The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amoebocyte lysate (CLAL) assay. When bacterial cells exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring 0-hydroxymyristic acid (3-OH tetradecanoic acid, D-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli OlKl strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their I-OHC14:0 contents. Four other E. coli strains (0 serotypes 014, 024, and 075) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli OlKl and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the jI-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the 05 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 lM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 lM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 lM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process.

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c+, CD8α−, CD11b−, B220−, and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4+ T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.

Free lipid A of Helicobater pylori was characterized with regard to chemical composition, reactivity with anti-lipid A antibodies, and activity in a Limulus lysate assay. The predominant fatty acids of H. pylori lipid A were 3-OH-18:0, 18:0, 3-OH-16:0, 16:0, and 14:0. Hexosamine was present in amounts similar to those in Campylobacter jejuni or SalmoneUla typhimunium lipid A. The lipopolysaccharide of H. pylori contained 2-keto-3-deoxyoctonic acid, a common constituent of enterobacterial and C. jejuni lipopolysaccharides. In the enzyme-linked immunosorbent assay, the doses of lipid A required to inhibit anti-lipid A by 50%o (EI5 values) by absorption of the immune (rabbit) serum were 7.9, 1.2, and 1.4 ,ug of 0-deacylated lipid A's from H. pylori, C.jejuni, and S. typhimunium per ml, respectively. The lower reactivity ofH. pylori lipid A compared with those of the other two lipid A preparations (as shown by the higher EIso value) was underscored by the use of a murine monoclonal anti-lipid A antibody in the inhibition assay. An EIso value was not obtained at the concentrations tested for H. pylori lipid A; the corresponding figures for C. jejuni and S. typhimunium lipid A's were 13 and 14 ,ug/ml, respectively. No inhibition was obtained with H. pylori lipopolysaccharide, which showed a low-molecular-weight profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activity of H. pylori lipid A in the Limulus assay was approximately 71 and 650 times lower than those of C. jejuni and S. typhimurium lipid A's, respectively. These findings suggest that lipid A is an integral part of the outer cell wall of H. pylori. The lower reactivity of H. pylori lipid A with anti-lipid A antibodies and in the Limulus assay compared with that of C. jejuni or S. typhimunium lipid A may be explained by a different composition of the fatty acids, especially the 3-hydroxy fatty acids, and a possible deviating phosphorylation pattern.Preparation of LPS and lipid A. For H. pylon and C. jejuni, the dried bacteria were first extracted with a chloroformmethanol mixture to remove the phospholipids. LPS was extracted by the phenol-water method of Westphal and Jann 4383 on August 1, 2020 by guest http://iai.asm.org/ Downloaded from