Experimental Biology and Medicine

In a previous communication 1 a method was described for producing bronchogenic lung abscess with human tubercle B. The present report is on the production of bronchogenic lung abscess in dogs by the same method, using pyogenic organisms.

Thirteen dogs were narcotized with morphine sulphate and divided into 2 groups. In each of the first 7 dogs, 0.25 cc. of a suspension of a mixture of Streptococcus hemolyticus and Staphylococcus aureus in lipiodol was deposited in the peripheral portion of the right lower lobe. In 2 of this group a similar amount was also placed in the left lower lobe. This was accomplished by means of a ureteral catheter introduced through a bronchoscope. The procedure was carried out under fluoroscopic control, thus the deposit could be made at any desired location. In each of the second 6 dogs 0.25 cc. of a broth suspension of Streptococcus hemolyticus and Staphylococcus aureus was deposited in the peripheral portion of the right lower lobe. A similar amount of lipiodol was placed in the peripheral portion of the left lower lobe.

Results. Group I. Three dogs were sacrificed on the 12th post-infected day. All presented indurated areas 1.5 to 2.5 cm. in diameter, one of which contained a cavity 0.33 cm. in diameter, and another a cavity of 1.0 cm. in diameter, each with a necrotic lining. The results in the other dogs are summarized in Table I. Staphylococcus aureus was cultured from one of the 2 cavities in animal 232.

Group II. One dog was sacrificed on the 14th post-infected day, 2 on the 15th and 3 on the 26th day. All lobes in which lipiodol had been deposited appeared normal except in one of the 15-day dogs. Here there was a slightly indurated area 0.5 x 1.0 cm. in dimension. All lobes in which micro-organisms had been placed appeared normal except the 14-day animal and one of the two 15-day dogs. In each of these a small indurated area 0.5 cm. in diameter was found, containing no cavity. In the 14-day animal, indurated areas were also noted in the right middle and right upper lobes. Microscopic sections through the indurated areas revealed infiltration with lymphocytes, endothelial cells and a few polymorphonuclear leucocytes.

Small colony variants of bacteria are regarded by some investigators as a filterable stage in a life cycle of the organisms. Other workers do not feel that there is sufficient evidence in support of this theory. Hadley 1 has been one of the chief exponents of the idea that bacterial variation represents a cyclical development of the organisms and in this connection he described a small colony filterable form of the Shiga dysentery bacillus and other bacteria.

In the present investigation small colony variants of Staphylococcus aureus were studied with respect to their occurrence, morphology, physiological and antigenic properties and filterability. The variants were obtained by plating ageing broth or agar cultures and from cultures in lithium chloride broth. Their occurrence was irregular and rare and no means were found by which this type of variation could be induced. Tiny colonies were not observed at any time in very young cultures except occasionally in broth containing lithium chloride. They appeared only after the cultures had aged for a week or two and were more numerous after 3 or 4 weeks.

Well-isolated colonies of Staph, aureus are usually from 1 to 2 mm. in diameter when grown on nutrient agar. Those of the small colony variants could scarcely be discerned without the aid of a microscope. In the various strains they ranged from .04 mm., the smallest ever observed, to 0.5 mm., but most frequently they were about 0.1 mm. in diameter. The small colony variants had lost the power of pigment production, the growth on agar slants being white and translucent.

Morphologically some of the variants were not strikingly different from the original strains. In other cultures, however, the cells were very irregular in size. Some were in tetrads, others in pairs or single. Within each cluster of cells there were marked contrasts in size, some being much larger and others smaller than those of normal cultures.

A series of strains of hemolytic streptococci and mucoid encapsulated streptococci was obtained from the throats of scarlet fever patients at Cook County Contagious Hospital.

Laboratory studies were made on a group of these strains to determine. (1) the results of their growth on chocolate agar, (2) their effect on the sugars sorbitol and trehalose and (3) the influence of mouse passage on capsule formation and colony characteristics.

Using chocolate agar Tunnicliff 1 found that hemolytic streptococci of scarlet fever produced no color change in this medium except occasionally a slight greening after several days'growth. The colonies almost always were conical in shape and slightly granular. Occasionally, however, a strain dissociated and produced atypical colonies that were large, moist, smooth and convex and turned chocolate agar green. These atypical colonies were not scarlatinal by the opsonic, agglutination or toxin neutralization tests. 2 , 3

A relation between dietary fats and thyroid activity has been reported by several investigators.^1-4 Jaffe 5 has reported interesting variations in the lipoid content of the thyroid in various diseases of the gland. Several reports have suggested the possibility that cod liver oil 1 or vitamins A and D in combination with iodine or iodized fat exert a favorable influence in Grave's disease. 6 ,⁷,⁸ These reports stimulated the author to test the effect of various oils including cod liver oil on rats rendered toxic by the administration of thyroid extract. The results are briefly reported here.

Methods. Both growing (3 months old and weighing from 100–110 gm.) and adult (150–195 gm.) rats were used in different groups of experiments. They were fed the standard Steenbock diet and kept in individual cages.

Dissociation of the gonococcus has been implied in the work of Herzog 1 and Warden 2 as early as 1913. However, very recently two contemporary schools of thought have appeared in the literature; the French (represented by Ramsine, 3 Milochevich, 4 and Lavrinowicz 5 ) and the German (represented by Kandyba 6 and Delbanco-Lorentz 7 ) who are convinced that changes in the morphology of the gonococcus exist in vitro and in vivo, while to the contrary, the Italian school (represented by Porcelli, 8 Sechi, 9 and Debiasi 10 ) firmly believes that such changes have very little in common with the gonococcus and are due to errors in technique and interpretation. In view of the conflicting literature the present report, as well as more detailed results to appear later, may prove significant.

Dissociation was attempted and accomplished with 4 stock strains of the gonococcus. During the entire study a modification of Hitchen's semi-solid medium was found satisfactory. Dissociation was produced by ageing and the use of immune serum.

Primary changes through ageing were noted when a sparsely inoculated ascitic agar plate was sealed and incubated for varying periods of time. These changes were evidenced as secondary or papillated colonies superimposed on the original colony or streak. Briefly, the sequence of events was as follows: The secondary growth was yellowish and opaque and when stained showed a beadlike bacillus (as though cocci were strung together to form a closely beaded chain). This form through further ageing and cultivation developed 2 types of growth, namely, a large opaque smooth colony, 3 mm. in diameter, composed of typical biscuit-shaped diplococci but taking the Gram counterstain very deeply and a small colony, 0.5 mm. in diameter, composed of tiny gram negative micrococci.

It has been demonstrated that bacteriophage injected intravenously into rats accumulates in the spleen and liver. These organs accumulate phage to the same general degree as they do bacteria.

The rôle of the reticulo-endothelial system in removing microorganisms from the blood stream has been generally recognized in recent years. The earlier work of Bull 1 in this field has been supported and extended by others, particularly Cannon 2 ,³ and his coworkers. The question arose as to whether or not a readily filterable biological principle such as bacteriophage, which is probably particulate and of the general order of magnitude of the filterable viruses, would be removed from the blood stream by organs of the reticulo-endothelial system to a greater degree than by organs with few fixed phagocytic cells.

Y. Shitate 4 reported on the fate of bacteriophage in the body. Since he made no attempt to obtain quantitative results, his findings do not aid in answering the above question. Smirnow and Goldin 5 made a similar investigation on the fate of phage in the guinea pig's body after inoculation into the blood stream. Unfortunately they selected a 24-hour period for their first examination of the organs. Phage was found in low concentrations in blood, liver, spleen and kidneys. Sullivan, Neckermann and Cannon 3 have recently pointed out that although bacteria are present in much greater numbers in the spleen than in the blood 1 hour after an intravenous inoculation, that organ may be sterile in 24 hours. This may explain why Smirnow and Goldin failed to find any significant difference in bacteriophage titer between the blood and the reticulo-endothelial organs after so long a period as 24 hours.

Method. 15 albino rats (150–200 gm.) were etherized and injected in the exposed femoral vein with 2 cc. of staphylococcus phage. (1 cc. was injected in rats 11, 12 and 13.) In the case of rats 12–15 0.5 cc. of blood was collected from the clipped tail within 5 minutes. Two hours later the animals were again anesthetized and 0.5 to 1.0 gm. of various tissues were removed, ground with sand and 5 cc. of saline in a test tube.

A few reports in the literature indicate that acidosis hinders the production of agglutinins. Various substances have been used to produce an acidosis. Ammonium chloride produces a prolonged acidosis in man. 1 ’ 2 Davesne and Haber 3 thought they obtained a decrease in agglutinin production while administering hydrochloric acid. Moen and Reimann 4 obtained a poor response or no agglutinin production to typhoid vaccine in uncontrolled diabetic patients with acidosis.

In the experiments reported here, rabbits were given three 0.5 cc. intravenous injections of suspensions of killed typhoid bacilli on alternate days. In 12 normal rabbits the agglutinin titer averaged 1:320 at the end of the first week, with variations from 1:60 to 1:5120 dilutions, 1:1450 at the end of the second week, 1:800 third and 1:750 at the end of the fourth week.

Another series of 12 rabbits were given, in addition, 0.3 to 1.3 cc. of a 10% solution of ammonium chloride intravenously daily. The average agglutinin titer at the end of the first week was 1:50 with variations of from 0 to 1:320, second week 1:20, third week 1:10 and at the end of the fourth week 1:10 dilution. Three rabbits died during the first and second week. The animal with a titer of 1:320 at the end of the first week, later dropping to 1:10, had a plasma CO₂ combining power of 46 volumes %, which progressively decreased later to 23.0 volumes %. In 3 rabbits with no agglutinin production throughout the plasma CO₂ was consistently below 33 volumes %.

The average CO₂ values in the animals receiving ammonium chloride was 27 volumes % with variations from 46 to 18.3 volumes %. The average plasma CO₂ combining power in normal rabbits was 50 volumes %.

The excessive loss of minerals from the body in diabetic acidosis has recently been presented quantitatively in detailed balance studies by Peters 1 and Atchley 2 and their co-workers. Kydd 3 has emphasized the depletion of chloride in excess of other electrolytes, as if the base lost with the ketone acids might be derived from NaCl rather than from bicarbonate. He stresses the need for replacement of chloride during recovery from coma. Diabetic children not infrequently manifest an unusual craving for salt, even when not excreting ketone bodies in abnormal amounts. This has been interpreted by Joslin as most likely being due to the high potassium content of their vegetable diets.

The present study was suggested by our experience in the case of a 15-year-old boy, who, as he emerged from diabetic coma, begged for salt and voraciously consumed 80 gm. of NaCl in the course of 24 hours. The water intake was proportionately increased. When it was observed that the craving for salt did not abate long after the disappearance of ketosis and that the patient continued to take 60 gm. daily before being satisfied, he was placed in the metabolism ward where complete balance studies could be carried out under standard conditions. He was given a constant diet containing protein 16, fat 33, carbohydrate 42, and water 1200 gm. every 6 hours.

This was made up from powdered milk, bread, butter, eggs, cane sugar, lemon juice and a small amount of powdered yeast. The insulin dosage was kept constant for each period, totaling 32 units per day. This dosage of insulin permitted the occurrence of glycosuria in every 6-hour period, thus making comparative measurements possible.

Shortly after the study was undertaken, it was discovered (1) that the high NaCl intake was accompanied by an elevation of arterial blood pressure from an average of 110 mm. of mercury systolic and 80 diastolic to 175 systolic and 115 diastolic and (2) that the urinary excretion of glucose fell from a control level of between 60 and 70 gm. to between 10 and 20 gm. daily under the same conditions. Reduction of the NaCl intake to the control value of about 4 gm. daily caused a return of the glucose excretion to the original level and a fall in the blood pressure to normal.

Cultures of the mesenteric lymph node of adult rabbits were exposed to the type of radiation usually used for treatment of lymph nodes. The high voltage therapy machine at the University Hospital was utilized.

The water cooled X-ray tube was run with a current of 30 milliamperes at 200 K.V. A focus-surface distance of 50 cm. was used. The filter used in the preliminary experiments consisted of 1/4 mm. copper plus 4.6 mm. aluminum (the aluminum in the ionization chamber).

The preliminary experiments consisted of cultures radiated with 1, 2, 4 and 8 erythema doses (800, 1600, 3200 and 6400 roentgens).

It became evident that higher doses were required to get the effects desired on the most resistant cells present.

The distance could not conveniently be reduced since it was desired to keep the cultures in a thermostat. In order to cut down the time of exposure the copper was removed from the filter, leaving the 4.6 mm. aluminum. The radiation was continued for 145 min. The absorption in the glass vessels was about 5%. With this taken into account the dose reaching the cultures is about 19 erythema doses or 15,000 roentgens, given at rate of 103 roentgens per min. The half value layer was found to he 0.395 mm. of copper. Under these conditions the radiation is quite inhomogeneous but quite comparable to that used during treatment of patients.

The data discussed here were derived from cultures radiated at this higher dose. The discussion is limited to the cells migrating from the explant as seen in the living preparation.

Wesson and Burr 1 showed that rats reared on the fat-deficient diet of Burr and Burr 2 gave respiratory quotients above unity and exceptionally high metabolic rates after a carbohydrate meal. Normal rats had lower basal rates and did not show the very high respiratory quotients or great specific dynamic action of the food.

This work has been continued over a period of 2 years with selected rats and their litter mate controls. A different technique has been used in order to follow gas exchange of the rats under normal conditions over long periods of time. An open-circuit apparatus with constant air stream has been used in conjunction with an automatic sampler (to be described elsewhere) which collects the dry samples over mercury and holds them for analysis. The Haldane-Carpenter 3 apparatus was used for determining the CO₂ and O₂.

The rats have been divided into 3 groups: those reared on stock diet; those reared on fat-deficient diet; and those reared on fat-deficient diet and then cured with certain fats. Three different methods of study have been applied. Some rats were starved 16 hours and then 2 periods of starvation metabolism were taken, followed by 4 periods while eating the normal diet. Other rats were run for 24 hours on the normal diet, then the food was removed and the behavior during 24 hours of starvation was followed. Still other rats were kept in the metabolism cage 48 hours on the normal diet. The periods were 4 hours long so that 6 samples were required for a 24-hour run. All runs were made at 28.5–29.0°C. The colony temperature is kept at 24–26°C. at all times.

The results show clearly that fat-deficient rats are very different from stock animals and that fat-deficient rats which have been cured with small doses of fats return to a much more nearly normal gas exchange. The most marked differences shown by the fat-deficient rats are higher basal rate, higher specific dynamic action of food, and higher respiratory quotients. These results are of especial interest since the runs were made over long periods of time under normal conditions. The respiratory quotients of the fat-deficient rats remain above unity for as long as 12 hours out of 24. They, therefore, synthesize every day large amounts of fat, but this synthetic fat does not prevent the fat deficiency.

The responses of the oral and pharyngeal mucous glands to injections of pilocarpine, atropine, and morphine and of the oral glands to epinephrine, resemble those of the salivary glands, an observation which can be explained on the basis of the similar dual innervation of both groups of glands through the cranial autonomic and sympathetic systems.

When Schaefer and Moore 1 gave pilocarpine to dogs without parotid and submaxillary glands, they observed the accumulation of a small quantity of exceedingly viscid mucus which appeared to be formed by the oral mucous glands. Langley, 2 in referring to the effects of suprarenal extracts found that they caused “a lively secretion from all the salivary glands and some secretion from the buccal, esophageal, and tracheal mucous glands.”

During the course of experiments upon the oral and pharyngeal mucous membranes, I observed the effect of the following drugs upon their secretory activity: pilocarpine, atropine, morphine, and epinephrine.

1. Pilocarpine sulfate. In Table I are listed the quantities of mucous secretion which I obtained at 5 minute intervals after intravenous injections of 0.2, 0.3, and 0.5 mg. per kg. of pilocarpine in 3 dogs each weighing 10 kilos. The salivary and orbital glands had been extirpated and the remaining secretion was collected from an esophageal fistula. A total of 35 experiments were performed upon these dogs. Pilocarpine injections result in the production of surprisingly large quantities of mucous secretion.

2. Atropine sulfate. Atropine in sufficient dosage abolished the reflex secretion of the mucous glands and their response to pilocarpine as well.

3. Morphine sulfate. The subcutaneous injection of 15 mg. of morphine produced, in the dogs described above, a transitory augmentation of the mucous secretion coinciding in time with the licking and retching movements and lasting 5 to 10 minutes. During the succeeding period of depression the secretion was normal or subnormal in quantity.

Dragstedt and Dragstedt 1 found in anesthetized animals prepared for the simultaneous collection of gastric, pancreatic and duodenal secretions that the intravenous injection of sublethal doses of fluid obtained from an obstructed duodenum or a closed intestinal loop produced “a striking increase in all of the secretions . . . . ” Ivy et al. 2 later reported that in 7 animals with Pavlov pouches of the stomach the production of a pyloric stenosis decreased at first the secretion of the pouch in response to a test meal. Later 4 of the 7 animals showed a moderate “hypersecretion”. Dragstedt, Montgomery, Ellis and Matthews 3 stated that the experiments referred to above 1 “suggest that under conditions similar to those existing in acute dilatation of the stomach, there occurs an absorption of toxic substances from the duodenum which have a pronounced secretogogue action”. Recently Sungchin Kim 4 observed an increase in the amount of gastric juice in dogs with a duodenal obstruction.

These experiments differ from the conditions seen in patients with acute intestinal obstruction in so far as the use of anesthesia and the intravenous injection of loop fluid 1 are abnormal and the influence of distention as a possible factor in secretion is absent in the Pavlov pouch experiments. We have, therefore, reexamined the subject under conditions which somewhat more naturally simulate the disorder.

In 7 healthy dogs weighing between 10 and 18 kg. the duodenum was divided below the lower pancreatic duct. A Dragstedt type of intestinal cannula was placed in the duodenum proximal to the division. From this cannula the combined gastric, pancreatic, biliary, and duodenal secretions were allowed to run freely into a clean rubber bag. Collections were made every one to 5 hours. The animals were muzzled or their salivary ducts were ligated in order to reduce or eliminate saliva as a factor. No food was given. Two of the animals were discarded because the ingestion of gauze and other materials caused a premature blocking of the cannula. The other 5 may be divided into 2 groups.

Soon after the discovery of latent psittacosis in the breeding establishments of California the sanitation of the aviaries became a paramount problem. A proposal was made to destroy the flocks which were proven to be infected and to resume breeding in the disinfected pens with a native stock imported from Australia. Such a plan might be feasible only provided the native birds should be free from disease. According to reliable reports no psittacosis has been observed or reported from the Australian continent. It was thus anticipated that the budgerigars would possess a high susceptibility to the virus and would therefore lend themselves for exposure and immunity tests.

Permission was obtained to export 200 shell parrakeets through the port of Sydney. The birds had been trapped during the month of June in the vicinity of Adelaide a few days before they left the country. They had no contact with other birds either on land or at sea. On their arrival in 8 large cages, in July, 1933, the green, partly mature and partly immature budgerigars appeared perfectly healthy and remained so under strict isolation in a special room in a separate building removed from any psittacosis work which was in progress.

One month after arrival one bird died in Cage 4. The autopsy was inconclusive but mice inoculated with the spleen and liver suspensions sickened on the 5th to the 7th day and when sacrificed presented enlarged spleens. Aerobic and anaerobic cultures remained sterile. By passage experiments and filtration tests a virus was obtained which produced the typical lesions in mice and infected rice birds by inoculation and per os. The inclusion bodies (L. C. L. bodies) were readily demonstrated. Following this unexpected discovery of the psittacosis virus 10 birds each from Cages 3, 4, 5, 6, 7, and 8 were sacrificed and examined by mouse inoculation tests. In 8 shell parrakeets the virus was present in the spleens and livers and in the nasal mucosa also of one other bird.

There are many reports on transmission experiments with fowl paralysis. Some workers have found a greater incidence of paralysis in chickens inoculated with tissues from paralyzed chickens than in uninoculated controls; e. g., Pappenheimer, Dunn and Seidlin 1 found paralysis in 25% of the inoculated chickens and 7% of the uninoculated controls. Similar but somewhat more successful were the transmission experiments of Dalling and Warrack. 2 Biely, Palmer and Asmundson 3 found that the incidence of paralysis depends on inherited susceptibility, and is not conspicuously influenced by inoculation with tissues from paralyzed chickens. There is no report of a readily transmissible strain of fowl paralysis and the relation of fowl paralysis to leukosis is not clearly defined in the literature.

Two transmissible strains of fowl paralysis have been isolated. Of one strain (Strain 5) 15 successful passages were made, of the other (Strain 6) 10 passages were made. Chart 1 shows only the results of inoculations of the first 5 passages of Strain 5.

The inoculations were made in most instances by intravenous injections of blood from paralyzed chickens, into healthy young chickens. The transmitting agent circulates in the blood of paralyzed chickens throughout the course of illness as shown by the following experiment:

No. 3946 was inoculated and showed beginning paralysis of legs and wings 42 days later. At this time three chickens were injected with 0.5 to 5 cc. of its blood. Two of these chickens developed paralysis and the third remained healthy. Eighteen days later the signs of paralysis were more conspicuous and another two chickens were injected with 0.5 to 5 cc. of blood of chicken No. 3946. Both of these chickens developed paralysis. Thirteen days later both extremities and one wing of No. 3946 were completely paralyzed, and the chicken was killed in extremis; two more chickens were injected with 0.5 cc. of its blood and both died with neurolymphomatosis.

Myeloid leukemia, the most common type of leukemia in man, is rare in animals. A survey of this disease leaves doubt whether myeloid leukemia has ever been observed in domesticated animals (Jármai 1 ). It has been described in mice by Simonds, 2 although neither the bone marrow nor blood smears were available to him.

Myeloid leukemia was rarely found among our mice until a large number of them had been exposed to a single dose of approximately 400 r-units of X-rays or to smaller doses repeated at approximately monthly intervals. Table I contains a preliminary survey of this experiment and shows the frequent occurrence of myeloid leukemia and mediastinal lymphosarcoma among the irradiated mice as compared to the unirradiated, closely related control mice.

Increase of spontaneous lymphomatosis in mice by exposure to X-rays was observed by Krebs, Rask-Nielsen and Wagner. 3 Aubertin 4 called attention to the frequent occurrence of myeloid leukemia in X-ray workers.

The mouse from which the first strain of myeloid leukemia of mice took its origin was an unirradiated male mouse (No. Ar117). At the age of 16 months, the spleen of this mouse was greatly enlarged while the lymph nodes were of normal size or only slightly enlarged. The leukocyte count was 105,000. The most significant feature of the differential count was the presence of 9.5% of immature myeloid cells with coarse basophile granules in the blood. Neutrophile polynuclear leukocytes were 31%, neutrophile myelocytes and metamyelocytes 20%, eosinophile granulocytes 0.5%, cells like monocytes 17%, cells like monocytes with basophile cytoplasm 11.5%, lymphocytes 10.5%. At post mortem examination the liver, bone marrow, and spleen showed extensive infiltrations by myeloid cells with basophile granules and the same cells filled the sinuses of many lymph nodes. Many of these myeloid cells were in the process of mitotic division.

The action of sodium salicylate on the biological functions of streptococci is of particular interest because of the obviously beneficial effects obtained through the administration of sodium salicylate in rheumatic fever. 1 , 2 The etiology of this disease is still considered to be doubtful, yet streptococci seem to be involved, at least in producing complications. Experiments on the direct streptococcidal action of salicylates indicated that this action is not sufficiently strong to explain any curative effect. 3 Curative effect was not found in animal experiments. 4 The opinion that salicylates act in clinical cases mainly by virtue of their antipyretic and analgesic properties has thus come to be almost generally accepted. 5 , 6 However, this is in contradiction to the fact that other antipyretic and analgesic drugs cannot as a rule replace salicylates in rheumatic fever.

Studies on the mechanism of the action of different chemotherapeutic agents (neoarsphenamine, Bayer 205) have indicated that in low therapeutic concentrations these do not kill the parasites but merely decrease their virulence. 7 While the activity of these agents can be easily demonstrated in animal experiments, salicylates do not seem to be active in the experimental infections so far studied. This may be due to the fact that there is no experimental infection of streptococci which is strictly comparable with rheumatic fever. The writers have studied skin lesions produced by the intradermal injection of streptococci (green producing and hemolytic) and by the same organisms treated in vitro with sodium salicylate. There was no appreciable difference between the lesions produced by salicylate-treated streptococci and controls. Neither was any effect observed on similar skin lesions in 2 rabbits which received 10 daily injections of one gram each (5 cc. of 20% solution) of sodium salicylate subcutaneously. Seven injections were given prior to and 3 following the intradermal injections of streptococci.

Mills 1 prepared fibrinogen and prothrombin solutions from platelet-free plasma; the fibrinogen remained unclotted by both calcium and cephalin for 24 hours at 40° C.; the prothrombin needed cephalin as well as calcium for its activation. Few other investigators have secured preparations which could fulfil these rigorous tests, and Howell, 2 in particular, has held to the idea that calcium alone is sufficient to activate prothrombin, the more especially since he and Cekada 3 were able to obtain active thrombins that were lipoid-free. In view of Mills'demonstration that (a) the prothrombin prepared by Howell's acetone method contained cephalin which could be extracted with cold benzene, leaving a preparation which needed cephalin as well as calcium for its activation, and that (b) an appreciable amount of cephalin-like lipoid could be extracted by boiling ether from platelet-free goose and dog plasma, we wondered whether Howell's pure thrombins had been tested on fibrinogen solutions free from lipoids. The following experiment emphasizes the need for satisfying this inquiry.

A dog, not fasted, was anesthetized with sodium amytal. Blood was collected, via a paraffined cannula in the carotid artery, into cooled paraffined centrifuge tubes containing sodium citrate (to a final concentration of 0.5%). After immediate low speed centrifugalization to remove red and white cells, the platelets were separated by prolonged high speed centrifugation. The plasma was salted with 15% (solid) NaCl, followed by 18% (NH₄)₂SO₄, the respective fibrinogen and prothrombin fractions being purified by washing and reprecipitation, and tested for their coagulation reactions. The final solution of prothrombin was found to be activated by calcium alone, and formed a firm clot with fibrinogen solution in 4–5 minutes. After extraction for a few minutes with cold benzene, calcium was unable to activate the coagulant.

Daily electrocardiograms were taken on patients suffering from acute disease who received no digitalis or quinidine. (Table I.) No such systematic electrocardiographic study has been made except in rheumatic fever, 1 pneumonia, 2 and acute rheumatoid (infectious) arthritis. 3 The recorded abnormalities were produced during the illness. Transient changes in lead III only were not included in the table.

For control data, 50 cases of acute rheumatoid (infectious) arthritis, 6 cases of ulcerative colitis and 9 cases of peptic ulcer were chosen. In the former 2 groups the patients were acutely ill with fever. In all the control patients no abnormalities in T-, QRS-waves, or in RS-T transitions, no auricular flutter or fibrillation, no heart-block were observed. Hence, one may conclude that an acute illness with fever and tachycardia does not produce significant arrhythmias, increased P-R intervals, T-wave inversions or marked RS-T changes unless the myocardium is involved.

Most marked heart-block and the most frequent arrhythmias were observed in acute rheumatic fever but a larger number of patients with this disease was studied than in any other. In acute disseminated lupus erythematosis, in periarteritis nodosa, in Malta fever, in capillary toxicosis, bronchopneumonia, typhoid and acute rheumatic fever, T-wave changes were most frequent. It is suggested that some of these diseases may have similar pathological alterations in the myocardium of vascular nature and that they may produce permanent heart damage.

A very fast pulse occurred in many cases with severe myocardial involvement. In 7 cases of pulmonary tuberculosis, for example, with rates above 120 beats per minute, 6 disclosed abnormal electrocardiograms. In this disease the occurrence of a heart rate of over 120 beats per minute should suggest myocardial involvement.

Bell, 1 using embryos of Rana esculenta, attempted to show the relation of the brain to the development of the olfactory placode. He transplanted “that part of the brain which forms the olfactory lobe” to other regions of the body without obtaining any induction of an olfactory placode. His conclusion was that the olfactory placode developed independently of the brain. He did not, however, preclude the possibility of an early stimulus for olfactory placode formation being imparted by the brain to the ectoderm above it.

Recently we have been able to show that the part of the fore-brain median and slightly anterior to the optic vesicle of the intermediate neurula stage of Rana pipiens has the power to induce an olfactory placode in the ectoderm of the flank (Fig. 1).

After one accidental induction (from a transplant of an optic vesicle which evidently included some of the rest of the fore-brain) 3 series of operations were performed in an attempt to localize the olfactory placode inducing part of the brain. The following stages were used for the operation:

Early neurula—equivalent to stage 17 on Harrison's scale for Ambystoma punctatum.

Intermediate neurula—equivalent to stage 20 on Harrison's scaie for Ambystoma punctatum.

Late neurula—equivalent to stage 22 on Harrison's scale for Ambystoma punctatum.

Early tail-bud—in which the olfactory placode is visible as a pigmented thickening of the ectoderm.

Several methods are available for measuring the circulation time from the antecubital veins through the pulmonary bed to the capillaries of the systemic circuit. One method is the decholin method proposed by Winternitz, Deutsch, and Bruell, 1 and recently studied by Tarr, Oppenheimer and Sager. 2 Another is described by Fishberg, Hitzig, and King, 3 in which saccharin injected into an antecubital vein is detected in the tongue. No method has been devised clinically applicable for measuring circulation time from the peripheral veins to the capillaries of the lung. This may be obtained by introducing ether into an antecubital vein and determining the time that elapses before the patient perceives its odor. Ether is peculiarly adapted to this purpose because: 1. Its volatility at blood temperature is so great that when a small quantity is injected, a sufficient amount volatilizes in the pulmonary capillaries during the first circuit of the blood to be invariably perceptible by smell. 2. The small volume needed permits rapid intravenous introduction, the advantage of this being that it will flow in a small blood volume, resulting in a sharp definition of both the time of injection and the time of arrival in the pulmonary capillaries. 3. It is harmless in the quantities used. In over 200 injections, no untoward constitutional reactions were encountered. The subject is usually aware of a “creeping” feeling along the course of the vein. About 25% of the patients complain of transient pain in this location. In 28 cases in which the veins were carefully followed, thrombosis developed on 3 occasions. Paravenous infiltration causes no necrosis. In many instances, the measurement was carried out 2 and even 3 times within a few minutes without causing unpleasant reactions. 4. Because of its volatility, the measurement can be repeated as soon as desired.

The dissimilations brought about by the propionic acid bacteria have received relatively little study. Phosphorylation appears to be a normal process with the intermediate formation of a hexose-monophosphate. 1 Methylglyoxal has been reported by Pett and Wynne 2 and Wood and Werkman have recently identified pyruvic acid 3 and a non-reducing carbohydrate 4 as intermediate products in the fermentation of glucose by Propionibacterium arabinosum. Virtanen 5 proposed the occurrence of lactic acid but was unable to detect its presence. Foote, Fred and Peterson 6 have reported the occurrence of lactic acid in the propionic acid fermentation. The intermediate mechanism leading to propionic acid is unknown. The fermentation of glycerol offers an opportunity of studying this phase. In this remarkable fermentation, glycerol is converted under anaerobic conditions practically quantitatively into propionic acid. No gas is produced (van Niel 10 ).

To detect intermediate compounds the following medium was fermented: Yeast extract (Difco) 0.5%, glycerol 2.0%, and calcium carbonate 2.0%. Calcium sulfite (1.0%) was included in the medium as a fixing reagent. P. arabinosum was the species employed. The fermentation took place under anaerobic conditions at 30°C. After 5 or 6 days'incubation, the fermentations were tested for aldehydes and ketones by addition of 2, 4-dinitrophenylhydrazine. An abundant precipitation was obtained. Distillation of the medium according to Peterson and Fred 7 gave a quantitative recovery of the unknown carbonyl compound. Derivatives were prepared from concentrated distillates and purified by crystallization. Table I gives the melting points obtained.

The finding of Anselmino and Hoffman, 1 , 2 that a fraction from the anterior pituitary, when injected into rats, markedly increases the acetone bodies in the blood, received support from the work of Funk, 3 , 4 who, using rats as test animals, showed that this peculiar “fat-metabolism” hormone is present in the urine of pregnancy and in normal urine. Funk records large increases in urinary acetone bodies. Katzman and Doisy, 5 using their method for getting the gonadotropic factor of pregnancy urine, find that hyperglycemia is produced by these extracts. They worked with rabbits.

Believing that Funk, on the one hand, and Katzman and Doisy, on the other, were working with an extract which contained the same active material, we prepared our extracts according to Funk's method and tested them on rabbits for blood-sugar, according to Katzman and Doisy. In addition, we also determined acetone bodies in the blood. The results are presented in Tables I and II.

Not only do we get hyperglycemia comparable to that obtained by Katzman and Doisy, but we also get a very definite increase in the acetone in the blood. We also get a decrease in CO₂-combining power and an increase in lactic acid. We may add that, aside from one result which appears anomalous, all attempts so far to obtain appreciable increases in urinary glucose and acetone have failed.

It is, perhaps, somewhat premature to interpret the significance of these results. However, as a tentative hypothesis, we may regard this anterior pituitary-like substance eliminated in the urine as being involved in carbohydrate metabolism, primarily, and in fat metabolism, secondarily.

It is possible to time the velocity of the blood current in man, starting, for example, from the median basilic vein in the elbow and ending at the alveoli of the lungs. The intervening distance may be considered as a constant circuit, part of the pulmonary circulation. The time needed for blood to traverse this intervening distance was carefully noted in conditions of health and disease. This report, based on studies in man, describes the technique of the method employed, results obtained by this method, and its application in physiological and clinical problems.

The subject is recumbent in bed. His mouth having been freely rinsed with tap water, directly before the test, he is instructed to breathe rapidly, about 40 excursions per minute, and to hold his mouth wide open. At ordinary room temperature (70° to 72°F.) and under usual atmospheric conditions (753 mm. Hg.) a measured quantity of a non-toxic substance possessing an odor easily identified is quickly injected, within one second, into the right or left median basilic vein at the elbow and the arrival of the odor in the subject's breath instantly noted. The time required for the odor to complete this circuit is carefully clocked.

For intravenous injections the following substances have been utilized: guaiacol, sodium cacodylate, allyl sulphide, methyl salicylate, ether, and perfumes, more especially ingredients in the preparation of jasmin perfume, benzyl acetate and linalyl acetate. The sharpest results are obtained with perfume and ether. Our earliest use of ether, October, 1932, was suggested by the work of Burkhardt, Kümmell, and Küttner, 1 , 2 , 3 who published results on the intravenous administration of ether as an anesthetic and commented on the prompt appearance of ether in the breath.

A study of the growth of pathogenic fungi on a mixture of barber shop hair and scalp products of adults was reported. 1 In this paper the results of a study of the growth of pathogenic fungi on the hair and scalp products of children under 12 years, before and after extraction with ether, are reported.

The hair was divided into 2 portions and one portion was extracted with ether for 24 hours in a Soxhlet. Portions of the material were placed in test tubes, moistened with distilled water and autoclaved. Plants were made on the extracted and unextracted hair and Sabourauds'proof medium with the following pathogenic fungi and saprophytes, Lichtheimia sp. and Scopulariopsis brevicaulis; Achorion schoenleinii, Acladium castellani, Candida candida, Endodermophyton tropicale, Endomyces capsulatus, Endomyces dermatidus, Epidermophyton inguinale, Glenospora gammeli, Geotrichum bachmann, Indiella americana, Monilia albicans, Microsporon apiospermum, Microsporon audouini, Oöspora humi, Trichophyton crateriforme, Trichophyton granulosum, Trichophyton japonicum, Trichophyton interdigitale, Willia anomala.

The results of the observations over a period of 30 days were as follows: Growth on Sabouraud's occurred in all instances within 3 days. Growth on extracted hair occurred in all instances with the exception of Endomyces capsulatus, while growth on plain hair was absent only in the case of Achorion schoenleinii. Growth was much more pronounced on extracted hair with Glenospora gammeli, Indiella americana, Microsporon apiospermum, Microsporon audouini, Monilia albicans, Trichophyton interdigitale. Growth occurred as early as the third day on both extracted and unextracted hair with Acladium castellani, Candida candida, Geotrichum bachmanni, Indiella americana, Lichthiemia sp., Microsporon apiospermum, Monilia albicans, Oöspora humi, Scopulariopsis brevicaulis, Trichophyton crateriforme, Trichophyton granulosum, Trichophyton interdigitale, Trichophyton japonicum.

It has been shown by Pearl and Curtis 1 that during the sojourn of the egg in the uterus the weight and nitrogen content of the albumen increases by the addition of a nitrogenous solution after the shell membrane has been formed. Data were collected to observe what kind of nitrogenous materials were added after the shell membrane was formed.

Mature eggs to be used as controls were obtained in the morning from hens just beginning to lay a clutch of eggs, so that it was likely that they would lay the next day. Four to 6 hours after the egg was laid the hens were sacrificed and the immature eggs removed from the uterus. At this time the eggs were found with the shell membrane formed. The mature egg of the morning and the later immature egg from the same hens were analyzed as pairs.

The eggs so obtained were separated into yolk, thick albumen and thin albumen. A screen, similar to that described by Holtz 2 and Almquist was used for the separation of the thick from the thin albumen. Thirteen pairs of eggs were separated in this manner. The results of the separation of the albumen and the yolk were found to confirm Pearl and Curtis in that only 60% of the albumen by weight, and 90% of the nitrogen in the albumen were present after the shell membrane was formed, and it was for the most part thick albumen.

The albumens were diluted, the ovomucins were removed with the centrifuge, and the supernatant fluid was made up to volume with a small amount of sodium chloride or sodium sulfate present to prevent precipitation of the ovoglobulins. The ovoglobulins and the ovalbumins were separated by precipitation of the ovoglobulins with 1.5 M sodium sulfate by a technique similar to that of Howe 3 for blood serum proteins. Non-protein nitrogen was determined by using Alman's tannic acid reagent or 2.12 sodium sulfate for protein precipitation. The ovomucoid content was determined by heat coagulation of the other proteins in the presence of acetate buffer approximately pH 4.7 and 0.2 M sodium sulfate. No attempt was made to separate the ovoglobulins or ovalbumins. Nitrogen was determined by macro-Kjeldahl.

During the course of studies concerning hypersensitiveness in patients with trachoma, it became necessary to determine the possibility of the existence of different immunological types among Staphylococci. Preliminary experiments undertaken with the agglutination reaction revealed that strains isolated from different sources and conditions are not differentiated satisfactorily by this technique, Consequently an effort was made to fractionate carbohydrates from this organism, and this was accomplished successfully.

The method finally adopted for the separation of carbohydrates from Staphylococci may be outlined as follows: (1) concentration of broth cultures (18–24 hours) by centrifugation in a Sharpies; (2) heating the thick suspension in N/16 HCl for 20 minutes in a boiling water bath; (3) after centrifuging, the supernatant is treated with 40% NaOH until flocculent precipitation occurs, which is at a slightly acid reaction; (4) to the supernatant N acetic acid and Na acetate, and 2 volumes of 95% alcohol are added; (5) after standing in refrigerator over night, the residue following centrifugation is taken up in H₂O, and the supernatant is reserved for further separation; (6) the water solution is treated with N acetic acid until precipitation occurs and to the supernatant after centrifugation are added 2 volumes of 95% alcohol and sufficient Na acetate to cause precipitation. The residue is re-extracted until all the soluble material is obtained; (7) up to 6 precipitations with acetic acid are sometimes required to cause complete removal of precipitable matter. The end product never precipitates in the usual protein precipitants; it is always ninhydrin negative, although from time to time biuret positive. The total nitrogen has varied up to 5%. The Molisch reaction is always strongly positive.

Various investigators have presented evidence to show that the hypophysis plays an important rôle in carbohydrate metabolism. The first conclusive evidence is that of Houssay and coworkers, who found (1) that extracts of the anterior lobe of the hypophysis injected into normal dogs produce hyperglycemia, glycosuria and ketonuria, and when injected into depancreatized dogs, an increase in severity of the diabetes; 1 , 2 (2) that removal of the hypophysis in normal dogs leads to a hypersensitivity to insulin, 3 hypoglycemic convulsions during fasting, and an increased tolerance to glucose; 4 , 5 (3) that in depancreatized dogs, removal of the hypophysis restores the carbohydrate metabolism to an approximately normal condition, but renders the animal hypersensitive to insulin; 6 , 7 that injection of active anterior-lobe extracts causes a reappearance of the symptoms of diabetes in hypophysectomized-depancreatized dogs. 8 These findings have been largely confirmed by the work of Evans et al., 9 Barnes and Regan, 10 Daggs and Eaton, 11 and Corkill et al. 12

Since the removal of the hypophysis decreases the symptoms of experimental diabetes, it was thought that destructive doses of X-ray to the hypophysis might produce similar results.

Six dogs were depancreatized and allowed to recover under insulin administration. About one week after operation they were placed on a basic diet of dog biscuits, cane sugar and freshly ground meat, to which was added liberal amounts of fresh or dried pancreas. Approximately 2 units of insulin per kilo body weight were given twice daily at the time of feeding. After the wound had healed completely, usually within 3 weeks, food and insulin were withheld for a period of 18 to 20 hours, and the tolerance of the animal to orally administered glucose was determined. After taking an initial blood sample, 1.75 gm. glucose per kilo was given by stomach tube, and further blood samples were taken at 15, 45, 75, and 105 minute intervals. Blood sugar was determined by Folin's method.

The primary purpose of this study is to determine whether the fetus can survive extensive damage to the brain, and the possible effects of brain injury on subsequent growth and development. Previous studies have been almost entirely confined to observations on teratological (anencephalic) human material (Preyer, 1 Bruns et al., 2 Nañagas, 3 Marcus and Nickman 4 ).

Destruction of portions of the fetal brain (rat∗) was accomplished by means of the electric cautery. No repair was found necessary other than suture of the maternal body wall (Nicholas 5 ). Fetuses recovered from the uterus or taken at birth subsequent to such procedure were examined as to behavior, measured, weighed and placed at once in 5% formalin preparatory to microscopic study of the injured area. Unoperated litter-mates served as controls, A series of control experiments involved (a) injection of carbon ink into the ventricles of the brain, and (b) extensive cautery of an extremity. Seventy fetuses from 21 litters comprise the experimental material, and 36 fetuses from 10 litters were used in the control experiments.

Of the 70 operated fetuses ranging between 11 and 37 mm. of crown-rump length at operation, 40 survived until term or were recovered alive from the uterus 1, 2, 3, 4, 5, or 6 days following cautery. The remainder were either aborted (2) or partially resorbed. At term (39 to 42 mm.) 11 fetuses died in utero and none of the animals survived parturition more than 24 hours. The rat fetus may survive brain cautery until term regardless of the stage of development at which the operation is performed.

Harvey and Taylor 1 have shown that concentrated heavy water markedly inhibits the respiration of 2 species of luminous bacteria and diminishes the luminescence of one form (Bacillus fischeri). The effect on the respiration of Bacillus fischeri was such that if the curve relating effect on respiration with concentration of heavy water is extrapolated, zero respiration is indicated in 100% heavy water. Reduction of respiration of Vibrio phosphorescens was not so great.

Experiments with yeast indicate that the effect of heavy water on respiration of this organism is also not so great. These are detailed below together with a study of the respiration in as near 100% heavy water as is experimentally feasible considering the hygroscopic properties of D₂O. Pacsu 2 has reported a 9-fold reduction of yeast fermentation (CO₂ production) in approximately 100% D₂O and 1.6 reduction in 60% D₂O).

The heavy water was provided by Professor H. S. Taylor, and had been prepared by electrolysis and burning of deuterium and oxygen in the manner described by Taylor, Eyring and Frost. 3 It had also been distilled from alkali and permanganate. High concentrations of such water is toxic for paramoecium, whereas low concentrations, (0.2%) although prepared in exactly the same manner and also distilled from alkali and permanganate in the same vessel, are not toxic, so that the diminution of respiration of cells in concentrated heavy water must be connected with the deuterium which it contains and not with impurities of any kind. Yeast in contact with 0.2% D₂O for 17 hours and control yeast in contact with distilled water for 17 hours both respire at the same rate (within 5%) when nutrient is added, showing that no deleterious impurities having a cumulative effect were present.

It has been shown that 2 antagonistic hormones are present in the anterior pituitary gland, one causing growth and maturation of follicles and the other atresia of follicles in the ovary of the guinea pig; the former predominates in the anterior pituitaries of rabbit, rat and guinea pig and the latter in the glands of cattle, sheep and hog, at least in the state in which these latter are accessible to us for experimentation. 1 , 2 , 3 When as the result of the action of the first hormone the follicle has reached full size and matured, either ovulation and formation of true corpora lutea may take place or there may be ingrowth of vessels and connective tissue into the granulosa and formation of pseudocorpora lutea not associated with ovulation. 2 There occurs a third effect common to the substances in which both of these hormones are found, a transformation of theca interna, and probably also of connective tissue cells into strands of interstitial gland and into interstitial gland bodies, which transformation is found in the course of follicular atresia. 4 Transitions from the interstitial gland bodies to small pseudocorpora lutea occur and such pseudocorpora lutea may be seen especially after several injections of extract of cattle anterior pituitary into the guinea pig. Smaller amounts of pseudolutein tissue may furthermore be produced in the ovary of the guinea pig in follicles undergoing connective tissue atresia as a result of cattle anterior pituitary extracts. However, we do not wish to exclude the possibility that under certain conditions also granulosa may participate in the formation of these structures, at least in a limited way.

Under these conditions it was of interest to study the combined effect of these 2 antagonistic principles in the guinea pig. Such investigations were begun several years ago; at that time we combined injections of extract of cattle anterior pituitary with implantations of guinea pig anterior pituitary and in one case with implantation of anterior pituitaries of rabbit. 4

To elucidate the relationship between the chemical structure and the pharmacological action in the barbital series, over 50 derivatives were investigated. These compounds were all 5, 5-substituted barbituric acids, having the general formula:

wherein R-alkyl radical (normal or secondary with 2 to 9 C-atoms). Several of them were new compounds synthesized for the first time. 1

Albino rats weighing from 71 to 126 gm. (average 96 gm.) were employed in this study. Solutions of the sodium salts of the compounds were injected intraperitoneally. The minimal anesthetic dose (M.A.D.), the duration of action, and the minimal lethal dose (M.L.D) were determined by using 5 animals for each dose level.

Since space does not permit a presentation of detailed results, only a few salient points will be discussed here. In Figs. 1 and 2, the “primary alkyls” refer to those compounds having a normal alkyl group. It should be noticed from Fig. 1 that with the increase in number of C-atoms in the alkyl group, either normal or secondary, both the M.A.D. and M.L.D. grow relatively smaller, but when the alkyl radical is longer than 5 C-atoms, the amount required to anesthetize or kill rats again increases. The therapeutic index, or the ratio between the M.L.D. and M.A.D., appears to be gradually greater as the alkyl chain lengthens.

In general, the duration of action shows similar features; that is, it is shorter when the alkyl group becomes lengthened (Fig. 2). In the series of normal alkyl derivatives, the critical compound is the one that possesses 6 C-atoms at R; and in the series of secondary alkyl derivatives, the critical compound is the one having 7 C-atoms at R, beyond which the duration of action begins to increase.

In a previous communication, 1 it was pointed out that there is obvious relationship between the pharmacological action and the chemical structure of certain barbituric acid derivatives, and that compounds with long alkyl groups have a shorter duration of action. The present report deals with the evaluation of 12 such compounds, all of which were prepared by Shonle, Waldo, Keltch, and Coles. 2

Typical hypnotic and anesthetic properties were observed with all the substances except one, injected intraperitoneally, in albino rats. Their minimal hypnotic doses (M.H.D.), minimal anesthetic doses (M.A.D.), and minimal lethal doses (M.L.D.) were determined, and their therapeutic indices calculated as shown in Table I. Compounds numbered 1, 2, 3, and 7 were also tested in dogs, being effective either by vein or by mouth. It is curious that compound numbered 12, 1, 3-dimethyl-butyl-ethyl barbituric acid, is devoid of any hypnotic or anesthetic action, but on the contrary, produces convulsions. The substance is also highly toxic.

Some unexpected differences in the distribution of blood groups observed by us a year ago 1 , 2 among the “Blackfeet'and “Blood” tribes of American Indians suggested that the different tribes may not have had a single origin and that at least some of the tribes may not have separated from the rest of the human family before the A agglutinogen developed in the race. Since the possibility of a degree of racial admixture among putative full blooded Indians always remains, regardless of their pedigree as given in the Agency records, the desirability of detecting the A agglutinogen in mummies of Indians who lived before the advent of the white man suggested itself.

Through the courtesy of Drs. G. D. Williams and H. A. McCordock, muscle tissue from 12 mummies from Peru, Arizona and Greenland, was placed at our disposal for such a study.

Because of the limited amount of material available in the preliminary experiments, dried cadaver tissues which had been previously washed and dried rapidly under a current of warm air were used in place of the mummy material.

The procedure followed at first was one in which O(αβ) serum was absorbed with cadaver tissues of groups O and A respectively by allowing mixtures of a small amount of the powdered tissue and varying dilutions of O(αβ) serum to remain over-night in the refrigerator. After centrifuging, the supernatnant fluid was tested with O, A and B cells to determine whether the absorption of the agglutinins by the tissues had occurred. In such a test it is considered that if the group A cells are not agglutinated while B cells are, that the α agglutinin in the O(αβ) serum has been absorbed by an A agglutinogen in the tissue; conversely, if B cells are not agglutinated whereas A cells are, the tissue would be considered to have contained the B agglutinogen which absorbed the β agglutinin from the O(αβ) serum. If, however, there is no inhibition of agglutination of either A or B cells, it may be assumed that the tissue contains neither A nor B agglutinogen and may consequently be considered to belong to group O.

The average alkalinity of the duodenal secretions was found to be approximately .04 normal and the chloride concentration 307 mg. per 100 cc. These values obtained on secretions from the intact duodenum are in excellent agreement with values previously reported on secretion obtained from isolated duodenal pouches.

In a previous communication 1 the results of studies on the composition of mixed duodenal secretions were reported. These previous studies were performed on healed duodenal pouches completely isolated from the stomach and every effort was made to avoid all complications which might alter the composition of the mixed duodenal secretions. However, it appeared necessary to confirm these results by different methods which would be free of certain hypothetical sources of error, based primarily on the operative procedures previously employed. A large number of analyses of the contents of the dogs'stomach obtained by lavage 24 hours after a light feeding of milk and syrup have shown that under these conditions hydrochloric acid is either absent or present in very small amounts. It therefore seemed that duodenal contents obtained from the intact duodenum, 24 hours after a light feeding of milk and syrup, would be practically free from contamination by acid gastric contents. Gastrostomy openings were made low on the body of the stomach through which a duodenal tube was inserted and the tip allowed to pass into the duodenum. When the dogs were lying on the side, the main cavity of the stomach was usually below the opening into the duodenum. At frequent intervals during the experiments a small rubber catheter was passed into the stomach and an attempt made to aspirate gastric secretions. Nothing but a small amount of tenacious mucus was ever obtained. The duodenum was stimulated by introducing a standard hydrochloric acid solution (approximately one-tenth normal) containing phenol red through the duodenal tube. After a latent period of several minutes'duration a copious flow of duodenal secretions occurred. The secretions aspirated from the duodenum often contained some of the acid-phenol red solution which had been introduced but the phenol red was too dilute to determine accurately, hence the duodenal secretions were added to approximately 25 cc. of the same strength acid-phenol red solution that had been introduced into the duodenum.

Over a period of a week or 10 days following intravenous injection of methylene blue into dogs poisoned with cyanide there occurs a decrease in the oxygen capacity of the blood and a corresponding decrease in red blood cell count. The same effect is observed when the dye alone is given. Results of a typical experiment are reported in this preliminary note.

Table I shows the changes in red blood cell count, oxyhemoglobin (by O₂ capacity method), and reticulocytes in a 17 kilogram dog following intravenous injection of 30 mg. of methylene blue per kilo body weight.

Each of 5 dogs which received methylene blue (in quantities ranging from 15 to 40 mg. per kilo body weight) developed an anemia the severity of which was greatest with the larger doses. In 2 experiments, otherwise less complete than that reported, the oxyhemoglobin (O₂ capacity) of the blood was determined on the first and second days following methylene blue injection and was found to be only slightly below the preinjection level, although several days later it had fallen to severely anemic levels. This fact and the absence of hemoglobinuria and hematinuria during the entire period following dye injection indicate that there is no immediate massive destruction of cells in the circulation.

Experiments designed to relate quantitatively dosage to response and to determine the mechanism of the methylene blue action are in progress. We also have in mind the possibility that methylene blue may find practical application in the treatment of polycythemia vera.

Clinical as well as experimental observations show that the cortical centers of vegetative organs are, in many instances, in close topical relation to those of adjacent skeletal muscles. The descending corticofugal tracts to the peripheral part of the autonomic system are topographically related to the pyramidal tract. They also lose their excitability after destruction of the motor cortex and frontal lobe, within the same time as do the corticofugal somatic fibers. (Spiegel, 1 Friedberg. 2 ) It seemed, therefore, of interest to ascertain whether the pyramidal tracts by themselves can conduct corticofugal impulses to the autonomic system. The experiments were performed on 30 cats. In a first series, the “extrapyramidal” fibers that arise from the hypothalamus were severed by deep incision into the floor of the 4th ventricle. The central grey matter and tegmentum in the pons and midbrain were destroyed where the centrifugal fibers of the hypothalamus are located. In a second series of experiments, the conduction of cortical impulses to vegetative organs was studied after transverse section of both pyramidal tracts on the base of the pons. After the lesion of either of these systems had been performed, the effects of stimulation of the motor cortex and the frontal lobe was noticed on the pupil, the blood vessels, the heart, the sweat glands and the urinary bladder.

In the study of the pupil, the lesion of the brain stem was combined with unilateral cervical sympathectomy. Thus, cortical stimulation elicited on the pupil ipsi-lateral to the sympathectomy only inhibition of the sphincter, on the other pupil inhibition of the oculomotor plus stimulation of the sympathetic.

Menkin 1 recently demonstrated that various materials are fixed in situ when injected into an acutely inflamed area, and that the mechanism of fixation of such substances as bacteria and trypan blue is primarily mechanical obstruction in the form of a fibrinous net-work and thrombosed lymphatics at the site of inflammation. Obviously the speed with which an inflammatory irritant causes the region to be blocked off is a significant factor in determining its ability to disseminate into the contiguous tissues or blood stream. Menkin 1 observed that Staphylococcus aureus caused the formation of thrombi in the lymphatics and fixation of trypan blue within about one hour after their introduction into the skin of rabbits, while the more invasive Streptococcus hemolyticus produced fixation only about 45 hours later. Menkin believed that the delayed fixation in the latter case was due to the “relatively mild local effects” of the streptococcus.

Using Menkin's 1 method, we have fully confirmed his observations of the behavior of Strep. hemolyticus and Staph, aureus in vivo, but our additional data show that the delayed inflammatory fixation of streptococci is due to the production by these organisms of a substance or substances, active both in vivo and in vitro, which when present in sufficient concentration is capable of inhibiting inflammatory fixation for many hours, thus enabling the organisms to disseminate relatively freely through the lymphatics.

Our in vivo observations have been carried out on more than 150 rabbits. The organisms studied were the Dochez and Griffith scarlatinal strains and Birkhaug's E₁ erysipelas strain of Strep. hemolyticus, a virulent strain of Strep, viridans, and 2 strains of Staph, aurevis. The “fixation time” for each strain was: Dochez 48–56 hr.; Griffith 48 hr.; E₁ 48 hr.; Strep, viridans 36 hr.; Staph. aureus 1–2 hr. The staphylococcus strains used always produced fixation of the dye in less than 2 hours.

The clinical observation that the beneficial effect of total thyroidectomy in angina pectoris is an almost immediate one, and so cannot be due entirely to the fall in the basal metabolic rate which does not begin to occur for over a week, has given rise to speculation as to the possible mechanism involved.

We investigated the possibility that this effect might be due to interruption of nerve impulses from the heart. Our method was that described by Sutton and Lueth 1 for producing “experimental angina pectoris” in the dog consisting in placing a ligature about the anterior descending branch of the left coronary artery and compressing the coronary vessels by traction on this ligature. This method was successfully employed by White and his associates 2 to test the efficacy of various surgical procedures in interrupting the afferent nervous pathways of the heart. Five dogs were tested in this manner after removal of both thyroids. One animal was tested 5 hours after thyroidectomy, and 4 dogs on the eleventh, thirteenth, fourteenth, and twenty-eighth day respectively. A definite discomfort response was elicited in each instance. Two of the dogs had been similarly tested previous to the thyroidectomy and in these no diminution was noted in the amount of traction required to produce the typical response.

The relief of angina by removal of the thyroid may be due to the diminished sensitiveness to adrenalin 3 which has been demonstrated clinically as early as 24 hours following the operation. This implies that the individual attacks of angina pectoris in a patient suffering from this disorder may be precipitated by a sudden increase in the patient's physiological output of adrenalin. We attempted to precipitate the discomfort response in dogs by injecting adrenalin. This was successfully accomplished in 9 dogs by applying a constant subminimal traction on the coronary suture by means of weights suspended over pulleys, and then raising the blood pressure suddenly by injecting adrenalin into the femoral vein.

Experimental data were presented 1 on 5 dogs in which each was sensitized with horse serum in the left cerebral motor area after injury to this region. Following an interval of 2 to 4 weeks they received intravenous injections of horse serum. These injections were followed in 4 of the 5 animals by transient right-sided hemiparesis.

The present communication concerns similar experiments on 35 dogs. Three, serving as controls and receiving normal saline into the brain instead of sensitizing protein, showed no subsequent focal reaction on repeated intravenous injection of the test protein. Twenty-one of the remaining 32 dogs showed evidence of local cerebral irritability after intravenous injection of the same protein with which the brain had been previously sensitized. In many of the dogs the local reaction could be repeated and was always confined to the side opposite the sensitized cerebral hemisphere. In a few animals which were kept alive over a long period of time the same reaction occurred almost as often as the intravenous injection was made, provided proper intervals between injections were allowed. One animal showed a focal convulsive seizure which became generalized and eventuated in death.

For the most part horse serum and egg albumen were used as the sensitizing proteins. A few protocols are cited to bring out special points:

DOG NO. 17. Small female Airedale.

1- 7–32. The left side of the brain was exposed and an agar disc containing horse serum, 1 mm. thick and 1.5 cm. in diameter, was placed against the exposed area and held in place by suturing the temporal muscle over it.

1-12–32. 1 cc. of horse serum was injected intravenously. In a few minutes symptoms of general anaphylaxis appeared (defecation and urination).

2-26–32. 2 cc. of horse serum were injected intravenously. In 2 minutes defecation and urin∗∗∗tion occurred. In 12 minutes slight hut definite dragging of the right forepaw was observed.

3- 9–32. Into a pocket formed by a small rubber ring placed over the right cerebrum 0.1 cc. of fresh egg albumen was injected between the dura and pia mater.

In order to determine whether the drop in natural resistance following the removal of the spleen is of a general character or is specific for certain types of infections and toxemias, the effect of the removal of the spleen in rats and mice on their resistance to histamine was observed. In these studies rats free of Bartonella muris infection and mice free of Bartonella muris, Eperythrozoon coccoides and Klossiella muris were used. 1 These latent infections are adversely influenced by splenectomy. 1

Ten splenectomized rats of Wistar stock received ergotamine acid phosphate one week after operation in amounts ranging from 700 to 1200 mg. per kg. of body weight. Those receiving 1000 to 1200 mg. per kg. died; the rest survived. Of 10 control rats the M.L.D. was found to lie between 1000 and 1200 mg. per kg. of body weight.

A similar number of splenectomized mice free of latent infections likewise survived as large an amount of ergotamine acid phosphate as the control mice. The M.L.D. of histamine in the mice of this strain was 1700 to 2000 mg. per kg. of body weight.

Splenectomy in rats and mice free of latent infections does not alter the resistance of these animals to histamine poisoning. In view of the fact that the removal of the spleen depresses the natural resistance to many infections, it is interesting that the resistance to a chemical poison such as histamine is unaffected.

Bilateral suprarenalectomy, on the other hand, lowers the natural resistance of rats to all toxemias, infections and poisons. In contrast to suprarenalectomy, splenectomy specifically affects the resistance to certain types of infections and toxemias, but the drop in natural resistance is not of a general character. 2

In rats that were carriers of Bartonella muris, certain changes were observed, following splenectomy and recovery from the anemia, in the hemocytoblastic tissue and in the reticular and endothelial elements. These changes appeared 3 to 5 months after splenectomy and were associated with immunity to further infection with Bartonella muris. The changes consisted primarily of hyperplasia of hemolymph tissue, hyperplasia of reticular and endothelial elements of lymph nodes, the formation of lymphoblastic foci periportally in the liver and peribronchially and perivascularly in the lung, regeneration of all elements of the thymus and marked hyperplasia of all elements of bone marrow (increased hematopoiesis). 1

In view of the presence of coincident latent infection in the rat, the compensatory changes described following splenectomy in rats of Bartonella carrier stock were difficult to evaluate. What was due to the absence of the spleen and what to previous Bartonella muris anemia remained uncertain. Studies were therefore made in rats in which the exacerbations of latent infections following splenectomy could be excluded. Six Bartonella-free rats, 3 months of age, of Wistar stock, were splenectomized and killed 6 months later. Six unsplenectomized rats of the same age and stock were killed at the same time. The presence of Bartonella infection was excluded by red blood cell counts and smears of peripheral blood at frequent intervals.

The anatomical changes observed were similar to those found in splenectomized Bartonella-carrier rats but less extensive. The liver showed little if any perivascular lymphocytic infiltration. The Kupffer cells were not enlarged, nor were they more prominent in phagocytic activity than in normal rats.

Adequate scientific proof of the existence of Trichophyton interdigitale (organism of “Athlete's Foot”) outside the body seems lacking. Statements in the literature, however, would lead one to judge otherwise. The U. S. Public Health Service states 1 : “Bath mats are justly blamed and it is probable that ringworm can be acquired from them just as warts on the soles of the feet can be . . . . . it can also originate in hotels and from the use of infected towels or soap.” The Public Health Engineering Section of the American Public Health Association 2 states: “Most if not all of these foot diseases are caused by a fungus which is spread by infection of the floors of dressing rooms, etc., at pools, bath houses, gymnasiums, and other places where persons go barefoot.” While these inferences may be true they are unaccompanied by cultural proof.

Bonar and Dryer 3 made 30–40 plants of scrapings of floors, hair, etc., in cracks in the floor and traps on the drains from showers. They were troubled by excessive overgrowth with other organisms. They failed to recover the Trichophyton interdigitale.

Because of scarcity of cultural work we felt such a study of showers, boat houses and gymnasia would be valuable.

Cotton applicators were moistened with normal saline, placed in test tubes which were plugged with cotton and autoclaved. The applicators were used to mop the area to be studied. They were then cut near the cotton end with a flamed scissors and this portion allowed to drop into a sterile petri dish. Melted Sabouraud's proof medium was poured over the swab and the medium agitated by sliding the dish back and forth on the table. In this manner an even distribution of colonies was obtained. The petri dishes were observed over a period of a month and transplants made on Sabouraud's proof slants of colonies which appeared the least suspicious.

In studying a group of fungi the inhibitory effects of varying concentrations of glycerine were tabulated from the point of view of possible differential features. Glycerine was incorporated in Sabouraud's proof medium and the pH adjusted to 5.5. Concentrations of 1, 5, 10, 15, 20 and 25% were used.

Growth of the following pathogenic fungi and the 2 saprophytes, Lichtheimia sp. and Scopulariopsis brevicaulis, was observed over a period of 30 days: Achorion schoenleinii, Acladium castellani, Candida Candida, Endodermophyton tropicale, Endomyces capsulatus, Endomyces dermatitidus, Epidermophyton inguinale, Gleno spore gammeli, Geotrichum bachmann, Indiella americana, Monilia albicans, Microsporon apiospernum, Microsporon audouini, Oöspora humi, Trichophyton crateriforme, Trichophyton granulosum, Trichophyton japonicum, Trichophyton interdigitale, Willia anomala.

Table I gives the days within which growth was observed. The d indicates a marked decrease in the amount of growth, and s a scant growth.

Achorion schoenleinii in addition to developing a smaller colony with increase in concentration of glycerine, became more translucent in concentrations of 15% and above.

Acladium castellani gradually lost its color with increase in concentration of glycerine, becoming less granular and more waxy in appearance.

Candida Candida gradually became more translucent with increase in concentration of glycerine.

Endodermophyton tropicale with increase in concentration of glycerine lost its granular white appearance and became more translucent.

Endomyces capsulatus became increasingly more translucent, its mycelia becoming subsurface.

Endomyces determatitidis. The mycelia became increasingly more sub-surface, starting to a marked degree with the 1% concentration of glycerine.

Two hypotheses were recently presented for the explanation of the mechanism of zoning occurring in complement fixation—one by Levine, 1 the other by Eagle. 2 The two hypotheses rest on principles which possess no element in common. Therefore, I undertook a critical study of the subject matter. In this paper are presented in brief the results of the study of the theory of Eagle.

The latter can be briefly summarized as follows: Serum in concentrations greater than 1:25 effects an inhibition of complement fixation through the adsorption of fixation-preventing substances presumably present in negative serum. 3 These inhibiting substances are adsorbed by the antigen-antibody complexes after they had become fully organized. The adsorption is more intense the more negative a serum is, and corresponds roughly to the concentration of the serum protein. Hence, in weakly positive serums a more pronounced fixation of complement will occur in the higher serum dilution, and hence, the zone phenomenon. This same inhibition, Eagle states, is less marked at lower temperatures, and hence, in an indirect manner it is also responsible for the greater sensitivity of the icebox test.

Experimental studies were carried out as follows: (1) Complement and amboceptor titrations in the presence of high concentrations of negative serum; (2) complement fixation tests with Kahn antigen precipitated from positive and negative reactions 4 in the presence of high negative serum concentrations; (3) the effect of the removal of the native antisheep amboceptor from negative serum and the bearing of this procedure on the assumption that the results of Eagle's original experiments were influenced by natural amboceptor.

Investigations of changes in the fat content of the blood have been handicapped greatly by the tedious methods available for determination of blood lipids. The method described in this paper is easily and quickly carried out and is thus adapted to work requiring the analysis of large numbers of samples. Duplicate samples check closely, and composite samples agree well with calculated values, indicating that the method is satisfactory for comparative studies.

The design and approximate dimensions of the test bottle used for the determination are shown in Fig. 1. An alkali reagent developed by Petersen and Herreid 1 for the purpose of testing buttermilk by a modified Babcock procedure is employed to free the fat from the blood plasma. This reagent consists of 110 gm. of sodium carbonate and 200 gm. of sodium salicylate dissolved in water and made up to a volume of one liter, to which are then added successively 30 cc. of 50% sodium hydroxide and 100 cc. of normal butyl alcohol. The samples should be tested in duplicate if the amount of blood plasma available permits.

Three cc. of blood plasma are measured into the bottle by means of a pipette. A very gentle suction applied to the opening of the reading neck will cause the sample to flow into the bottle readily. Five cc. of reagent are then added in the same manner and thoroughly mixed with the plasma by allowing air to be drawn through the mixture by the suction used in filling the bottle. The filling neck is then closed tightly by means of a small stopper, the bottle is placed in a water bath, and digested for 20 minutes at 180° to 190° F., with occasional shaking.

Blalock, 1 Parsons and Phemister, 2 and others have shown that in traumatic shock and in severe burns there is a marked shift of fluid from the blood into the injured tissues. This fluid shift has been measured in the case of burns by burning one lateral half of the animal and then after midline sagittal section, measuring directly the difference in weight of the 2 halves. The amount of fluid shift has been found sufficient to explain the resultant shock. This method allows only one determination of the amount of shift in each animal. Because of variation in the extent of the burn and in the reaction of individual animals, a complete record of the rate of shift of fluid will not be obtained.

In all experiments completely anesthetized animals who suffered no pain whatsoever were used and no burned animals were allowed to recover.

The author devised the apparatus shown in Fig. 1. An animal placed on this apparatus is in a state of balance and any lateral shift in fluid will cause a tipping of the apparatus and resultant change in the position of the marker on the kymographic drum. When one-half of the animal is burned, the fluid shift to the burned side results in such a tipping and this in turn is recorded on the drum as a tracing.

Large rabbits or small dogs anesthetized with barbital and ether were used in all experiments. The animal was completely encased in a plaster cast except an opening on one side for the fore leg, axilla and upper chest and another opening for the lower abdomen and hind leg, which were burned. The tracing was continued until death. The animal was then divided into 2 lateral halves and weighed, the head, tail, viscera and spinal column being discarded.

The results in 4 animals burned on the balanced apparatus are given in Table I. It is seen that in general about half of the total shift occurred in the first hour. In the first 2 experiments the total shift was obtained only approximately by placing weights on the apparatus at a distance from the fulcrum corresponding to the center of the burned area until the apparatus was again in balance.

Undissociated 2–4 dinitrophenol (alpha-DNP) stimulates yeast respiration 1 ,²,³ and fermentation. 1 We have undertaken a survey of the action of the isomers of this substance, the first studied being 2–6 dinitrophenol (beta-DNP). The experimental procedure has been described elsewhere. 1 , 2

Beta-DNP resembles alpha-DNP in that only the undissociated acid affects yeast metabolism. The action is less intense, in that optimal doses of alpha-DNP cause approximately 100% increase in the rate of oxygen consumption, while the beta form raises the respiratory rate by about 50% in optimal doses. Similar relations have been demonstrated in the capacities of these substances to induce hyperthermia in the pigeon. 4 The optimal concentration of beta-DNP at pH 5.4 and at pH 6.0 in glucose phosphate solutions is about 1.4 mg. of the undissociated acid per liter.

Like alpha-DNP, beta-DNP rapidly induces either stimulation or inhibition, depending upon concentration. The effect develops within 5 minutes.

Inhibition of respiration by beta-DNP is reversible, as is the similar inhibition by alpha-DNP.

A slight further increase over the beta-DNP optimum can be evoked by addition of alpha-DNP. Addition of optimal concentration of beta-DNP to yeast suspensions in which the alpha-DNP concentration is optimal causes a decrease in the alpha-DNP effect. Further studies on these interrelations are in progress.

Like alpha-DNP, the action of beta-DNP does not depend upon the presence of glucose, but unlike alpha-DNP the usual stimulation pattern in phosphate buffer at pH 5.4 is not obtained in acetate buffer at DH 5.4.

Tainter and coworkers 1 , 2 and others 3 have shown that 2–4 dinitrophenol (alpha-DNP) stimulates respiration in animals by direct action on the tissue cells. We have shown previously that this substance stimulates or inhibits respiration and fermentation of yeast (Saccharomyces cerevisiae, Race F) depending upon the concentration of the undissociated acid form. 4 , 5

The experimental procedure used in the present study has been described elsewhere. 4 , 5

Treatment of yeast suspended in glucose phosphate media with M/1000 NaCN of the same pH reduces respiration to about 14% of the control rate. If alpha-DNP be now added in a dose which causes optimal stimulation of the cyanide-free yeast, i. e., in amount giving a free acid alpha-DNP concentration of 0.64 mg. per liter at pH 6.8, 5 it further depresses the respiration of the yeast poisoned with cyanide.

However, the respiration of yeast poisoned with cyanide can be stimulated by alpha-DNP, provided that the concentration of the undissociated acid form of this agent be approximately 0.032 mg. per liter, i. e., about 1/20 of the optimal dose for unpoisoned yeast. Under these conditions the respiratory rate of the poisoned yeast may be increased by about 20%. This relation has been demonstrated at pH 5.9 and pH 6.8. Further experiments on the action of alpha-DNP on respiration of organisms and tissues poisoned by cyanide are in progress.

Previous work 1 on certain nitrogenous substances in liquid ammonia has shown that some of them tend to ammonolyze with changes in their physical, chemical, and biological properties. Ammonolyzed edestin was reported 2 not to have given an anaphylactic reaction with a guinea pig previously sensitized to the untreated edestin. Recent observations on changes in some physical constants of certain amino acids in liquid ammonia indicate an ammonolysis. Epstein, Gunn and Virden 3 have shown changes brought about in the physiological activity of phenylethylamines by methylation. These facts, in addition to the observations of Kraus and White 4 that the hydroxyl groups of alcohols and phenols are quite reactive in liquid ammonia and to the report of Roberts and Miller 5 that the phenolic group of tyrosine seems to be acidic in liquid ammonia have led us to believe that epinephrine could be ammonolyzed and thus altered in its physiological activity.

The chemical treatment consisted of adding 60 mg. of Parke, Davis adrenalin to 150 cc. of liquid ammonia contained in a Dewar flask connected by gas-tight stoppers and tubing to a mercury seal allowing the ammonia to vaporize without admitting moisture to the adrenalin. The liquid ammonia, previously dried over sodium by the method of Fernelius and Johnson, boils off in about 24 hours, and the drug is ready for use. The yellowish adrenalin base darkens to a reddish-brown on standing in liquid ammonia, and its solubility in water and in glycerine decreases.

The flaky material is powdered, and used in the form of an aqueous sol. With glycerine used as a vehicle, the suspension is more uniform, and the base has less tendency to settle out. When water is used, the injection is made at once since the ammonolyzed base hydrolyzes on standing. With glycerine, a pressor activity characteristic of the new compound was obtained after 30 days, but after standing for 6 months an adrenalin curve was again obtained on injection.

In an effort to determine whether the formation of the comparatively soluble disodium salt accounts for the well-marked increase in basal metabolism recently observed by Thompson et al 1 following the oral administration of thyroxine in alkaline solution, we have prepared a disodium salt of thyroxine according to the method of Kendall 2 and have determined its calorigenic action in patients with myxedema. In one patient a single oral dose in distilled water of 15.1 mg. of the product containing 6.5 mg. of iodine, produced an increase in basal metabolism only from —-29% to —21%, and in another patient the same dose produced an increase only from —26% to —20%. These increases are about the same as those produced by single doses of the monosodium salt containing the same amount of iodine and only about 1/3 as great as those produced by thyroxine in alkaline solution. This small effect may be explained in at least 2 ways: (1) The method as used by us may not actually yield the disodium salt. (2) A large part of the disodium salt may hydrolyze when treated with distilled water to form sodium hydroxide and thyroxine. Although the disodium salt is said to be soluble to the extent of 4% and the monosodium salt only slightly soluble, the well-marked effect of thyroxine in alkaline solution by mouth must not be attributed to the formation of the disodium salt without more evidence.

N-acetyl thyroxine, prepared according to the method of Kendall and Osterberg, 3 has been given intravenously in alkaline solution to 3 patients with myxedema in doses of from 10.5 mg. to 31.5 mg., containing from 6.5 mg. to 19.5 mg. of iodine; subcutaneously, suspended in 10% glucose, to 2 patients with myxedema in doses of 10.5 and 21.0 mg. respectively; and orally to one patient with myxedema in a dose of 10.5 mg. The patient who received 31.5 mg. intravenously showed a slight but fairly prolonged increase in basal metabolism of from —41% to —35%. If this slight increase may be considered definite, then this compound when administered intravenously in alkaline solution produces only about 1/16 of the effect of thyroxine. When given subcutaneously in suspension, the effect was greater and amounted in one patient to about 1/4 and in another to about 1/6 of the effect of thyroxine given intravenously. These data suggest that the amino group is important for the calorigenic action of thyroxine.

Todd^1a described a method of titrating antistreptolysin in blood serum by which a diagnosis of a preceding hemolytic streptococcus infection may be made. Coburn and Pauli 2 considered the antistreptolysin titre of 200 units or higher at the onset of rheumatic fever as immunologic evidence that the hemolytic streptococcus initiates the disease. Myers and Keefer, 3 however, obtained an average of 200 units in normal subjects with a higher average (500 units) in rheumatic fever.

The majority of these observations were made in adults. They represent either single determinations or repeated tests over a short period and give a wide normal range of from 50 to 500 units.

In view of these findings it seemed of interest to obtain information as to the antistreptolysin titre in children of various ages, including: (1) Subjects apparently well; (2) Convalescents following streptococcus and miscellaneous infections; and (3) Rheumatic children during active and inactive periods.

Method. Todd's technic^1b was closely followed. A standard horse serum and patients'serums of known titre were used as controls in each series of tests.

Control series included 150 subjects from 3 months to 16 years of age; a group of 67 subjects in apparent health (no history of streptococcus infection); 51 convalescing from miscellaneous infections (varicella, pertussis, pneumonia); and 32 convalescing from hemolytic streptococcus infections (scarlet fever, erysipelas, respiratory infections). The rheumatic series included 133 subjects under weekly observation for from 9 to 12 months; 41 patients with active rheumatic fever (chorea, carditis, arthritis); and 92 subjects in the inactive stage.

Observations are summarized in Table I.

The qualitative iodoxybenzoate test of Leake has been found suitable for quantitative adaptation to the estimation of morphine. Methods for determining morphine in biological fluids and a rapid method of accurately checking the morphine content of morphine hydrochloride are suggested.

Development of a satisfactorily accurate quantitative application of the usual color reactions for morphine has been retarded because of their general lack of (a) proper selectivity for this single opium alkaloid, (b) permanence of hue produced or (c) sufficient sensitivity. In addition such reactions are in most instances dependent to an undesirable degree on the temperature and pH of the test solution and the presence of commonly occurring contaminants. This situation is not surprising since most of these reactions are supposedly due to the formation of complex unstable chromogenic substances. A more ideal quantitative test should be the stoichiometric conversion of morphine to a simple colored form.

Leake 1 suggested that such an effect could be obtained for many substances by reacting their free phenolic groups with an aqueous solution of ammonium iodoxybenzoate, the oxidative powers of which had been studied in another regard by Loevenhart 2 and others. Morphine rapidly develops the same color when treated with this agent as is produced by slow spontaneous oxidation of morphine solutions in air, and it was recommended 1 that this reaction be used not only as a qualitative test to differentiate morphine from codeine, heroin, dionin and apomorphine, but also as the basis of a quantitative colorimetric estimation. We have found that when 25 to 50 mg. of powdered ammonium iodoxybenzoate are added to 5 to 10 cc. of 0.005 M to 0.02 M aqueous solutions of morphine salts sufficient agreement with Beer's Law holds so that colorimeter readings yield results accurate to ±3% if the comparison with a standard is made after 30 minutes are allowed for full development of the color. Spectroscopic examination of color absorption by the treated solutions reveals that in concentrations higher than 0.03 M extinction extends further toward the yellow band than with the lower concentrations, which is represented grossly by a change in shade from orange to garnet and considerable deviation in colorimetric measurements unless a suitable filter is used.

One of us 1 demonstrated that red blood cells are hemolyzed by solutions of fluorescein dyes which have been previously irradiated with the visible portion of sunlight. Finding that such solutions. contained a small concentration of peroxide, 1 and for reasons which seemed consistent with the then known facts of photodynamic action, we assumed that the hemolysis was probably due to the activity of this peroxide, which we suspected of being an organic peroxide. Recently, 2 however, we have offered evidence to show that this peroxide is H₂O₂.

We have determined the quantity of peroxide and dye in solutions which are just capable of producing hemolysis, and have found that mixtures of eosine and H₂O₂ do not produce hemolysis at nearly such low concentrations of these reactants as do irradiated eosine solutions. Table I indicates this discrepancy.

Furthermore, in most cases, we have found that when Na₂S₂O₃ is added to the irradiated solutions in concentrations just equivalent to the concentration of H₂O₂ present, hemolysis is not abolished nor in most cases perceptibly diminished.

An amount of bleaching of the dye solutions occurs which is detectable spectrophotometrically; the quantities are indicated in Table I. We have been forced to conclude that the products of this bleaching, and not peroxide are responsible for at least the greater part of the increased hemolytic properties of our solutions. Further experiments are in progress.

Prolonged experience with a large breeding colony of rabbits has revealed that a small proportion of sexually mature females repeatedly fail to become pregnant after being served by known fertile bucks. In general, such does are in excellent physical condition and show no external evidence of disease, although a few have a tendency to obesity characterized by a shoulder girdle adiposity and an abnormal accumulation of fat in the abdomen. Recently, certain of these persistently infertile does carried genetic factors which were under investigation, and progeny from them were desired in order to carry on the genetic studies. Accordingly, they were submitted to different therapeutic procedures, with a view to developing a method which would render them fertile. Since all the animals were of value because of their genetic constitution, operative interference and experiments hazardous to life were of necessity avoided. The present report is concerned with one of the methods employed in the treatment of these does.

In the rabbit, ovulation occurs about 10 hours after copulation. It was thought that the threshold necessary to induce ovulation in refractory females was higher than the stimulus afforded by the act of coitus. A simple method of augmenting the copulation stimulus lay in the observation of Friedman 1 that the parenteral administration of human pregnancy urine induces ovulation in the rabbit. The method employed in the present investigation was to inject a single dose of 40 Rat Units per kilo body weight∗ of Antuitrin “S” into the marginal ear vein of the doe. The time of mating with a known fertile buck with reference to the injection varied from 22 hours before to 24 hours after the administration of Antuitrin “S”.

The reproductive activities of certain rabbits in our breeding colony have been observed on repeated occasions to be abnormal. These abnormalities may be classified, for present consideration, in 3 phases, namely, infertility, desertion or neglect of the young, and cannibalism. Observations indicated that some of these conditions were associated with nutritional disorders and it had been found that some improvement could be obtained by a general increase in vitamin intake. Accordingly, an attempt was made to correct the abnormalities of a selected series of animals by the administration of vitamin concentrates singly or in various combinations. The results thus far obtained have been highly encouraging and these form the basis of the present communication.

A group of 42 rabbits was selected for this study. These included 38 females and 4 males of pure bred and hybrid stocks, chiefly Rex, Belgian hare, English, Havana, Dutch, Polish, Himalayan and a highly inbred Albino family. In each case previous observations had indicated that the reproductive behavior was abnormal. Following vitaminization for various periods of time, the treated animals were mated with known fertile animals and the results were measured by subsequent observations made with regard to the particular abnormality presented. In all cases except one, dried brewer's yeast, representing the B complex was used either alone or in combination with halibut liver oil or viosterol or with both. Halibut liver oil alone was employed in one case. All vitamin concentrates were administered by hand. A few preliminary tests were made during the Winter of 1932-33 and the Spring of 1933, but most of the experiments were carried out between December, 1933, and February, 1934, inclusive. The test animals have lived indoors from birth and were distributed among normals in several rooms, and all the rabbits of the colony were fed a standard diet.

It was found, on the basis of 3 biologic tests, that the gonad-stimulating hormone in the urine of a patient with a teratoma testis had the same characteristics as the substance occurring in the urine of pregnant women.

Zondek, 1 Ferguson, 2 and others have shown that the urine of men with various testicular tumors may contain a gonad-stimulating hormone. Evans et al. 3 reported that this substance may have characteristics of both the anterior-pituitary-sex hormone and the factor occurring in the urine of pregnant women. Hamburger 4 found a hormone in the urine of 5 men with testicular tumors and one woman with a teratoma of the ovary, which produced the same histologic picture in the ovaries of immature mice as that resulting from the injection of urine from pregnant women. In a seventh case, a man with an embryoma testis, he obtained results which he felt were due to the presence of 2 hormones. Since the patient had been castrated as a consequence of radium therapy, he believed that one of these substances was due to the presence of the tumor and that the other appeared as a post-castration effect.

The urine examined in this study was from a man 34 years of age, with metastases from a teratoma testis, for which he was under the care of Doctor J. R. Dillon, of the Department of Urology.

A 24-hour specimen of urine contained approximately 2500 rat units per litre. The histologic picture in the ovaries of more than 20 immature rats injected with the urine or with an extract, corresponded to that induced by the gonad-stimulating hormone of pregnancy (Fluhmann 5 ) and showed corpora lutea, lutein cysts, and developing follicles. The injection of a known dosage of an extract to 5 immature rats resulted in an average increase in ovarian weight of 190% in 5 days, while the ovaries of 5 littermates given the same total dosage over a period of 10 days produced an average increase in ovarian weight of 460%.

The gonad-stimulating hormone found in the urine of a patient with a chorioepithelioma uteri had certain biologic characteristics similar to those of the substance occurring in women during normal gestation.

Although the discovery of Rössler, 1 that the urine of women with hydatidiform mole or chorioepithelioma uteri contains large amounts of a gonad-stimulating factor, has been repeatedly confirmed, there is as yet no information available as to the nature of this hormone.

The specimens examined in this study were from a patient under the care of Doctors Reginald Knight Smith and T. Henshaw Kelly. She was a woman of 29, who developed a chorioepithelioma uteri following a hydatidiform mole, and died with extensive metastases in the liver and in the lungs.

A number of extracts were prepared by alcohol precipitation of the urine, and various dosages were injected into more than 25 immature rats. The histologic examination of the ovaries of these animals showed the presence of a few corpora lutea, lutein cysts, and developing graafian follicles. The injection of a known dosage of the extract to a group of 5 rats over a period of 5 days produced an average increase in ovarian weight of 63%, while the administration of the same total dosage over a period of 10 days to 5 litter-mates gave an average increase of 90%. It has been shown previously that these 2 findings are characteristic of the gonad-stimulating hormone found in pregnant women and not of anterior pituitary extracts (Fluhmann 2 , 3 ).

Administration of the untreated urine failed to induce any demonstrable changes in the atrophied ovaries of 2 hypophysectomized rats, but a concentrated extract induced an extensive development of the theca and interstitial tissue in 4 other instances. It has been shown by Noguchi, 4 Collip et al., 5 and Smith and Leonard 6 that this is a characteristic of the gonad-stimulating hormone of pregnant women.

When I injected poliomyelitis virus in Macacus rhesus monkeys either intrasciatically, intracerebrally or directly into the cord in the region of the first lumbar segment, periods of 4, 7 and 2 1/2 to 3 days, respectively, elapsed before paralysis came on. Such latent intervals are considered the incubation periods of the virus. It is curious that the time elapsing before paralysis appeared was shorter the nearer the injection was made to the lumbar area. Unlike the virus of smallpox, for instance, which multiplies in situ, the virus of poliomyelitis spreads away from the cortex and down the cord, 1 practically disappearing from the site of the injection in the cerebrum. It does not cause a great amount of functional damage in the animal until a few days after it reaches the lower part of the cord. In its manner of progress, it acts like a catalyst, a catalyst with nearly an obligate affinity for grey fibers or for axis cylinders of medullated nerves.

In previous experiments, I tried to recover the virus from the stools of humans but failed. However, the stools obtained from monkeys, before and after paralysis, showed a difference in that the feces obtained at the time of paralysis contained a toxic factor that was not present before the onset of paralysis when the animals were in normal condition. 2 The appearance of increased toxicity in the stools of these monkeys coincided somewhat with a marked drop in the agglutinin titer value of their blood serums withdrawn during the stages of paralysis against various enteric organisms. 3

The discovery by Tillett and Garner 1 of an antihuman fibrinolytic function in S. hemolyticus isolated from human cases and the absence of this enzyme from apparently identical streptococci isolated from the environment or from lower animals, is of suggestive epidemiologic interest. We have, therefore, attempted to confirm their findings.

To do this, 193 cultures of typical hemolytic streptococci were kindly furnished by various clinics and veterinary institutions of Northern California. Antihuman fibrinolytic tests were made by the Tillett-Garner technic, each test being repeated 3 times with different samples of normal human blood. Control tests were made with stock cultures of known lytic powers.

For convenience in recording our data, the 193 cultures were divided into 3 groups: (a) 32 cultures originally isolated from internal human tissues, representing such diseases as pneumonia, septicemia, empyemia, endocarditis, osteomyelitis, and meningitis; (b) 123 typical hemolytic strains originally isolated from superficial human tissues, representing such diseases as erysipelas, furunculosis, and fistula, septic sore throat, sinusitis, and acute gastric disturbance; and (c) 38 cultures of veterinary origin, representing such animals as cow, horse, hog, dog, deer, and rabbit. The lytic tests thus grouped are recorded in Table I.

Groups I and III are in practical accord with the Tillett-Garner data. Group II, however, suggests a much less sharp differentiation between antihuman and antiveterinary streptococci than their data indicate. The low percentage of fibrinolytic hemolytic streptococci in this group suggests that a fibrinolytic typing of superficial human infections might be of clinical interest.

Thirty-three strains of S. viridans were tested in connection with this work. All were non-fibrinolytic.

The type of degradation of lactose-fermentation here considered is that in which bacterial growth in lactose broth produces no fermentation the first few days of incubation. After 2 to 14 days, acidity and gas production may appear. In other strains it is only after repeated subculture on lactose that this sugar is fermented. As a result of lactose-fermenting deficiency, degraded organisms are entirely missed in water analysis. Since dextrose fermentation is normal, degraded strains grow on Endo and Russell's medium like paratyphoids and complicate the examination of suspect material for members of the Salmonella group. Many, but not all, strains ferment saccharose promptly. This led Krumwiede 1 to add saccharose to Russell's medium to exclude these degraded forms designated in Park and Williams as “paratyphoid-like intermediates”. Kennedy et al. 2 have presented data for 22 strains of colon bacilli showing delayed lactose fermentation, in urine, stools and water.

We have been interested in the Escherichia-Citrobacter-Aerobacter ratio in saline suspensions of feces, fresh and after storage at 37° and icebox temperature. Lactose degraded organisms have been encountered. The fecal suspensions were first plated on Endo from which representatives of all colony types were fished to dextrose broth. From these tubes plates were again made on Endo and a well isolated colony picked to lactose broth. From this growth a third Endo plate was made from which again a well isolated colony was fished to lactose. By such technique we were reasonably assured of pure cultures and were able to exclude growth which failed to ferment dextrose. The strains isolated were tested for their gelatin liquefaction, action on litmus milk, growth on Simmons'citrate agar, production of indole and hydrogen sulfide, their methyl red and Voges-Proskauer reactions and for fermentation of saccharose, dulcite, cellobiose, α-methyl-d-glucoside and Levine's boric acid lactose broth.

On December 12, 1933, a nurse became suddenly ill and repeatedly vomited bile stained material. The following morning she had a severe chill, a temperature of 101 °F., severe headache, some rigidity of the neck and a doubtful Kernig's sign. A day later her temperature reached 105.3°F. and the meningeal symptoms were more marked. During the first 10 days of her illness the more prominent symptoms were: fever, headache, occipital tenderness, rigidity of the neck, strabismus, ptosis of left eyelid, diplopia, twitching muscle fasciculi and lethargy from which she could be fairly easily roused. There was gradual improvement after about the tenth day. Ten weeks after the onset there remained some speech defect, difficulty in feeding herself, dysmetria, sprawling gait, slight nodding of head, emotional instability and impaired memory.

Spinal fluid obtained December 15 was hazy and contained 812 cells per cu. mm. (95% small lymphocytes). The sugar content was 37 mg. per 100 cc. and the gold curve 0023321111. Subsequent specimens gave a paretic curve. Repeated Wassermann tests have been negative. Inoculated guinea pigs failed to develop tuberculosis.

Of special interest is the fact that an apparently new Gram-positive bacillus was obtained in pure culture from the spinal fluid on December 18, 19, 21, 22, 26, 28, 1933; January 3, 8, 10, 18, March 8, April 12, 1934. Cultures made May 10 were negative. A rabbit inoculated intracerebrally with 0.5 cc. of the spinal fluid obtained on December 28 developed a severe encephalitis 2 days after inoculation. The same organism was cultivated from the brain tissue of this animal.

Robison 1 has pointed out that the enzyme phosphatase is an important factor in the calcification of bone. Kay 2 has more recently summarized the evidence for its activity in connection with studies on a wide variety of clinical conditions. Since osteogenesis imperfecta is an outstanding example of defective bone formation for which no pathogenesis has as yet been discovered, a comprehensive study of the various metabolic factors which might be involved in this condition was begun by the writer in 1931. The present report deals with the phase of that study which concerns the phosphatase activity of the blood serum and fixed tissues.

Kay reported the serum phosphatase activity to be slightly increased in some cases of osteogenesis imperfecta, while Bodansky and Jaffe 3 found it to be normal in 6 cases.

Periodic determinations of the serum phosphatase by the method of Kay have now been made in 4 cases of osteogenesis imperfecta under a variety of experimental conditions. In one case the fixed tissue phosphatase has also been studied postmortem.

Repeated determinations in the 4 cases showed values almost identical with those found for normal control subjects and for a miscellaneous group of patients in the same age period. Variations in the ordinary constituents of the diet were found to have no definite effect on the serum phosphatase values. Following the administration of vitamin D in relatively enormous doses in the form of viosterol 10,000X, however, the phosphatase activity of the serum was uniformly reduced. Parathormone administration over a period of several days had a similar but less marked depressing effect on the enzyme.

Inasmuch as intracranial complications following pneumococcus Type III mastoiditis are known to occur, as a rule, not earlier than several weeks after the initial infection, serious attempts to induce a state of active acquired immunity should receive special consideration. The use of prophylactic vaccination was suggested by Kolmer and Amano on the basis of their work in rabbits in which immunization with pneumococcus vaccines elicited protection against experimental meningitis. 1 Barach found that type-specific active acquired immunity appeared within 6 days after intradermal or intravenous injection of pneumococcus type-heterologous vaccines in cases of lobar pneumonia. 2

Accordingly, in September, 1931, we undertook to immunize every case of pneumococcus Type III mastoiditis admitted to the Otological service of The Mount Sinai Hospital as soon as the bacteriological diagnosis was made, whether that be before or after the operation. The following method of vaccination was employed:

The patients were given a course of 6 intradermal injections, 2 a week, in the following increasing doses: 0.1 cc., 0.2 cc., 0.3 cc., 0.5 cc., 0.7 cc., and 1 cc. For doses larger than 0.2 cc. several simultaneous injections of 0.2 cc. each were made. In order to avoid loss of time, a stock vaccine was used for the initial injection. This was done never later than 48 hours after the operation and sometimes before the operation. For the remaining 5 injections an autogenous vaccine was used. The autogenous vaccine was prepared by growing a pure culture of pneumococcus Type III in 100 cc. of 1% glucose broth for 24 hours. The organisms were centrifuged, washed once and then resuspended in 0.85% NaCl solution to obtain a concentration of six hundred million organisms per cc. The bacteria were then killed by heating at 60° for one-half hour.