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As commonly used the phrase ‘early influences’ denotes the conditioning of behavior by the experiences of very early life. Early experiences however do more than conditioning behavioural patterns; they also affect profoundly and lastingly many biological characteristics of the adult. I shall show that in animals events occurring during the very first days of life determine the initial growth rate the maximum adult size the efficiency in utilization of food and the resistance to infection malnutrition and other stressful stimuli. Early influences are of course at least [as] important in human life as they are in animal life. In fact the experiments to be reported here were designed to provide experimental models for the study of socio-medical problems first recognized in human populations. (excerpt)

Tubercle bacilli, growing diffusely in liquid media containing a water dispersible ester of oleic acid (Tween 80), retain unaltered many of their morphological and biological properties.1,2 The present paper describes the virulence of these cultures for mice of different genetic backgrounds inoculated under various experimental conditions. The cultures used in the infection tests to be reported were grown for 7-10 days in a medium containing 0.05% Tween 80 and 0.2% bovine albumin (serum Fraction V).2 Macroscopically, these cultures appear homogeneous, but in reality consist of microscopical clumps. Their density corresponds to approximately 0.20 mg/cc in terms of dry weight of bacilli.Mice, 3 to 6 weeks old, of the Rockefeller Institute strain inoculated intravenously with 0.01 cc of whole culture begin to lose weight during the second week after infection and die in 3 to 4 weeks with a disease primarily pulmonary. The pulmonary lesions consist of discrete and confluent nodules varying in size, pearly gra...

We have shown elsewhere that certain water soluble lipids promote diffuse growth of tubercle bacilli in synthetic media, especially in the presence of serum albumin.1-2 These same substances have now been found to enhance the growth of other microbial species, in particular of an unidentified micrococcus (strain C), recently isolated in our laboratory. Some understanding has also been gained of the conditions under which long chain fatty acids can stimulate bacterial growth.It is known that the soaps of fatty acids exert a bacteriostatic and bactericidal effect on certain micro-organisms, particularly on the Gram-positive and acid-fast species, and that unsaturated acids are more toxic than the corresponding saturated compounds.3-9 For example, concentrations of oleic acid as low as 0.000001-0.00001% are sufficient to cause inhibition or retardation of growth of small inocula of human tubercle bacilli in synthetic liquid media. On the other hand, fatty acid esters (methyl oleate, triethan-olamine oleate, ...

NCS mice gained weight rapidly when fed a gluten diet deficient in several amino acids, but their weight gain on the same regimen was very much retarded if they were given antibacterial drugs, even for a short period of time. This retardation of growth could not be entirely corrected by supplementing the gluten diet with lysine and threonine. The decrease in growth rate brought about by antibacterial drugs could probably be traced to the alteration in intestinal flora resulting from drug treatment. The intensity and duration of both types of changes were related to the dose of drug administered, and to the length of the treatment period. Whatever the nutritional regimen, treatment with penicillin caused a retardation of weight gain in NCS mice. The retardation was more pronounced, and longer lasting, when the animals were fed semisynthetic regimens (containing casein or gluten) than when they were fed crude diets (pellets) containing natural materials of ill defined composition. These differences probably had their origin in the fact that the changes in fecal flora induced by the drugs were profoundly influenced by the composition of the diet. Antibacterial drugs which retarded weight gain of Swiss NCS mice, in contrast increased weight gain in ordinary Swiss mice raised under usual conditions. It is probable that this difference in response to the antibacterial drugs resulted from the fact that ordinary Swiss mice have a much more complex intestinal flora than NCS animals.

There are present in commercial peptones substances which exhibit bacteriostatic properties for certain bacterial species. These substances are bacteriostatic in the oxidized form, but not in the reduced form. Their bacteriostatic action can be overcome, and their concentration titrated, by the addition of reduced thiol compounds to the media in which they are present. Different brands of peptone differ greatly in the amount of bacteriostatic substances they contain; these differences account, in part at least, for the fact that media prepared from the same meat infusion, but with different kinds of peptone, vary in their ability to support bacterial growth. The bacteriostatic fraction of a certain peptone solution can be completely removed by precipitation with acid and acetone. A peptone which has thus been purified becomes capable of supporting the growth of very small inocula of Pneumococcus. The significance of the sensitiveness of certain bacterial species to substances which are bacteriostatic in the oxidized but not in the reduced form is considered with reference to (a) the mechanism of bacteriostasis, (b) the growth of bacterial species in artificial media, (c) the problem of infection.

Serum albumin is a protective bacterial growth factor; by binding traces of fatty acid in the media it permits initiation of growth by the smallest possible inocula of tubercle bacilli. Each molecule of albumin binds 3 to 6 molecules of oleic acid (1 to 2 per cent of the weight of the albumin) tightly enough to prevent bacteriostasis, and 9 molecules of oleic acid in equilibrium with a saturated neutral solution. The property requires undenatured albumin. Crystalline ß-lactoglobulin has a smaller capacity, and a number of other proteins no perceptible capacity to bind oleic acid. The inhibitory effect of the commercial product Tween 80 (polyoxyethylene sorbitan monooleate) on the growth of small inocula of tubercle bacilli in liquid media is caused by its content of unesterified oleic acid (0.6 per cent by weight). Purified Tween 80, freed of this contaminating fatty acid, not only permits growth of small inocula, but protects against small amounts of added oleic acid. The implications of the binding capacity of albumin for its possible physiological significance in the animal body (transport; protection against cytotoxins), and for the structure of the protein, are briefly discussed.

The bacterial enzyme which decomposes the purified capsular polysaccharide of Type III Pneumococcus in vitro also destroys the capsules of the living organisms growing in media and in the animal body. Potent preparations of this same enzyme protect mice against infection with virulent Type III Pneumococcus. The protective action is type-specific. The protective activity of the specific enzyme is destroyed by heat (70°C. for 10 minutes). The enzyme remains in an effective concentration 24 to 48 hours after its injection into normal mice. The enzyme has been found to exert a favorable influence on the outcome of an infection already established at the time of treatment. A definite relationship has been found to exist between the activity of the enzyme in vitro and its protective power in the animal body. The mechanism of the protective action is discussed with special reference to the relation between the decapsulation of the bacteria by the enzyme and the phagocytic response of the host.

Several species of aerobic sporulating bacilli recently isolated from soil, sewage, manure, and cheese, as well as authentic strains obtained from type culture collections, have been found to exhibit antagonistic activity against unrelated microorganisms. Cultures of these aerobic sporulating bacilli yield an alcohol-soluble, water-insoluble fraction,—tyrothricin,—which is bactericidal for most Gram-positive and Gram-negative microbial species. Two different crystalline products have been separated from tyrothricin. One, which may be called tyrocidine, is bactericidal in vitro for both Gram-positive and Gram-negative species; the other substance, gramicidin, is effective only against Gram-positive microorganisms. In general, tyrocidine behaves like a protoplasmic poison and like other antiseptics, loses much of its activity in the presence of animal tissues. Gramicidin on the contrary exerts a much more subtle physiological effect on the susceptible bacterial cells and, when applied locally at the site of the infection, retains in vivo a striking activity against Gram-positive microorganisms.

1. The enzyme which decomposes the capsular polysaccharide of Type III Pneumococcus is associated with a protein which under optimal conditions of filtration passes through membranes with an average pore size of 10.6 mµ but is held back by pores having a diameter of 8.2 mµ. 2. When enzyme solutions are filtered to dryness through membranes of such porosity as to hold back the active principle, and when proper precautions are taken to prevent or minimize adsorption, the enzyme can be completely recovered in solution by immersing the membrane in distilled water or physiological salt solution. 3. These results are discussed with reference to the dimensions of the enzyme particle, and to the purification obtained in the course of ultrafiltration. 4. A practical method is described for the concentration and purification of the crude enzyme preparation by the use of graded collodion membranes.

The action of the enzyme which specifically decomposes the capsular polysaccharide of Type III Pneumococcus has been tested in Type III pneumococcus dermal infections in rabbits. When injected in sufficient amounts, this enzyme is capable of bringing about a favorable and early termination of the experimental disease which ordinarily is fatal in nearly all instances. The results of the present study yield further evidence that the capsular substance is of great importance in pneumococcus infection, since, in so far as known, the only action of which the specific enzyme is capable is that of decomposing the capsular polysaccharide.

A study was made of the effect of certain dietary regimens on the lactobacillus flora in the stools of mice and on their resistance to infection. Semi-synthetic diets with purified casein or wheat gluten as sole source of protein, gave rise to much smaller numbers of viable lactobacilli in the stools than did other diets containing unidentified natural products—as present for example in mixtures of whole wheat and whole milk, or in certain commercial pellets. Furthermore, one of the lactobacillus types with rhizoid morphology disappeared completely from the stools of animals fed the semi-synthetic diet. The change in the lactobacillus flora became apparent within a very few days after the animals had been shifted from the complex to the synthetic diet Moreover, this change was not completely reversible. Whereas the total numbers of lactobacilli increased when the animals were shifted back from the synthetic to the complex diets, the rhizoid lactobacilli which had disappeared completely from the stools reappeared only slowly or not at all. In twelve consecutive experiments the three diets which gave rise to the large numbers of lactobacilli in the stools also conferred on the mice a much higher resistance to experimental infection with Staphylococcus aureus and Klebsiella pneumoniae, than did the semi-synthetic diets. However, direct evidence has not yet been obtained that the two kinds of phenomena were causally related. Following administration of endotoxin there was a rapid and very large increase in the numbers of enterococci and coliform bacilli in mice fed the semi-synthetic casein diet, but not in those fed the pellets. In two preliminary experiments carried out with another colony of mice, not pathogen-free, it was also found that the rhizoid type of lactobacilli disappeared from animals fed the semi-synthetic casein diet while enterococci and coliform bacilli progressively increased in numbers under the same conditions. The dietary effects on the lactobacillus flora, and on resistance to experimental infection, were equally pronounced whether the mice were housed in individual cages on wire grids, or grouped in larger cages with wood shavings as litter. This was true even if the bedding was changed only once weekly and became therefore grossly soiled.

Groups of young albino mice were fed continuously four different types of diets and were compared with regard to (1) rate of weight gain; (2) resistance to experimental bacterial infections. The protein content of the four diets was as follows: (a) pellets: a minimum of 21 per cent "crude" protein (according to the manufacturer); (b) diet 20 C: 20 per cent casein; (c) diet 8 C: 8 per cent casein; (d) diet 8 C + AA: 8 per cent casein supplemented with 12 per cent of a mixture of essential amino acids. All diets provided an adequate supply of minerals and vitamins. They were administered ad lib. Three strains of pathogens virulent for mice were used for the infection tests, namely: Staphylococcus aureus, Mycobacterium fortuitum, and Mycobacterium tuberculosis bovis. The bacteria were injected by the intravenous route. The experimental regimens were begun at different times before infection, and were continued until death of the animal, or until termination of the experiment. It was found that mice on the 8 C diet exhibited much greater susceptibility to infection than did mice on the 20 C diet; mice receiving pellets were intermediate between these two groups. The infection-enhancing effect of the 8 C diet could be entirely corrected by amino acid supplementation (diet 8 C + AA). Indeed, mice fed diet 8 C + AA proved the most resistant to infection. The fact that animals fed pellets (which contain a minimum of 21 per cent protein) consistently died faster following infection than did animals fed diets 20 C or 8 C + AA suggests that qualitative characteristics of the protein in the regimen are as important as the quantity of protein fed in determining susceptibility to infection. The differences in susceptibility exhibited by the mice on the four experimental diets were the same whatever the species of bacterial pathogen used for the infection test, the size of the infective dose, and the duration of the disease. There was no apparent relation between the effects of the diets on the weight curves of the animals, and on resistance to infection. Mice on diet 8 C (which were most susceptible) gained weight as rapidly as those on 20 C and more rapidly than those fed 8 C + AA (which were most resistant). All the tests reported in the present paper were carried out with young mice, which were placed on experimental diets within 1 to 2 weeks after weaning. Preliminary experiments suggest that the relation between dietary factors and susceptibility to infection was more difficult to bring out in older animals. There was evidence also that this relation was most apparent during the first weeks that the animals were fed the experimental diets, and became less striking after several weeks.

Addition of penicillin, Terramycin, or kanamycin to the drinking water of adult mice rapidly induced in them an enlargement of the cecum. In all animals, this occurred within 12 hr after the beginning of drug administration—the effect being most pronounced with penicillin. The cecums remained enlarged and generally continued to increase in size as long as the antibacterial drugs were administered. The increase in wet weight of the cecums was due primarily to an accumulation of water in the lumens during the first 24–48 hr of drug administration. At that time, there were no detectable histological changes in any case, but the bacteriological picture differed from drug to drug. The cecums were free of bacteria in animals receiving penicillin, fusiform-shaped bacteria and bacteroides were present in those receiving Terramycin, and lactobacilli and bacteroides in those receiving kanamycin. After the initial 48 hr, an abundant and complex secondary microflora developed in all treated animals, its composition being characteristic for each type of antibacterial drug. When penicillin was administered for 2 wk, the cecal weights and microbial populations did not return to normal levels for over 14 days after discontinuance of the drug. This recovery period could be shortened to 10 days by giving the mice food contaminated with cecal homogenates prepared from normal animals. A period of 7 or 8 days was required for the cecal weights and microflora to reach normal levels when the administration of penicillin lasted only 24 hr; this period could not be shortened by giving the animals contaminated food. The effects of drugs on the size and bacterial contents of the cecum have been discussed in the light of earlier findings concerning the characteristics of the huge cecums uniformly found in germfree mice. Taken together, these observations support the hypothesis that certain elements of the intestinal microflora—not yet completely identified—play an essential role in maintaining the integrity of the water-transport mechanism in the intestinal epithelium.

1. Living pneumococcus cells contain a group of enzymes, the bacteriolytic system, capable of causing the lysis of heat-killed pneumococci (R and S variants irrespective of type derivation). This lysis expresses itself by a loss of the Gram staining reaction, a disintegration of the cell body, and a clearing of the bacterial suspension. 2. Under certain conditions of treatment with the bacteriolytic complex, it is possible to render the cocci Gram-negative without changing their characteristic morphology, or causing any appreciable clearing of the cell suspension. 3. The enzyme responsible for this change has been partially purified, and some of its properties described. 4. The cellular structure which is responsible for the Gram-positive reaction of pneumococci is resistant to proteolytic enzymes, and is still present when tryptic digestion has reduced the heat-killed cell to a body which has lost 75 per cent of its original weight, and contains only 8 per cent nitrogen. 5. The same enzyme preparation which attacks pneumococci is also capable of liberating reducing sugars from some acetyl amino glucose glucuronides of animal and bacterial origin. The possibility is considered, and discussed, that one and the same enzyme in the autolytic complex is capable of attacking both types of substrates.

1. The conditions under which a certain strain of staphylococcus (OH 172) causes in rabbits the development of bone inflammation have been described. 2. The virulence of the strain for rabbits was markedly raised by passage through this animal species, and especially after the culture had been recovered from a bone abscess. 3. The results indicate that it is possible to produce consistently inflammation of the bones of rabbits by the mere intravenous injection of a suitable strain of staphylococcus, without resorting to any elaborate operative technique designed to localize the organisms in the bones. It appears also that the inflammatory process so produced bears a close resemblance to staphylococcal osteomyelitis as occurring in human beings.

The legend of the Golden Age is so universal and ancient that it must have a base of truth. From the beginning of recorded time, men have be lieved in some form of Arcadia, a happy land where their ancestors led an Orphic life devoted to the enjoyment of natural pleasures and of ex ternal nature. In Works and Days, written almost 3,000 years ago, Hesiod evokes the Golden Age when "men feasted gaily, undarkened by sufferings ... and all good things were theirs." The name Arcadia might symbolize the pastoral scenery and semi tropical climate of the East African plateaux, where the human species probably emerged under environmental conditions that most people still find enjoyable. Arcadia could have been located also in one of many other regions that were occupied by man during prehistory and where abundance of game, nuts, and fruit assured him a life of plenty without much effort. As recently reported in the symposium devoted to "Man the Hunt er/'* there still exist a few archaic populations that derive adequate sus tenance by hunting small game and collecting wild plants. The bush

The immunity induced in mice by vaccination with living attenuated cultures of tubercle bacilli was measured by two criteria. (a) Increase in survival time of the vaccinated animals after infection with a dose of virulent bacilli sufficient to kill all the unvaccinated controls within 10 to 20 days. (b) Difference in the number of living bacilli recovered from the spleen and lungs of vaccinated and normal animals infected with a small dose of virulent bacilli. The level of immunity induced was found to depend upon the extent of multiplication in vivo of the bacilli used for vaccination. This in turn was conditioned by the degree of attenuation characteristic of the bacterial strain used in the preparation of the vaccine, the amount of vaccine injected, the route of vaccination, and the time interval between vaccination and challenge infection. It was possible to prevent or retard the development of immunity by treating the mice in course of immunization with a drug, isoniazid, capable of interrupting the multiplication in vivo of the bacilli used as vaccine. Although immunity regularly developed and lasted for many weeks when the proper conditions of vaccination were used, the immune response was never sufficient to protect the animals against ultimate death from infection with virulent tubercle bacilli. The prolongation of life in the vaccinated mice was not consequent on a direct bactericidal effect but rather on a retarded or interrupted multiplication of the virulent bacilli in vivo. The quantitative bacteriological techniques used in the present study would appear to be of value for the analysis of certain problems of immunity, and for the appraisal of vaccines and techniques of vaccination.

1. Shigella dysenteriae (Shiga) can, under the proper cultural conditions, produce a soluble toxin which is independent of the specific somatic polysaccharide antigen. A method is described for the rapid production of this toxin by an avirulent R variant of this organism. 2. The amount of bacterial protoplasm synthesized, and the yield of toxin produced, are very much increased when the culture is grown under conditions which favor aerobic metabolism and which do not permit accumulation of organic acids. 3. Although addition of an excess of inorganic iron to the medium does not interfere with the synthesis of bacterial protoplasm, maximal toxin production is obtained only in media which have been freed of the metal. 4. R Shiga bacilli grown under conditions of extreme aerobiosis at pH 7.0 in a simple medium free of inorganic iron and containing fumaric acid, glucose, meat extract, and peptone, give rise within 24 to 36 hours to cultures exhibiting a high degree of toxicity. The toxic principle rapidly becomes soluble as the cells die; it can be concentrated, purified, and freed of phosphorus by selective precipitation methods. 5. The toxin is fairly resistant to heat at pH 6.0 but rapidly denatured at pH 9.0. It is only slowly inactivated by trypsin. It filters readily and without loss of activity through anionic exchange resins but is precipitated and adsorbed by cationic resins which retain the nitrogen and toxic activity of the preparation. 6. The most active preparations of toxin available possess an LD50 of 1 to 10 µg. for mice and rabbits. Young mice (up to 5 weeks of age) are more resistant than older animals. 7. The soluble toxin can be detoxified by treatment with 0.5 per cent formalin at pH 8.5; detoxification does not take place at lower pH whereas treatment at higher pH destroys specific antigenicity. Mice immunized with Shiga toxoid (in solution, or in the form of an alum precipitate) develop active immunity to the toxin.

Summary1) Female albino mice of two different breeds were treated intraperitoneally with a mixture of bovine serum and pertussis vaccine. In confirmation of findings of other investigators, it was found that a large percentage of the animals so sensitized died within 30-60 minutes following the intravenous injection of very small amounts of serum 2-4 weeks after sensitization. The sensitizing effect appeared specific as no deaths occurred when either rabbit serum or egg albumin was used for the challenge injection. 2) Mice pretreated by intraperitoneal injection of bovine serum and pertussis vaccine, or of pertussis vaccine alone, were infected intravenously several weeks later with staphylococci resuspended in 1/2000 bovine serum. When they were sacrificed 24 hours after infection, it was found that many more staphylococcal colonies could be recovered from the kidneys, liver and lungs of the animals that had been sensitized to bovine serum, than from the organs of animals of the control group.

Albino mice lose weight within 24 hours following administration of bacterial endotoxin. The initial weight loss is proportional to the dose of endotoxin injected only when this dose is very small. The loss during the 1st day reaches a maximum with 10 to 30 µg of endotoxin; larger doses increase the duration of the overall effect. The rate at which mice regain weight after administration of endotoxin is markedly influenced by the composition of the diet. Recovery was rapid and complete within a few days when the animals were fed commercial pellets or a semisynthetic diet containing casein. In contrast, recovery was slow and incomplete when wheat gluten was used instead of casein in the diet. The deleterious effect of the gluten diet was less marked in older than in younger animals, probably because the latter have less exacting nutritional requirements. It was postulated that the failure of endotoxin-treated mice to regain weight when fed the gluten diet was due to the fact that this protein is low in certain amino acids. In fact, rapid and complete recovery from the weight loss uniformly occurred when the gluten diet was supplemented with proper amounts of lysine and threonine. The composition of the diet did not influence the extent of the initial loss of weight caused by endotoxin, nor did it prevent the animals from developing tolerance to this substance.

Zuchtet man Tuberkelbazillen nach den bis heute ublichen Methoden, so begegnet man immer wieder gewissen Schwierigkeiten, die sich nicht leicht beheben lassen: Die Bakterien wachsen nur sehr langsam; sie bilden Klumpen oder kompakte, an der Oberflache der Kultur schwimmende Haute, die aus einem uneinheitlichen Gemisch verschieden alter, lebender und toter Bakterien bestehen; sie lassen sich nicht gut homogen in einer Aufschwemmung verteilen. Auserdem kann man nur sehr grose Inocula mit Erfolg verimpfen, da kleinere Bakterienmengen in der Regel nicht angehen.

Latent corynebactenai infection occurs naturally in many strains of mice. It can be evoked into the active disease, pseudotuberculosis, by a single injection of 10 mg of cortisone. The cortisone effect was tested in 21 colonies, representing 11 genetically different strains of mice. Animals of the C57B1/6, DBA/2, and RIII strains were shown to be latently infected with Corynebacterium kutscheri by the fact that they developed fatal pseudotuberculosis following cortisone treatment. Virulent C. kutscheri could not be isolated from homogenates of organs obtained from latently infected animals before cortisone administration; however, these homogenates yielded small translucent colonies of avirulent organisms. Recovery of these atypical colonies was facilitated by preincubating the organ homogenates before plating. The organisms constituting such colonies differed morphologically and immunologically from C. kutscheri, but had similar biochemical properties with the exception that they lacked urease and catalase activity. Mice treated with cortisone yielded both the avirulent bacteria and virulent C. kutscheri. The latter was the predominant organism present in the organs at the height of infection. Injection of avirulent organisms into Swiss Lynch mice, which are normally free of latent corynebacteria, occasionally established a latent infection which could be converted into corynebacterial pseudotuberculosis by cortisone. Cultures of fully virulent C. kutscheri were then obtained from the lesions. Latency was produced experimentally with a streptomycin-resistant strain of virulent C. kutscheri (CKsr) derived from the stock culture. When sublethal doses of CKsr were injected into NCS mice (Institut Pasteur colony), they induced a latent infection characterized by the presence of avirulent organisms possessing the streptomycin resistance marker. These were isolated in the form of small translucent colonies from the livers of the infected animals. Administration of cortisone to these animals subsequently evoked active infection from which virulent CKsr could be obtained. Injection of the avirulent streptomycin-resistant organisms into normal NCS mice established a latent infection which could be uniformly converted into corynebacterial pseudotuberculosis by cortisone. The virulent C. kutscheri obtained from the lesions bore the genetic marker of streptomycin resistance, thus being identical with CKsr. Except for streptomycin resistance, the avirulent organisms isolated from the experimentally induced latent infections were identical with those found in the naturally occurring latent infections. These results suggest that C. kutscheri can persist in vitro in an avirulent form which is resistant to the defense mechanisms of the host, and can thus establish a latent infection. Treatment of the animal with cortisone results in the conversion of the avirulent form into virulent C. kutscheri, and of the latent infection into active corynebacterial pseudotuberculosis. The findings are discussed with regard to their relevance to infection immunity, and to the conversion of latent infection into overt disease.

Treatment of elementary bodies of vaccinia with dilute solutions of sodium hydroxide resulted in the extraction of certain soluble materials accounting for half of the dry weight of the virus. Elementary bodies contained about 0.4 per cent inorganic phosphorus, practically all of which occurred in the form of a nucleoprotein containing thymus nucleic acid. In addition, a substance was recovered that reacted with S antibodies. From past experience one is led to believe that S antigen, as ordinarily encountered, is a protein which is not in combination with nucleic acid.

It proved possible to increase the resistance of mice to tuberculous infection by vaccinating them with a suspension of avirulent tubercle bacilli killed by exposure to 2 per cent phenol. This increase in resistance was demonstrated by two different techniques: (a) observation of survival time of vaccinated animals following challenge infection with a large dose of virulent bacilli, and (b) determination of numbers of virulent bacilli in the spleens of animals 2 weeks after injection of a small infective dose. The minimum protective dose of vaccine corresponded to approximately one-tenth the acutely toxic dose. Addition of an adjuvant to the bacillary suspension markedly increased both the protective effectiveness of the vaccine and the duration of the immunity. It enhanced also the toxicity of the vaccine in approximately the same proportion. However, other lines of evidence suggested that toxicity and protective activity were independent one from the other and were the manifestations of different bacillary constituents. Extraction with absolute methanol released from the bacillary bodies a crude soluble fraction possessing low, if any, toxicity, yet capable of eliciting in mice a state of increased resistance to virulent infection. The protective activity of this methanol-soluble fraction was low; it accounted for only a small part of the total protective activity of the original material.

Mice were given daily per os amounts of dinitrophenol or of thyroid extract sufficient to prevent or retard the normal weight gain of uninfected animals, but not large enough to cause their death. When mice maintained on these regimens for 1 or 2 weeks were infected with staphylococci, most of them died within 12 days—much more rapidly than did mice fed a normal diet. Deaths occurred even when the organism injected was a non-virulent staphylococcus, unable to cause fatal disease in mice fed a normal diet. There was some suggestion that thyroid treatment interfered with the bactericidal mechanism in the liver, spleen, and kidneys of mice during the initial phase of infection. In contrast there was no clear evidence at any time thereafter that either thyroid extract or dinitrophenol caused the staphylococci to multiply more rapidly in the various organs.

When used in low concentration, formaldehyde increases the rate of autolytic disintegration of pneumococci whereas in large concentrations it completely inhibits autolysis and preserves both the morphological and staining characteristics of the cells. Pneumococci treated with large concentrations of formaldehyde, then washed free of the antiseptic and resuspended in physiological solutions, rapidly undergo a change which renders them Gram-negative and smaller. The lysis is only partial, however, and is not accompanied by an actual disintegration of the cell. It is caused by the autolytic enzyme of the cell which remains inactive in the presence of an excess of formaldehyde but recovers its activity when the cells are resuspended in a neutral medium after removal of the antiseptic. If the autolytic enzyme is irreversibly inactivated by heating, or maintained inactive in acid or alkaline reaction, the formolized cells retain their staining characteristics and morphological integrity. Formolized pneumococci which have become Gram-negative owing to the action of their autolytic enzyme, fail to elicit the type specific carbohydrate antibodies in rabbits. Formolized pneumococci in which the autolytic enzyme has been destroyed or maintained inactive, and which have retained their Gram-positive character, function as a very effective type specific antigen in the rabbit. These observations emphasize once more the close relation between the Gram-positive structure of pneumococci and the capsular polysaccharide antigen of the cell. They can be used as a basis for the preparation of suspensions of formolized pneumococci which are stable and very effective as type specific antigens.

Injection of endotoxin of Gram-negative bacilli into NCS mice caused an immediate reduction or interruption of water intake by these animals, with a resultant loss of body weight. Endotoxins prepared by three different techniques from four different cultures of Gram-negative bacilli yielded products having approximately the same activity in inhibiting water intake. The minimum effective dose was 0.1 µg. or less. With all toxin preparations tested, the duration of the effect was directly related to the dose injected. The heat-killed cells of Esch. coli proved approximately as effective as the endotoxins prepared from Gram-negative bacilli. In contrast, heat-killed cells of Mycobacterium tuberculosis (BCG) were much less active, and heat-killed cells of Staphylococcus aureus were essentially inactive. Mice previously treated with endotoxin exhibited a marked degree of tolerance to the inhibition of water intake caused in normal animals by a subsequent treatment with the same material. Tolerance could also be induced by vaccination with heat-killed Gram-negative bacilli. Tolerance overlapped from one bacterial species to another but was more pronounced toward the endotoxin prepared from the bacterial culture with which the animal was vaccinated. The duration of the inhibitory effect of endotoxin on water intake was much shorter with mice fed a complete diet than with mice fed a deficient diet (corn). It took approximately the same dose of endotoxin (0.1 µg.) to inhibit water intake, reduce the influx of polymorphonuclear leucocytes, and enhance staphylococcal infection.

Groups of guinea pigs were vaccinated by the intracutaneous route with cultures of BCG grown in a liquid medium containing Tween 80 and the soluble fraction of heated human serum. After the cultures had been stored at 4°C. for various periods of time, the antigenic response was compared with that of another group of guinea pigs receiving standard BCG vaccine prepared by the conventional technique. The local lesions occurring at the site of injection of cultures in Tween-serum filtrate medium were more severe than those produced by the standard vaccine. It was shown that this difference was probably due to the much larger number of viable bacilli in the former preparations. A marked degree of sensitization could be produced with culture dilutions containing as few as 10 viable units (single bacilli or small clumps). Slightly larger doses of BCG led to the highest degree of tuberculin allergy detectable by the technique employed. Further increases in the dose of vaccine failed to alter the level of sensibility when the animals were tested with tuberculin 5 weeks after vaccination. The same degree of sensitization was achieved by vaccination with 0.1 cc. of either the standard vaccine or any of the fresh or stored cultures in Tween-serum filtrate medium. It was shown that these doses contained numbers of living bacilli far greater than the minimal number required to induce maximal sensitization. Under the conditions used, the guinea pigs vaccinated with cultures of BCG (fresh or stored) grown in the Tween-serum filtrate medium exhibited a marked degree of resistance to subcutaneous infection with virulent tubercle bacilli.

In the first paper of this series, a description was given of a cell-free extract, obtained from autolysates of a particular strain of a soil bacillus, which selectively inhibits the growth of all the Gram-positive microorganisms so far tested, and exerts on them a bactericidal effect in vitro. In the present study it is shown that the same agent protects white mice against infection with large numbers of virulent pneumococci. It also exerts a curative effect when administered to mice several hours after injection of the infecting organisms. The degree of protection afforded, and the minimal effective dose of bactericidal agent, are approximately the same for all virulent pneumococci, irrespective of type specificity. The bactericidal agent is entirely ineffective against infection with virulent Friediänder bacilli (type B). This agrees with the fact that the agent does not affect Gram-negative bacilli in vitro. The protective action exerted by the bactericidal agent against experimental pneumococcus infection depends upon the same mechanism which determines its bactericidal effect in vitro.

Specific pathogen-free mice were exposed to three different kinds of environmental stress during early life: (a) by infecting them with a mouse enterovirus on the second day after birth; (b) by placing the mother during pregnancy and lactation on a mildly deficient diet containing wheat gluten supplemented with See PDF for Structure small amounts of lysine and threonine; (c) by combining a (neonatal infection) and b (early malnutrition). All animals survived the three types of stresses, but all exhibited marked depressions of metabolic activity, and of body weights and organ weights. These depressions lasted throughout the experimental period even though all animals were placed under optimum conditions of nutrition and husbandry after weaning, and maintained under these same conditions thereafter. Metabolic activity was determined by measuring the turnover of 14C-acetate and 14C-glucose in respiratory CO2, and their incorporation in total lipids of liver and brain. The utilization of 14C-acetate was profoundly depressed in all experimental groups with regard to both elimination in respiratory CO2 and their incorporation in total lipids of liver and brain. In contrast, the utilization of 14C-glucose was much less affected; its incorporation into lipids was not decreased and its elimination in respiratory CO2 was depressed only in animals having experienced both neonatal infection and early malnutrition. The extent of weight depression per 100 g of body weight differed according to the organ and the type of stress. Irrespective of the organ, however, depression of weight was largest in animals having experienced both neonatal infection and early malnutrition. And irrespective of the type of stress, the brain exhibited the smallest depression of weight relative to total body weight.

The growth of tubercle bacilli in vitro was inhibited by the addition of the sodium salts of very low concentrations of certain organic acids to a variety of liquid and agar culture media containing whole serum or serum albumin. Capric acid was the most active of the compounds tested, but inhibition of growth occurred also with the shorter aliphatic acids. Lactic acid was also growth-inhibitory, whereas the keto and dicarboxylic acids tested were inactive in this respect. The inhibitory activity of the aliphatic acids and of lactic acid increased as the pH of the medium was lowered by addition of HCl. It was greater in media enriched with serum or with oleic acid-albumin complex, but was otherwise fairly independent of the composition of the medium. The inhibitory effect appears to be bacteriostatic rather than bactericidal and to depend upon a disturbance of the normal metabolic processes of the bacilli. Some of the long chain fatty acids caused a marked enhancement of growth when used in low concentrations and in admixture with enough serum albumin to overcome their toxicity. The significance of these findings is discussed with reference to the survival and multiplication of tubercle bacilli in vivo within inflammatory and caseous areas, which are known to be often acidic and to contain high concentrations of organic acids.

A cell-free extract of cultures of an unidentified soil bacillus, which exerts a bactericidal effect on Gram-positive microorganisms, has been described in previous reports; the first active preparations which were obtained were found to contain a protein precipitable at pH 4.5. It is shown in the present report that the bactericidal agent can be obtained in an active form free of protein. The new purified preparations retain all the activity of the original material, both in vitro and in vivo.

Fractionation procedures yielding partially purified vaccine preparations from a 60°C. methanol extract of tubercle bacilli have been described. Some of the preparations have the characteristics of lipopolysaccharides. Certain ones have been found capable of increasing resistance to experimental tuberculosis in albino mice of the Rockefeller Swiss strain. The levels of resistance elicited by these preparations are equivalent to those following vaccination with BCG (Phipps) in this strain of mice as reported by other authors. The admixture of two of the crude fractions in amounts as small as 0.05 mg. each per dose per mouse affords an even greater increase in resistance. Neither of these substances alone in larger doses can approach this degree of efficacy in mouse protection experiments. The protective activity appears to involve the stimulation of two supplementary mechanisms, one providing a peak resistance between 1 and 3 weeks post vaccination but falling off to a lower level thereafter, the other not responding fully until approximately 6 weeks but continuing undiminished through a 12 week post-vaccination period. The first of these peaks corresponds to an increase in resistance against staphylococci as well as tubercle bacilli. The possibility that the term "broad specificity," rather than "non-specificity," might best describe this phenomenon permits the implication of classical immune mechanisms.

A substance possessing antimycobacterial activity under certain conditions in vitro has been prepared from aqueous extracts of calf thymus. Chemical studies have demonstrated that the activity of this substance is due to a basic peptide or a mixture of basic peptides. Although this thymus fraction has been shown to be essentially free of compounds other than peptides, it has not been obtained in a homogeneous state. The thymus peptide preparation is soluble in water and in the lower alcohols. Its solubility is minimal between pH 10 and 11, suggesting that its isoelectric point may be in this vicinity. The microbiological activity of thymus peptide is destroyed by acid or alkaline hydrolysis and also by trypsin digestion, but is unaffected by pepsin digestion. Cellulose membranes are permeable to thymus peptide. The most noteworthy finding concerning the amino acid composition of thymus peptide is the preponderance of the basic amino acids lysine and arginine, which together account for about 40 per cent of the weight of this substance. No cystine, and only trace amounts of other amino acids containing sulfur, are present in the thymus peptide preparation.

Three different cultures of BCG propagated for over 2 years in a liquid medium containing Tween 80 and albumin were found to differ in several of their intrinsic properties. Cultures of the three strains were found to consist of morphologically heterogeneous populations—each culture being made up of three main colonial types—spreading, intermediate, and non-spreading. The percentage distribution of colonial types was characteristic for each culture and remained constant during cultivation in liquid media. Injection of the various cultures into mice and guinea pigs resulted in a self-limited disease. The distribution, extent, and duration of the lesions were also characteristic for each culture. Both the spreading and non-spreading substrains derived from the various cultures exhibited the degree of attenuation of virulence characteristic of the parent strain.