1887

Abstract

Summary

An assay has been developed for heat-labile toxin (HLT) based on morphological alterations in certain human embryonic lung (HEL) cell lines. Eighteen cell lines from human and other sources were tested but only two, MRC-5 and HELu2, were responsive to HLT. Confluent monolayers of the cells contracted within 24 h of exposure to the toxin, but without loss of viability during incubation for a further 3 days. The effect of HLT was quantitated by scoring the extent of morphological change, and by the decrease in Giemsa staining of the cell monolayers, as measured on an ELISA plate reader. This cell culture assay for HLT was more sensitive than lethality titration in mice but the dose-response curve had a lower slope. The specificity of the response was established by comparing unheated HLT with HLT heated at 56°C, and with extracts from transposon-insertion mutants of which were deficient in HLT. Purified preparations of pertussis toxin and lipopolysaccharide gave no morphological response even at high doses.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-34-1-45
1991-01-01
2023-02-08
Loading full text...

Full text loading...

/deliver/fulltext/jmm/34/1/medmicro-34-1-45.html?itemId=/content/journal/jmm/10.1099/00222615-34-1-45&mimeType=html&fmt=ahah

References

  1. Wardlaw A. C., Parton R. (eds) Pathogenesis and immunity in pertussis. Chichester, Wiley: 1988482
    [Google Scholar]
  2. Bordet J., Gengou O. L’endotoxine coquelucheuse. Ann Inst Pasteur 1909; 23:415–419
    [Google Scholar]
  3. Wardlaw A. C., Parton R. Bordetella pertussis toxins. Pharmacol Ther 1983; 19:1–53
    [Google Scholar]
  4. Střížová V, Trlifajova J. The neutralization of B. pertussis toxin in a tissue culture. J Hyg Epidemol Microbiol Immunol 1964; 8:428–432
    [Google Scholar]
  5. Kume K., Nakai T., Samejima Y., Sugimoto C. Properties of dermonecrotic toxin prepared from sonic extracts of Bordetella bronchiseptica. Infect Immun 1986; 52:370–377
    [Google Scholar]
  6. Endoh M., Nagai M., Ueda T, Yoshida Y., Nakase Y. Cytopathic effect of heat-labile toxin of Bordetella parapertussis on aortic smooth muscle cells from pigs or guinea pigs. Microbiol Immunol 1988; 32:423–428
    [Google Scholar]
  7. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun 1983; 42:33–41
    [Google Scholar]
  8. Arico B., Miller J. F., Roy C. Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins. Proc Natl Acad Sci USA 1989; 86:6671–6675
    [Google Scholar]
  9. Stainer D. W., Scholte M. J. A simple chemically defined medium for the production of phase I Bordetella pertussis. J Gen Microbiol 1970; 63:211–220
    [Google Scholar]
  10. Livey I., Wardlaw A. C. Production and properties of Bordetella pertussis heat-labile toxin. J Med Microbiol 1984; 17:91–103
    [Google Scholar]
  11. Bradford M. M. A rapid and sensitive method for the quantita tion of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248–254
    [Google Scholar]
  12. Imaizumi A., Suzuki Y., Ono S., Sato H., Sato Y. Heptakis (2, 6-0-dimethyl) β-cyclodextrin: a novel growth stimulant for Bordetella pertussis phase I. J Clin Microbiol 1983; 17:781–786
    [Google Scholar]
  13. Christodoulides M., Sidey F. M., Parton R. Stewart-Tull DES. An acellular pertussis vaccine prepared by a simple extraction and toxoiding procedure. Vaccine 1987; 5:199–207
    [Google Scholar]
  14. Jacobs J. P., Jones C. M., Baillie J. P. Characteristics of a human diploid cell designated MRC-S. Nature; 1970; 227:168–170
    [Google Scholar]
  15. Greaves M. D., Potter C. W., McEntegart M. G. A comparison of the sensitivity of cell cultures to diphtheria toxin by dye-uptake methods. J Med Microbiol 1971; 4:519–527
    [Google Scholar]
  16. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55–63
    [Google Scholar]
  17. Takano S., Kondo H. Quantitative method for determination of hemolytic activity of Clostridium septicum toxin. Jpn J Med Sci Bio 1987; 40:47–59
    [Google Scholar]
  18. Fairweather D. S., Fox M., Margison G. P. The in-vitro lifespan of MRC-5 cells is shortened by S-azacytidine-induced de-methylation. Exp Cell Res 1987; 168:153–159
    [Google Scholar]
  19. Felton H. M., Gaggero A., Pomerat C. M. Reactions of cells in tissue culture to Hemophilus pertussis. Tex Rep Biol Med 1954; 12:960–971
    [Google Scholar]
  20. Angela G. C., Rosso C., Guiliani G. Effetto della tossina pertossica sulle cellule coltivate in vitro. Minerva Med Roma 1962; 53:778–784
    [Google Scholar]
  21. Rutter J. M., Luther P. D. Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of pigs. Vet Rec 1984; 114:393–396
    [Google Scholar]
  22. Nakase Y, Endoh M. Heat-labile toxin of Bordetella pertussis. In Wardlaw A. C, Parton R. (eds) Pathogenesis and immunity in pertussis Chichester, Wiley: 1988211–229
    [Google Scholar]
  23. Freshney R. I. Introduction: principles of sterile technique and cell propagation. In Freshney R. I. (ed) Animal cell culture: a practical approach Oxford: IRL Press; 19861–11
    [Google Scholar]
  24. Zhang Y. L, Sekura R. D. Isolation and characterization of heat labile toxin (HLT) from Bordetella pertussis. Federation Proc 1985; 44:674
    [Google Scholar]
  25. Endoh M, Amitani M., Nakase Y. Purification and characteri zation of heat-labile toxin from Bordetella bronchiseptica. Microbiol Immunol 1986; 30:659–673
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-34-1-45
Loading
/content/journal/jmm/10.1099/00222615-34-1-45
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error