Edwin Carleton MacDowell

The experimental transmission of leukemia in mice

The only report in the literature of the successful transmission of leukemia in mammals, is that of Snijders, 1 who succeeded in transmitting the disease by the inoculation of emulsions of tissues from spontaneously affected guinea pigs into normal animals of the same species. Unsuccessful attempts to transmit leukemia in mice were made by Haaland 2 and Tyzzer. 3 In a strain of mice designated hereafter as C58, which has been inbred by brother-sister matings since 1921, it was observed that a considerable number of those that lived more than 8 months had enlargements of their spleens and lymph nodes. Microscopic examination by Dr. Alwin M. Pappenheimer in 1926 disclosed that these enlargements were due to lymphatic leukemia. The present cooperative investigation was undertaken early in 1928, at which time the number of matings in this strain was increased. Of 42 mice in the 16th and 17th generations that lived more than 8 months, 4 29 have had splenomegaly. Of these, 19 have been diagnosed as leukemia by microscopic examination, 2 presented the gross anatomical lesions of leukemia and are awaiting microscopic diagnosis, 2 had the gross lesions of leukemia but are not available for microscopic study, and 6 are still alive. Four mice died or were killed at about 10 months of age, but were not leukemic. Nine mice are still living and at present do not have leukemic symptoms. Tumors other than those occurring as part of the leukemic syndrome are uncommon in this strain. Saline emulsions of spleens from 6 mice spontaneously affected with lymphatic leukemia were inoculated intraperitoneally and sub-cutaneously into non-leukemic mice from the same and also from other strains. The non-leukemic mice were young animals of both sexes, from 1 to 2 months old.

Transfer of Acquired Resistance to Transplantable Leukemia in Mice.

It has been demonstrated that graduated doses (cell-immunization) of leukemic cells 1 and certain normal tissues 2 , 3 can induce resistance to transplantable leukemia in normally susceptible mice. Following cell-immunization injected leukemic cells immediately develop small lesions which soon become necrotic. 4 Lesions do not form immediately following leukemic inoculation of strain C58 mice made resistant by Sto-Li foetal tissues. Once resistance to line I leukemia has been established by means of graduated cell doses it is permanent, while resistance caused by normal tissue implantation may be only temporary with a peak of effectiveness in C58 mice 3 days after implantation of Sto-Li tissue (unpublished data). Of further interest in this problem is the question: Can resistance induced by these 2 methods be transferred to normally susceptible mice? Accordingly, spleen and liver were removed from cell-immunized mice, minced and injected into susceptible C58 mice. Another group of C58 mice was given injections of Sto-Li foetal tissues and 3 days later the spleens were removed from these animals, minced and injected into susceptible C58 mice. Three days after receiving transplants from resistant animals both groups of mice were injected with a normally lethal dose of line I transplantable leukemia. C58 mice treated with normal C58 spleen were also given leukemic inoculations to verify the observed lack of resistance to line I leukemia following implantation of C58 tissue in C58 mice. 2 Control inoculations of normal C58 mice were made to check the potency of the dose of leukemic cells. A summary of the results of these experiments is given in Table I. These preliminary results demonstrate that resistance to a transplantable leukemia may be transferred from cell-immunized mice to normal mice by implantation of tissue from actively immunized mice. Under the conditions of this experiment resistance induced by normal tissues was not transferable. Quantitative effects, duration of the transferred resistance and use of cell-free material are questions for further experiments.

Immunization of Mice Naturally Susceptible to a Transplantable Leukemia

The transplantable leukemia designated as line I 1 was started from a spontaneous case of lymphatic leukemia in April, 1929. The line has passed through 441 transfer generations and, by routine technique, has been inoculated in massive doses into 3625 mice of the highly inbred strain C 58; all but one of these died with the leukemic indications characteristic of this particular line of cells. The single survivor, which at no time showed clinical effects of the inoculation, was inoculated in the 77th transfer generation, in December, 1930. In the 3 ¾ years since that time, the 2925 mice from strain C 58 that have been inoculated with the massive standard dose of cells of line I have all developed leukemia. In the light of this record the natural susceptibility of strain C 58 to leukemic cells of line I appears to be fully established. The present experiments, started in April, 1934, are based on 100% susceptibility to the standard dose. It has been shown previously 2 that between certain limits, reduction of the dosage lengthens the interval before death, and in the early transfers of line I that were used for these experiments (transfers 27-34, Jan.-Mar., 1930) the 18 mice given doses of 6000-9000 cells did not die with leukemia. However, these mice were not tested for an immunizing effect of surviving the small doses. In returning to the study of small doses of the leukemic cells of line I, 346 transfer generations later, the virulence of the cells had become considerably enhanced and it now is found that the minimum dose that will kill is reduced to the order of magnitude of 200 cells. In successive dilutions of the massive standard dose the interval before death is progressively lengthened, as previously reported, 2 but a dilution is reached (1/1024th of standard) that permits a few of the mice to survive and as the dose is further reduced the proportion of survivors increases until every mouse survives (1/524,000th of standard).

On the absence of isoagglutinins in mice

The number of units of inheritance, or genes that are known in mice, is already so much in excess of the number of those known in any other mammal that there is offered a strong inducement to make intensive studies in order to increase the number of genes to the point where the deeper study of the genetic mechanism of a mammal may be undertaken. A possible field for the search for new characters was suggested by the claims of Von Dungern and Hirschfeld 1 , Learmonth 2 , and Ottenberg 3 that blood groups in man depend in their inheritance upon simple Mendelian factors. Besides being of interest from the standpoint of mouse genetics and of the technique of experimental transplantations, the discovery of blood groups in mice would lead to a genetic investigation that could provide an experimental basis for the study of the inheritance of blood groups in man. The tests made upon rabbits and steer by Ottenberg and Friedman 4 are said to reveal the presence of blood groups in these animals. On the other hand, other investigators (Hektoen 5 , Ingebristen 6 , Fischbein 7 , and Rohdenburg 8 ), using a variety of animals (cats, dogs, sheep, swine, cattle, horses, rabbits, guinea-pigs, rats, and frogs), have failed to find any evidence of blood groups. In certain cases agglutinations were found, but these did not appear to be grouped. Such negative results did not promise well for the discovery of blood groups in mice, but the availability of a greater number of different races than had been used in testing the other animals favored the chanlces of a positive result. The following races of mice were used in the present experiments: (1) Japanese Waltzers (Lambert strain), which originated from a single pair of mice isolated in 1906 and intensively inbred ever since; (2) ten lines inbred three or four generations from Lathrop stock by Dr. C. C.

Studies on mouse leukemia. IV: Specificity of susceptibility to different lines of inoculated leukemia

Susceptibility and resistance to a particular line of inoculable mouse leukemia may, for present purposes, be defined in terms of the presence or absence of conditions necessary for the survival of the active agent. A group of 11 such lines of agent, each of which originated in a different case of spontaneous lymphatic leukemia in a highly inbred strain of mice designated as C58, found the necessary conditions for survival in mice of the same strain. 1 Although different lines of agent can grow in the same pure bred strain of mice, these different lines do not all have the same requirements for survival, as has been shown by hybridization experiments. 2 The present report gives direct evidence of the specificity of requirements of different lines of agent, without the use of hybridization, by means of a new line that originated from a spontaneous case in another highly inbred strain of mice. The new line of agent is designated line L. The spontaneous case that gave rise to this line was a mouse in strain 89; the line is carried in mice of this same strain. During the course of 13 transfers 75 mice of this strain have been inoculated. Of these all but 4 (94.6%) have died with unmistakable lesions of leukemia, mostly between the 15th and 25th days after inoculation. The agent of line L has been inoculated into 64 mice of strain Storrs-Little with completely negative results and into 82 mice of strain C58, of which all but one were negative. The active agent of line I originated in a spontaneous case in strain C58 in April, 1929. It has now reached its 116th transfer. Barring experiments designed to study the nature of the agent, 859 mice of this strain have been so inoculated; all have proved susceptible with the exception of a single mouse whose identity is questionable.

Heavy alcoholization and prenatal mortality in mice

Summary Brief reference is made to the more important earlier studies on the action of a utero-stimulating substance in mammals. In a small group of tests of the utero-stimulating action of a placental extract, and of a substance prepared from the liquor folliculi of the sow by the Allen-Doisy method, we have obtained a few positive results-enlargement and hyperemia of the oviduct of virgin doves. This supplies the last necessary fact in proof of the lack of specificity, as between birds and mammals, of this substance. Heavy dosage failed to affect a response in the virgin oviduct in some cases. Responses in the ovary and on sex behavior in virgin and mature doves have not been observed. An insufficient number of cases was studied but our results indicate a less ready, and a less varied, response of the bird to this substance than has been found for mammals.

The sex ratio in litters of mice classified by the total amount of prenatal mortality.

From studies of the normal sex ratio of living young and of abortions and still-births in man and the rat, King 1 and others have concluded that the male fœtus is less viable than the female. If this is true for the entire period of gestation there should be a negative correlation between the amount of prenatal mortality and the sex ratio. From counts of the corpora lutea of pregnancy of about 20 mice (sectioned material) Parkes 2 concludes that this is the case. Data showing the percent of prenatal mortality for 445 litters of mice have been obtained by subtracting the number of young born from the number of ova as indicated by the number of corpora lutea. In the last week of pregnancy the corpora lutea corresponding to the litter in utero are strikingly differentiated as large hyperemic bodies protruding from the surface of the otherwise pale colored ovary. These corpora were counted in the living animals under a low power binocular microscope by means of an operation which has been shown 3 to have no influence upon the fœtuses in utero or upon the subsequent reproduction. Table I gives the total number of males and females in litters classified, according to the per cent of prenatal mortality, into five classes, each 20 per cent in width. The sex ratio shows no tendency to decline as the percentage of prenatal mortality increases; the highest ratio is found in the next to highest prenatal mortality class. The small numbers in the fifth class indicate that its low sex ratio is probably not significant. These results indicate that when the total prenatal mortality is revealed there is found no selective elimination of males.

The sex ratio of mice from alcoholized fathers

Bluhm 1 and Danforth, 2 using somewhat different experimental methods, have reported a rise in the sex ratio of mice from alcohol treated fathers. However, these ratios are based on small numbers (182 ♂ : 149♀ Bluhm; 210 :164♀ Danforth) and, although the probable errors of the deviations from the control ratios (+10.60 per cent ±2.03 and +5.36 per cent ±1.99) may be taken to mean that random sampling alone is not responsible for the results, various other possible influences besides alcohol, such as season, mothers age and parity, have not been eliminated by the methods of these investigators. Moreover, Gyllenswärds 3 more completely controlled experiments with alcoholized male mice show as great a change in the opposite direction (—10.4 per cent ±3.4), and MacDowell and Lord 4 have called attention to the fact that when all question of the modification of the sex ratio by prenatal mortality was removed by using only complete litters (so judged by the number of the corresponding corpora lutea) the primary sex ratio given by litters from heavily alcoholized fathers and normal mothers (50.3 per cent males, based on 308 mice) showed no significant deviation from the primary sex ratio from normal parents (49.9 per cent males, based on 523 mice). The present report gives the sexes of 2133 mice from normal mothers by fathers given completely anesthetizing doses of alcohol fumes five days a week, beginning at the age of four weeks and continued over a year, to the end of the experiment; and 2322 mice from the same normal mothers by normal fathers, brothers of those treated. The treatment was given by inhalation, in one pint milk bottles; for each treatment 3 cc. 95 per cent alcohol was poured on a piece of atisorbent paper which was placed in the bottle with the mouse, a regular milk bottle cap inserted and the bottle inverted.

The effect of light closes of alcohol upon the estrus cycle, and on the number of corpora lutea and prenatal mortality in the mouse.

Stockard and Papanicolaou, Arlitt, Stockard, Hanson, Mac-Dowell 1 have found that alcohol may cause partial or complete sterility in laboratory mammals. This has been explained both as a result of the elimination of less vigorous germ cells or zygotes, and as an immediate effect of the alcohol upon the activity of the ovary. The present experiments offer a method for analyzing more fully than before the effects of alcohol upon the reproductive processes of a mammal. The following criteria are used here for the first time in a study of this type: (1) the length of the estrus cycle, (2) the number of corpora lutea for given pregnancies, and (3) the proportion of these represented at the subsequent births by living young. The mice used came from lines that had been inbred for three and four generations by brother to sister mating. Tests and controls from the same litter were kept in the same pen to equalize environmental conditions as far as possible. The alcohol was given by inhalation in pint milk bottles. Before treatment each mouse from one pen was placed in its own bottle; into each test bottle was then inserted a strip of absorbent paper, approximately 37 by 15 cm., wet with 3 cc. commercial (95 per cent) alcohol. The bottle was capped and inverted at once. Into each control bottle was inserted a similar piece of paper without the alcohol; these bottles also were closed, and inverted. Each mouse remained in its bottle for 45 minutes. This treatment, starting at weaning (4 to 5 weeks), was given daily. The controls seemed to be unaffected by confinement in the closed bottles. For the first week the alcohol treatment left most mice flat on their sides; later, when most mice could turn over and run off, their behavior still showed an unquestionable effect of the alcohol.