Experimental Biology and Medicine

The diazotization of bilirubin in the van den Bergh estimation is strictly proportioned to the intensity of the color produced. 1 However, the use of the artificial color standards and the stratification of the plasma-ammonium sulphate and alcohol in the determination afford a valid criticism of the procedures ordinarily employed. Some three years ago we adapted the Thannhauser-Anderson 2 method to avoid these difficulties. A strongly acidified solution of azobilirubin, which remains unchanged for 3 to 6 months if kept in a cold refrigerator, is employed as a standard. A proportional concentration of acid is added to the unknown. There is no stratification of the mixture, and the red reaction changes to an intense blue. 3 Publication of our procedure has been delayed until a ready source of bilirubin for the standard could be located. Failing in this, we have prepared our own bilirubin from bile rather simply. Foweather 4 recently has given a short procedure for obtaining bilirubin from gall stones. Our method of preparing bilirubin and the technique for the quantitative test follow:

Preparation of bilirubin. 500 cc. of bile from surgical drainage patients or 50 cc. of post mortem gall bladder bile (diluted 2–3 times) is allowed to stand a few hours in the refrigerator and the supernatant fluid is decanted. This is diluted several times with water. Barium chloride solution is added with stirring. If the precipitate of barium bilirubinate does not flocculate immediately, addition of a few drops of alkali usually suffices. When the precipitate settles the supernatant fluid is siphoned off. The precipitate is poured on a filter and is washed with water on the paper; it is air dried and pulverized in a mortar. The powder is extracted with warm alcohol and with ether or chloroform and again air dried.

The partition of phosphorus in whole blood and serum, the serum calcium and plasma phosphatase have been studied in 2 patients during the slow healing of late rickets of the low phosphorus type. The evidence obtained indicates that the organic acid-soluble (ester) phosphorus of corpuscles is markedly decreased in active late rickets. The lowest value observed was 41.5 mg. per 100 cc. (normal for the age, 66 mg.). This fraction of the blood phosphorus responds very quickly when antirachitic treatment is given, and may reach the normal value before any definite change in serum inorganic phosphorus is observed. The intake of vitamin D by one patient was apparently insufficient to maintain the increased level of corpuscle ester phosphorus. Relatively much greater increases in vitamin D intake were necessary to induce a second rise, and the normal level was not attained until after the serum inorganic phosphorus had been normal for a period of about 4 weeks.

The plasma phosphatase values of both patients were 5 to 6 times the normal at the beginning of treatment. The amounts decreased during healing but were still high when roentgenological healing was complete. In one of the subjects, the phosphatase remained at double the normal value for more than a year after complete healing occurred.

It was shown 1 that the concentration of creatine in the human myocardium parallels the heart's ability to maintain an adequate circulation. It follows that if, as is commonly supposed, hypertrophy increases the power of cardiac muscle to do work, the total amount of creatine in a hypertrophied heart should exceed the amount found in one of normal size.

Cardiac hypertrophy was produced in growing rats by the production of nutritional anemia as described by Forman and Daniels. 2 A litter mate of each diet rat was maintained on a standard ration of rat biscuits and water. Occasional bleeding of the diet rats (for hemoglobin estimation) doubtlessly hastened the production of anemia in these animals.

At the end of from 66 to 75 days the anemic rats, together with their litter mate controls, were killed by a blow on the head followed by section of the neck vessels. Hemoglobin estimations were done with a Sahli hemoglobinometer on samples of blood thus collected. Each rat was opened immediately after death, and the ventricles severed from the body by careful section through the atrioventricular ring. The ventricles were then opened, blotted between filters to remove all free blood, weighed, and analyzed for creatine by the method of Rose, Helmer, and Chanutin. 3 A second group of controls was picked from stock, each animal of this series weighing within 5 gm. of the anemic rat with which he was compared. This size control group was necessary since the anemic rats did not maintain normal growth curves. This group also allowed comparison of creatine concentration at different ages, since they were younger animals. There were 11 anemic rats, 10 litter mate controls, and 11 size controls. The results are shown in Table I.

The author has described 1 the condition of overripeness that develops in frog eggs if fertilization is delayed 3 to 5 days after ovulation. Among the abnormalities of embryonic development that follow, one of the most striking is the frequent appearance of reduplications. In the early gastrulation stage instead of the normal upper blastopore lip of limited extent and regular curvature there often appear long and irregular clefts and folds in the equatorial region. Attempts at gastrulation become visible not only on one side but all around the whole circumference of the embryo. As time progresses multiple blastopores and multiple gastrulation lead to the development of twin embryos. As we have pointed out previously, 1 this effect indicates that overripeness has brought about 2 concomitant changes in the embryos. First, the physiological correlations by which an embryo develops as a balanced organism are impaired. These correlations largely rest on inhibitory functions by which established “organizers” (inductors) suppress either the formation or the effective functioning of competing organizers. Secondly, “organizers” appear in lateral and ventral regions of embryos, where normally they never occur. We have described this as self-differentiation or merogenesis of physiologically isolated parts. The mechanism of this formation of secondary organizers remained a complete puzzle until the recent discovery of Holtfreter 2 that material from the ventral region of an early embryo which has no inductive qualities in living condition, acquires such potencies after being killed by heat, dryness, etc. We are inclined now to assume that the possibility to develop into organizer is inherent to any part of the embryo, probably even to every cell. The inhibitors that suppress their realization are destroyed by such factors as heat and dryness but also by the process of overripeness.

A property common to many enzymes 1 , 2 is the ease with which mercury bichloride inactivation may be quantitatively reversed. Such reactivation after bichloride inactivation has also been demonstrated with bacteriophage 3 , 4 and since some workers have postulated an analogy between phage and viruses the following experiments were undertaken to determine whether or not bichloride inactivated vaccinia virus could be reactivated.

The least amount of mercury bichloride which would extinguish the reaction of the viruses in rabbit skin was first determined. A calf-skin strain (dermovaccine) and a rabbit-brain strain (neurolapine) of virus were used. Heated and unheated virus-saline mixtures were used as controls. Portions of a 1–500 suspension of the viruses (known to be active in a 1–1,000 suspension) were exposed to equal amounts of various mercury bichloride dilutions for 24 hours at room temperature. One-tenth cubic centimeter of each virus-bichloride or virus-saline mixture was injected intradermally into corresponding shaved skin areas on 2 albino rabbits. The heated virus-saline or heated virus-bichloride mixtures caused no reaction on the skin of rabbits. The reaction of the dermovaccine was extinguished by the 1–10,000 dilution but not by the 1–100,000 dilution of bichloride. The reaction of the neurolapine was extinguished by a 1–1,000 but not by any higher dilution of bichloride.

It was pointed out 1 that no significant fall of serum protein concentration occurred in rats kept for a period of 5 months on a diet extremely low in protein, provided the diet was otherwise adequate. These results are at variance with the conclusions of Kohman, 2 and of Frisch, Mendel and Peters. 3 However, the diet used by these observers consisted in large part of carrots and the question therefore came up whether carrots alone may exercise some specific blood protein lowering effect. The present experiments were planned to obtain information about this possibility.

Eighty-eight young adult white rats were placed on a diet consisting exclusively of fresh carrots. No salts or vitamins were added. After varying intervals groups of 5 or 6 animals were exsanguinated under ether anesthesia, aliquot portions of serum were pooled and the total serum proteins were measured. 4 The experiment lasted 145 days during which time the rats remained well and active and there were none of the usual signs of vitamin deficiency. There was progressive weight loss.

In the majority of the animals there was a definite fall in serum protein concentration and values as low as 4.28, 4.44, 4.72 were obtained with some of the groups (control level 6.30–6.50 gm. %). Five rats among the whole group of 88 developed gross watery effusions into the serous cavities and in one of these animals the serum protein concentration was only 2.99 gm. %. It is of interest that the serum protein level bore no close relation to the duration of carrot feeding. Serum protein from a group of rats on the 61st day averaged 4.44 gm. %, from another group on the 82d day 5.06 gm. % and from the final group on the 145th day 4.28 gm. %.

The mechanism of the lowering of serum protein concentration after carrot feeding appears to be extremely complex. Neither lack of protein alone nor lack of vitamins seem to offer a complete explanation.

Compensatory hypertrophy may follow a deficiency in the total cell mass induced by operative removal of one of the paired organs. The process is manifested in some but not in all paired organs. The present study was undertaken because no data are available with respect to compensatory hypertrophy in Cowper's glands.

Albino rats of 250 days of age were used. The age of the rat excluded the possible influence of growth on the size of the gland. The stimulus to compensatory hypertrophy was given by the removal of one Cowper's gland from each rat. Under ether anesthesia and through the perineal route, alternate glands were removed from each successive rat. Immediately after removal each gland was freed from any adherent fat or tissue and weighed to within a milligram on a torsion balance. The weights of these glands were taken as controls.

Twenty days was arbitrarily chosen as the period of time during which to determine the ability of the remaining Cowper's gland to undergo compensatory hypertrophy. At the end of this period the rats were etherized and the remaining Cowper's glands removed and carefully weighed as before.

Two methods of approach have been used by investigators for the extirpation of the hypophysis of the rat. The parapharyngeal approach proposed by Smith 1 which has been modified by several investigators is the one most commonly used. The second method, suggested by Koyama, 2 is through the ear. The temporal approach used successfully on dogs by Dandy and Reichert 3 has not been, to our knowledge, used on rats. This method is particularly adapted for operating on rats, (1) because the sella turcica is shallow and a satisfactory approach can be obtained from the side; (2) the rat is very resistant to infection; (3) the anterior and the posterior lobe of the hypophysis in the rat can be removed separately by this approach. This cannot be done with dogs. (4) The lobes can be removed intact and may be used for microscopic examination; (5) the operation can be carried out on very young animals.

The work of Weed and McKibben 4 on the experimental alteration of the brain bulk has been useful in this operation. The decrease in the brain bulk of rats given intravenous injection of hypertonic sodium chloride solution is very striking.

Under light ether anesthesia sufficient hypertonic salt solution is injected intravenously in order to obtain the maximum amount of brain shrinkage. We have been using 400 mg. of sodium chloride per kilo of body weight. This is slightly more than the amount used by Weed and McKibben on cats and by Dandy and Reichert on dogs. In rats weighing over 100 gm., the sodium chloride was injected very slowly into the jugular vein in a 20% solution. Rats weighing less than 100 gm. were given sodium chloride in a 5% solution.

The close chemical relationship between arsenic and antimony suggests that the latter substance might be used in place of arsenic in the treatment of abnormal leucocytoses, Meneghetti 1 concluded that a “true hemopathy” characterized by leucocytosis followed by leucopenia, a profound anemia, and marked normoblastosis could be produced after a single intravenous inoculation of colloidal antimony sulfide. His experiments with tartar emetic, however, were incomplete. In a series of preliminary experiments using a freshly prepared 1% solution of potassium antimony tartrate we established a dose of 6 mg. per kilo body weight as the maximum tolerated initial dose for rabbits. When inoculated intravenously this dose produced a characteristic leucopenia without subsequent leucocytosis.

Normal adult rabbits were inoculated in the marginal ear vein with a freshly prepared 1% solution of potassium antimonyl tartrate. A complete blood count was done before and after each experiment. The total leucocyte and differential counts were done at intervals of one-half, one, 2, 4, 7, and 28 hours after inoculation. The blood films were stained with Wright's stain, and the platelet counts were done using Rees and Ecker's diluting fluid. After the completion of each experiment the animals were sacrificed and a histological study of the hematopoietic organs was made.

Little emphasis has been placed upon the in vitro bactericidal activity against Mycobacterium leprae of compounds other than the fixed and essential oils and various fatty acids examined by Walker and Sweeney, Schöbl, and Adams and his co-workers, 1 In view of the recent interest in the clinical use of non-chaulmoogryl compounds, particularly certain dyes, as antileprosy drugs, a report of our accumulated findings on the action of these and other agents against M. leprae is made herewith. These observations have been made during the past year as a tentative guide to therapeutic efforts on experimentally infected leprous rats using a standardized technique previously described.²

The leprocidal activity of over 200 chemical agents falling into 4 general types has been studied by the 2 appropriate techniques described by Adams et al., 1 with careful adherence to the precautions noted by these authors. A single strain of test organism was used, this being M. leprae hominis, Mary Puhulahula strain, isolated by Dr. E. L. Walker.† The media used as substrate in the tests was that of Long and Seibert, 3 recommended for our use by Dr. Walker, rather than a 5% glycerol broth as used by Adams 1 in his tests. No other variations from Adams'method were made. Most of the tests were carried out in Dr. Walker's laboratory and in conjunction with the Bacteriological Laboratory of the University of California. By closely following the bacteriological technique worked out by Adams, his estimate of a probable error of ±10% appears justified.

In a report 1 dealing with the effect of Antuitrin-S on the mature human ovary, it was pointed out that the response was not similar to that obtained in the rodent. It was therefore planned to inject a series of immature human females and examine their pelvic organs. Naturally such an opportunity is limited, for the cases must be selected from patients who are suffering from diseases that are of themselves fatal.

We have had opportunity to study 2 cases, one an infant of 10 months and one of 18 months. Both were suffering from tuberculous meningitis, proved by bacteriological examination of the spinal fluid and later confirmed at autopsy.

In the first case a 10 months infant received a total of 2,500 rat units of Antuitrin-S† in 5 divided doses over a period of 96 hours. The injections were given intramuscularly in the buttocks. The last injection was given 12 hours before death. At autopsy the ovaries both macroscopically and microscopically presented no evidence of follicular maturation or luteinization. The uterine mucosa showed no evidence of hypertrophy or cystic glandular hyperplasia. The second case, an infant of 18 months, also received a total of 2,500 rat units of Antuitrin-S. The last injection in this case was 16 hours before death. At autopsy the ovaries both macroscopically and microscopically presented no evidence of follicle maturation or luteinization. One ovary showed a tiny pinpoint area of hemorrhage on the surface. The uterine mucosa did not show any variation from the normal.

Several patients desperately ill with gastro-intestinal and brain tumors received similar or greater amounts, but recovered from their illnesses.

The examination of a complete series of cases up to the age of puberty will of necessity require much time.

The problem of obtaining suitable material for the determination of the antibody content of tissues or organs has usually been met by grinding the tissues with sand and extracting in a solvent, either physiological saline alone or equal parts of the saline solution and glycerine. The method is tedious and the material obtained has already undergone dilution which is not always desirable. The ease with which tissue juices are obtained by pressure in certain experiments carried out by Dr. Foster of the Department of Biochemistry suggested to us the possibility of extracting organs by this procedure for use in immunological experiments. The method consists in placing the tissue in an hydraulic press and extracting its fluid content under a pressure of approximately 16,000 pounds per square inch. The method may be used for any organ but is particularly serviceable in extracting skin and subcutaneous tissue where the large amount of connective tissue makes other methods of extraction difficult.

The tissue to be extracted is cut into pieces and mixed with a large volume of portions of filter paper about 1 cm. square. The whole mixture is cut up further until it consists of a grumous mass of filter paper which has absorbed all the bits of tissue to it. The filter paper offers traction and prevents the tissues from sliding out of the press. This mixture of filter paper and tissue is then wrapped in a piece of cheese cloth and is ready to be pressed for the extraction of its fluid content. The press used is an hydraulic press manufactured by Fred S. Carver of New York City, capable of exerting a pressure of 20,000 pounds per square inch.

The injection of a foreign protein into the anterior chamber of the rabbit's eye results in a local sensitization of this organ. Weeks or months later when the eye has returned to a normal appearance the intravenous injection of the same protein produces a marked inflammation of the iris and conjunctiva. The opposite, control eye shows no reaction or only a slight hyperemia. 1 This local hypersensitivity has the characteristics of an antigen-antibody reaction.^{1, 2} The presence of antibodies in the tissues of the hypersensitive eye was not demonstrated, however, by the technique previously employed. 2

This paper reports further studies on the antibody content of the juices extracted from the tissues of the injected eye. By an alteration in the technique of extracting the tissues and the utilization of a particulate antigen which stimulates the production of agglutinins it has been possible to demonstrate the concentration of antibody in the tissues of an eye injected with typhoid vaccine.

A series of 12 adult albino rabbits were injected into the anterior chamber of the right eye with 0.1 or 0.15 cc. of New York City Board of Health typhoid vaccine which contained approximately 3 billion organisms per cc. The injection was given under cocaine anesthesia by the method already described. 1 The animals were sacrificed 4 to 102 days later by bleeding from the heart. Sixty to 120 cc. of blood was obtained. The anterior chamber fluids were removed and the palpebral and scleral conjunctiva together with the tissues from the anterior two-thirds of the eyeball from each eye were dissected out. The lens was expelled and the excess aqueous and vitreous humors were blotted off. The tissue juice from these fragments of tissues, consisting of conjunctiva, iris, sclera and cornea, was extracted with pressure by a method previously described.∗, 3 The fluids so obtained were tested for their agglutinin titer and the titers compared with those obtained in the serum and the aqueous humors. A series of 11 control animals was similarly tested. The treatment of the controls is indicated in Table I.

The increased interest in chest and precordial leads, which are semi-direct leads tapping the cardiac action current from points close to the anterior surface of the heart, prompted the authors to seek an equivalent lead portraying more intimately the electrical phenomena at the posterior surface and base of the heart. A lead taken from the back of the chest fails to do this, for unlike the front, where there is only 1/2 inch of tissue intervening between heart and electrode, there is usually 4 inches of intervening tissue posteriorly.

The difficulty was overcome by making use of the fact that the esophagus is closely attached to the posterior pericardium throughout its extent. A non-polarizable German Silver electrode, shaped somewhat like the Rehfuss tip, to which a thin flexible German Silver cable was clamped, served as an internal electrode. The cable was insulated throughout its length by rubber tubing. This electrode was swallowed in turn by 6 normal subjects, very much like the Rehfuss tube, its descent being checked by fluoroscopic examination. The esophageal electrode was connected to the right-arm side of the galvanometer, and the left-arm electrode (a flat 1 inch disc) lead to some point distant from the heart, such as the left leg or right arm of the subject.

When the internal or exploring electrode lay at the level of the auricles, the electrocardiogram demonstrated uniformly a peculiar deflection, a very sharp, steep, rapidly moving deflection, which arose from the rounded portion of the P-wave, and which was almost as tall as the QRS deflection, which it resembled. (See Fig. 1.)

The report of Avery and Dubos 1 that the specific carbohydrate of pneumococcus type III was split by an enzyme from a soil microorganism appeared to offer a new approach to the study of relationships and differences between chemically and immunologically different preparations of the specific carbohydrates.

Organisms which decompose the specific carbohydrates of pneumococcus types II and III were described in a previous report. 2 We now have in pure culture a microorganism which utilizes to some extent the specific carbohydrate of pneumococcus type I. As in the study of the bacteria that decomposed the carbohydrates of pneumococcus types II and III, the precipitation test with antipneumococcus serum was used as an indication of the presence or absence of the carbohydrate, but, unlike the results obtained with these microorganisms, the precipitation reaction never entirely disappeared. It has not yet been ascertained whether this residual reaction is due to an unused portion of the original carbohydrate or to products of decomposition, which might either be present in the original sample or be formed as a result of the action of the microorganism from the soil.

In mixed culture, as it was first obtained from soil, this microorganism could not be cultivated on the purified specific carbohydrate, and, until it was isolated in pure culture, it had to be maintained on a mineral medium to which specific carbohydrate only partly purified had been added. In pure culture, the microorganism utilized the soluble specific substance obtained from broth culture and the specific carbohydrate isolated from pneumococcus type-I cells as well.

Extracts of lens 1 derived from any of the vertebrates, with the exception of rabbit lens, produce, when injected into rabbits, precipitins for all lens extracts Although a high precipitin titer can be developed neither an Arthus reaction nor an anaphylactic state can be produced in the rabbit. In a previous report 2 it was shown that Staphylococcus toxin, injected intracutaneously into rabbits, produces not only an antitoxin but also a hypersensitive state to the beef bouillon in which the toxin is produced and to beef serum. This result suggested that if such a poor antigen as beef broth could be made antigenically active, a similar antigenic effect could be developed for lens substance if this material were added to the broth. Accordingly the following experiments were undertaken.

Beef eyes, trimmed of all extraocular tissue, were dropped into boiling water for 30 seconds. With a sterile scissors a transverse corneal incision was made and the lens expressed into a tube containing 20 cc. of hormone bouillon. After a preliminary incubation period of 7 days to eliminate the contaminated tubes, the sterile tubes were inoculated with toxin-forming Staph. aureus, strain Ha. After 10 days'incubation at 37°C. the contents of the tubes were pooled, 1/2% trikresol added and filtered through a Berkefeld V filter. Injected intravenously, 0.3 cc. killed a 3000 gm. rabbit within 24 hours. This lens broth toxin precipitated with lens immune serum in dilutions as high as 1–50,000.

Rabbits were injected intracutaneously with 0.1 cc. amounts of lens broth toxin and lens extract, each in a different skin area at intervals of one week. Two rabbits were injected intravenously with 5 cc. amounts of lens extracts once a week. Other details of technique have been outlined elsewhere. 1 , 2

Considerable experimental work has been done within the past few years on the phosphatase activity of various bodily tissues. Robison 1 showed that soluble salts of hexosemonophosphoric esters were hydrolyzed by an enzyme present in ossifying cartilage, kidney, and the shafts of long bones. Kay, 2 Robison, 1 and others have shown that the intestinal mucosa, kidney, and whole bone contain the greatest concentration of this enzyme. Furthermore, the plasma and tissue phosphatase activity has been shown to vary from the normal in certain pathological conditions. Page 3 has shown that bone phosphatase is diminished if an excess of vitamin D is fed to an animal, along with the coincident decalcification of bone and metastatic calcification of other tissues. Parathormone has also been shown 4 to depress the activity of the bone enzyme in rats, though not to the same extent as the administration of vitamin D.

It is known that the thyroid gland also profoundly influences calcium and phosphorus metabolism. In exophthalmic goiter the excretion of calcium and phosphorus is markedly increased and there is decalcification of bone though the level of calcium and phosphorus in the blood remains normal. 5 Kay 6 and Hunter 7 have demonstrated an increase in the phosphatase activity of the plasma in hyperthyroidism but a correlation of the increased excretion of calcium and phosphorus and the decalcification of bone with the phosphatase activity of the kidneys and bones, respectively, has not been attempted. The present study was undertaken to determine whether such a correlation exists by measuring the phosphatase content of the bones and kidneys of animals rendered thyrotoxic by injections of thyroxin.

In several previous communications 1 , 2 , 3 we brought forth some evidence showing that diuretic measures increased neither the rate nor the percentage of elimination of barbital, nor did they shorten the recovery period from barbital depression. Since Gower and Van de Erve 4 recently again stated definitely that diuresis hastens recovery from barbital poisoning, and because of the practical therapeutic importance of the matter involved we decided to reinvestigate the problem.

Dogs were used throughout the experiments. Sodium barbital was administered in doses ranging from 225 to 600 mg. per kg. intravenously. Diuresis was produced by intravenous injection of appropriate doses of 10% glucose, physiological salt solution, 3% calcium chloride, and from 0.5 to 1.0% ammonium chloride. In contrast to the diuretic measures, dogs were dehydrated by withholding of fluids for 48 hours and by bleeding (12 cc. of blood per kg. withdrawn from the saphenous vein) immediately before barbital administration.

These intravenous injections were carried out by slow infusion with a burette, the total volume being injected during a period of from 10 to 12 hours. The animals, if they survived either procedure, were again given the same dose of barbital about 2 weeks following the first injection.

One of us (Duval¹) reported the observation that the acid-fast bacillus of Hansen in removed pieces of leprous tissue continued slowly, over a period of months, to multiply and that the microdetail by Holst and Frölich, 2 Hess, 3 Höjer and Westin, 4 Meyer and McCormick, 5 and others. Changes in the organs have received less attention.

Guinea pigs were fed on the basal vitamin C free diet of Sherman, 6 supplemented by cod liver oil and yeast to supply adequate quantities of vitamins A, B, D, and G. After a preliminary survey of the histopathology of the organs and tissues, work was concentrated on adrenals, heart, testes and liver in which evidence of scorbutic effects was most marked. The pathologic bone changes studied by us did not differ from those reported by other investigators.

Microchemical preparations of formalin fixed and fresh tissue, both teased and cut with freezing microtome, were compared after treatment with Sudan iv, osmic acid, Nile blue sulphate and Nile pink 7 for distribution of fat. Since practically identical pictures were obtained with all of these reagents, in later work Sudan iv and formalin fixed tissue was chosen. Hematoxylin and eosin stained sections were used for controls.

The distribution of cholesterol esters was indicated by the presence of doubly refractive materials in warmed formalin sections when examined with Nicol's prisms. The distribution and relative concentration of vitamin C in the tissues, particularly in the adrenals, was followed by the intensity of the reduction of 0.4% neutral silver nitrate.

We have found that a fair degree of accuracy can be obtained in testing for pregnancy by oxidizing 1 cc. of urine with 1 drop of 0.5% hydrogen peroxide, after which 5 drops of a 1% aqueous solution of phenylhydrazine hydrochloride are added and 5 drops of a 5% aqueous solution of methyl cyanide, followed by 5 drops of concentrated hydrochloric acid. It is then put into a boiling water bath and allowed to react for 25 minutes. After this time the reaction is said to be positive if a russet color develops and a flocculent precipitate appears. A negative reaction is shown by the absence of a precipitate or the presence of a powdery one and usually a straw color. All reagents should be fresh at the time the tests are made.

It was found that a modification of this test, which appears to be more sensitive for certain of the hormones, could be adapted to titration methods. This modification is carried out as follows:

To 1 cc. of urine, one drop of 0.5% hydrogen peroxide is added and this allowed to stand for 3 minutes. Five drops of a 1% solution of phenylhydrazine hydrochloride are added, followed by 5 drops of a 5% aqueous solution of potassium ferricyanide. This is allowed to stand for 10 minutes at room temperature and then put in a boiling water bath for 15 minutes. Upon removal from the water bath, one drop of concentrated hydrochloric acid is added and this is followed by an excess of sodium hydroxide. Dilute hydrochloric acid is then added by titration until a definite end point is obtained. (The color changes from an orange to a green to a prussian blue.) It was found that it required much less dilute hydrochloric acid to obtain the end point in pregnancy specimens than was required in the case of non-pregnancy female urine specimens, and that various pregnancy tests fall in a given range, while normal tests fall in another more restricted range.

Active movements, as opposed to the passive movements due to heart-beat, begin in embryos of the pigeon of 105 ± 5 hours'incubation. These earliest movements, weak and infrequent, are extensions and flexions of the head. By 115 ± 5 hours these movements have come to involve the neck, trunk, rump and tail in the order mentioned. All these movements are spontaneous, and the wings and legs move only in perfect integration with the trunk. These total movements increase in frequency and amplitude, and eventually several parts of the axial musculature move discretely. Even at this stage the embryos do not respond to chemical or tactile stimulation, except possibly in rare cases in the latter part of the period.

The first reactions to tactile stimuli are in response to stimulation on the snout at 125 ± 5 hours, although in rare cases sensitivity to touch on the upper part of the neck may appear at about the same time. The response is at first flexion of the head and neck, and sometimes the trunk. After several hours the reaction comes to involve the tail and rump, and then the wings and legs, which, under these circumstances also, move only in complete integration with the trunk. Meanwhile the reflexogenous zone extends to the shoulder, trunk and rump in cephalocaudal progression. In embryos of 135 ± 5 hours the tail moves with the rump, sometimes also with the leg, in response to touch on the base of the tail; and a little later the wing and the leg respond to stimulation on the shoulder or lower part of the neck, but embryos of this age do not respond to stimulation on the wing or leg. Embryos of 155 ± 10 hours exhibit local reflexes of the wings, legs and tail in response to stimulation on the respective appendages.

The development of reflexes of the eyelid, eyeball, digits, mandibles and anus, together with hatching, will be described in the final paper.

Webster and Armour 1 reported that artificially fed dogs with oesophagotomy, gastric fistula, obstructed pylorus and duodenal fistula, in which a condition of practically total avitaminosis had been induced, developed a complete achlorhydria without anorexia and inanition. Administration of powdered brewer's yeast restored the normal secretory activity of the stomach in 3 days. This indicated that for the normal activity of the gastric glands it is necessary that the vitamins contained in yeast, presumably vitamin B complex, be present in the body.

Two experiments on a similarly operated animal are here reported. The aim was to exclude as causative of achlorhydria other vitamins than vitamins B, and to determine if possible which of the latter is essential for the normal functioning of the gastric. mucosa.

First Experiment. The dog, kept on Scott and Ivy's 2 food mixture, was in excellent condition 40 days after the pylorus had been obstructed. This diet was replaced by a diet of glucose, casein, sodium chloride and olive oil. As in the previous experiments the secretion of gastric juice in response to food introduced into the duodenum, subcutaneous administration of 1 mg. histamine and sham feeding gradually diminished. On the 17th day of experiment 50 cc. of olive oil in the diet were replaced by 50 cc. of cod liver oil in order to supply the body with vitamins A and D. In spite of this change in the diet, 2 days later 1 mg. of histamine provoked only 3.0 cc. of mucoid secretion as compared with 40.5 cc. under normal conditions, and the next day only 2.6 cc. On the 21st day daily administration of 10 gm. of brewer's powdered yeast was begun and the special diet was replaced by Scott and Ivy's food mixture. Four days later 1 mg. of histamine provoked an enormous secretion (93.0 cc.) of regular gastric juice. Two days after this, 4 minutes'sham feeding gave 31.5 cc. in 40 minutes. During the whole experimental period the animal lost only 1.6 kg.

Macht, Dunning and Stickels 1 described the preparation and some of the pharmacological properties of a large number of derivatives of hydroxy-benzyl alcohol, commonly known as saligenin. Further investigation of the compounds pointed to the mono-brom-hydroxy-benzyl alcohol as being the most interesting of the series, and a more intensive and extensive study of that compound was accordingly begun.

Mono-brom-hydroxy-benzyl alcohol, named bromsalizol for convenience, is a white crystalline compound, melting at 107.5°–109°C., which on tasting, produces a numbness of the mucous membranes of the mouth and tongue. Its formula is with the bromine in the para-position. The analogous compounds with halogen atoms in the ortho- and meta-positions in the benzene ring have also been prepared in our laboratories. Bromsalizol is soluble in water to the extent of 0.5% and is freely soluble in alcohol and olive oil.

When studied on lower and higher animals, the toxicity of bromsalizol was found to be quite low. The minimal lethal dosage for some of the higher animals is shown in Table I. Doses of from 5 to 10 grains (0.3 to 0.6 gm.) have been repeatedly ingested by a large number of men without any discomfort or untoward effect.

The two salient pharmacodynamic properties exhibited by bromsalizol, even when employed in small doses or low dilutions, are (1) its antispasmodic or relaxant effect on smooth muscle tissues and organs, and (2) its local anesthetic effect. Its action on smooth muscle was studied in situ on intestines of cats, rabbits and rats, and much more extensively on all kinds of smooth muscle preparations from excised organs of the cat, rabbit, guinea pig, rat, bear, woodchuck and other animals. Experiments on excised muscle strips from the intestines, uterus, gall bladder, urinary bladder, fallopian tubes, ureters and other organs showed that the drug in dilutions of from 1 :10,000 to 1 :50,000 lowered the tonus of smooth muscle and slowed or completely inhibited the rhythmic contractions without killing the tissue, however, as could be proven by subsequent application of a suitable pharmacological stimulus. (Fig. 1.)

The attention of investigators has been so centered on the peripheral effects of ergotoxine that the central action of this drug has received scant notice. Most of the studies of ergotoxine have been conducted upon anesthetized animals. In these its depressant action upon the respiratory center has been repeatedly reported. There have been few references, however, to central stimulation. Barger and Dale, 1 after intramuscular injection of ergotoxine in cats, observed vomiting and “hyperexcitability”. They have recorded similar findings including fever in rabbits. Githens 2 considered this fever to be of central origin. Rothlin 3 found clonic and tonic convulsions 3 hours after large intravenous doses of ergotamine in rabbits. Koppanyi and Evans 4 reported vomiting and defecation of central origin in dogs and cats after the administration of small doses of ergotamine. Recent studies 5 in which ergotamine has been intramuscularly administered to unanesthetized animals in amounts sufficient to affect the sympathetic system make no mention of central action.

The observations here reported were made upon cats. Unanesthetized animals were tied to boards. No injection was made until the subjects had been calmed. Ergotoxine ethanesulphonate (1 mg./kilo)∗ was administered by intracardiac injection. This procedure can be accomplished without any excitement of the cat. The immediate manifestations of the action of the drug were those of sympathetic stimulation, wide dilation of the pupil and pilo-erection, especially in the tail. One minute after injection, the central effects became evident. The reflexes were hyperactive, the slightest touch causing a single spastic response in all voluntary muscles. More dramatic, however, were the emotional changes in the animal. The cats previously calm and unmindful of the injection, showed marked rage and apprehension. The slightest stimulation, visual, auditory or tactual, caused a savage hissing and snarling. Objects, brought near the head of the cat were ferociously attacked and bitten.

The cause of the extensive hemorrhage which occurs in hemorrhagic pancreatitis has long been a matter of speculation. Diapedesis of red cells from capillaries has been said to occur as a result of the action of trypsin, but no one has demonstrated that trypsin has the power to destroy the walls of previously intact arteries and veins, and in the only histological study of the question which we have been able to find it is stated that the ferment does not attack vessels larger than capillaries. 1 Our microscopical studies of the pancreas in cases of hemorrhagic pancreatitis have revealed the constant and, as far as we can determine, hitherto unrecognized presence of a specific form of necrosis of the walls of arteries and veins, leading to the rupture of the vessels with consequent hemorrhage. We have found the same vascular lesions in experimental hemorrhagic pancreatitis produced by the injection of irritants into the pancreatic ducts of dogs, following the procedure of Opie 2 and Flexner³; and we have determined experimentally that this vascular necrosis is the direct result of the action of pancreatic juice upon the walls of the vessels, and that the agent in the juice responsible for the necrosis is trypsin. Pancreatic juice, or pancreatic trypsin freed from lipase, when injected subcutaneously produces in the vessels at the site precisely the same vascular necrosis with hemorrhage as that which occurs regularly in hemorrhagic pancreatitis, and when trypsin is injected into the pancreas hemorrhagic pancreatitis results. The activation of trypsinogen by enterokinase is not necessary for the production of the vascular necrosis, for pancreatic juice obtained by cannulating the pancreatic duct outside the duodenum produces local vascular necrosis and hemorrhage when injected into the subcutaneous tissue. We have not yet determined precisely what it is that serves to activate trypsinogen when it is injected into the tissue. It is, however, clear from our experiments that if pancreatic juice escapes about blood-vessels in the substance of the pancreas, necrosis of the vessels with hemorrhage (hemorrhagic pancreatitis) can occur.

In a previous communication 1 a simple method for producing spinal anesthesia in the frog was described. In addition, a series of 10 local anesthetics were studied in summer frogs, kept at temperatures of from 26° to 29°C. The data obtained on each drug included the determination of the minimal anesthetic dose, minimal lethal dose, duration of anesthesia produced by a sublethal dose, and the calculation of a therapeutic coefficient from the fraction M.L.D./M.A.D. Comparatively, this coefficient gives evidence of the usable therapeutic range for each local anesthetic.

In this paper similar results are described in which 7 local anesthetics were injected intraspinally into winter frogs kept before, during and after the experiments at 10° to 12°C. This series includes those local anesthetics showing, experimentally, the best possibilities for use in human spinal anesthesia. Five of these drugs had been studied previously in summer frogs, but because of the relatively high temperature prevailing at that time, it was thought advisable to repeat the work at a lower temperature and perhaps a more satisfactory one for frogs. Because of great variations noted in frogs, which are ascribed to seasonal and temperature changes, a further check on uniformity was attempted by carrying out the complete series during two winters. Consequently the first series was carried out between October 10, 1931, and February 20, 1932, and the second, between the same periods of 1932–33. As can be seen from the table no great variations are to be observed between the 2 series. It might also be added that the results obtained on winter frogs show a close parallel to similar data obtained on the rabbit, These will be reported later.

The work of which this contribution forms a part was carried on in Puerto Rico, where Manson's blood fluke infection is an important and extensive clinical entity. The lateral-spined eggs of the parasite were obtained from the feces of human and experimental animals. The stools were washed and concentrated either by centrifugation or by sedimentation. The numbers of these eggs obtained from a 24-hour specimen varied not only with the age of the infection in the mammalian host but also fluctuated from day to day. The majority were viable, and contained ciliated larvae (miracidia), which were studied for detailed morphological features, including the number and pattern of the epithelial plates.

Hatching was found to be due to several factors, including the maturity and vitality of the miracidium, the tonicity of the medium and mechanical agitation of the eggs. Some larvae hatch immediately after washing and concentration but the majority require from 12 to 24 hours. After swimming around for several minutes in or near the bottom sediment the miracidia rise to the top of the water level and swim energetically in the top inch of the water. Under laboratory conditions their length of free-living existence was not over 24 hours, usually considerably less. If specimens of laboratory bred snails of the species Helisoma (Planorbina) guadeloupense, the appropriate intermediate host in Puerto Rico, are placed in a pan containing miracidia hatched from washed viable eggs of S. mansoni, they are soon attacked by the miracidia, which are attracted to the soft parts of the snail, particularly the tentacles. Some of these larvae secure a permanent hold on the tissues and bore their way inward. The compatibility of this host-parasite relationship was confirmed by the high percentage of snails which usually became infected (75–100%). Exposed snails were sacrificed at suitable intervals and dissected to determine the stage of development of the intramolluscan phase and the position of the parasite in the snail's body.

In Puerto Rico man is the only known definitive host of Schistosoma mansoni infection. The experimental data included in this report are based on infections of laboratory rats and rabbits and rhesus monkeys, as well as on one accidental human infection. Twenty-three rats, 8 rabbits and 4 monkeys were submitted to inoculation, either by placing the animal for one to 2 hours in a bath of water swarming with cercaria, or by placing desirable food in such water, or by spraying infected water into the animal's mouth. Evidence that cercariae were attacking the animal's skin within an hour after exposure was furnished by the extreme nervousness and later by intense pruritus of the exposed tissue of the animal. This continued for several hours after the animals were removed from the infective bath. All animals exposed became infected. The animals were sacrificed under anesthesia at intervals varying from 16 hours to 70 days, the cavities were opened, the various organs and tissues carefully inspected for gross evidence of infection and then the several organs were separated by ligation and removed from the body into isotonic citrate solution. These tissues and organs were thoroughly perfused and/or chopped up and the solution centrifuged and then examined for migrating larvae. Likewise, suitable portions of each organ were fixed in Zenker's fluid and sectioned. By these methods it was found possible to trace the route of migration and development of the young worms day by day through the prepatent period.

In general the development follows that described by various workers for Schistosoma japonicum, the Oriental blood fluke, but with important exceptions. The migration route is as follows. Entry of the cercariae through the skin takes place within a few hours after exposure. Within 16–20 hours after inoculation the larvae have left the skin, most usually via the peripheral veins, but at times passing through peripheral lymph nodes.

The blood picture in experimental mammals infected with S. mansoni shows first a leukocytosis, paralleling the migration of the larvae through the lungs. This is followed by a gradually developing leukopenia, with or without an eosinophilia, which, if present, is most pronounced about the end of the prepatent period. From about the 40th day there is an excess of euglobulin in the blood plasma. Moderate to late clinical cases usually give a positive precipitation test for serum globulin. The total erythrocyte counts are little altered in experimental and clinical cases, but there is definite evidence of regeneration of these cells, as indicated by a hyperplastic bone marrow and an increased reticulocyte count.

Smith and Engle 1 pointed out that in mice more than 10 days old, sexual maturity may be induced by hypophysis transplants, but at this early date 5 daily transplants are required. After the 17th day 2 or 3 daily transplants are sufficient. Hill 2 has shown that this age difference holds for injections of pregnancy urine. However, the ovary of the new-born rat responds neither to the amount required for the 15–20-day-old rat nor to a much higher dosage (Wiesner 3 and Pfeiffer, unpublished work). It remained to be tested how early the ovary of the new-born might mature if it were subjected to gonadotrophic hormones from the time of birth. This could be done by injections. However, if the ovaries are transplanted into an adult female at the time of castration, they will be supplied with a constant, or possibly a steadily increasing amount of anterior pituitary hormone.

Young, healthy, mature female rats whose cycles had been followed for some time previous to the operation were used for hosts. The ovaries were obtained from new-born young and one ovary was transplanted under the dorsal surface of each kidney of the host, on which bilateral ovariotomy was performed at the time of implantation. Seven animals were used and the grafting was successful in 5 cases. The changes in the ovary were followed by daily vaginal smears which also indicated the extent to which the immature ovary was able to fulfill the endocrine functions of the adult ovary. After 4½ to 8½ months the grafts and the hypophyses were removed, sectioned, and studied, in order to correlate the oestrus condition with the histological structure in the ovary and in the hypophysis. Proof that no regeneration of the ovaries had taken place was obtained by a study of sections through the region of the ovarian stalk and by comparison with a series of control castrates.

By means of an adaptation of the method reported by Andrus and Carter, 1 the authors have determined the refractory period of extrasystoles induced at various points in the cardiac cycle in the normally beating dog's auricle. A typical experiment in which extrasystoles were induced soon after the end of the refractory period of the spontaneous excitation is summarized in Table I.

Columns I and II depict the determination of the refractory period of the spontaneous beat. The intervals, which are given in seconds, were measured between a spontaneous excitation and the induced stimulus which followed (R₁-S₁ interval). Those stimuli represented above the “refractory period” line were followed by auricular extrasystoles; those below were not. The refractory period, therefore, lay between the shortest interval which was followed by a response and the longest which was not.

Each series is made up of consecutive observations; those of Column I with successively decreasing, and those of Column II with successively increasing, R-S intervals. Identical supra-maximal stimuli were used throughout.

Columns III and IV illustrate the estimation of the refractory period of extrasystoles induced 0.1470 sec. ± 0.0031 sec. after spontaneous excitations. The intervals are those between a stimulus which produced an extrasystole and a succeeding identical stimulus (S₁-S₂ interval). The stimuli above the “refractory period” line were followed by a second auricular systole, those below were not. The refractory period of the auricular extrasystoles, therefore, lay between the extremes as defined above. These results are also from consecutive observations with successive S₁-S₂ intervals shorter in column III and longer in column IV.

It has recently been shown that the complete inactivation of phage following treatment with HgCl₂ can be reversed by precipitation of Hg⁺⁺ with restoration of the phage to its original titre. 1 , 2 The phenomenon is interesting in that reactivation occurs after exposure to very concentrated HgCl₂ solutions for long periods of time, conditions which produce irreversible loss of the growth function in even the most resistant spore forms of bacterial life. The suggestion was advanced 2 that this behavior spoke for the enzyme concept of phage rather than for the view that phage is composed of living protoplasm.

Further work seemed indicated to determine whether the reversible inactivation of phage with HgCl₂ represented a special case or whether, as seemed likely, it was merely one example of a general property of phage. We wish to summarize here certain experiments on the cyanide-phage reaction.

1. Inactivation. Standard antistaphylococcal phage 3 suspended in beef infusion broth of pH 7.6 was mixed with an equal volume of 1/5,000 KCN (M/325) solution. The mixture was kept at room temperature for 24 hours after which time the following samples were taken: (a) 1 ml. immediately diluted 1/1,000 with broth for determination of residual [P]. (b) 10 ml. for reactivation.

2. Reactivation. To 10 ml. of inactivated phage was added 1 ml. of 2.74% AgNO₃ and 2 ml. of 1% KCN, the purpose being to remove the CN ions from their union with phage and to combine them in the complex double ion Ag(CN)₂ ^-. The latter ion does not form insoluble compounds with the cations present and therefore obviates the dangers of phage adsorption on a precipitate. Dilutions of 1 part of the reactivated mixture to 1000 parts of broth were made at once for subsequent titration.

The following observations have been carried out as a part of the study concerning the therapeutic value of total thyroidectomy in angina pectoris. It was noted a few years ago 1 that intramuscular injections of adrenalin, when given to patients suffering with angina pectoris would provoke attacks of anginal pain. As a result of clinical observation, it was found that when total thyroidectomy was performed in patients who were having daily attacks of angina, these attacks disappeared immediately following the operation and long before any changes had taken place either in the basal metabolism or in the rate of blood flow. It was therefore suspected and, in fact, predicted 2 that removal of the thyroid gland might be producing a fundamental alteration in the response of the circulation to adrenalin and that a previously positive response might possibly become negative after such an operation.

Five patients were given intramuscular injections of adrenalin varying from 0.3 to 0.5 cc. of a 1–1000 solution before and 2–3 days after operation. In the first of these cases, which was operated upon August 22, 1933, 0.5 cc. before the operation induced a maximum rise of the pulse rate of about 20 and the blood pressure rose from 188/88 to a maximum of 226/94 in a few minutes. Typical substernal anginal pain also resulted. On August 23, 1933, 0.5 cc. of adrenalin produced no pain, a rise in the pulse rate of only 10 and the blood pressure was hardly changed. On August 26 0.5 cc. of adrenalin again produced no chest pain, although the systolic blood pressure rose about 30 mm. On August 27, 1.0 cc. of adrenalin again failed to produce anginal pain and the effect on the pulse and blood pressure was slight. Here, although twice the original amount of adrenalin was given, the effect on the pulse rate and blood pressure was less and there was no chest pain.

It has been shown that the greatly enlarged ovaries of rats injected with extract containing the anterior pituitary-like hormone of pregnancy urine (A.P.L.) will decrease in weight after a certain period even though administration of the hormone be continued. Such ovaries will finally return to a normal weight level. 1 , 2 , 3

We have recently repeated these experiments on a series of 12 rats, continuing the A.P.L. injections for a longer time, and we found that after prolonged administration of the hormone the ovaries of some of these rats became actually subnormal in size, weighing as little as 7 mg. after 4 months'treatment (increasing doses of 25–200 units of A.P.L. daily). The ovaries of 8 out of 10 rats, however, were still normal in size at this time (30–45 mg.), but they lost their sensitivity to A.P.L., for 200 units per day of this hormone did not have any luteinizing effect upon them. Histologically they showed numerous corpora lutea in retrogression, but no mature follicles.

In 4 similarly treated rats we performed a laparotomy, inspected the ovaries after 4 months of treatment with A.P.L. and found them normal or subnormal in size. We then injected these rats over a period of 14 days with an aqueous ammoniacal gonadotropic extract of pig pituitary in addition to A.P.L. At autopsy all of these ovaries were greatly enlarged and contained numerous fresh corpora lutea. Their weights ranged from 99–170 mg.

These experiments show that even if the ovary loses its sensitivity to the anterior pituitary-like hormone of pregnancy urine, it still responds readily to extracts obtained from the hypophysis.

While Caesarian section normally initiates lactation in the rat, it has been found that milk secretion does not occur if the uterus is filled with paraffin immediately after the foetuses have been removed. 1 Furthermore, no oestrus is usually observed in such rats for several weeks after the operation; while in the controls, the uteri of which have not been distended, normal cycles set in 3 days after the interruption of pregnancy. We have recently repeated these experiments, and it is the object of this paper to describe the morphological changes observed in the ovary, pituitary and mammary gland.

In 10 pregnant rats, the uteri were emptied through a small incision during the second half of gestation; then each horn of the uterus was injected separately with melted paraffin (melting point 42°C.) after tying the lower end with silk. When the uterus was well distended, the upper end, through which the injection was made, was also tied.

During the 6 weeks following the operation, only 2 of these animals came into oestrus—one had 2 cycles, the other, 4. At autopsy it was found that the ovaries were greatly enlarged (75–110 mg. as compared with 40–45 mg. in the normal controls), and contained many large corpora lutea similar in appearance to those observed during gestation (Fig. 1).

In a control series where the foetuses were removed, but the uterus was not filled with paraffin, the corpora lutea of gestation shrank rapidly after the operation, and were as small as the corpus luteum of a normal cycle within 6 days after operation.

Histologically, the corpora lutea of the experimental animals consisted of cells rich in cytoplasm with a large vesicular nucleus; they showed no signs of round cell infiltration or of invasion by connective tissue, and resembled closely the corpora lutea of gestation. Sections stained with Sudan III showed, however, that besides the small lipoid granules characteristic of the active corpus luteum of gestation, larger fat globules appear in many of the cells. In none of these ovaries did we find mature follicles.

Within the last few years much work has been done concerning the survival of rats after adrenalectomy. 1 We had supposed that animals kept alive by injections of the cortical hormone following adrenalectomy would show approximately the same life-span after injections were stopped as those untreated after removal of the adrenals. However, in the course of other investigations in which we made prolonged cortical hormone injections into adrenalectomized rats, we found that rats show a much higher percentage of indefinite survival after extract treatment is withdrawn than they do when untreated following adrenalectomy. (Table I.)

The animals used were of a colony which had previously been shown to survive adrenalectomy for 30 days or longer in only 5% of the cases. 1 The average life-span of those not surviving adrenalectomy we had previously found to be 7 days. (A later control series of 15 animals showed an average survival of 10 days, with none surviving indefinitely.) Technique of operation and care was as previously described, except in cases to be subsequently mentioned. In all cases the injections of extract were begun at the time of operation, and given generally in divided doses twice daily—in a few cases once daily. Only the healthiest of animals were used. Ages ranged from 2.5 to 4.5 months. The cortical extract was kindly furnished by Dr. W. W. Swingle of Princeton University and Dr. Oliver Kamm of Parke, Davis & Co., to whom the authors are grateful for making this work possible. The dosage of extract given was probably in all cases far in excess of the minimum needed to maintain life.

We have observed the effects of extract treatment for varying lengths of time on 16 rats. Seven of these 16 cases survived in good condition after extract was discontinued, showed no sign of adrenal insufficiency, and were consistently gaining weight when they were killed for autopsy. In all 7 cases macroscopic cortical accessories, determined to be such by histological examination, were found on the left side near the junction of the adrenal and renal veins.

Francis and Tillett 1 demonstrated that following the intradermal injection of minute amounts of the type-specific polysaccharide of Type I, II or III Pneumococcus in humans, there developed in the serum of these individuals homologous type-specific agglutinins. In addition, the serum of individuals so treated was found to possess the capacity of conferring passive protection upon mice against infection with pneumococci of homologous type. These results were confirmed by Finland and Sutliff. 2

Recently, it has been shown that the specific polysaccharide of Type I Pneumococcus exists as an acetyl polysaccharide. 3 The acetyl compound is capable of completely absorbing the type-specific antibodies from an homologous immune serum and of inducing type-specific active immunity in mice. The acetyl polysaccharide is readily converted into its deacetylated derivative by treatment with dilute alkali. The chemical and immunological properties of the deacetylated polysaccharide are identical with those of the soluble specific substance in the form in which it was originally isolated⁴; it is non-antigenic in mice and only partially absorbs the type-specific antibodies from Type I anti-pneumococcus serum.

The recognition of the specific polysaccharide in acetyl form raised the question as to whether the antigenicity of the pneumococcus carbohydrates previously observed in humans had been due to the deacetylated product or to contaminations of that material with the acetyl polysaccharide. Consequently, 2 groups of normal humans, each 7 in number, were given intradermal injections of the acetyl polysaccharide and the deacetylated polysaccharide of Type I Pneumococcus, respectively. The deacetylated product was prepared by Dr. W. F. Goebel from the same lot of acetyl polysaccharide used in the experiment. An injection of 0.01 mg. in 0.1 cc. of physiological saline was made intradermally in each individual at weekly intervals for 3 weeks. Serum was obtained from each person before the first injection and again one week after the third injection. These sera were then tested for the presence of type-specific agglutinins, precipitins and mouse-protective antibodies.

In experiments performed at the Army Medical School, Kelser 1 has shown that Borna disease, or epidemic equine encephalomyelitis, can be transmitted by the mosquito Aedes aegypti. This suggested the advisability of reconsidering the abandoned theory of insect transmission in poliomyelitis, using mosquitoes of the genus Aedes or other insects not previously considered.

Poliomyelitis presents certain epidemiological features which have suggested insect transmission: (1) it is claimed that all outbreaks, even those that extend into the winter months, begin during the summer; (2) relatively few cases can be traced to previous cases or carriers, and the incidence of infection among contacts is low; (3) the rural distribution of the disease usually equals or exceeds urban incidence; and (4) many outbreaks occur in the neighborhood of water or water systems. Arguments advanced against this possibility include: (1) the virus has not been demonstrated in the blood of human patients, and only in small amounts in the blood of monkeys; (2) large amounts of virus have been required to infect monkeys injected by the intravenous route; (3) with one exception virus has not been demonstrated in filtered saline suspensions of insects previously fed on infected human beings or monkeys; and (4) such transmission experiments as have been reported indicate that biological transmission does not occur.

In experiments based on the inoculation into normal monkeys of filtered saline extracts of insects which had previously fed on materials containing virus, it has been shown that the virus survived in Musca domestica as long as 48 hours (Flexner and Clark 2 ; Howard and Clark 3 ; that adult Musca domestica and Calliphora vomitoria developed from larvae grown on infected tissues gave negative results (Noguchi and Kudo 4 ); and that virus was not demonstrable in Culex pipiens, C. sollicitans, or C. cantator (Howard and Clark 3 ).

The problem of the immunological relationship of the viruses of the encephalitis prevailing in St. Louis and Kansas City in 1933 and the viruses of equine encephalomyelitis and vesicular stomatitis has been studied by a series of cross-protection tests with the viruses associated with these diseases and their respective immune sera. A mouse strain of encephalitis virus obtained from Webster and Fite, 1 a neurotropic guinea pig strain of equine encephalomyelitis virus of the western type, 2 as well as similar mouse strains of the New Jersey and Indiana types 3 of vesicular stomatitis virus were employed, together with antisera from a monkey convalescent from experimental encephalitis, antisera from a rabbit and a horse convalescent from encephalomyelitis of the eastern type, 4 serum from a rabbit immunized against the western type of encephalomyelitis virus, and similar sera from rabbits immunized against the 2 strains of vesicular stomatitis virus.

Hormone broth suspensions of the stomatitis and encephalitis materials were mixed with equal volumes of antisera so as to make final dilutions of 10^-3, 10^-4, 10^-5, and 10⁻⁶. In the case of encephalomyelitis material, final dilutions of only 10^-3 and 10^-4 were used. Normal rabbit serum was substituted for antisera in similar series as controls. The mixtures were incubated at 37.5°C. for 2 hours, placed in the icechest overnight, and then examined for the presence of virus by intracerebral injection into animals.† For the stomatitis and the encephalitis virus mice were employed, and for the encephalomyelitis virus guinea pigs.

The results as tabulated showed that a particular virus was neutralized only by its homologous antiserum. Hence no cross-immunity reactions occur between any of the viruses studied. Webster and Fite 1 have stated that the results of animal inoculation tests indicate that encephalitis virus is not related to equine encephalomyelitis or vesicular stomatitis viruses. The results of the serological tests here reported are in accord with their observations.

In energy metabolism experiments in which the same dogs are used for a year or more, it is desirable to have masks which are sufficiently durable to be used for all of the experiments, and which fit the individual animal, therefore causing a minimum of dead space. These ends are readily accomplished by making the masks on casts of the heads to be fitted, by a method similar to that which is used by makers of orthopedic appliances.

The lower jaw of the animal is padded from the angles to the snout with a piece of felt, one half inch in thickness. The felt is secured by means of a gauze bandage which also binds down the ears. Stockinette is drawn over the animal's head, and plaster bandages are then applied until a mould varying from one-eighth to one-fourth inch in thickness has been made. After the plaster has set sufficiently the mould is readily pulled off if its posterior portion has not been applied too tightly. The felt pad and stockinette are removed from the mould, which is allowed to harden and is then filled with plaster of Paris mortar. Into the mortar a piece of gas pipe is inserted, to serve later as a handle. When the plaster has hardened, the mould is cut away, leaving a cast of the dog's head. Any modifications desired in the final mask, such as providing more space for the ears, or lengthening the mask posteriorly, can be made at this time by applying fresh plaster to the cast in the appropriate places. Space to permit the animal to open its mouth slightly has already been provided by padding the lower jaw before making the mould.

Flexner and Lewis 1 were the first to show that poliomyelitis could be produced in monkeys by the intranasal instillation of the virus. Faber 2 states that the virus is absorbed by way of the olfactory bulb along the base of the brain, thence proceeding to the medullary area and to the cord. After intracerebral injection, the spread of the virus would presumably be along the same route after it reaches the base of the brain. The axones are the main transmitting structures (Hurst³) and the spinal fluid plays no part in the spread (Jungeblut and Spring⁴). Monkeys may contract poliomyelitis after intraperitoneal and subcutaneous injections of massive doses of virus, 5 but, to my knowledge, never after the injection of the small doses that produce the disease when the virus has been intracerebrally and intrasciatically injected or intranasally instilled. The intrasciatic, intracerebral, intraperitoneal and subcutaneous routes are highly artificial portals of entry. When the virus is intranasally instilled, it enters the system through a natural portal of entry, and the spread along nerve fiber connections can easily be traced to the brain. Many workers believe that the nasal area is the usual and natural portal of entry in the human.

The following experiments were performed to show that the virus may spread by way of the sympathetic fibers in monkeys experimentally infected with poliomyelitis.

Our purpose was to isolate the cord carrying the virus from its axones, and thus to leave the sympathetic system as the only means of communication between the upper cord and the injected peripheral nerve.

Transabdominal operations were technically impossible and the operations to expose the sympathetic chain posteriorly were usually followed by death of the animal. The only feasible method was to cut out the cord at some strategic point so that all the communications between the somatic nerves and upper cord would be destroyed and then to inject a peripheral nerve, which in these experiments was the sciatic.

Experiments previously reported 1 have shown a loss in nutritive value of wheat gluten and casein when these proteins had been exposed to a temperature of 150 C. for 30 minutes. In an attempt to determine the site of injury to the protein molecule thus indicated new experiments have been made using supplements of various amino-acids to the diets containing raw and heated casein.

The casein in the present series was heated uniformly at 140°C. in a thin layer for 30 minutes. The growth of young rats fed diets containing various levels of the raw and heated casein as sole sources of protein and the nitrogen balance or biological value of such diets for mature rats were used as criteria of the nutritive efficiency of the proteins. These procedures were then repeated, the diets being supplemented with various amino-acids. The methods are described in a former publication. 1

Results of Growth Experiments. Young rats fed for 28 days from weaning diets containing raw and heated casein at 9, 12, 15, 18, and 22% levels grew better in all cases on the raw casein. Eight to 16 rats were used for each diet tested. The food intakes were quite similar but the resulting growth was in all cases less when the heated casein was used. The gain in weight per gram of protein eaten was greatest at the 12% level for both raw and heated casein and decreased slightly with each increase in level. However, the difference between the two types of diet, raw and heated, remained nearly the same, 0.20 to 0.34, at all levels, the values being 2.10 to 1.61 for the raw, and 1.76 to 1.40 for the heated diet.

The 12 and 15% raw and heated casein diets were then fed, supplemented by 0.2% lysine, histidine, tyrosine, cystine and tryptophane respectively.

The deposition of fat and sterol-ester in the livers of animals fed diets rich in cholesterol has been reported by a number of workers. The following is a report of one of a series of experiments made to determine the influence of other dietary constituents than sterol on this lipid deposition in the livers of rats.

The experimental diet fed contained dried egg yolk powder 25.3%, extracted casein 12.6%, agar 4%, Osborne Mendel salts 3%, starch 55.1% with supplements of yeast and cod liver oil. This represented egg yolk protein 8.4%, egg yolk lipids 16%, of which 1/16 was cholesterol, 1/4 lecithin. For the control diet the egg yolk powder was first extracted with alcohol-ether and the egg yolk lipid replaced by Crisco. Each experimental group consisted of 8 rats, while there were 12 in the control group, littermates of the experimental animals.

The rats were placed on the diet at weaning time. Both experimental and control groups grew extremely well. They were killed after approximately 60 days on the diet. The livers of the experimental animals presented the typical whitish coloration of sterol-fed animals. The average total fatty acid content of the livers was for the males of the experimental group, 9.3%; for the females, 11.7%. For the control groups the corresponding figures were: males 7.5%; females 7.4%. Total cholesterol (digitonid precipitation) for the males of the experimental group was 2.58%; for the females 3.86%. For the control groups the corresponding figures were: males .48%; females .30%. There was very little difference in the free cholesterol in the control and experimental groups. The average for both groups was approximately 0.3%. Likewise the figures for lecithin were not very different, the average for males in both groups being 2.7% and for females in both groups 2.25%. It was noted, however, that the odor of choline, presumably from decomposition of lecithin, was very strong in the tissues of the animals of the experimental group and absent in those of the control group.

During the examination of about 100 Black Tail deer of the California Coast Range approximately 25 were found to be affected with a lungworm-disease as yet unknown among ruminants. Heavily infested lungs showed macroscopically the picture of an induration. Microscopically this was found due to innumerable small nodules, invisible to the unaided eye. Each tubercle ordinarily enclosed several eggs of a nematode or embryos or both. In no case could adult stages be found, either in the parenchyma or in the blood vessels of the lungs. The regular distribution of the nodules on the other hand indicated that an embolisation of the eggs had taken place. Larvae, of course, were present in large numbers throughout the tubulous system of the lungs.

In a single case 2 adult nemas could be seen twisted around a trabecle of the right pro ventricle of the heart, thus indicating that the regular localization of the worms was distant from lungs and heart.

Finally, eggs as well as the nematodes themselves could be detected embedded in the connective tissues of the vessels beneath the spine and their nearby branches. They could be extracted even from between the muscles surrounding the body cavity and from along the vessels of the upper part of the hind legs. No traces of the worms, however, or of the eggs were seen on the subcutaneous tissue of the entire body, nor between the muscles of the head, neck, and frontlegs, nor between the mesenterium. Ordinarily they were coiled up in the lymphatic spaces of the connective tissue. Several specimens, however, were secured which had penetrated the wall of larger venous vessels of the hind legs.

The nematodes presented different stages of growth. Sexual larvae have as yet not been found. Worms recovered from the subserosa were usually smaller than those found in remote muscles. Inconspicuous accumulations of hemoglobin or hemosiderin deposited under the peritoneum or small hemorrhages between the connective tissue of the muscles together with the brown colored intestine of the worms were sometimes of assistance in the detection of the minute nematodes.