Andrew B. Maksymowych

Binding and Transcytosis of Botulinum Neurotoxin by Polarized Human Colon Carcinoma Cells

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 toxicin 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 μm brefeldin A, decreased 42% when microtubules were disrupted by 10 μm nocodazole, and decreased 74% at 18 °C. 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.

Cloning and sequence analysis of a cDNA encoding human syntaxin 1A, a polypeptide essential for exocytosis

A clone encoding human syntaxin 1A was isolated and sequenced from a human brain library. The deduced sequence encodes a 288-amino-acid (aa) protein that presumably plays a critical role in neurotransmitter exocytosis and is 98.0% identical to the aa sequence of rat syntaxin 1A.

589. Repeated Recombinant Adenovirus (rAd) Administration To Generate Mucosal Antibodies (Abs) vs Botulinum Toxin A (BoNT/A)

Many infectious agents enter the body via the mucosae. Thus, mucosal immunity is an important early defense and a logical vaccine target. Mucosal Abs (mainly secretory IgA, sIgA) are typically made after mucosal immunization. Adjuvants are often required to elicit a strong response. BoNT/A, one of the most potent poisons known, is a potential biological weapon. Since it can poison by inhalation, it could be aerosolized, easily disseminated and hard to inactivate.