PUBLIC HEALTH REPORTS VOL 39 MAY 30, 1924 No. 22 SOME EXPERIMENTS ON THE ANTIGENIC PRINCIPLES OF RAGWEED POLLEN EXTRACT (AMBROSI ELATIOR AND AMBROSIA TRIFIDA). By W. T. HARRISON, Passed Assistant Surgeon, and CHARLES ARMSTRONG, Passed Assistant Surgeon, United States Public Health Service. In view of the differences in opinion regarding the possibility of rendering laboratory animals hypersensitive to the protein of rag- weed pollen, some experiments to determine this point were under- taken in the course of our efforts to establish an official standard for pollen extracts. -Coca (1) did not find pollen extracts to be antigenic and suggested placing human hypersensitiveness to pollens in a class distinct from general protein hypersensitiveness. Parker (2), using the Schultz-Dale method, was able to demon- strate hypersensitiveness in the horn of the isolated uterus from a giant-sensitive guinea pig, using giant ragweed extract and mixed giant and s/tort ragweed extract as exciting agents, the latter pre- pared in a different laboratory. Alexander (3) has shown conclusively that guinea pigs may be ren- dered sensitive to the pollen extract of rye pollen (Secale cereale). The pollen extracts used in the experiments herein reported were made as follows: The glycerin extract was made in the proportion of 1 gm. of mature giant ragweed pollen to 100 c. c. of extractive fluid composed of 661 per cent glycerin and 33k per cent of a solution containing 2.5 gm. sodium chloride and 2.7 gm. sodium bicarbonate to 1,000 c. c. dis- tilled water. More concentrated extracts were made by extracting 1 gm. of pollen, giant or short, in 10 c. c. or 20 c. c. of the saline solution, which forms 33k per cent or the extractive described above. The pollen in each case was extracted with occasional shaking for seven days at room temperature and filtered through a Berkefeld filter. As a preservative in saline extracts when glycerin was omitted, 0.5 per cent phenol was used. The pollens were collected in the city of Washington. Experiments on eight guinea pigs are included in this report, these animals being selected from a number of typical examples. Guinea 9440"-24t-1 (1261) May 30, 1924 1262 pigs Nos. 1, 2, and 3 were sensitized by injection intraperitoneally witlh 1 c. c. giantt ragweed glycerin extract, 1:100, diluted 5 times with physiological salt solution. Injections were made every second day, with slight variations, beginning on June 12, 1923, and continu- ing. until August 1, 1923. Twenty-three c. c. of the 1:100 extract were given during this period (protocol, Table 1). TABLE 1.-Sixty-six and two-thirds per cent glycerin poUen extract (giant) 1:100- Intraperitoneal injections. Guinea pigs Nos. 1, 2, and 3 given 1 c. c. of extract on the following dates-June 12 (1923), 14, 16, 18, 20 22, 24, 26, 28, 30, July 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 27, August 1. Guinea pig No. 1: Dccember 27, 1923, 1 c. c. giant ragweed extract 1:10 given intravenously, followed in 30 seconds by symptoms, and death in 3 minutes. Guinea pig No. 2: December 28, 1923, given 0.75 c. c. short ragweed extract 1:10 intravenously, folowed by symptoms in 1 minute, and death in 5 minutes. Guinea pig No. 3: December 27, 1923, given 1 c. c. giant ragweed extract 1:10 intravenously, followed by symptoms (marked) in 2 minutes. Recovery. December 28, I923, given 1 c. c. giant ragweed extract 1:10 intravenously. No symptoms. December 23, 1923, given 0.5 c. c. 8hort ragweed extract 1:10 intra- venously. No symptoms. January 22, 1924, given 0.5 c. c. giant ragweed extract 1:10 intravenously. Marked symptoms in 2 minutes. Recovery. February 14, 1924, given 0.7 c. c. short ragweed extract intravenously. Symptoms marked in 2 minutes. Recovery. On December 27, 1923, guinea. pig No. 1 was shocked by injecting intravenously 1 c. c. of giant ragweed extract 1:10. 'Typical ana- phylactic symptoms (coughing, rubbing of nose) appeared in 30 seconds, rapidly proceeding to convulsive seizures with' death in 3 minutes. On December 28, 1923, guinea pig No. 2 was shocked by injecting intravenously 0.75 c. c. short ragweed extract 1:10. Typical symp- toms of anaphylaxis were manifested in 1 minute, with convulsions and dea.th in 5 minutes. On December 27, 1923, guinea pig No. 3 was shocked with 1 c. c. giant ragweed extract 1:10 inltravenously, followed in 2 minutes by coughing, rubbing of nose, and difficult breathing. At the end of 10 minutes the pig was huddled in a corner of the cage, the coat was rough, breathing was difficult, and 15 pellets of feces had been expelled during this period. On the day following, December 28, 1923, the same shocking dose of giant extract was repeated followed by no symptoms; 0.5 c. c. short ragweed extract 1:10 was then given, with no symptoms. On January 22, 1924, 0.5 c. c. giant ragweed extract 1:10, and on February 14, 1924, 0.7 c. c. short ragweed extract 1:10 were given, both injections being followed by the same symp- toms of anaphylaxis as those observed on December 27, 1923. Guinea pig No. 4 was sensitized by intraperitoneal injections of gzant ragweed extract 1:10. This concentrated extract was injected in doses of 2 c. c. on December 19 and 24, 1923 (protocol, Table 2). On February 21, 1924, 0.25 c. c. of the same extract was given intravenously, followed by typical anaphylactic symptoms in 30 seconds and death in 5 minutes. 1263 May 30, 1924 TABLE 2.-Saline extract giant ragweed pollen 1:10, 0.5 per cent phenol preservative. Intraperitoneal injections. Date and amount. (uinea Pig. Dcc. 19, Dec. 24, 1923. 1923. No. 4- 2 c. c_.. 2 c. c-- Feb. 21, 1924, 0.25 giant ragweed extract 1:10 intravenously, followed by symptoms in 30 seconds and death in 5 minutes. Saline extract short ragweed pollen 1:10, 0.5 per cent phenol preservative. Intra- peritoneal injections. No. 5- 2 c. c---- 2 c. c---- Jan. 22, 1924, short ragweed extract 1:10, 0.5 c. c. intravenously, followed by marked symptoms; recovery. Feb. 21, 1924, same dose as on Jan. 22, modlerately severe symptoms in 1 minute; recovery, Apr. 22, 1924, giant ragweed extract 1:10, 1 c. c. intravenously; marked symptoms in 1 minute; recovery. No. 6- 2 c. c---- 2 c. c Mar. 10, i923, 0.5 c. c. giant ragweed extract 1:10, intravenously. Severe symptoms in 2 minutes; coughing, rubbing of nose, rough coat; recovery. No. 7 ------- 2 c. c---- None---- pr. 24, 1924, short ragweed extract 1:10, 0.05 c. c. intravenously. Se- vere symptoms in 30 seconds, with recovery. Apr. 26, 1921, giant ragweed extract 1:10, 0.2 c. c. intravenously. No symptoms. Guinea pig No. 5 was sensitized by intraperitoneal injections of short ragweed extract 1:10 in doses of 2 c. c. on December 19 and 24, 1923 (protocol. Table 2). On January 22, 1924, 0.5 c. c. of the same extract was given intravenously, followed by marked symptoms, with recovery. On February 21, 1924, the same dose was repeated, and on April 22, 1924, 1 c. c. giant extract 1:10 was given. The last two injections were both followed by marked symptoms in 1 minute with recovery. Guinea pig No. 6 was sensitized with short ragweed extract in the same manner as No. 5 (protocol, Table 2), and on March 10, 1924, was injected intravenously with 0.5 c. c. giant extract 1:10 followed by severe symptoms of anaphylaxis in 2 minutes with recovery. Guinea pig No. 7 was sensitized with a single intraperitoneal injection on December 19, 1923, of 2 c. c. of short ragweed extract 1:10 (protocol, Table 2). On April 24, 1924, 0.05 c. c. of the same extract was injected intravenously followed in 30 seconds by severe symptoms with recovery. Two days later, on April 26, 1924, giant extract 1:10, 0.2 c. c. was injected intravenously with no symp- toms resulting. No symptoms were observed in nonsensitive pigs when twice tho maximum dose used above was injected. Mayr 30, 1924 1264 TABLE 3.-Sixty-six and two-thirds per cent glycerin poUen extract (giant) 1:100; intraperitoneal injections. Date and amount of injection (c. c.). Guinea l pig. o -_ g - C 6 06 No.8-_ 0.01002. 04 0.80.16 0.25 04 1.0 2.0 3.0 4.0 4.0 4. 0 Apr. 19, 1924, pig killed and uterus removed to Locke's solution. Figures 1 and 2 represent the kymograph tracings made by adding 0.5 c. c. giant and extracts 1:20 to bath in whichshort horns of uterus were suspended. Bath contained 100 c. c. Locke's solution at 370 C. Autopsies were done on all pigs which died following shocking and in each instance the typical lesions of anaphylactic death were observed, viz, inflated lungs and capillary injection of viscera without other apparent cause of death. Guinea pig No. 8 was sensitized with giant ragweed extract pre- pared as was that for pigs 1, 2, and 3. The injections varied, however, in that the first dose on February 7, 1924, was 1/100 c. c. Intra- peritoneal iinjections were made every second day, with slight varia- tions, and the dose was doubled each time until 2 c. c. were given on February 25. Three c. c.