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.

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.

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.

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.

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.

Comparison of interferon action in interferon resistant and sensitive L1210 cells.

Translation inhibition, leu-tRNA aminoacylation and double-stranded RNA and ATP dependent phosphorylation were examined in interferon-treated and control cell-free lysates of leukaemic mouse L 1210 R and L 1210 S cells. No differences were observed between the respective interferon-treated and control cell-free extracts, except for the presence of an enhanced 67K dalton phosphoprotein fraction in interferon-treated L 1210 S cell-free extracts. In non-responding cell-free lysates, the lack of stimulation of a 67K dalton phosphoprotein fraction cannot be explained by the presence of an increased level of some inhibitory activity, such as a phosphatase.

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.

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.

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.

Susceptibility of bovine rotavirus to interferon. Brief report.

Using the plaque assay or the CPE test (cytopathogenic effect), we investigated the action of human, simian (rhesus), bovine and murine interferons on bovine rotavirus 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.

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.

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.

[Attempt at the experimental reproduction of rotavirus diarrhea in newborn calves deprived of colostrum]. / Tentative de reproduction expérimentale de la diarrhée à rotavirus chez des veaux nouveau-nés privés de colostrum.

Colostrum-deprived newborn calves were orally inoculated with different doses of cell-culture bovine rotavirus or with bacterium-free filtrates of calf stools containing rotavirus. None of the animals that received high doses of cell-culture rotavirus developed diarrhoea or any other clinical sign, although all of them excreted virus for several days and produced specific antibodies; calves inoculated with lower doses of cell-culture virus or with stool filtrates showed a transient diarrhoea 48 h after inoculation. Such paradoxical results might be due to a phenomenon of interference, as bovine rotavirus is susceptible to interferon. In experimental conditions, rotavirus produces only a mild and transient diarrhoea: this contrasts with the situation observed in farms, where that virus may provoke important problems. In association with the virus itself, numerous other factors such as the environmental conditions or the response of the calf to the infection also play a role in the evolution of the disease.

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. With lower doses of virus, interferon production was delayed and preceded by a severe but transient diarrhoea. In all cases, several waves of interferon production were observed. Our data indicate that interferon plays an important role in the control of viral diseases in calves and in their natural recovery.

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.