Cloning, characterization, and expression of a cDNA encoding an inducible nitric oxide synthase from the human chondrocyte.

Incubation of human articular chondrocytes with interleukin 1 beta results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.

Biologic and protective properties of the 69-kDa outer membrane protein of Bordetella pertussis: a novel formulation for an acellular pertussis vaccine.

A combination of the 69-kDa outer membrane protein and filamentous hemagglutinin (FHA), both isolated from lymphocytosis promoting factor (LPF; pertussis toxin) minus mutants of Bordetella pertussis, is protective in the mouse intracerebral challenge potency (Kendrick) test. A combination of the same 69-kDa protein and LPF is approximately 15 times less effective. These data suggest that, surprisingly, the 69-kDa protein in tandem with FHA is the most relevant combination for mouse protection; consequently such a combination may be a more suitable acellular pertussis vaccine candidate than the LPF/FHA combinations, which have never been satisfactorily protective in the mouse test. Preparation of samples of the 69-kDa protein of acceptable protective quality remains difficult. Attempts were made to screen the most suitable batches of the preparations by exploiting some recently discovered properties of the 69-kDa protein: the characteristic chromatofocusing pattern of the protein, the affinity for lymphocytes, and the ability to bind to nicotinamide adenine dinucleotide. None of these tests was able to replace the mouse intracerebral challenge potency test for final quality assessment.

A novel bivalent acellular pertussis vaccine based on the 69 kDa protein and FHA.

A combination of the 69 kDa outer membrane protein and filamentous hemagglutinin (FHA), both isolated from pertussis toxin (ptx) minus mutants of B. pertussis, is protective in the mouse intracerebral challenge potency test ("Kendrick test"). A combination of the same 69 kDa protein and lymphocytosis promoting factor (LPF, pertussis toxin, PTX) is approximately 15 times less effective. The data show that the 69 kDa protein in tandem with FHA is the most relevant combination for mouse protection, and may be a more suitable acellular pertussis vaccine candidate than the LPF/FHA combination which has never been satisfactorily protective in the mouse test. However, the preparation of the 69 kDa protein of acceptable quality is still difficult. Attempts were made at screening the most suitable batches of the preparation by exploiting some recently discovered properties of the 69 kDa protein, i.e. its chromatofocusing pattern and lymphocyte affinity. Although both these properties may differentiate 69 kDa preparations, none of them was able to replace the mouse intracerebral challenge potency test for the final quality assessment.

Evaluation of Bordetella bronchiseptica vaccines in specific-pathogen-free piglets with bacterial cell surface antigens in enzyme-linked immunosorbent assay.

The progenies of specific-pathogen-free sows which had been immunized with Bordetella bronchiseptica vaccines of various origin before parturition were challenged intranasally with B. bronchiseptica within 5 days of birth. Sera of piglets were taken weekly and investigated by enzyme-linked immunosorbent assay against a mixture of B. bronchiseptica cell surface antigens containing curled fibers and fimbriae, lipopolysaccharide, and a mixture of proteins mostly derived from the outer membrane. The serological response to this antigenic mixture was paradoxical; the highest titers were obtained with the least effective vaccines. Antibodies which did relate to protection were oriented against the outer-membrane-derived proteins, one of which, of 68,000 molecular weight, appeared to be particularly important for two reasons. First, its concentration within the antigenic mixture was dependent upon cultural conditions; of all the proteins present in virulent strains, it was the first to disappear upon modulation. Second, it was absent from a strain which was unable to induce atrophic rhinitis in specific-pathogen-free piglets. Although all vaccines tested had some beneficial effect on the various clinical manifestations of the disease, only two vaccines were effective (P less than 0.001) in the prevention of nasal pathological changes. These two vaccines also stimulated the highest titers against the 68,000-molecular-weight protein. A mouse protection test utilizing a lethal intraperitoneal challenge failed to monitor the efficacy of vaccines for protection against atrophic rhinitis.

Adenylate cyclase activity of a 68,000-molecular-weight protein isolated from the outer membrane of Bordetella bronchiseptica.

A method was developed which is suitable for the isolation of substantial quantities of outer membrane proteins of Bordetella species in a water-soluble form. The extracted material may then be further fractionated in the absence of detergents by ion-exchange chromatography and preparative flat-bed isoelectrofocusing. These procedures facilitated the isolation of one of the proteins, of molecular weight 68,000, for which the antibody titer correlated with the degree of protection against nasal changes induced in specific-pathogen-free piglets by Bordetella bronchiseptica infection (P. Novotny, M. Kobisch, K. Cownley, A. P. Chubb, and J. A. Montaraz, Infect. Immun. 50:190-198). This protein, which banded between 7.0 and 7.6 pH in preparative isoelectrofocusing, may be further purified with a monoclonal immunosorbent. Immunopurified protein showed adenylate cyclase activity. The enzymatic activity was found to be unstable during processing; i.e., although the crude extract showed up to 150 nmol of cyclic AMP per mg/min, the immunopurified protein showed a maximum of only 200 nmol of cyclic AMP per mg/min. Two strains of B. bronchiseptica, isolated from herds of healthy pigs showing no signs of atrophic rhinitis, did not produce the 68,000-molecular-weight protein and were negative for adenylate cyclase. However, it is not known whether the 68,000-molecular-weight protein is a component of adenylate cyclase or whether it is an unrelated protein associated with this enzyme in some unknown way. Adenylate cyclase activity from culture supernatants of B. bronchiseptica, B. pertussis, and B. parapertussis can be absorbed equally to the same monoclonal immunosorbent.

Bordetella adenylate cyclase: a genus specific protective antigen and virulence factor.

Most of the adenylate cyclase (AC) present in Bordetella species is localized in the outer membrane, partly exposed to the cell surface. An isolation procedure to obtain the cell-bound enzyme was applied to Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis. Passive transfer of B. bronchiseptica anti-AC antibody, either in the form of monoclonal antibody or antibody transferred to offspring from female mice previously immunized with immunopurified B. bronchiseptica AC, prevented both death and the development of "atrophic rhinitis" in mice infected by an aerosol of virulent B. bronchiseptica. Immunopurified AC from both B. bronchiseptica and B. pertussis protected mice against intracerebral challenge with strain 18323, although five to six times more protein of B. bronchiseptica was necessary to produce the same level of protection as with B. pertussis-derived protein. The AC of Bordetella species differ in molecular weight and, despite sharing at least two common non-overlapping epitopes (BB05, BB07), antigenic differences are likely: sera from piglets artificially infected with virulent B. bronchiseptica developed high ELISA titers against AC from B. bronchiseptica but low titers against B. pertussis-derived antigen. Strains of B. bronchiseptica which did not induce "atrophic rhinitis" in pigs were found to be AC-less variants. When tested in our laboratory model, such a strain was also unable to induce "atrophic rhinitis" in mice. We conclude that AC is a mandatory component of any future subcellular vaccine against Bordetella species.