Experimental Biology and Medicine

The dialysis of serum containing Rh antibodies permits the division of such a serum into two fractions, the precipitate, containing mostly globulins, and the supernatant, containing the albumin fraction and a certain amount of the globulin fraction. The sediment, when dissolved in physiological saline solution using one-tenth of the original volume of the serum prior to treatment, is shown to contain the major portion of the Rh agglutinins. A rather potent Rh testing serum can be obtained by this procedure, where in many instances anti-Rh sera contain agglutinins of such weak potency that the native serum would not be strong enough to serve as a diagnostic test serum. The method is especially recommended for this purpose. The “incomplete” or “blocking antibody” has been found to be associated to a large extent with the supernatant, indicating that at least a partial separation of the Rh agglutinin from the “blocking antibody” has been accomplished by dialysis. The effectiveness of the resulting Rh agglutination by the globulin fraction is therefore not only attributed to simple concentration of the Rh agglutinins, but also to the partial removal of the Rh “blocking antibody.” The isoagglutinins anti-A and anti-B are not concentrated to the same extent as the Rh agglutinins which constitutes an additional advantage of the globulin preparation over the untreated serum.

It has been demonstrated that an Okinawan horse was susceptible to infection by the virus of Japanese B encephalitis and that the virus was present in the circulating blood 3 and 6 days after inoculation. It could not be demonstrated 16 hours after inoculation, nor after 9 days, at which time a small amount of neutralizing antibody had appeared. In 3 other horses which possessed high titers of neutralizing antibodies prior to the injection, virus could not be demonstrated in the blood stream following inoculation. These results, when considered in conjunction with the previously demonstrated presence of high titers of antibodies in the sera of many Okinawan horses,1 constitute further evidence that this species of animal may play a role in the epidemiology of Japanese B encephalitis.

Vomiting initiated by digitoxin is not abolished by removal of the carotid bodies and sinuses and by denervation of the aortic mechanisms. Assumign the mechanism to be relex in nature, the site of origin of the impulses causing digitalis vomiting is still undetermined. The possibility remains that afferent impulses may be set up in a structure or structures not yet eliminated. It is perhaps more probable that impulses may arise from any one of several structures all of which would have to be denervated in the same animal to abolish the emetic response to digitalis.

Shadowed electron micrographs of mixtures of purified southern bean mosaic and bushy stunt viruses with their specific antisera show their spherical elementary bodies aggregated into microflocs in which the particle-separations seem to be about twice their normal values. More detailed electron micrographic study of these systems is underway.

Thirteen samples of commercial lecithin were fatal to rabbits when administered intravenously at a level of 1.75 g per kilo body weight.

A method for preparing lecithin from egg yolk is described and it is shown that lecithin so prepared is harmless to rabbits when given by vein even at a level of 3.5 g per kilo body weight. Lecithin so prepared will acquire toxic properties if exposed to the air. Elevated temperatures accelerate this process. If the lecithin is stored under high vacuum, toxic properties will not develop within at least 228 days.

During treatment of 3 cases of histoplasmosis in the early months of 1945, it was decided to carry out in vitro susceptibility studies on the yeast phase of Histoplasma capsulatum.

Methods. Five-day-old cultures of Histoplasma capsulatum in heart-brain infusion broth (Difco) were used as the test organism.

The drugs tested were neoarsphenamine, neostam, sulfadiazine, sulfathiazol, stilbamidine.∗ Two antibiotics, streptomycin∗ and penicillin were likewise tested. The drugs were added to the media, before inoculation of the organism, in the following mg% concentrations: 100, 50, 25, 20, 10, 1, 0.1, 0.01, and 0.001.

The streptomycin was employed in the same manner, but in concentrations of 2500, 1250, 750, 250, 125, 100, 75, 50, 25, 12.5, 10, 7.5, and 5 units per ml. Penicillin was used in concentrations of 10, 5, and 1 units per ml.

Culture tubes of heart-brain infusion at pH 7.4, containing the desired concentrations of drugs in 10 ml, were inoculated, in duplicate, with 0.5 ml of a 5-day broth culture of the yeast phase of the test organism and incubated at 37°C. These cultures were examined for growth at the end of 7 days of incubation. Similar tests were performed in which the amount of inoculum was reduced to 0.2 ml of the 5-day culture of Histoplasma capsulatum. Each of these series, in duplicate, was repeated once with identical results.

A control series, in which drug-free culture medium was inoculated with the same quantity of inoculum as used in the drug series, was performed with each experiment.

The cultures were examined at the end of 7 days, and subcultures were made from all tubes which did not show growth or which were very cloudy. In tests using neoarsphenamine, stilbamidine, and streptomycin, some precipitate was formed when these agents were added to the heart-brain infusion broth.

Search for an effective therapeutic agent against viral infections has been stimulated by the success attained in treating bacterial diseases with sulfonamides and antibiotics. However, since the problem is complicated by the intracellular growth of viruses, it is perhaps not remarkable that the results up to the present time have been almost consistently negative. 1 Recently a small quantity of streptomycin 2 ,∗ became available to us. Because of the effectiveness of this agent against a number of bacteria which have been resistant to previously studied antibiotics and chemo-therapeutic drugs, and its low toxicity for the chick embryo, 3 a laboratory trial against viruses which would grow in the chick embryo seemed warranted.

Method of Study. The allantoic cavities of 11- to 13-day-old embryonated chicken eggs were inoculated with various strains of influenza combined with varying amounts of streptomycin. The PR-8 and Olsen strains were used as representative of Type A and the Lee strain as an example of Type B influenza.† Each embryo received an original total inoculum of 0.15 cc; fo this 0.05 cc was a 1:1000 dilution of infected allantoic fluid, and 0.10 cc was either saline or the antibiotic reconstituted and diluted with sufficient saline to give the desired concentration. The virus and streptomycin were mixed immediately before inoculation. In those instances where more than one “dose” of streptomycin was given, the subsequent injections were made in 0.15 cc quantities within the first 24 hours. In the interim, the shellholes were sealed with a small tent of Scotch tape. The eggs were incubated at 36° to 37°C for 48 hours. After this period the allantoic fluids were collected and their hemagglutinating titers for chicken red cells were determined. 4

Large doses of penicillin repeatedly administered by subcutaneous injection protected a significant proportion of mice against the lethal action of a crude preparation of gonococcal endotoxin.

Houssay and Lascano Gonzalez 1 found that in the testis of toads, in which anterior pituitary glands were implanted, spermatozoa were released into the lumen of the seminiferous tubules. Rugh 2 , 3 confirmed this observation finding that the infection of pituitary extract produces, in the testis of hibernating frog, the release of the spermatozoa, normally attached to the Sertoli cells. The author gives no explanation of the mechanism by which the spermatozoa are released into the lumen, but suggests that: “Possibly smooth muscle fibers are involved in the expulsion of spermatozoa from the tubules” (Rugh 3 ).

In order to investigate the actual mechanism of this release, toads (Bufo arenarum Hensel) in sexual rest (end of summer) were injected intravenously with a total extract of 3 anterior lobes of the pituitary gland of male toads.

Thirty minutes, one, 2,3, and 5 hours after the injection, the testes were fixed in Bouin or in Zenker-formol fluids; and also in the fixative of Champy, followed for 7 days by immersion in osmic acid at 38°C. Testes of normal control animals showed almost all mature spermatozoa clustered about their Sertoli cells (Fig. 1). Thirty minutes after the injection of the pituitary extract some of the spermatozoa have been liberated. The Sertoli cells showed big increase and vacuolization of cytoplasm (Fig. 2). After one hour, clear hydropic vacuoles were more conspicuous and also some acidophil droplets were found (Fig. 3). The vacuoles have the tendency to aggregate and seem to push the spermatozoa away from the tubule wall. Three hours after the injection almost all mature spermatozoa have been released. Most of the Sertoli cells appear enormously vacuolized and with the apical border completely irregular, due to the rupture of some vacuoles (Fig. 4).

Herein is described a method for the removal of adult S. mansoni worms from experimentally infected rabbits. The method has been used with good results in rabbits although there is no apparent reason why it could not be applied to other experimental animals susceptible to schistosomiasis.

The animal is tied to a board and a large dose (100 mg) of Heparin (Abbott-10 mg per cc) in 30 to 40 cc of physiological saline is injected very slowly into the heart or intravenously. Between 15 to 30 minutes are allowed to elapse at which time the animal is sacrificed in any desired fashion, the abdomen opened and the portal vein exposed. An 18-gauge needle with attached 50-cc syringe is inserted into the portal vein with the point of the needle directed towards the liver. The heparinized blood in the portal vein is aspirated while the syringe and needle are rotated to direct the bevel of the needle toward the worms which will be seen flowing into the syringe with the blood. About 15 to 20 cc can thus be aspirated before the portal vein collapses.

An additional 10 to 15 cc of blood can be aspirated by exerting a gentle squeezing pressure on the lobes of the liver-this will force more blood into the portal vein, and the liver will be seen to blanch slightly. The syringe is disconnected from the needle and put aside without fear of the blood clotting. A clean dry syringe may be attached to the needle and additional aspirations carried out in the manner described.

If it is desired to keep the worms alive, the aspirated blood is poured into a solution of 0.5% saponin in physiological salt solution (0.85%)-this will lake the blood cells but have no apparent effect on the worms.

Subtilin, an antibiotic extracted from Bacillus subtilis, was found to be antagonistic chiefly against Gram-positive organisms, including Mycobacterium tuberculosis and other acid-fast bacteria. Two notable exceptions were Neisseria catarrhalls and N. gonorrhoeae, both Gram-negative but also antagonized by subtilin. The antibiotic showed an extremely low toxicity for embryonic chick heart tissue fragments cultivated in vitro. Under the conditions of the test subtilin was approximately 20 times more toxic to Staphylococcus aureus than to chick heart tissue, a remarkably low tissue toxicity. A unit of subtilin is defined as the amount contained in 1 cc of the highest dilution capable of killing Staphylococcus aureus in 10 minutes at 37°C (F.D.A. phenol coefficient method).

Forty-six days after successful inoculation of L. donovani into hamsters, there appeared edema associated with amyloidosis. There was marked anasarca at 60 and 77 days after infection with extensive deposition of amyloid in the glomeruli and the adrenal cortex. The hypoalbuminemia, which coincided with the edema and a proteinuria, is believed to have been caused by the impairment of glomerular function.

Polysaccharides have been isolated from Listeria monocytogenes which precipitate in the presence of their specific immune sera. The immunological studies presented indicate the existence of 2 distinct animal groups, namely a rodent group and a ruminant group, thus corroborating the classification suggested by Schultz 2 and Julianelle. 3 . The precipitin test affords a simplified technic for the identification and immunological differentiation of the members of the genus Listeria.

1. Arthritis of the tibio-tarsal joint produced in cats by injecting silver nitrate, causes an atrophy of the soleus and tibialis anticus muscles. Tables I and II, Fig. 1 and 2.

2. Immobilization of the inflamed tibiotarsal joint in hyperflexion results in a loss of weight of the tibialis that is similar or slightly less marked than that caused by arthritis alone. Table III, Fig. 1. In these conditions the soleus keeps its normal weight. Table IV, Fig. 2.

3. Immobilization of the inflamed joint in hyperextension results in an increase of weight of the tibialis. Table V, Fig. 1. The soleus, on the other hand, shows a greater loss of weight than by arthritis alone. Table VI, Fig. 2.

The units to be described were developed in this laboratory for the purpose of collecting expired air of unanesthetized, trained dogs. The avoidance of leaks, minimization of resistance to breathing and of dead space were prime factors considered in designing the apparatus. No new fundamental principles are involved; rather, the apparatus embodies good features of older designs, eliminates some of the bad and utilizes new devices, to our knowledge never previously described.

Mask. The mask consists of a copper wire frame, on the order of carefully shaped commercial wire muzzle, made to fit loosely the snout of a 15 kg mongrel hound. The nasal end is projected about 1” beyond the nose and fitted with a No. 9 stopper with a single 3/8” bore. The open end is bent to fit the snout near the lip corners with about 1/4” clearance. Eight longitudinal wires spaced evenly around the frame serve to give good support and shape to the covering material. The finished frame (Fig. 1) is wrapped with 3 or 4 thicknesses of plaster bandage, allowed to dry overnight and soaked for an hour in melted paraffin. A latex surgeon's glove, size 8, from which the fingers are removed is stretched over the open end of the mask and secured by wrapping tightly with string and brushing with molten paraffin; about 3” of the wrist end of the glove is allowed to project as a sleeve. In use, the sleeve is stretched over the dog's muzzle to beyond the lip corners and invaginated into the mask by sliding the latter toward the eyes. Two rubber strips cemented to the end of the sleeve and tied together behind the ears prevent rolling of the glove and exposure of the lip corners during a measurement.

In 10 of 15 patients with elevated sedimentation rates, suffering from nonrheumatic and rheumatic disorders, the sedimentation rate and concentration of fibrinogen decreased following salicylate therapy. It is suggested that the change in the fibrinogen may represent an effect of salicylate on the liver.

The results of penicillin sensitivity tests for staphylococci are influenced by the size of the inoculum on two accounts: (a) the presence in sensitive strains of a minority of resistant individuals originating by mutation; and (b) the occurrence of penicillinase-producing strains whose cells are individually sensitive to penicillin. A simple laboratory test is proposed for the detection and quantitative measurement of penicillin resistance in staphylococcus.

1. Both free and total acetylcholine content of the whole rat brain are higher after nembutal or chloroform anesthesia (diffuse diminution of activity) than in the unanesthetized animal. The free acetylcholine change is more convincing after chloroform, whereas the total acetylcholine change is more convincing after nembutal. The total acetylcholine content of the frog brain is similarly elevated after nembutal.

2. Neither free nor total acetylcholine content of rat brain differed significantly after the onset of strychnine or picrotoxin convulsions (diffuse increase of activity) from that found in quiet animals awake. Nor did strychnine alter total acetylcholine content of the frog brain.

3. With the methods used, normal whole rat brain has been found to contain about 0.7 μg of free and 2.9 μg of total acetylcholine per gram wet weight; frog brain about 4.9 μg of total acetylcholine per gram.

The resistance of mice to the intraperitoneal injection of Type I pneu-mococcus was found to be markedly dependent on the type of diet on which they were maintained. Those on a purified diet survived the equivalent of 100,000 lethal doses for those on a commercial laboratory diet. This difference could not be ascribed to any known dietary essential.

A method is described for the preparation of follicle-stimulating preparations by tryptic digestion and the removal of a fraction which caused reactions in the human being at the site of injection.

Preparations made by the tryptic digestion method before and after it was changed to remove the local reacting factor were assayed both in normal and hypophysectomized rats. By macroscopic observation the ovaries from both kinds of rats appeared to contain only follicles but microscopic examination showed that a few contained lutein tissue indicating that not all the preparations were entirely devoid of luteinizing activity.

The levels of excretion of estrogens and 17-ketosteroids in the urine have been determined in 11 young adult males with acute infectious hepatitis, at frequent intervals, from onset of the disease to convalescence. In 9 moderately severe cases there was a significant elevation in the level of excretion of biologically active estrogens in the urine which persisted at a slowly diminishing elevation until convalescence was considerably advanced. There was no significant increase in the concentration of the urinary estrogens in 2 cases of hepatitis which presented only mild clinical symptoms.

Marked to moderate depression occurred in the level of excretion of urinary 17-ketosteroids early in the course of acute hepatitis, with a return to normal values considerably in advance of the period of diminished estrogen excretion and of clinical recovery.

Black Leghorn chicks were raised on a basal diet designed to contain the previously recognized chromotrichial factors, except the L. casei factor. Synthetic L. casei factor injected 5 times weekly at a level calculated to supply 10 μg per day showed feathering and pigmentation comparable to that of chicks receiving 10% brewers′ yeast; supplement. Growth was somewhat better om the latter. At a level of 5 μg, feathering was fairly good and depigmentation was marked. At 1 and 2.5 μg levels, feathering was very poor. Growth and feathering were in proportion to the level of L. casei factor used.

Evidence is presented suggesting that the effect of Radon ointment applied to the skin of men and animals is: (1) To produce active hyperemia. (2) To increase lymph formation and lymph flow without appreciably increasing capillary permeability to colloids. (3) It is suggested that the therapeutic effect of Radon ointment may be apparent in these cases where lymph flow is diminished, per- haps due to partial block. Evidence is offered suggesting that lymph flow is diminished in a limb that has, in the past, been exposed to excessive x-ray irradiation. Also, the result of treating acute lesions thought to represent partial lymphatic block following penicillin injections is described, with results that are suggestively good although the series is too small for absolute certainty.

Under the conditions of these experiments glutathione and thioglycollic acid are capable of reversing the growth inhibiting effect of atabrine on the Lactobacillus arabinosus. Once the concentration of atabrine for complete inhibition had been significantly exceeded, no reversal of bac-teriostasis was attained by increased concentration of glutathione and thioglycollic acid. Several additional compounds were tested under comparable conditions with negative results.

Adult stock rats maintained on stock diets show no changes in the white blood cells during the 3-week lactation period. Maintenance on a purified diet during the same period results in a marked leucopenia and granulocytopenia by the twenty-first day. Daily supplements of brewers′ yeast or liver extract result in improvement but not entire prevention of the blood dyscrasia.

The specific complement fixation test, using purified rickettsial suspensions as antigen, is recommended as a routine test to differentiate infections in guinea pigs caused by rickettsiae of murine and epidemic typhus and Rocky Mountain spotted fever.

In a comparative study of several strains of Ducrey bacillus with H. influenzal and H. parainfluenzal a number of significant biologic differences have been noted. The Ducrey bacillus will not grow in a medium which supplies both the X and V factors. Unlike these other members of the genus Hemophilus, the Ducrey bacillus will grow in a medium enriched only with blood serum. It will also grow in a medium enriched only with erythrocytes. The agents in serum and in erythrocytes which support growth of the Ducrey bacillus differ in heat stability; that in serum withstands 100° C but not auto-claving, while that in erythrocytes is inactivated by heating to 65 ° C. The addition of both erythrocytes and serum to the base medium provides a more favorable medium for the growth of the Ducrey bacillus than either one alone. Ducrey colonies will not develop on the surface of a solid medium unless the humidity of the atmosphere is very high. The “satellite phenomenon” is not observed in colonies of Ducrey bacillus. Attempts to separate Ducrey growth factors from whole blood or serum by dialysis have not been successful. The Ducrey bacillus differs significantly from H. influenza and H. parainfluenzce in susceptibility to inhibition by penicillin and by tyrothricin.

It was found that in some patients with chronic poliomyelitis very weak muscles can be found which seem to consist of one remaining motor unit. A study of their properties has been made by recording the action potentials through skin leads.

During the course of a survey for brucella agglutinins made on sera discarded from the Wassermann laboratory (C. W. E., N. B. McC. and G. A. B.), a blood bank donor was found to have a brucella agglutinin titer of 1:200. This individual was a healthy male who, 3 months previously, had been experimentally vaccinated by one of us (W. B.) with U. S. Army cholera vaccine. The possibility that this finding might be more than a coincidental occurrence led us to examine the stored sera from the cholera studies for brucella agglutinins.

Methods and Material. Thirty-four individuals were given either regular U. S. Army cholera vaccination or varying amounts of an experimental cholera vaccine consisting of extracted cell residue. Whereas the Army vaccine contained both H and O antigens, the experimental vaccine contained primarily the O antigen. The vaccines were given in 2 doses of 0.5 and 1.0 cc with an interval of one week between inoculations. Sera were collected before immunization and at 1. 2, 5 and 9 weeks after completion of the vaccination. Brucella agglutinin titers were determined by the rapid slide method of Huddleson, using the standard dilutions from 1:25 to 1:500. The subjects were divided into 4 groups:

Group I-7 subjects, routine U. S. Army cholera vaccination, 8,000 million organisms per cc.

Group II-8 subjects, experimental vaccine, 8,000 million organisms per cc.

Group III-10 subjects, experimental vaccine, 24,000 million organisms per cc.

Group IV-9 subjects, experimental vaccine, 40,000 million organisms per cc.

Five post-immunization sera from 3 individuals were further studied by reciprocal agglutinin absorption tests, using as antigens a live virulent strain of Br. abortus and a live 24-hour culture of Vibrio comma. After absorption with brucella, the sera were tested with cholera antigens 1 containing HO and containing only O antigen.

Although electrical stimulation is used widely in diagnosis and in physical therapy and is the only practical procedure to test muscle function in animals, the effects of prolonged direct stimulation have not been studied extensively.

Alternating 60-cycle current was used in 3 independent but in phase circuits for stimulation through 3 pairs of electrodes, the first pair being inserted into the knee and tendon of the gastrocnemius muscle in situ of anesthetized rats (nembutal) and cats (dial), (D₁ electrodes); the second pair (D₂) inserted into the muscle itself at its upper and lower ends; the third pair (I) being employed for stimulation of the peripheral end of the cut sciatic nerve. The strength of the stimulus was adjusted with a rheostat and measured by a volt meter; in addition, the resistance in the muscle was measured by means of an oscilloscope in a shunt parallel to the muscle. Stimulation was continued either with D₁ or with D₂ until the muscle length reached the pre-stimulus resting value. During and after the continued stimulation, brief tetanic stimuli were applied at regular intervals through the 2 other pairs of electrodes. In all cases maximum stimuli were applied through a 10,000-ohm resistance placed in series with muscle and nerve. The muscle was loaded with 200 g and contraction was isotonic. Before continued stimulation the effect of summation was tested for all combinations of D₁, D₂, and I. There was no summation between D 1 and I, or D₂ and I, and a tendency to slight summation of D₁ + D₂.

Steel, platinum, silver, and nonpolarizable (Cu-CuSO_4l.0M - K₂S0_4sat. - NaCl_sat. NaCl_{0 9%}-Tissue) electrodes were employed in various experiments.

Vitamin B₁₀ and B₁₁ concentrates were inactive as a source of folic acid for the monkey.

Crystalline vitamin B_c and the synthetic Lactobacillus casei factor were active as a source of folic acid for the monkey when fed at a level of 100 γ per day. The B_c conjugate had less activity when fed at levels of 200 and 300 γ per clay.

Folic acid deficiency precipitates a deficiency for the monkey anti-anemia factor which is characterized by lack of growth, sub-optimal hemoglobin levels and a reversal in the lymphocyte-neutrophile ratio.

A differential microbiological assay for O-heterobiotin with S. faecalis R and L. arabinosus 17-5 has been developed. Assays, of several natural materials indicated that O-heterobiotin does not occur generally, but that a metabolite does exist in animal tissues which has biotin activity for S. faecalis and not for L. arabinosus.