142 D. W. H. Barnes and J. F. Loutit 



came later from San Francisco. Cole and co-workers (1952) 

 showed that homogenization, with its attendant severe 

 damage to cells, did not destroy the therapeutic effect. 

 Furthermore, fractionation of these homogenates in sucrose 

 by ultracentrifugation (Cole et a/., 1953) localized the effective 

 principle in the layer of greatest density composed of cell 

 nuclei (and a few whole cells). The activity of this nuclear 

 fraction could be destroyed by preparations made from 

 crystalline desoxyribonuclease and trypsin (Cole and Ellis, 

 1954) which were said to be without action on intact cells 

 (Cole and Ellis, 1955). 



Against the humoral theory we have argued on the follow- 

 ing grounds. 



(1) On repeating the original observations of Jacobson 

 and co-workers, we were impressed that, in those mice 

 which survived the critical month but died subse- 

 quently, some of the spleens implanted had "taken" 

 and had become accessory spleens. 



(2) We confirmed that an intact organ was not an essential, 

 that suspensions were effective on injection, and that 

 the intravenous route gave results superior to the 

 intraperitoneal (Barnes and Loutit, 1953). (We were 

 not at that time able to obtain improved survival from 

 heterologous material from guinea pigs or rabbits.) 

 This superiority of the intravenous route would not be 

 expected if a soluble hormone were involved but would 

 be in the case of cells. 



(3) When we adopted intravenous injections as a routine 

 we noted that there was an approximate threshold of 

 material above which no further improvement was 

 obtained. Jacobson and co-workers (1955) have made 

 similar observations. Depending on the material used 

 this threshold may be 10^-10^ cells. 



(4) The active principle is extremely thermolabile : it is 

 inactivated in a few minutes at 50°C (Cole, Fishier and 

 Ellis, 1955) and in a few hours at room temperature, at 

 4° C and at - 15° C (Barnes and Loutit, 1954). This is 



