A physiological product-release model for baculovirus infected insect cells

Existing models describe the product release from baculovirus infected insect cells as an unspecific protein leakage occurring in parallel with protein production. The model presented here shows that the observed product release of normally non-secreted proteins can be described through cell death alone. This model avoids the implicit non-physiological assumption of previous models that cells permeable to recombinant protein as well as trypan blue continue to produce protein. (c) 2005 Wiley Periodicals, Inc.

Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8(+) T-cell function in Hodgkin lymphoma patients

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg (HRS) cells constitute only 0.5% of 10% of the diseased tissue. The surrounding cellular infiltrate is enriched with T cells that are hypothesized to modulate antitumor immunity. We show that a marker of regulatory T cells, LAG-3, is strongly expressed on infiltrating lymphocytes present in proximity to HRS cells. Circulating regulatory T cells (CD4(+) CD25(hi) CD45 ROhi, CD4(+) CTLA4(hi), and CD4(+) LAG-3(hi)) were elevated in HL patients with active disease when compared with remission. Longitudinal profiling of EBV-specific CD8(+) T-cell responses in 94 HL patients revealed a selective loss of interferon-gamma expression by CD8(+) T cells specific for latent membrane proteins 1 and 2 (LMP1/2), irrespective of EBV tissue status. Intratumoral LAG-3 expression was associated with EBV tissue positivity, whereas FOXP3 was linked with neither LAG-3 nor EBV tissue status. The level of LAG-3 and FOXP3 expression on the tumor-infiltrating lymphocytes was coincident with impairment of LMP1/2-specific T-cell function. In vitro pre-exposure of peripheral blood mono-nuclear cells to HRS cell line supernatant significantly increased the expansion of regulatory T cells and suppressed LMP-specific T-cell responses. Deletion of CD4(+) LAG-3(+) T cells enhanced LMP-specific reactivity. These findings indicate a pivotal role for regulatory T cells and LAG-3 in the suppression of EBV-specific cell-mediated immunity in HL.

Heterogeneity of MUC1 expression by human breast carcinoma cell lines in vivo and in vitro

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. Recent studies have demonstrated that overexpression of MUC1 interferes with cell-substrate and cell-cell adhesion by masking cell surface integrins and E-cadherin. Additionally, the cytoplasmic tail of MUC1 is involved in signal transduction and interactions with catenins. In the present study, we have examined the in vitro expression of MUC1 mRNA and protein in a panel of 14 human breast cancer cell lines using northern blotting, western blotting, immunocytochemistry, and flow cytometry. Considerable variability of expression was noted not only between cell lines but also within several individual lines. Many cell lines such as BT 20, KPL-1, and T47D expressed abundant MUC1 whilst others such as MDA-MB-231 and MCF-7 showed intermediate expression, and MDA-MB-435 and MDA-MB-453 expressed very low levels. Low levels of MUC1 expression were associated with decreased expression of cytokeratin and increased expression of vimentin. Additionally, 12 of the cell lines were established as xenografts in immunocompromised (SCID) mice, and MUC1 expression in both the primary tumors as well as metastases was assessed immunohistochemically. In general, in vivo expression mirrored in vitro expression, although there was reduced in vivo expression in T47D and ZR-75-1 xenografts. Although we showed no correlation between tumorigenicity or metastasis and MUC1 expression, this study will assist development of experimental models to assess the influence of MUC1 of on breast cancer progression.

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination. Copyright © 2003 S. Karger AG, Basel

Comparison of the expression of calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) with CGRP and adrenomedullin binding in cell lines

1. The calcitonin receptor-like receptor (CRLR) and specific receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2. There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Specific human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3. We confirmed CGRP subtype 1 receptor (CGRP(1)) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP(1) and adrenomedullin receptors whereas Rat-2 fibroblasts expressed only adrenomedullin receptors. In contrast we could not confirm CGRP(2) receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP(1)-like in this study. 4. L6, SK-N-MC and Col-29 cells expressed mRNA for RAMP1 and RAMP2 but Rat-2 fibroblasts had only RAMP2. No cell line had detectable RAMP3 mRNA. 5. SK-N-MC, Col-29 and Rat-2 fibroblast cells expressed CRLR mRNA. By contrast, CRLR mRNA was undetectable by Northern analysis in one source of L6 cells. Conversely, a different source of L6 cells had mRNA for CRLR. All of the cell lines expressed CRLR protein. Thus circumstances where CRLR mRNA is apparently absent by Northern analysis do not exclude the presence of this receptor. 6. These data strongly support CRLR, together with appropriate RAMPs as binding sites for CGRP and adrenomedullin in cultured cells.

Cell passage-associated transient high oxygenation causes a transient decrease in cellular glutathione and affects T cell responses to apoptotic and mitogenic stimuli

Routine cell line maintenance involves removal of waste products and replenishment of nutrients via replacement of cell culture media. Here, we report that routine maintenance of three discrete cell lines (HSB-CCRF-2 and Jurkat T cells, and phaeo-chromocytoma PC12 cells) decreases the principal cellular antioxidant, glutathione, by up to 42% in HSB-CCRF-2 cells between 60 and 120 min after media replenishment. However, cellular glutathione levels returned to baseline within 5 h after passage. The decrease in glutathione was associated with modulation of the response of Jurkat T cells to apoptotic and mitogenic signals. Methotrexate-induced apoptosis over 16 h, measured as accumulation of apoptotic nucleoids, was decreased from 22 to 17% if cells were exposed to cytotoxic agent 30 min after passage compared with cells exposed to MTX in the absence of passage. In contrast, interleukin-2 (IL-2) production over 24 h in response to the toxin phytohaemagglutinin (PHA), was increased by 34% if cells were challenged 2 h after passage compared with PHA treatment in the absence of passage. This research highlights the presence of a window of time after cell passage of non-adherent cells that may lead to over- or under-estimation of subsequent cell responses to toxins, which is dependent on cellular antioxidant capacity or redox state. © 2007 Elsevier B.V. All rights reserved.

A comparison of high-content screening versus manual analysis to assay the effects of mesenchymal stem cell-conditioned medium on neurite outgrowth in vitro

Bone marrow mesenchymal stem cells (MSCs) promote nerve growth and functional recovery in animal models of spinal cord injury (SCI) to varying levels. The authors have tested high-content screening to examine the effects of MSC-conditioned medium (MSC-CM) on neurite outgrowth from the human neuroblastoma cell line SH-SY5Y and from explants of chick dorsal root ganglia (DRG). These analyses were compared to previously published methods that involved hand-tracing individual neurites. Both methods demonstrated that MSC-CM promoted neurite outgrowth. Each showed the proportion of SH-SY5Y cells with neurites increased by ~200% in MSC-CM within 48 h, and the number of neurites/SH-SY5Y cells was significantly increased in MSC-CM compared with control medium. For high-content screening, the analysis was performed within minutes, testing multiple samples of MSC-CM and in each case measuring >15,000 SH-SY5Y cells. In contrast, the manual measurement of neurite outgrowth from >200 SH-SY5Y cells in a single sample of MSC-CM took at least 1 h. High-content analysis provided additional measures of increased neurite branching in MSC-CM compared with control medium. MSC-CM was also found to stimulate neurite outgrowth in DRG explants using either method. The application of the high-content analysis was less well optimized for measuring neurite outgrowth from DRG explants than from SH-SY5Y cells.

Induction of inflammatory cytokines and nitric oxide in J774.2 cells and murine macrophages by lipoteichoic acid and related cell wall antigens from Staphylococcus epidermidis

Staphylococcus epidermidis causes infections associated with medical devices including central venous catheters, orthopaedic prosthetic joints and artificial heart valves. This coagulase-negative Staphylococcus produces a conventional cellular lipoteichoic acid (LTA) and also releases a short-glycerophosphate-chain-length form of LTA (previously termed lipid S) into the medium during growth. The relative pro-inflammatory activities of cellular and short-chain-length exocellular LTA were investigated in comparison with peptidoglycan and wall teichoic acid from S. epidermidis and LPS from Escherichia coli O111. The ability of these components to stimulate the production of proinflammatory cytokines [interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α] and nitric oxide was investigated in a murine macrophage-like cell line (J774.2), and in peritoneal and splenic macrophages. On a weight-for-weight basis the short-chain-length exocellular LTA was the most active of the S. epidermidis products, stimulating significant amounts of each of the inflammatory cytokines and nitric oxide, although it was approximately 100-fold less active than LPS from E. coli. By comparison the full-chain-length cellular LTA and peptidoglycan were less active and the wall teichoic acid had no activity. As an exocellular product potentially released from S. epidermidis biofilms, the short-chain-length exocellular LTA may act as the prime mediator of the host inflammatory response to device-related infection by this organism and act as the Gram-positive equivalent of LPS in Gram-negative sepsis. The understanding of the role of short-chain-length exocellular LTA in Gram-positive sepsis may lead to improved treatment strategies. © 2005 SGM.

Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems

Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

Uptake and transport of orally-deliverable drugs across caco-2 cell monolayers:the effect of lipid formulations

The aim of this thesis is to investigate the physicochemical parameters which can influence drug loading within liposomes and to characterise the effect such formulations have on drug uptake and transport across in vitro epithelial barrier models. Liposomes composed of phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) and cholesterol (0, 4, 8, 16 µM) were prepared and optimised in terms of drug loading using the hand-shaking method (Bangham et al., 1965). Subsequently, liposomes composed of 16 µM PC or DSPC and cholesterol (4 µM) were used to monitor hydroxybenzoate release and transport from Iiposomes. The MIT (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and crystal violet assays were employed to determine toxicity of the Iiposome. formulations towards the Caco-2 cell line, employed to model the epithelial barrier in vitro. Uptake and transport of mannitol, propranolol, glutamine and digoxin was measured in the presence and absence of Iiposome formulations to establish changes in absorption resulting from the presence of lipid formulations. Incorporation of the four hydroxybenzoates was shown to be influenced by a number of factors, including liposome composition and drug conformation. Methyl hydroxybenzo.ate (MP) was incorporated into the bilayer most effectively with percentage incorporation of 68% compared to 45% for butyl hydroxybenzoate (BP), despite its increased Iipophilicity. This was attributed to the decreased packing ability of BP within the hydrocarbon core of the lipid bilayer compared to MP. Release studies also suggested that the smaller MP was more strongly incorporated within the lipid bilayer with only 8% of the incorporated solute being released after 48-hours compared to 17% in the case of BP. Model transport studies were seen to reflect drug release profiles from the liposome bilayers with significantly (p < 0.01) higher amounts of BP partitioning from the liposome compared to MP, Caco-2 cell viability was maintained above 86% in the presence of all Iiposome formulations tested indicating the liposome formulations are non-toxic towards Caco-2 cells. Paracellular (apical-to-basolateral) transport of mannitol was significantly increased in the presence of DSPC, PC / DSPC:Cholesterol (16:4 µM; 1000 µg). Glutamine uptake and transport via the carrier-mediated route was Significantly (p < 0.01) increased in the presence of PC I DSPC:Cholesterol (16:0; 16:4 µM). Digoxin apical-to-basolateral transport was significantly increased (p < 0,01) in the presence of PC / DSPC:Cholesterol (16:0; 16:4 µM); thus reducing digoxin efflux via P-glycoprotein. In contrast, PC:ChoJesterol (16:0; 16:4 µM) significantly (p < 0.01) decreased propranolol uptake via the passive transcellular route. Bi-directional transport of propranolol was significantly (p < 0,01) decreased in the presence of PC/DSPC:Cholesterol (16:0; 16:4 µM). The structure of a solute is an important determinant for the incorporation and release of a solute from liposome formulations. PC, DSPC and cholesterol liposome formulations are nontoxic towards Caco-2 cell monolayers and improved uptake and transport of mannitol, glutamine. and digoxin across Caco-2 cell monolayers; thus providing a potential alternative delivery vehicle.

Development of a post-mitotic, neuronal-astrocytic cell system, for the prediction of acute neurotoxicity in humans

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

The importance of Na(plus)-K(plus)-Cl- cotransport in nitrogen mustard induced cell death

The incubation of murine leukaemic L1210 cells in vitro for 4 hours (hr) with 10uM nitrogen mustard (HN2), a bifunctional alkylating agent, inhibited the influx of the potassium congener, 88rubidium+ ( 86Rb+) by the selective inhibition of the Na+-K+-CI- cotransporter. The aim of this project was to investigate the importance of this lesion in HN2-induced cytotoxicity. 86Rb+ uptake in human erythrocytes was inhibited by high concentrations of HN2 (2mM) and occurred in two phases.In the first hour both the Na+/K+ ATPase pump and the Na+-K+-CI- cotransporter were equally inhibited but after 2 hrs exposure to 2mM HN2, the Na+ -K+ -CI- cotransporter was significantly more inhibited than the Na+/K+ ATPase pump. In contrast, both potassium transport systems were equally inhibited in L1210 cells incubated for 10 minutes with 1mM HN2. The selective inhibition of the Na+-K+-CI- cotransporter, after a 3 hrs exposure to 10uM HN2, was not absolved by coincubation with 5ug/ml cycloheximide (CHX), an inhibitor of protein synthesis. Incubation of L1210 cells with concentrations of diuretics which completely inhibited Na+-K+-CI- cotransport did not enhance the cytotoxicity of either HN2 or its monofunctional analogue 2-chloroethyldimethylamine (Me-HN1). The incubation of L1210 cells with a twice strength Rosewell Park Memorial Institute 1640 media did not enhance the toxicity of HN2. An L1210 cell line (L1210FR) was prepared which was able to grow in toxic concentrations of furosemide and exhibited a similiar sensitivity to HN2 as parental L1210 cells. Treatment of L1210 cells with 10uM HN2 resulted in a decrease in cell volume which was concurrent with the inhibition of the Na+-K+-CI- cotransporter. This was not observed in L1210 cells treated with either 1 or O.SuM HN2. Thus, possible differences in the cell death, in terms of necrosis and apoptosis, induced by the different concentrations of HN2 was investigated. The cell cycle of L1210 cells appeared to be blocked non-specifically by 10uM HN2 and in S and G2/M by either 1 or 0.5uM HN2. There were no significant changes in the cytosolic calcium concentrations of L1210 cells for up to 48 hrs after exposure to the three concentrations of HN2. No protection against th_ toxic effects of HN2 was observed in L1210 cells incubated with 5ug/ml CHX for up to 6 hrs. Incubation for 12 or 18 hrs with a non-toxic concentration (5mM) of L-Azetidine-2- carboxylic acid (ACA) enhanced the toxicity of low concentrations (<0.5uM) of HN2.

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

A preliminary investigation into the impact of a pesticide combination on human neuronal and glial cell lines in vitro

Many pesticides are used increasingly in combinations during crop protection and their stability ensures the presence of such combinations in foodstuffs. The effects of three fungicides, pyrimethanil, cyprodinil and fludioxonil, were investigated together and separately on U251 and SH-SY5Y cells, which can be representative of human CNS glial and neuronal cells respectively. Over 48h, all three agents showed significant reductions in cellular ATP, at concentrations that were more than tenfold lower than those which significantly impaired cellular viability. The effects on energy metabolism were reflected in their marked toxic effects on mitochondrial membrane potential. In addition, evidence of oxidative stress was seen in terms of a fall in cellular thiols coupled with increases in the expression of enzymes associated with reactive species formation, such as GSH peroxidase and superoxide dismutase. The glial cell line showed significant responsiveness to the toxin challenge in terms of changes in antioxidant gene expression, although the neuronal SH-SY5Y line exhibited greater vulnerability to toxicity, which was reflected in significant increases in caspase-3 expression, which is indicative of the initiation of apoptosis. Cyprodinil was the most toxic agent individually, although oxidative stress-related enzyme gene expression increases appeared to demonstrate some degree of synergy in the presence of the combination of agents. This report suggests that the impact of some pesticides, both individually and in combinations, merits further study in terms of their impact on human cellular health.