Immunostimulatory in vitro and in vivo effects of a water-soluble extract from kale.

The water-soluble fraction of kale (Brassica oleracea L. var. acephala DC.) had immunoglobulin (Ig) production stimulating activity in human hybridoma HB4C5 cells and human peripheral blood lymphocytes. The biochemical and physical properties of the main active substance in kale were found to be a heat-stable protein with a molecular weight higher than 50 kDa. The Ig production-stimulating factors were assumed to act on the translational and/or secreting processes of Igs. This Ig production-stimulating effect was also observed in lymphocytes from the mesenteric lymph node and Peyer's patches of mice that had been administered with the kale extract for 14 d. The partially purified kale extract was analyzed by LC-ESI-MS/MS, the result indicating ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) as an active substance. Rubisco from spinach indeed exhibited Ig production-stimulating activity in HB4C5 cells. These findings provide another beneficial aspect of kale as a health-promoting foodstuff.

GADD153 expression does not necessarily correlate with changes in culture behavior of hybridoma cells.

BACKGROUND: The acute sensitivity of some hybridoma cell lines to culture-related stresses severely limits their productivity. Recent developments in the characterization of the stress signals modulating the cellular phenotype revealed that the pro-apoptotic transcription factor Gadd153 could be used as a marker to facilitate the optimization of mammalian cell cultures. In this report, we analyzed the expression of Gadd153 in Sp2/0-Ag14 murine hybridoma cells grown in stationary batch culture and subjected to two different culture optimization paradigms: L-glutamine supplementation and ectopic expression of Bcl-xL, an anti-apoptotic gene. RESULTS: The expression of Gadd153 was found to increase in Sp2/0-Ag14 cells in a manner which coincided with the decline in cell viability. L-glutamine supplementation prolonged Sp2/0-Ag14 cell survival and greatly suppressed Gadd153 expression both at the mRNA and protein level. However, Gadd153 levels remained low after L-glutamine supplementation even as cell viability declined. Bcl-xL overexpression also extended Sp2/0-Ag14 cell viability, initially delayed the induction of Gadd153, but did not prevent the increase in Gadd153 protein levels during the later phase of the culture, when cell viability was declining. Interestingly, L-glutamine supplementation prevented Gadd153 up-regulation in cells ectopically expressing Bcl-xL, but had no effect on cell viability. CONCLUSION: This study highlights important limitations to the use of Gadd153 as an indicator of cell stress in hybridoma cells.

Zinc as an insulin replacement in hybridoma cultures.

There are many advantages to the use of protein-free media for biologics production, including a reduced risk of viral contamination from animal-derived proteins and simplification of downstream purification. In the course of developing protein-free media for hybridoma and myeloma cells, zinc was found to be an effective replacement for insulin, with no negative impact on viable cell density and antibody production. Transcript profiling using DNA microarrays indicated no major change in the global expression profile between the insulin and zinc-supplemented cultures, which is consistent with their similar growth and metabolic characteristics. Both DNA microarray and quantitative RT-PCR analysis showed increase in insulin receptor substrate 1 (Irs1) expression in zinc-supplemented cultures, while several key genes downstream of Irs1 in the insulin-signaling pathway, such as protein kinase B (PKB/Akt) and 3-phosphoinositide dependent protein kinase 1 (Pdpk1) did not show significant differences at the transcript level. Comparison of transcript profiles from cultures with low versus optimal zinc supplementation implicated the involvement of the insulin-related genes Pax6 and Phas1. Subtle differences were also observed between insulin and zinc in the serine-473 phosphorylation of Akt. Zinc increased serine-473 phosphorylation of Akt, but to a lesser extent than insulin. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, totally blocked the effect of both zinc and insulin on Akt activation, indicating the involvement of PI3K in the activation of Akt by zinc, rather than zinc acting on Akt directly. Our results highlight the impact of trace metal supplementation as protein-free media formulations move towards greater chemical definition.

Effect of ajoene, a natural antitumor small molecule, on human 20S proteasome activity in vitro and in human leukemic HL60 cells.

The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

Enhancement of monoclonal antibody production by lysine-containing peptides.

In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine-containing peptides was compared. The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth-suppressing activity. No correlation of the production-enhancing activity with the character of the distribution of cell-cycle phases could be found. All of the tested peptides, including the negative control peptide Gly-Phe-Gly, altered the cell-cycle phases distribution in favor of the proportion of the S phase. The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids. Among the set of tested lysine-containing di- to pentapeptides, the best results were obtained with the tripeptide Gly-Lys-Gly. The growth-suppressing and production-enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect-a slight promotion of cell growth.

Measurement of hydrophobic interactions of mammalian cells grown in culture.

An assay was developed to measure the hydrophobic interactions of commonly used mammalian cell lines grown in culture. The assay depends on the loss of cells from an aqueous suspension following vortexing with a hydrophobic oil phase. This allowed the determination of a hydrophobicity index, which was significantly higher for Chinese Hamster Ovary (CHO) cells than either a murine hybridoma (CC9C10) or a myeloma (SP2/0). This suggests that CHO cells may have a higher intrinsic cell surface hydrophobicity. The assay was also used to study the effect of different additives on the hydrophobic interactions of the cells. A dose-dependent effect was shown for the non-ionic surfactant, Pluronic F68, in reducing the hydrophobic interaction of the CHO cells. However, the pattern of the decrease due to Pluronic F68 was different for each cell line. A higher concentration of Pluronic F68 (0.2%) was required to eliminate the hydrophobic interactions of CHO cells compared to either myelomas or hybridomas, where only 0.05% was required to reduce these interactions to a similar level. Several oils were found suitable for this assay although canola oil maximized the sensitivity of the measured changes. The assay may be useful in monitoring changes in the hydrophobic interactions of mammalian cells during growth in bioreactors. This may be important in optimizing the concentration of cell protectants such as Pluronic F68 in agitated cultures.

Antioxidant capability and efficacy of Mega-H silica hydride, an antioxidant dietary supplement, by in vitro cellular analysis using photosensitization and fluorescence detection.

Treatment of Chinese hamster ovary and mouse hybridoma cells with Mega-H brand silica hydride, a marketed antioxidant, after photosensitization with singlet oxygen and hydroxyl/superoxide reactive oxygen species through the use of rose bengal diacetate and malachite green resulted in an effective method of reducing free radical activity by more than 96% against singlet oxygen species and more than 86% for hydroxyl and superoxide free radicals with the dosage recommended by the manufacturer. The analysis used a combinational spectrafluorometric technique to determine cell viability and cytotoxicity through the mechanism of intracellular esterase activity and plasma membrane integrity. Photosensitized controls not treated with silica hydride showed less than 1% viability under the same conditions. The reduction of the introduced free radicals and singlet oxygen species and the consequent high levels of cell viability may be the result of effective and efficient antioxidant and radical scavenging properties of silica hydride.

The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties.

The tryptic meat digest Primatone RL is a low-cost medium supplement of a complex nature which serves as a source of amino acids, oligopeptides, iron salts, some lipids and other trace low molecular weight substances. Its addition to mammalian and insect cell culture media significantly improves the cell growth properties of many cell lines. In this work the growth promoting effects of Primatone RL are described in more detail using different mouse hybridomas, a mouse myeloma cell line, and human promyelocytic leukemia HL-60 cells. The positive effects on cell growth induced by Primatone were observed in the presence of serum but were even more pronounced in serum-free culture. In addition the adaptation time from high serum to low (1%) or serum-free growth in the presence of Primatone is also significantly reduced. Primatone RL, when added to HL and DHI medium, improves cell growth under low serum or serum-free conditions by increasing the maximum cell numbers and in particular the viability of the culture. The observed decrease in cell death (apoptosis) induction leads to a significant improvement in antibody (recombinant protein) production by increasing the volumetric yields during long-term batch culture. The so-called anti-apoptotic effects of Primatone RL for mouse hybridomas, which is concentration dependent, is not fully understood.

Calcineurin activation protects T cells from glucocorticoid-induced apoptosis.

In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway.

Hybridoma cells producing antibodies against A-chain of mistletoe lectin I are resistant to this toxin.

The cytotoxic effect of mistletoe lectin I (MLI) on TA5 hybridoma cells which produce monoclonal antibodies (mAb) to MLI A-chain (MLA) was investigated. In vitro cytotoxic tests with colorimetric assay were carried out for LD50 determination. TA5 hybridoma cells were 100 times more resistant to MLI and 30 times to chimeric toxin consisting of MLA and ricin B-chain (MLA/RTB) than control cells. The TA5 mAb (IgG1) recognized MLI A-chain in Western blotting and bound 125I-labeled MLI with Ka of 0.43 x 10(8) M-1. The TA5 and control hybridomas had the same number of 125I-labeled MLI binding sites. Therefore cell-surface TA5 antibodies did not influence MLI binding with the cell. The cytotoxic effect and binding of MLI were completely blocked in the presence of 20 mM lactose. Thus, MLI cytotoxicity was mediated only by cell-surface galactosyl residues; intracellular mAb molecules block MLI toxicity. Our data suggest that MLA molecules mediating cytotoxicity pass through an anti-MLA antibody-containing vesicular compartment and that mAbs inhibit the translocation activity of MLI A-chain from intracellular vesicles into the cytosol.

The effect of fatty acids on hybridoma cell growth and antibody productivity in serum-free cultures.

A murine B-lymphocyte hybridoma (CC9C10) was adapted for growth in a serum-free medium. Supplementation of the medium with cis-unsaturated fatty acids (10-50 microM) improved the cell yield in the order oleic/linoleic > linoleic > oleic. Initial supplementation with the fatty acids also caused a significant increase (58%) in the volumetric Mab titre. Continued growth of the cells in the fatty acid supplemented media over five culture passages resulted in a gradual deterioration of the Mab yield concomitant with the appearance of lipid inclusions in the cytosol. The higher Mab yield could be restored by a limited period of growth of the lipid-loaded cells in fatty acid-free medium. These effects were independent of growth rate. This suggests that the optimal intracellular lipid content is finely balanced between a reduced and an overloaded state. Specific glucose and glutamine utilisation rates were unaffected by the presence of fatty acids. Also, the optimal glucose and glutamine concentrations for growth were independent of the fatty acids.

Rat antibodies bearing idiotypes of mouse antibodies against bromelain-treated mouse RBC.

Part of mouse antibodies reactive with bromelain-treated mouse RBC (BrMRBC) use VH12 and VK4 genes, and VH12/VK4-type antibodies are detectable specifically with rabbit anti-idiotype (Id) antibodies (here referred to as RAIb). This study showed that normal rat sera contained RAIb+ IgM at concentrations of 1-6 micrograms/ml. Rat spleens had many anti-BrMRBC B cells, most of which secreted RAIb+ IgM. Hybridomas of spleen cells from LPS-injected rats were screened with RAIb-binding and BrMRBC-hemolytic activity. We found 48 BrMRBC-hemolytic wells, which included all of 39 RAIb(+)-wells. From anti-BrMRBC wells, 7 RAIb+ monoclonal antibodies (mAb) were isolated. All the RAIb+ mAb could react with phospholipid antigens. Rat RAIb+ antibodies, as well as mouse RAIb+ antibodies, can be regarded as antiphospholipid antibodies reactive with BrMRBC. The interspecies expression of RAIb-Id on mouse and rat anti-BrMRBC antibodies indicates that some (antigenic) selective forces may act strongly to conserve the Id (V genes).

A plant extract which enhances the plating efficiency of lymphoid cell lines and enhances the survival of normal lymphoid cells in vitro.

A water soluble extract from the bark of the Samoan medicinal plant Alphitonia zizyphoides A. Gray (Rhamnaceae), enhances the plating efficiency in vitro of lymphoid cell lines as well as the survival of bone marrow cells and normal T and B lymphocytes. Furthermore, the inclusion of bark-extract into culture media enhances the cloning efficiency of a T-hybridoma cell line by more than 30 times at otherwise unsuitably low serum concentrations, but does not completely substitute for serum. The enhanced growth of a B-cell hybridoma is also paralleled by an increased production of monoclonal antibodies in cultures containing low cell densities.

Adaptation of mammalian cells to non-ammoniagenic media.

Although glutamine is used as a major substrate for the growth of mammalian cells in culture, it suffers from some disadvantages. Glutamine is deaminated through storage or by cellular metabolism, leading to the formation of ammonia which can result in growth inhibition. Non-ammoniagenic alternatives to glutamine have been investigated in an attempt to develop strategies for obtaining improved cell yields for ammonia sensitive cell lines. Glutamate is a suitable substitute for glutamine in some culture systems. A period of adaptation to glutamate is required during which the activity of glutamine synthetase and the rate of transport of glutamate both increase. The cell yield increases when the ammonia accumulation is decreased following culture supplementation with glutamate rather than glutamine. However some cell lines fail to adapt to growth in glutamate and this may be due to a low efficiency transport system. The glutamine-based dipeptides, ala-gln and gly-gln can substitute for glutamine in cultures of antibody-secreting hybridomas. The accumulation of ammonia in these cultures is less and cell yields in dipeptide-based media may be improved compared to glutamine-based controls. In murine hybridomas, a higher concentration of gly-gln is required to obtain comparable cell growth to ala-gln or gln-based cultures. This is attributed to a requirement for dipeptide hydrolysis catalyzed by an enzyme with higher affinity for ala-gln than gly-gln.

Effects of peptone on hybridoma growth and monoclonal antibody formation.

Hybridoma WuT3 secreting a monoclonal antibody against T lymphocytes was grown in RPMI 1640 medium supplemented with 1% human serum. The effect of the concentration of peptone, as an additive, was investigated on cell growth, monoclonal antibody formation, and cell metabolism over 0-10 g l-1 range. It was found that 1-5 g l-1 peptone can significantly promote the growth of cells and increase the formation of monoclonal antibody, especially at 3-5 g l-1, when both the accumulating level and secretion rate of monoclonal antibody are higher than that at other peptone concentrations. Based on glucose, lactate and ammonia analysis data, the efficiency of glycolysis was assessed and the utilization of amino acids was more efficient at 3-5 g l-1 peptone. The cell growth and monoclonal antibody formation were inhibited at higher peptone concentrations, e.g. 10 g l-1.

Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations.

A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.

Optimization of culture conditions for feline x murine heterohybridomas.

Feline splenocytes were fused to the murine myeloma lines NSO or Ag8. Autologous serum and taurine were used as media supplements for the cat x mouse heterohybridomas. The best results were obtained by the use of NSO as fusion line with taurine-supported media.

Three-dimensional electric field traps for manipulation of cells--calculation and experimental verification.

The forces acting on dielectric particles and living cells exposed to alternating and rotating fields generated by three-dimensional multi-electrode arrangements are investigated. Numerical procedures are described for the calculation of the electric field distribution and forces. The physical treatment considers electrodes of any shape and dielectric particles of complex structure. Particle and cell trapping are based on negative dielectrophoretic forces produced by high-frequency a.c. or rotating electric fields up to 400 MHz. Various multi-electrode systems were realised in commercially fabricated microelectrode systems, and tested for their ability to move and assemble microparticles or living cells without contact with the electrodes. The field distribution and accuracy of phase-controlled power application was tested using individual artificial particles trapped in the electric field cage. Position and trajectories of particle motion were measured. The paper gives an overview of electrode and field cage design in the microscale range.

Kinetics of development of spontaneous apoptosis in B cell hybridoma cultures.

The kinetics of the development of apoptosis was studied in mouse B cell hybridoma batch cultures carried out in the iron-rich protein-free medium. One of the markers of apoptosis, the apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride, was found to rise significantly at 144 h, i.e., in the late stationary phase. At the decline of the culture (216 h) the value of the apoptotic index reached 29.1%. Analysis of another marker, the degree of DNA fragmentation determined on the basis of chromatographic resolution of isolated cellular DNA, revealed a significant increase as early as 96 h, i.e., at the end of the exponential phase. At 216 h the net value of the fragmented DNA fraction was about 30% of cellular DNA. The values of both markers were found to be very similar when the iron-rich protein-free supplement was replaced with conventional 10% foetal calf serum. This finding suggested that the growth factors present in the serum were not able to abolish the tendency of the hybridoma culture to undergo spontaneous apoptosis. The timing of the spontaneous onset of apoptosis in the exponential phase indicated that B cell hybridoma apoptosis was a process associated with cell proliferation and full metabolic activity rather than with the decline of cell vitality.