Experimental Biology and Medicine

The carbohydrate and phosphatide fractions of the tubercle bacillus (M. tuberculosis, H-37) exert a selective inhibitory action upon the endocellular enzyme, Cathep-sin II, of tuberculous tissue. This phenomenon may help to explain the cytotoxic action of tuberculin in vitro and the failure of autolysis of caseous, tuberculous tissue.

Though it is generally agreed that the cholera vibrio contains a potent endotoxin, the nature of the toxin is by no means clear. The isolation of a toxic protein has been reported by some workers such as Galeotti, 1 Sanarelli, 2 and Hahn and Hirsch. 3 More recently the trichloracetic acid extraction method of Boivin 4 has been applied to the cholera vibrio by Checcacci, 5 Raynal, Lieou, and Feissole, 6

Damboviceanu and Barber 7 and Gallut, 8 all of whom have reported successful extraction of a toxic fraction. It has been assumed by these workers that the active material is a polysaccharide-lipid complex, though in no instance is this conclusion supported by unequivocal evidence.

In the course of a study of active immunity to infection with Vibrio cholerœ we have had occasion to isolate the toxic principle from both Inaba and Ogawa types and partially purify it. Toxic solutions of the vibrio cell substance have been prepared in the following ways: (a) The cells are readily disintegrated in 4-5 hours by high speed grinding with sand, and the cellular debris is spun off, leaving a toxic opalescent supernate. (b) The cells may be dissolved in 6 M urea to give a similar toxic solution, (c) The cells may be digested with pepsin 3-5 days without inactivation of the toxicity, and the insoluble material removed by centrifugation to leave a toxic supernate. (d) The intact cells may be extracted in the cold with M/2 trichloracetic acid, all the toxicity going into solution, (e) Lyophilized cells may be extracted with methyl alcohol, ethyl alcohol, chloroform, or ethyl ether in a Soxhlet apparatus to give toxic extracts. Attempts to extract the toxicity from the intact cells with glycols have not been successful.

The toxicity of the cholera vibrio seems to play an important role in the pathology of Asiatic cholera in man and the immune response to this toxicity is of some interest. The isolation and purification of the vibrio endotoxin reported in the preceding paper 1 has allowed a more precise investigation of this matter than has hitherto been possible. Certain aspects of these studies are herein reported in preliminary form.

There appears to be considerable confusion regarding the antigenicity of the cholera toxin since many workers have designated hemolytic strains as “toxic” and have worked with anti-hemolysins. Since it is now established that the “true” cholera vibrio is non-hemolytic, 2 such studies would seem to be irrelevant. Some workers, including Pottevin, 3 Horowitz, 4 Bail, 5 Hahn and Hirsch, 6 and Andu and van Niekerk 7 have reported the preparation of antisera which were protective in vivo against the lethal action of toxic extracts of the vibrios. It is generally admitted, however, that the antitoxic properties of antisera are only feeble at best. For example, Bail 8 attempted to account for the unaltered toxicity of serum-treated vibrios by the demonstration of free complement-fixing substances in immune guinea pigs infected with vibrios, these substances presumably representing uncombined toxin. According to Eisler and Kovacs 9 the toxin is not antigenic in that it is not specifically precipitated by antiserum, but adsorbed on the flocculating antigen-antibody complex.

In our experiments various preparations of endotoxin have been studied. The immunological activity of endotoxin prepared by preliminary extraction with alcohol and 3 subsequent precipitations with chilled acetone was indicated by skin reactions in immune rabbits and by specific precipitation and complement fixation with rabbit immune sera. The intradermal inoculation of 100 to 300 μg of endotoxin in 0.1 ml 25% alcoholic solution gave a skin reaction of the delayed type, appearing in 24 to 36 hours and fading appreciably in 48 hours, in immune rabbits but not in normal rabbits. An immediate toxic reaction, attributable only in small part to the alcohol and characterized by a circumscribed erythema followed by superficial necrosis, appeared in 1 to 3 hours and persisted for several days.

The profuse diarrhea, a prominent feature of Asiatic cholera in man, is usually attributed to the action of the endotoxin of the vibrios; whether the pathology of organs such as the kidney is produced by absorbed toxin or is a consequence of the marked dehydration and hypochloremia is not clear. Though in general the pharmacological activity of the bacterial endotoxins is not characteristic, some workers have reported that intravenous inoculation of experimental animals with toxic extracts produces a symptom complex analogous to that of the early stages of human cholera. Hahn and Hirsch, 1 for example, reported that a profuse diarrhea with a loss of 10-17% of the body weight of fluids is produced in young rabbits and Ghosh 2 has made similar observations. Pham 3 has produced a diarrhea accompanied by marked albuminuria leading to emaciation and death in guinea pigs and rabbits; postmortem examination showed hemorrhagic infiltration of the terminal portion of the small intestine, congestion of Peyer's patches and desquamation of the mucosa together with some pathology of the kidney. Less characteristic findings have been reported by Sanarelli 4 and by Basu, Chaudhury, Basu. 5

In a few instances the effect of toxic extracts on isolated tissues, heart and intestine, has been studied. Perfusion of the heart with toxic extracts is reported to produce paralysis 6 and peristalsis of the intestine is said to be stimulated by small doses of toxin and inhibited by large doses. 1 , 6

In our investigation of the pharmacological activity of purified endotoxin isolated from the cholera vibrio, 7 the effect of the toxin on the permeability of living membranes to fluid has been of some interest. Frog skin from the ventral surface and small intestine of the guinea pig and rabbit have been used; other membranes such as rabbit omentum proved too fragile or otherwise unsatisfactory.

Von Euler and Högberg 1 reported that the administration of citric acid produces a delayed decrease in the concentration of pyruvic acid in the blood. The principal evidence was from a small series of experiments with rats receiving oral or subcutaneous dosages of sodium citrate. Emphasis, however, was placed on a single experiment with a normal man whose blood pyruvate was lowered to 22% of the original level 12 to 15 hours after the oral ingestion of 2 g of citric acid as the sodium salt. The blood pyruvate change in this man was stated to be accompanied by indications of a profound general disturbance including sweating, shivering, and fever.

Apart from the suggestion of important theoretical relations this report is of interest because of the growing use of blood pyruvate measurements in the study of thiamine deficiency and the fact that the citric acid dosage employed is scarcely heroic. Two grams of citric acid would be supplied by one large lemon or by 200 cc of orange juice. 2

In this Laboratory this question was studied in 5 experiments on 3 normal young men who had been maintained on a carefully standardized dietary for several months. In each experiment a blood sample was drawn before breakfast at 8:00 a.m. and 3 g of citric acid as Na citrate were administered orally in 200 cc of orange juice on the same day at 6.00 p.m. The final blood sample was taken before breakfast on the following morning at 8:00 a.m., that is, 14 hours after the citric acid administration. The blood samples were drawn from veins in the antecubital fossa, Pyruvate was estimated in duplicate by the method of Friedemann and Haugen. 3

The results are summarized in Table I.

In an earlier study 1 it was observed that in male patients with pernicious anemia the rate of evacuation of the gall bladder was not significantly retarded by the disease, but that in a surprisingly large proportion of the cases studied roentgenologically, the gall bladder could not be visualized, notwithstanding the use of the intravenous method and the absence of any gall bladder history. Accordingly it seemed desirable to examine a comparable number of female patients.

The present report is based upon cholecystographic studies of 48 consecutive, unselected patients with pernicious anemia (23 males and 25 females). In this group, 42% could not be visualized (35% of the males and 48% of the females). Also, a review of the 31, 311 necropsies recorded by the Department of Pathology for the 15-year period between 1926 and 1940, showed that of the 105 individuals having pernicious anemia, 32.4% had had either cholecystitis or cholelithiasis (or both) or had had the gall bladder removed. Furthermore the incidence of gall bladder disease increased progressively with age to the 8th decade—whereas Blalock 2 has shown that the greatest percentage of cases of biliary disease occurs in the fifth decade. These figures suggest that pernicious anemia increases the incidence of gall bladder disease and may have an etiological relation to it.

Evacuation of the gall bladder in 12 female patients (average age, 58 years) showed marked retardation over the group of female controls 3 (average age, 65 years); for in the first 40 minutes after the standard meal, the pernicious anemia group had evacuated only 71.5% of the contents of the gall bladder as against 84% by the controls. The difference is statistically significant, being nearly 3 times the standard error.

1. Negative pressure can be produced by ciliary activity within a normal sinus after a considerable quantity of mucinous secretion (from the same species of animal) has been injected. 2. This negative pressure occurs even after death and decapitation of the animal and is therefore not due to absorption of air by the blood stream.

Mice that had been permitted access to a mixed food before exposure to high tensions of oxygen with a subsequent sudden fall of total pressure to 70 mm were found much more likely to succumb to such treatment than were mice that had been deprived of food for a day. This effect of food was also observed after the administration of glucose, intragastrically or intraperi-toneally, but not after the intragastric administration of olive oil.

Mice not previously treated with high tensions of oxygen were nearly all killed by the sudden exposure to 70 mm pressure, but those that had been deprived of food did not show the marked congestion of the lungs that was observed in mice that had been fed a mixed food. Mice that had received glucose or starch showed this congestion, but those that had received xylose, olive oil, egg albumin, leucine, or sodium chloride did not.

Much of the data presented here cannot be correlated to show a relationship between antibacterial activity and rate of deacylation of the conjugated compounds, and it seems clear that the antibacterial activity of these compounds is not due solely to the liberation of the free sulfonamides. A considerable part of the antibacterial activity of these substances must, therefore, reside in the conjugated molecule. The node of action is complex and cannot be defined at this time.

1. The effect of steroid substances on the synthesis of acetylcholine was investigated. 2. Cholesterol did not modify the synthesis of acetylcholine. 3. Bile salts and vitamin D did not modify the synthesis in low concentrations and depressed it in higher. 4. Hormones of the adrenal cortex, male sex hormones, and corpus luteum hormones, except pregnenolone, depressed the synthesis in low and increasing concentrations. 5. Estrogenic hormones and pregnenolone increased the synthesis of acetylcholine.

1. Hepatic cirrhosis without ceroid has been produced in rats by feeding certain purified diets. 2. The type of fat given to rats in low-choline, low-protein diets appears to have an important influence on the deposition of ceroid. It seems probable that some substance or substances which are present in cod liver oil causes the appearance of this pigment. We have not observed ceroid in rats receiving palmitric, stearic, oleic, linoleic, or linolenic acids. 3. Cirrhosis may be produced on a completely fat-free diet.

In the thyroprived cow with pronounced myxedema no mammary development was obtained by diethylstilbestrol or thyroprotein administration. Negative results were also obtained with the simultaneous administration of these two substances as long as myxedema persisted. Substantial mammary development and lactation resulted from diethylstilbestrol administration following recovery from myxedematous symptoms by thyroprotein therapy. It is suggested that the failure of mammary development in the myxedematous animal is due to some phase of the myxedema and that the response obtained following thyroid therapy is not due to a synergistic action of thyroxine and diethylstilbestrol.

The potassium content of respiratory tract fluid was found to average 7.6 mg per 100 ml in decerebrate cats, and 27 mg per 100 ml in urethanized cats. The results could not be related to serum potassium, the level of which declined slightly during urethane anesthesia; it is suggested that they may have some relation to the reputed value of urethane in bronchial asthma and the sympathomimetic action of potassium.

Serological studies on convalescent specimens from cases of Rocky Mountain spotted fever show the presence of epidemic and murine agglutinins and epidemic neutralizing antibodies although there is no evidence of cross complement fixation. This evidence fits in with the observations of Castaneda and Silva and those of Parker, and indicates that there is a serologic relationship between epidemic typhus fever and Rocky Mountain spotted fever. The data presented in this paper must be considered in evaluating the results obtained with rickettsial agglutination or the neutralizing antibody when these tests are used as diagnostic procedures.

Glass electrodes were constructed for measuring the nasal pH of hamsters, ferrets, and rats in situ. The normal pH was found to be 7.2 at 35°C.

The agglutination test, using standardized, type specific, heated and phenolized antigens, prepared from stock culture strains, representative of the prevalent types of meningococci, offers a simple method for the demonstration of agglutinins in the serums of individuals suffering from meningococcic infection. The test may be useful as an additional diagnostic procedure in cases where other laboratory confirmation is lacking.

Following repeated injections of phenylhydrazine in rabbits, varying degrees of reticulocytosis, up to 100%, occur. With the increasing reticulocytosis the concentration of adenosine triphosphate in the erythrocytes increases from initial concentrations of 10 to 15 mg to 30 mg or more per 100 cc. The mean volume of the cells is greatly increased from around 60 n3 up to 150 ft3, and the corpuscular hemoglobin concentration is decreased from 34 to 22 g per 100 cc of cells.

By means of the method of unstained cell counts both distilled water and urea were found to be more toxic to testicular cells than to lymphocytes and polymorphonuclear leukocytes. Ethyl alcohol was not cytocidal even in relatively high concentrations. The cell membrane of viable thymic, testicular and other cells appeared readily permeable to urea and ethyl alcohol but not to sodium chloride or sucrose. The addition of distilled water or freezing and thawing produced a characteristic gelatinous precipitate only in suspensions of thymic cells but not in suspensions of splenic or testicular cells.

In view of the wide antibiotic activity of penicillin, it is of interest to learn whether it has any effect on the growth of viruses. The present paper deals with experiments designed to gain information on this subject.

Certain representative viruses were used as follows: Vaccinia BH,1 a strain of vaccine virus derived originally from the New York Board of Health Strain and maintained for several years by rabbit testicular passage; Vaccinia CV II, 1 derived from BH by serial passage in Rivers-Li culture medium now of apparently stable virulence; St. Louis Encephalitis, Hubbard strain, obtained from Dr. Margaret E. Smith, and maintained by serial intracerebral mouse passage; Psittacosis 6 BC, obtained through the courtesy of Dr. J. E. Smadel, maintained by serial yolk sac passages; Meningo-pneumonitis virus, M.P., obtained from Dr. T. Francis, Jr., and maintained by mouse lung and chick embryo yolk sac passage; Equine Encephalomyelitis virus, the Eastern strain, secured through the courtesy of Dr. J. E. Beard, was used in its third chick embryo passage.

Penicillin used was prepared by the Reichel and Abbott Laboratories and was part of a supply allocated to the University Hospitals of Cleveland by the Committee on Therapeutics and Other Agents of the National Research Council. Assays were made by the Oxford cup method using spores of Bacillus subtilis for inoculating the solid medium as suggested by Foster and Woodruff. 2 It was found that a much greater yield of spores could be obtained under conditions of partial desiccation of the culture than in the liquid medium originally proposed. The culture was accordingly seeded on the surface of nutrient agar, and incubated for 7 days. It was then harvested and pasteurized and the spore suspension stored at 4°C. If assays were not carried out immediately, the sample was stored at −15°C. Appropriate controls were always included. It was then harvested and pasteurized and the spore suspension stored at 4°C. If assays were not carried out immediately, the sample was stored at −15°C. Appropriate controls were always included.

By the direct intra-allantoic inoculation of unfiltered throat washings influenza A virus was isolated from 5 of 13 apparently normal contacts during an epidemic of influenza A. Significant increases in neutralizing antibodies were demonstrated in the sera of 9 of 13 such contacts by means of the red blood cell agglutination inhibition test.

The discovery by Thomas, Mirick, Ziegler, Curnen, and Horsfallr 1 that sera of patients convalescent from primary atypical pneumonia frequently agglutinate an indifferent streptococcus, labeled No. 344, has provided yet another method for investigating this disease. The fact that the streptococcus agglutinins persist for a long time when stored at 4°C has made it possible to investigate sera from patients with primary atypical pneumonia received at this laboratory since the summer of 1941. Results of the tests, together with certain observations correlating laboratory and clinical findings, form the subject of this report.

Methods and Materials. The method described in the original report was followed without modification. Titers of 10 or more were considered significant.

Sera were collected from students admitted to Cowell Memorial Hospital, University of California, Berkeley, and to the University of California Hospital, San Francisco; from patients admitted to certain west coast military establishments; and from scattered patients in the San Francisco area. Both early and late specimens were available in most cases. Those specimens taken during the first week of illness were considered acute. Convalescent specimens were collected between the twelfth and forty-fifth days.

Clinical Observations. Cases were classified prior to this investigation on the basis of complete clinical records. The first day on which constitutional symptoms were noted was considered the day of onset. Criteria used in making a diagnosis of primary atypical pneumonia were (1) the history and course of the disease, (2) X-ray evidence of pneumonia, (3) a white blood count of less than 12,000 during the first week of illness, and (4) the absence of large numbers of pneumo-cocci in direct sputum examination. Cases in which only small numbers of pneumococci were seen and in which typing was accomplished only after mouse inoculation were not excluded if the course of illness suggested primary atypical pneumonia rather than pneumococcal pneumonia.

Ultracentrifugally sedimented preparations of Rous sarcoma virus contain antigenic groupings similar to that of the similarly sedimentable normal tissue components. 1 It would seem desirable that other newly isolated viruses be routinely examined for their content of groupings common to the tissue from which they have been derived. The demonstration of apparent physical homogeneity and unique particle size is not sufficient to warrant the assumption of alien chemical uniqueness implicit in the designation of “virus” to the isolated body. This is so because it may be expected that a stage in the synthesis of a virus would require the union of the stimulating virus particle and the protein-synthesizing mechanisms of the host. Thus the liberated particle bearing virus activity may also contain considerable portions of the host, the combination possessing new physical properties. In any case, it may be assumed that the orientation of the chemical work on these virus preparations would be affected if it became clear that a considerable portion of the isolated entity was common to a non-infected host. Virus preparations of influenza∗ were used as a model for an immunochemical assay, since so much chemical, physical, and biological data have been published on purified preparations.∗2, 3 , 4 The virus in the chorioallantoic fluid presumably is liberated into the fluid by the cytolysis of infected cells of the allantoic sac.

The chorioallantoic membranes of normal 11- to 13-day-old chick embryos were rinsed frequently in cold saline containing 0.01% merthiolate and were finely suspended in a final volume of 1 cc per membrane. The suspension was centrifuged at 4000 RPM for 1 hour and the resulting supernate at 30,000 RPM for 20 minutes. The pellets were permitted to soak at 4°C in 0.1 M borate buffer at pH 7.8 for 18 hours and emulsified.

As far as we are aware, antiperistalsis of the descending colon of rabbit has never been studied by direct inspection, yet this can be readily accomplished. The rabbit is narcotized by the subcutaneous injection of 150 mg sodium barbital per kilo; after 45 minutes the descending colon is exposed by a cut through the linea alba from the ensiform cartilage to the pubis; a segment of the descending colon is selected and prepared by making two cuts about 9 cm apart; these cuts sever 60% to 80% of the gut, opposite to the mesenteric border; the segment and adjacent portions of the colon are milked clear of scybala. Now by introducing dry, normal scybala either at the oral or aboral cut, peristaltic and antiperistaltic waves may be produced. Sluggish motor activity is readily improved by subcutaneous injection of 0.1 to 0.3 mg of physostigmine sulfate per kilo.

Results. The introduction of a dry scyba-lum 1 to 2 cm into the segment at the oral or aboral end generally produces a peristaltic contraction after a short period of time. If the progress of the scybalum is prevented by a gentle digital compression of the segment, the peristaltic wave of contraction sooner or later relaxes and the formerly relaxed, compressed area contracts, the scybalum now moves antiperistaltically and may be expelled at the oral cut or enter the colon oral to the cut.

The antiperistaltic wave shows the following characteristics:

1. A contraction wave of the circularis 0.5 to 1 cm long, produces a grayish-white bloodless cord, while its surface longitudinal layer appears pink. The length of the progressing contraction remains more or less constant. The contraction wave passes without noticeable pause through the cut to the oral colon.

Evidence has been provided for the assumption that the energy released by the breakdown of phosphocreatine is adequate to account for the electric energy released by the nerve action potential 1 Hence if the primary event responsible for the alterations of the nerve membrane during the passage of the impulse is the release of acetylcholine, as recently suggested, 3 , 4 energy rich phosphate bonds should be used for reversing the process and rebuilding the ester. In accordance with this hypothesis an enzyme, choline acetylase, has been extracted from brain which in presence of adenosine-triphosphate under strictly anaerobic conditions synthesizes acetylcholine in cell free solution. From 1 g fresh rat brain an enzyme solution may be prepared which forms 100-150 μg of acetylcholine (ACh) per hour. Presence of eserine and fluoride is necessary to inhibit the action of choline esterase and adenosinetriphosphatase. The enzyme contains active sulfhydryl groups which may be easily oxidized and are readily inactivated by monoiodoacetic acid or Cu in low concentrations. 5 On dialysis choline acetylase rapidly loses its activity. Addition of the natural l (+) glutamic acid reactivates it partly. 6 With potassium + glutamic acid 50-80% of the original activity may be restored; further addition of cyanide or replacement of glutamic acid by cysteine may reactivate the enzyme nearly completely. 7 , 8 The experiments suggest that the enzyme may require besides active -SH groups and potassium the presence of an amino acid group.

It has now been found that oxidized amino acids, i.e., α-keto acids, are strong inhibitors of choline acetylase. Pyruvic acid, a-keto-glutaric acid, phenyl- and oxyphenyl pyruvic acid have been tested so far. They inhibit choline acetylase in concentrations of 10^-3 to 10^-4 M. Table I gives a few significant data.

A simplified medium has been described which supported the growth of 26 out of 29 strains of pneumococci. All strains required biotin, choline, nicotinic acid, and pantothenic acid for growth, but varied in their response to thiamin, asparagine, adenine, guanine, and uracil. The extent of this variation makes apparent the necessity of considering the growth requirements of each strain in the study of pneumococcal nutrition. In such a study the importance of concentration and interaction of medium constituents, size of inoculum, and the time of measurement of growth cannot be overemphasized.

Daily administration of 0.3 to 0.5 g of thiouracil in Brown Leghorn capons and cockerels brought about changes in plumage similar to those observed following thyroidectomy in this breed. Black pigments were replaced by red, barbulation was reduced, and growth rates retarded. Changes in feather color were first apparent 24 to 28 days following initiation of medication and in capons continued for approximately 18 to 20 days after medication ceased.

In a recent note 7 Juhn reported similar modifications of breast feather pattern in the Brown Leghorn capon following thiouracil administration ad libitum by mixing with food. No record was made of observations on growth rate.