William Burrows

Studies of Prolactin in the Fowl Pituitary. I. Broody Hens Compared With Laying Hens and Males

Conclusions It has been found that single fowl pituitaries implanted over the crop glands of 8- to 10-weeks-old pigeons (age computed from time of conception) will, in many instances, cause a prolactin-like reaction. It is assumed that this reaction is caused by prolactin in the pituitaries of the donating fowls. Pituitaries from broody hens cause a greater reaction of the pigeon crop gland than the pituitaries of laying hens. Pituitaries from males cause a reaction about equal to, or slightly less, than the pituitaries from laying hens. It was noted in a few cases that the pituitaries of hens just becoming broody gave a greater reaction than those of hens nearly over their broody period. More data are being gathered on this point.

Detection of antibody to urease by hemagglutination.

Summary A new method for determination of the antibody to the enzyme urease is described which can be correlated with the precipitin method for antiurease.

The Endotoxin of the Cholera Vibrio : Action on Living Semipermeable Membranes.

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 Peyers 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.

DISCUSSION: THE CHOLERA ENTEROTOXIN AND LOCAL IMMUNITY*

The demonstration by De and coworkers that the ligated loop in the small bowel of the adult rabbit responds to infection with Vibrio cholerae with the intralumenal accumulation of fluid provided an invaluable model for cholera. Their later observation ~ 3 9 that the same reaction is produced by cell-free supernatants of cultures of Vibrio cholerae or lysates of the bacteria emphasized the probable importance of an enterotoxin in the pathogenesis of this disease. These, in turn, led to the quantitative titration of the toxic activity, and the demonstration that the pathogenesis of the experimental infection may be accounted for, apparently entirely, by the activity of toxin produced in vivo.2 It appeared to us highly improbable that the toxin-induced, or accentuated, movement of water and ions from the tissues into the bowel to give a net secretion rests upon a unique basis in cholera, and that different mechanisms are operative in all other diarrheal diseases of infectious etiology. Substantially concurrent studies with enteropathogenic coliforms, beginning with the work of De in India and of Taylor lo, z1 in England with coliform strains of human origin, and by others in this country, Canada, and England 7 * 12* 13* 16-18 with strains isolated from swine were, therefore, of particular interest. Dr. Taylor’s *O observation that chloroform-killed bacteria also produced the reaction suggested that the water and ion movement into the lumen of the bowel here too resulted from the action of a toxin, and this has been amply confirmed by others using cell-free preparations of coliforms from porcine and human sources. Studies on the cholera enterotoxin have shown that it behaves as if it were a part of a dissociable complex, and it has been possible to separate, by column chromatography and other methods, a toxic moiety and a specific immunogenic protein antigen eliciting the formation of toxin-neutralizing antibody.1° While in these studies emphasis was placed on the preparation of the nontoxic antigen with reference to its possible use as an immunizing agent against the disease, in the present context the occurrence of a separable toxic moiety is of greater interest. The possibility that the formation of such a toxic element may be common to more than one kind of enteropathogenic bacteria, perhaps occumng in different kinds of complexes, is obvious. 11* 14*

Protoplast Formation as the Mechanism for Immune Lysis of Vibrio cholerae.

Summary It is shown that cholera vibrio is converted to a protoplast-like, osmotically fragile form in the presence of specific antibody and complement. When osmotic protection is provided by 0.5 m lactose in the reaction mixture, the bacteria are protected from the vibriocidal effect of antibody and complement. Protection is conferred during the course of the reaction as indicated by cessation of killing when lactose is added; and disappears with onset of the vibriocidal reaction when osmotic protection is removed by dilution of the lactose-containing reaction mixture. In view of the apparent intimate relationship between the vibriocidal reaction and osmotic fragility, and the substantial identity of the rates of killing and protoplast formation, it is suggested that the primary consequence of the antigen-antibody-complement union is the formation of osmotically fragile, protoplast-like forms to give, in the absence of osmotic protection, the vibriocidal and vibriolytic reactions as secondary phenomena.

The Endotoxin of the Cholera Vibrio : Isolation and Properties.

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.

Studies on the growth of chilomonas paramecium in inorganic and acetate solutions

SummaryCultures ofChilomonas paramecium in sodium acetate and inorganic salt solutions were examined for contaminating bacteria by cultural and microscopic methods. No bacteria could be found. Qualitative tests for nitrite and nitrate and quantitative estimation of the former indicated thatChilomonas cannot oxidize ammonia to nitrite or nitrate.

Nature of the Substance Causing Staphylococcus Food Poisoning

The difficulties experienced in studying staphylococcus food poisoning when the only available subject for experimentation was the human volunteer have been in part overcome by the observation that monkeys can be fed staphylococcus filtrates in such a way as to insure a certain proportion of definite and characteristic gastrointestinal reactions. 1 Utilizing monkey feeding as a crude method of determining the presence or absence of the significant principle, some data bearing on the nature of this principle have been obtained. In all the tests here reported only animals showing actual vomiting have been set down as yielding a positive reaction; other symptoms such as pallor, diarrhea, and evident distress have been noted but not recorded as indicating an unmistakable positive result. Not all monkeys react to the active principle inducing vomiting and consequently, before using an animal for experiment we have fed it with a filtrate of known potency. Negative results were controlled by subsequent feeding with potent filtrate. The possible development of tolerance has also been controlled. The following results have been obtained: 1. The active principle will not distill. 2. It is not readily dialyzable. 3. It is markedly unstable to N/100 NaOH. 4. It is unstable to heat in N/100 HCl solution. 5. It is not identical with the hemolytic substance present in many staphylococcus filtrates nor does it produce a skin reaction. 6. It is completely removed from acid aqueous solution by extraction with ethyl ether or chloroform as judged by our method of assay. 7. It may be extracted from alkaline solution with ethyl ether or chloroform but the deleterious effect of alkali tends to mask such removal. Feeding with a neutral saline or aqueous solution of the ether or chloroform extracts from which all traces of the solvent had been removed by warming in vacuo caused typical vomiting in monkeys.

Oxidation-Reduction Potentials of Some Non-Sporulating Obligate Anaerobes

Oxidation-reduction potential studies have been made on cultures of many aerobic and some sporulating anaerobic bacteria, but to our knowledge none has been made on the non-sporulating obligate anaerobes. The potentials produced by the latter group of organisms are of particular interest in connection with current theories as to the relation of such potentials to the growth of obligate anaerobes. A certain degree of negativity was thought to be necessary before the obligate anaerobes are able to initiate their growth processes. Experimental evidence of a positive limit of oxidation-reduction potential required for the germination of spores of Cl. tetani has been presented by Fildes 1 and by Knight and Fildes. 2 This positive limit was found to lie in the vicinity of Eh O at pHs close to neutrality. The potentials produced in cultures of the sporulating anaerobes are considerably more negative than this positive limit, usually being about 350 millivolts negative at reactions at or near neutrality. 3 In unpublished experiments one of us (W.B.) has found that several strains of Cl. botulinum produce potentials around 400 millivolts negative. That anaerobiosis is a matter of oxidation-reduction potential rather than simple absence of oxygen is a theory that has been generally accepted, 4 although there has been some dissent. 5 The technique used in the present experiment has been previously described. 6 The organisms were grown in a cystine glucose beef infusion broth, the pH varying between 6.4 and 6.9. Anaerobic conditions were produced and maintained by constant bubbling of oxygen-free nitrogen through the medium. Very heavy inoculums were used, usually 1 to 2 cc. of young cultures. Under these conditions we had no difficulty in growing the organisms. With 2 exceptions, the strains used were isolated from the colons of patients suffering with ulcerative colitis. Strain 39 was isolated from the ulcerated colon of a monkey dying from bacillary dysentery (Flexner) and strain M1 was isolated from the colon of a healthy monkey.

The Endotoxin of the Cholera Vibrio : Immunological Properties.

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.