Toward prevention of cowdriosis.

Mass production of Ehrlichia ruminantium variants from different regions of sub-Saharan Africa is one of the difficulties that must be overcome in producing a heartwater vaccine. Vaccine productivity can be limited by endogenous induction of interferon (IFN), which inhibits the propagation of Ehrlichia ruminantium (ER) in cell culture. Different kinds of endothelial cells, in which ER multiply efficiently, could be grown in a scalable way in VueLife Teflon bags on Cytodex 3 microcarriers where bead-to-bead transfer of cells occurs.

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.

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.

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.

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.

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.

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).

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.

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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

Interferon response in colostrum-deprived newborn calves infected with bovine rotavirus: its possible role in the control of the pathogenicity.

Colostrum-deprived newborn calves were experimentally infected with cell-culture rotavirus. A similar process of infection was observed when the animals were inoculated immediately after birth or at the age of three days, with a corresponding delay in the onset of virus excretion and interferon production in the later case. With high doses of virus, interferon was produced very early and no symptoms were observed.