James W. Jobling

Bactericidal Function of the Liver.

During the course of our studies on the effects of intestinal stasis on blood destruction and regeneration, some experiments were undertaken to determine the rôle of the liver and bile in the elimination of bacteria in normal fasting dogs. Several investigators 1 have found organisms in the tissues of apparently normal animals. Wolbach and Tadasu 2 found an anaerobic spore-bearing bacterium in the livers of 21 out of 23 healthy dogs. These authors obtained their material from dogs which were killed by chloroform. The interval between the last feeding and the death of the animal was not stated. They were unable to grow the organisms obtained from the liver in sterile bile, or bile containing media. The material for our studies was obtained under strict aseptic conditions from 11 normal dogs which had been kept without food for 18 hours. Ether anesthesia was used. The specimens consisted of blood from the portal vein (obtained in 5 dogs), bile from the gallbladder, and a wedge-shaped piece of liver measuring approximately 3 cm. × 2 cm. In addition, the organisms which were obtained from the liver tissue were incubated in undiluted and diluted bile (1:10 to 1:1,000,000). The organisms and bile were obtained from the same animal in each instance. Cooked meat dextrose broth was used as the culture medium. Strict anaerobiosis was maintained. Aerobes as well as anaerobes of intestinal origin develop satisfactorily under these conditions. 3 The liver tissue was also cultured aerobically. In every dog the bile was sterile. The liver tissue from all the animals yielded a pure culture of an anaerobic spore-bearing bacterium which was similar to the one described by Wolbach. In 3 dogs the organism was identified as B. welchii; in the others the organism was not identified.

Demonstration of a Tumor-Inhibiting Substance in Filtrate of Rous Chicken Sarcoma and in Normal Chicken Sera.

In a previous communication 1 we reported that the active agent in the Rous chicken sarcoma filtrate could be concentrated and partially purified by adjusting the filtrate, normally about pH 7.2, to a reaction of pH 4. When brought to this reaction with a phthalate buffer, the tumor-producing agent is carried down with the precipitated portion. When this precipitate is extracted at pH 8, the carcinogenic agent is not only set free, but the activity of the extract seems definitely greater than that of the original filtrate. To account for the increased activity of the first extract at pH 8, several possibilities suggested themselves; among others, that the active agent in the filtrate may be in combination with substances that restrain its activity to some extent, and that in the process of precipitation by the method described above, there is brought about dissociation between the restraining or protective agent and the tumor-producing element. If true, this would explain why the extract of the first precipitate is considerably stronger than the filtrate from which it was prepared. To test for the presence of a protective agent, we examined the supernatant fluid obtained after precipitating at pH 4 the active agent from a 10% filtrate. After centrifuging, the supernatant fluid was decanted, neutralized and then concentrated in vacuo at room temperature to 1/2 its original volume. Two cubic centimeters of this concentrated supernatant fluid were now mixed with 0.5 cc. of the original filtrate, containing approximately 2 infective doses, and allowed to stand for 30-40 minutes prior to injection. Chicks, 10-14 days old, were used in all the experiments, for they have been found to give more uniform results. Of 80 chickens inoculated with this mixture, 75% failed to develop tumors, while tumors developed in all the controls which had received this amount of filtrate alone.

Effect of Organ Extracts on Blood Regeneration.

We wish to report the results of some experiments made to determine the effects of various organ extracts on blood regeneration in animals made anemic by bleeding. This work was begun three years ago, but the contradictory findings at different times made it seem unwise to make an earlier report. The preparations used consisted of (1) 75 per cent alcoholic extracts of the stomach, liver and spleens of calves; (2) saline suspensions of the cells of the same organs of rabbits; (3) hemoglobin solution; (4) fat-soluble non-saponifiable material from calves livers; (5) saline extracts of these organs of rabbits which had been made anemic by bleeding. Rabbits were used in all the experiments. They were made anemic by bleeding from the heart. The blood removed constituted about 3 per cent of the body weight, about 60 per cent of the total volume. Several blood counts and hemoglobin determinations were made before the bleeding and at frequent intervals afterwards. Our interpretation of the results of the injections of the different substances was based upon the rapidity with which the normal count was restored, as compared to a series of similar untreated anemic animals. The results obtained with these various extracts were not very promising. At times we felt that the liver extracts were active in stimulating blood regeneration, but subsequent experiments made it evident that we were not justified in this assumption. Our most positive findings were with the alcoholic spleen extracts. In every instance this extract prevented blood regeneration and if the injections were continued the animal died. Our results are particularly interesting in view of those obtained with the feeding of liver by Whipple 1 in the experimental anemias of dogs, and by Minot and Murphy 2 in pernicious anemia. Several explanations of the discrepancies are possible.

The Effect of Division and Transplantation of the Common Duct Upon Gall-Bladder Function.

Recent studies 1 , 2 tend to minimize the importance of the sphincter of Oddi in the regulation of gall-bladder activity. However, the methods which have been employed to eliminate the action of the sphincter are open to a number of criticisms. We have attempted to avoid them by completely dividing the common duct proximal to the sphincter and implanting the stump into another part of the duodenum. The effect of this procedure upon the action of the gall-bladder was determined by means of cholecystograms which were made according to the method described by Graham and his co-workers. 3 Under ether anesthesia, the common duct in 2 dogs was isolated and doubly ligated just above its entrance into the duodenum. A linear incision 2 cm. long was made in the anterior surface of the duodenum about 8 cm. from the pyloric sphincter. The duct was divided 1/2 cm. proximal to the ligature, and fixed at the lower angle of the opening in the duodenum, which was then closed by two sutures. Six weeks and 8 weeks respectively, after the operation, cholecystograms were obtained in the following manner: 0.12 gms. sodium tetraiodophthalein per kilo body weight were injected intravenously. Eighteen hours after the injection of the dye, the first X-ray film was taken. Then the dogs were fed with the yolks of 3 eggs in 200 cc. of cream 4 and films were taken at intervals of 1, 3, 6 and 24 hours after the meal. In both dogs, the shadow was normal in size and density at the 18th hour observation. Following the meal, in one dog, there was progressive shrinkage and increased density of the shadow, with complete disappearance at the 24 hour period.

Attempt to Produce Atherosclerosis in Chickens by Feeding Cholesterol.

Conclusion The aorta of the young adult rooster commonly exhibits areas of intimal fibrosis, with an infiltration of lipid into the thickened intima and the underlying media. Feeding of cholesterol for periods up to 4 months fails to increase the incidence of such lesions, but may slightly augment the deposition of lipid, particularly in the media. For the production of intimal lipid deposits in the aorta by feeding cholesterol, the chicken, therefore, appears to be an unsatisfactory animal.

Fate of the Active Agent in the Chicken Sarcoma in Mixtures Containing Inhibiting Substances.

We have previously described 1 the presence of a tumor inhibiting substance in the filtrate of the Rous chicken sarcoma and in normal chicken sera. It was found that this inhibiting substance is retained in the supernatant fluid when the tumor filtrate is brought to pH 4, while the active agent is carried down in the precipitate. In a more recent report, Murphy 2 and his associates report results that would seem to confirm these observations. The fact that the activity of the agent can be inhibited by the supernatant fluid and sera made it of interest to ascertain whether the agent was actually destroyed in such mixtures or whether its tumor producing properties were merely inactivated. In order to answer this question it became necessary to determine if it is possible to recover the agent in an active state from these non-infective mixtures. The supernatant fluid used was prepared by adding an equal amount of a phthalate buffer solution at pH 4 to a 20% filtrate of the Rous chicken sarcoma. After standing for 30 minutes, the mixture was centrifuged, and the supernatant fluid decanted, neutralized and concentrated in vacuo to 1/2 its original volume. To 2 cc. of the concentrated supernatant fluid was added 0.5 cc. of filtrate, and the mixture allowed to stand for 30 minutes at room temperature. Such a mixture when injected into chickens fails to produce tumors in 75% of the inoculations. If, however, the mixture is brought to pH 4, and the precipitate extracted at pH 8, the active agent is recovered, for the extract is now able to induce tumor growth. Similar results were obtained when blood was used instead of the supernatant fluid.

Effects of Repeated Injections of Toxins of Intestinal Bacteria on Blood Regeneration in Rabbits.

There is doubt as to how much significance should be attached to the belief that certain pathological conditions are due to the absorption of intestinal toxins. Similar doubts are expressed concerning the value of interpretations based on the experimental work done, since most of it has been done with either a pure culture of bacterium isolated from the intestinal tract, or with extracts of the feces. It seems possible that mixtures of bacteria grown in the presence of protein or carbohydrates may produce toxic substances not to be found in pure cultures, and it is also possible that some unknown organism may grow under such symbiotic conditions and produce its specific toxin. The following experiments were conducted to determine the effect of toxins of the intestinal bacteria on blood regeneration. The culture medium consisted of bouillon containing either finely chopped beef, or brain. To half of each series was added 1 per cent soluble starch. One half of the flasks were inoculated with saline emulsions of feces from a patient with pernicious anemia, and the other half with feces from a normal individual. Control uninoculated flasks were incubated for the same periods. The flasks were removed from the incubator at intervals of 1 week, 2 weeks and 4 weeks, and the contents passed through Berkefeld filters. Only those filtrates found to be sterile by aerobic and anaerobic methods were used. The filtrates were kept in the ice-box, but even then the toxicity soon decreased. Rabbits were used in all experiments. The filtrates were given intravenously in amounts of 0.25 to 0.5 cc., diluted 1 to 3 with salt solution, or subcutaneously in 1 cc. doses. The injections were given at intervals of 48 hours.

The effects of repeated intravenous injections of India ink on the blood picture in rabbits.

The following investigation was undertaken in order to study the effects of the repeated intravenous injection of India ink upon the blood picture in rabbits. Higgins waterproof India ink was used. It was filtered and suspended in physiological salt solution in the proportion of one part of ink to three parts of salt solution. The suspensions were sterilized before each injection. With few exceptions, ten cubic centimeters of the ink suspension were injected into the ear veins of the rabbits, at intervals of forty-eight hours. The number of injections varied between seventeen and fifty-six. The rabbits weighed between two and one-half and three kilos. The following constituents of the blood were studied: erythrocytes, hemoglobin, leucocytes and reticulated cells. Examinations were made immediately preceding injections. Similar examinations were made on a series of normal control rabbits kept under the same conditions. In all the rabbits, as a result of the injections, there was a gradual fall in the number of erythrocytes, which at the lowest levels was forty to fifty per cent of the original count. Following this drop, in spite of continued injections, there was a rise in the erythrocyte count which reached approximately normal values at about the twentieth injection. Determinations of hemoglobin were made with the Newcomer apparatus, using daylight as the source of illumination. The values obtained with this apparatus are only relative, but they tended to follow the fluctuations in erythrocyte counts except in one rabbit, in which the fluctuations were less pronounced than those of the other three rabbits. Normoblasts were usually observed in every smear. The normoblasts became more numerous as the number of injections increased and hemacytoblasts appeared. The reticulated cells were moderately increased during the period of the anemia, but the count remained within normal limits during the period of recovery.

The effects of repeated intravenous injections of distilled water on the blood picture in rabbits.

The effect of repeated induced intravascular hemolysis upon the blood of rabbits was studied in the following manner: Ten to 12 cc. of sterile distilled water were injected slowly into the marginal ear veins of 6 rabbits at 48 hour intervals. The number of injections varied, the maximum being 34. Recently, as a control experiment, Patterson and Kast 1 described an anemia in rabbits produced by the intravenous injection of sterile distilled water, but as their experiments were only continued for 19 days, they apparently did not observe that the animals subsequently developed an increased resistance to such injections. The following constituents of the blood were studied; erythrocytes, hemoglobin, leucocytes and reticulocytes. The resistance of the erythrocytes against hypotonic salt solution was also determined. The examinations were made immediately preceding each injection. Control observations were made upon normal rabbits and rabbits which had received physiological salt solution in doses corresponding to the distilled water. After various intervals following the repeated injections of water, there was a diminution of 40 per cent to 50 per cent in the number of erythrocytes. However, despite continued repeated injections the erythrocyte count returned approximately to the original figures. There was a slight to moderate poly-chromatophilia and anisolcytosis with an occasional normoblast and hemacytoblast. The number of reticulocytes showed a slight increase; the highest figure was 7 per cent. The variations in the percentage of hemoglobin were less marked than in the number of erythrocytes. The total number and differential count of the leucocytes remained within normal limits. The resistance of the erythrocytes against hypotonic salt solution did not change beyond the limits of normal variations in rabbits.

Phenoltetrachlorphthalein test of a liver function in Eck fistula dogs kept upon meat diet.

Conclusions The ability of the liver to remove the di-sodium salt of phenoltetrachlorphthalein from the blood stream was not impaired by the establishment of Eck fistulas in dogs. Definite lesions in the liver (to be described later) followed the establishment of the fistulas. A prolonged meat diet did not induce toxic symptoms in these dogs.