periphery is it possible to demonstrate the entrance of either fibroblast or capillary. Robbed in this way of nourishment, the implanted cells have succumbed to their unfavourable environment, save only those of the outlying layer, which, by virtue of their position, have been able to absorb a certain amount of nourishment from the tissues of the host, and even to proliferate, if only in small degree. But the vitality of these elements themselves is at a low ebb, and in specimens removed a full day earlier it is not difficult to find certain of them with from two to five mitosing nuclei, witnesses—as Russell has indicated for mouse material-of a gradual paralysis of the protoplasm preventing the completion of division. All traces of alveolar arrangement have been lost, and in many cases the fibrinous exudate still encloses the graft, making its isolation even more complete.

Although among the cells bordering the dying graft in immune animals there may occasionally be found a few undergoing division as long after transplantation as eight days, most of them show signs of serious damage at the end of this period. By the tenth (fig. 9), most, if not all, of the epithelial elements have been entirely destroyed. The implant is undergoing penetration by fibroblasts from the host, phagocytosis is in active progression, and the whole process, in short, represents the efforts of the tissues to rid themselves of the defeated tumour fragment.

Conclusions.

(1) The investigation which has been described in the preceding pages leads inevitably to the decision that the phenomena described by Russell as characterizing the immunity of mice to tumour implantation occur also in the case of the resistance offered by rats.

(2) Furthermore, as no difference could be detected between grafts taken from rats treated with embryo skin, and those removed from animals which had undergone a previous unsuccessful inoculation with tumour, it is concluded that resistance is similar in the two cases.

(3) Finally, both of these types are the outcome of a failure on the part of the new host to furnish to the implanted fragment the proper blood vessel supply and connective tissue scaffolding.

The expenses of this investigation were borne in part by the Crocker Cancer Research Fund of the College of Physicians and Surgeons, Columbia University, New York City.

LITERATURE.

1. ANITSCHKOW, N. N.: Beitr. zur path. Anat. etc. (Ziegler), 1912, lii. p. 235. 2. BASHFORD, E. F., & MURRAY, J. A.: Sci. Reports, Cancer Research Fund,

London, 1904, No. 1, p. 13.

3. BASHFORD, E. F., MURRAY, J. A., & CRAMER, W.: Sci. Reports, Imperial Cancer Research Fund, London, 1905, No. 2, Part ii, p. 24.

BASHFORD, E. F., MURRAY, J. A., CRAMER, W.: Proc. Roy. Soc., Series B, 1907, lxxix. P. 164; Third Sci. Report, Imperial Cancer Research Fund, London, 1908, 315.

5. BASHFORD, E. F., & RUSSELL, B. R. G.: Proc. Roy. Soc., Series B, 1910, lxxxii. p. 298; Lancet, 1910, i. p. 782.

6. BURGESS, A. M.: Jour. Med. Research, 1909, N. S. xvi. 575.

7. EHRLICH, P.: Zeitschrft. f. ärztliche Fortbildung, 1906, vii. p. 208; Arb. a.d. königl. Inst. f. Exp. Therap., 1906, Heft i. p. 77.

8.

: Zeitschrft. f. Krebsforsch., 1907, v. p. 59.

9. DA FANO, C.: Zeitschrft. f. Immunitätsforsch. etc., 1910, v. p. 1.

10. FLEXNER, S., & JOBLING, J. W.: Monographs on Medical and Allied Subjects, Rockefeller Institute, New York, 1910, No. 1, p. 1.

11. GIERKE, E.: Beitr. zur path. Anat. etc. (Ziegler), 1908, xliii. p. 328; Third Sci Report, Imperial Cancer Research Fund, London, 1908, p. 115.

12. GOLDMANN, E.: Zeitschrft. f. Krebsforsch., 1907, v. p. 39.

13.

14.

·: Münch. med. Woch., 1907, liv. p. 2161.

Beitr. zur klin. Chir., 1911, lxxii. P. 1.

15. JENSEN, C. O.: Centralbl. f. Bakt. etc., 1903, Orig. xxxiv. p. 28.

16. LOEB, I..: Jour. Med. Research, 1901, N. S. i. p. 28.

17. Lowenthal, L., & MICHAELIS, W.: Zeitschrft. f. Krebsforsch., 1906, iv. p. 18. MAXIMOW, A.: Beitr. zur path. Anat., etc. (Ziegler), 1904, xxxv. p. 93.

639.

19. RUSSELL, B. R. G.: Third Sci. Report, Imperial Cancer Research Fund, London,

1908, p. 341.

[Fifth Scientific Report of the Imperial Cancer Research Fund.]

A

CYTOLOGICAL ANALYSIS OF THE

REACTION IN ANIMALS RESISTANT TO IMPLANTED CARCINOMATA *.

BY C. DA FANO, M.D. (MILAN).

THAT small experimental animals, rats and mice, can be rendered immune to implanted cancer, is one of the many facts that have been brought to light by the experimental investigation of tumours, and, moreover, is one which has been corroborated by various observers in different laboratories. Attempts to demonstrate an analogy with other forms of immunity, especially with a view to demonstrating antibodies in the blood serum by analysis in vitro, have been fruitless, and all that has been accomplished so far, has been to follow the changes occurring during spontaneous healing, and to establish the differences between the connective tissue changes observed in the early stages of growth in grafts from normal and immune animals. These investigations build the foundation for the present series of experiments, which have been designed to elucidate the histological details of these processes.

The connective tissue changes occurring in carcinomata undergoing spontaneous absorption were first studied and compared with those observed in immune animals. The investigation was later extended to a study of the reactions following inoculation of embryo skin, blood, and spontaneous tumour, as well as tumour previously killed by freezing and grinding.

The material and the methods of transplantation were those usually employed in this laboratory, and will be found fully discussed in various papers in previous Reports. Of the special stains utilised, Azur II solution (Grübler), with which very satisfactory results were obtained, may be recommended. As a differentiating fluid, a mixture of nine

This paper is a somewhat abridged translation of the author's "Zelluläre Analyse der Geschwulstimmunitätsreaktionen " (Zeitschrift für Immunitätsforschung, 1910, Bd. v. Heft 1), and gives the result of work carried out in the laboratory of the Imperial Cancer Research Fund.—E. F. B.

parts of 90 per cent, alcohol with one part of aniline oil was employed, followed by careful washing in 90 per cent. alcohol. The best results were obtained after alcohol fixation. To avoid confusion, the structure and nomenclature of the normal connective tissue elements will be discussed first.

The Loose Connective Tissue of the Mouse, and the Nomenclature
of its Elements.

The subcutaneous and intermuscular connective tissues have been especially examined, because they are the usual sites selected for the inoculation of tumours. While a complete review of the literature. pertaining to this subject is rendered unnecessary by the recent monographs of Marchand (12) and Maximow (13), the work of the latter author will be discussed fully, as the nomenclature adopted by him has found wide acceptance. He described the following elements in the connective tissue of the rabbit :—(1) Fibroblasts, (2) Wandering cells, (3) Clasmatocytes, (4) Fat cells, (5) Clasmatocyte-like adventitial

cells.

By fibroblasts he understands the distinctive connective tissue corpuscles. The wandering cells are small elements analogous to the blood lymphocytes, which in the course of development forsake the blood stream and wander into the tissues.

The clasmatocytes, described first by Ranvier (20), exhibit a denser nuclear network than the fibroblasts, and contain coarse refractile granules in the cytoplasm. Maximow accepts Ranvier's view that they develop from the wandering cells, and may in turn become changed into fibroblasts. Altnough the clasmatocytes of mammalia are readily distinguished from mast cells by their staining reactions, this distinction does not hold for the amphibia.

The clasmatocyte-like adventitial cells are identical with the elements described by Marchand as adventitial cells, an 1 some of them, according to Maximow, are atypical fibroblasts, while others are clasmatocytes. Marchand's category of adventitial cells certainly includes elements indistinguishable from the wandering cells. Marchand (12) ascribed to the adventitial cells an important part in all inflammatory and reparative processes, and noted their early proliferation under those conditions and their transformation into the following forms :(1) round cells with varying amounts of protoplasm, and possessing amoeboid movement and phagocytic properties, (2) lymphocyte-like cells which form the chief part of the "small cell infiltration," (3) mast

cells, and (4) plasma cells. To all these various forms of the adventitial cell Marchand applied the name "leucocytoid-cells."

In Maximow's opinion cells from the blood stream also played an important part in reparative inflammations. The clasmatocytes and clasmatocyte-like cells rounded themselves off in the first few hours of an inflammatory process, and became converted into large amoeboid cells. In this same period, Maximow found an active emigration from the blood stream of small lymphocytes, which mingled with the pre-existing wandering cells, altered clasmatocytes, and clasmatocyte-like adventitial cells. To all these various cells he gave the name "polyblasts," and as in his opinion plasma cells are simply emigrated and metamorphosed lymphocytes and mononuclear leucocytes, these also were included under the above term. The so-called macrophages he regarded as products of the evolution of poly blasts. The term "polyblast" includes rather a large variety of elements, and Maximow himself later restricted its application by excluding the plasma cell.

It would appear that Maximow's " polyblast" and Marchand's "leucocytoid" cell are interchangeable terms, but with this restriction in view of the respective authors' opinions, that the former are of histogenetic and hæmatogenetic origin, the latter of histogenetic origin only.

The loose connective tissue of the rat was later the subject of a special article by Maximow (14). Two of the histological points in which this animal differs from the rabbit are that the clasmatocytes do not contain specifically staining granules, and that polymorphonuclear leucocytes are present under normal conditions in the connective tissues. In inflammatory reactions the polyblasts take an important part, but plasma cells are never involved in the scar formation.

In a more recent communication, Maximow (15) returned to the classification of the normal connective tissue elements, and gave a review of the rôle played by the various cells in reparative inflammatory processes. He found the connective tissues of all mammalia to contain the following elements :-(1) Fibroblasts, (2) Mast cells, (3) Quiescent wandering cells †, (4) Small amoeboid wandering cells,

*In view of the complexity of the question, and the wide differences of opinion on the subject, it has been thought advisable to use in the present article the definition of plasma cells given by Maximow (13). Special attention was paid to the question whether these cells are to be found in the normal sulcutaneous or fatty tissues of the mouse. The source of these cells has not been fixed, and the present investigation of the cellular reactions in cancer-immune animals does not decide whether lymphocytes can undergo diapedesis, although it will be seen from the experiments that it is a possibility to be taken into account.

† Quiescent wandering cell is used throughout for "ruhende Wanderzelle" in the original.