J. Werenne

Increased productivity of recombinant tissular plasminogen activator (t-PA) by butyrate and shift of temperature: a cell cycle phases analysis.

Directed control of cell metabolism by a modification of the physicochemical conditions (presence of Na-butyrate and modification of the temperature) was used to modulate the productivity of human recombinant tissular plasminogen activator (t-PA) expressed under control of SV40 promoter in Chinese Hamster Ovary (CHO) cell lines. We showed that both by adding Na-butyrate or lowering temperature from 37 °C to 32 °C there is an increase in the amount of t-PA excreted, while cell growth is significantly reduced. The treatments also increased the intracellular amount of t-PA. We measured the distribution of cell cycle phases by cytometry and used a modification of the equations of Kromenaker and Srienc (1991, 1994 a, b) to analyse the intracellular t-PA production rate in the different cell cycle phases. Intracellular t-PA was shown to accumulate in G1 phase in all conditions (at 37 °C, at 32 °C and in presence of butyrate). Moreover, we have shown that the distribution of the time cells treated by butyrate are maintained in the G1cell cycle phase is significantly increased. t-PA produced in the different cell culture conditions tested was analysed by zymogram and western blotting: neither butyrate, neither the shift of temperature changed significantly the overall quality of the protein. The N-glycan patterns of recombinant human t-PA was also analysed with carbohydrate-specific lectins. Butyrate caused a transitory increase in N-linked complex high-mannose oligosaccharides without any effect on the sialic acid content of t-PA.

Constitutive release of metalloproteinase-9 (92-kd type IV collagenase) by Kaposi's sarcoma cells

Kaposis sarcoma (KS) is an angioproliferative disease characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Several lines of evidence suggest that KS is a multifocal cytokine-mediated disease of vascular origin. Because metalloproteinases (MMPs) are important enzymes involved in angiogenesis, we studied their activity in five different KS-derived cell lines and compared these data with those obtained with human umbilical vein endothelial cells (HUVEC). We focused on the activity of the 72- and 92-kd type IV collagenases because these enzymes are thought to play an important role in the process of tumoral invasion. Nonstimulated HUVEC released a weak 72-kd collagenase activity and no 92-kd collagenase activity, as determined by zymographic analysis. Stimulation of HUVEC with phorbol myristate acetate (PMA) or TNF-alpha increased the 72-kd collagenase activity and also induced a 92-kd collagenase activity. By contrast, KS-derived cells constitutively released significant 72- and 92-kd collagenase activities. The basal release of these enzymes by KS cells was further enhanced by TNF-alpha or PMA. Conversely after in vivo exposure to chemotherapy, KS-derived cells showed a downregulation of the production of MMPS that could be reversed by the addition of TNF or PMA. These results suggest that KS cells have constitutive features of activated cells that have an invasive and metastasizing potential.

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

Adhesion, growth and detachment of cells on modified polystyrene surface

By adsorbing poly(N-isopropylacrylamide) (PNIPAAm) from an aqueous solution onto oxidised polystyrene without the need for grafting the polymer to the surface, we showed here that cells(CHO-K1) adhere and grow well at 37 °C and are detached by lowering the temperature to 10 °C without any other deleterious treatment. Both bacterial culture grade polystyrene Petri dishes and polystyrene beads (120 to 250μm diameters) commercially available used in static conditions of growth were tested with similar results. The contact angle of modified Petri dishes with a water droplet increases from 36 to 58° when the temperature is raised from 25 to 37 °C indicating change in hydrophilicity of the surface as a function of temperature.

Susceptibility of bovine rotavirus to interferon

SummaryUsing the plaque assay or the CPE test (cytopathogenic effect), we investigated the action of human, simian (rhesus), bovine and murine interferons on bovinerotavirus adapted to grow on established Rhesus monkey kidney cell line (MA 104) or Georgia Bovine Kidney cell line (GBK). Except for the murine interferon we used, which had no antiviral effect at all, the different interferons tested were clearly active.

A general artificial neural network for the modelization of culture kinetics of different CHO strains

Animal cell cultures are characterized by very complex nonlinear behaviors, difficult to simulate by analytical modeling. Artificial Neural Networks, while being black box models, possess learning and generalizing capacities that could lead to better results. We first trained a three-layer perceptron to simulate the kinetics of five important parameters (biomass, lactate, glucose, glutamine and ammonia concentrations) for a series of CHO K1(Chinese Hamster Ovary, type K1) batch cultures. We then tried to use the same trained model to simulate the behavior of recombinant CHO TF70R. This was achieved, but necessitated to synchronize the time-scales of the two cell lines to compensate for their different specific growth rates.

Protection against #Cowdria ruminantium# infection in mice with gamma interferon produced in animal cells

We report here that y interferon produced in animal cell culture injected intraperitoneally, efficiently protects mice against Cowdria ruminantium infection. None of the other cytokines tested exerted any effect. Neutralizing antibodies against the cytokines (anti IL6, anti TNF, anti γIFN), did not modify the course of the disease, indicating that these cytokines do not play any crucial role in the pathology. The results concerning the protective effect of interferon pave the way towards the establishment of a rational selection method for protective antigens.

Biological response of endothelial cells and its modulation by cytokines: prospects for therapy and bioprocesses.

Endothelial cells are involved in important pathological situations. They could be the target for infectious processes as for example in Cowdriosis, an important disease in cattle due to the rickettsia Cowdria ruminantium prevalent in the south of the Sahara. They are also connected to angiogenic processes related to tumor invasion.Our results indicate that AIDS related Kaposi sarcoma cells may be of endothelial origin. We conclude from our data the mobility of those cells, related to the expression of the metalloproteinases (especially the 92 kD form of the enzyme), is an important factor in Kaposi saroma dissemination and is the main factor limiting the scale up of Cowdriosis vaccine production in Bovine Umbilical Endothelial Cell line. We showed that PMA and TNF increased the 92 kD Metallaproteinase and that TGFβ, produced in an inactive form in cultures of Human Umbilical Vein Endothelial Cells, is a potential inhibitor of Kaposi sarcoma spreading, and could also be useful in improving our process for Cowdria ruminantium vaccine production, since it reduces the sensitivity of the cells to mechanical stress without affecting significantly the overall infectious process.

Mesenchymal cells: metalloproteinases and adhesion on microcarriers

The purpose of this work was to approach the development of a safe and validable process to grow the amounts of adult Mesenchymal Stem Cells (MSC) necessary for future biomedical applications. Using a single use Vue Life bag system integrated in a simple agitations bioreactor we constructed, we have studied the adhesion, migration and growth properties of MSC on different carriers (Cytodex and Bionoc) in comparison to other cells (endothelial/Kaposi sarcoma cells, CHO and HeLa) in relation to the expression of endogenous Metalloproteinases.

Modulation of Cell Cycle for Optimal Recombinant Protein Production

Efficient t-PA production in recombinant CHO cells is of major interest for pharmaceutical industry. Contrary to the multigene metabolic engineering approach, our strategy allows investigations of recombinant cell lines already validated. Compared to 37°C, 32°C showed that t-PA productivity was significantly increased. The specific rate of t-PA secretion was enhanced at the lower temperature, in relation to the cell cycle modification. At this temperature, glycosylation is not significantly altered while serine protease activity is reduced. A similar study involving cytofluorimetric data and mathematical analysis was made for the other factors tested (PMA, TGF-β and butyrate). Our data not only emphasize the interest of a two step process for t-PA production (involving 1. a cell biomass production phase 2. a high protein productivity phase), but showed moreover that productivity can be furthermodulated by the extracellular environmental factors affecting cell cycle. On the basis of results obtained by our rapid screening method a multigen engineering strategy could be decided on a rational basis.