Twentieth Century History of Findlay and Hancock County, Ohio, and Representative Citizens, Part 131

Author: Jacob Anthony Kimmell
Publication date: 1910
Publisher:
Number of Pages: 1189

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Since every theory that has tried to explain the so-cal: tuberculin reaction is based on evidence that has denied t. possibility of sensitizing non-tuberculous animals with - rivatives of the bacillus, Baldwin's announcement, made i year ago, that he had succeeded in doing just this thing ": of the greatest importance. It is this work and further !:

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vwawo vi tuveremosis that I wish to go over with you ·night.

Three years before Baldwin, in 1907, Rosenau and Ander- 1 had intoxicated previously sensitized guinea-pigs with an tract of human tubercle bacilli, so that after a subcutaneous intraperitoneal injection a few of their animals showed Id symptoms of illness that consisted of restlessness, catching the nose, irregular respiration, tremor and weak- 38. They did not follow up this work with further experi- entation.

Baldwin sensitized guinea-pigs by the ordinary methods intraperitoneal or subcutaneous injections of a derivative the tubercle bacillus. As a rule his preparation was a ter-extract-filtrate of the micro-organisms. He obtained s by digesting at 50° C. in distilled water the powder de- ed from thoroughly washed, dried, pulverized cultures, that d been grown in broth media. After extracting the powder · a day or two, he filtered the supernatant extract-emulsion :ough a Berkefeld bougie. The filtrate was a material that s freed from bacilli and which very likely contained no ier organic constituents except bacillary proteins. Since organisms were very carefully washed when they were parated from the culture media it is hardly possible that any tein constituents of the broth were carried over with the illi except in very negligible quantity. By using this ma- ial the contingency of a broth protein sensitization was ided, and the results of the subsequent experiments could interpreted as being due to the effect of bacillary protein. If now a normal animal was treated with a single dose of a all amount of such a water-extract it was found that two three weeks later it would respond to a second or toxic ection of bacillary protein with all the symptoms of acute aphylaxis, dying as a rule in from three to six minutes. The terial that was used for this second or toxic injection was tally the unfiltered, supernatant extract-emulsion which I 'e already mentioned. This is the product which Koch cribed at T. O. It is an extract of the bacilli, and, since contains the ground-up bodies of the organisms, it is an ulsion as well : we have for this reason usually designated s extract-emulsion in our work.

.Ithough Baldwin and I found that a small amount of tu- ulo-protein would sensitize an animal if it was introduced almost any parenteral route, it soon appeared that not every was available for the second injection if toxic symptoms e to be elicited. Baldwin practised toxic injections by of the peritoneum and subcutaneous tissue without result, on account of operative shock he got obscure or question- results with intracerebral and intracardial injections. By g the postorbital method of injection, which had been al- y described by Gay and Southard, he succeeded in pro- ng acute anaphylaxis almost invariably in sensitized ani- . The symptoms were sharp and definite. For a minute > following the injection the animal was apparently well normal. It then began to scratch its nose, to cough nodically, and to go through "bucking" movements.

and in a very few minutes the animal was dead. Respiratory distress dominated the picture throughout the attack. At first deep and labored and perceptibly slowed, the thoracic ex- cursions became greater and greater and more and more de- layed, until approaching death was signalized by a few tre- mendous inspirations which toward the end were as much as thirty or forty-five seconds apart. There was no mistaking the character of the symptoms. They were an exact replica of what had been so often described as the anaphylactic shock caused by other proteins. Nor were the pathological findings any less typical. All the animals, that died in this way, had the large, white, blown-out lungs that Auer and Lewis had proved to be characteristic, the pathological anatomy of which has so recently been worked out by Schultz and Jordan. The epicardial hemorrhages to which attention was called by Gay and Southard were also a constant feature.

The amounts that Baldwin usually used were one or two cubic centimeters of water extract intraperitoneally to sensi- tize a full-grown guinea-pig, and one-half to one cubic centi- meter of extract-emulsion postorbitally to intoxicate it fatally. I found later that I could satisfactorily sensitize an animal with 0.01 cc. water extract given intradermically, bring on fatal anaphylaxis with 0.05 cc. extract-emulsion postorbitally, and cause symptoms with 0.03 cc. extract-emulsion post- orbitally. The last named quantities are represented by 0.05 mg., 0.9 mg. and 1.6 mg. dry weight respectively.

Such experiments showed us at once that we had succeeded in putting non-tuberculous animals in such a condition that in one respect they resembled tuberculous guinea-pigs : that is, that a fraction of a milligram of tuberculo-protein would make them violently ill. Let us see how the similarity worked out after further experimentation. Let us remember too, that up to this point our information comprised the following facts: the normal, non-tuberculous animal can tolerate the injection of large amounts of our tuberculo-preparations- 1.0 cc. extract-emulsion postorbitally, at least 5.0 cc. in- travenously, and at least 10 or 15 cc. intraperitoneally without effect; less than a milligram postorbitally will bring about an acute and fatal intoxication in a sensitized non-tuberculous animal; and a fraction of a milligram, introduced subcutane- ously or intraperitoneally, will kill a tuberculous guinea-pig with a focal and general reaction that comes on several hours after the injection of the antigen.

I have said that Baldwin failed to poison sensitized guinea- pigs by intraperitoneal injections. As a general thing it is im- possible to do so, but in a few very highly sensitive animals I . have succeeded in inducing symptoms of illness. I used moderate-sized toxic doses. A citation of a couple of the experiments will show what occurred.

ANIMAL 1 .- Sensitized 82 days previously. Injected intraperi- toneally with 1.5 cc. E. E. 161.

Result .- The animal seems normal and runs around for about 10 minutes after the injection. Then symptoms of illness begin to develop rapidly. It drags its hind legs, becomes very weak and lies down on its side. These symptoms continue for 10 minutes,

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when they begin to subside. Within a half hour after the in- jection the animal has almost regained its normal condition.

ANIMAL 2 .- Sensitized 126 days previously. Injected intraperi- toneally with 2.5 cc. E. E. 161.

Result .- No symptoms for 10 minutes. Then the animal be- comes weak and staggers around on the floor. The respira- tion is slow and labored with marked heaving of the chest. The animal slowly recovers from the symptoms.

Here in certain selected animals I get symptoms of illness from an intraperitoneal injection. The fundamental char- acter of the reaction must be the same as that which occurs when I inject postorbitally. But the reaction expresses itself differently. In the latter case, there is a fulminant process that ends in death in a very few minutes; in the former, there is a prolonged chain of symptoms that come on gradually, are much less severe and terminate in recovery. If, now, I make an intravenous, toxic injection on a sensitized animal, I find that it will have the same result as a postorbital one. If, however, I inject subcutaneously, I elicit no symptoms. I am taught, therefore, that given a sensitive animal, the nature of its response to a toxic injection of tuberculo-protein will de- pend altogether on my method of introducing the antigen. If the preparation enters the body in such a way that it will get into the circulation rapidly, acute and fatal anaphylaxis will result. If it is absorbed more slowly, there will either be no symptoms or the symptoms will tend to approximate the com- paratively gradual illness that occurs when we inject the tu- berculous animal subcutaneously. It further seems as though we have to do with an antibody that is present in the circu- lation which reacts with the antigen to set free a powerful toxin-the so-called anaphylotoxin of Friedberger. In fur- ther support of this idea that the antibody that is concerned in the production of the acute anaphylaxis is free in the cir- culation, is the circumstance that in some forms of protein anaphylaxis, the serum of a sensitized animal will render a second animal sensitive to the particular protein if trans- ferred to the latter.

After demonstrating that the non-tuberculous animal that had been treated with tuberculo-protein was unlike the normal animal but similar to the tuberculous animal in that it would react to very small amounts of this antigen, Baldwin showed further that a large proportion of tuberculous animals that had never before received tuberculo-protein would react to it exactly like the sensitized animals that were not tuberculous. If he injected such tuberculous animals subcutaneously or in- traperitoneally with extract-emulsion, they would suffer the · well-known general tuberculin reaction, which manifested it- self several hours later and ended in death if the dose was sufficiently large. If, however, he introduced his extract- emulsion postorbitally, many of these tuberculous animals would die in a few minutes of acute anaphylaxis and would show the typical lesions of the heart and lungs. Not all tu- berculous animals, picked at random, will react in this way and the reason why they differ from one another is not yet clear, but probably depends upon the nature and extent of the tuberculous foci, the conditions of absorption from them, and

the animals' inherent ability to create antibodies. But after: injecting say fifteen tuberculous guinea-pigs of one lot, I her- had sixty per cent show acute anaphylaxis.

This event brought out plainly that all that was necesse" to prove one point of similarity between tuberculous guines- pigs and sensitized, non-tuberculous ones was to treat es: kind with a toxic dose of antigen postorbitally-that is, i: a rapidly absorbable way. The first logical deduction was the: the tuberculous animal had a circulating anaphylactic art- body ; another, that it was identical with that of the sens- tized, non-tuberculous animal; a third, that it was produce. in response to the same antigen as in the latter; and a fouri. that the antigen arose in the tuberculous focus and na- probably the tubercle bacillus.

* * *

After these relations had been worked out the first probla that naturally suggested itself was to find out what wow !! happen to a non-tuberculous animal if it were treated wit tuberculo-protein like tuberculous animals that are unde: going tuberculin therapy. The specific treatment of tube- culosis after a long period of disuse and even disrepute bs: of late years been gradually coming into its own again. Cit- ician after clinician is now taking up the method, and de majority of capable observers are reporting strikingly gori: results in many cases. However, the action of the bacille" preparations so far as their therapeutic uses are concerta. still remains as mysterious as ever. Their application the- far has been purely empirical and limited only by the clinks! judgment of the practitioner. The general opinion of t .: latter has been, I believe, that the introduction of careful.i graded doses rouses the tuberculous organism to the ore: production of defensive substances-antibodies-that neutri- ize the poisons elaborated in the focus or attack the bac. themselves or perform both of these functions.

The idea that the bacillary products that are ordina. used contain primary toxins, does not seem tenable. If we ba. to do with such toxins, they should poison the normal, Die- tuberculous as well as the tuberculous animal. But we har: seen that extremely large doses are without untoward effi on the normal animals. If there are toxins, too, they sher." stimulate the animal organism to the production of antiter: in amounts directly proportional to the quantity of tubercu .- protein introduced. As a matter of fact, no one has eff: succeeded beyond dispute in producing an antitoxin to z derivative of the bacillus.

But how the picture changes when we consider animals thr have already absorbed antigen ! If an animal be infected es- perimentally it begins to react to tuberculin on or about the fifteenth day; in like manner, the non-tuberculous anins treated with protein will react to a second injection gir. about two weeks after the first. Both classes of animal: F. then be made violently ill by exceedingly small doses of => protein. What other explanation can we have but the: : both there have been produced antibodies that react with : tigen to set free a poison-the anaphylactic poison?

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e disease-the weakness, the anorexia, the tachycardia, the naciation, the fever that are so often observed. Indeed it is ore rational, in the light of our present knowledge, to as- ibe these to the continual absorption of antigen by an al- ady sensitized system. If an extract of the entire contents several tubes of cultures of tubercle bacilli will not make normal animal the least bit ill, why should the products of mparatively few bacilli, as many as would be enclosed in a bercle the size of a walnut, bring about so severe an intox- ition of an animal? It is hard to explain this on the toxin ea, but the conception of hypersensitiveness makes it much arer.

The method of tuberculin therapy that is in most common e to-day is that of treating the individual with small doses d gradually raising them until the patient can tolerate quite ge amounts without reaction. Every therapeutist comes to ilize that there is no exact guide in this treatment and that ; sole index in proceeding with tuberculin injections is the inner in which the patient responds to them. He finds that cannot even predict which particular case will do well and ich one will lose ground under treatment. He learns that far as the results are concerned each individual patient is aw to himself; and that, if the latter holds his ground or proves, it is probably safe to continue with tuberculin, but his health retrogrades it is better to discontinue it. Out the tremendous mass of conflicting evidence that clinicians ve given us, I think that several generalizations can fairly made. These are that the chronic, afebrile case, who is in od condition and whose disease is not too far advanced, is : most favorable case for treatment; that such a patient's dition is often improved by tuberculin therapy after he long ceased to show improvement under the other well- own methods of treatment; that those patients do best who : tolerate relatively large amounts of tuberculin, and con- sely, those patients can best tolerate tuberculin who are st free from symptoms of their disease; and that in many es the patient's tolerance to tuberculin is apparently greatly reased by spaced, gradually increasing doses of the antigen. d it is this latter principle-to diminish hypersensitiveness 1 its consequent intoxication-that has perhaps been the ling aim in therapy.

therefore attempted to imitate the conditions of tuber- n therapy on non-tuberculous animals and to follow the ilts so far as sensitiveness was concerned. I wished to find whether I could render them tolerant or immune to tu- ulo-protein. In the experiment I used thirty-six normal lea-pigs, which I divided into three sets of twelve each. h set received spaced, gradually increasing amounts of r-extract at various intervals of time-one set every two , another every four days, and a third every seven days. r they had reached fairly high dosage the animals were ved to go without treatment for from seven to sixteen , when they were given postorbital injections of extract- Ision. The animals that were treated every two days

nine days were getting 5 cc. intraperitoneally ; those treated every four days began with 0.25 cc. subcutaneously and ended on the thirty-ninth day with 4 cc. intraperitoneally; those treated every seven days began with 0.25 cc. subcutaneously and ended on the thirty-ninth day with 4 cc. intraperitoneally. The first set received a total of 25 cc. W. E. in seventeen doses ; the second, a total of 13 cc. in ten doses; the third, a total of 8 cc. in six doses. Seven days after treatment stopped, I tested several members of each series postorbitally, and again sixteen days afterwards. All of the animals except one were very sensitive to the ordinary toxic doses for sensitized guinea-pigs (0.75 cc. E. E.), while most of them reacted strongly to very small doses (0.15 cc. to 0.3 cc.). The general result was to indicate that after repeated doses sensitiveness was much greater than after single injections. There was no evidence that tuberculo-protein toleration could be obtained in non- tuberculous animals by repeated dosing,-there was, at least, no noticeable tolerance seven days after treatment stopped. A further endeavor was then made to discover whether there was any tolerance whatever for a very short time after regu- lar treatment with the protein. I therefore repeated the ex- periment I have just cited, making the toxic, postorbital in- jection three days after the last subcutaneous treatment. I thus tested four animals and found them all sensitive, three of them fatally so. I have since treated many animals with more than a single sensitizing injection and have found that the best way to attain a very high degree of sensitiveness is to give them repeated injections of antigen-and that the larger the individual doses of antigen, the greater the degree of sen- sitiveness.

These experiments led to two conclusions; namely, first, once sensitiveness to tuberculo-protein is acquired by a non- tuberculous animal, the repeated absorption of protein tends only to increase this condition; and second, tolerance to tu- berculo-protein cannot be attained in these animals by grad- ually raising the dosage or diminishing the intervals of time between doses. Other experiments, which I shall cite later, showed that perceptible sensitiveness persists for a long time after a single injection-often for more than a year-but that there is a tendency for it to diminish as time goes on unless it is renewed by further applications of antigen.

The results of the experiments which I have just described do not seem to square with the clinical idea of the method of gaining tolerance in the tuberculous, and it is this contra- diction to which we must now address ourselves. As the first step, let us examine the nature of the different forms of sen- sitiveness as they exist in the tuberculous organism.

If we treat the skin with a derivative of the tubercle bacil- lus either by applying the latter to a break in the surface or injecting it between the layers of the skin, we find that some hours afterwards an inflammation will occur at the site of or in the immediate neighborhood of the injection. All of our present knowledge indicates that this inflammation is the ulti- mate result of an interaction between the injected antigen and antibodies that are present at the point of injection, and that

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these antibodies are produced by the diseased organism in re- sponse to antigen that has arisen from the tuberculous focus. According to von Pirquet's view all of the cells of the body take part in this response to stimulation by antigen. At the Saranac laboratory we have never been able to elicit a skin reaction in guinea-pigs unless the animal has tubercles caused by living or dead bacilli; that is, a first injection of extracted protein will not sensitize to a later skin reaction. Moss, however, has found that individuals sensitized with horse serum acquire a skin sensitiveness to the serum. The possi- bility of sensitizing the skin by injecting an animal with pure tuberculo-protein is therefore not so remote if we once hit upon the proper conditions; and we are still working along this line.

If a small amount of tuberculo-protein enters the body sub- cutaneously or intraperitoneally or even intravenously (pro- vided in the last instance that not too much anaphylactic an- tibody is present in the circulation) the tuberculous focus will suffer a reaction. This reaction also comes on several hours after the injection and expresses itself symptomatically by evi- dences of increased activity of the disease process, such as cough and expectoration in pulmonary tuberculosis, and ana- tomically by inflammation. As in the skin reactions, this in- flammation is also most likely the end result of the union of antigen and anaphylactic antibody at the focus. Now the inflammation of the focus introduces a factor altogether dif- ferent from any that can exist in the sensitized, non-tuber- culous animal. By bringing about circulatory changes and soft- ening the focus it renders the absorption of the focal material much easier than it was before. This focal material thrown rapidly into the circulation then reacts on an organism that has already been sensitized to it during the course of the dis- ease and brings about an acute type of illness, the so-called general fever reaction, the manifestations of which are not at all unlike those met with in any acute exacerbation of the disease. Indeed, there is great similarity between what occurs during the general reaction and the course of events in or- dinary acute tuberculosis. In both there are signs and symp- toms of increased inflammatory focal changes: in both, the symptoms are very much alike. I have already said that the conception that the symptoms of tuberculosis are due to the absorption of focal material is more reasonable than the pri- mary toxin idea; and it is evidence such as this that leads me to think that the general reaction is a phenomenon that de- pends upon what happens at the focus after a tuberculin in- jection. I am at the present time engaged in experiments in an attempt to discover what the sensitizing material of the focus is: whether it is the bacilli or the necrotic material or a substance that is formed by the effect of one acting on the other. My results are still very incomplete, but I may say here that I have not been able to confirm Kraus and Volk's observation that extracts of focal material are primarily toxic for guinea-pigs while those of normal organs are not.

Besides these three forms of reaction there is the so-called puncture reaction, which is essentially a local reaction and similar to the skin reactions, and the acute anaphylactic re- action, which already discussed.

Let us take such a tuberculous animal with local, focal ex: what I may call acute anaphylactic sensitiveness and wander into more or less hypothetical ground. If we should inje: it subcutaneously with tuberculin we might get & punctu: reaction and no other perceptible response to the injectie Here I should say that at the point of injection there w: enough anaphylactic antibody present to bind all or most e: the antigen, so that if there was a residuum of the latter i: was not enough to combine with sufficient antibody to care reaction elsewhere. Most of the protein would be neutralize. as it were, at the surface of the body. If, however, the p ripheral antibody were absent, or small in amount, or if the doses were considerably larger than what the peripheral ant. body could take care of, the antigen would get into the circ. lation. Here it would meet with circulating anaphylactic an :- body if the latter were present. The acute anaphylactic shoef would not result, however, because on account of slow absor- tion the units of antigen would be picked up one by one, and we have already seen that the fulminant symptoms are on. brought on when we introduce the antigen in such a mante: that it gets into the circulation very rapidly. Whatere: antigen is bound by circulating anaphylactic antibody be comes incapable of reacting with the anaphylactic ant: body in the focus and thus the focus is protected to : degree that is directly proportional to the amount of cir- culating antibody that exists at any one time. We should bare here a very unusual condition. The circulating anaphylact: antibody would be a great menace to an animal if the antiges were injected intravenously, but it is just as great & bulwark against focal and general reactions if the antigen is so injecta! that absorption is slow. With the combination proceeding gradually unit by unit all the circulating antibody is used up without the production of symptoms, and the only way this the focus will be affected will be by the unbound residua! antigen. Thus we should have two zones of defense, which I may call peripheral and circulating zones; and the contin- gency of a focal reaction would depend upon quantitative relations between peripheral and circulating antibody and antigen, as well as upon time relations that would govern the binding of the two opposing substances, which in turn would depend largely upon the method of injection and the degne of solubility of the antigen that is employed. I have ap- proached this matter of absorption experimentally in the fol- lowing manner. I first injected sensitized, non-tuberculous guinea-pigs with very large doses of extract-emulsion intra- peritoneally and followed this up some time later with & post- orbital injection. When the second injection had been given some time (one-half to one hour and a half) after the first injection it very often failed to bring on the anaphylactic symptoms, showing that the circulating antibody had disep- peared. These observations have been sufficiently controlled to be of some value, and I have experiments now under way in which I am trying to establish the time relations more definitely.

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Twentieth Century History of Findlay and Hancock County, Ohio, and Representative Citizens, Part 131

Author: Jacob Anthony Kimmell Publication date: 1910 Publisher: Number of Pages: 1189

Author: Jacob Anthony Kimmell
Publication date: 1910
Publisher:
Number of Pages: 1189