made of normal tissue in plasma taken from animals previously infected with various bacteria. Pyocyaneus-infected plasma showed an interesting result in that the pigment, as it formed, had a tendency to stain the growing tissue.

While the experiments upon the behavior of tissues in various infected plasmas performed here were limited, they were sufficient to suggest great possibilities in future study on the actions of infection upon living cells.

TRANSPLANTATION OF CULTURE TISSUES.

Cultures of various tissues after two weeks of incubation were transplanted to animals in order to determine the behavior of these new-formed cells under normal conditions. Before considering the results obtained a review of present literature upon transplantation is briefly given.

It has been known for a long time that certain tissues proliferate in parts of the body quite foreign to their normal environment. Not only do certain tissues of an animal grow when transplanted to other parts of its own body (autoplastic transplantation), but tissues of one animal may be transplanted to another animal with success (heteroplastic transplantation). Grafting, as this latter process is better known, has been practiced by surgeons as well as by horticulturists for centuries. In lower organisms heteroplastic transplantation, even between different species, is possible to a remarkable extent. Joest,121 experimenting with the earthworm, and Born,122 with larvæ of amphibians, joined individuals of different species. Segments of different species were interposed and alternated with

success.

In mammals heteroplastic transplantation, and that, too, of the same species, has proven unsuccessful in most instances. Even auto plastic transplantation is very much limited. It is most successful when the blood vessel originally supplying the transplanted tissue is left in contact until complete union takes place.

Ribbert 123 and Lubarsch 124 have done much work on experimental transplantation. The former has transposed many tissues to the liver and the latter to the lymph gland. Bone, periosteum, cartilage, and even ducts of glands proliferated for awhile in these new environments, but after a short time the transplanted tissue disappeared as a result of absorption and cicatrization.

Fere 125 had considerable success with heteroplastic transplantation in fowls. Forty-eight-hour embryo chicks were transplanted underneath the skin of young chickens. A mesodermic growth of the embryo persisted in one case for five months, but in most instances embryos were completely absorbed in from two to three months,

Ribbert,126 as well as Birch-Hirschfeld and Garten, 127 obtained the same results in transposed rabbit embryos. They were soon absorbed. Hunter's well-known cockspur transplantations were, as a rule, never permanently successful, for after a few weeks the spurs usually dropped off.

Von Eiselberg 128 successfully transplanted the two lobes of the thyroid glands of a cat to the abdominal wall. One lobe was transplanted several days before the other, so that its union with the abdominal wall tissues had taken place before the second was transplanted. Here they functioned sufficiently and when removed later the cat died, showing all symptoms of athyroidism.

Munk, 129 Enderlein,130 Sultan,131 and Christiani 132 confirmed, in cats and dogs, the observations of Von Eiselberg. The center of the thyroid underwent necrosis, which was followed by granulation. The epithelia grew in solid processes which later separated off. Colloid was then secreted by these cells and lumena were formed. These transposed thyroids have remained active for months. Christiani reports functioning thyroids two years after transplantation.

Portions of the mammary glands have been transplanted by Ribbert into the ear of guinea pigs, and here they even secreted milk after parturition.

Both autoplastic and heteroplastic transplantation of ovaries have been successfully accomplished in humans as well as in other mammals. Knauer 133 and Grigorieff 134 transplanted ovaries of rabbits to the abdominal wall; these later became pregnant. Morris 135 grafted an ovary of a woman into a girl who had never menstruated; this function followed immediately.

Skin grafting is so well known that no further comment is needed. In the case of periosteum Saltykoff 136 observed that most of the periosteal cells necrosed and that only a certain portion of the inner layer proliferated and covered the bone. Bruns 137 and Kolliker 138 found that cartilage, osteod tissue, and true bone formed in transplanted bone marrow.

In recent years Carrel, Guthrie, and others have had great success in transplanting entire organs and limbs. In these experiments accurate anastomosis of all vessels was made.

Grafting of skin, mucous membrane, periosteum, and ovaries have thus far been the most successful.

Marchand discusses the subject of transplantation in his book, reference to which has been made. (See Der Process der Wündheilung, 1901.)

In the Hygienic Laboratory the following culture tissues were transplanted back to animals:

1. Cultures of hypophysis, transplanted to thyroidectomized guinea pig.

2. Cultures of suprarenal, transplanted to thyroidectomized guinea pig.

3. Cultures of spleen, transplanted to splenectomized guinea pig. 4. Cultures of thyroid, transplanted to freshly thyroidectomized guinea pig.

5. Cultures of bone, transplanted to normal guinea pig.

6. Cultures of spleen, transplanted to normal guinea pig.

In every instance watch-glass cultures of the above tissues, after two weeks' incubation, were transferred to the subperitoneal pockets previously made by careful aseptic dissecting.

The possibility suggested itself that these new cells which had proliferated under conditions so different to those experienced by normal cells might be free from the restraint exercised by normal cells upon each other, and when transplanted back to the body might go on proliferating, thus throwing further light on the origin and behavior of tumors.

In each case complete absorption of these transferred cultures occurred within from one to three months. So far the degree of proliferation of these transferred cultures has not been minutely determined, but it was very slight. The cells which developed in cultures had apparently less capacity to proliferate when transferred back to the body than do normal transposed tissues. It is possible, however, that by further experimentation cells may be grown in plasma somewhat altered by the addition of certain accelerating substances or other favorable media, which will continue to proliferate upon inoculation into the body. If this can be done successfully it may add much to our knowledge of the etiology and behavior of tumors.

PART III.

GENERAL DISCUSSION AND CONCLUSIONS.

In the description of the behavior of the various culture tissues in plasma, no attempt was made to give the minute changes which occurred hour by hour in each culture taken from the animals of different ages. To do so would require many pages of unimportant data, descriptions, and drawings. The chief purpose of the experiments was to ascertain, as far as possible, the nature and degree of proliferation in normal plasma as an essential preliminary to further study of the numerous possibilities that this method presented. The following facts have been gained by the experiments:

1. Mammalian tissue does proliferate to some extent in plasma when incubated, the degree of proliferation depending upon the age of the animal from which the cultures are taken. Tissues removed from young embryos are most prolific, the degree of proliferation decreasing as the age of the animal increases. In old animals the proliferative capacity is very much limited.

2. Generally speaking, the degrees of proliferation of the various tissues in plasma bear a close relation to the regenerative capacities of the same tissues within the body-that is, those tissues which regenerate the greatest within the body show, as a rule, the greatest proliferation in cultures, tissues from animals of corresponding ages being used for the comparisons. This was the case in stratified epithelium of skin, endothelia of the various serous cavities and connective tissue, and was especially true of glandular epithelium. Spleen and bone marrow were found to be exceptions to this rule, as both possessed a greater degree of proliferation in cultures than of regeneration in the body. There is no doubt, however, but that this new growth in both spleen and marrow was connective tissue.

3. Two types of cells are usually found in the new growths of tissues which normally possess both connective tissue and parenchymatous cells, while only one general type is seen in the proliferation of tissues, such as skin, which are composed of one type of cell. The two types of cells in the former are: (a) Elongated cells, doubtless of connective-tissue origin; and (b) polygonal or spherical cells, or parenchymatous epithelial origin. This has been observed by Carrel and Burrows and others.

4. The new cells are characterized by both normal and atypical nuclei, the latter predominating especially in all postnatal mammalian tissue cultures. The cytoplasm is generally composed of many small fat globules which resemble the fatty infiltration of cells seen in certain pathological tissue. As a consequence of these

characteristics (atypical nuclei, giant cells, and fat infiltrations) in the majority of growing cells it would seem that they can not be considered as normal.

The new cells in all glandular epithelial proliferation, whether of hypophysis, kidney, liver, etc., do not bear the differential characteristics of the cells from which they have had origin. All are similar in morphology, i. e., more or less spherical or polygonal, possessing centrally placed nuclei, which usually show various forms of amitotic and mitotic figures, the amitotic predominating, and a cytoplasm which shows a very simple structure of reticular network or is filled with lipoid bodies. In no instance was it possible to demonstrate structures within the cells that would characterize them as being of any particular origin. These new glandular epithelial cells are of the most simple structure and probably represent a reversionary embryonal type. Possibly metaplasia plays an important rôle here. The same may be stated of the new connective-tissue cell. Practically all such new proliferations are similar in structure, i. e., elongated, irregular, or stellate, with more or less oval atypical and normal nuclei and cytoplasm, filled with fat bodies. These new cells resembled in form the very simple embryonal connective-tissue cells, hence a reversionary new growth or possibly metaplasia.

6. The presence of numerous fat globules in the proliferating cells suggests that the cells themselves are undergoing degenerative changes. Virchow's necrobiosis or necrobiotic growth would seem a satisfactory term for this new growth.

Let us examine briefly the processes of degeneration that occur within the body and then compare them with the conditions present in tissue-culture growths.

Degeneration may be subdivided for convenience into two types: 1. The histolytic processes, which include (a) the atrophies, such as senile, pathological, disease, etc.; and (b) necrosis, such as mummification or dry gangrene, coagulation necrosis, liquefaction, and granular disintegration.

2. The second type of degeneration is known as metamorphic processes. The metabolism of the cell in degeneration does not come to a standstill gradually, but is previously turned into a perverse course in such a way that substances which in the normal cell are either not manufactured at all or appear only at intermediate stages are produced in quantity as a result of disturbed metabolism and accumulate within the cell until it perishes (Verworn139). The most common type of these metamorphic processes are (a) fat, (b) mucoid, (c) amyloid degeneration, and (d) calcifications. As Verworn points out, we must distinguish between (a) fatty infiltration, which occurs without disturbance of cell metabolism but finds

a Adami discourages, with good reasons, the use of the term "infiltration of fat."