We have examined the efficiency of coexpression of two heterologous genes from a recombinant Bombyx mori nuclear polyhedrosis virus for the production of antibodies in silkworm larvae. The cDNAs encoding the light and the heavy chains of a murine immunoglobulin, directed against lipoprotein I of Pseudomonas aeruginosa, were brought under the control of two separate copies of the viral polyhedrin promotor. Infection of silkworm cells with the recombinant baculovirus yielded a maximum of 6.4 micrograms/ml IgG2A in the culture supernatant 72 hours post infection, while 800 micrograms/ml IgG2A was found in the hemolymph of infected fifth instar silkworm larvae seven days after infection with the same construct. The recombinant antibody exhibited a similar antigen specificity and avidity to that of the monoclonal antibody derived from ascites fluid.
A synthetic peptide of human recombinant interleukin 1 beta (hrIL-1 beta) 165-186, which exhibits biological activity in the human fibroblast assay, was used as an immunizing antigen to obtain a murine monoclonal antibody (MoAb) termed FIB 1. This MoAb, an IgG1, reacts specifically with hrIL-1 beta, but not with hrIL-1 alpha, as measured in enzyme-linked immunosorbent assays (ELISA). The MoAb FIB 1 detects the characteristic 17 kDa IL-1 protein in Western blots. Binding to the antigen is specific, as deduced also from the close correlation of ELISA immunoreactivity with IL-1 biological activity. The anti-IL-1 beta 165-186 Ab specifically neutralizes the biological activity of hrIL-1 beta and native IL-1, as measured by the IL-1-induced proliferation of murine thymocytes and human fibroblasts and the IL-1-dependent IL-2 production by murine T cells (EL4-6.1). Fifty per cent of hrIL-1 beta activity (25 U/ml, or 0.25 ng/ml) has neutralized by less than 30 micrograms/ml of MoAb. Furthermore, FIB 1 recognizes intracellular IL-1 in lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The anti-IL-1 beta 165-186 Ab does not react with the shorter IL-1 beta fragment 161-173 in solid-phase ELISA, therefore the binding region seems to be localized in the amino acid sequence VALGLKEKNLYLS. A sandwich-ELISA, using a polyclonal sheep anti-IL-1 beta 251-269 Ab as the capture antibody and an anti-IL-1 beta 165-186 MoAb as the detecting probe, allowed the determination of IL-1 beta from crude culture supernatants.
Recent experiments have shown that gamma interferon (IFN-␥), either administered or induced in vivo, e.g., by certain bacteria, is a key mediator in inducing hypersensitivity to bacterial lipopolysaccharides. The source of endogenous IFN-␥ in this context (natural killer versus T H 1 cells) has not been investigated yet. In order to investigate the role of antigen-specific, IFN-␥-producing T H 1 cells in murine Pseudomonas aeruginosa infection, a murine T H 1 cell line was propagated in vitro by using recombinant P. aeruginosa outer membrane protein I. Adoptive transfer experiments were performed by intravenous injection of various amounts of T H 1 cells into P. aeruginosa-challenged SCID mice. Adoptive transfer of 5 ؋ 10 6 T cells into SCID mice followed by an intraperitoneal challenge with 1.4 ؋ 10 6 CFU of live P. aeruginosa resulted in the rapid death of the animals within 12 h postchallenge, whereas transfer of lower T-cell doses and saline as a control did not cause any detrimental effects. After challenge with 2.8 ؋ 10 6 CFU of P. aeruginosa, similar results were obtained 18 h postchallenge; however, at the end of the 72-h observation period, no significant differences in survival rates were obtained between the groups treated with different amounts of T cells. The rapid death of mice treated with 5 ؋ 10 6 T cells was reflected by 860-fold-elevated levels of tumor necrosis factor alpha (TNF-␣) present in serum 2 h postchallenge, whereas no significant differences in TNF-␣ serum levels were detectable in mice treated with lower doses of T cells or with saline. Pretreatment of T-cell-reconstituted SCID mice with neutralizing anti-IFN-␥ monoclonal antibodies completely protected mice from bacterial challenge and reduced TNF-␣ levels in serum. We conclude that under the experimental conditions described here, IFN-␥-and interleukin-2-producing T H 1 cells represent an important trigger mechanism inducing TNF-␣-mediated hypersensitivity to bacterial endotoxin.
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