Twentieth Century History of Findlay and Hancock County, Ohio, and Representative Citizens, Part 132

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

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But there is a third defensive zone with which we have to do. This is of a mechanical nature and is comprised in the extent of fibrosis that any given tuberculous focus has undergoli.

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fully than if no such protection exists. As healing occurs, and more and more scarring takes place, and the tubercle be- comes less and less vascular, it certainly must happen that less extraneous matter can be carried into the tubercle. This factor must surely be of prime importance in many cases. We know that as a general thing the longer a given case has been healed and the more completely a lesion has been closed in, the larger our dose of tuberculin must be to induce reactions. There are some exceptions to this rule but I believe the gener- alization can safely be made. Now the non-tuberculous ani- nal, once sensitized, gradually loses this condition unless we nject more antigen-in other words, its circulating antibody diminishes in amount. The case that has been healed for a ong time would so far be comparable to the former. We should assume, therefore, that it had less circulating anaphy- lactic antibody to protect its old focus. The tubercle, how- ever, has a more or less efficient protection of its own, namely, its fibrous capsule.

We now begin tuberculin therapy with a tuberculous animal, such as we have been considering. If the tissues of the tuber- culous animal respond to injections of tuberculo-protein like hose of the non-tuberculous animal-and I see no reason why they should not-they will gradually heap up anaphy- actic antibody in the circulation. If there is very little of ;his antibody to begin with, or if the focus is naked, as it were, with but slight fibrosis, we have manifestly a more delicate ask ahead of us than if the reverse is the case. We can con- ceive of several possible conditions of the patient at the time le starts treatment. He may have much circulating antibody with a well invested tuberculosis, or much circulating antibody vith more or less softened tubercles, or little circulating anti- Jody combined with either focal condition. It is therefore lot hard to foresee how every case may respond in its own vay to tuberculin. But if we have a favorable case to begin rith, the tendency would be, as stated, to stimulate the organ- 3m to the production of circulating antibody, which, as we ave assumed, protects the focus against subsequent doses of ntigen and is one of the factors in the so-called acquired lerance after treatment. I now have experiments under way > test the validity of this hypothesis. By repeated doses of ntigen I sensitized normal animals to a high degree. I have itely rendered these sensitive animals tuberculous, together ith normal, non-sensitive control animals. In a couple : months, after the disease is well established, I shall give th sets lethal doses of tuberculo-protein subcutaneously. or traperitoneally. If my hypothesis is correct I shall expect

find the initially non-sensitive animals reacting focally to haller doses than those that were sensitized before inoc- ation.

The other factor in the production of tolerance in therapy ould enter in the direct action of the tuberculo-protein on e focus itself. Since we know that tuberculo-protein in- mes the tubercle, it is more than likely that the end result every dose that reaches the tubercle, no matter how small,

sient ny peræima to the most intense Inflammation of the focus. It seems quite reasonable that slight inflammations could be of real service in assisting the healing process of the tuber- cle and many clinical observations, which it is unnecessary to go into here, support this view. Certainly by the increased stimulation of fibrous reaction the scarring process would be hastened. The amount of tuberculo-protein which would react with the focus would depend upon the conditions that I have already mentioned. With a good defense of circulating anti- body the focus would pick out only the residual tuberculo- protein and would be saved from the dangers of too violent a reaction-the excessive inflammation and a dissemination of the disease that might result were the tubercle subjected to all the protein of a given dose. Hyperæmia and inflam- mations during the course of treatment might be so slight as to be imperceptible, yet their end effects-the resultant focal sclerosis-might be marked. And as the focus gradually becomes closed in during the healing process, the third zone of defense that we have considered would become strengthened and assist appreciably in producing tolerance to the antigen.

I think that you have already become impressed by the fact that the problems of the tuberculin reaction and of tu- berculin therapy are extremely complicated ones and that any adequate theories of them must take into consideration a great many factors. In the above recital I have tried to do no more than elaborate a working hypothesis that is based on well au- thenticated clinical observations and on experimental work that has been carried on at the Saranac laboratory. I have tried to separate what has been demonstrated from what re- mains yet to be proved before these conceptions can be lifted from the realms of hypothesis to those of theory. And if I have been successful in suggesting fruitful paths of experi- mentation I shall be more than satisfied.

* * *

A problem of the first importance is what relation the hyper- sensitiveness to the products of a given microorganism bears to susceptibility to infection from it. It has long been known that during the course of an infection the diseased individual becomes sensitive to the organic products of the infecting agent, but thus far we have had practically no experimental information concerning the part that the sensitive state plays in the defensive processes of the organism against bacillary implantation. It was our problem, therefore, to discover whether the tuberculo-protein sensitized animal acquired increased or lowered resistance to infection by the tubercle bacillus.

I accordingly undertook experiments in which I aimed, first, to sensitize normal animals; next, to inoculate these together with normal, non-sensitive controls; and finally, to sacrifice the whole number after the tuberculosis was well established and compare the progress of the disease in the initially sensi- tive animals with that in the control animals. The animals that I sensitized before inoculation comprised forty-two guinea-pigs which were divided into four sets. The members

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of set A received a subcutaneous or intraperitoneal injection of water-extract every two days over a period of thirty-nine days in which they got a total of 25 cc. Those of set B were similarly treated every four days and received a total of 13 cc. in the thirty-nine days. Each animal of set C, treated every seven days, had in all 8 cc. during the thirty-nine days. The individuals of set D were given only one sensitizing injec- tion,-1.25 cc. of water-extract intraperitoneally. There were originally twelve animals in set A, twelve in set B, twelve in set C and six in set D. Sixteen days after the preliminary treatment of sets A, B and C ended, I tested individuals from each lot and found that a high degree of sensitiveness to extract-emulsion existed in all of the tested animals. At the same time I gave two members of each set A, B and C sub- lethal doses. These last named animals all showed severe anaphylactic symptoms but did not die. I did not test the animals of set D, because I had sensitized animals in a similar manner many times previously and had always found them hypersensitive.

I was now ready for my inoculation. For this purpose I took six animals, that were sensitive but had not been tested, from each of the sets, A, B, C, and D; two from each of the sets A, B, and C that had received a toxic postorbital injec- tion an hour before, had shown symptoms but had to a cer- tain extent recovered from them; and six normal non-sensi- tized animals as controls. I thus had six different lots for inoculation, made up as follows: Lot 1, six guinea-pigs that had been sensitized, then given sub-lethal toxic injections, and were more or less intoxicated at the time of inoculation. These animals were in a refractory condition to further immediate injections of protein, since their first non-lethal toxic injection protected them against a second. Lot 2, six guinea-pigs, sen- sitized by treatment every two days, and not given toxic in- jections. Lot 3, six guinea-pigs, sensitized by treatment every four days, not given toxic injections. Lot 4, six guinea-pigs, sensitized by treatment every seven days, not given toxic in- jections. Lot 5, six guinea-pigs, sensitized by one injection thirty-seven days before inoculation. Lot 6, six normal un- treated guinea-pigs.

I injected each of the above animals in the same spot sub- cutaneously in the right groin with one-tenth of a cubic cen- timeter of an emulsion of human tubercle bacilli prepared as follows : Several spadefuls of a beef serum culture were rubbed up in a mortar with a minimal amount of physiological salt solution until the mixture was a fairly homogeneous paste. This was then diluted and centrifugalized several times. The super- natant emulsion was pipetted off, further diluted and again centrifugalized. The resultant supernatant emulsion was then filtered several times through cotton. This filtrate was the final emulsion that was used for inoculation. Examination of the filtrate showed no clumps and an average of but one tubercle bacillus to every one or two fields of the oil immer- sion lens. Before inoculating each animal the beaker contain- ing the stock emulsion was given the same number of whirls to ensure an even mixture and but one-tenth of a cubic centi- meter was withdrawn at any one time.

Sixty-two 1 .... afterwards the animals were killed and their

condition noted. The results of the inoculation can be sur- marized as follows : "

Refractory Animals, 5 guinea pigs .- Total weight at time c. inoculation, 1795 grams; average weight, 369 grams; total weight: 62 days later, 2200 grams; average weight, 440 grams; average net gain, 81 grams. None pregnant. There is advanced tuber culosis in all. The lungs and spleen are involved in every anim' and the liver in two.

Sensitized Animals, treated every 2 days, 6 guinea pigs .- Ton weight at time of inoculation, 2265 grams; average weight, 3. grams; total weight, 62 days later, 2690 grams; average weight . 448 grams; average net gain, 71 grams. Three were pregnan: The disease has reached the spleen in only one of the six and bx advanced no further in any.

Sensitized Animals, treated every four days, 6 guinea pigi .- Total weight at time of inoculation, 2100 grams; average weight: 350 grams; total weight, 62 days later, 2650 grams; avere weight, 442 grams; average net gain, 92 grams. Two were preg nant, one of which littered 31 days after inoculation. The splet is involved in three of the six guinea pigs. The process ha- affected other viscera in only one which was pregnant and : which the lungs were diseased.

Sensitized Animals, treated every 7 days, 6 guinea pigs .- Total weight at time of inoculation, 2185 grams; average weight, MA grams; total weight, 62 days later, 2950 grams; average weigh: 492 grams; average net gain, 128 grams. Two were pregnac: The spleen was involved in three, and the lungs in two of the three.

Sensitized Animals, given only one dose, 6 guinea pigs .- Tota' weight at time of inoculation, 2140 grams; average weight, 35% grams; total weight, 62 days later, 2680 grams; average weight 447 grams; average net gain, 90 grams. One was pregnant. Th spleen was diseased in every animal and the lungs in two.

Control, Non-sensitized Animals, 6 guinea pigs, 5 autopsied .- Total weight at time of inoculation, 2180 grams; average weight: 436 grams; total weight 62 days later, 2830 grams; aversg- weight, 566 grams; average net gain, 130 grams. None wer pregnant. The spleen was involved in four of the five animal and the lungs in two.

So far as known, all of the pregnant animals became pre !- nant after inoculation. Macroscopic tuberculosis is meant is the above descriptions when an organ is said to be involved.

The refractory animals suffered most. The disease w2: pretty well disseminated in all of these and they exhibited far more tuberculosis than any of the animals that had not been intoxicated and than any of the controls. It will be remem- bered that these refractory animals were given toxic injection: about an hour before inoculation. The widespread extent : the process was very likely due to the fact that the bacili were implanted in animals that were still more or less poisone. at the moment of infection. And it would appear that the animal organism is less resistant to infection than normal during the period that it is undergoing symptoms of anaphy- laxis or before it has completely recovered from their effects. Whether this event is specific or whether toxicity from on- bacterio-protein would fail to render an animal more su :- ceptible to infection by another microorganism is a matter that has not yet been determined. We know that some infa- tious diseases undoubtedly increase the susceptibility t- tuberculosis : or, at any rate, that tuberculosis often active !:

2 Details of these experiments are to be found in an article ir the Journal of Medical Research, 1911, XXIV, 2, 361.

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po zowy an important to und out whether this dimin- ished resistance to tuberculosis is due to the intoxication that is brought about by hypersensitiveness to another bacterio- protein, and I am now trying to solve this problem by experi- mentation.

The animals that were sensitized in various ways all became diseased. As a general thing, we may say that the more pro- tein the animal received during preliminary treatment, the less the resultant infection. So far as I could tell from the toxic symptoms of the tested animals, there was very little difference in the average degree of sensitization between mem- bers of the several sets. Nevertheless, the reaction is a gross one and the mere fact that animals from three different series reacted fatally does not imply that their degree of sensitization is the same. You will easily see that if one thousand anti- body units, say, or 0.3 cc. of antigen, were enough to produce death in a given guinea-pig, then ten thousand antibody units or 0.5 cc. of antigen would bring about the same result in an- other guinea-pig. Both die but are not equally sensitive, for guinea-pig B would have ten times as much antibody as guinea-pig A. We can, however, determine the degree of sen- sitiveness by graduating the dose of antigen which would be the constant. The guinea-pig would be the variable and its symptoms in response to a fixed, moderate dose of antigen would determine its sensitiveness. Unfortunately in these experiments I did not have enough animals to determine these variations. Since the preparation of an article that has lately been published and in which I assumed that the animals of the different series were probably equally sensitive, I have had occasion to deduce. from a number of experiments that sensi- tiveness does fluctuate-tending to die out, though very slowly indeed, if the antigen is not renewed and being markedly in- creased by repeated injections. I am glad therefore to take this opportunity to qualify my former assumption and point out its fallacy.

However, it is very likely that the differences of tuberculous nvolvement were altogether independent of any degree of 'aised or lowered resistance conferred by the sensitive state, out that they were due to a heightened immunity that followed he protein injections and was brought about by other un- nown factors. The changes that occurred in those guinea- vigs that had only one sensitizing dose support this view. These were inoculated thirty-seven days after sensitization. Experiments covering a period of two years have shown us hat thirty-seven days after an intraperitoneal injection of .25 cc. of water extract, a guinea-pig is almost invariably atally sensitive. So we are pretty safe in assuming that at ne time of inoculation these were highly sensitive. Yet these one dose " animals suffered in just about the same measure 3 did the controls. The one dose of water-extract was not ifficient to make them immune to infection. But it was rough to sensitize them, and while sensitive they contracted >out as much tuberculosis as did the normal animals. This idence would therefore strengthen the opinion that sensi- zation to a bacterio-protein of itself does not convey im- unity to infection, and also that hypersensitiveness and in-

experiment also shows definitely that they can exist side by side in the same animal.

But we must remember that in our experiments we have very likely introduced into the animal body relatively large amounts of the infecting agent compared to what is taken in under natural conditions of infection. It is conceivable that the degree of sensitiveness that would express itself by fatal anaphylaxis might confer upon the animal the capacity to get rid of ten or fifty or one hundred tubercle bacilli, numbers that probably represent the amount of infecting material that is naturally introduced more nearly than the number contained in our inoculated emulsion. The question is certainly an open one and the point must be emphasized. At the Saranac laboratory we are now employing the more exact method of Barber in our inoculation of sensitized animals. This as you know consists in counting out the living microorganisms one by one, and thus enables us to infect with any desired number from one up. By the use of this method we hope to determine definitely whether sensitization carries with it any increased resistance to infection.

There remains one phase of our work on sensitization which I should like to bring before you. This is the matter of the inheritance of tuberculo-protein sensitiveness. Most of you know that Rosenau and Anderson first demonstrated that sen- sitiveness to horse serum was transmitted from the female guinea-pig to its offspring and that the male parent has no in- fluence in this transmission. Their work has been confirmed by Gay and Southard, Lewis, and Otto. Schenck has brought forward some evidence that serum sensitiveness may also to a slight degree be handed down from the male parent. In his work on tuberculo-protein anaphylaxis, Baldwin found that the law of maternal transmission also held good with this bacillary derivative. Guinea-pigs, the offspring of actively sensitized mothers, reacted when one or two months old.

Although Baldwin was unable to show in a few animals that the tuberculous mother, which has not been treated with tuberculo-protein, transmits sensitiveness to her young, the chances are in favor of the possibility that in some instances she actually does so. You will remember that a certain pro- portion of tuberculous animals react to postorbital injection of bacillary protein with acute anaphylaxis in exactly the same manner as non-tuberculous sensitized ones; and that the simi- larity of the reaction compelled the conclusion that the reaction is in both instances the resultant of the same factors; namely, an interaction of antigen and circulating antibody. Since therefore the non-treated tuberculous animal which will react and the treated, hypersensitive animal are in exactly the same anaphylactic condition which results from the same funda- mental cause, it would seem certain that in some cases tuber- culous females would bear offspring that are perceptibly sen- sitive. This would necessarily follow in an animal that would conceive and carry young to term during a period in which she herself had considerable circulating antibody and was very sensitive. Thus far in our laboratory we have come across no

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sensitive offspring of tuberculous mothers, but we are still testing all such young, in the belief that sooner or later we shall encounter a sensitive guinea-pig.

There is probably no other disease that has occasioned 80 much discussion in regard to hereditary influences as tubercu- losis, and any addition to our knowledge on this subject further than mere surmise is manifestly important. Bald- win's discovery of the maternal transmission of tuberculo- protein hypersensitiveness is, I believe, the first satisfactory evidence of the inheritance of any specific property in relation to tuberculosis. While we do not yet know what part the hypersensitive condition to bacterial products plays in im- munity-whether it protects against natural infection or lays the animal organism more open to invasion-it seemed worth while to try to find out how constantly the tuberculo-protein sensitized mother will transmit the condition, and how long the animal that is born sensitive will retain the anaphylactic state. Because, too, of the logical inference, as outlined above, that under certain conditions tuberculous mothers must con- fer a sensitiveness on their offspring that may be perceptible or imperceptible, I thought it well to continue Baldwin's ex- periments.

I tested a total of sixty-one young, giving them single post- orbital or intravenous injections of extract-emulsion. All were the offspring of presumably sensitive mothers which were not tuberculous. When they were tested they varied greatly in age as well as in the time that had elapsed between the mother's last dose of antigen and the birth of the different animals. I shall not burden you with the details of the ex- periments, which may be found in another publication, but shall merely state their results, which may be summed up as follows :

1. The possibility of maternal inheritance is more or less irregular and inconstant.

2. It depends largely on the degree of sensitiveness of the mother at the time of pregnancy.

3. The degree of sensitiveness of the offspring as a no- varies directly with that of the mother.

4. The mother's sensitiveness is heightened by repeat: applications of antigen, and, conversely, tends to die out wi2 time unless renewed by subsequent injections.

5. The degree of sensitiveness of the offspring according: varies, depending on the time that has elapsed between t: mother's last dose and the birth of the offspring.

6. The degree of sensitiveness that an animal inherits ten's to diminish as it increases in age and size.

7. Guinea-pigs have been born sensitive 379 days afte their mother's last injection of antigen. They have also t- mained sensitive as long as 404 days after birth, although the: had never been tested before.

8. Animals of the same litter may vary greatly in the de gree of sensitiveness which they inherit.

9. The fact that a number of animals were born sensitive over a hundred days, and several 379 days, after their mothers last injection, is direct evidence that the transmission may ? one of anaphylactic antibody and not of the antigen.

10. The transmission by inheritance is probably always !: mainly one of antibodies.

11. Hypersensitiveness to tuberculo-protein is most likely never handed down to the third generation. This conclusion is based on tests on nine animals, the grandchildren of sens- tized females through the female line.

All this evidence concerning inheritance will probably be . more tangible value after we find out what rôle hypersensitive ness plays in infection. Meanwhile it furnishes encourage- ment for more experimentation in this direction.

In the above recital of experimental work performed an. in the discussion of where such investigations lead, I have doce little more than state the problem of tuberculo-protein hype :- sensitiveness in its relation to several of the phases of tuber- culous infection. I thank you heartily for the forbearance with which you have followed me, and trust that the futur: will bring forth still more fruitful results than the past.

VARIATIONS IN THE LEUCOCYTE COUNT IN NORMAL RABBITS, IN RABBITS FOLLOWING THE INJECTION OF NORMAL HORSE SERUM, AND DURING A CUTANEOUS ANAPHYLACTIC REACTION. By W. L. Moss, M. D., Associate in Medicine, Johns Hopkins University.

AND G. L. BROWN, M. D., Research Worker.

(From the Research Laboratory, The Phipps Tuberculosis Dispensary, The Johns Hopkins Hospital, Baltimore, Md.)

The observations to be reported in this paper were made in connection with some recent work of Knox, Moss and Brown (The Journal of Experimental Medicine, 1910, XII, No. 4) on a local anaphylactic reaction in rabbits. Healthy rabbits were sensitized by the intravenous injection of normal horse serum in doses of 0.1 cc. to 5 cc. After an interval of

12 days or longer their sensitiveness was tested by an in- tradermal injection of 0.01 cc. normal horse serum giren is the skin of the abdomen. A positive reaction consists .. localized inflammation and swelling, varying from 0.5 cm. 1: 2 cm. in diameter which appears within 24 hours and usually subsides in three or four days.

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Twentieth Century History of Findlay and Hancock County, Ohio, and Representative Citizens, Part 132

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

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