A cell line infectible by prion strains from different species.

It has been shown previously that ovine prion protein (PrP(C)) renders rabbit epithelial RK13 cells permissive to the multiplication of ovine prions, thus providing evidence that species barriers can be crossed in cultured cells through the expression of a relevant PrP(C). The present study significantly extended this observation by showing that mouse and bank vole prions can be propagated in RK13 cells that express the corresponding PrP(C). Importantly, the respective molecular patterns of abnormal PrP (PrP(res)) and, where examined, the neuropathological features of the infecting strains appeared to be maintained during the propagation in cell culture. These findings indicate that RK13 cells can be genetically engineered to replicate prion strains faithfully from different species. Such an approach may facilitate investigations of the molecular basis of strain identity and prion diversity.

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp.

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.

Lipopolysaccharide complexed with soluble CD14 binds to normal human monocytes.

Using flow cytometry we have compared the binding of Neisseria meningitidis lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) to normal human monocytes in whole blood with the binding to chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO cells). Binding of FITC-LPS to cells was dose dependent, saturable and enhanced in the presence of increasing concentrations of serum. Blockade of membrane CD14 with saturating concentrations of anti-CD14 monoclonal antibody (mAb) My4 inhibited 50% of the binding of FITC-LPS to monocytes and 100% to hCD14-CHO cells. Similarly, removal of membrane CD14 by phosphatidylinositol phospholipase C (PI-PLC) treatment of the cells partially decreased the binding of FITC-LPS to monocytes but totally inhibited the binding to hCD14-CHO-transfected cells. These results suggest that binding of FITC-LPS to monocytes is not only mediated by membrane CD14. Using two-color flow cytometry, we observed that FITC-LPS binds to My4-saturated monocytes in association with soluble (s)CD14 present in serum as revealed by staining with rhodamine-labeled My4 mAb. The binding of FITC-LPS/sCD14 complexes to monocytes treated with saturating amounts of unlabeled My4 prior to addition of the complexes was completely inhibited by anti-CD14 mAb 10G33. When cells were first saturated with a mixture of My4 and 10G33 mAb, washed and further incubated with FITC-LPS/sCD14, inhibition of the binding of LPS was similar to that observed with cells saturated with My4 alone, showing that the binding of FITC-LPS is not mediated by the 10G33 epitope present on mCD14. These results suggest that either the 10G33 epitope on sCD14 is involved in the binding of LPS/sCD14 complexes to the cells, or that 10G33 mAb inhibits the binding of FITC-LPS to sCD14. Taken together, these data indicate that sCD14 which is present in normal serum, in addition to membrane CD14, enables LPS to bind monocytes through an as yet unidentified molecule and that sCD14 does not simply serve as a shuttle for transfer of LPS to membrane CD14.

Specific binding of lipopolysaccharides to mouse macrophages--II. Involvement of distinct lipid a substructures.

The interaction of lipopolysaccharide-binding sites of mouse macrophages with the Lipid A region of endotoxins (LPS) was demonstrated by direct binding of labeled Lipid A conjugates, by inhibition of the binding of labeled LPS with anti-Lipid A monoclonal antibodies, and by the considerable reduction of this binding after chemical and enzymatic removal of the fatty acid esters of the LPS. The substructures of Lipid A required for the specific binding of LPS to macrophages were analyzed by the use of synthetic lipids consisting of mono- or disaccharide derivatives of glucosamine. The two phosphate groups of Lipid A (at positions 1 and 4') as well as certain hydroxyl groups, appeared to play a critical role in the binding. However, the reactivities of the synthetic lipids with the macrophage surface, as compared with those with anti-Lipid A antibodies, could hardly be explained by the existence of a single LPS receptor, and suggested the presence, on the macrophage surface, of different LPS-binding sites that recognize different substructures or spatial configurations of the lipid moiety of endotoxins.

Isolation of two protein-free and chemically different lipopolysaccharides from Bordetella pertussis phenol-extracted endotoxin.

Endotoxin prepared from several Bordetella pertussis strains in both immunological phases I and IV gave two lipopolysaccharide peaks (LPS-I and LPS-II) when analyzed on hydroxylapatite columns in a phosphate buffer containing 0.1% sodium dodecyl sulfate; these lipopolysaccharides, present in the ratio of 2:3, are true endotoxins by both chemical and biological criteria. Endotoxins isolated from Escherichia coli, Salmonella typhimurium, and Shigella flexneri gave single lipopolysaccharide peaks when analyzed by the same procedure. Upon hydrolysis with acetic acid (pH 3.4) at 100 degrees C for 1 h, LPS-I released a polysaccharide (PS-I); the linkage broken was that of the glycosidic bond of a non-phosphorylated 3-deoxy-oct-2-ulosonic acid. Treatment with 0.25 M mineral acid at 100 degrees C for 30 min was required to free the polysaccharide moiety (PS-II) of LPS-II, the linkage broken being the glycosidic bond of a phosphorylated 3-deoxy-oct-2-ulosonic acid. Chemical and physical differences of the polysaccharide moieties PS-I and PS-II present in LPS-I and LPS-II have been described previously (25). By using the technique of 125I labeling, it was shown that the totality of labeled proteins present in the endotoxin extracted from Bordetella pertussis by the phenol-water procedure could be separated from the lipopolysaccharide by column chromatography on hydroxylapatite; it follows that these proteins are not linked by covalent bonds to the lipopolysaccharide.

Biological activities of fragments derived from Bordetella pertussis endotoxin: isolation of a nontoxic, Shwartzman-negative lipid A possessing high adjuvant properties.

Endotoxin from fresly sedimented Bordetella pertussis cells, isolated by the phenol/water procedure when submitted to kinetically controlled, mild acidic hydrolysis released a polysaccharide (polysaccharide 1), a complex lipid (lipid X), and a glycolipid. When treated with somewhat stronger acid, the glycolipid yielded a second polysaccharide (polysaccharide 2) and another complex lipid (lipid A). The intact pertussis endotoxin had all the usual properties of endotoxins extracted from enteric bacteria. Lipid X and the intermediary glycolipid retained all the endotoxic properties of the unfractionated endotoxin. In lipid A, pyrogenicity was reduced to a very low level and toxicity and Shwartzman reactivity were absent; however, this fraction retained most of the endotoxin's antiviral activity, and its adjuvant power was considerably higher than that of the intact endotoxin. Lipid A elicited nonspecific resistance against challenge with certain bacteria, but not against others.

A novel type of endotoxin structure present in Bordetella pertussis. Isolation of two different polysaccharides bound to lipid A.

The endotoxin of Bordetella pertussis was cleaved by mild acidic hydrolysis to yield a polysaccharide (polysaccharide I, 15%), a glycolipid (63%) and lipid X (2%). Further treatment of the glycolipid with stronger acid released a second polysaccharide (polysaccharide II, 9%) and material similar to lipid A present in enterobacterial endotoxins. Both polysaccharides possess a single molecule of 3-deoxy-2-octulosonic acid as the reducing, terminal sugar. In polysaccharide II the octulosonic acid is phosphorylated in position 5 and presumably substituted in position 4; in polysaccharide I the octulosonic acid is not phosphorylated, but is substituted in position 5. Following treatment of the endotoxin with strong base, a fragment was isolated that contained bound, non-phosphorylated 3-deoxy-2-octulosonic acid, glucosamine phosphate and fatty acids. This indicated that polysaccharide I, like polysaccharide II, was bound to the lipid region of the endotoxin. The endotoxin structure thus defined is different from that proposed for the lipopolysaccharides of enterobacteria.