Peripheral Blood Leukocyte Cultures Research Articles

Single-cell atlas of rainbow trout peripheral blood leukocytes and profiling of their early response to infectious pancreatic necrosis virus.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.

RESEARCH ON VARIOUS MODELS OF THE BIOLOGICAL EFFECTS OF COMPONENTS OF A COMPLEX EFFECT (PHOTOIRRADIATION; EXOSOMES OF MESENCHYMAL STEM CELLS AND NANOPARTICLES) FOR THE CORRECTION OF THE INFLAMMATORY PROCESS

RESEARCH ON VARIOUS MODELS OF THE BIOLOGICAL EFFECTS OF COMPONENTS OF A COMPLEX EFFECT (PHOTOIRRADIATION; EXOSOMES OF MESENCHYMAL STEM CELLS AND NANOPARTICLES) FOR THE CORRECTION OF THE INFLAMMATORY PROCESS

The Effect of Phytochemical Extracts on Cytokine Gene Expression

Background: In the last century, nutritional supplements have shown a wide spectrum of biochemical effects, most notably about immunomodulation and countering inflammation. Objective: This study investigates the impact of phytochemical compounds that are present in different quantities of pomegranate, grape seeds and garlic extracts on the expression of inflammatory (IL1β and IL6) and anti-inflammatory (IL10) genes, the effects of polymorphisms in these genes on this response. Methods: Human peripheral blood leukocyte cultures were treated with pomegranate (1.2% or 2.4%), garlic (0.5% or 1.2%), or grape seed (1.2% or 2.4%) extracts. Gene expression was assessed with real-time polymerase chain reaction (PCR). Polymorphisms of the cytokine genes were analyzed using allele-specific PCR. Results: Pomegranate extract (2.4%) reduced the transcription of IL1β by 16-fold in comparison to control. The expression of IL6 relative to the control after the addition of grape seed extract (1.2%) was reduced by 100-fold. The grape seeds extract (1.2%) showed the effect of increasing transcription for IL10 compared to the control. The level of IL1β transcription in culture with garlic extract depends on the genotype of the cell for -31T>C polymorphism (r = 0.67 p = 0.03). There is correlation between polymorphism -174G>C and level gene expression IL6 (r=-0.66, p = 0.04) after adding grape seeds extract. Conclusion: The phytochemical compounds in pomegranate extracts and grape seed extracts play the role of anti-inflammatory by decreasing the gene expression of IL1β, IL6 and increasing the transcription of IL10.

The Effect of Three Different Suture Anchors for Rotator Cuff Repair on Primary Cultures of Human Bone Marrow Mesenchymal Stem Cells.

Purpose The purpose of this study is to investigate the in vitro biocompatibility of three different suture anchors (all-suture anchor, metal anchor, and polyetheretherketone anchor), commonly used for the rotator cuff repair. Methods To assess the biocompatibility of the anchors, the possible cytotoxicity and the immunogenicity of the devices were assessed by cell viability assay and cell count on cultures of bone marrow stem cells (BMSCs) and peripheral blood leucocytes (PBLs), respectively. The possible inhibitory effect of the devices on BMSCs osteogenic potential was evaluated by alkaline phosphatase activity and matrix deposition assay. Results The viability of BMSCs was slightly reduced when cultured in the presence of the devices (−24 ± 3%). Nevertheless, they were able to differentiate toward the osteogenic lineage in all culture conditions. The proliferation of PBLs and the production of interleukin-2 were not enhanced by the presence of any device. Conclusion The analyzed devices did not significantly affect the normal cells functions when directly cultured with human primary BMSCs or PBLs, in terms of osteogenic differentiation and inflammatory reaction. Clinical Relevance A deeper knowledge of the biological reactions to different devices used in rotator cuff surgeries would improve the clinical outcome of these procedures.

Aging in Ghana

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.

Carica papaya induces in vitro thrombopoietic cytokines secretion by mesenchymal stem cells and haematopoietic cells.

BackgroundUse of Carica papaya leaf extracts, reported to improve thrombocyte counts in dengue patients, demands further analysis on the underlying mechanism of its thrombopoietic cytokines inductionMethodsIn vitro cultures of peripheral blood leukocytes (PBL) and stem cells from human exfoliated deciduous teeth (SHED) were treated with unripe papaya pulp juice (UPJ) to evaluate its potential to induce thrombopoietic cytokines (IL-6 and SCF)ResultsIn vitro scratch gap closure was significantly faster (p < .05) in SHED culture treated with UPJ. IL-6 concentration was significantly increased (p < .05) in SHED and PBL culture supernatant when treated with UPJ. SCF synthesis in SHED culture was also significantly increased (p < .05) when treated with UPJConclusionIn vitro upregulated synthesis of IL −6 and SCF both in PBL and SHED reveals the potential mechanism of unripe papaya to induce thrombopoietic cytokines synthesis in cells of hematopoietic and mesenchymal origin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0749-6) contains supplementary material, which is available to authorized users.

Essential oil from Ageratum fastigiatum reduces expression of the pro-inflammatory cytokine tumor necrosis factor-alpha in peripheral blood leukocytes subjected to in vitro stimulation with phorbol myristate acetate

Ageratum fastigiatum (Gardner) R.M. King & H. Rob., a member of the Asteraceae family popularly known in Brazil as “matapasto”, is indicated in folk medicine as anti-inflammatory and analgesic. Despite its popular use, little is known about its potential effect on the parameters involved in an inflammatory response. The objective of this study was to characterize the chemical composition of the essential oil from A. fastigiatum and to evaluate the frequency of tumor necrosis factor alpha and interferon gamma producing cells in peripheral blood lymphocytes stimulated with phorbol myristate acetate in the presence of essential oil from A. fastigiatum. Non-toxic concentrations of essential oil from A. fastigiatum were evaluated in cultures of peripheral blood leucocytes using the trypan blue exclusion assay by flow cytometry. GC–MS analysis revealed that the prevalent compounds identified in the essential oil from A. fastigiatum sample were α-pinene, limonene, trans-caryophyllene, α-humulene, caryophyllene oxide, 1,2-humulene-epoxide, 1,6-humulanodien-3-ol, and α-cadinol. Results showed that exposure to essential oil from A. fastigiatum at concentrations of 0.5×10−2 and 1×10−2μl/ml caused no alterations in leukocyte viability as compared to the control group. Both concentrations lowered the percentage of tumor necrosis factor alpha (+)-lymphocytes and neutrophils. There were no changes in the percentage of lymphocytes positive for the interferon gamma cytokine. Our results suggest that part of the anti-inflammatory activity attributed to A. fastigiatum may be due to the effect of some of its components in decreasing the number of cells that produce the pro-inflammatory cytokine tumor necrosis factor alpha.

SIMULATION OF PROTECTIVE IMMUNE MECHANISMS AT AFRICAN SWINE FEVER in vitro

S u m m a r y Pigs that survive after infection with African swine fever virus are resistant to reinfection with seroimmunotype-homologous virulent isolates, indicating an immune protection development. Nevertheless, the points regarding the protective importance of various immune mechanisms at African swine fever still remain controversial. Peripheral blood leukocyte cultures prepared from both intact or vaccinated pigs were used as models of implemented virus-specific reactions in vivo. The maximum amount of ASF virus was determined in the cultures with adherent cells from intact or immunized gilt, non-adherent white blood cells and the serum of an intact animal. Replacement of non-adherent cells from intact gilt by those from the immunized one led to a significant decrease in ASF virus titres. It was shown that combination of A-cells from intact or immunized gilt, non-adherent cells from the intact animal and the serum from immunized animal limited the viral propagation more strictly, under the replacement of non-adherent cells from the intact gilt by those from the immunized animal at the most. The results determine the role of cellular and humoral immunity in limiting African swine fever virus replication. The protection mechanisms mediated by cytotoxic T lymphocytes and antibody-mediated cellular anti-effectors are found to have an integrated effect, suggesting their targeting against different epitopes.

МОДЕЛИРОВАНИЕ in vitro ЗАЩИТНЫХ ИММУННЫХ МЕХАНИЗМОВ ПРИ АФРИКАНСКОЙ ЧУМЕ СВИНЕЙ

Pigs that survive after infection with African swine fever virus are resistant to reinfection with seroimmunotype-homologous virulent isolates, indicating an immune protection development. Nevertheless, the points regarding the protective importance of various immune mechanisms at African swine fever still remain controversial. Peripheral blood leukocyte cultures prepared from both intact or vaccinated pigs were used as models of implemented virus-specific reactions in vivo. The maximum amount of ASF virus was determined in the cultures with adherent cells from intact or immunized gilt, non-adherent white blood cells and the serum of an intact animal. Replacement of non-adherent cells from intact gilt by those from the immunized one led to a significant decrease in ASF virus titres. It was shown that combination of A-cells from intact or immunized gilt, non-adherent cells from the intact animal and the serum from immunized animal limited the viral propagation more strictly, under the replacement of non-adherent cells from the intact gilt by those from the immunized animal at the most. The results determine the role of cellular and humoral immunity in limiting African swine fever virus replication. The protection mechanisms mediated by cytotoxic T lymphocytes and antibody-mediated cellular anti-effectors are found to have an integrated effect, suggesting their targeting against different epitopes.

Release of reactive oxygen species and production of nitric oxide in the cultures of peripheral blood leukocytes of young athletes

Release of reactive oxygen species and production of nitric oxide in the cultures of peripheral blood leukocytes of young athletes

Sour Cherry Seed Kernel Extract Increases Heme Oxygenase‐1 Expression and Decreases Representation of CD3+ TNF‐α + and CD3 + IL‐8+ Subpopulations in Peripheral Blood Leukocyte Cultures from Type 2 Diabetes Patients

The present study evaluates a hypothesis that sour cherry (Prunus cerasus) seed extracts (SCE) modulate CD3+ T lymphocyte activity in ways predictive of potential for uses of SCE in management of inflammatory diseases. Peripheral blood mononuclear cells (PBMC) from 12 type 2 diabetes (T2DM) patients and eight healthy control subjects were cultured 24 h with 100 ng/ml lipopolysaccharide (LPS) to increase inflammatory signaling and co-incubated with 0.5-100 µg/ml SCE. Cultures were evaluated by two-color flow cytometry for percent representation of CD3+ IL8+ and CD3+TNF-α cells which express interleukin-8 (IL-8), and tumor necrosis factor-α, (TNF-α+) respectively, and by enzyme-linked immunoassay for lymphocyte-associated heme oxygenase-1 (HO-1, known to be induced by SCE). SCE dosage ranges of 0.5-100 µg/ml in cell cultures significantly suppressed LPS-increased CD3+TNF-α+ and CD3+IL8+ representation from all participants (p < 0.05), with greater pharmacological effect noted in suppression of CD3+TNF-α+ noted in cells from T2DM patients versus healthy control subjects. These effects correlated with increased HO-1 expression in SCE-treated PBMC from all subjects (p < 0.05). Since TNF-α and IL-8 are diagnostic/prognostic biomarkers for many inflammatory syndromes, the capacity of SCE to down-regulate representation of cells that express them suggests potential for therapeutic use of SCE in T2DM and other diseases.

An inflammatory CC chemokine of Cynoglossus semilaevis is involved in immune defense against bacterial infection

Chemokines are a family of small cytokines that regulate leukocyte migration. Based on the arrangement of the first two cysteine residues, chemokines are classified into four groups called CXC(α), CC(β), C, and CX 3C. In this study, we identified a CC chemokine, CsCCK1, from half-smooth tongue sole ( Cynoglossus semilaevis) and analyzed its biological activity. The deduced amino acid sequence of CsCCK1 contains 111 amino acid residues and is phylogenetically belonging to the CCL19/21/25 group of CC chemokines. CsCCK1 possesses a DCCL motif that is highly conserved among CC chemokines. Quantitative real time RT-PCR analysis showed that the expression of CsCCK1 was relatively abundant in immune organs under normal physiological conditions and was upregulated by experimental infection of a bacterial pathogen. Purified recombinant CsCCK1 (rCsCCK1) induced chemotaxis in peripheral blood leukocytes (PBL) of both tongue sole and turbot ( Scophthalmus maximus) in a dose-dependent manner. Mutation of the CC residues in the DCCL motif by serine substitution completely abolished the biological activity of rCsCCK1. When rCsCCK1, but not the mutant protein, was added to the cell culture of PBL, it enhanced cellular resistance against intracellular bacterial infection. Taken together, these results indicate that CsCCK1 is a functional CC chemokine whose biological activity depends on the DCCL motif and that CsCCK1 plays a role in host immune defense against bacterial infection.

CXCL8 of Scophthalmus maximus: Expression, biological activity and immunoregulatory effect

CXCL8, or interleukin-8, is a CXC chemokine that promotes neutrophil migration in response to inflammatory stimuli. In this study, we identified and analyzed a CXCL8 orthologue, SmCXCL8, from turbot (Scophthalmus maximus). The deduced amino acid sequence of SmCXCL8 is 99-residue in length and shares 52–83% overall identities with the lineage 1 CXCL8 of a number of teleost. SmCXCL8 possesses a CXC chemokine domain that contains the conserved CXC motif preceded by the tripeptide sequence EMH. Purified recombinant SmCXCL8 (rSmCXCL8) induced chemotaxis in peripheral blood neutrophils and, to lesser extents, head kidney (HK) lymphocytes and macrophages in a dose-dependent manner. Mutation of the EMH motif by alanine substitution reduced the chemoattractive effect of rSmCXCL8. Expression of SmCXCL8 as determined by quantitative real time RT-PCR (qRT-PCR) was detected mainly in immune organs under normal physiological conditions and was upregulated by experiment challenges with bacterial pathogen and poly(I:C). In addition, SmCXCL8 expression was also induced to significant extents following vaccination of turbot with a subunit vaccine. When rSmCXCL8 was added to the cell cultures of peripheral blood leukocytes and HK lymphocytes and macrophages, it stimulated the proliferation of these cells and enhanced cellular resistance against intracellular bacterial survival. qRT-PCR analysis showed that rSmCXCL8 induced the expression of TNF-α and suppressor of cytokine signaling 3 in HK lymphocytes in different time frames. On the other hand, SmCXCL8 expression was also upregulated by TNF-α. Taken together, these results indicate that SmCXCL8 is a functional CXC chemokine with immunomodulatory effect and plays a role in inflammatory response induced by bacterial infection.

Genotoxic Effect of Dye Effluents in Chromosomes of Indigenous Goats (Capra hircus)

The genotoxic effect of dye effluents was assessed in indigenous goats inhabiting textile industry areas encompassing dyeing and bleaching units by means of mitotic drive, mitotic index, chromosome aberrations and the mean SCEs frequency. Peripheral blood leucocyte cultures were set up with culture medium and pokeweed mitogen. The cultures were supplemented without bromodeoxyuridine (BrdU) for chromosome aberrations and with BrdU for the mean SCEs frequency for 2 cell cycles. Air-dried slides were stained with Giemsa for chromosome aberrations and the fluorescent plus Giemsa (FPG) technique was adopted for the display of sister chromatid differentiation and the estimation of the mean SCEs frequency. No gross structural and numerical chromosome abnormalities were noticed in all animals screened based on G-banding and karyotyping. The mitotic drive for the control and exposed populations was 37.58±1.29 and 26.13±0.78%, respectively. The mitotic drive in exposed population was significantly (p<0.01) lower when compared to control animals. The mitotic index for the control and exposed populations was 12.49±0.42 and 11.49±0.43%, respectively. Though there was a decrease in mitotic index in the exposed population, it was not statistically significant. The frequency of SCEs did not follow the Poisson distribution in both the control and exposed populations of goats. The pooled mean±S.E (range) of SCEs frequency for the control and exposed populations of goats were 4.83±0.16 (1–11) and 12.98±0.16 (8–19), respectively. There was significant (p<0.01) increase in the mean SCEs frequency of goats (exposed) reared in industrial areas. The number of SCEs in chromosome 1 was significantly (p<0.05) higher than that expected from relative chromosomal length.

In Vitro Culture of Peripheral Blood Leukocytes of &lt;i&gt;Channa punctatus&lt;/i&gt;

Cytokines are the most studied immunological molecules which play a crucial role in immunomodulatory processes involving functional T leukocytes. Cytokines are small cell-signaling protein molecules that are secreted by numerous cells mostly by peripheral blood leucocytes. In vitro culture of leucocytes plays an important role in the study of different cytokines and its role in immune system. We developed and optimized a simple and efficient method for in vitro culture of peripheral blood leukocytes of snakehead fish (Channa punctatus), using various parameters including culture medium, different mitogens and its concentration and incubation time. In the present study, peripheral blood leukocytes (PBLs) were isolated from the blood of C. punctatus using percoll and ficoll gradient solutions and were cultured in vitro in different culture media. The PBLs were stimulated with different mitogens such as phytohaemagglutinin (PHA), concanavalin A (ConA) and phorbol myristate acetate (PMA) to optimize the growth of peripheral blood leukocytes. Growth kinetics was observed to be comparatively more in PHA induced culture media. This study established an in vitro culture model that allowed us to study the growth kinetics of peripheral blood leukocytes in different media in response to different mitogens and incubation period.

Chronic Intestinal Helminth Infections Are Associated with Immune Hyporesponsiveness and Induction of a Regulatory Network

Helminth infections have been associated with protection against allergy and autoimmune diseases. We investigated the effects of chronic infections with Ascaris lumbricoides and Trichuris trichiura (measured twice over a 5-year period) on cytokine and antibody responses. We collected blood from 1,060 children aged 4 to 11 years living in a poor urban area of Brazil and measured Th1 (gamma interferon [IFN-gamma]) and Th2 (interleukin-5 [IL-5] and IL-13) cytokines and the regulatory cytokine IL-10 in unstimulated and stimulated (with mitogen or A. lumbricoides antigens) cultures of peripheral blood leukocytes and levels of total IgE and anti-A. lumbricoides IgG4 and IgE in serum. Intestinal helminth infections were associated with an increased proportion of children producing IL-5 in response to A. lumbricoides and producing IL-10 spontaneously, especially among coinfected and chronically infected children. Helminth infections were associated with a generalized suppression of cytokine responses to mitogen. Levels of total IgE and anti-A. lumbricoides IgG4 and IgE were especially elevated in chronically infected children. In conclusion, intestinal helminth infections were associated with a typical Th2 immune response profile and with the induction of immune hyporesponsiveness that was associated with greater frequencies of the production of spontaneous IL-10.

Neutrophil Influx and Chemokine Production during the Early Phases of the Antitumor Response to the Vascular Disrupting Agent DMXAA (ASA404)

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G+CD11b+F4/80−. cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b+ cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor á, interleukin 6 (IL-6), and macrophage inflammatory 1α (MIP-1α). CD49b+ natural killer cells produced IP-10, whereas CD45R+ B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1α (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.

Mixture of betel leaf, areca nut and tobacco chewing is a risk factor for cytogenetic damage in construction workers from south India

Aim: To determine the cytogenetic effect of betel leaf, areca nut and tobacco mixture usage among female construction workers in Tamilnadu, Southern India. Methods: Totally 236 buccal cells and blood samples were collected from 80 betel quid users and 76 users with tobacco snuffing habit which were compared with 80 healthy subjects. Peripheral blood leukocyte cultures were analyzed for chromosomal aberrations (CA) and exfoliated cells from the buccal mucosa were examined for micronucleus (MN). Results: Statistically significant (p<0.01) increase in CA and MN were observed in users with snuffing habit when compared to users without snuffing habit and controls as confirmed by chi-square test. Therefore, specific biomarkers on cytogenetic endpoints might help in planning precautionary measures to reduce oral cancer risks. Conclusions: The present study can be concluded that a mixture of betel quid, areca nut and tobacco chewing/snuffing is unsafe for oral health. The genotoxic effect of smokeless tobacco should be considered in addition to other known hazards for assessing health risks.

Cell culture conditions potentiate differences in the response to ionising radiation of peripheral blood leukocytes isolated from breast cancer patients and healthy subjects

To compare the effects of ionising radiation on leukocytes from breast cancer patients and healthy subjects ex vivo, the level of NF-κB and the antioxidant enzymes manganese-containing superoxide dismutase (Mn-SOD), copper/zinc-containing superoxide dismutase (CuZn-SOD) and catalase (CAT) in combination with flow cytometric analysis of CD4+ lymphocytes was performed. The level of Mn-SOD protein was significantly increased in the breast cancer study group both before (P < 0.001) and after (P < 0.001) irradiation when compared with healthy subjects. Measurements in parallel indicated that the level of CAT protein was significantly higher in the breast cancer study group after irradiation (2 Gy [P < 0.001] and 9 Gy [P < 0.05]) when compared with healthy subjects. Although the initial number of lymphocytes in the blood of breast cancer patients was not different from healthy subjects, the percentage of apoptotic CD4+ cells was significantly (P < 0.001) lower both before and after irradiation indicating that cell culture conditions induced radioresistance of CD4+ cells in the blood of breast cancer patients. The data presented in this current study indicate that brief ex vivo culture of peripheral blood leukocytes potentiates oxidative stress imposed by a breast cancer tumour.

DEVELOPMENT OF A CONTINUOUS CELL LINE, PBLE, FROM AN AMERICAN EEL PERIPHERAL BLOOD LEUKOCYTE PREPARATION

A continuous cell line, PBLE, was developed from the adherent cells in a culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. The cells were grown in Leibovitz's L-15 basal medium supplemented with 20% fetal bovine serum (FBS). Under normal culture conditions at 18 degrees C, the morphology of PBLE was fibroblast-like. The cultures have been subcultured over 80 times and have been cryopreserved successfully. These cells have a diploid karyotype of 38 chromosomes, survived temperatures from 5 to 36 degrees C, and proliferated at temperatures from 5 degrees C to at least 30 degrees C. PBLE underwent apoptosis in response to gliotoxin, but did not show a respiratory burst. Results suggest that PBLE may have arisen from a circulating mesenchymal stem cell. PBLE was susceptible to Chum salmon reovirus (CSV) and supported CSV replication. Therefore this cell line should be useful in studying eel specific virus-host interactions.