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Initial velocities of energy-dependent Ca++ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca++ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca++ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca++. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca++ transport in cardiac mitochondria. Comparable rates of Ca++ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca++ uptake by frog heart mitochondria were higher at any Ca++ concentrations. The half-maximal rate of Ca++ transport was observed at 30, 60, 72, 87, 92 µM Ca++ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent Km render mitochondrial Ca++ uptake slow below 10 µM. At these concentrations the rate of Ca++ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca++ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca++ by mitochondria is inadequate for regulating the beat-to-beat Ca++ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca++ uptake are discussed in terms of physiological regulation of intracellular Ca++ homeostasis in cardiac cells.

We have examined the capacity of the major secretory protein (MSP) of Legionella pneumophila to induce humoral, cell-mediated, and protective immunity in a guinea pig model of Legionnaires' disease. MSP was purified to homogeneity by ammonium sulfate precipitation, molecular sieve chromatography, and ion-exchange chromatography. The purified MSP was nonlethal and nontoxic to guinea pigs upon subcutaneous administration. Guinea pigs immunized with a sublethal dose of aerosolized L. pneumophila or a subcutaneous dose of MSP developed a strong cell-mediated immune response to MSP. Such guinea pigs exhibited marked splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity to MSP in comparison with control animals. Guinea pigs immunized with MSP also developed a strong humoral immune response to MSP, as assayed by an ELISA. The median reciprocal antibody titer was 362 (range 45 to greater than 2,048) for immunized animals compared with less than 8 for controls. In contrast, guinea pigs immunized with a sublethal dose of L. pneumophila failed to develop anti-MSP antibody. Guinea pigs immunized with MSP and then challenged with a lethal aerosol dose of L. pneumophila exhibited highly significant protective immunity in each of five consecutive experiments. MSP induced protective immunity in dose-dependent fashion (40 greater than 10 greater than 2.5 greater than 0.6 micrograms MSP); vaccination with two doses of as little as 2.5 micrograms MSP induced significant protective immunity (p = 0.01, Fisher's Exact Test, two-tailed). Altogether, 21 (81%) of 26 animals immunized with 40 micrograms MSP survived challenge compared with 0 (0%) of 26 sham-immunized control animals (p = 7 x 10(-10), Fisher's Exact Test, two-tailed). MSP-immunized but not control guinea pigs were able to limit L. pneumophila multiplication in their lungs. This study demonstrates that (a) guinea pigs sublethally infected with L. pneumophila develop a strong cell-mediated immune response to MSP; (b) guinea pigs immunized with MSP develop a strong humoral and cell-mediated immune response to MSP; (c) guinea pigs immunized with MSP develop a very high level of protective immunity to lethal aerosol challenge with L. pneumophila; and (d) MSP-immunized animals are able to limit L. pneumophila multiplication in their lungs. MSP, an extracellular protein of an intracellular pathogen, has potential as a vaccine for the prevention of Legionnaires' disease. Secretory molecules of other intracellular pathogens may also have vaccine potential.

Guinea pig periotoneal exudate (PE) cells incubated overnight in vitro with heat-killed Staphylococci released an endogenous pyrogen (EP) that could be assayed by intravenous injection in rabbits. The febrile responses were linearly related to the dosage of EP over an eightfold range. PE cells derived from guinea pigs with delayed hypersensitivity (DH) to bovine gamma globulin (BGG), also released EP when incubated with antigen in vitro. This reaction was specific and did not occur withe PE cells from normal or complete Freund's adjuvant-sensitized guinea pigs. Studies indicated that monos and/or polymorphonuclear leukocytes rather than lymphocytes were the source of EP. However, when incubated with BGG and sufficient dosages of BGG-sensitized lymphocytes, normal PE cells released EP over a 42 h period. These results suggest that antigen stimulates specifically sensitized lymphocytes to release an agent (perhaps a lymphokine) that activates phagocytic cells to release EP. This model offers unique advantages for investigating in vitro the role of the lymphocyte in antigen-induced fever in DH as well as the relationship of this lymphocyte-induced activity to other known biologic activities mediated by antigen stimulated lymphocytes.

Previous work by Germuth and McKinnon (1), Trapani et al. (2), and ourselves (3) has established the fact that soluble antigen-antibody complexes formed in excess antigen can, (a) induce symptoms similar to anaphylaxis, (b) cause contraction of isolated smooth muscle from normal guinea pigs, and (c) increase the permeability of skin capillaries in a manner similar to that obtained in passive cutaneous anaphylaxis. These findings immediately raise many questions as to the fundamental mechanisms involved. For example, is the free antigen playing some role; is the toxicity dependent upon some change in the molecular structure of either antigen or antibody upon combination; is the complex itself toxic without any change in the molecular structure of the components; is the antigen-antibody ratio important; and, is complement involved? The work reported here involves a study of the possible role of free antigen and the nature of the complex. Some study was also made of untreated and decomplemented antiserums and, although there was no difference, this cannot rule out the possible participation of the test animal's (guinea pig's) own complement.

Guinea pigs have been rendered relatively immune against infection with virulent tubercle bacilli by preliminary inoculation with a suitable quantity of avirulent tubercle bacilli. Blood counts on these animals show that associated with the immune reaction there is a definite general leucocytic response characterized by an absolute increase in the total count, with an absolute and relative increase in the lymphocytes. The period of greatest activity coincides with that known to be the period of greatest reaction, based on anatomical evidence during the course of infection following this method of immunization. Moreover, blood counts made on animals inoculated with avirulent tubercle bacilli alone show an increase in the circulating lymphocytes during the period of greatest reaction to the infection, while blood counts on guinea pigs inoculated with virulent bacilli alone show an erratic course in which the polymorphonuclear forms are much increased, though not regularly so. These results indicate a parallelism between lymphoid activity and resistance of the animals to tuberculous infection, and suggest an association of lymphocytes with the factors determining this resistance, a relation which warrants consideration of the blood picture in the clinical study of tuberculous infection.

The suppression of antibody formation by passively administered antibody is influenced by the dose and nature of the antigen, type of immunization procedure, ratio of antibody to antigen, species origin and characteristics of the antiserum used, as well as the species selected for immunization. In guinea pigs, diphtheria antitoxin formation can be effectively suppressed by an intravenous injection of excess homologous or heterologous antitoxin as long as 5 days after toxoid immunization and after delayed-type hypersensitivity to toxoid has developed. Following the period of antibody suppression which lasts 2 to 7 weeks, serum antibody can usually be demonstrated. It is proposed that this delayed immunization results from dissociation of antigen, since diphtheritic paralysis and death can be produced in guinea pigs and rabbits by the intravenous injection of toxin-antitoxin precipitates formed in antitoxin excess. This syndrome is prevented by injection of excess horse antitoxin 1 hour after injection of the toxin-antitoxin complexes.

The heat-stable, homocytotropic antibody synthesized by guinea pigs to highly purified dextran has been studied by passive cutaneous anaphylaxis (PCA). Antibody arising in single guinea pigs was found to give PCA in some homologous recipients but not in others. Data are presented which indicate that the antibodies are bound in the skin of some animals to a greater degree than in that of others. This suggests some type of variability, perhaps allotypic, in the Fc portion of these immunoglobulins which relates to specificities of the loci where Fc becomes fixed in skin.

Intravenous administration of 5-hydroxytryptamine to rabbits and guinea pigs is shown to bring about changes very similar to those produced by (+) air ions, including (1) decreased ciliary rate, (2) contraction of the posterior tracheal wall, (3) exaggerated response of the tracheal mucosa to trauma, (4) marked vasoconstriction in the tracheal wall, and (5) increased respiratory rate. These effects are reversed by (-) air ions. Iproniazid, which raises 5-hydroxytryptamine levels in the animal by blocking monamine oxidase, produces similar but non-reversible effects. Reserpine, which depletes 5-hydroxytryptamine in the animal, causes changes that resemble those produced by (-) air ions, including (1) increased ciliary rate, (2) relaxed posterior sulcus, (3) hyperemia of the tracheal mucosa, (4) lowered respiratory rate, and (5) increased volume and rate of mucus flow. On the basis of these facts, the hypothesis is advanced that (+) air ion effects are mediated by the release of free 5-hydroxytryptamine, while (-) air ion effects depend on the ability of (-) ions to accelerate the enzymatic oxidation of 5-hydroxytryptamine.

It is conceivable that a change from the virulent, non-phagocytable S form of Pneumococcus to the avirulent phagocytable R form may take place in pneumococcus disease, but the experiments here reported do not settle the question whether or not this is an important factor in determining the outcome in natural infection. It has been shown experimentally that the degradation from the S form to the R form actually does take place in cultures of Pneumococcus growing in agar subcutaneously embedded in guinea pigs, in agar enclosed in vials subcutaneously embedded in rabbits, and spontaneously in the blood stream of infected horses. However, it was not possible in any of the experiments here cited to demonstrate the complete change from S to R pneumococci before the bacteria disappeared from the body. When the intermediate or R forms did appear, they were always accompanied and usually exceeded in number by the S forms and all three forms disappeared together. S organisms may disappear entirely without evidence of first going through the intermediate and R stages. On the other hand, contrary to expectations, pure cultures of R forms remained viable in subcutaneous foci for weeks although apparently freely accessible to the action of phagocytes. It seems of some significance that the R forms appeared early in the vials (inoculated with S pneumococci) in immunized and normal rabbits alike, indicating that the presence of demonstrable specific immune bodies was not alone responsible for the variation of the bacteria. Of some importance also is the fact that R forms were never derived from similarly prepared control cultures growing in vitro at the same temperature and immersed in normal serum, although the S forms remained viable and unaltered for 6 weeks. It is likely that variations of pneumococci do not occur readily when S cultures are exposed to normal serum in vitro, especially when growing in closed vials under a diminished oxygen supply, for it has previously been shown (2) that only slight variation occurs even after prolonged (240) transfers in heterologous serum broth in the test-tube. It is possible, therefore, that the variation which occurred among pneumococci growing in agar vials embedded in normal rabbits was actually provoked by unknown influences in the living tissue fluids. Although R forms have been shown to occur in vivo, no positive evidence can be derived from these experiments to prove that recovery from pneumococcus infection depends upon the degradation of the virulent S forms of pneumococci to the avirulent R forms and the subsequent destruction of the latter by phagocytes.

A technique is described for measuring objectively and quantitatively the reaction of sensitized guinea pigs when exposed to an aerosol of specific antigen. The principle involves registration by semi-automatic means of the number of coughs produced in animals passively sensitized with known amounts of antibody. The number of coughs is shown to be linearly related to log dose of antibody within a limited range and a dose-response curve is presented. The cough produced by this procedure is not inhibited by the antitussive drugs, codeine and propadrine, but can be inhibited by anti-allergic agents, such as cortisone and an antihistaminic drug. It is also inhibited by narcotine. The last is the only compound so far tested which suppresses both the cough produced by this procedure and that produced by a simple irritant. The action of cortisone and the antihistaminic drug, pyrilamine, is shown to be synergistic. A small dose of pyrilamine in animals pretreated with cortisone gives a degree of inhibition which cannot be obtained by increasing the dose of pyrilamine in animals not treated with cortisone.

Injection of sodium atoxyl-diazoamino-sulfoanthranilate into guinea pigs produces an anaphylactic hypersensitiveness to the corresponding azoprotein (Schultz-Dale test). This leads to the conclusion that the injected sodium atoxyl-diazoamino-sulfoanthranilate first decomposes and then couples in vivo with the body protein to form the corresponding azoprotein and that therefore it is this compound, produced within the organism itself, which sensitizes.

1. Alkali (pH 7.6)-denatured pepsins from swine, cattle, and guinea pigs precipitate in swine pepsin antiserum. Similarly treated pepsins from the rabbit, chicken, and shark do not. 2. Pepsin antisera react with both pepsin and pepsinogen, but do not react with the serum proteins from the homologous species. 3. Pepsinogen antisera react with pepsinogen, but not with twice crystallized pepsin, nor with the serum proteins from the homologous species. Positive reactions between activated pepsinogen and pepsinogen antiserum have been observed. It was possible to remove the reacting material from either the pepsinogen or the activated pepsinogen mixture. 4. Antisera made with serum proteins do not react with the homologous pepsin or pepsinogen.

1. Guinea pigs injected intravenously with massive doses of India ink before active sensitization exhibited occasionally a more or less marked, decreased sensitiveness to the reinjection of the antigen. 2. The serum of rabbits which had received massive doses of India ink before the sensitizing injections, showed approximately the same titer of anaphylactic antibodies as that of sensitized normal control animals, as demonstrated by the degree of passive sensitization induced in guinea pigs. 3. The precipitin titer of sensitized rabbits blocked with massive doses of India ink was somewhat lower than that of sensitized normal controls. In one instance, the intravenous injection of smaller doses of India ink was followed by a higher precipitin titer. No uniform relation was found between the height of the precipitin titer and the anaphylactic power of the antiserums. 4. One blocking injection of India ink given to guinea pigs before passive sensitization did not interfere with the occurrence of anaphylactic shock nor alter its nature.

The soluble antigen of lymphocytic choriomeningitis which is readily separable from the virus is a relatively stable substance and appears to be of a protein nature. A specific precipitin reaction can be demonstrated when immune serum is added to solutions of antigen which have been freed of certain serologically inactive substances. The complement-fixation and precipitation reactions which occur in the presence of immune serum and non-infectious extracts of splenic tissue obtained from guinea pigs moribund with lymphocytic choriomeningitis seem to be manifestations of union of the same soluble antigen and its antibody. On the other hand, the antisoluble substance antibodies and neutralizing substances appear to be different entities.

A bacillus of the paratypnoid-enteritidis group associated with epizootics among laboratory mice has been identified with Bacillus festis caviæ Smith which produces similar affections in guinea pigs and has been very closely related to the type "mutton" aertrycke strain of Schütze. This identification is based upon the cultural reactions of the organism, direct and cross-agglutinations, and similar absorptive capacities of the unknown and type strains.

The effect of crystalline diphtheria toxin on protein synthesis in vivo was evaluated in guinea pigs and mice. By two independent methods of analysis (microdensitometry of tissue radioautograms and radioactivity of tissue proteins), it was established that inhibition of protein synthesis was not a widespread metabolic effect of diphtheria toxin. In the sensitive guinea pig, only the heart and the pancreas showed any demonstrable reduction in the quantity of tritiated leucine incorporated into proteins following challenge of the animals with the crystalline toxin. No such inhibition was noted in mice which are resistant to the action of diphtheria toxin. The effect on the pancreas involved a decrease in the synthesis of pancreatic enzymes and their subsequent secretion. For reasons discussed, it was concluded that this lesion was not as significant as the inhibition of protein synthesis in the heart tissues. Although the rate of protein turnover in heart muscle is relatively low, an inhibition of 73% was noted when the exchange period with the tritiated leucine was 6 hr. It was suggested that the inhibition of protein synthesis in heart tissues could provide a biochemical rationale for the site and mode of action of diphtheria toxin in the sensitive mammalian host. An attempt was also made to correlate the biochemical heart lesion described here with past clinical evidence of cardiac failure and tissue pathology noted in many cases of fatal diphtheria infections of humans.

Experiments carried out with several well defined antigenic systems (hapten conjugates of poly-L-lysine and guinea pig serum albumin) in guinea pigs demonstrated that: 1. Arthus reactions also manifest carrier specificity, although to a smaller extent than do delayed hypersensitivity reactions. 2. Desensitization by injection of minute doses of antigen results in moderate specific desensitization of delayed hypersensitivity without desensitization of Arthus reactivity to the same antigenic determinant. 3. Insoluble antigen-antibody complexes prepared from high affinity guinea pig antibodies can elicit specific delayed skin reactions in sensitized guinea pigs. 4. Homologous conjugates of structurally similar haptens show considerably less cross-reactivity in delayed reactions than in immediate hypersensitivity reactions to the same antigenic determinant. These experimental results are interpreted as indicating that delayed hypersensitivity reactions in the guinea pig are mediated by "antibodies" of comparatively high binding affinities. High binding affinities are achieved for these antibodies more likely by closer structural adaptation between antigen and antibody than by a larger area of specific contact.

1. Two cases of rat-bite fever with a typical fever course and a maculopapular exanthem came under my observation in Mexico. 2. A spiral organism morphologically identical with one known in Japan as the causative agent of Sodoku was found in a Mus decumanus caught in Mexico City. 3. The organism was found to be pathogenic for white rats and guinea pigs, the infection being fatal for guinea pigs. 4. The bite of an infected guinea pig led in one of two instances to infection and death of the animal bitten. 5. The microscopical findings in the guinea pigs infected with this microorganism closely resemble in many respects the findings in Blake's case and in Kaneko and Okuda's first case, the only ones of human rat-bite fever which have been closely studied at post mortem. 6. Eye lesions were found in all the infected guinea pigs and the spiral organisms were demonstrated in the discharge from the eyes and also within the cornea. 7. Similar eye lesions were found in most of the infected rats and the secretion from the eyes of one of them was demonstrated to be infectious for guinea pigs. 8. The eye lesions constitute an evident source for the spiral organisms transmitted by biting, and not improbably the only one. My experiments may give an explanation for the transmission of some other spirochetal diseases besides rat-bite fever. Cases of Weil's diseases are reported to be caused by the bite of a rat and it is noteworthy that eye lesions occur in spirochætosis icterohæmorrhagica (Manson).

While passaging a recently isolated strain of poliomyelitis virus through a rhesus monkey, another virus was procured from its central nervous system. After intracerebral inoculation, the virus produced meningoencephalitis in monkeys, cotton rats, hamsters, guinea pigs, and rabbits; after intracutaneous inoculation a necrotic skin lesion was produced in the monkey and rabbit and this was often followed by myelitis. The virus could also be passed in newborn mice less than 48 hours old and in chick embryos by inoculation of the chorioallantoic membrane. Immunological and host range studies revealed this virus to be related to the B virus originally described by Sabin and Wright in 1934 (1). To our knowledge this is the first record of B virus having been isolated from a monkey, and lends support to the inclusion of this agent as the simian member of the herpes group. The infection is not uncommon in monkey stocks, as revealed by the finding of antibodies to the virus in their sera. In the present series 9 of 44 monkeys gave positive antibody tests. Gamma globulin prepared in the United States in 1945, 1951, and 1953, as well as a certain proportion of sera from normal individuals in Bombay, India, and elsewhere, showed neutralizing activity against the new strain of B virus, and also to herpes simplex virus. This may be the result of the partial crossing which exists between the two viruses.

The method of filter paper electrophoresis was used to study proteins and protein-bound polysaccharides in sera obtained from subjects before and after a single booster dose of diphtheria toxoid, and in sera from allergic subjects. The electrophoretic patterns of precipitating antitoxic sera resembled those found in normal non-immune sera. However, skin-sensitizing antitoxic sera were distinguished by a relatively large beta globulin component and a small or indistinct alpha2 globulin. Fusion of both components was present in some sera containing this variety of antitoxin. Considerable amounts of serum-bound polysaccharides in these sera migrated relatively slowly in contrast to the behavior of polysaccharides of precipitating antitoxic sera which migrated faster when tested under similar conditions. Alterations in proteins and carbohydrates were most readily observed in specimens containing high titers of antitoxin. There were no demonstrable differences between the electrophoretic behavior of sera obtained from subjects before or after immunization with toxoid. Electrophoretic patterns of serum from allergic subjects who developed marked eosinophilia showed attenuation of the alphas globulin associated with a relative preponderance of slow migrating protein-bound polysaccharides. These alterations were not present in sera obtained from the same persons before and after the development of eosinophilia. Changes in the proteins and polysaccharides could not be demonstrated with consistency in subjects with mild to moderate hay-fever symptoms. One person who developed severe acute hay-fever symptoms showed alterations in the beta and alpha2 globulins. Rheumatic fever subjects showed no unusual changes in the distribution of serum components. However, transition from the acute process to convalescence is graphically demonstrated by the marked decreases in gamma and alpha globulins and in protein-bound carbohydrates.

1. Bartonella muris has been isolated in pure culture on Noguchi's leptospira medium from the blood of the splenectomized adult rat suffering with Bartonella muris anemia. 2. Bartonella muris is a small, actively motile, Gram-negative bacillus. It grows best on media containing blood and on Noguchi's leptospira medium. The optimal temperature for growth is 20–25°C. It produces neither gas nor acid on media containing sugars. It does not hemolyze blood in artificial media. Viability of the cultures in leptospira media was maintained for 36 days. 3. With this culture a severe anemia was produced in rats weighing 30 gm., with the occasional appearance of Bartonella muris bodies on the red cells. The anemia occurred within 24 hours, and lasted for 3 to 5 days with recovery of the rat. Bartonella muris was recovered in pure culture from the blood of these animals. The blood of these rats was infectious for other 30 gm. rats. 4. 3 week old rabbits were infected with cultures of Bartonella muris, a severe anemia resulting after an incubation period of 48 hours. The organism was recovered from the blood on the 5th day after injection. Bartonella muris is non-pathogenic for adult rabbits. 5. A severe anemia was produced in young guinea pigs with cultures of Bartonella muris within 48 hours. The organism was recovered on the 2nd and 5th days after injection. Postmortem examination revealed changes in the organs similar to those found in splenectomized rats suffering with spontaneous Bartonella muris anemia. 6. The infection was reproduced in white mice. In one instance a severe anemia developed on the 5th and 6th days. The organism was recovered on the 6th day. 7. The anemia was produced in splenectomized adult rats of non-carrier stock. The organism was recovered from the blood stream of these rats. A marked leukocytosis was noted (65,000) at the peak of the anemia as is found in the spontaneous disease in infected splenectomized adult rats. 8. Serological tests have thus far failed to demonstrate the production of agglutinins, though complement-fixing antibodies are present in homologous sera in low dilutions.

Diphtheritic neuritis was produced in rabbits and guinea pigs by injection of underneutralized toxin-antitoxin mixtures. The disease is a progressive, ataxic paresis related in severity to the dose of injected toxin-antitoxin. Cerebrospinal fluids of rabbits with this disease showed "albuminocytologic dissociation" similar to that found in the same disease in man. Histologically the disease is a progressive demyelination of the peripheral nervous system, un-accompanied by axis cylinder damage or inflammation. It involves the spinal roots and sensory ganglia in the rabbit, the peripheral nerves in the guinea pig. Diphtheritic neuritis can be distinguished from experimental allergic neuritis in the rabbit and guinea pig on both clinical and histological grounds. The rabbit disease is however an excellent model of diphtheritic neuritis in man. In the animals with diphtheritic neuritis, studied in the present work, there was vigorous production of circulating antitoxin and infrequently complement-fixing antibody to horse serum proteins. No antibody against rabbit spinal cord or sciatic nerve was demonstrated. Skin reactivity to both rabbit nerve suspension and diphtheria toxoid was present at 1 to 2 weeks following inoculation and reached a maximum well before the peak of antitoxin response. These reactions did not seem to be typical delayed reactions. No correlation existed between the development of diphtheritic polyneuritis and any of these immunologic events or the circulating complement level, either in time or in degree. Treatment of rabbits with 400 r whole body irradiation 48 hours before inoculation resulted in severe leukopenia lasting about 3 weeks, delay of antitoxin formation with considerable reduction of peak titers, and some decreased skin reactivity to toxoid. It had no effect on the disease process. It is concluded that diphtheritic polyneuritis is produced by a non-immunologic mechanism, e.g., direct toxicity.

The above experiments demonstrate that guinea pigs and rats subjected to vitamin-deficient diets to a point at which deficiency symptoms appear, and then inoculated with typhus virus, exhibit clinical pictures which indicate a far more severe infection than that observed in normal animals after inoculation. There is also a wider distribution of Rickettsiae and a concentration of organisms which, in pleural and peritoneal exudates, amounts to almost cultural proportions. Important from our point of view is the fact that these experiments furnished a step toward the accomplishment of our purpose, which was to obtain amounts and concentrations of Rickettsiae suitable for immunological studies until such a time when tissue culture may have developed to a practically useful stage. The experiments are of immediate importance in that they furnish us a method for improving our technique of active immunization reported upon in the preceding paper, No. V (8). From the epidemiological point of view these experiments at least suggest an explanation of one of the important factors which enter into the historical association of high typhus mortality with war and famine.

Acute lethal anaphylaxis in the intact rabbit is caused by a failure of the heart. This failure of the heart is due to a change in the heart itself; it is peripheral and independent of the central nervous system for its production. This change in the heart is shown anatomically and functionally by decreased translucency, change in consistency, and by failure to respond to stimuli, and is probably to be classed as a chemical rigor. The rigor of the heart is most pronounced in the right ventricle, the wall of which may be gray, stiff, very tough to the finger nail, and non-irritable. Cardiac stimulants of the digitalis group seem to exert a harmful effect when injected in acute anaphylaxis. Blood coagulation is delayed; a loose clot forms after one half to two hours. Anti-anaphylaxis is produced when the animal does not succumb to the injection. When anaphylactic death is delayed for about one hour, a well developed rigor of the white muscles of the thigh, and of the diaphragm may occur while the animal is still alive. Reasons are brought forward to show the necessity of more caution in employing the word anaphylaxis. Friedberger's statement, that the lungs of guinea pigs dead from acute anaphylaxis are not characteristic of anaphylaxis for this animal, is shown to be baseless.

Separate groups of rats, mice, hamsters, and guinea pigs were caused to inhale virulent tubercle bacilli, of human or bovine strains, as single cells in fine droplet nuclei. Members of each of these eight host-parasite combinations were killed for study at stated intervals after infection. For approximately 3 weeks after the bacilli were deposited in the lungs the progress of the infection, and the reaction of all species to it, followed a highly uniform developmental pattern. During the 4th week the rate and pattern of tubercle formation became distinctive for the species of host and the strain of parasite, but within any host-parasite combination this rate and pattern continued uniform for a time. The duration of this period of homogeneous response after the 4th week varied with the host-parasite combination, ranging from less than 5 weeks to more than 12 weeks after the induction of infection. It is concluded that the highly uniform initial response is evidence that these animals do not differ in their inherent resistance to inhaled infection by the bacilli of human or bovine tuberculosis. Instead, they differ widely in their capacity to acquire resistance to these organisms, as shown by the variation in the later stages of the disease.

The outbreak of infectious abortion in swine, probably the first reported from the eastern United States, was associated with a strain of Bacillus abortus growing rapidly on ordinary nutrient agar slopes without seal and presenting certain slight pathological deviations from the bovine form of disease in guinea pigs such as the occurrence of necrotic, suppurating foci in spleen and lymph nodes. Agglutination tests, comprising both cross-agglutination and absorption procedures failed to distinguish the strain from the bovine type. The gross appearance of the fetuses from this outbreak was normal. The shreds of placentas obtainable indicated slight erosion of the chorionic epithelium and some exudation. The specific bacilli were quite widely disseminated in the tissues of the fetuses. The pathogenic action of this swine strain on guinea pigs was evidently much feebler than that of most earlier swine strains as reported and it approached more closely that of bovine strains. The culture fed to a pregnant sow failed to produce abortion, possibly because of the advanced stage of pregnancy. The organism was not recovered from the uterus but was found in the sow's milk.

Concentrated culture filtrates of two strains of human tubercle bacilli, a virulent and a slightly virulent one, have been fractionated to give fourteen fractions in each case. Chemical determinations and sedimentation velocity measurements have been carried out on those fractions for which significant results could be obtained. The evidence is that two distinct proteins are present, in addition to a polysaccharide and nucleic acid. The physical measurements have not demonstrated the presence of any other proteins. One of the proteins has been isolated in pure form, and found to have a molecular weight of 44,000 ± 5,000, based on measurements of partial specific volume, sedimentation velocity, and diffusion rate. This protein is believed to be the same as one previously isolated by Seibert et al. (6), who assigned it a molecular weight of 32,000. The other protein was not obtained sufficiently free from polysaccharide so that its molecular weight could be determined, but it is believed to have a sedimentation constant of about 2 S. Sedimentation and diffusion constants have been obtained for the polysaccharide, which appears to be a homogeneous molecular species with a molecular weight of about 20,000. The source in unheated tuberculin of the proteins obtained from heated preparations is discussed.

Quantitative relationships between the amounts of encephalitogenic neural tissue and mycobacterial adjuvant are presented for the guinea pig, for comparison with similar results obtained in the mouse and presented in an accompanying paper by Lee and Schneider (27). Definite threshold amounts of both neural encephalitogenic and mycobacterial adjuvant can be defined. With the proper amounts of each component, 100 per cent of guinea pigs can be made to develop EAE, 97 per cent dying of it, and over 50 per cent becoming paralyzed by the 12th day after challenge. With moderate amounts of encephalitogen the severity and incidence of EAE can be very great, but this encephalitogenic potential can be masked if large amounts of mycobacteria are employed. The mechanism of this masking effect by excess adjuvant is not known, but speculation centers upon the possibility of competition of antigens of the mycobacteria at the expense of those of the encephalitogen.

AKR mice with spontaneous leukemia were infused with normal serum from a variety of species. Leukemia cell destruction was produced by serum from strains of mice possessing the full spectrum of complement components, but not by serum from strains with a genetically determined deficiency of C5. Serum from guinea pigs, horses, and humans also causes destruction of leukemia cells. The antileukemic factor in normal serum was heat labile (56°C for 35 min) and could be inactivated by cobra venom factor (CVF). Tests of individual complement factors from guinea pig serum and from human serum suggest that C5 is the antileukemic complement component in normal serum. Evidence was obtained that complement also plays a role in the antileukemic effect of interferon and endotoxin.

Immunization of mice or guinea pigs with BCG rendered all or most of the histiocytes of these animals resistant to necrotization by virulent H37Rv; this cellular resistance was mediated by immune serum. Immune mouse histiocytes (from BCG-immunized animals) were able to induce cellular resistance in normal homologous and heterologous (rabbit) animal species; mouse histiocytic ribosomes were also tested in the homologous species and found to be active. Immune guinea pig histiocytes (from BCG-immunized guinea pigs) were ineffective in transferring cellular resistance to either homologous or heterologous (mouse and rabbit) animal species. Immune rabbit histiocytes were capable of inducing cellular resistance in mice and guinea pigs; rabbit histiocytic ribosomes were also tested in normal mice and found to be active in induction of cellular resistance. Recipient guinea pig histiocytes (from guinea pigs inoculated with immune rabbit histiocytes) were capable of inducing cellular resistance in normal guinea pigs and rabbits. Cultivation of lysed immune histiocytes of all three animal species on glycerol-blood agar medium failed to reveal any viable BCG; this provided one additional bit of evidence against the idea that induction of cellular resistance is due to viable bacilli.

A new species of spirochete which we have called Spirochæta hebdomadis has been described as the specific etiological agent of seven day fever, a disease prevailing in the autumn in Fukuoka and other parts of Japan. This spirochete is distinguishable from Spirochæta icterohæmorrhagiæ to which it presents certain similarities. Young guinea pigs are susceptible to inoculation with the blood of patients and to pure cultures of the spirochete, and those developing infection exhibit definite symptoms suggestive of those of seven day fever in man. The blood serum of convalescents from seven day fever contains specific immune bodies acting spirochetolytically and spirocheticidally against the specific spirochetes, but not against Spirochæta icterohæmorrhagiæ. The field mouse (Microtus montebelli) is the normal host of the spirochetes, which have been detected in the kidneys and urine of 3.3 per cent of the animals examined. The endemic area of prevalence of seven day fever corresponds with the region in which field mice abound.

The kidney of guinea pigs infected with the H37Rv and BCG strains of M. tuberculosis showed a diminution in succinic dehydrogenase activity when measured by the tetrazolium technique. This effect was also seen in the liver and spleen of animals infected with the BCG strain. Sensitized animals showed similar results when given tuberculin in sublethal doses. The succinic oxidase was also low in the kidneys of animals infected with the H37Rv strain. The depressed enzyme activity of the tissues of infected animals could be restored to normal by addition of normal tissue extract or dialysate. This suggests that the alteration in tissue metabolism observed in tuberculosis may depend upon the loss of some as yet unidentified factor important for succinic dehydrogenase activity.

The parameningococci of Dopter are culturally indistinguishable from true or normal meningococci, but serologically they exhibit differences as regards agglutination, opsonization, and complement deviation. Because of the variations and irregularities of serum reactions existing among otherwise normal strains of meningococci it does not seem either possible or desirable to separate the parameningococci into a strictly definite class. It appears desirable to consider them as constituting a special strain among meningococci not, however, wholly consistent in itself. The distinctions in serum reactions between normal and para meningococci are supported by the differences in protective effects of the monovalent immune sera upon infection in guinea pigs and monkeys. It is therefore concluded that it is highly desirable to employ strains of parameningococcus in the preparation of the usual polyvalent antimeningococcic serum. It remains to be determined whether it is better to employ the parameningococci along with normal meningococci in immunizing horses, or to employ normal and para strains separately in the immunization process and to combine afterwards, in certain proportions, the sera from the two kinds of immunized horses.

When a strain of human tuberculosis was carried from guinea pig to guinea pig by intracerebral inoculation the bacillus of tuberculosis could be readily recovered in the semisolid "Leptospira" medium of Noguchi simply modified by the substitution of guinea pig for rabbit serum. Whether even this modification is required remains to be determined. The tubercle bacillus in these cultures and possibly in the nervous tissue as well differed from the classical type in that it was less acid-fast and quite lacking in alcohol fastness. However, it retained the Gram stain as modified by Much. Staining reactions, as this culture of the tubercle bacillus shows, are not absolute qualities but are variable with conditions, a consideration which it may be important to recognize. Efforts to define these conditions more precisely are likely to be productive.

Prior observations on the cellular reactions to tuberculo-phosphatide are confirmed and compared with reactions induced by this material in tuberculous animals. In the latter the response is accelerated and augmented and simulates the Koch phenomenon. Tuberculo-protein produces no macroscopic reaction in normal animals. The microscopic reaction of neutrophiles and monocytes regresses in less than a week. The same material in tuberculous animals causes a response characterized by more or less hemorrhage and necrosis, tissue degeneration, and infiltration of neutrophiles and monocytes. Late in the reaction there may be a few epithelioid cells and foreign body giant cells. Preparations of tuberculo-phosphatide which contain no tubercle bacilli, or only a few, induce the typical cellular response but do not induce hypersensitiveness to tuberculin. Repeated intradermal skin-test injections of tuberculo-protein MA-100 in normal guinea pigs may be followed by a mild hypersensitiveness to subsequent injections.

The virus of vesicular stomatitis is not readily killed by formalin. This chemical is one of a group which coagulates the proteins of the medium in which the virus is usually contained. It has already been found7 that other reagents of the protein-coagulating group are not actively virucidal) and the effect of formalin in this instance is therefore characteristic of the group. The so called formalinized vaccines which give rise to immunity can be shown to have done so because of the presence of living virus. A single injection of such so called "vaccine," or of other material containing living virus, is capable of inducing immunity in guinea pigs. No protection, however, follows a single injection of dead virus. Furthermore, repeated inoculations of virus killed by formalin likewise fail to induce resistance against subsequent injections of the living virus. It is concluded, with respect to the virus of vesicular stomatitis, that the use of formalin has failed to solve the problem of active immunization with dead virus.

1. Following thyroidectomy in guinea pigs, there is a gradual elevation of the surface tension of the blood plasma which reaches its height in from 19 to 22 days after operation. This elevation is probably permanent, since we have found it to persist for as long as 120 days. 2. In our experiments, we found a few thyroidectomized animals in which the surface tension of the plasma was still within normal limits 24 and 120 days after operation. We suggest that these exceptions are due to three possible factors: (1) incomplete thyroidectomy; (2) presence of accessory thyroid tissue; (3) compensatory activity on the part of other organs. 3. The surface tension of the plasma from operated animals is higher than that from controls in both the initial and 20 minute determinations, but the difference is greater at the 20 minute period. 4. The time-drop (difference between the initial and 20 minute determinations) is somewhat greater in the plasma from normal than in that from operated animals. 5. It is suggested that these changes are due to a decrease in the amount of certain normally occurring surface-active substances, the production of which is directly or indirectly dependent upon the thyroid gland.

The PR8 strain of human influenza virus was found to proliferate and disseminate widely in the tissues of fetal guinea pigs inoculated in utero. Large quantities of virus free of bacteria were recovered from lung, liver, and placenta, and smaller quantities from blood and brain, after incubation periods ranging from 2 to 6 days. Although the fetuses proved to constitute an excellent medium for the propagation of influenza virus, they evinced little gross reaction to the infection. Several series of passages from fetus to fetus were accomplished; one consisted of 10 transfers, another of 16. For serial passage the virus was inoculated intracerebrally into half-grown fetuses and the fetal lungs were harvested 48 hours later as a source of virus for subinoculation. It is concluded that multiplication of the virus occurred particularly in the lungs, which may be considered a significant reaffirmation of the pneumotropic tendencies of this virus. Following passage in series the virus was found, on the basis of cross-immunity and cross-neutralization tests, to be immunologically identical with the mouse passage virus from which it was derived. Other properties also appeared to be unaltered by passage of the virus under these conditions.

1. Infection with tubercle bacilli of bovine type produced no alteration in the serum cholesterol content in guinea pigs. 2. Certain controlled variations in diet produced no changes in the serum cholesterol content in guinea pigs. 3. Inbred families of guinea pigs known to manifest differing resistances to tuberculosis gave differing serum cholesterol values, but no direct relationship between the two sets of phenomena could be established. 4. It seems probable that in the guinea pig the cholesterol content of the blood serum is influenced by inherited factors.

The Rickettsia of Rocky Mountain spotted fever were easily differentiated from mitochondria, phagocytosed blood pigment, nuclear debris, and all other known cellular constituents. Although they were lodged within the cytoplasm of endothelial cells, they were not observed to establish any definite relations with the nucleus or with other cellular components. Their number varied in contiguous cells which sustained the same degree of injury as evidenced by nuclear changes, and alterations in their mitochondria content. The mitochondria, on the other hand, showed similar modifications, characterized by a decrease in number and a rounding up into spherules, in all the endothelial cells seen in a section of an affected blood vessel. Diplobacillary forms were most abundant in the early stages of the reaction and single bacillary bodies towards its termination. Other slight differences in morphology from Wolbach's account were noted in the organisms as seen in the tissues of both ticks and guinea pigs. His study of the distribution of specific lesions with accompanying organisms in the tissues of guinea pigs was confirmed and extended.

Following the divisions before used, the results presented in the preceding pages may be briefly stated. I. The particular method of sensitization and the place where the test injection is made have an important bearing on the results obtained by various workers. Comparing the results obtained by the various methods, we may conclude that the incubation period of the hypersensitive reaction is not sharply limited, but that there is a progressive increase in sensitiveness from the sixth day, and presumably before that, extending over a period of several weeks. It seems very probable that the degree of hypersensitiveness attained where the sensitizing dose consists of a mixture of diphtheria toxin and serum is greater than when a single dose of the same small quantity of serum is given alone. II. Our early experiments, the first in this field, are in thorough agreement with those first reported by Otto, and shortly after him by Rosenau and Anderson. III. This hypersensitive reaction is transmissible from mother to offspring. The transmission is probably not equally effective in all cases, and individual young guinea-pigs probably vary greatly in the rate with which they lose their ability to react. As a result not all of the young of a hypersensitive mother react to a subcutaneous dose of five cubic centimeters of serum given when they are four or five weeks old. The reaction in the young animals differs quite markedly from that in those actively sensitized. These differences are such as to indicate that in the mother there is a considerable localization of the reaction in tissues and organs whose destruction does not cause sudden death. This local reaction is a protective factor and is not transmitted to the same degree as the factors involved in the fatal acute reaction. IV. The hypersensitive reaction to horse serum depends on the development of a special anti-body during the incubation period, which anti-body may be passively transferred to a fresh animal. If the dose of hypersensitive serum be sufficient, and the intoxicating injection be given directly into the circulation, this passive hypersensitiveness may be enough so that the animal will die when tested. There is also in the serum of hypersensitive guinea-pigs an uneliminated horse serum element or "rest," which is distinct from this antibody, and probably without influence on the course of the acute reaction. V. The anti-body on which the hypersensitive reaction depends may be entirely neutralized by horse serum without causing symptoms. The gradual introduction of increasing doses over a total period of twenty-four hours suffices for this. The animal is then, properly speaking, neither immune nor refractory, but is essentially in the condition of a normal animal which has recently had a large dose of horse serum. This rapid neutralization is made possible by the great binding power which the subcutaneous and other relatively unimportant tissues have for the toxic element of the serum. The so-called "Phenomenon of Arthus" is probably the same reaction for the rabbit that we have here dealt with in the guinea-pig. The fact that the manifestation is more prominently a local one depends on racial differences. I have encountered cases in the guinea-pig in which the conditions in the rabbit are closely simulated.

1. Normal horse serum was irradiated for periods of 3 to 4 days, with visible light or with ultraviolet light of known intensity and wave length. The photosensitizer hematoporphyrin was employed in some instances. The serum was exposed to the air in thin layers, and thoroughly agitated throughout irradiation. 2. The irradiated sera were unchanged in color, and over 90 per cent of the original protein content remained precipitable by phosphotungstic acid. 3. Studies of the antigenicity of the sera were carried out on guinea pigs and rabbits. Fresh antigenicities of deviated specificity and of an activity of the order of 1/50th, 1/1,000th, and less than 1/20,000th that of normal horse serum were obtained. The residual content of material having the same antigenic specificity as normal horse serum was estimated as approximately equivalent in activity to dilutions of normal horse serum of 1 cc., 1/10 cc., and less than 1/100 cc. per litre respectively.

Guinea pigs given three subcutaneous injections of triton WR-1339, 300 mg./kg., at 3 day intervals develop an intense lipemia as evidenced by marked increases in serum phospholipide, free cholesterol, ester cholesterol, neutral fat, and total fatty acids. The total fatty acid content of the lung, liver, and spleen, however, remains unchanged. No qualitative change in the fatty acids of the lung and liver from treated animals could be detected by the methods used. The injection of triton was found to diminish slightly the cholesterol content of the adrenal glands. The implications of these findings are discussed in relation to an understanding of the mechanism involved in the therapeutic action of triton in experimental tuberculosis.

Pseudorabies virus has been cultivated in series in rabbit testicle, guinea pig testicle, and chick embryo media, and its growth requirements have been studied. Intranuclear inclusions, similar to those produced by pseudorabies virus in vivo, have been found in rabbit testicle cultures. The virus has not changed its pathogenic properties for rabbits, guinea pigs, or mice during the course of cultivation.

Lymphoid cells, prepared from the thymus and lymph glands of rats, when suspended in the serum of x-rayed rats and incubated for 2 hours, increase in number from 15 to 30 per cent, and mitotic figures are found among these cells in fairly large numbers. A like suspension of cells in normal serum undergoes rapid disintegration and in only one instance among a large number of films examined was a mitotic figure found. The stimulative effect of the serum from x-rayed rats endures from 1 to 2 hours after the exposure but is not detectable in the serum taken 17 hours or later after the treatment. Serum x-rayed in vitro is devoid of stimulative action. The lymphoid cells of rabbits and guinea pigs are so fragile as to make impossible the obtaining of counts accurate enough for experimental purposes. The serum of one species caused such rapid disintegration of the cells of another that it was impossible to determine the specificity of the reaction.

The capacity to develop the delayed type of hypersensitivity to diphtheria toxoid and ovalbumin may persist in guinea pigs and rabbits that have received doses of x-ray sufficient to eliminate a detectable antibody response. Larger doses of x-irradiation can prevent development of delayed-type hypersensitivity in rabbits.

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.

Anaphylaxis in the isolated, perfused hearts of cats has been shown to be accompanied by a considerable, though transient, increase in coronary flow. This result is contrasted with that observed in the hearts of guinea pigs and rabbits in which the coronary arteries are constricted during anaphylaxis. Attention is directed to the fact that, in the hearts of these three species, the effects of anaphylaxis and of histamine are qualitatively parallel. The characteristic anaphylactic response in the isolated hearts of guinea pigs has been evoked: (a) in the organs removed from immune animals, (b) by each of two antigens (horse serum and egg albumen) under conditions of double sensitization, and (c) upon exposure of the hearts of passively sensitized animals to the type-specific polysaccharide of the pneumococcus. It is evident that, among the effects of anaphylaxis upon smooth muscle in various organs, there must be considered that upon the coronary arteries.

Outbred Hartley strain guinea pigs capable of responding immunologically to 2,4-dinitrophenylated poly-L-lysine were shown to display a histocompatibility specificity in common with inbred strain 2 guinea pigs. This histocompatibility specificity was not detected in guinea pigs unable to respond immunologically to DNP-PLL. The result suggests that the poly-L-lysine specific immune response gene is very closely linked or even identical with a gene determining a major histocompatibility antigen in guinea pigs.

As determined by the intracutaneous test in guinea pigs, diphtheria toxin is not altered in the presence of cardiac tissue obtained from the fetal or from the adult heart of the guinea pig. Tissue cultures were apparently uninjured by the presence of the toxin in the dilutions used in these experiments, and, when washed with embryo extract after removal of the diluted toxin, continued to grow. Embryonic guinea pig cardiac muscle tissue growing in cultures in vitro possesses the power of neutralizing, binding, or destroying diphtheria toxin so that it is no longer toxic for normal guinea pigs. Such neutralization takes place through the intervention of growing tissue and is a property which is lacking in similar surviving tissue not in a state of cultivation. Thus, it appears that the living, growing cells of the tissues neutralize or destroy limited quantities of toxin; only when the quantity of toxin exceeds a certain limit is its action injurious.