Abstract
T-84 and Caco-2 human colon carcinoma cells and Madin-Darby canine kidney (MDCK) cells were used to study binding and transcytosis of iodinated Clostridium botulinum neurotoxin serotypes A, B, and C, as well as tetanus toxin. Specific binding and transcytosis were demonstrated for serotypes A and B in intestinal cells. Using serotype A as an example, the rate of transcytosis by T-84 cells was determined in both apical to basolateral (11.34 fmol/h/cm2) as well as basolateral to apical (8.98 fmol/h/cm2) directions, and by Caco-2 cells in the apical to basolateral (8.42 fmol/h/cm2) direction. Serotype A retained intact di-chain structure during transit through T-84 or Caco-2 cells, and when released on the basolateral side was toxic in vivo to mice and in vitro on mouse phrenic nerve-hemidiaphragm preparations. Serotype C and tetanus toxin did not bind effectively to T-84 cells, nor were they efficiently transcytosed (8-10% of serotype A). MDCK cells did not bind or efficiently transcytose (0.32 fmol/h/cm2) botulinum toxin. Further characterization demonstrated that the rate of transcytosis for serotype A in T-84 cells was increased 66% when vesicle sorting was disrupted by 5 microM brefeldin A, decreased 42% when microtubules were disrupted by 10 microM nocodazole, and decreased 74% at 18 degreesC. Drugs that antagonize toxin action at the nerve terminal, such as bafilomycin A1 (which prevents acidification of endosomes) and methylamine HCl (which neutralizes acidification of endosomes), produced only a modest inhibitory effect on the rate of transcytosis (17-22%). These results may provide an explanation for the mechanism by which botulinum toxin escapes the human gastrointestinal tract, and they may also explain why specific serotypes cause human disease and others do not.
Highlights
Botulinum neurotoxin (BoNT),1 which is the etiologic agent responsible for the disease botulism, can enter the body by several different routes, but the most common of these is the gastrointestinal system [1,2,3,4]
The average values obtained in three experiments indicated that the rate of [3H]inulin flux from the apical to the serosal side of T-84 cells typically fell within the range of 0.01- 0.07 nmol/h/cm2
Identical experiments were done on cultures that had been pretreated with 1 ϫ 10Ϫ8 M BoNT type A (BoNT/A) for 18 h
Summary
BoNT/A, botulinum neurotoxin serotype A; Caco-2, human colon adenocarcinoma; MDCK, Madin-Darby canine kidney; T-84, human colon carcinoma. In contrast to our knowledge of events at the nerve ending, very little is known about the cellular and subcellular events that account for the ability of the toxin to cross membranes in the gastrointestinal system (14 –16). Work has been done to test the hypothesis that BoNT crosses the gut wall by the process of specific binding and transcytosis According to this hypothesis, there should be little or no pH-induced translocation of toxin molecules across intracellular membranes. This hypothesis requires that toxin bound on the lumenal side of cells must be delivered to the serosal side of cells, and the transcytosed toxin must be in a conformation that can act upon cholinergic nerves This hypothesis has been tested by studying the binding, movement, and residual toxicity of BoNT type A (BoNT/A) added to a human epithelial cell line of gut origin (T-84). The resulting data provide the first insights into the cellular and subcellular events that account for toxin movement from the gastrointestinal system to the general circulation
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call