evaluate any cytotoxicity in Mioblast Muscle C2C12 Cells (ATCC® CRL-1772™) and Bovine Coronary Artery Endothelial Cells (BCAEC); <em>Escherichia coli </em>and <em>Staphylococcus aureus </em>were used to evaluate bacterial adhesion. All variants of scaffolds provoked a mild inflammatory response, without showing necrosis. No evidence of cytotoxicity was presented in cell viability tests and good bacterial cell adhesion was visualized for all of the materials studied.</p>

scholarly journals Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus

2020 ◽  
Vol 21 (7) ◽  
pp. 2549 ◽  
Author(s):  
Asghar Ali ◽  
Mark Stenglein ◽  
Thomas Spencer ◽  
Gerrit Bouma ◽  
Russell Anthony ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Critical Period ◽  
In Vitro Experiments ◽  
Placental Development ◽  
Expression Of Genes ◽  
Trophectoderm Cells ◽  
Successful Establishment

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28-let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28-let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

scholarly journals Antiplasmodial profile of selected compounds from Malaria Box: in vitro evaluation, speed of action and drug combination studies

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Guilherme Eduardo de Souza ◽  
Renata Vieira Bueno ◽  
Juliana Oliveira de Souza ◽  
Camila Lima Zanini ◽  
Fábio Cardoso Cruz ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Inhibitory Activity ◽  
Similarity Index ◽  
Structural Similarity ◽  
Neutral Red ◽  
Molecular Target ◽  
Benzodiazepine Derivatives ◽  
Malaria Box

Abstract Background Artemisinin-based combination therapy (ACT) is used as the first-line treatment of uncomplicated malaria caused by the Plasmodium falciparum parasite and chloroquine-resistant Plasmodium vivax parasites. Evidence of resistance to ACT has been reported in Cambodia, and without new and effective anti-malarial agents, malaria burden and mortality will rise. Methods The used MolPrint 2D fingerprints and the Tanimoto similarity index were used to perform a structural similarity search within the Malaria Box collection to select diverse molecular scaffolds that are different from artesunate. Next, the inhibitory potency against the P. falciparum 3D7 strain (SYBR Green I inhibition assay) and the cytotoxicity against HepG2 cells (MTT and neutral red assays) were evaluated. Then, the speed of action, the combination profile of selected inhibitors with artesunate, and the P. berghei in vivo activity of the best compounds were assessed. Results A set of 11 structurally diverse compounds from the Malaria Box with a similarity threshold of less than 0.05 was selected and compared with artesunate. The in vitro inhibitory activity of each compound confirmed the reported potencies (IC50 values ranging from 0.005 to 1 µM). The cytotoxicity of each selected compound was evaluated and used to calculate the selectivity index (SI values ranging from 15.1 to 6100). Next, both the speed of action and the combination profile of each compound with artesunate was assessed. Acridine, thiazolopyrimidine, quinoxaline, benzimidazole, thiophene, benzodiazepine, isoxazole and pyrimidoindole derivatives showed fast in vitro inhibitory activity of parasite growth, whereas hydrazinobenzimidazole, indenopyridazinone and naphthalenone derivatives were slow-acting in vitro inhibitors. Combinatory profile evaluation indicated that thiazolopyrimidinone and benzodiazepine derivatives have an additive profile, suggesting that the combination of these inhibitors with artesunate is favourable for in vitro inhibitory activity. The remaining compounds showed an antagonistic combinatory profile with artesunate. The collected data indicated that the indenopyridazinone derivative, a bc1 complex inhibitor, had a similar association profile in combination with proguanil when compared to atovaquone combined with proguanil, thereby corroborating the correlation between the molecular target and the combination profile. Lastly, the in vivo activity of the thiazolopyrimidinone and benzodiazepine derivatives were assessed. Both compounds showed oral efficacy at 50 mg/kg in a mouse model of Plasmodium berghei malaria (64% and 40% reduction in parasitaemia on day 5 post-infection, respectively). Conclusions The findings in this paper shed light on the relationship among the speed of action, molecular target and combinatory profile and identified new hits with in vivo activity as candidates for anti-malarial combination therapy.

Triphenyltin acetate toxicity : a biochemical and ultrastructural study on mouse thymocytes

1996 ◽  
Vol 15 (3) ◽  
pp. 219-225 ◽  
Author(s):  
E. Bollo ◽  
L. Ceppa ◽  
E. Cornaglia ◽  
C. Nebbia ◽  
B. Biolatti ◽  
...  
Keyword(s):  
Colorimetric Assay ◽  
Immunosuppressive Agents ◽  
Primary Cultures ◽  
Lactic Dehydrogenase ◽  
Neutral Red ◽  
Transmission Electron ◽  
Dose Dependent ◽  
Triphenyltin Acetate

1 Triphenyltin acetate (TPTA) has been shown to exert in vivo a selective toxic effect on the immune system. To assess in vitro possible alterations induced by TPTA exposure, primary cultures of mouse thymocytes were incubated up to 24 h with graded amounts (1-12 μM) ofthe organotin. 2 The cytotoxic activity has been evaluated with the MTT colorimetric assay, the neutral red (NR) assay and the lactic dehydrogenase (LDH) cellular release. Cell pellets were fixed with 2.5% glutaraldehyde, resin-embedded and ultrathin sections were observed through transmission electron microscopy. 3 After 2 h of incubation, dose-dependent increases of cytotoxicity were observed in thymocytes submitted to MTT and NR tests (up to 41.43% and 18.9%, respectively), while 22 h later this overt effect on cell viability was noticed merely in cells exposed to 12 μM TPTA. Dose- dependent increases of LDH leakage in the culture medium were observed all throughout the study. 4 Morphological investigations revealed features (chro matin condensation, cell membranes fragmentation and formation of membrane bound apoptotic bodies) sugges tive of apoptosis. 5 This study indicates that TPTA is cytotoxic to mouse thymocytes: morphologically, the rising of apoptosis is likely to be recognized, as previously reported in different in vitro studies with other immunosuppressive agents as dioxin and corticosteroids.

scholarly journals 260. Detection of Aspergillus fumigatus Infection in Mice with 2-Deoxy-2-[18F]fluorosorbitol

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S144-S145
Author(s):  
Yohan Yu ◽  
Seung ji Kang ◽  
Dong-Yeon Kim ◽  
Ayoung Pyo ◽  
Sehyeon Ji ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Pet Imaging ◽  
Ph Value ◽  
Diagnostic Methods ◽  
Bacterial Strains ◽  
Brain Infection ◽  
Uptake Assay ◽  
Uptake Test

Abstract Background Invasive aspergillosis is a major cause of infectious morbidity and mortality in immunocompromised patients.However, definitive diagnosis of invasive Aspergillus infection is still difficult due to the lack of a rapid, sensitive and specific diagnostic methods. In this studies, we investigated 2-deoxy-2-[18F]fluorosorbitol ([18F]FDS) which has been reported to be accumulated in Gram-negative bacteria but not in Gram-positive bacteria or healthy mammalian or cancer cells, for the imaging detection of Aspergullus fumigatus infections with PET in vivo. Methods [18F]FDS was synthesized by reduction of 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) using NaBH4. When the reaction was complete, the mixture was adjusted to a pH value to 6.5–7.5. Subsequently, the solution was filtered directly into a sterile product vial through a Sep-Pak Alumina N cartridge with a sterile filter. The probe uptake assay was performed by incubating bacterial cell and fungi with [18F]FDS (20 µCi) at 37°C for 2 h. Female BALB/c were immunosuppressed with cyclophosphamide and cortisone acetate prior to A. fumigatus intranasal, intramuscular, brain infection. The mircoPET images were obtained at 2 h after i.v. injection of [18F]FDS in infected mice. Results In vitro uptake test revealed significantly higher accumulation of [18F]FDS at 2 hin A. fumigatus, C. albicans and R. oryzae rather than with bacterial strains (Figure 1). PET imaging of BALB/c mice with pulmonary A. fumigatus infections showed obvious accumulation of [18F]FDS in the infected lungs compared with control (Figure 2). [18F]FDS PET imaging also detected A. fumigatus muscle and brain infection in mice. In infected shoulder muscle of mice, [18F]FDS PET imaging showed high legion-to-background ratio at 2 h. (4.05 ± 1.59, Figure 3). Conclusion [18F]FDS PET study demonstrated stable uptake in infected tissue with A. fumigatus and rapid clearance from the blood and other organs. [18F]FDS could be a useful imaging probe visualizing the invasive aspergillosis in vivo. Disclosures All authors: No reported disclosures.

In Vitro and In Vivo Immunomodulatory Activities of an Acidic Polysaccharide from Gracilaria lemaneiformis

2012 ◽  
Vol 468-471 ◽  
pp. 1941-1945 ◽  
Author(s):  
Yan Li Fan ◽  
Wen Hang Wang ◽  
Hong Shuo Chen ◽  
Nian Liu ◽  
An Jun Liu
Keyword(s):  
Neutral Red ◽  
Gracilaria Lemaneiformis ◽  
Splenocyte Proliferation ◽  
Cd4 Cells ◽  
Acidic Polysaccharide ◽  
Biomedical Material ◽  
Macrophage Phagocytosis ◽  
Potential Use

The effects of an acidic polysaccharide from Gracilaria lemaneiformis (GLSPs) on the immunomodulation in vitro and in vivo were investigated. It was shown that GLSPs with different concentrations significantly promoted the splenocyte proliferation and macrophage phagocytosis in vitro. In addition, GLSPs also remarkably enhanced the splenocyte proliferation induced by ConA or LPS in mice, notably improved the macrophage phagocytosis towards neutral red, and increased the percentages of CD3+ and CD4+ cells in peripheral blood of mice. The results suggested that GLSPs has significant effect on immunomodulation, and may have biomedical material applications and potential use in stimulating the immune system.

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