D. W. H. BARNES AND J. F. LOUTIT 



of mice surviving 30 days was 500 days. The data are summarized in 

 Figure 1 which shows in addition the times to death of unirradiated normal 

 mice of the same colony. 



Furthermore, whilst spleen of mouse strain A has some temporary recuper- 

 ative effect, even this temporary benefit was lost if the CBA recipient had 

 been previously immunized with strain A spleen. All these. results are more 

 easily explicable in terms of the active principle acting as a tissue graft 

 than being a chemical growth hormone. 



The active agent whatever its nature is extremely labile. In our hands 

 it becomes inactivated by a few hours' storage at room temperature. It 

 cannot be kept chilled in the refrigerator at 4°C or frozen at — 15° C for 

 48 hours-. One might have anticipated that the frozen state at least would 

 have preserved a hypothetical hormone but not an intact cell or tissue. 

 On the other hand Parkes and his collaborators^ have demonstrated that 

 many tissues can be preserved at — 79°C (alcohol — dry ice) after imbibing 

 glycerol to prevent formation of ice crystals. We have adopted the Smith- 

 Parkes technique and preserved finely chopped CBA spleen for 48 hours. 

 On homogenizing this after quick thawing and injecting the homogenate 

 intravenously we have just obtained the following results [Table I). 



While the initial results suggest some loss on storage, they are at least 

 promising in that some activity has been preserved. 



Nevertheless the unequivocal answer to the main problem — is the spleen 

 factor a living cell or a chemical agent ? — will come from one of two experi- 

 ments, the demonstration either of positive survival after treatment with a 

 proven cell-free material, or of the persistence in the surviving host of the 

 donated cells or their descendants. 



Our first essay at the latter experiment was by genetic tagging of the 

 donated spleen. In this case the donor spleen had of necessity to be hetero- 

 logous. It proved to be one of the less effective heterologous materials. 

 The median survival time of the 10 recipients was increased only from 8 days 

 to 23 days and no tagged descendants of these cells were identified. 



REFERENCES 



1 Barnes, D. W. H. and Loutit, J. F. Proc. R. Soc. Med. 1953, 46 25 1 . 



2 Barnes. D. W. H. and Loutit. J. F. Nucleonics. 1954. 12 No. 5, 68. 



3 Smith, A. U. Proc. R. Soc. Med. 1954, 47 57. 



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