A high density culture system for the in-vitro production of human and mouse monoclonal antibodies

A simple high density cell culture system is described which demonstrates many of the features of commercially available hollow-fibre systems, but without the need to invest in a dedicated system. The system has been shown to achieve product concentrations of up to 40-fold greater than that obtained in batch culture making gram production of MAbs possible with considerable saving on serum costs.List of Symbols ELISA enzyme-linked immunosorbent assay - FBS foetal bovine serum - IgG immunoglobulin G - IgM immunoglobulin M - MAb monoclonal antibody - pH negative common logarithm of the hydrogen ion concentration - pO₂ dissolved oxygen concentration - RPMI 1640 Roswell Park Memorial Institute medium 1640     

Effect of environment, conformation, sequence and base substituents on the imino proton exchange rates in guanine and inosine-containing DNA, RNA, and DNA-RNA duplexes

High-resolution 1H nuclear magnetic resonance in H2O has been used to study the effect of sequence, conformation, environmental factors and base substituents on the exchange behavior of the hydrogen-bonded imino protons of guainine X cytosine and inosine X cytosine base-pairs in DNA, RNA, and DNA-RNA duplexes. The exchange rates were determined by measurement of the spin-lattice relaxation rates of the imino protons as a function of temperature. The exchange was not altered by the presence of high concentrations of salt, and the inability of phosphate to catalyze the exchange indicates that the exchange is limited by formation of a solvent-accessible "open" state. The exchange behavior depends on the duplex conformation and sequence. Exchange from the Z form polymers was orders of magnitude slower than the corresponding duplexes in the B conformation, and the A form RNA duplexes exchanged more slowly than the B form DNA polymers with the same sequence. The exchange behavior of the DNA-RNA hybrids was dependent on whether the purine or the pyrimidine strand contained the deoxyribose sugar. For both the guanine and inosine-containing duplexes, the homopolymer duplexes exchange more slowly than the more stable alternating copolymers. For the alternating duplexes, substitution of cytosine with 5-bromo- or 5-methylcytosine slowed the exchange and increased the activation energy for exchange. The inosine-containing duplexes exchanged more rapidly than the guanosine-containing duplexes, but both showed similar changes in exchange behavior in response to changes in sequence and base substituents. The activation energies for base-pair opening in B form DNA are correlated with the van der Waals contribution to the base-base interaction energy, suggesting that the purine base is partially unstacked in the open state. Using the relaxation measurements to set an upper limit on the exchange rate in poly(dG-dC) and the tritium exchange behavior at low temperature, we find that even though Z-DNA exchanges very slowly, the activation energy is similar to that observed in the A and B form duplexes, suggesting that exchange occurs from a similar open state.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Preparation,Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more “temperate swarmers” that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. “Wettability”, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.     

Modulation of natural killer activity in mice following infection with Listeria monocytogenes

Mice that received a sublethal, intraperitoneal dose of viable Listeria monocytogenes, virulent strain 10403, exhibited a systemic increase in natural killer (NK) activity. The kinetics of the response differed with respect to the various effector cell populations analyzed. Resident peritoneal cells and peripheral blood leukocytes demonstrated high NK activity on Days 3, 7, and 10. Peak spleen and bone marrow NK activity was observed on Day 3, returning to normal levels by Day 7. In contrast, peritoneal exudate cells, elicited with proteose peptone, expressed enhanced NK activity for 60 days following infection with viable Listeria. Augmented NK activity was detected with all cell types as early as 12 hr after infection. The intraperitoneal injection of nonviable antigenic preparations derived from L. monocytogenes, strain 10403, resulted in the enhancement of peritoneal and splenic NK activity. In contrast, mice that received an intraperitoneal injection of avirulent Listeria, strain 19113, failed to express enhanced levels of NK activity. The genetic trait of anti-listerial resistance which is associated with non-H-2 linked genes was of no importance with respect to enhanced NK activity. Listeria-resistant C57BL/6J and Listeria-susceptible DBA/2J mice both produced systemic augmentation of NK activity following infection. NK activity was not abrogated by macrophage depletion or by treatment with anti-Thy 1.2 serum plus complement. These results confirm the potent immunostimulatory capacity of virulent Listeria for NK activity and provide further insight into the kinetics of this response in various lymphoid compartments. The protracted augmentation of NK activity of elicited peritoneal exudate cells as compared to nonelicited peritoneal cells in Listeria-primed mice suggests that the influx of inflammatory cells may provide NK-enriched and/or accessory populations for immunopotentiation of NK activity in inflammatory sites.