This section is from the book "Research In Physiopathology As Basis Of Guided Chemotherapy With Special Application To Cancer", by Emanuel Revici. Also available from amazon: Research In Physiopathology
Three groups of five rabbits each, of the same sex and weight, were injected intravenously on two consecutive days with the same amount of a suspension of killed Eb. Thyphi. One group was kept as control, receiving daily injections of 1 cc. of cottonseed oil. Of the other groups, one was injected subcutaneously daily with 1 cc. of a 5% solution in cottonseed oil of the acid lipids mixture obtained from human placenta. The third group received daily 1 cc. of a 5% solution in cottonseed oil of the insaponifiable fraction of the same origin. Every second day, venous blood was obtained and the agglutinating power of the serum determined. Figure 265A shows the average values for each group.
The injection of microbes killed by heat and treated in vitro with various lipids and lipoids had an interesting effect on the appearance of antibodies. Eb. typhi, cultivated on agar and suspended in saline so as to give nephelometrical values corresponding to 30 mil. per cc., were killed by heating for 1 hour at 62°C. Different portions of this suspension were treated by mixing them with preparations of the acid lipidic or insaponifiable fractions of various origins, such as human placenta, cow, carp or rabbit organs; entire bodies of guinea pigs or rats; entire bodies of squid; seeds of Bixa orellana; microbes such as Esch. coli, B. subtilis, and tubercle bacilli. Fatty acids such as oleic, linoleic, eleostearic or mixtures of acids obtained from cod liver oil or butanol or heptanol also were used. 5 cc. of a 2% alcoholic solution of the lipoids were introduced in 110 cc. distilled water and the solvent eliminated by boiling the mixture to bring the amount to 100 cc. 1-5 cc. of these milky preparations were added to 5 cc. of the microbe suspension and incubated at 37°C for 24 hours. The treated microbes were then separated by centrifugation, the fluid decanted and the microbes resuspended in the same amount of saline. They were used in doses from 1/10-1 cc. for subcutaneous injections in rabbits, repeated every third day for two weeks. As controls, animals were injected with the same amoun's of suspension of untreated microbes.
Fig. 265A. Lipids and agglutinines. Influence exerted by the administration of unsa ponifiable lipid fraction and the acid lipid fraction of human placenta upon the appearance of agglutinines against Eb. typhi vaccines injected intravenously. Each curve corresponds to the average value of 5 rabbits. The agglutinines appear earlier and their amount increases more rapidly in the animals treated with lipids than in the controls.
For the first two weeks small amounts of blood were obtained every third day from the ear veins of the treated animals. Blood was obtained from the heart every second week for 8 weeks. The agglutinins were determined for all the samples. In the two week samples, the presence of immune antibodies was determined by the capacity of different amounts of the sera to prevent a lethal infection in mice given intraperitoneal injections of standard amounts of living microbes. The agglutinins were seen to appear earlier than in controls—in a manner similar to that seen when lipids were injected in the animal as related in Chapter 7 Note 5. It was especially in the immune protective antibodies where the difference was manifest. It was not only an earlier appearance of these antibodies, but the protection against a lethal injection was obtained with much smaller amounts of serum.
An aqueous extract of squid body was prepared by blending it and extracting with saline in a proportion of 1 /20. After filtration, the mixture was centrifuged and the supernatant fluid put in ampules with methiolate added as a preservative. Some of the ampules were tyndallized by heating them at 56°C one hour daily for four consecutive days.
1/10 cc. of these preparations was injected intradermically in various subjects who also received control injections of saline. Immediate and 24 hour reactions were noted. An induration present the second day was considered as a positive cellular response, while the immediate appearance of a hive was considered as a reaction taking place in the metazoic compartment.
Twelve days after a first injection in exactly the same place, a second injection was given with the same material. The immediate and the 24 hour reactions were judged. If the reaction was negative, a third injection was made, ten days after the second, in the same place. It could be seen that in normal individuals, the second sometimes, and the third injection always induced second day induration which persisted for several days. In cancer patients, including those in terminal condition, the injection of this antigen induced virtually the same reaction as in normal individuals.