In the preceding paper of this series we have described an efficient and versatile apparatus for freeze-drying by vacuum sublimation. This apparatus makes it possible to dehydrate biological materials at low temperatures and, by the use of ionization gauges, to determine the end-point of dehydration of material maintained at temperatures ranging down to --80°C. It is so designed that different samples of the same material can be subjected simultaneously to various experimental conditions. Time, rate, and degree of drying have been shown to have a marked influence on the physical properties of lyophilized biological materials other than viruses by Elser, Thomas, and Steffen (1); Greaves and Adair (2); Flosdorf, Hull, and Mudd (3); Greaves (4) and others. Proom and Hemmons (5) have published working details for the freeze-drying preservation of a collection of more than 1500 strains of bacteria. Various methods of freezing, the degree of drying, and the effect of storage were tested by viability counts. Recently, Hutton, Hilmoe, and Roberts (6) have reported on the effects of these factors on the quantitative survival of Brucella abortus. Similar studies dealing with the survival of viruses have not been reported.The present investigations deal with the quantitative survival of influenz a virus suspensions, after repeated freeze-thaw cycles, freezing at various speeds, storage in the frozen state at various temperatures, and vacuum sublimation at various temperatures following different types of preliminary treatment. Subsequent investigations are planned which will be concerned with the effects of these factors on the survival of complex cells.

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The study objectives were to determine; (1) whether activated T cells could be generated from peripheral blood of patients immunized with their own cancer cells, (2) whether adoptive transfer of the activated T cells to patients had toxic effects and (3) whether the infused cells produced clinical responses. Study patients had recurrent, surgically accessible grade III/IV astrocytomas. The patients were tapered off steroids after total surgical resection and immunized with autologous cancer cells plus Bacillus, Calmette and Guerin (BCG). Peripheral blood mononuclear cells were activated with anti-CD3, expanded with interleukin-2 (IL-2) and reinfused to patients. The number of activated T cells that was given back to patients varied between 10(10) and 10(11). Side effects that were observed following immunization and adoptive cell transfer included mainly transient flu-like symptoms. One patient's tumor partially regressed, but there was no effect on survival. Two other patients' tumors regressed, and the patients are apparently disease-free more than 5 and 4 years later. The other six patients' tumors were apparently unaffected by the treatment. Patient age, tumor grade and CD4/CD8 composition of infused cells were positively correlated with clinical responses. Cellular immunotherapy is feasible and is associated with minimal toxicity. Additional appropriately controlled studies will be required to determine whether cellular immunotherapy could be used as a treatment for central nervous system malignancy. Additional studies also will be required to determine the underlying immunological mechanisms.