Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus.

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected. A BRSV specific antibody response was observed for all the BRSV infected calves. These antibodies were shown to have neutralizing activity. No lymphocyte proliferation response was detected in the mock-infected group whereas three animals in the infected group were positive three weeks after the infection. All BRSV-infected calves, except one, produced interferon-gamma (IFN-gamma) one week post-infection and IFN-gamma was observed in all six infected calves after three weeks. The control group showed no IFN-gamma synthesis. In spite of the limits of the BRSV infection model, humoral and cellular immune responses were actively developed by all the calves against this pathogen.

Immunological response of mice to the bovine respiratory syncytial virus fusion glycoprotein expressed in recombinant baculovirus infected insect cells.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves. The BRSV genome encodes two major glycoproteins, G and F, which are the major targets for the host antibody response. We have expressed the F glycoprotein in insect cells (Sf9) using a recombinant baculovirus vector. A comparison of the F protein expressed in mammalian and insect cells by SDS-PAGE showed that only part of the baculovirus-produced protein was soluble and processed like the native protein. The antigenicity of the soluble form of the F protein expressed in insect cells was identical to that of the F protein expressed in mammalian cells. Immunization with the F protein expressed in insect cells induced neutralizing antibodies in mice. This antigenic preparation adjuvanted with Quil-A produced an increased neutralizing antibody titer and induced protection.

Cathepsin B and N-acetyl-beta-D-glucosaminidase in human synovial cells in culture: effects of interleukin-1.

Human synovial cells were cultured in vitro and tested for the activities of two lysosomal enzymes, cathepsin B and N-acetyl-beta-D-glucosaminidase (NAGA) under various conditions. Unstimulated synovial cells display intracellular and extracellular activities of both enzymes. However, cathepsin B was secreted in a latent pepsin-activatable form, whereas NAGA was secreted in an active form. Most of the cell strains analysed secreted rather limited amounts of the enzymes (less than 25% of total activity); some strains, however were highly secretory, the secreted activity reaching up to 50% of total activity. Cells were then stimulated with human recombinant interleukin-1 alpha (rhIL-1 alpha) or beta. Only the levels of secreted NAGA were clearly increased. Results are to be interpreted in view of the role played by synovial cells and by the lysosomal enzymes they release in inflammatory joint diseases and it would be worthwhile in the future to check for secreted NAGA in various body fluids, such as the synovial fluid of the inflamed joint.

Induction of cyclooxygenase by interleukin 1: comparative study between human synovial cells and chondrocytes.

OBJECTIVE: To investigate the regulation of the prostaglandin (PG) synthesis by interleukin 1 (IL-1) in human synovial cells and chondrocytes. METHODS: Both cell types stimulated by human recombinant IL-1 synthesized PGE2, PGF2 alpha and 6-keto-PGF1 alpha. RESULTS: PGE2 was the major PG synthesized. When arachidonic acid was added exogenously at the end of the stimulation, an increase in the prostaglandin synthesis was observed after 6 and 24 h suggesting that cyclooxygenase is the limiting enzyme. Using actinomycin D and cycloheximide, PG synthesis was shown to be protein synthesis dependent. Inhibition of the constitutive cyclooxygenase by aspirin before the IL-1 stimulation confirmed that the increased prostaglandin synthesis was due to a de novo synthesis of cyclooxygenase. CONCLUSION: This enzyme induction by IL-1 was found to be similar in both cell types.

Differential effects of interleukin-1 alpha and beta on the arachidonic acid cascade in human synovial cells and chondrocytes in culture.

The effects of interleukin-1 alpha and beta were tested on the [3H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 beta was more potent than IL-1 alpha. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 alpha or beta: prostaglandin E2, F2 alpha and 6-keto-prostaglandin F1 alpha. Regarding the synthesis of these prostaglandins, IL-1 beta was again more potent than IL-1 alpha. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.

Routine prostaglandin assay by GC-MS in multiwell tissue culture plates: application to human synoviocytes and chondrocytes.

Prostanoids can be assayed by the highly sensitive and reproducible gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) technique. This paper describes a simplified method applied to the quantification of prostaglandins (PG) in the supernatants of limited amounts of cells, cultured in multiwell plates (30,000 cells/well). The culture medium was extracted on C2 columns and the derivatization was performed in three steps. Prostanoids were then analyzed by GC-NICI-MS within 10 min. The storage conditions at each step were tested in order to achieve maximal yields. This procedure allowed the determination and quantification of PG produced by cultured human synovial cells and chondrocytes at the basal level as well as after interleukin-1 stimulation. PGE2, PGF2 alpha, and 6-keto-PGF1 alpha, the stable form of PGI2, were the main PG detected. Derivatization and GC-MS analyses of 18 samples could easily be performed in 1 day. The simplified GC-NICI-MS method can thus be routinely applied for assaying PG in any cell line cultured in multiwells.

Stimulation of prostaglandin synthesis by human endothelial cells exposed to hypoxia.

In ischemic organs, arachidonic acid (AA) metabolites and mostly prostaglandins (PGs) have been found to be released in high amounts. The mechanism for this AA metabolism activation and its physiological implications are not clear. Because endothelial cells are an important source of PGs and because they seem to be very rapidly affected by ischemia, we developed an in vitro model where human endothelial cells were submitted to hypoxia. An important specific activation of phospholipase A2 was observed during hypoxia, which was concomitant with a rise in cytosolic calcium concentration. Endothelial cells synthetize in normal conditions as a mean 1.42, 1.00, 7.69, and 26.92 ng/mg proteins of, respectively, PGE2, PGD2, PGF2 alpha, PGI2. An important increase of about five- to ninefold in the synthesis of the four PGs was observed during hypoxia, which followed the same kinetics as the PLA2 activation. This increase in PG synthesis was sensitive to cyclooxygenase inhibitors. During reoxygenation, PG synthesis decreased back to the basal level of resting cells, suggesting that cells were able to recover their homeostasis after hypoxia. These observations indicate that endothelial cells exposed to oxygen deprivation are a major source of PGs and could contribute to the high amounts of PG released in vivo in ischemic organs.