Control of 92 kDa collagenase secretion in mammalian cells by modulation of AP-1 activity: an experimentally based theoretical study.

Collagenolytic enzymes control cell migration through connective tissues. They appear to be of crucial importance for angiogenesis, tumor metastasis or wound repair. A well-documented stimulation pathway of collagenase secretion, either by natural (cytokines) or synthetic (phorbol esters) molecules, acts through activation of the proto-oncogene activating protein 1 (AP-1). Interestingly, this nuclear factor enhances its own synthesis. It also modulates the activity of different genes, including the one coding for 92 kDa gelatinase. We developed a mathematical model to describe this pathway. It led us to conjecture the existence of an hysteresis cycle for PMA-stimulated collagenase secretion, which was experimentally demonstrated later in MDBK cells in culture. We also modified our model to simulate the behavior of tumoral cells expressing AP-1. In this case, the system becomes highly unstable and, once stimulated, cannot be brought back to rest. This approach paved the way for the understanding and the control of mammalian cell processes, connective tissue maintenance or metastasis dissemination.

Tips and step-by-step protocol for the optimization of important factors affecting cellular enzyme-linked immunosorbent assay (CELISA).

CELISA, or cellular enzyme-linked immunosorbent assay, is a powerful and easy to use technique to study cell surface antigens under different stimulations. Nevertheless, some factors must be discussed and optimized prior to reaching a reproducible CELISA. These include the choice of cell density, fixative agent, blocking agent, culture medium, optimal antibody dilutions, and incubation time. In this paper, we first present a short review of some references devoted to CELISA by means of a comparison of these parameters, followed by their description. Then, we describe and study these different parameters using practical examples comparing TNF-induced ICAM-1 expression as an end point, on HBL melanoma and HUVEC. These cell lines were also chosen because they differ in their ability to grow as discontinuous and continuous layers, respectively. Furthermore, we designed a comprehensive flow chart, as well as a complete step-by-step protocol for CELISA optimization.

Detection and quantification of ATP and heavy metal with electronical micro-circuits.

In order to use whole eukaryotic cells as an active element in the detection and amplification of biological signals, for both in vitro and in vivo applications, we have undertaken a first approach to interface live cells and integrated circuit, and evaluate the possibility to develop a microbioreactor. An amplified photodiode system was designed and built as an electronical circuit in a way that it could easily be miniaturised. In parallel micro-chips with silicium chambers were used as microbioreactors to adhere cells. We showed here that this etched silicon chamber allows endothelial and CHO cells spreading, permitting determination of a number of cell properties {\it on line} providing appropriate integrated circuits are designed to perform the desired functions. The photodiode system reacting to the luminescent luciferase system permitted, through the use of appropriate software from a personal computer (PC) connected on line in vitro, the determination of ATP concentration, and using different luciferase transfected bacteria permitted the detection of constitutive or induced luminescence.

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.

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 degrees C and are detached by lowering the temperature to 10 degrees C without any other deleterious treatment. Both bacterial culture grade polystyrene Petri dishes and polystyrene beads (120 to 250mum 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 degrees when the temperature is raised from 25 to 37 degrees C indicating change in hydrophilicity of the surface as a function of temperature.

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 degrees C to 32 degrees 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 degrees C, at 32 degrees 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.

Biological activities of a human amniotic membrane interferon.

In order to characterize further the human amniotic membrane interferon (IFN-AM), an interferon antigenically unrelated to human IFN-alpha, -beta, and -gamma or TNF, we analysed its biological activities. Here, we present direct evidence of its ability to affect cell growth and to induce the IFN-stimulated genes (ISGs) 6-16 and 2'-5' oligoadenylate synthetase (OAS), in addition to its crossed anti-viral activity. The cellular growth arrest effect of IFN-AM was dose-dependent and paralleled that of IFN-beta. IFN-AM was also able to inhibit thymidine incorporation into DNA, similar to IFN-beta. The mRNA induction of 6-16 gene with IFN-AM treatment reached its highest level at 500 IU/ml and remained constant up to 2000 IU/ml. Conversely, 2'-5' OAS mRNA induction was dose-dependent, with the maximum level detected at 2000 IU/ml of IFN-AM treatment. The time course of mRNA accumulation by ISGs with IFN-AM (500 IU/ml) stimulation was also investigated. Gene induction reached a maximum at 16 h after IFN treatment for 2'-5' OAS and at 48 h for the 6-16 gene. IFN-AM and human IFN-alpha induced similar levels of the OAS enzyme. IFN-AM also showed small but significant activity in bovine cells. In conclusion, the amniotic membrane IFN here studied showed both anti-cellular activity and the ability to stimulate ISG-transcriptional activation in a similar manner to IFN-beta. In addition, IFN-AM was also as able to induce the expression of the enzyme 2'-5' OAS, as did IFN-alpha. Lastly, amniotic IFN showed a significant cross-species anti-viral activity, which was different from both human IFN-alpha and -beta. Taken together, these data strongly suggest that IFN-AM is a novel sub-type I IFN.

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

Kaposi's 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.

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 TGFß, 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.

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.

Uptake of recombinant myeloperoxidase, free or fused to Fc gamma, by macrophages enhances killing activity toward micro-organisms.

A chimeric antibody-like molecule consisting of the human myeloperoxidase (rMPO) fused to the second and third constant-sequence (CH2 and CH3) Fc domains of human immunoglobulin G-1 has been constructed and expressed in Chinese hamster ovary (CHO) cells. This fusion molecule was designed to combine the binding specificity of Fc with the antimicrobial properties of rMPO. The rMPO-Fc fusion dimerized through the Fc fragment, while retaining the enzymatic activity of rMPO. The chimeric molecule was glycosylated and most of the propeptide was eliminated, indicating a better processing of the polypeptide than for rMPO alone. Both rMPO and rMPO-Fc bound to and were internalized by macrophage-like U937 promonocytic cells. Unexpectedly, the chimera failed to bind to the Fc receptor but interacted with a higher affinity than rMPO with the same binding sites. The presence of the Fc fragment in the chimera, in addition, did not extend the plasma half-life of the fusion protein. In vitro, rMPO-Fc exhibited a stronger killing effect than rMPO toward Candida albicans in the presence of either H202 alone or human macrophages. In vivo, rMPO-Fc similarly conferred a better protection than rMPO in a lethal model of murine cowdriosis. These properties could be related to the Fc-induced dimerization of the fusion protein in CHO cells.

Production of alpha interferon in Cowdria ruminantium-infected cattle and its effect on infected endothelial cell cultures.

Cattle that resisted experimental heartwater infection caused by the rickettsia Cowdria ruminantium produced significant levels of circulating alpha interferon (IFN-alpha), whereas animals that died from heartwater did not. In vitro, recombinant bovine IFN-alpha was found to significantly reduce the yield of Cowdria organisms in bovine endothelial cells, but even at a high concentration (1,000 U/ml), IFN-alpha did not completely prevent the growth of Cowdria organisms in these cells. This limited inhibitory effect of IFN-alpha is in agreement with the in vivo situation where an infectious process has to take place to induce a protective immune response. Our results suggest that IFN-alpha produced in vivo in response to Cowdria infection may represent an efficient way to slow down the infection and allow the animal to mount a protective immune response. IFN-alpha is the first endogenously produced factor shown to have anti-Cowdria activity.

Bovine and human endothelial cell growth on collagen microspheres and their infection with the rickettsia Cowdria ruminantium: prospects for cells and vaccine production.

We successfully cultivated the rickettsia Cowdria ruminantium, in bovine endothelial cell lines (Bovine Umbilical Endothelial Cells/BUEC and Bovine microvasculature Cells/BMC) and also in primary endothelial cells of bovine origin (Bovine Aorta Endothelial cells/BAEC) and more surprisingly in cells of human origin--Human Umbilical Vein Endothelial Cells/HUVEC--and Human Endothelial Cells from the Microvasculature/HEMEC. This first evidence of the pathogenicity of this bovine rickettsia in the human cell system gene-rates new interest as regards its possible relevance for human health. It provides also further possibilities for the attenuation of Cowdria ruminantium isolates, and therefore brings new prospects for vaccine preparation. In vaccine production, mass cell culture is essential. Our results indicate that endothelial cells attach efficiently on collagen microspheres. As BAEC cells grow well on them in a batch mode, and if the process could be optimized for the different cell types (using appropriate adhesion and growth factors) our observations offer interesting prospects for the future development of a Cowdria ruminantium vaccine production in the fluidized-bed reactor VERAX System one, which provides easy control of growth conditions.

Inhibition of Cowdria ruminantium infectious yield by interferons alpha and gamma in endothelial cells.

We have shown before that there is a positive correlation between resistance of cattle against Cowdria infection and early IFN production. Our in vitro studies demonstrated an activity of rBoIFN alpha 2C and rBoIFN gamma against Cowdria in bovine endothelial cells of brain microvasculature (BMEC). rBoIFN gamma is much more active in this respect than rBoIFN alpha 2C. These results suggest a role of IFNs in the resistance against the disease. Strikingly, in the same conditions rBoIFN alpha 2C has no effect on the yield of Cowdria from infected bovine endothelial cells of umbilical artery origin (BUEC). Similarly we showed that HuIFNa had no effect on the multiplication of Cowdria in human vein umbilical endothelial cells (HUVEC). We found no differences in the capacity of BUE and BME cells to bind rBoIFN alpha 2C. This may reflect a true difference between capillary and large blood vessels.

Role of interferons in infectious diseases in the bovine species: effect on viruses and rickettsias.

Successful protection was obtained with interferon treatment in experimental viral infections in the bovine species in a number of cases. The efficacy of the treatment against vaccinia virus infection and against rotavirus infection have been demonstrated. On the contrary, bovine herpes virus 1 (BHV 1-causing rhinotracheitis and part of the shipping fever complex) infections were not inhibited by interferon (IFN). The authors have undertaken a study in cattle in Zimbabwe to assess the role of interferon in the resistance of the animals to Cowdria ruminantium. A good correlation between production of interferon by the animal following the infection, and the resistance of the animals against the rickettsia was demonstrated. This pointed out the possible "adjuvant" role of interferons and other cytokines.

Antiviral effect of bacterially produced human interferon (Hu-IFN alpha 2) against experimental vaccinia infection in calves.

The heterologous antiviral efficiency of bacterially produced human interferon (Hu-IFN alpha 2) in the bovine species was studied, using vaccinia infection as experimental model. In a double blind experiment, young calves were intramuscularly injected daily for seven consecutive days with different doses of Hu-IFN alpha 2 or placebo, the treatment starting 24 h before intradermal inoculation of vaccinia virus. A clear protection by interferon was observed in all the IFN treated animals, although individual variations in the sensitivity to IFN were recorded. The efficiency of treatment varied according to the dose of IFN used: With the highest dose (10(6) IU/kg), complete protection could be obtained. The only side-effect observed was hyperthermia. Circulating antiviral activity appeared quite early after each IFN injection, presented a more or less biphasic kinetics, and was completely cleared after 24 h, justifying the daily treatment schedule. The first evidence of an in vivo antiviral effect of human interferon in the bovine species opens broad perspectives for a future use of interferon in veterinary medicine.

Experimental rotavirus diarrhoea in colostrum-deprived newborn calves: assay of treatment by administration of bacterially produced human interferon (Hu-IFN alpha 2).

Seven colostrum-deprived newborn calves were orally inoculated within 24 hours after birth with bovine rotavirus. Three of them were intramuscularly injected with bacterially produced human interferon (Hu-IFN alpha 2). The four control animals presented a severe diarrhoea for at least 48 hours, while only one of the treated calves suffered from a transient diarrhoea for a few hours. Hu-IFN alpha 2 seems therefore able to control rotavirus diarrhoea in newborn calves, although it did not inhibit virus excretion and seroconversion in the treated animals. Moreover, the administration of endogenous interferon appeared to be well tolerated by newborn calves. The efficacy of human alpha 2 interferon for the treatment of this important virus infection of cattle seems thus well established.