Experimental Biology and Medicine

Necrosis, hemorrhage and cirrhosis of the liver in rats were described by György and Goldblatt. 1 They attributed these lesions to a deficiency of a part of the vitamin B₂ complex. By modifying the diet they were later 2 able to increase the incidence of the liver injury. In the report on this work, they stated that the pathogenesis of the necrosis and cirrhosis was related to the lipotropic effect of casein. Blumberg and McCollum 3 reported the production of liver cirrhosis in rats with or without accompanying necrosis and the prevention of the cirrhosis with choline.

Daft, Sebrell and Lillie 4 described the production of liver cirrhosis in rats and its prevention by choline, methionine or casein. Lowry, Daft, Sebrell, Ashburn and Lillie 5 reported that treatment by choline or casein of rats with experimentally produced liver cirrhosis resulted in hyperplastic regeneration of liver cells and clinical improvement of the animals. In a paper describing the histology and histogenesis of this liver cirrhosis, Lillie, Ashburn, Sebrell, Daft and Lowry 6 stated that among the noteworthy features is the absence of hemorrhage and, in the earlier stages, of evident necrotic liver cells.

The composition of the diets used in these 3 laboratories is given in Table I. It will be noted that our cirrhosis-producing diet No. 545 contains a much lower percentage both of casein and of fat than the diets of György and Goldblatt 1 2 and those of Blumberg and McCollum 3 and that our diet contains cornstarch while the others contain sucrose. Diet No. 545 also contains free cystine while the others do not. In view of the classic observations of Osborne and Mendel, 7 it is apparent that the 10% casein diets are deficient in the sulfur-containing amino acids.

1. Phosphatase activity and localization in developing tooth germs of rats (1 and 4 days old) is correlated with the degree of cellular differentiation and the status of the calcification process. 2. The enzyme is shown to be localized (a) in the stratum intermedium, (b) in the outer enamel epithelium of the incisors, (c) to a lesser extent in the stellate reticulum about the first molar in the more advanced stages (4 days old), and (d) in the pulp contiguous to those odontoblasts in the regions of dentin calcification. 3. No phosphatase activity was demonstrated in the ameloblasts or odontoblasts. 4. The connective tissue surrounding the crypt bone showed extensive phosphatase activity.

Seventy-seven percent of 17 eclamptic women had electroencephalograms indicative of cerebral dysrhythmia. Fifty-eight percent of this series gave a family history of convulsive disorders.

It may be stated that the increased efficiency of formaldehyde as a germicide was not due to any effect of the vacuum on the gas but rather to the ability of the vacuum to increase the penetration of the gas. Formaldehyde is chiefly a surface disinfectant. The increased penetration in the presence of a vacuum made it possible for the gas to reach the organisms. The vacuum principle is capable of producing a very deep penetration of the gas into porous materials. The method is very efficient, cheap, easily performed, and indispensable for the sterilization of objects likely to be injured by the application of dry or moist heat.

When added to whole, unlaked blood, menadione causes a marked methemoglobin formation, and the drug is rapidly converted to some other substance (or substances). Assays with vitamin K-deficient chicks showed that these reactions cause a marked loss in the antihemorrhagic activity of the menadione. Vitamin K_1, unlike menadione, does not produce methemoglobin under comparable conditions, and the vitamin is remarkably stable in whole blood.

Young rats weighing 65 to 90 g were maintained for 14 to 21 days on a vitamin B-free diet (casein 10, sucrose 80, butter 4, salts, agar and vitamins A and D). At this time these animals survived fasting but 2 or 3 days. Substitution of a diet of dextrose, supplemented by dextrose solution by stomach tube, permitted survival of from 7 to 16 days.

Vitamin-depleted rats receiving dextrose and thiamine survived 2 to 4 times as long as those receiving only dextrose. Nicotinamide appeared to have some effect, but riboflavin, pyridoxine hydrochloride, pantothenic acid and choline had no appreciable effect in prolonging the life of the dextrose-fed rats. All of the foregoing vitamins given together had no further beneficial effect than when thiamine alone was given.

When dextrose was substituted immediately following an adequate vitamin-containing diet, rats survived 19 to 29 days. The injection of thiamine hydrochloride or the vitamin mixture did not appreciably alter the survival time of these animals.

A rapid and heavy growth of meningococcus developed on an agar medium containing tryptic digest of casein, dextrose and buffer at pH 7.4-7.6. However, this medium failed to support growth of the gonococcus. Addition to the medium of a small amount of cystine, however, stimulated growth of this organism. The growth of gonococcus on this casein digest medium, with cystine, was as heavy as that of meningococcus. The growth of meningococcus did not seem to be improved by this addition of cystine to the medium.

The intraperitoneal injection of bile results in the production of convulsive seizures in rats up to 10 days of age and not thereafter. The intracerebral injection of bile produces convulsions in adult rats. The hypothesis is developed that young animals are more susceptible to convulsive seizures because of an inherent cerebral dysrhythmia and a deficient hematoencephalic barrier.

1. Subarachnoid injection of thiamine causes rapid recovery of thiamine-deficient cats. Recovery is equally rapid when the vitamin is given intravenously or intramuscularly. 2. Moderate signs of meningeal irritation follow introduction of thiamine chloride into the subarachnoid space. 3. Thiamine deficiency may unmask symptoms due to an organic defect in the brain.

1. Two samples of cozymase were tested, and each could replace both thiamin chloride and nicotinic acid, essential growth requirements for the staphylococcus; however, these preparations contained small quantities of thiamin. It has been shown that an adequate amount of thiamin was contained in the cozymase, which explains why maximum growth of staphylococci was obtained in the presence of cozymase, but in the absence of nicotinic acid. 2. When certain experimental conditions are fulfilled, cozymase, but not thiamin chloride and nicotinic acid, will inhibit the bacteriostatic action of sulfanilamide and sulfapyridine against staphylococci.

1. Sulfathiazole and, to a slightly lesser extent, sulfanilamide accelerated the clotting action in citrated plasma of staphylocoagulase contained in a sterile broth filtrate. Sulfadiazine markedly inhibited the coagulating effect, while sulfapyridine had only slight inhibitory activity. 2. Para-aminobenzoic acid operated like sulfanilamide and accelerated the coagulating action. 3. Broth filtrates with staphylocoagulase did not cause coagulation of every sample of normal, citrated human plasma. The degree of coagulation also varied.

The bacteriostatic effect of paranitrobenzoate on the pneumococcus is markedly reduced by the products of disintegrating tissue.

1. The penetration of sodium sulfathiazole (NaSAT) into the intact skin of rats, rabbits, and humans, up to 6 hours'application by iontophoresis and from wet dressings, is essentially equal, while that from an ointment base is less than either of these. Penetration is somewht better from wet dressings than by iontophoresis after longer times.

2. Saturated (1.8%) calcium sulfathiazole (CaSAT) penetrates the intact skin of rats to the same extent by iontophoresis as from 5% NaSAT ointment.

3. Iontophoresis of NaSAT through the intact skin of rabbits produces a greater concentration of SAT in subcutaneous muscle than wet dressing application, but the concentrations in deep muscle and blood are negligibly small.

4. Impaired circulation in rats by ligation of the limbs, has little effect on penetration of CaSAT and NaSAT into these limbs by iontophoresis and from ointment.

5. Penetration of CaSAT and NaSAT by iontophoresis and from wet dressings into rabbit skin is essentially the same. Penetration of NaSAT into the skin of dead rabbits by iontophoresis is greater than from wet dressings.

An investigation has been made of certain aspects of Van Slyke's laws concerning the distribution of ions between red cells and plasma at different pH values. When hydrochloric acid was used to bring about variations in blood pH, losses of CO₂-from plasma water and cell water were equal, so that the ratio (HCO₃)_c/(HCO₃)_s remained unchanged and equal to unity at all pH values. The values of (Cl)_c/(Cl)_s at different pH values did not parallel the values of (HCO₃)_c/(HCO₃)_s but gradually rose to unity at values of pH which varied, the actual value appearing to depend upon and to vary with the red cell content of the blood.

In the course of studies on experimental subacute bacterial ednocarditis, attempts were made to produce the disease in heparinized rabbits. The methods for heparinizing humans, namely, by continuous venoclysis or by repeated intravenous injections, were virtually impossible of adaptation for the rabbit. However, rabbits will develop and maintain a prolonged coagulation time over a protracted period with repeated subcutaneous injections of heparin, the average daily requirement being about 45 mg of commercial heparin† administered in fractional doses. 1

In order to eliminate the disturbing abrupt rises, sharp peaks and unpredictable drops in coagulation time as well as the technical difficulties incidental to fractional daily dosage, a simpler means had to be devised for continuous heparinization by the subcutaneous route.

In view of the success obtained with pellet and capsule implantation of hormones, 2 similar experiments were pursued with heparin. Pellets containing 50 mg and glass capsules tightly packed with 100 mg of heparin were implanted under sterile precautions subcutaneously or subfascially through a small skin incision. While rabbits could be heparinized in this fashion, this approach had to be abandoned for the following reasons: The results with glass capsules were erratic. On some occasions a spectacular but shortlived effect was obtained: at other times, the results were negative and when the capsule was removed subsequently it was discovered that practically none of the heparin had been acted upon.

Additional shortcomings were: The necessity for instrumental procedure; the rapid absorption of pellets with resultant inordinately delayed coagulation time and subsequent precipitate drop; the presence at times of local hemorrhage and the obvious impracticability of sustaining heparin effects over prolonged periods.

The inability of prolonged administration of purified renin to change materially the blood pressure of the 3 hypertensive dogs reported herein does not necessarily refute the findings of Wakerlin, for he used a crude preparation of renin, and the reductions in pressure in his hypertensive dogs may have been due to some other factor present in his kidney extracts. The present observations indicate, however, that the prolonged administration of purified renin neither reduces the blood pressure of hypertensive dogs nor endows their plasma with the ability to inhibit the vasopressor response of renin.

The classical operation for preparing an innervated pouch (“Pavlov Pouch”) of a portion of the stomach 1 2 is difficult and time-consuming and there is some doubt whether the pouch retains its full vagus innervation. 3 Modifications have been suggested which were designed to simplify the technic 4 5 or to preserve a larger proportion of the nerve supply. 5 6 1 7 1 8 However, with one exception 8 no procedure has been described which does not involve the complete separation of a part of the wall of the pouch from that of the main stomach with the consequent risk of severance of at least some of the nerve fibers.

The operation to be described leaves intact the entire muscular wall connecting the pouch with the main stomach. This has the double advantage of preserving the entire nerve supply and of avoiding the necessity of severing any of the larger blood vessels. Because no time is lost controlling hemorrhage, the operation can be performed more quickly, and therefore with less shock to the animal than the classical operation. This method exclusively has been used in our laboratory for the past several years with excellent results.

The general plan of the operation involves turning a portion of the stomach inside out through a small incision, thus making the mucosa accessible for the building of a mucosal wall between the projected pouch and the main stomach.

Detailed Procedure. The special instruments required are 2 pairs of vulsellum forceps and a set of gastroenterostomy forceps with 3 blades. The fundic portion of the stomach is delivered through an abdominal incision in the usual manner and the site of the pouch selected. An incision about two centimeters long is made in the center of the pouch area, through the entire gastric wall including the mucosa.

Small pieces of heart tissue from young chick embryos coalesce to form a spherical body when brought into actual contact with each other in a mixture of chicken serum and Ringer's solution. The pieces must touch each other. A preliminary treatment of these fragments with “trypsin” increases the rate and percentage of coalescence. The method is serviceable for titration of cyto-antibodies in a medium, as these inhibit the coalescence in low concentrations.

The method is described, and its use is demonstrated by examples.

A prolongation in the coagulation time and a lowering of the prothrombin index of the blood for approximately 6 days beginning between 24 and 72 hours after the drug has been ingested will usually be produced by oral administration of a single dose of 400 mg of 3,3′-methylenebis (4-hydroxycoumarin) to a patient weighing 130 lb or less, or 600 mg to a patient weighing over 160 lb.

The order of the toxicity of the proteins investigated is as follows: protamine > histone > globin. Globin was found to be devoid of appreciable toxicity when injected into animals or tested on tissues and on monocellular organisms in vitro. Protamine and histone were found to be highly toxic to trypanosomes. Factors which may contribute to the toxicity of these proteins were discussed.

1. The amount of labile iron derived from preformed pseudohemoglobins is not more than 2% of the total blood iron. 2. Under the influence of very dilute acids additional iron is split from the hemoglobin molecule itself. 3. Carbon monoxide inhibits the liberation of iron. 4. This inhibitory effect can be used for diagnostic purposes and confirms the presence of carbon monoxide in the blood of tobacco smokers.

In many institutions where it has been necessary to devocalize the laboratory dogs the results have not been satisfactory. In 1932 the writer made a study of the usual devocalizing methods with the object in view of improving the results obtained from such methods.

By using a pair of Miles nasal cutting forceps it was found that the true vocal cords could be rapidly and completely removed. 1 The procedure was carried out on 20 dogs under morphine-ether anesthesia. There were no complications and the animals recovered within 24 hours. After about 3 months, however, each dog developed a hoarse, unpleasant bark. The immediate assumption was that the operative technic had been faulty and that the vocal cords had not been completely removed. Later, when some of the same animals came to autopsy, laryngeal examinations showed a complete absence of the true vocal cords. Further studies of the autopsy material revealed in each case a well developed pair of soft mucous folds or pockets at the base of the epiglottis and just above the true vocal cords. These folds were identified as false vocal cords and seemed to be the only remaining structures by the use of which sound might be made.

The surviving dogs whose vocal cords had been removed with poor results were again prepared for operation. The soft folds or pockets of the false cords were removed as completely as possible with a pair of straight, back-cutting Ostrom forceps. Upon recovery these animals were no longer able to produce any noise other than soft whispered sounds even with their most vigorous barking efforts. Since that time approximately 200 animals have been devocalized by the method of removing both the true and false vocal cords.

A simple and rapid method is suggested for ascertaining the presence or absence of iodine concentrating tissue (thyroid gland) in thyroidectomized animals. Injected radioactive iodine is assayed by radioautography of the entire neck region, either in the fresh or the fixed state.

The difference between prothrombin times of whole and diluted plasma is a more reliable guide to prothrombin activity of the blood than is the prothrombin time of either alone. The difference has been found to be fairly constant in normal individuals. When prolonged, it indicates hypoprothrombinemia. When reduced, it may signify hyperprothrombinemia or an excess of anticoagulants in the blood. The procedure recommended is well suited for following the therapeutic effects of agents which influence the prothrombin level (or activity) such as dicumarol or vitamin K.

The 3H2 strain of P. cathemerium is a very virulent agent when transmitted as indicated in the designation; i. e., exclusively through the bite of infected C. pipiens mosquitoes.

Hagemann 1 reported the superiority of a fluorescence method using berberine sulfate stain for demonstrating leprosy bacilli in nasal mucus and thick blood smears. The same year he 2 proposed an auramin-fluorescence technic for the routine identification of acid-fast fast bacteria, especially tubercle bacilli. The suitability of this method for cultures of Myco. leprae has been confirmed by Küster 3 and for leproma smears by Kline and Leach. 4

Recently one of us (H.J.H.) has investigated the separation and concentration of acid-fast organisms from the tissues of leprosy patients. To cryochemed spleen† distilled water was added and direct impression smears were made from a piece of the tissue. One set of smears was stained with auramin O and examined according to the fluorescence procedure described by Richards and Miller. 5

A representative field is shown in the accompanying photomicrographs. The faint material in the background is non-acid-fast splenic cellular substance. The acid-fast bacilli have a characteristic granular appearance.

Although no accurate comparison was made with Ziehl-Neelsen stained smears, the fluorescence technic was clearly superior for demonstrating globi and leprosy bacilli. It is of interest to note that Richards, Kline and Leach 6 believed more tubercle bacilli could be demonstrated in the same microscopic fields by fluorescence than by the conventional method.

In a recent investigation the author 1 attempted to obtain from spleens of lepers an antigen or product analogous to tuberculin which might be used in the diagnosis of leprosy. The product, obtained by buffer solution extraction of finely ground leprous spleens rich in acid-fast bacilli was tested on leprosy patients by a committee in Manila, P. I. 2 Specific tuberculin-like action could not be demonstrated. There are at least two possible explanations for this negative result. One is that there is no hypersensitiveness to leprosy bacilli. The other is that the acid-fast bacilli in the spleen after the grinding were adsorbed or protected by the preponderantly great amount of normal splenic cells present, and therefore not well extracted.

It seemed preferable, if possible, to separate the bacilli from leprous tissues before grinding and extraction. Previously, attempts have been made to separate acid-fast bacilli from the tissue of the lesions in which they are found. Cowdry 3 and his coworkers dissected bacilli-laden cells from tissues in rat leprosy and then ruptured them with a Mueller press. The expressed tissue fluid, cytoplasm, nucleoplasm and some unruptured cells were ground with sand and centrifuged at low speed. The supernatant fluid, which contained acid-fast bacilli, was then removed and recentrifuged at high speed. The sediment contained a large number of acid-fast organisms. In the experiments here reported advantage was taken of homogenizing chambers 4 for tissue cells and the interface studies of Mudd and Mudd 5 which showed that certain acid-fast organisms, including stock strains of leprosy bacilli, pass readily from the watery into the oil phase.

These results indicate that exposure of Paramecium caudatum to the carcinogens employed in these experiments, causes the production of certain abnormalities regardless of the different concentrations of the carcinogenic agents used. The order of potency for these carcinogens, as demonstrated by the time of appearance of abnormalities, is as follows: 3:4 benzpyrene: methylcholanthrene, and Scharlach red.

Succinic, lactic and malic dehydrogenases and hydrogenase were demonstrated in cell-free juices of Asotobacter vinelandii by the use of the methylene blue reduction technic. Oxygen uptake with sodium succinate as substrate and hydrogen-oxygen uptake with hydrogen as substrate were observed in the Warburg respirometer. Cell-free juices contained oxalacetic acid and alpha-ketoglutaric acid decarboxylases, but not pyruvic acid decarboxylase. Though succinate oxidation was cyanide-sensitive, addition of heart muscle cytochrome c did not stimulate the oxidation of ascorbic acid, suggesting that the cytochrome system differs from the animal cytochrome system.

In a consecutive series of cultures from the genital tract of 129 unselected patients, pleuropneumonia-like (L) organisms were recovered from 23 of 77 cervical cultures, from 1 of 8 vaginal cultures, from 3 of 36 prostatic cultures, and from 1 of 8 cultures of urethral discharges from males. 1

The 4 male patients who yielded an abundant growth of these organisms all had chronic prostatitis. Gonococci were not demonstrable. One patient had rheumatoid arthritis, one had polyarthritis clinically resembling gonococcal arthritis, one complained of soreness of feet and knees and swelling of fingers. Joint effusions were obtained from the first 2 of these patients, but no organisms could be found in them by smear or culture.

Among the females, considering only those cases in which pleuropneumonia-like organisms were the predominating flora and, excluding cases in which only a few colonies were observed, we find that 5 had chronic cervicitis, 7 had gonorrhea, while in 4 the cervix appeared clean. Among the 9 from whom gonococci were not recovered, 1 had rheumatoid arthritis and 3 complained of various skeletal aches and pains. One of these 3 was the wife of the male patient who complained of prostatitis, soreness of feet and knees and swelling of fingers, a finding which suggests the possibility of transfer of the organisms by sexual intercourse.

This mode of transmission is further indicated by another finding, not included in the above series. In this case, a man was admitted to the hospital because of acute tenosynovitis of the left wrist and chronic prostatitis.

Evidence is presented to show that biotin is essential for normal embryonic development in the hen's egg. A satisfactory synthetic ration for use in studies of the vitamin requirements of laying and breeding hens is also presented.

Atropinesterase of rabbit serum was found to be activated to the same degree by equimolar concentrations of KCl, NaCl, NaBr, and NaI. Activations were also produced by KCNS, MgSO_4, and CaCl_2. Trisodium citrate had no effect. Cyanide, fluoride, physostigmine and sulfhydryl compounds inhibited the enzyme. Compounds capable of eliminating sulfhydryl groups present in the enzyme preparation had no influence, nor did Congo red or methylene blue.

1. The pressor activity of certain aromatic amines is destroyed by aeration in vitro in the presence of quinones. 2. Simultaneous injection of dihydroxyphenyl-alanine and small amounts of copper salts in hypertensive rats causes a considerable lowering of blood pressure. 3. It is suggested that tyrosinase reduces the blood pressure by its oxidative effect on acceptor substances of the catechol type such as dopa and epinephrine, which in turn, upon oxidation to ortho-quinones, destroy the pressor amines in vivo.

Differential characters of two strains of Clostridium botulinum, type E, are described. One strain was isolated from German-canned sprats; the other from Nova Scotian smoked salmon. Culturally they can be differentiated by colony formation on horse blood agar plates. Agglutination and absorption tests indicate them to be of distinct serologic groups.

The toxins of both strains were neutralized reciprocally by their antitoxins. Tests for toxigenic activity demonstrated, however, that the toxins were not identical. Subcutaneous injections of both toxins uniformly gave rise to symptoms characteristic of botulism in guinea pigs, mice, rabbits, and kittens. White Leghorn chickens were highly susceptible to the toxin of the salmon strain but not to that of the sprat strain. In chickens 4 to 12 weeks old, approximately 6 to 8 guinea pig M.L.D. of the salmon toxin gave rise to fatal botulism, while doses of the sprat toxin several hundred times as large failed to kill or induce symptoms of botulism. The sprat toxin was demonstrable in the blood and also in the excreta of some of the chickens 72 hours after inoculation.

In previous reports 1 2 we have shown that extracts of the urine of patients with leucemia contained substances capable of inciting cellular proliferation in the organs of guinea pigs similar to those found in human leucemias. In one of these reports 2 it was shown that the substances obtained were specific for the type of leucemia from which the urinary extracts were made.

It has seemed obvious that the chloroform extracts described in the earlier communication contained many inert materials as well as the active principles. Because of this we decided to fractionate the chloroform extracts and tried to trace the active principles preparatory to chemical analysis.

The following method has been developed to fractionate the extracts of urine of patients with chronic myeloid and chronic lymphoid leucemia. It applies particularly to the fractionation of the former.

The urine is extracted with chloroform as before. 2 The chloroform is removed by distillation and the residue is suspended in 200 to 300 cc of 10% sodium hydroxide. This suspension is extracted 8 to 10 times with 100 cc portions of ethyl ether to remove any substances which will not react with sodium hydroxide. The fraction soluble in ethyl ether (A) is recovered by removal of the solvent. The residual alkaline solution (B) is reacidified with 30 to 50 cc of concentrated hydrochloric acid. The acid solution is extracted with chloroform, the extract after removal of the chloroform is extracted with successive portions of petroleum ether (C); these extracts are combined, distilled in vacuo. The residue crystallizes readily in the cold.

Sulfanilamide if applied percutaneously is resorbed by the skin of rabbits and men. (a) The concentration of sulfanilamide in the blood of rabbits following its percutaneous administration is as high as that obtained after its oral administration, but inferior to the concentration following subcutaneous or intramuscular injections. (b) The blood concentration of sulfanilamide in men following its percutaneous administration is lower than that after oral administration, but vomiting is much more rare and less severe.

The percutaneous use of sulfanilamide may serve as an auxiliary method of chemotherapentic treatment in certain cases.

A simple autosomal recessive lethal mutation that causes crowding together of the vertebrae with consequent shortening of the neck and body is described. The percentage of ash in the wing and leg bones was approximately the same as in the normal embryos, but the percentage of ash was less in the scapula, ilium, and ischium than in the corresponding bones of the normal embryos.

1. The Harderian glands of female mice of C₃H strain at 75 days of life appear to differ in at least two ways from the Harderian glands of male mice of the same age and strain, as follows: (a) They appear to contain under normal light a greater degree of gray pigmentation; (b) they contain approximately 25% more protoporphyrins, in spite of the fact that the glands are smaller and the body weights are also smaller than in males. 2. These differences between sexes are more pronounced at 75 days of age than they are at 250 days.

It appears that certain species of Culex mosquitoes may act as vectors of the virus of St. Louis encephalitis. This is supported by the following evidence: 1. In certain areas Culex mosquitoes have fitted into the epidemiological picture to the extent that several workers have stressed their probable importance as vectors. 2. Virus is available to mosquitoes in the blood of doves and a number of other species of experimentally infected vertebrates. Evidence of naturally acquired infection has been demonstrated in the serum of many vertebrate hosts. 3. Infected Culex tarsalis have been collected in nature. 4. Culex pipiens has been shown to act as an experimental vector, transmission having been demonstrated to occur between the 4th and the 11th day after an infective meal. Virus persists in the mosquito for at least 13 days.

In a recent communication from this laboratory 1 a number of different influenza vaccines were described, and their relative antigenicity in man was compared. The highest mean post-vaccination antibody levels were obtained with those preparations which because of concentration contained the greatest amounts of virus. The concentrated virus was obtained from allantoic fluid by centrifugation at 11,000 rpm for 2 hours. In the present paper another method of virus concentration is described, together with the data showing the antigenic potency of such concentrates when given to man.

Preparation of Virus Suspensions. All of the virus suspensions used, both for concentration experiments and for testing of sera, were prepared from the allantoic fluid of eggs infected with either the PR8 strain of influenza A virus 2 or the Lee strain of influenza B virus. 3 The method of preparation used was described in the previous report on the series of vaccinations. 1

Virus titrations in mice, measurements of red cell agglutination titer, and agglutination inhibition antibody titrations in human sera were all done as described in the previous report. 1 The only change in the method was that the degree of agglutination in the red cell tests was determined by means of a photoelectric cell rather than by comparison with visual standards. In order that the antibody levels of the human sera in this series might be compared directly with those previously reported, the same standard ferret immune sera for each virus (PR8 and Lee) which had been used as controls in the former tests were again titrated. Seven duplicate titrations of each standard serum were distributed throughout the tests as a check on the uniformity of the technic.

A rapidly increasing number of data show that biotin is of wide-spread metabolic significance for a wide range of living things, and the fact that this vitamin markedly increases the growth rate of yeast cells and bacteria indicates that it might have similar effects on other plant or animal cells. Our present limited knowledge about many of the viruses, particularly those of the neurotropic group, is due in large part to the difficulties encountered in trying to grow them in vitro. In view of the important relationship between the metabolic state of cells and the ability of viruses to infect them, 1 experiments were devised to test the effects of biotin on various cell types, especially nerve tissue, in an effort to apply the data thus obtained for initiating or improving the growth of viruses and rickettsiae in vitro.

The method used for studying the effects of biotin on living cells was a slight modification of a technic previously described, using hanging drop cultures. 2 A piece of tissue was removed, preferably from the midline of an embryo, and divided into two equally-sized (and usually, bilateral) halves. One piece was explanted into a clot made up of plasma, embryonic extract, and biotin in Tyrode solution. A control culture was made of the other half in a clot consisting of the same proportions of plasma, extract, and plain Tyrode solution. Since the biotin (obtained from the S. M. A. Corporation) was furnished in alcoholic solution, it was necessary to add an equivalent amount of alcohol to the Tyrode solution used in control cultures. In most of the concentrations (1/6 γ to 5/6 γ of biotin per cc), the amount of alcohol was too small to affect the results appreciably.

By the use of a precold treatment injection schedule it has been demonstrated that desoxycorticosterone acetate at dose levels of 250 γ and 500 γ and progesterone at 500 γ prolongs the life of adrenalectomized mice subjected to low temperatures. Peanut oil at the dose level of 0.2 cc has been shown to possess a significant toxic action.

In the present studies, attempts are made to determine what effects the removal of the hormones of the pituitary and of those glands under its control has upon the ability of an animal to exert reparative epithelial growth or reparative fibroplasia.

Rats of the Wisconsin strain, obtained from the local Anheuser-Busch laboratories were used. At the age of approximately 14 weeks they were hypophysectomized, and then used 7-10 weeks later, at which time they showed a 10-20% loss of weight, loose thin skin, emaciation, appearance of fine silky hair, atrophy of testes, and atrophy of the adrenals, gonads and thyroid at postmortem.

In the first part of the experiment an area of 2 sq cm was resected from the right flank of 19 normal and 18 hypophysectomized animals under nembutal-ether anesthesia. This area was measured on the first and every other subsequent day by tracing on cellophane and measuring with a planimeter. The reduction in wound areas for the hypophysectomized animals is identical with that of the controls.

The wounds in the normals were slightly larger than those in the experimentals for the first 4 days. This is probably due to a greater amount of elastic tissue and less stretching of the normal skin before the area was demarcated, for in the normals the skin is more tightly bound down by the subcutaneous tissue that is strikingly lacking in the hypophysectomized animals.

In the second part, we attempted to test the rate of fibroplasia. Harvey had shown that fibroplasia is proportional to the return of strength in wounds of the stomach. Using his technic, incisions 1 cm long were made in the stomachs of 29 normals and 18 hypophysectomized rats.

Calvin 1 reported that the total circulating proteins of normal dogs could be increased by relatively large infusions of normal saline. During the course of other experimental work² it seemed pertinent to study the effect of massive infusions of saline upon the total amount of circulating protein in both normal and hypoproteinemic dogs.

Three dogs in varying states of nutritional hypoproteinemia and one normal dog were used in this work. After a preliminary determination of blood volume and serum proteins had been made, the experimental animals were given relatively large intravenous infusions of normal saline solution during short periods of 6 to 9 minutes. The blood volumes and proteins were studied in these animals at varying intervals. The methods used for determining blood volume, serum proteins and non-protein nitrogen have been described. 2

The data for the plasma volumes and serum proteins are presented in Table 1. The total circulating protein is calculated from plasma volume and serum protein concentration. After relatively large saline infusions the absolute amount of circulating protein in the hypoproteinemic animals shows practically no change during the first thirty minutes and at the end of 6 hours there is a slight but definite increase. On the other hand, infusions in normal animals are followed immediately by a relatively large increase in the circulating protein. At the end of 6 hours, total circulating protein decreases but does not return to the preinfusion level.

In 3 different experiments the maximum increases in total circulating protein in a normal dog were 8.7, 15, and 16% respectively at 30 minutes.

A recent quantitative analysis of bacteriostasis by sulfanilamide, sulfapyridine, sulfadiazine and sulfathiazole showed that for inhibition of Es. coli decreasing amounts of each drug in the order named were required. 1 Actually over six hundred times more sulfanilamide is required than sulfathiazole or sulfadiazine, and similar results have also been obtained with a variety of other microörganisms, 2 indicating that this regular increasing order of bacteriostatic potency is characteristic of these N₁ sulfonamides (Col. A, Table I) and not attributable to the selective action of one drug against a certain organism. Furthermore, the quantitative relationships between these sulfonamides and para aminobenzoic acid (PAB) differ just as widely; e. g., over 600 times more PAB is needed to block sulfathiazole and sulfadiazine than to block sulfanilamide. (Col. F.) When the minimum effective concentration of each drug was tested, the same amount of PAB was required to block each drug. (Col. E.) In other words, going from sulfanilamide to sulfadiazine a progressively larger proportion of each drug actually present in the cultures appears to possess activity against either bacteria or PAB; the active moiety presumably being similar for each drug.

It seems possible that such a partition into active and inactive portions might be controlled by the extent to which the drugs are ionized in the cultures. Accordingly the dissociation constants were measured∗ (Col. B) and the percent of each drug ionized at pH 7.0 (Col. C) was computed. (Some shift in the apparent pK may be expected to occur in complex biological media.) Based on these values and using the minimum effective amounts of drug added to the culture (Col. A), the concentration of ionized drug was estimated (Col. D).

From the nature of the function relating the effectiveness of sulfonamides to the hydrogen ion concentration the conclusion can be drawn that the active agent in a sulfonamide solution is an anionic species.

Studies of the effect of certain media on the action of sulfonamides indicate that a medium which may seem to be inhibitory is one which favors the growth and multiplication of bacteria so that an ultimate large bacterial population results. Our data also indicate that a medium, which by comparison may seem not to be inhibitory, is one which is not favorable to the development of large numbers of the bacteria under study. It is also apparent that the type of medium, time of incubation and number of bacteria inoculated are important factors which should influence conclusions drawn from sulfonamide studies in vitro. Prolonged incubation may result in adaptation of the bacteria to unusually high concentrations of the sulfonamide.

It has been found that p-aminohenzoic acid prevents the inhibitory action of sulfanilamide on the growth of hemolytic streptococci in vitro 1 2 and in vivo. 3 This compound also inhibits the therapeutic effect of sulfanilamide on mice inoculated intracerebrally with the virus of lymphogranuloma venereum. 4 It has been suggested 2 3 4 that sulfanilamide exerts its action by competing for an enzyme associated with a metabolite similar in structure to p-aminobenzoic acid and essential to the organism.

Coggeshall 5 showed that sulfanilamide had a marked effect against plasmodia (Plasmodium knowlesi in rhesus monkeys). Other sulfonamides 6 were shown to be effective against P. knowlesi and against Plasmodium cynomolgi and Plasmodium inui in rhesus monkeys. Sulfadiazine was shown 7 to be effective against human malaria (Plasmodium vivax and Plasmodium falciparum). The present paper reports the effect of p-aminobenzoic acid in inhibiting the action of sulfanilamide against plasmodia.

Plasmodium gallinaceum was used as the test infection in Leghorn or Rhode Island Red chicks weighing 50-100 g. Five birds were used for each test and 10 for each control group. The drugs used were mixed with a powdered chick mash so that the stated daily dose of drug was contained in 10 g of mash. This amount of drug-food mixture was consumed over a 24-hour period. Blood concentrations of sulfanilamide, determined by the method of Marshall and Litchfield 8 with a photoelectric colorimeter, were shown to be maintained at a uniform level over a 24-hour period by this method of administration. Drug feeding was continued for 10 days with blood level determinations on the 2nd and 3rd days.

Starvation of the albino rat is fatal within 8 to 15 days and produces a structural detachment of about 20% of the motor nerves in skeletal muscle, morphologically manifested by: (1) progressive retraction and eventual uprooting of the hypolemmal end plate from within the atrophic muscle fiber and loss of the granular sole plate of Kühne, and (2) enormous distention of the epilemmal axons which stain very intensely with gold chloride.

Unlike in the adult animal, estradiol in high doses causes no pituitary or adrenal cortical hypertrophy in the immature rat, in fact it tends to decrease the size of these organs. This difference in hormone responsiveness is not due to the presence of a mature or immature gonad respectively since it is demonstrable even in castrates.

Recent experiments with radioactive isotopes have demonstrated the permeability of red blood cells to cations. The results obtained by different experimenters are, however, not directly comparable with each other or with the permeability of other systems. Permeability and diffusion phenomena ought to be expressed in standard c g s units. 1 With the aid of published measurements on cells of the same species 2 and with certain assumptions, it is possible to evaluate permeabilities in terms of standard units. Following Rashevsky 1 one may define the permeability, h, of a membrane to a given solute (provided the membrane is the limiting factor in the rate of exchange of solute) as where Q is the rate at which the solute crosses the membrane in mols per second, S is the area of the membrane and C₁—C₂ the difference in concentration of the diffusing substance between the two sides of the membrane in mols per cc.

The permeability constant, h, of a membrane is defined on the assumption that the rate of diffusion, Q, is the difference between the rate of exit which is proportional to C_1, and the rate of entry, which is proportional to C₂ In the red cell, however, Q, the net rate of ion change, is zero. The permeability factor, b, 3 measures the fraction of the ions inside the cell which cross the membrane each minute. We may assume, for example, that loss of potassium from the cell is entirely passive and may be regarded as a leak, while the uptake of potassium takes place by another path motivated by an unspecified mechanism. If so, the rate of loss, b, should be proportional to C₁ and not to C₁—C_2. In some experiments, b is measured by the rate of uptake of radioactive potassium; however, the rate of active uptake equals the rate of passive loss, if there is no net change in total potassium, so conversion of b to permeability units still entails dividing b by C₁.

The permeability of human blood clot to various sulfonamide drugs under conditions of increased temperature was determined in an in vitro study. Negligible penetration of the clot occurred even in the presence of temperatures up to 41°C. Heparin had no effect on the permeability of the blood clot.

Evidence is presented indicating the presence of two antibodies for crystalline insulin in the serum of a patient who was both allergic and resistant to crystalline insulin. It was also shown that the patient was allergic but not resistant to a preparation of human insulin. The relation of the two antibodies to the patient's response to crystalline and human insulin is discussed.

After several of the causes for false positive reactions in this method were eliminated, about 4% of human tuberculous sera gave specific positive reactions.

Two new Salmonella types with undescribed flagellar antigens were reported. Salmonella tennessee was represented by the antigenic formula, VI, VII:z_29. The antigens of Salmonella simsbury were characterized as I, III, XIX :z_27. Each type is represented by a single strain isolated from the feces of normal human carriers.

Purulent meningitis was induced in rabbits, employing a technical procedure essentially the same as that of Felton 1 and Wegeforth, by intracisternal injection of Staphylococcus aureus, using cultures of a strain (Sca) derived from human meningitis and also cultures of the same strain after rabbit passage (237). Some of these animals were then subjected to treatment with bacteriophage, sulfathiazole or with both these agents.

Table I presents briefly the results in a preliminary group of 6 rabbits. The untreated controls died on the second day. The other 4 animals were treated with bacteriophage, beginning a few hours after inoculation. Only one survived, Rabbit 261, apparently the pioneer observation of recovery from experimental staphylococcal meningitis. The culture strain Sca, maintained on agar slants, gradually lost virulence so that not every control animal inoculated with a minute dose would die of meningitis.

The results in a group of 10 rabbits inoculated with strain 237 on March 31 are shown in Table II. The 4 untreated control animals all died of meningitis by the 5th day. The other 6 animals were treated with sulfathiazole beginning on the day of inoculation, and of these 5 died of meningitis and one survived.

Other experiments, in which this same culture strain was used and both bacteriophage and sulfathiazole were administered revealed a sharper contrast between treated and untreated animals.

Table III indicates briefly the result in one such experiment. All 4 of the untreated controls developed purulent meningitis and were dead on the 4th day.

The importance of mumps as a disease among military personnel is considerable. In the last war it stood third among the diseases causing time lost from duty, being surpassed only by venereal diseases and influenza. 1 2

Although the work of Johnson and Goodpasture, 3 using Macaccus mulatta as the experimental animal, established the etiological agent of mumps as a filterable virus, the practical implications of their findings have not been realized. Thus, no laboratory tests for the diagnosis of the infection or tests for susceptibility are available. Moreover, no means are at hand by which the potency of convalescent sera which have been employed as prophylaxis may be assayed. Accordingly, we undertook the repetition of the basic experiments of Johnson and Goodpasture with the object of extending them in these directions. In this paper we report briefly on a complement-fixation test for the detection and estimation of antibody in the sera of man and monkey convalescent from mumps.

About 3 cc of saliva collected on the 1st and 2nd days of the disease from a typical case of epidemic parotitis was introduced into Stensen's duct of a monkey (Macaccus mulatta). On the 5th day the gland, which was somewhat enlarged, was removed under ether anesthesia, weighed and emulsified by grinding in infusion broth to make a 20% suspension. This was then inoculated into both ducts of another monkey. The animal, on the 7th day following inoculation, developed marked swelling of both parotid glands with considerable subcutaneous edema of the cheeks. In this way the virus has been passed in series through 5 monkeys—the last of which received material obtained by nitration of the parotid suspension through an Elford collodion membrane of 515 mμ pore diameter.

Single rats rendered diuretic by water, and alcohol in sedative dosage, constitute a satisfactory biologic test for the antidiuretic action of pitressin. It is 10 times more sensitive than Walker's 3 test, in which he used the diuretic rabbit; however, the required dose of pitressin per kilogram of animal is essentially the same. As an assay technic, using the same number of injections, ours is at least as reliable as the multiple rat test of Burn, ¹ and as other tests available for antidiuretic substances.