22 
(2) The cloning vehicle is to be an EKl vector preferrably belonging 
to the class of poorly mobilizable plasmids such as pBR322, pBR328, 
and pBR325. 
Dr. Landy asked if the working group recommendation specified that both the 
host-vector system and strain 933 coli 0157H7 were to be grown under 
optimal conditions. Dr. Gottesman replied that both strains should be 
grown under optimal toxin producing conditions. 
Dr. Gottesman said the working group recommended approval of the third 
item of the April 4 request with the clarification that the modified 
organism will not contain overlapping fragments which together would 
encompass the structural gene(s). This specification will eliminate the 
possibility that the structural gene might be regenerated through 
r ecombinat ional events. 
In regard to the fourth item in the" April 4, 1984, proposal. Dr. Gottesman 
said it was the consensus of the working group that these experiments 
would not fall under Appendix F of the NIH Guidelines, and no action need 
be taken by the RAC. 
In regard to the fifth item in the April 4, 1984, letter from Drs. O’Brien 
and Holmes, Dr. Gottesman said it was the consensus of the group that no 
working group could predict all potential scenarios; thus, each specific 
nontoxinogenic allele should be considered individually on a case-by-case 
basis. A system is in place within the NIH to perform this type of evalua- 
tion, so no specific action need be taken by the RAC. 
Dr. King Holmes said the preposed research is extremely important and 
should be pursued. He had, however, several concerns which he felt should 
be addressed: (1) He noted that only four individual animals of one primate 
species had been tested by Branham, Dack, and Riggs. He asked whether 
primate species might differ in their response to the toxin. (2) He also 
questioned the calculations developed by the working group in a worst case 
scenario; he wondered whether this scenario would correspond to the in 
vivo situation. (3) He noted that data presented at an earlier RAC meeting 
by Dr. O'Brien suggested a toxin dose-effect; i.e., E. coli isolates frcm 
patients who have hemorrhagic colitis produced more toxin in vitro than 
did E. coli isolates frem patients who did not have hemorrhagic colitis. 
(4) He questioned what would be the effect of feeding "non-healthy” individ- 
uals Eh_ coli K-12 producing Shiga toxin. 
Dr. Holmes felt the apparent lack of toxicity for intestinal epithelial 
cells is not entirely reassuring in terms of toxicities for other epithelial 
cell types such as HeLa cells. He pointed out that the toxin is presumed 
to be toxic for endothelial vascular cells. He asked what would be the 
effect on humans if toxin producing E . coli is inhaled? What if toxin 
producing Eh_ coli colonizes the skin or urogenital tract? 
[ 475 ] 
