TRANSACTIONSOF THE

AMERICAN SURGICALASSOCIATION

MEETING HELD IN CLEVELAND, OHIO

APRIL 6-8, 1942

ADDRESS OF THE PRESIDENT

THE DEFENSE OF THE HUMAN BODY AGAINSTLIVING MAMMALIAN CELLS*

HARVEY B. STONE, M.D.BALTIMORE, MD.

EVERY LIVING ORGANISM depends for its survival on the possession of atleast two powers: The power of adaptation and, if necessary, resistance toits external environment; and the power of maintaining stability, within cer-tain limits, of its own internal economy. These two abilities are found in allanimals from ameba to man, but apparently the problems involved grow incomplexity with the increasing complexity of structure and function of thehigher forms of life. This is well illustrated by the response to trauma. Thecutting in half of certain of the simpler living creatures may result in eachhalf forming a complete new survivor. Such mechanical insult to any of theadult higher animals would be invariably fatal, but note that the earlieststage of the fertilized ovum, even of man, can also apparently survive com-plete fission. But the response to trauma is only one of many reactions ofdefense against the external environment. Another great field of reaction isdependent upon the intimate presence of other living organisms or theirproducts, and includes within its scope such conditions as infection, para-sitism and intoxications. In the higher animals, and particularly in man, thepsychic or emotional reaction to the environment is another manifestation ofresponse of the greatest interest and importance. Now much fruitful studyhas been devoted to these and other particular forms of response-behavior inmany different animals, and while, no doubt, much remains to be learned, atleast the problems have been recognized, methods of attack have been devised,and many substantial results have been obtained. As much cannot be saidfor our knowledge of the defense mechanism against the introduction of alienliving mammalian cells.

The second great power of the organism, that of regulating and controllingits initernal functions, also, has many specific phases that have been the subject

* Delivered before the American Surgical Association, Cleveland, Ohio, April 6-8,I942.

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of long and intense study. Digestion, absorption, secretion, the maintenanceof the circulation, the function of the nervous system, are but partial aspectsof the subject that we have called general physiology. Physiology, in itsclassical sense, does not cover the whole field. The physical and chemicalprocesses that regulate and maintain the composition and osmotic pressure ofbody fluids, the phenomena related to membranes, surfaces and molecularbehavior fall within the scope of biochemistry and biophysics. The consciousand subconscious states that affect the internal activities of the body are ma-terial for the student of psychology. All of these varied and numerous func-tions must be coordinated and equitably controlled if the higher animal, par-ticularly man, is to survive and prosper. To this regulated interrelationshipCannon' has given the name "homeostasis." His numerous writings on thesubject have greatly helped to establish and popularize this concept of theliving being as a mechanism dependent upon a highly complex system of in-ternal checks and balances. Here again, a vast deal has been learned andmuch still remains to be learned. At any rate, methods and techniques ofstudy, many of them intricate and highly specialized, have been evolved andsuccessfully applied.

There is a special phase of this whole problem that has apparently laggedbehind, at least so far as the interest of the medical profession and of surgeonsis concerned. To be sure, the biologists have engaged themselves with it,but often the living material with which they work is so different from thehuman, that their results may not be transferable. I refer to the reactionof the higher animals, particularly man, to the living cells of mammals. Theproblem has two distinct aspects that correspond to the two properties ofliving creatures just now set forth, anid each of them is rich in theoretical and(possibly practical interest. The first conicerns the reaction of lhumani beingsto implanted livinig mammlialiani cells-a defenise reaction. The seconid cotn-cernis the control of the humiian being over the growth, destruction anld repairof its ownI cells-a special phase of honmeostasis. Later, a developmenlt ofthese two statements will be attempted. Perhaps here it may be well to glancebriefly at some of the biological studies bearing on the problem.

Dr. Ross G. Harrison2 has employed heteroplastic grafting extensively inhis experimental studies of embryology. By this technique he has made manyfundamental contributions to his choseni field, particularly the cystologicsources and relationships in the development of many structures. What con-cerns us here is the remarkable success of some of these grafting experiments.In the amphibia during the larval stage, not only may organs such as theeye, and whole limbs, but, indeed, large parts of one animal may be success-fully grafted onto another animal. These grafts will take between individualsof different species, and some of such composite animals, called chimeras,have developed through the stage of metamorphosis into living adult forms.This tolerance for foreign tissues does not exist in the same degree, of course,for the higher animals among the vertebrates, in which respect there is agraded series, the higher forms showing increasing resistance to such alien

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grafts. It has been stated that successful cross-grafts between species arelimited to cold-blooded animals, and that in mammals even grafting betweenindividuals of the same species and variety encounters great intolerance ex-cept in cases of close blood relationship.

That these statements are not universally true is evidenced by the workof Eastlick,3 who transplanted limbs between embryos of various avian specieswith a certain number of successful experiments that survived hatching andlived with the foreign limb also surviving. We are fortunate in having on ourprogram to-day Doctor Willier, whose intensely interesting original work isin the same general field. Leo Loeb4 has done a great deal of interesting workupon the general subject of transplantation and, in I930, published an exten-sive review of it. In this he records observations of many writers, in additionto his own investigations, and presents a philosophical discussion of the wholeproblem. He introduces a special terminology, using the expression "organismdifferential" to indicate the basic differences between different animals and"individuality differential" to mean the total of physical and chemical charac-teristics peculiar to the separate individuals of the higher animal series. Thesehe attributes to the specific genes and chromosomes. Except perhaps for iden-tical twins, no two higher animals are exactly alike in these endowments.There is a graded scale of increasing diversity beginning with siblings, ex-tending through blood relatives, variety, species and genera. Because of thisinherent endowment an individual possesses a chemical constitution to whichcells from any other source are alien in varying degrees, and in which theyprovoke a reaction that leads to their destruction. He does not believe thatthis inherent reaction can be modified or broken down by any method yetsuggested.

There has been, of course, an enormous amount of clinical work done onthe general subject of grafting, and a great deal on cross-grafting. Most ofthis has been confined to the reporting of the introduction of various tissuesinto human beings, with an overwhelming incidence of failure, but withoutserious effort to explain or prevent the failure. Among the few publicationsthat deal with underlying conditions affecting cross-grafts is Holman's5 workon protein sensitization in isoskin grafting. Holman doubts that cross-graftsof human skin are ever successful, finds that taking grafts from donors ofthe same blood group as the host makes little difference in the generally poorresults, and attributes these results to the chemical differences between thetissues of host and graft. He thinks the grafts serve as sources of foreignprotein intoxication, and lead to the development of a reaction like anaphy-laxis. He further cites observations that suggest that this reaction is highlyspecific against the donor tissue causing it, and does not affect grafts fromanother donor, which, however, soon develop their own specific destructivechemical reaction.

The control of the multicellular animal over its own cells is obviously aninstance of the general principle of homeostasis. However, it has received farless consideration, or, at least, efforts to study it have been far less successful

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than in the case of other mechanisms of homeostasis. The movements of water,salts and proteins between the blood, the lymph, the intercellular spaces, andthe cells themselves have been the subject of much study and impressiveresults. But who knows why the thyroid gland reaches an optimum size andthen stops growing? Why do certain tissues throughout life possess the powerof regeneration after injury, and other tissues have this power only in a verylimited way, or only in embryonic life? What stops the regeneration of tissueswhen they reach the optimum stage, and prevents abnormal overgrowth?There are many such questions that we have all asked ourselves many times.They lie at the root of this problem of cellular homeostasis. The investigativeattack on them has so far been very unproductive. We take refuge in specula-tive phraseology. We say an organ's size is conditioned by the physiologicneed for it. But what mechanism implements that- need, and what brakeskeep it in check? Is it a balancing of hormonal stimulants? Is tissue pressurea factor? Does the metabolic product of a certain type of cell exert an in-hibitory effect upon the growth of that type of cell? These things shouldbe susceptible of investigation, if we can evolve proper technic. This matterof normal balance and restraint of cells and organs has its highly practicalimportance as well as its biologic interest. It concerns the healing of everywound, the response of work hypertrophy on the muscles, the developmentof compensations for damages done to various structures. It also, of course,is fundamental to the whole cancer problem. Every pathologic new growthrepresents an escape of a special tissue from the general restraints of cellularhomeostasis. A great deal of study has been devoted to the discovery ofcarcinogenic agencies, and most valuable progress has resulted. Less attentionhas been given the defense mechanism against cellular overgrowth. Perhapsthe problem is much harder to grapple with. At the present time, one mightgeneralize the situation somewhat as follows: A normal cell is subject to thenormal restraints on its growth and reproduction. A greatly abnormal cell,badly damaged or perverted greatly in its internal mechanism, becomes essen-tially a foreign cell, and hence the victim of the normal defense reactionagainst alien cells and is thrown off or destroyed. But a cell somewhat modi-fied from normal by some agency-chemical action, radiation effect, or what-not-may be near enough to normal to escape the destructive reaction againstalien cells, yet sufficiently diverted from normal to escape the restrainingmechanism of normal cellular homeostasis. That is cancer. Should we notthen concern ourselves more with these two great basic protective mechanismswhich perhaps are really different phases of the same mechanism and tryto discover how they work? We may learn how to sharpen the body's dis-crimination against damaged cells, in other words, heighten its cancer resist-ance, if we know the method of discrimination against alien cells. It is helpful,no doubt, to state a problem, but far more difficult to solve it, and, so far, novery promising line of attack has been developed in the study of the mecha-nism of cellular homeostasis. Perhaps the approach may be less arduous toan understanding of the defense of the body against alien mammalian cells.

That the human body does possess such a defense mechanism against886

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these cells will be generally admitted. All the evidence of clinical medicineand experimental investigation indicates this. Is this a specific property or isit merely a special aspect of the general resistance of living organisms to for-eign substances? There are certain well known processes by which an animalreacts to such stimuli. An inert sterile foreign body may be encysted byfibrous tissue. An irritating foreign body, such as a chemically active sub-stance, or living cells of the bacterial or protozoan order, may induce and beresisted by a suppurative response. Certain other chemical substances maybe combated by a specific chemical response in the nature of an allergic orimmunity reaction. It has not been proved that the reaction to living mam-malian cells fits exactly into any of these pictures. Certain inferences, how-ever, nmay be made from the known facts. The increasing complexity ofstructure, function, and chemical internal environment met in the ascendinganimal series is parallel to the increasing difficulty of cross-grafting. Thisincreasing complexity is, no doubt, associated with more and more exactspecialization and individualization of the body cells to fit them to a highlyspecialized environment, and a corresponding further removal from the toti-potency ascribed to such cells as the ameba and the early fertilized ovum.These changes affect cross-grafting in two ways. The cells grafted from anothermammal have been highly adapted to their own original environment, andhence are less well equipped to adjust themselves to changes, even of com-paratively slight degree. On the other hand, the internal environment of thelhost animal is a highly complex affair that is provided with a mechanism tomaintain itself constant, which apparently includes defenses against evenslightly alien chemical substances, such as might be brought in by the graftingof foreign mammalian cells. When one recalls the size, complexity, andvariety of protein molecules, for example, it is quite conceivable that certainproteins in the living mammal and in its cells may exhibit slight modificationsthat are specific for that one individual, and that would excite intoleranceand reaction in other individuals, even of the same species. There are someobservations that suggest such a possible explanation of tlle resistance to cross-grafting. When human skin is grafted to another person, it often seems to dowell for a number of days, and then usually rapidly dissolves and disappears.This strongly suggests a defense mechanism that requires an appreciabletime to develop, and that is a direct response to the stimulus of the foreigntissue. In short, it looks like the development of something like a specificimmunity reaction. Such an explanation fits better than simpler reasons thatmight be given, such as lack of nourishment to the graft, inflammatory re-action caused by it, or incidental complications like bacterial infection. Ifthe defense mechanism proves to be in the nature of an acquired immunologicreaction, there are many further questions that arise. Is such a reactionmediated through the circulating body fluids or is it a property of the hostcells, or do both cells and fluids take part in it? Is it a local or a generalresponse? Is it specific against the type of grafted tissue alone, or is it equallyeffective against all tissues of the donor animal? Is it limited to cells fromthe original donor, or has there been set up a reaction that will now function

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promptly against the same type of cells from any donor? Most important ofall, if such a defense mechanism explains the intolerance of the mammal forforeign living cells, is there any way in which the defense may be brokendown or set aside? There is also a possibility that nerve relations may playsome part in the question of survival of cross-grafts. Indirect evidence of thismay perhaps be read into the experiments of Schotte and Butler.6 Theseexperiments indicated that when a limb of certain amphibian larvae is keptdenervated, the capacity for regeneration after injury is lost. Not only doesthe amputated extremity of such a limb fail to grow out anew, as it normallywould, but regression and resorption start from the level of the amputationand proceed proximally, often bringing about complete disappearance of thelimb. There is so much superficial resemblance in this process to what oftenhappens to foreign tissue grafts in adult mammals that it stimulates suspicionthat the lack of nerve supply to the implant might play a part in its failure.

Our work includes experimental studies on laboratory and clinical mate-rial. It is being carried on by a group of my associates on the Surgical Serviceof the Johns Hopkins Medical School-Drs. James C. Owings, George 0.Gey, Kenneth Pickrell, and Douglas H. Stone, and enjoys the hearty supportof Dr. Alfred Blalock, head of the department. As sufficient material is devel-oped, detailed scientific reports will be published. The purpose of this paperis to establish the problem and to lay down certain generalizations based onour own previous work and that of many others. The first point of emphasisis the effort to understand why implants of alien cells die, rather than thedevelopment of a specific successful grafting technic, which may be hopedfor as a later result. Certain conditions essential for the success of implantsdo concern the details of technique, however, and may be summarized as fol-lows, from one of our earlier publications7: (i) Implants must be small, a fewmillimneters in diameter, otherwise the central portion dies before it can bevascularized. (2) The technique must be surgically sterile. (3) The bed to re-ceive it must be near a good blood supply, but must not be too richly vascular,otherwise hematoma formation will choke the graft. (4) The bed should be ofloose structure, otherwise tissue pressure will compress the graft. These pointsmay be considered as established. Much more doubtful are two conditionsthat certain workers have thought to be necessary: (5) A physiologic needon the part of the host for the tissue grafted. (6) Anatomic correspondenceof the bed with the site from which the graft is taken, such as thyroid graftsinto the thyroid region of the host. So much for present knowledge of tech-nical conditions.

The large experience of many zoologists permits certain conclusions of ageneral biologic nature: (i) Resistance to alien living cells is lowest in thelower forms of animal life and increases as one moves upward in the phylo-genetic scale, this resistance being greatest in the mammalian group. (2) Re-sistance in any single species is lowest in the youngest forms and increasesfrom embryo to old age. (3) Grafts are more apt to succeed the youngerthe donor individual, the best source being embryonic tissue. (4) The wider

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the biologic diversity of host and donor the less likely is success of grafting,the best opportunity being between identical twins.

In addition to the technical and general biologic conditions that affect theproblem, there is finally the specific reaction of the recipient as related to thealien cells. At least two views have been advanced concerning the reactionof the host to the graft. One of these, on which much stress is laid by Loeb,4emphasizes the more or less specific local tissue response about the graft; andthe other regards the host reaction as essentially specific chemical in nature,similar to anaphylaxis or immunity. This brings us to the very kernel of theproblem, and our work is far from that stage of advancement that wouldpermit an answer as yet. However, enough has been accomplished to give uscertain impressions. Thus, studies of autografts of thyroid in dogs show widedifferences in grafted fragments made in the same way and at the same time.Some of these fragments are so well preserved as to look almost normal, andabout them there is very little infiltration with leukocytes, lymphocytes, orother wandering cells. A short distance away, another fragment may showdefective staining properties of the cells, breaking up of acini, and an ac-companying active infiltration with leukocytes and particularly lymphocytes.Still another fragment may be completely hyalinized and imbedded in granu-lation and early scar tissue. To us it seems likely that the inflammatory re-action is not a defense against the grafts, per se, but merely the usual phago-cytic response to damaged tissue. In other words, thie cellular local reactiondid not kill the grafts. Otherwise, all of them should be equally and simul-taneously attacked. But some of the mechanical or technical factors, perhapsfailure to establish blood supply in time, led to the death of certain fragmentswhich are then removed by the well-recognized inflammatory local reactions.In short, we would interpret the local tissue reaction, not as a defense againstliving grafted cells, but as a scavenger reaction against dead cells.

There are a number of observations that have a bearing on the possibilitythat defense against alien grafts is ini the nature of a chemical immunity inmany cases. It was with this idea in mind that the writer, and his associates.7some years ago, conducted a series of experiments. These consisted in grow-ing for a considerable time fragments of the tissue to be grafted in tissueculture, on media containing the serum and plasma of the anticipated host.In this way it was hoped to adapt the grafts to their future chemical environ-ment. Following these experiments on dogs, a number of human cross-graftswere performed in a similar manner, with apparently some definite success.8Others have undertaken similar experiments with conflicting results-somesuccessful, many not.9

It now appears to us, however, that a much better method is possible,and we are in process of developing it. As has been said, there have beenmany observations, both clinical and experimental, that alien grafts oftenseem healthy for a considerable time, perhaps two to three weeks, and thensuddenly disintegrate. Such a phenomenon does not fit into our ideas eitherof an inflammatory reaction defense or of an immediately hostile chemicalenvironment. In either, such form of resistance to the graft one would expect

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a prompt and continuous destructive effect. It does suggest that the graft isthe source of alien chemical substances, probably proteins that differ, perhapsnot greatly, from those of the host, but sufficiently to act as antigens. In re-sponse to this stimulus, a defense mechanism is set in motion that producesantibodies, not originally present, that in time lead to the destruction of thealien cells of the graft. There are a number of experiments that suggestthemselves as methods of testing the truth of this theory, and that we expectto carry out. There is also an obvious extension of our idea of adaptationof the graft to its environment that we also are planning to test. It consistsin grafting a host with a given tissue and awaiting the development of thespecific defense meclhanism. Then we shall attempt to adapt the same tissueto the nlow stimulated host; in otlher words, to immunize the graft againstthe immunized host. We regret that much of this work is still only in thestage of development and that specific detailed reports must be deferred tillsome future time. Whether any of these experiments prove fruitful or notin leading to concrete results, the purpose of this paper will be attainied ifan aroused interest in the general problem develops.

Let us take a little time to consider the importance of this field and theopportunity it offers at the present. It would be an error to assume thatother fields of surgical development have been exhausted; an error similarto that attributed to Pare, Billroth, Moynihan and Halsted, each of whomis said to have lamented the future of the young surgeons succeeding him,because all the great advances had already been made. Nevertheless, it istrue that many fields of surgery are already extensively developed. Inablative surgery, the operative removal of offending lesions or organs, thegreatest attainments have been achieved. Now that the skull and the chestare successfully invaded, not as a rare adventure, but as a routine, every-dayperformance, further progress would seem to be largely a matter of detail.No doubt better technique, better instruments, better preparation beforeoperation, and better after-care will greatly improve results, but there areno remaining great anatomic areas for the expansion of ablative surgery.Reconstructive surgery in its various subdivisions-plastic, orthopedic, pros-thetic-is still far from the stage of advancement that ablative surgery hasreached. Here there are constant announcements of new achievements, andwithout doubt there still remains much opportunity for original develop-ment. But for those seeking a new frontier, a wide virgin country for ex-ploitation, I would enlist your interest, your thinking, and your hard workin what may be called replacement surgery. As on every frontier, thegoing may be hard. As in every virgin territory, the clearing of the terrainmay be backbreaking and heartbreaking, with delays, disappointments, hopedeferred. It will call for toil and sweat, if not for blood and tears. Butthe promised reward is great. To take a single example, consider the possi-bilities, if we can overcome resistance to alien mammalian cells, in the wholegroup of endocrine deficiency diseases-a definite cure for parathyroidtetany, for myxedema, for Addison's disease, perhaps for diabetes, and possi-bly for other conditions not now understood. The replacement of entire

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organs, useful anid functioniing, is not inmpossible. The vasctular anid ureteralhook-up of a new kidney could be accomplished, if the kidney would liveafter it had been implanted. These are obvious potential results of a conquestof the resistance of the human body to alien cells, and if the imagination beallowed free rein, they are but the beginning. There is no need, however, toexaggerate the results that might follow such a success. My purpose isaccomplished if you give this plea the importance I think it deserves.

We hear much these days of military matters. There is a certain analogybetween the methods of scienitific attack and military offensive. The armyfeels out its opponent, finds weak spots, drives in a wedge here and a columnthere, develops a pincer movement, surrounds an era, and then reduces it.So in science, a lead in one field, an original idea in another, a new technique,a better instrument result in advances that stimulate each other. But in war,field tactics are not enough. One may win battles and lose the war unlessthere is a broad view, a general strategy. In science also, it behooves us tothink at timnes beyond the day's work and the immediate problem, to surveythe whole front and its hinterland. In such a survey, local issues will fallinto their proper proportions and the larger strategic problems will emerge.One of the functions of such a body as the American Surgical Associationis to act as a general board of strategy in its own field. The object of thispaper is not to record a local advance, but to bring up for consideration amajor problem-that of the reaction of the human body to its own and othermammnalian living cells. Perhaps such a purpose today may seem visionary,but let us not forget that often in the story of mankind the visionary of to-dayhas proved in some distant tomorrow to have been the man of vision.

REFERENCES1 Cannoni, WV. B.: The Wisdom of the Body. W. W. Norton, New York, 1932.2 Harrison, Ross G.: Heteroplastic Grafting in Embryology. Harvey Lectures, 1933-1934.3 Eastlick, Herbert L.: Study of Feather Characteristics in Limbs Transplanted Be-

tweeni Embryos of Different Bird Species. Proc. Nat. Acad. Sci., 25, 551, 1939.4 Loeh, Leo: Transplantation and Individuality. Physiol. Rev., IO, 547, I930.Holman, E.: Protein Sensitization in Isoskin Grafting. Surg., Gynec., and Obstet.,

38, IOO, I924.6 Schotte and Butler: Morphological Effects of Denervation and Amputation of Limbs

of Urodile Larvae. Jour. Exper. Zool., 87, 279, 194I.7 Stone, Owings, and Gey: Living Grafts of Endocrine Glands. Calif. and Western Med.,

38, 409; 39, IO; 39, 8i, respectively, June, July and August, 1933.Ident: Living Grafts of Endocrine Glands. Amer. Jour. Surg., 24, 380, May, I934.Idemii: Cross-Grafting of Endocrine Tissues. Surg., Gynec., and Obstet., 59, 683, Oc-

tober, 1934.Ideom: Transplantation of Living Grafts of Thyroid and Parathyroid Glands. ANNALS

OF SURGERY, IOO, 6I3, October, I934.Idemil: Living Grafts of Thyroid and Parathyroid Glands. Surg., Gynec., and Obstet.,

60, 390, February, I935.8 Stone, Owings, and Gey: Further Reports on Grafting of Endocrine Glands. Read

at meeting of Mid-South Post-Grad. Med. Assembly, Memphis, Tenn., Februaryi 6, I 938.

9 Houghton, Klassen, and Curtis: Studies in Human Parathyroid Transplantation. OhioState Med. Jour., 35, May, I939.

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