Experimental Biology and Medicine

In the blood of an experimental monkey (Macacus rhesus), Dr. Richard Lamar found on October 28, 1911, an actively motile trypanosome. This discovery led to my examining the blood of all of the monkeys at the Rockefeller Institute.

In examining 130 monkeys, 28 were found infected with trypanosomes. The infected monkeys had been used for experiments between July 17 and October 6. Six monkeys used on or before July 17 were negative, and 80 others, some normal, the rest used after October 6, were also negative.

With but one exception, all of the monkeys examined belonged to the Macacus rhesus species.

The trypanosomes found in the 28 monkeys were apparently of the same kind. They have been successfully inoculated into one monkey (Macacus rhesus), six mice, two rats, one guinea pig, and one young rabbit. In none of these animals has a rich infection been seen. The two rats were infected on the 8th day, the six mice between the 9th and the 52d day, the guinea pig and rabbit on the 16th day, and the monkey between the 16th and the 24th day. The trypanosomes do not appear to be very pathogenic.

The micronucleus is usually at the estrerne posterior end of the parasite, is strikingly large, measures 1μ or more in diameter, and often projects on either side of the parasite. The nucleus is oval, measures 1⅔ to 2⅔μ in its long diameter, and is situated near the juncture of the anterior ⅓ and the posterior ⅔ of the body. The flagellum is very long, the free part measuring 10 to 12⅓μ. The entire length of the parasites thus far measured has varied between 25 and 28μ, the breadth between 2 and 2¼μ.

That the injection of a suitable quantity of homologous tissue induces in susceptible mice a certain degree of resistance to the inoculation of their transplantable cancers is a well-established fact. There appeared recently a paper by Woglom 1 in which experiments were described showing that autogenous tissue (spleen) injected subcutaneously was also capable of inducing this immunity. Woglom's work has been questioned by Apolant 2 who repeated the experiments with negative results. Apolant maintains further that the spleen of a mouse does not afford sufficient tissue for immunization, and that the question as to the possibility of immunizing with an animal's own tissues is still an open one.

In the experiments herewith reported, blood has been used as the immunizing agent. The quantity of defibrinated blood necessary for immunizing young mice was shown by Bashford to be about .3 c.c. In order to eliminate, however, the possibility of insufficient dosage, it was considered desirable to use at least .5 c.c. As Woglom stated in his paper, mice cannot be bled this amount plus the loss attending the bleeding without causing death. Interval bleedings from the jugulars were therefore resorted to. In nearly all of the experiments two bleedings only, on successive days, were necessary. About ten drops of blood in citrate solution were taken each time. The corpuscles were preserved in the ice box and injected subcutaneously on the day after the last bleeding. Ten mice were treated in this way: ten controls were injected with a similar quantity of homologous blood; ten normal mice were set aside as controls on the two treated groups. The mice were all young and thrifty; average weight, 18 grams. The three series were inoculated with carcinoma ten days later.

To the four or five described cases of sex-limited heredity in poultry another is added.

The Jungle-fowl and its derivative, the Brown Leghorn, have the hackle and saddle feathered “laced” with red. The upper wing coverts of the cock are also red, forming the “wing bar.” In the Dark Brahma, on the other hand, the red is not formed on the hackle and saddle and is nearly absent on the wing bar of the male, so that the red is replaced by white.

If, now, a male Dark Brahma be crossed with a female Brown Leghorn, or if the cross be made in the opposite direction, all sons are white-laced; but the wing bar is red. Thus, in these sons the white lacing is dominant but the white wing bar appears to be recessive. The most important point, however, is that the sons derived from the reciprocal crosses are practically indistinguishable.

With the daughters this is by no means the case. When the father is white-laced, the daughter is also; but if the father is redlaced the daughter is likewise; i. e., the daughter's lacing comes from the father's side of the house only.

The explanation is simple on the assumption that the lacing is linked with the sex-chromosome, following the scheme of transmission of the sex-chromosome as worked out by Stevens, Wilson and Morgan. While the male of poultry must have two somatic sex-chromosomes the female has only one. Therefore, while all sperm possess a sex-chromosome, only half of the eggs do. In the fertilized egg or zygote that has only one sex-chromosome, this is derived from the father and the zygote becomes a daughter. Hence the daughter “inherits” from the father only.

I have been able by means of a simple duodenal catheter to obtain for the first time access to the duodenum in the living infant. This has enabled me to investigate the secretion of the upper part of the small intestine. This catheter is, in brief, merely a Nelaton soft rubber catheter No. 14 (F). That I actually do reach the intestine is proved by X ray photographs which I show.

The present report concerns solely newborn infants, which had never obtained any nourishment. I have found some interesting conditions not only as concerns intestinal secretions but also regarding the secretion of the gastric juice. It is noteworthy that there have been no previous investigations in this regard; in fact I have been able to find note of only one test of the gastric secretion of the newborn before it has been given food.

I found that in almost all cases hydrochloric acid is present in the stomach of these newborn infants whether they are examined one half hour or twenty hours after birth. The hydrochloric acid varies from 1/2 cu. cm. to about 7 cu. cm., and is not in direct proportion to the age of the child. This hydrochloric acid is not stimulated merely by the passage of the tube, for it was obtained within a minute or two after the catheter was introduced. It, therefore, must have another origin. I believe that it is a reflex due to the sucking of the tube. The longer the catheter is kept in the stomach the more juice is obtained; in one hour 14 cu. cm. was obtained; in an hour and 50 minutes 17 cu. cm. This gastric juice has an acidity on an average of about 50.

A series of experiments were performed upon dogs with the object of determining the effects on the output of nitrogen and sodium chloride in nephritis produced by the subcutaneous injection of uranium nitrate. These dogs were fed the usual standard diets, also diets containing much meat, as well as food to which considerable amounts of urea and sodium chloride had been added.

The results of these experiments appear to justify the following conclusions regarding uranium nephritis.

1. The nitrogen secreted by the small intestine, as determined by the Thiry fistula method, is somewhat diminished.

2. The fecal nitrogen remains approximately unchanged.

3. The urinary nitrogen is not diminished even when the diet demands an excretion of one gram of urinary nitrogen per kilo of body weight of the dog.

4. In some cases the urinary nitrogen is increased. The source of this excess of nitrogen is problematical. It may be due to protein destruction of the body tissues caused by the same toxic agent as the nephritis. If this be true, the “rest” nitrogen of the blood serum should be higher than normal. Experimental attempts have been made to ascertain the facts in this connection but have not been pushed far enough to warrant a definite statement.

5. The sodium chloride excretion in the urine keeps pace with the intake even when considerable quantities are added to the food.

6. The above statements have held true for dogs after the first as well as after several large single injections of the drug at long intervals. One animal was tested after he had received as many as seven injections.

Both the immediate cause and seat of tetany are still obscure. It has been suggested, though never proven, that a poison must circulate in the blood to cause the tetanic twitchings. It has also been suggested that these twitchings may result from a lack of calcium in the circulating fluids. It has been shown that tetany does not appear in a limb to which the nerves have been cut. It is conceivable that this is due to the fact that no impulses reach that limb from the spinal cord. The present experiments show that if the nerves be cut during tetany, their electrical excitability remains the same as that of the intact nerves on the opposite side. Further, it is shown that if the nerves be cut before the development of tetany, they become hyperexcitable to an extent which equals that of the intact nerve on the opposite side, although they are quite separate from the spinal cord. Since degeneration occurs within two or three days, such observations must be made with due regard to this fact. The peculiar character of this rise in the excitability is seen in the figures obtained for the cathode and anode opening shocks to which the nerves become especially excitable. In complete anemia or after the death of the animal there is a period of about one half hour during which this characteristic rise in the excitability of the nerve to the cathode and anode opening shocks appears. Nevertheless, the excitability of the nerves is, throughout this period, very much lowered and the curve is in no way comparable, therefore, to that in tetany.

Transfusion of the blood of an animal in tetany into the vessels of a normal animal has not succeeded in producing a characteristic tetany nor even a marked change in the excitability of the nerve.

The patient from whom the curves were taken has been under observation since August, 1910. He was at that time suffering from cardiac decompensation. It was clear from the physical examination that he had a valvular defect. Combined arterial and venous curves were made. These showed that for short periods every impulse from the auricles was answered by a ventricular contraction, while at others the ventricular contractions responded to every second beat of the auricles. Curves were taken in February and March, 1911. These were volume curves from the jugular vein and radial artery and likewise curves of the cardiac action current, registered with an Edelmann stringgalvanometer. At first the ventricles responded to every second auricular beat, but somewhat later there was complete dissociation between the two pairs of cavities. Before the patient left the hospital, the relation between the auricles and ventricles was normal. Electric curves were taken on December 19, 1911, when the ventricles responded to every auricular contraction.

The curves which are of interest date from the period of complete auriculo-ventricular dissociation. Each ventricular complex is represented by R-, S-, and T-waves. They vary from each other in that, when the R- is large, the S-wave is small; and that when the S-wave is large, the R-wave is small. The R-wave gradually increases in size and then gradually diminishes, when the S-wave gradually increases in size and as gradually diminishes. If the apexes of succeeding R- and S-waves, i. e., the significant wave in each complex were joined, a waved line would result. So diagrammatic a sequence as this did not occur frequently, but there was a tendency to approximate to this description. Sometimes the transition from complexes of one type to those of another was abrupt.

In the New York Medical Journal of November 26 and at the New Haven meeting of December, 1910, we gave observations on the quantities of gas (air) tolerated and the apparent relation of the adrenals to this tolerance. We have further determined that section of the cord between the fifth and sixth cervical vertebræ interferes with this tolerance of gas (air). After the removal of the adrenals from two dogs carbon dioxide was tolerated up to 942 c.c. in one case and 952 c.c. in the other, in 1 hour and 23 minutes.

Two dogs were injected with nitrogen. 1 One received 32 c.c. in six minutes with marked depressions; the animal lived. Panting occurred. The other received 272 c.c. within 22 minutes and died; panting was not noticed. The lungs were collapsed at autopsy.

The tolerance of oxygen was tested on two dogs. One received 150 c.c. slowly and survived. The other received 312 c.c. in 231/2 minutes and died. During the oxygen experiments, panting occurred. The lungs were practically normal.

The tolerance of hydrogen was tested in two dogs. In one 184 c.c. in 8 c.c. volumes were injected in 13½ minutes. Each injection caused a moderate depression. 60 c.c. were given in 20 c.c. volumes in 3½ minutes, and 20 c.c. in 5 c.c. volumes. The large volumes caused great depression and almost death. In the other dog 90 c.c. were injected in 7 minutes in 8 c.c. volumes without harmful results.

Sulphuretted hydrogen was used to study elimination on two dogs. Three C.C. saturated H₂S water were injected into the femoral vein. The breath almost immediately blackened lead acetate. Twenty-five minutes after tying and clamping off the adrenals 3 C.C. more of saturated H₂S water was injected and a similar result was obtained.

Soon after the discovery of the profound effect of adrenal extract upon blood-pressure the question arose as to the fate of this extract in the blood. The rise of the blood-pressure after an intravenous injection of adrenin passes off in a few minutes and none of the adrenin is found to persist in the blood, no matter how large the injected dose has been. The quite natural explanation of this phenomenon was, that the blood destroys adrenin. But Oliver and Schäfer found that in a mixture of adrenin and blood, even after standing for 22 hours, the adrenin remained unaffected. It has been confirmed since by several investigators, that neither blood nor serum is capable of destroying adrenal extract. I shall not discuss for the present the problem of the fate of adrenin in the body in general. I wish only to report the discovery of the fact that there is at least one body fluid which is capable of destroying adrenin and that is spinal fluid. The observation was made by mixing human spinal fluid with adrenalin. The spinal fluids were obtained in the first place from a number of cases of poliomyelitis of the Rockefeller Hospital and from two cases of tuberculous meningitis, obtained for me by Dr. Flexner. But this destructive action is not specific to these diseases. I found it to be possessed by spinal fluids from cases of resolving pneumonia, gastro-enteritis and eczema, obtained through the kindness of Dr. Wollstein. Evidently it is a physiologic property of normal spinal fluids, although there seems to be a difference in degree of action between some pathologic cases; for instance the spinal fluid from poliomyelitis seems to destroy adrenalin definitely more readily than that from tuberculous meningitis.

While the liver generally plays a subordinate part in the synthesis of glucuronic acid, it would seem from the experiments of Pohl (1), that upon the incorporation of chloral-hydrate or camphor, the conjugation of these substances with glucuronic acid does take place largely in that organ.

We have been interested in seeking a measure of the functional activity of the liver before and after experimental duodenal obstruction. By giving a dog camphor and determining the output of camphor-glucuronic acid, both before and after obstruction, we hoped to measure at least in a relative way any impairment in liver function which may follow this intestinal lesion.

The experiments were carried out on dogs—the operative portion under complete ether anesthesia. The animals were fed for fully a week on an exclusive meat diet in order to free the urine as much as possible from pentoses.

For the glucuronic acid determinations we employed one of the two methods described by C. Tollens (2, 3), viz., distillation of the glucuronic acid lacton with dilute hydrochloric acid, and precipitation of the resulting furfurol with phlorglucin. This method seems open to less objection that most of the other quantitative methods which have been proposed. In the hope of further determining the accuracy of our results, we are now experimenting with the CO₂ method described by Tollens.

The experiments here reported were carried out as follows: The glucuronic acid excreted during one or two 24-hour periods was determined and used as a normal. According to Tollens, the variations in the daily quantities of glucuronic acid are very slight, if the diet is uniform.

From 2 to 5 gm. of commercial camphor dissolved in olive oil were given subcutaneously and the camphor-glucuronic acid excretion in the urine determined for three to four 24-hour periods.

Last April it was reported from this laboratory that (1) substances which are capable of causing a definite biological reaction in frogs cause the characteristic reaction also when injected after the removal of the heart, and (2) that some substances, like morphin and acid fuchsin, appear to be even more effective in cardiectomized than in normal frogs. The first phenomenon was explained by the theory that distribution in frogs deprived of the cardio-vascular circulation is accomplished by a peripheral mechanism, namely, by the tissue spaces, which present a connected system throughout the body. The second phenomenon was interpreted by the hypothesis that the fresh blood in the cardiovascular mechanism continually antagonizes the convulsant action of such substances as morphin and acid fuchsin. The study of the last mentioned hypothesis and the underlying phenomenon has been hampered by the fact that the distribution by the peripheral mechanism is necessarily a slow one and since, at the warmer seasons, frogs survive cardiectomy only a short time, it happens that the animals die before the convulsant effect could make its appearance. We have therefore tried to study the hypothesis by the reversed method, that is, morphin injected first and the heart removed later. This was carried out in several series, the doses varying from 0.1 to 0.5 mg. of morphin per gram frog, and the intervals between the injection and the subsequent removal of the heart varying from a few minutes to 4 hours. We shall not enter upon details: we shall merely state that the result was strikingly positive. While the morphin frogs, which were kept with hearts intact, remained normal, all the frogs which received proper doses of morphin and had their hearts removed at different intervals, developed tetanic convulsions, which in many cases had to be characterized as very violent.

Experiments were performed by introducing sterilized (1) untreated, (2) oiled silk, and (3) human hair into arteries and veins and observing the results. Twelve common carotid arteries and twelve external jugular veins were employed. The number of silk strands varied from the smallest single strands used in blood vessel suture to twenty-four such strands. Where a number were used they were threaded into suitably large needles and were not twisted. Cambric needles were used.

The experiments were performed by exposing the vessels of anesthetized dogs, transversely piercing the vessel with the needle as near the mid-line as possible, drawing the ligatures through and loosely tying the free ends together. Three weeks later specimens were taken and examined.

In no instance was there occlusion of the lumen nor was there any evidence to indicate that the vessels would have subsequently become occluded through thrombus formation. In general the ligatures were found dividing the lumen and coated with a substance closely resembling the intima in gross appearance.

In the case of one artery and one vein, an ordinary occluding ligature was first tied about the vessel, after which a transverse suture was introduced about one centimeter on each side of the ligature. The results in these cases did not materially differ from those observed on'the non-occluded vessels. No marked differences were observed with different kinds or sizes of sutures.

Microscopical results, particularly as regards the character of the deposit on the foreign surfaces and the presence or absence of intimal covering, will be reported later.

It is the intention of the present paper to summarize briefly experiments bearing on the problem of the acquired immunity of rats to the implantation of tumors. In rats, as indeed in practically all the other species of animals in which similar experiments have been practicable, it has been found that the absorption of an implanted tumor is succeeded by a period during which the animal is refractory to further implantations. The same refractory condition can be induced by the injection of tumor autolysates (Levin), or by the injection of suspensions of normal tissue, or even of blood. The relative specificity and effectiveness of these procedures vary in their details; the underlying principle has been thought to be the same throughout.

In a series of experiments with the Flexner-Jobling rat tumor, I have observed certain phenomena which seem in a measure to support the belief that the mechanism of immunity to the tumor is identical in its biological manifestations with that found in infectious disease, and is therefore mediated by immune substances carried by the serum. These observations may be summarized as follows:

I. Inoculation of the Flexner-Jobling adeno-carcinoma into normals rats is not followed by any macroscopic evidence of inflammation or reaction, if the grafts are made from a small, non-necrotic tumor.

2. Rats specifically immunized to the Flexner-Jobling adenocarcinoma respond to an implantation by an intense local inflammatory reaction, often with necrosis.

3. Animals bearing necrotic tumors present this reaction on reinoculation.

4. After removal of the necrotic tumor, the animal still reacts to reimplantation by local inflammation.

These phenomena seem analogous to those instances of local anaphylaxis known as the “Arthus” phenomenon in rabbits, and to the local allergetic reactions described in human beings by Pirquet.