STUDIES ON THE CHEMISTRY AND IMMUNOCHEMISTRY OF CELL WALLS OF STAPHYLOCOCCUS AUREUS

The cell walls of an 80/81 strain of Staphylococcus aureus (NYH-6) contain alanine, glycine, glutamic acid, lysine, muramic acid, glucosamine, and ribitol phosphate. 94 per cent of the phosphorus and 41 per cent of the glucosamine are removed by extraction of the cell walls with hot 5 per cent TCA, but significant amounts of the other constituents are not extracted by this procedure. The residue after hot TCA extraction (mucopeptide) is susceptible to lysozyme whereas the intact cell walls are resistant. Staphylococcus aureus cell walls are agglutinated by S. aureus antisera. Agglutination of the cell walls of one S. aureus strain is inhibited by absorption of antisera with cell walls of other S. aureus strains but not by absorption with S. albus cell walls. The ribitol teichoic acid can be isolated from cold TCA extracts of the cell walls. This compound consists almost entirely of ribitol phosphate and glucosamine. The isolated teichoic acid of strain NYH-6 is readily fixed to tanned sheep erythrocytes and these sensitized cells are agglutinated by S. aureus antisera. Cold TCA extracts of cell walls of other strains of S. aureus inhibit hemagglutination whereas extracts of S. albus walls do not. Studies on the inhibition of both hemagglutination and precipitation indicate that the antigenic determinant of S. aureus NYH-6 teichoic acid is β-N-acetylglucosamine.     

EFFECT OF DIETARY PROTEINS AND AMINO ACIDS ON THE SUSCEPTIBILITY OF MICE TO BACTERIAL INFECTIONS

Young mice were maintained for periods of 1 to 6 weeks on experimental diets containing all known growth factors, but differing in their protein and amino acid contents. All diets were supplemented with L-cystine. The effect of the nutritional regimen on infection was tested by inoculating the animals with either one of four pathogens (Mycobacterium tuberculosis var. bovis, Mycobacterium fortuitum, Staphylococcus aureus, Klebsiella pneumoniae type C), and by observing the survival time. The infective dose was administered by either one of three routes: intravenous, intraperitoneal, or air-borne (aerosol). In some experiments, the animals were maintained in groups of five throughout the tests. In other experiments they were housed in individual cages. This difference in housing did not affect the results in a detectable manner. Mice fed diets containing 5 or 8 per cent casein as sole source of amino acid (except for cystine supplementation) proved more susceptible to the experimental diseases than did mice fed diets containing 15 or 20 per cent of the same protein. Susceptibility to infection developed when wheat gluten, or soybean α-protein, was substituted for casein—even in high concentrations (15 or 20 per cent). In one experiment, mice were fed a diet containing as sole source of amino acids a mixture of soybean and rice flour, so designed as to provide a protein concentration of 15 per cent, with an amino acid pattern similar to that of casein. These animals gained weight at the same rate as those fed a diet containing 15 per cent casein and they exhibited a satisfactory level of resistance to bacterial infection. The infection-enhancing effect of low casein concentration (5 and 8 per cent) could be corrected by supplementing the diet with the proper mixture of amino acids. This could be done using either synthetic or natural amino acids. In contrast, susceptibility to infection developed when low casein diets were supplemented with unbalanced mixtures of amino acids. The infection-enhancing effect of gluten diets could not be corrected by supplementing the latter with lysine even though this supplementation markedly improved weight gains in uninfected animals. It appears in conclusion that the relative proportion of the various amino acids in the diet is as important a factor as their total amount in conditioning resistance to bacterial infections. This effect of nutrition on resistance can be detected irrespective of the route of infection: intravenous, intraperitoneal, or air-borne. Moreover, the effect has been observed with two strains of mice differing markedly in their natural resistance to bacterial infection.     

FACTORS RELATING TO THE VIRULENCE OF STAPHYLOCOCCI : III. ANTIBACTERIAL VERSUS ANTITOXIC IMMUNITY

Antitoxic and antibacterial immunity have been clearly differentiated in the experimental mouse infection produced by the diffuse colonial variant of the Smith strain of Staphylococcus aureus. Immunization with crude toxoid protected mice from otherwise lethal doses of alpha hemolysin, but did not alter mortality following intraperitoneal infection with living staphylococci. Conversely, animals immunized with heat killed vaccines were readily killed by culture supernates containing alpha hemolysin, but were strikingly protected from otherwise fatal intraperitoneal infection with viable staphylococci. Protection was directly related to the ability of the immunizing substance to promote early intraperitoneal phagocytosis of the infecting inoculum. In these studies with the Smith diffuse variant, rapid intraperitoneal phagocytosis was induced by vaccination with whole cell bacterial vaccines but not by alpha hemolysin toxoid.     

Studies on the generalized Shwartzman reaction. III. Lesions of the myocardium and coronary arteries accompanying the reaction in rabbits prepared by infection with group A streptococci

Cutaneous and systemic infections of rabbits by Group A streptococci bring about a state of preparation for, respectively, the local and generalized Shwartzman reactions, produced by intravenous injection of meningococcal or S. marcescens toxin. With maximal systemic streptococcal infections, the lesions of the generalized Shwartzman reaction do not differ from those caused by two successive intravenous injections of Gram-negative bacterial toxins. The characteristic lesions of the reaction are bilateral cortical necrosis of the kidneys, hemorrhagic necrosis in the lungs, liver, and spleen, and myofiber necrosis in the myocardium. Under optimal conditions involving the dosages of streptococci and toxin, and the time interval between the injections, a new lesion consisting of necrosis and the accumulation of fibrinoid material in the walls of the coronary arteries occurred in approximately 50 per cent of animals within 48 hours after the injection of meningococcal toxin. Fibrinoid necrosis was not observed in the arteries of tissues other than the heart. It did not occur in control rabbits injected with streptococci alone or toxin alone, nor in animals with the generalized Shwartzman reaction produced by two intravenous injections of toxin. Streptococcal bacteriemia was present at the time of death in one-third of the animals with fibrinoid necrosis. In one animal, a group of bodies resembling cocci in chains was seen within the wall of a coronary artery with fibrinoid necrosis. A series of photomicrographs to illustrate the pathological changes in the hearts and kidneys of streptococcus-infected rabbits subjected to the Shwartzman reaction is presented.     

FACTORS RELATING TO THE VIRULENCE OF STAPHYLOCOCCI : II. OBSERVATIONS ON FOUR MOUSE-PATHOGENIC STRAINS

Four clumping factor-negative strains of Staphylococcus aureus were found to closely resemble the diffuse colonial variant of the Smith strain. All produced fatal intraperitoneal infections in mice, all grew in diffuse, streaming colonies in plasma or serum soft agar, and all behaved like encapsulated microorganisms in in vitro opsonic systems. These staphylococci were resistant to phagocytosis in the peritoneal cavities of normal mice. When mice were immunized with heat-killed vaccines prepared from the Smith diffuse variant these strains were rapidly ingested by peritoneal leukocytes and the animals survived. This observation suggests that these strains share the same or a similar phagocytosis-retarding antigen. While most pathogenic staphylococci isolated from human material do not behave like these unusual mouse-virulent strains, indirect evidence is cited to support the suggestion that other staphylococci may acquire similar phagocytosis-resisting characteristics during in vivo multiplication. Studies to support or refute this thesis are in progress.     

THE EFFECT OF STAPHYLOCOCCUS AUREUS TOXIN ON THE KIDNEY

1. The hemolytic Staphylococcus aureus elaborates a toxin in vitro that when injected intravenously produces lesions in the kidneys of rabbits and cats. 2. The toxin injures primarily the blood vessels of the kidney.     

Effect of dietary proteins and amino acids on the susceptibility of mice to bacterial infections

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.     

MICROBIC VIRULENCE AND HOST SUSCEPTIBILITY IN PARATYPHOID-ENTERITIDIS INFECTION OF WHITE MICE : IX. THE RELATIONSHIP OF DOSAGE TO MORTALITY RATE,SURVIVAL TIME,AND CAGE POPULATION.

Epidemics of mouse typhoid set up among the Rockefeller Institute strain of mice were studied over a period of 6 months. During this time the relationship of cage number of mouse typhoid bacilli to mortality, total population, and survival time was determined. A single virulence titration of the epidemic strain was made, and at the end of the experiment all survivors were examined for evidence of infection. The following conclusions may be drawn for the data here presented. 1. The available dosage of mouse typhoid bacilli varied directly with the mortality (plus a time constant of 6 to 8 days) and inversely with the survival time. 2. The virulence of the epidemic strain appeared to be practically the same as that of the original stock culture. 3. About 53.5 per cent of the survivors of one epidemic and 68 per cent of those in the other showed, at the end of the experiment, no signs of infection; the others had either specific blood agglutinins, or living bacteria in their heart''s blood, spleen, feces, or gall bladder. 4. During the course of the epidemic, the original infecting strain (mouse typhoid Type II—Bacillus pestis caviæ) was almost entirely replaced by an antigenically dissimilar strain (mouse typhoid Type I—Bacillus enteritidis), probably introduced through the inadvertent inclusion of fecal carriers among the normal mice added as contacts.     

THE EFFECT OF DIET ON THE FECAL BACTERIAL FLORA OF MICE AND ON THEIR RESISTANCE TO INFECTION

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.     

MICROBIC VIRULENCE AND HOST SUSCEPTIBILITY IN PARATYPHOID-ENTERITIDIS INFECTION OF WHITE MICE : V. THE EFFECT OF DIET ON HOST RESISTANCE.

White mice from the Rockefeller Institute breeding room fed on a McCollum complete diet, consisting of whole wheat (67.5 per cent), casein (15 per cent), milk powder (10 per cent), NaCl (1 per cent), CaCO₃(1.5 per cent), and butter fat (5 per cent) are more resistant to mouse typhoid infection, mercury bichloride intoxication, and botulinus toxin than are similar mice fed on bread and pasteurized milk supplemented by an oatmeal and buckwheat mixture and dog biscuit.     

Assessment of an Anti-Alpha-Toxin Monoclonal Antibody for Prevention and Treatment of Staphylococcus aureus-Induced Pneumonia

Alpha-toxin (AT) is a major virulence factor in the disease pathogenesis of Staphylococcus aureus. We previously identified a monoclonal antibody (MAb) against AT that reduced disease severity in a mouse dermonecrosis model. Here, we evaluate the activity of an affinity-optimized variant, LC10, in a mouse model of S. aureus pneumonia. Passive immunization with LC10 increased survival and reduced bacterial numbers in the lungs and kidneys of infected mice and showed protection against diverse S. aureus clinical isolates. The lungs of S. aureus-infected mice exhibited bacterial pneumonia, including widespread inflammation, whereas the lungs of mice that received LC10 exhibited minimal inflammation and retained healthy architecture. Consistent with reduced immune cell infiltration, LC10-treated animals had significantly lower (P < 0.05) proinflammatory cytokine and chemokine levels in the bronchoalveolar lavage fluid than did those of the control animals. This reduction in inflammation and damage to the LC10-treated animals resulted in reduced vascular protein leakage and CO₂ levels in the blood. LC10 was also assessed for its therapeutic activity in combination with vancomycin or linezolid. Treatment with a combination of LC10 and vancomycin or linezolid resulted in a significant increase (P < 0.05) in survival relative to the monotherapies and was deemed additive to synergistic by isobologram analysis. Consistent with improved survival, the lungs of animals treated with antibiotic plus LC10 exhibited less inflammatory tissue damage than those that received monotherapy. These data provide insight into the mechanisms of protection provided by AT inhibition and support AT as a promising target for immunoprophylaxis or adjunctive therapy against S. aureus pneumonia.     

IRON METABOLISM IN EXPERIMENTAL ANEMIA : "AVAILABILITY OF IRON"

In experimental anemia in dogs due to blood loss the term "available iron" as determined by the dipyridyl test has no physiological significance. Iron salts (100 per cent available by dipyridyl) given in optimum dose (560 mg. per 2 weeks) will cause a net production of 50 to 55 gm. hemoglobin above the control base line in anemic dogs. This means that an iron salt which is rated as 100 per cent available by the dipyridyl test is only 35 per cent physiologically available. The term "available iron (dipyridyl)" simmers down to iron not in the form of hematin compounds. The absorption of this "available iron" is conditioned by a great variety of factors, many unknown at this time. Iron is indeed an elusive sprite whose "availability" or comings and goings cannot be determined in dogs by dipyridyl—perhaps only in part by studies of absorption and excretion. Liver contains "available iron (dipyridyl)" but also organic factors influencing hemoglobin regeneration in anemia as liver ash contains only about 50 per cent the potency of the whole liver. One can readily dissociate the iron from other potent factors in various tissues. Fractions of heart, liver, spleen, and kidney may contain very little iron yet cause much hemoglobin regeneration in anemic dogs. No investigator has reported any condition of copper deficiency in man or dog. In fact, in anemias copper is usually above normal concentration in the liver. It is unlikely, therefore, that in experimental anemia in dogs and in the various anemias of man, any significance attaches to the intake of copper.     

INHERITANCE OF RESISTANCE OF MICE TO ENTERIC BACTERIAL AND NEUROTROPIC VIRUS INFECTIONS

Under the conditions specified, there may be selected promptly from a hybrid stock of mice, of which 40 to 50 per cent die following a standard dose of B. enteritidis or St. Louis encephalitis virus, lines in which as high as 95 per cent and as low as 15 per cent succumb. Three lines,—one bacteria-susceptible-virus-susceptible, one bacteria-susceptible-virus-resistant, and one bacteria-resistant-virus-susceptible,—are regarded as remaining relatively stable after approximately twelve generations of selection and brother to sister or line inbreeding. Crossing susceptible with resistant lines and testing F₁, F₂, F₃, and backcross progeny resulted in mortality percentages in the neighborhood of those expected on the basis that resistance to B. enteritidis and to encephalitis virus is each inherited independently on a single factor basis with resistance dominant over susceptibility. A bacteria-resistant-virus-resistant line is being developed from a cross between bacteria-susceptible-virus-resistant and bacteria-resistant-virus-susceptible lines. All selected lines proved uniformly susceptible to a strain of mouse passage rabies virus.     

STUDIES ON THE MODE OF ACTION OF DIPHTHERIA TOXIN : III. EFFECT ON SUBCELLULAR COMPONENTS OF PROTEIN SYNTHESIS FROM THE TISSUES OF INTOXICATED GUINEA PIGS AND RATS

The effect of diphtheria toxin on subcellular components of protein synthesis was determined. Polyribosomes prepared from intoxicated guinea pigs functioned normally in an in vitro assay system, while the activity of soluble enzymes (transferases) from toxin-treated animals was significantly reduced. At high toxin dosages, this reduction was widespread, but when levels of toxin comparable to those which might be generated in a natural infection were given, inhibition of soluble enzyme activity was found only in extracts from heart and skeletal muscle. Possible nonspecific inhibition in the assay system due to interference by free toxin or by a serum component was eliminated. Since it was possible to demonstrate reactivation of soluble enzyme activity with nicotinamide and toxin, it was suggested that diphtheria toxin acts in the intact sensitive animal in a manner analogous to its action in tissue culture or in cell-free systems. It was hypothesized that the lethal biochemical lesion of the toxin in sensitive animals was the inactivation of transferase enzymes, principally in the heart. It was also suggested that the lethal lesion induced in diphtheria-sensitive and resistant species may not be identical.     

STUDIES ON THE ROLE OF THE THYMUS IN IMMUNOBIOLOGY : RECONSTITUTION OF IMMUNOLOGIC CAPACITY IN MICE THYMECTOMIZED AT BIRTH

The immunologic competence of spleen cells of mice, as assessed by their graft versus host capabilities, increases to 35 days of age and beyond. Thymectomy at any point along this continuum of development produces "immunologic arrest;" the peripheral lymphoid tissues of such mice do not show significant increases in activity as the animals mature, nor is there appreciable loss of activity up to 6 months after surgery. Adult spleen cells from mice thymectomized at 1 to 24 hours of age have a greatly reduced ability to induce runt disease. Five million spleen cells from immunologically mature animals will uniformly cause fatal runt disease in neonatal recipients, but this same number of cells from neonatally thymectomized animals produces almost no runt disease. When the dosage of cells from neonatally thymectomized C₅₇Bl mice is increased to 20 million, about half of the A recipients develop runt disease. Thus, the defect is a quantitative one. Spleen cells from neonatally thymectomized mice will induce tolerance of skin grafts from the donor strain. In one recalcitrant strain combination, C₅₇Bl to A, use of spleen cells from neonatally thymectomized donors as the tolerance-inducing inoculum permits survival of the recipients, which usually die with severe runt disease, but does not induce tolerance. Cell free extracts of spleen and thymus tissue, including "promine" of Szent-Gyorgyi et al., did not affect the runting syndrome or the immunologic reactivity of neonatally thymectomized mice. When syngeneic thymic tissue is grafted into neonatally thymectomized mice, or the animals are given viable syngeneic spleen or thymus cells, the majority of the animals escape the early mortality characteristic of this group. Administration of syngeneic spleen cells from adult donors and grafting of syngeneic neonatal thymus provide restoration of homograft immunity and graft versus host reactivity of the peripheral lymphoid tissues in most of the neonatally thymectomized animals. Thymus cells rarely provide significant restoration of these parameters. Allogeneic thymus grafts also restore neonatally thymectomized mice. Such animals are chimeric: the immunologically competent cells of their peripheral lymphoid tissues are chiefly of host origin as determined by the discriminant spleen assay, but in many instances a significant donor component is also demonstrable in this system. These chimeric animals accept skin grafts from both donor and host strains. A degree of reconstitution has also been attained by grafting of allogeneic adult spleen in neonatally thymectomized animals. The discriminant spleen assay indicates that cells of the donor strain predominate in the peripheral lymphoid tissues of such mice.     

ISOLATION AND PROPERTIES OF A SURFACE ANTIGEN OF STAPHYLOCOCCUS AUREUS

A technique is described for the isolation and purification of an antigen released into the culture medium by Staphylococcus aureus strain Smith. The antigen was found to be homogeneous when examined by free electrophoresis and analytic ultracentrifugation. Immunologic homogeneity was established by immunoelectrophoresis and quantitative precipitin tests using high titer antiserum prepared against the homologous organism. Chemical analysis showed that the antigen contained 70 per cent carbohydrate, of which approximately 30 to 35 per cent was believed to be glucosamine. The analytic data suggested that another amino sugar, probably carboxylated, was also present, but extreme lability of this compound to mild hydrolytic procedures has thus far precluded further identification. The remainder of the antigen was composed of alanine, glutamic acid, aspartic acid, lysine, glycine, serine, and threonine. No muramic acid was found. The chemical and physical data indicate that the antigen described herein is a previously unrecognized component of Staphylococcus aureus. The purified compound was capable of absorbing agglutinating antibody from antiserum prepared against S. aureus Smith, indicating that it was a surface component of this encapsulated staphylococcus. It is proposed that the antigen be known as the Smith surface antigen (SSA). The injection of SSA into rabbits did not produce precipitating antibodies. However, SSA did precipitate at low concentrations (0.5 µg/ml) with antiserum prepared against S. aureus Smith and one other strain of S. aureus tested. Antiserum against two other aureus strains reacted only with high concentrations of SSA. SSA did not react with S. albus antiserum or with normal sera from several animal species. Experiments are in progress to define further the distribution of SSA. Intradermal injection of small quantities of SSA into rabbits immunized with S. aureus Smith evoked a reaction of cutaneous hypersensitivity, which was maximal in 8 to 12 hours. SSA appeared to be the substance responsible for the ability of S. aureus Smith to resist engulfment by phagocytes, since absorption of Smith antiserum with SSA effectively removed opsonizing antibodies. SSA induced protection in mice against experimental staphylococcal disease. The subcutaneous injection of 0.1 µg resulted in protection against a subsequent intraperitoneal challenge with 50 to 100 LD₅₀'s of S. aureus Smith suspended in mucin. Increasing as well as decreasing the immunizing dose resulted in significantly less protection.     

Characterization and Quantitation of Experimental Surgical-Wound Infections Used to Evaluate Topical Antibacterial Agents

Reproducible experimental surgical-wound infections in mice for use in the evaluation of topical antibacterial agents are described. The experimental would was created on the backs of mice by means of a midline incision and was infected by means of cotton sutures monocontaminated with Staphylococcus aureus or Pseudomonas aeruginosa. The course of these wound infections was followed by quantitation of surface bacteria through use of a surface rinse technique. Surface wound counts of the infecting organisms thus obtained appeared to reflect the dynamics of the total wound count, as determined by homogenization of biopsied tissue. Treatment of infected wounds with a placebo cream had only a slight effect on surface wound counts and on mortality in the case of the S. aureus infection but enhanced markedly the lethality of the P. aeruginosa infection.     

THE PRODUCTION OF RUNT DISEASE IN RATS THYMECTOMIZED AT BIRTH

Thymectomy of Sprague-Dawley rats on the 3rd day of life failed to influence the time of onset, incidence, clinical, or histologic picture of runt disease produced by the intraperitoneal injection of adult Long-Evans spleen cells. The fact that severe immunologic impairment of the host by thymectomy does not modify runt disease was felt to be consistent with the current view that the if direction of the immunologic event in this syndrome is graft versus host. Following the injection of 800 to 1000 million Long-Evans spleen cells into adult Sprague-Dawley rats, a severe illness comprised of dermatitis, gastrointestinal bleeding, arthritis, weight loss, and death ensued in 37 per cent of adults thymectomized neonatally and 13 per cent of normal controls. Histologic lesions were observed in 69 per cent of adequately thymectomized animals and 17 per cent of normal controls, and involved lymph nodes, spleen, liver, lungs, kidneys, joints, heart, and skin. The time of onset and the histologic and clinical pictures are consistent with the adult disease being a typical graft versus host reaction.     

THE EFFECT OF BACTERIAL ENDOTOXINS ON THE WATER INTAKE AND BODY WEIGHT OF MICE

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.     

THE IN VIVO INTERACTION BETWEEN STAPHYLOCOCCUS BACTERIOPHAGE AND STAPHYLOCOCCUS AUREUS

Staphylococcus bacteriophage 81 is capable of in vivo interaction with Staphylococcus aureus, Type 80/81. This is immediately made evident by increased levels of bacteriophage and concomitant survival of 81 per cent infected mice. The reaction is dependent upon the use of active, type-specific bacteriophage. The maximal protective effect is observed at a bacteriophage to bacteria ratio of 1:2 and decreased quantities of bacteriophage result in decreased protection. Time and sequence of administration are also determining factors. It is evident that bacteriophage administered intravenously is capable of interaction with the infecting bacterial cell at the site of infection. In vivo produced bacteriophage is apparently eliminated or otherwise rendered nondetectable fairly rapidly, occurring within a period of 5 to 10 days. However, it appears that host defense mechanisms are stimulated in the process and actively play a protective role against subsequent challenge inocula administered up to 3 weeks later.