Leukocyte Viability Research Articles

Phytochemical Compounds and Biological Properties of Carob Pods (Ceratonia siliqua L.) Extracts at Different Ripening Stages

Ceratonia siliqua L. pods have been classically used in Mediterranean folk medicine as a potential source of natural antibacterial and antioxidant agents. The in vitro antiradical, immunostimulant, antibacterial and cytotoxic properties, as well as phytochemical composition of the ethanolic and aqueous extracts obtained from carob pods harvested at three ripening stages were assessed. Fructose, glucose, and sucrose, as quantified in both extracts, showed varied yields according to the ripening stage and solvent type. Likewise, significant differences were shown in bioactive compounds content depending on extraction solvent and ripening stage. Moreover, gilthead seabream (Sparus aurata L.) leucocyte viability and functions were not altered after being incubated for 24 h with the extracts of carob pods. On the other hand, the ethanolic extracts (0.75 and 1 mg mL−1), exhibited an interesting cytotoxic effect on a tumoricidal fish cell line (PLHC-1 cell). Besides, the ethanolic extract (1 mg mL−1) displayed a moderate bactericidal activity against different examined bacteria. Photobacterium damselae was found to be the most sensitive to different carob pod extracts, except for the aqueous extract obtained from ripened pods. While Vibrio harveyi and Vibrio anguillarum exhibited a strong resistance against the aqueous extracts. Our data indicate that the carob pods are an excellent source of natural bioactive molecules that would be used to prevent and treat several marine animal diseases.

An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses.

Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC. Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

Fragile neutrophils in surgical patients: A phenomenon associated with critical illness.

Leukocyte viability (determined by e.g. propidium iodide [PI] staining) is automatically measured by hematology analyzers to check for delayed bench time. Incidental findings in fresh blood samples revealed the existence of leukocytes with decreased viability in critically ill surgical patients. Not much is known about these cells and their functional and/or clinical implications. Therefore, we investigated the incidence of decreased leukocyte viability, the implications for leukocyte functioning and its relation with clinical outcomes. An automated alarm was set in a routine hematology analyzer (Cell-Dyn Sapphire) for the presence of non-viable leukocytes characterized by increased fluorescence in the PI-channel (FL3:630±30nm). Patients with non-viable leukocytes were prospectively included and blood samples were drawn to investigate leukocyte viability in detail and to investigate leukocyte functioning (phagocytosis and responsiveness to a bacterial stimulus). Then, a retrospective analysis was conducted to investigate the incidence of fragile neutrophils in the circulation and clinical outcomes of surgical patients with fragile neutrophils hospitalized between 2013-2017. A high FL3 signal was either caused by 1) neutrophil autofluorescence which was considered false positive, or by 2) actual non-viable PI-positive neutrophils in the blood sample. These two causes could be distinguished using automatically generated data from the hematology analyzer. The non-viable (PI-positive) neutrophils proved to be viable (PI-negative) in non-lysed blood samples, and were therefore referred to as 'fragile neutrophils'. Overall leukocyte functioning was not impaired in patients with fragile neutrophils. Of the 11 872 retrospectively included surgical patients, 75 (0.63%) were identified to have fragile neutrophils during hospitalization. Of all patients with fragile neutrophils, 75.7% developed an infection, 70.3% were admitted to the ICU and 31.3% died during hospitalization. In conclusion, fragile neutrophils occur in the circulation of critically ill surgical patients. These cells can be automatically detected during routine blood analyses and are an indicator of critical illness.

Death of guppy fish (Poecilia reticulata) leukocytes induced by in vivo exposure to temephos and spinosad

ABSTRACT Temephos and spinosad are pesticides used for control of vector-borne diseases such as dengue, chikungunya and zika. However, the inadequate use of these substances has affected the health of non-target organisms. The aim of this study was to evaluate and compare, the effects of temephos and spinosad on leukocyte viability and death, using guppy fish (Poecilia reticulate) as a model organism. Guppies were exposed to temephos (10 mg/L) and spinosad (0.5 mg/L) for 7, 14, and 21 days. Afterwards, they were placed in pesticide-free fish tanks (7, 35, and 70 days) for recovery. The results showed that exposure to temephos caused leukocyte death, even at 35 days of recovery. Contrarily, the exposure to spinosad did not cause leukocyte death. This research show, for the first time, that a single dose of temephos causes apoptosis up to 56 days post-exposition, indicating that this pesticide induces chronic effects on immune response cells.

Variability in peripheral blood enrichment techniques can alter equine leukocyte cellularity, viability and function

Peripheral blood is commonly sampled to assess the health status of human and veterinary patients. Venous blood collection is a minimally invasive procedure, and in the horse, the common collection site is the jugular vein. Post blood collection, sample processing for leukocyte enrichment can vary by research laboratory with the potential to yield different effects on the enriched cells and their function. The focus of the present study was to compare a common blood dilution-leukocyte enrichment technique using a Histopaque gradient medium (His) to a modified leukocyte buffy coat syringe-lymphocyte separation medium technique (Syr- LSM) with peripheral blood from 12 healthy horses. The endpoints examined included cell recovery/mL of blood, cell viability, leukocyte enrichment purity, leukocyte cell marker subset phenotype, leukocyte spontaneous and mitogen-induced proliferation and secretory TNFα concentrations. Leukocyte cell recovery/mL of whole blood and cell viability was significantly increased in enriched leukocytes from the Syr-LSM technique. Interestingly, the percentage of CD8+ and CD21+ were significantly increased with the His technique as was Con A-induced proliferation. Still, leukocyte cell purity and TNFα concentrations from the 72 h cell culture supernatants were comparable across the two enrichment techniques. To summarize, the type of whole blood leukocyte enrichment technique employed can affect the results of immunologic assay endpoints possibly altering data interpretation.

Diclofenac-induced cytotoxicity in cultured carp leukocytes.

Diclofenac is a drug commonly used in human and veterinary medicine for the treatment of diseases associated with inflammation and pain. Medicinal products enter waste and surface waters on an everyday basis and contaminate the aquatic environment. Fish are therefore permanently exposed to these chemicals dissolved in their aquatic environment. To simulate variable environmental conditions, the aim of our study was to examine adverse effects of diclofenac under different temperatures of cell incubation (18, 21, 24, 27 and 30 °C). Cyto-toxic and -static effects of diclofenac in concentrations of 0.001 mcg/ml, 0.01 microg/ml, 0.1 mcg/ml, 1 mcg/ml, 10 mcg/ml and 100 mcg/ml for the carp (Cyprinuscarpio) cultured leukocytes were quantified using detection of lactate dehydrogenase released from damaged cells. Overall DCF cytotoxicity was relatively low and its impact was pronounced at higher temperature and DCF concentration. Cells growth inhibition is changing more rapidly but it is high mainly at the highest concentration from low temperature. DNA fragmentation was not detected in tested leukocyte cell line. CYP450 increased diclofenac cytotoxicity only at the highest concentration but at incubation temperatures 18 and 27 °C. Leukocyte viability is essential for immune functions and any change can lead to reduction of resistance against pathogens, mainly in cold year seasons, when the immune system is naturally suppressed.

Investigation of Leukocyte Viability and Damage in Spiral Microchannel and Contraction-Expansion Array.

Inertial separation techniques in a microfluidic system have been widely employed in the field of medical diagnosis for a long time. Despite no requirement of external forces, it requires strong hydrodynamic forces that could potentially cause cell damage or loss during the separation process. This might lead to the wrong interpretation of laboratory results since the change of structures and functional characteristics of cells due to the hydrodynamic forces that occur are not taken into account. Therefore, it is important to investigate the cell viability and damage along with the separation efficacy of the device in the design process. In this study, two inertial separation techniques—spiral microchannel and contraction-expansion array (CEA)—were examined to evaluate cell viability, morphology and intracellular structures using a trypan blue assay (TB), Scanning Electron Microscopy (SEM) and Wright-Giemsa stain. We discovered that cell loss was not significantly found in a feeding system, i.e., syringe, needle and tube, but mostly occurred in the inertial separation devices while the change of cell morphology and intracellular structures were found in the feeding system and inertial separation devices. Furthermore, percentage of cell loss was not significant in both devices (7–10%). However, the change of cell morphology was considerably increased (30%) in spiral microchannel (shear stress dominated) rather than in CEA (12%). In contrast, the disruption of intracellular structures was increased (14%) in CEA (extensional and shear stress dominated equally) rather than spiral microchannel (2%). In these experiments, leukocytes of canine were used as samples because their sizes are varied in a range between 7–12 µm, and they are commonly used as a biomarker in many clinical and medical applications.

The role of Kuniran (U. moluccensis) and Gurami (O. goramy) fish thorns and scales in increasing salivary leukocyte and monocyte cells viability against Streptococcus mutans

Background: Kuniran thorns and Gurami fish scales are rich in protein and minerals such as dentin believed to increase cell viability against Streptococcus mutans (S. mutans) that causes dental caries. These, in turn, can cause systemic diseases if left untreated. Purpose: This study aims to analyze the influence of Kuniran thorns and Gurami fishes scales on the viability of monocytes and salivary leukocytes against S. mutans. Methods: Monocytes and leukocytes salivary cells were placed on a microtiter plate and treated according to the nature of each group. This study comprised the following groups: control group: untreated; S. mutans group: induced by S. Mutans; Gurami thorn group: thorns + S. Mutans; Gurami scales group: scales + S. Mutans; Kuniran thorn group: thorns + S. Mutans; Kuniran scales group: scales + S. mutans. Viability analysis involved staining with Tripan Blue. Furthermore, the number of viable cells (white) was calculated under an inverted microscope at 200 times magnification from five fields of view. Data was analyzed by means of an ANOVA test followed by LSD test. Results: The ANOVA and LSD tests confirmed significant differences (0.01<P). Kuniran thorns and Gurami fish scales increased the viability of monocytes and salivary leukocytes, but not significantly. The content of flavonoids, amino acids, omega 3, omega 6 and antioxidants increased leukocyte metabolism, thereby increasing cell resistance to S. mutans infection. Conclusion: Kuniran thorns (U. moluccensis) and Gurami (O. goramy) fish scales increase the viability of salivary leukocyte and monocyte cells against Streptococcus mutans.

In vitro effects of the neuroactive substances serotonin and γ-aminobutyric acid on leucocytes from sticklebacks (Gasterosteus aculeatus)

The majority of parasites have evolved strategies to evade the immune responses of their hosts. Neuroactive substances produced by cestodes are possible candidate molecules for regulating host immune responses. The neurons of helminths can synthesize a wide range of molecules that are identical to the ones functioning in their host organisms, and host lymphocytes have receptors for these neuroactive substances. We hypothesized that in teleost fish, antihelminthic immune responses are regulated via 5-hydroxytryptamine (5-HT, or serotonin) and γ-aminobutyric acid (GABA). In the present study, we investigated the in vitro influence of serotonin, GABA and Schistocephalus solidus (helminth) antigens on basic characteristics of the three-spined stickleback Schistocephalus solidus cellular immune response. Head kidney leucocytes (HKLs) were analysed by flow cytometry for cell viability and the frequency of leucocyte subsets (the granulocyte-to-lymphocyte ratio) and by a chemiluminescence assay for the production of reactive oxygen species (ROS). In short-term (2-h) HKL cultures, 5-HT did not change the total numbers of live HKLs, but the production of ROS decreased significantly with all 5-HT concentrations. In long-term (96-h) cultures, high 5-HT concentrations induced a decrease in leucocyte viability. This coincided with elevated ROS production in cultures with all 5-HT concentrations. In short-term (2-h) HKL cultures, GABA did not change the total numbers of live HKLs, but the production of ROS decreased significantly with high (100 nmol L−1) GABA concentrations. In long-term (96-h) cultures, high and medium concentrations of GABA (100 nmol L−1 and 10 nmol L−1) elevated the numbers of live HKLs compared to controls. The granulocyte-to-lymphocyte ratios generally increased upon exposure to GABA at all concentrations. All concentrations of GABA alone elevated the ROS production of HKLs compared to controls. In the present work, we showed that the neuroactive substances serotonin and GABA regulate the teleost immune system. Our study supports the hypothesis that these substances might be immunomodulators in tapeworm–fish parasite-host interactions.

Debaryomyces hansenii CBS 8339 β-glucan enhances immune responses and down-stream gene signaling pathways in goat peripheral blood leukocytes

Debaryomyces hansenii-derived β-glucan has shown immunostimulant effect on aquaculture species and recently on goat peripheral blood leukocytes. Moreover, the marine yeast D. hansenii CBS 8339 has demonstrated to enhance fish immune response. Nonetheless, the associated immune signaling pathways induced by β-glucan from this marine yeast have not been characterized yet. This study described the effects of β-glucan from D. hansenii CBS 8339 against challenge with Escherichia coli and activation of possible mechanisms on goat peripheral blood leukocytes. The proton nuclear magnetic resonance spectra showed that D. hansenii had β-(1,3)(1,6)-glucan. The phagocytic ability enhanced after E. coli challenge, and nitric oxide production increased before and after challenge in leukocytes stimulated with D. hansenii β-glucan. In addition, an early gene expression stimulation was found related to β-glucan recognition by TLR2 and Dectin-1 receptors, intracellular regulation by Syk, TRAF6, MyD88 and transcription factor NFκB, and effector functions of pro-inflammatory cytokine, such as IL-1β and TNF-α. Interestingly, simulation with D. hansenii-derived β-glucan increased leukocyte viability after E. coli challenge. In conclusion, β-glucan from D. hansenii CBS 8339 reduced cytotoxic effects of E. coli and modulated signaling pathways and innate immune response in goat peripheral blood leukocytes.

Centrifuge-less red blood cell lysis and immunostaining of whole blood for flow cytometry using DA-Cell washer and plate

Abstract Blood cells are prime indicators of immuno-surveillance, and the ease of blood sampling makes blood analysis a key interest for clinical and research applications. While current flow cytometry methods are high-throughput and provide fine resolution in the segregation of white blood cell (WBC) populations, WBC enrichment involving red blood cell (RBC) lysis are laborious and typically performed manually, contributing to experimental variability especially as blood cells are sensitive to physical and chemical stress. We describe RBC lysis and leukocyte immunostaining on a centrifuge-less platform DA-Cell™, using a novel wall-less plate and laminar flow washer. The DropArray (DA) plate consists of an array of 96 hydrophilic spots surrounded by hydrophobic surface, which functions as a virtual wall that separates each spot. The maximum volume of each spot can be increased to 300μL with an insert. During lysis, WBC settle to the surface of the spot, allowing the spent lysis buffer to be removed. After removal of the insert, the plate goes through a gentle 3–4min laminar-flow washing process in the DA-Cell washer, decreasing cell washing time by at least 50% while eliminating centrifugation that stresses cells and disrupts antibody binding. In studies comparing mouse whole blood lysis (1X RBC Lysis buffer, eBioscience) and antibody staining by conventional tube centrifuge and DA-Cell, DA-Cell achieved dramatically higher staining index and improved resolution of cell cluster by flow cytometry. CD45+ leukocyte recovery and viability was uncompromised compared to conventional tube centrifuge. In summary, DA-Cell system provides gentle, fast and convenient blood lysis, while improving data quality with superior antibody staining.

The effect of 1.5 T cardiac magnetic resonance on human circulating leucocytes.

Investigators have proposed that cardiovascular magnetic resonance (CMR) should have restrictions similar to those of ionizing imaging techniques. We aimed to investigate the acute effect of 1.5 T CMR on leucocyte DNA integrity, cell counts, and function in vitro, and in a large cohort of patients in vivo. In vitro study: peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, and histone H2AX phosphorylation (γ-H2AX) expression, leucocyte counts, and functional parameters were quantified using flow cytometry under the following conditions: (i) immediately following PBMC isolation, (ii) after standing on the benchside as a temperature and time control, (iii) after a standard CMR scan. In vivo study: blood samples were taken from 64 consecutive consenting patients immediately before and after a standard clinical scan. Samples were analysed for γ-H2AX expression and leucocyte counts. CMR was not associated with a significant change in γ-H2AX expression in vitro or in vivo, although there were significant inter-patient variations. In vitro cell integrity and function did not change with CMR. There was a significant reduction in circulating T cells in vivo following CMR. 1.5 T CMR was not associated with DNA damage in vitro or in vivo. Histone H2AX phosphorylation expression varied markedly between individuals; therefore, small studies using γ-H2AX as a marker of DNA damage should be interpreted with caution. Cardiovascular magnetic resonance was not associated with loss of leucocyte viability or function in vitro. Cardiovascular magnetic resonance was associated with a statistically significant reduction in viable leucocytes in vivo.

Localized Electroporation With Dielectrophoretic Field Flow Fractionation: Toward Removal of Circulating Tumour Cells From Human Blood.

This paper presents the design and experimental performance of a microelectrode-based device to selectively lyse cells in a flow in a microfluidic channel. Localized cell lysis is achieved by utilizing "irreversible electroporation," in which cells are exposed to high magnitude electric pulses. Localized cell lysis in a flow has research applications and may allow for the removal of harmful cells, such as circulating tumor cells from blood. Due to the dependence of this technique on the magnitude of the applied electric field, lethal electric field regions can be localized in the channel by the calibration of the applied voltage. Dielectrophoresis field flow fractionation is used to levitate target cancer cells in the lethal region of the device microchannel. Experiments are performed to demonstrate the localized lysis of MCF7 cancer cells in a mixture of blood cells. Due to their larger size, these circulating tumor cell analogues levitate to a greater height in the channel than erythrocytes. MCF7 lysis is observed to increase from 4.6% in control experiments to 57.3% in active experiments. Leukocyte viability was unaffected in active experiments. These results demonstrate the feasibility of localizing cell lysis in a microfluidic flow environment.

Preservative solution that stabilizes erythrocyte morphology and leukocyte viability under ambient conditions

The deterioration of whole blood ex vivo represents a logistical hurdle in clinical and research settings. Here, a cocktail preservative is described that stabilizes leukocyte viability and erythrocyte morphology in whole blood under ambient storage. Neutrophil biostabilization was explored using a sophisticated microfluidic assay to examine the effectiveness of caspase inhibition to stabilize purified neutrophils. Following 72 h ambient storage, neutrophils remained fully functional to migrate towards chemical cues and maintained their ability to undergo NETosis after stimulation. Furthermore, stored neutrophils exhibited improved CD45 biomarker retention and reduced apoptosis and mortality compared to untreated controls. To stabilize erythrocyte morphology, a preservative solution was formulated using Taguchi methods of experimental design, and combined with the caspase inhibitor to form a whole blood cocktail solution, CSWB. CSWB was evaluated in blood from healthy donors and from women with metastatic breast cancer stored under ambient conditions for 72 h. CSWB-treated samples showed a significant improvement in erythrocyte morphology compared to untreated controls. Leukocytes in CSWB-treated blood exhibited significantly higher viability and CD45 biomarker retention compared to untreated controls. This 72 h shelf life under ambient conditions represents an opportunity to transport isolates or simply ease experimental timelines where blood degradation is problematic.

Immune response after an experimental intramammary challenge with killed Staphylococcus aureus in cows and heifers vaccinated and not vaccinated with Startvac, a polyvalent mastitis vaccine

An experimental trial was conducted to explore the effect of vaccination with a polyvalent vaccine against mastitis (Startvac) on the early immune response after experimental intramammary challenge with a heterologous killed Staphylococcus aureus strain. The effect of vaccination on milk production, clinical signs, quarter milk somatic cell count, milk polymorphonuclear neutrophilic leukocyte (PMN) concentration and viability, the concentration of antigen-specific antibodies [slime associated antigenic complex (SAAC) and J5] and their IgG1 and IgG2 subtypes in both serum and whey, and the antigen-specific IFN-γ, IL-4, and IL-17 production by blood lymphocytes after in vitro stimulation with S. aureus and Escherichia coli extracts were determined. A cohort of 8 clinically healthy end-term cows and heifers were conveniently selected, of which half was vaccinated with Startvac at 45 and 10 d before the expected calving date and half served as nonvaccinated control animals. At 15 d in milk, 2 contralateral quarters of each of the 8 animals were challenged with 2×109 cfu/mL of the formaldehyde-killed S. aureusC195strain. The 2 other quarters were infused with phosphate-buffered saline and served as control quarters. The increase in both quarter milk somatic cell count and PMN concentration and the drop in milk production after S. aureus inoculation was less pronounced in the vaccinates than in the nonvaccinates, reflecting a less severe inflammatory response. No significant differences in PMN viability between vaccinates and nonvaccinates could be demonstrated. The serum SAAC- and J5-specific antibody concentration significantly increased across the dry period in the vaccinated animals only. The whey concentration of SAAC-specific antibodies was significantly higher in vaccinates than in nonvaccinates at both 15 and 17 d in milk, independent from the challenge status of the quarters. No significant differences in the whey J5-specific antibody concentration were observed. Vaccination with Startvac seems to primarily evoke a Th2 response for S. aureus characterized by a shift toward the IgG1 antibody subtype and accompanied by a less pronounced Th1 response. The type of response against E. coli was less clear, though a weak but significant shift toward the IgG2 antibody subtype after vaccination and high IFN-γ levels after in vitro stimulation suggest a Th1 response. The increased SAAC-specific antibody concentration in whey in vaccinates compared with nonvaccinates most probably triggers the opsonization of the inoculated S. aureus bacteria, resulting in a more efficient elimination of the bacteria from the mammary gland.

The potential toxicological insights about the anti-HIV drug azidothymidine-derived monoselenides in human leukocytes: Toxicological insights of new selenium-azidothymidine analogs.

Acquired immunodeficiency syndrome (AIDS) is a worldwide disease characterized by impairments of immune function. AIDS can be associated with oxidative stress (OS) that can be linked to selenium (Se) deficiency. Se is fundamental for the synthesis of selenoproteins, such as glutathione peroxidase and thioredoxin reductase. These enzymes catalyze the decomposition of reactive oxygen species and contribute to maintain equilibrium in cell redox status. Literature data indicate that organoselenium compounds, such as ebselen and diphenyl diselenide, have antioxidant properties in vitro and in vivo models associated with OS. Nevertheless, selenocompounds can also react and oxidize thiols groups, inducing toxicity in mammals. Here, we tested the potential cytotoxic and genotoxic properties of six analogs of the prototypal anti-HIV drug azidothymidine (AZT) containing Se (5'-Se-(phenyl)zidovudine; 5'-Se-(1,3,5-trimethylphenyl)zidovudine; 5'-Se-(1-naphtyl)zidovudine; 5'-Se-(4-chlorophenyl)zidovudine) (C4); 5'-Se-(4-methylphenyl)zidovudine (C5); and 5'-(4-methylbenzoselenoate)zidovudine). C5 increased the rate of dithiothreitol oxidation (thiol oxidase activity) and C2-C4 and C6 (at 100 µM) increased DNA damage index (DI) in human leukocytes. Moreover, C5 (200 µM) decreased human leukocyte viability to about 50%. Taken together, these results indicated the low in vitro toxicity in human leukocytes of some Se-containing analogs of AZT.

Acclimation capacity of the three-spined stickleback (Gasterosteus aculeatus, L.) to a sudden biological stress following a polymetallic exposure.

To get closer to the environmental reality, ecotoxicological studies should no longer consider the evaluation of a single pollutant, but rather combination of stress and their interaction. The aim of this study was to determine if responses of a fish to a sudden biological stress could be modified by a prior exposure to a chemical stress (a polymetallic contamination). For this purpose, in situ experiment was conducted in three ponds in the Haute-Vienne department (France). One pond was chosen for its high uranium concentration due to uranium mine tailings, and the two other ponds, which were not submitted to these tailings. Three-spined sticklebacks (Gasterosteus aculeatus) were caged in these ponds for 14days. After this period, fish were submitted to a biological stress, exerted by lipopolysaccharides injection after anesthesia, and were sacrificed 4days after these injections for multi-biomarkers analyses (leucocyte viability, phagocytic capacity and reactive oxygen species production, antioxidant peptide and enzymes, lipid peroxidation and DNA damage). The pond which received uranium mine tailings had higher metallic concentrations. Without biological stress, sticklebacks caged in this pond presented an oxidative stress, with increasing of reactive oxygen species levels, modification of some parts of the antioxidant system, and lipid peroxidation. Caging in the two most metal-contaminated ponds resulted in an increase of susceptibility of sticklebacks to the biological stress, preventing their phagocytic responses to lipopolysaccharides and modifying their glutathione contents and glutathione-S-transferase activity.

Detergent Sclerosants Stimulate Leukocyte Apoptosis and Oncosis

The objective was to investigate the effects of the detergent sclerosants sodium tetradecyl sulfate (STS) and polidocanol (POL) on human leukocytes at sublytic concentrations. Leukocytes were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated leukocyte count and viability was assessed using trypan blue, and propidium iodide staining. Phosphatidylserine (PS) exposure, a universal hallmark to measure cell apoptosis, was identified by flow cytometry using lactadherin. Caspases 3, 8, and 9, and Bax activation, as well as inhibitory assays with pan-caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to determine apoptotic pathways. Porimin activation was used to assess cell permeability. Up to 40% of leukocytes maintained membrane integrity at sublytic concentrations (≤0.15%) of sclerosants. The remaining 60% did not maintain membrane integrity but were not completely lysed. PS exposure was increased with both STS and POL exhibiting a dose- and time-dependant trend. While activation of caspases 3, 8, and 9, as well as Bax activation, were increased in leukocytes stimulated with low concentrations of STS, only caspases 3 and 9 and Bax were increased with POL. Inhibitory assays demonstrated caspases 3, 8, and 9, and Bax inhibition at low concentrations with both STS and POL. Both agents increased the leukocyte activation of porimin at all concentrations. On confocal microscopy, stains for caspases 3, 8, and 9, and Bax were increased for both STS and POL. Porimin stain was markedly positive for both STS and POL. Both sclerosants induced leukocyte apoptosis at sublytic concentrations. STS activated both extrinsic and intrinsic pathways of apoptosis, while POL stimulated the intrinsic pathway of apoptosis only. Both agents induced oncosis. Based on these results, STS appears to have a greater effect than POL.

The Role of Physical Stabilization in Whole Blood Preservation.

The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase—a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.

Immunotoxicity of Zinc Oxide Nanoparticles and Municipal Effluents to Fathead Minnows

The presence of nanoparticles in sewage water has raised concerns about the cumulative toxicity of municipal effluent to aquatic organisms. The purpose of this study was to examine the effects of dietary zinc oxide nanoparticles (nano-ZnO) and ZnCl2 (at 100 ng total Zn/g food) in adult fathead minnows Pimephales promelas during continuous exposure to a physico-chemically treated effluent for 21 days. Immunocompetence was determined by assessing leucocyte viability, phagocytosis activity, oxidative stress (lipid peroxidation) and DNA strand breaks in gills. The results revealed that leucocyte viability decreased with increasing effluent concentration, while it increased somewhat in fish fed either form of Zn. The decrease in viability was not observed in fish exposed to the municipal effluent that were fed either form of Zn. Phagocytosis activity decreased after an initial increase at a low concentration of the effluent (5% v/v), while it readily decreased in fish fed either form of Zn. The decrease was also observed in fish fed either form of Zn that were exposed to the effluent. The data revealed that nano-ZnO toxicity differed from ZnCl2 effects, but when the fish were both exposed to the effluent and fed a nano-ZnOcontaminated diet, the overall effects closely resembled the effects in fish fed a ZnCl2 -supplemented diet. In conclusion, ingested nanoparticles in food could affect the immune system of fish exposed to municipal wastewaters differently than non-exposed fish, rendering the exposed fish more vulnerable to microorganisms.