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The antimycobacterial activity of thymus peptide under certain conditions in vitro can be partially neutralized by increasing the concentration of sulfate ions in the medium, and to a lesser extent by the addition of certain organic compounds which contain sulfur. It is suggested that thymus peptide suppresses the growth of tubercle bacilli by interfering with the normal sulfur metabolism of these microorganisms. Polylysine peptide and pituitary adrenocorticotropic hormone, other basic peptides derived from animal tissues, also inhibit the multiplication of tubercle bacilli in vitro, and their antimycobacterial activity is also antagonized by sulfate ions. Basic peptide hormones prepared from the posterior pituitary gland do not affect the growth of acid-fast bacteria under the conditions of the test.

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of beta-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.

Fractionation of rabbit heterophil leukocyte homogenates by isopycnic centrifugation as well as by zonal sedimentation has helped to characterize further the particulate components of these cells. Four classes have been identified: (A) Large (0.5-0.8 microm) and dense (1.26) azurophil or primary granules, containing all the myeloperoxidase, one-third of the lysozyme, and a major proportion of the lysosomal acid hydrolase activities of the cells. (B) Smaller (0.25-0.40 microm) and less dense (1.23) specific or secondary granules, containing 90% of the alkaline phosphatase and the remainder of the lysozyme activities, but very little if any acid hydrolases. (C) Particles of low density (1.20), containing the remainder of the lysosomal acid hydrolases. This fraction was heterogeneous, but showed abundant small rod- or dumbbell-shaped particles of moderate electron opacity, surrounded by a single membrane (tertiary granules?). The possible origin of these lysosomes from contaminating macrophages could not be ruled out but appeared unlikely. (D) Slowly sedimenting material of very low density (1.14), made up of large, empty vesicular membrane structures, and containing 10% of the alkaline phosphatase, and all of a thiol-dependent acid p-nitrophenyl phosphatase, an enzyme clearly different from the lysosomal acid phosphatase.

Nonadherent mouse spleen cells exhibited poor viability and little or no capacity to form antibodies to sheep red cells in the Mishell-Dutton culture system. Viability and antibody-forming capacity could be restored by addition to these cultures of low concentrations of mercaptoethanol (10(-4)-10(-5)M), or by addition of appropriate numbers of mouse peritoneal macrophages. Macrophage concentrations lower than optimal resulted in lower lymphoid cell viability and correspondingly fewer plaque-forming cells, whereas excess macrophages resulted in marked inhibition of antibody formation despite good viability of the lymphocytes. Restoration of the nonadherent cells with mercaptoethanol was thus much simpler and more reproducible than it was with macrophages; furthermore, the number of plaque-forming cells developed in cultures restored with mercaptoethanol was approximately fourfold higher than it was in cultures restored with optimal numbers of macrophages. In the presence of mercaptoethanol, the plaque-forming capacity of the nonadherent spleen cells was not increased when small numbers of macrophages were added to the system, nor was it decreased when the few macrophages present in the nonadherent cells were further reduced or eliminated. Excess macrophages inhibited antibody formation in the cultures containing mercaptoethanol as they did in control cultures. Optimal restoration of plaque-forming capacity to the nonadherent spleen cells with mercaptoethanol required the reducing agent to be present throughout the 4 or 5 day culture period. Addition of mercaptoethanol 1 or more days after initiation of culture, or transfer of the cells to a medium free of mercaptoethanol before completion of the culture resulted in a reduction in the numbers of plaque-forming cells. The results suggest that mouse lymphoid cells do not require macrophages in order to form antibodies to sheep red cells in vitro, provided mercaptoethanol is present in the culture medium. The mechanism of action of mercaptoethanol under these conditions is not completely clear, but one of its effects is to promote the viability of lymphoid cells in the cultures.

The arginine-rich fraction of calf thymus histone (histone B) exerts bactericidal activity on various coliform bacilli and micrococci under certain conditions in vitro. Final concentrations of less than 1 microg. histone per ml. kill susceptible microbes without detectable morphological alteration or lysis. Among the microorganisms highly susceptible to histone are Escherichia, Salmonella, Shigella, Pseudomonas, Klebsiella, and Micrococcus pyogenes var. albus. Less susceptible or completely resistant are Proteus, Serratia, Micrococcus pyogenes var. aureus, and various types of hemolytic streptococci. Coliforms grown on solid media are much more resistant to the lethal effect of histone than are those cultured in liquid media. This difference is apparently related to the physiological state of the bacteria; agar grown microorganisms washed with water remain resistant to histone, whereas incubation in broth rapidly renders them more susceptible. Histone is adsorbed onto heat-killed E. coli K-12 under conditions suitable for lethal action on this organism. The bactericidal activity of histone is but little affected by pH of the test system, but ionic strength of the medium exerts a marked influence, the lethal action being reduced or blocked as the salt concentration reaches levels higher than that of 0.15-0.2 M NaCl. Relatively high concentrations of rabbit serum or of bovine plasma albumin reduce the bactericidal activity of histone in a medium at pH 7; these serum preparations are, however, essentially without effect in the test system at pH 5.6. The bactericidal effect of histone is antagonized by addition to the medium of small amounts of certain basic substances (protamine, spermine), or of various acid polysaccharides (heparin, nucleic acid, bacterial lipopolysaccharides). The rate of killing of E. coli K-12 by histone increases as the temperature and the concentration of histone are raised. Within the limits studied, this rate also appears to be directly proportional to the concentration of bacteria in the system.

The initial morphologic alteration in rabbit polymorphonuclear leucocytes exposed to streptolysin is rapid and extensive lysis of cytoplasmic granules. The granules appear to rupture directly into the cell sap. Within a few minutes following degranulation, the leucocyte rounds up, filamentous processes appear on the cell membrane, the cytoplasm liquefies, and finally the nuclear lobes swell and fuse. Streptolysin O causes these changes in intact leucocytes when added in concentrations only slightly higher than those required for release of hydrolases from isolated liver lysosomes, and furthermore exerts its action on granulocytes promptly. On the other hand streptolysin S acts on white cells only after a 15 to 30 minute delay, and the levels necessary to disrupt granules in leucocytes are considerably higher than those which act on lysosome suspensions. Exposure of rabbit alveolar macrophages to streptolysin O also results in lysis of granules, soon followed by alterations in the cytoplasm and membrane. The observations are in accord with the hypothesis that streptolysins penetrate the leucocyte membrane and bring about lysis of granules. Autolytic enzymes released from the granules might then be responsible for the subsequent damage seen in various other cell structures.

This study attempted to refine the assessment of the effectance of four-year-old children through observation of their play. Two scales were developed; the first scale defined organization, involvement, and interpersonal responsiveness in play, and the second operationally defined psychosexual content of play. The validity and reliability of both scales and the inter correlations among the four major variables were explored. The results confirmed the validity and reliability of the instruments as measures of the children's effectance and important indicators of a child's capacity as a learner at age four.

Rabbit and human plasma can be prepared without resort to anticoagulants by employing low temperatures and non-wetting surfaces. Serum formed after clotting of rabbit plasma devoid of cells and platelets manifests essentially no bactericidal activity on Bacillus subtilis, Bacillus megaterium, and a strain of Staphylococcus aureus. In contrast, rabbit blood serum exhibits this activity to a high degree. Rabbit plasma rich in platelets gives rise to a serum with capacity to kill these Gram-positive microbes equal to that of serum prepared from whole blood. This heat-stable antibacterial agent is efficiently formed or released when platelets are present during the coagulation of plasma, but not on incubation of platelets in heparinized or citrated plasma or in saline. Leucocytes and red cells appear to play no significant role in its production. Rabbit blood serum and plasma serum have similar heat-labile lethal effects on enteric bacilli. Human serum, whether from blood or plasma, manifests much less bactericidal activity on Bacillus subtilis than does rabbit blood serum. These findings serve to emphasize the fact that substances formed or released during coagulation of whole blood may impart to the serum activities not present in circulating plasma.

A stable, water-soluble substance which possesses potent antimycobacterial activity under certain conditions in vitro has been prepared from calf thymus. This substance has been tentatively named thymus peptide. In final concentrations of 1 to 10 microg. per ml. of an albumin medium it inhibits the growth of various strains of mammalian mycobacteria, but manifests only little or no inhibitory activity against a variety of other microbial species. The ability of thymus peptide to inhibit the multiplication of tubercle bacilli diminishes when the inoculum is large, or when the medium is acidic. It is also markedly antagonized by addition of enzymatic hydrolysate of casein or beef heart infusion broth to the culture medium. Thymus peptide does not exert a rapid bactericidal action on tubercle bacilli, but organisms exposed to this compound for longer than 2 weeks could not be made to multiply in ordinary culture media. Substances similar or identical to the thymus peptide preparation could be extracted from calf spleen, sheep thymus, beef lymph nodes, and calf pancreas, but not from calf lung or calf liver.

Electron microscopic observations have established the mechanisms underlying degranulation of neutrophils and eosinophils during phagocytosis. The content of the granules is released into the phagocytic vacuole when their membranes fuse with the membrane lining the vacuole. The discharge mechanism appears to be applicable to the various morphological types of granules seen in rabbit polymorphs. It has been proposed that the release of material to the outside of the cell or the discharge of material into intracellular vacuoles, events involving membrane fusion, be termed "exoplasmosis."

A technique has been developed for collecting large numbers of polymorphonuclear leucocytes from peritoneal exudates in rabbits. These cells are obtained essentially free from other cell types and from debris. When microphages so procured are disrupted by physical methods and extracted with aqueous salt solutions, the soluble fraction manifests striking bactericidal activity, especially on Gram-negative enteric bacilli. The susceptible microorganisms are not lysed. This bactericidal substance, which has been called phagocytin, appears to be limited in distribution mainly to the polymorphonuclear leucocyte. No phagocytin is present in extracts of rabbit heart, kidney, or skeletal muscle, and rabbit liver and spleen contain much less than do packed leucocytes. Extracts of human and of guinea pig microphages show less bactericidal activity than rabbit cell preparations. Similar extracts of rat and mouse polymorphonuclear leucocytes contain no demonstrable phagocytin. As indicated by its behavior on dialysis, on exposure to proteolytic enzymes, and on salt fractionation, phagocytin appears to be a protein with general properties characteristic of a globulin. It is clearly different from lysozyme and from properdin. Although phagocytin is reasonably stable at temperatures of 65 degrees C. and lower for several hours, solutions of it gradually lose bactericidal activity on standing for prolonged periods at 4 degrees C. This instability, and also the ease with which phagocytin is inactivated, presumably by adsorption, on exposure to a variety of materials, have thus far rendered fruitless efforts to isolate it.

Plasma membranes have been isolated from pure populations of rabbit alveolar macrophages which were swollen in water, fixed briefly with glutaraldehyde, disrupted by Dounce homogenization, and separated by sucrose gradient centrifugation. The recovered membranes exhibited good structural preservation and enzymatic activity; both morphologic and biochemical evidence indicated a high degree of purity (>90%) of the membrane preparation. Interiorized plasma membranes were also prepared without exposure to glutaraldehyde from phagocytic vacuoles recovered from alveolar macrophages which had ingested large numbers of polystyrene spheres. These membranes were contaminated with lysosomal constituents, but they were nevertheless of value for comparison to the "pure" membranes isolated by the glutaraldehyde procedure. Acrylamide gel electrophoresis of the solubilized plasma membranes and phagolysosomal membranes revealed similar protein patterns, with seven to nine individual components ranging in molecular weight from 70,000 to 140,000. The two most rapidly migrating components gave positive reactions for lipid as well as protein. A band containing carbohydrate was detected near the origin of the plasma membrane gels. Antisera were made by injecting guinea pigs with the purified rabbit alveolar macrophage plasma membranes. Gel diffusion and immunoelectrophoretic study of these antisera established the presence of rabbit immunoglobulin G and of one or two other antigenic constituents in the membrane preparation.

The structure of unstimulated mouse peritoneal phagocytes has been examined by electron microscopy and compared to cells obtained from the inflamed peritoneum and from cultures maintained in vitro. The unstimulated cell resembles the blood monocyte and contains a moderate amount of rough surfaced endoplasmic reticulum, a small but well defined Golgi apparatus and a few, small, electron-opaque granules in the cytoplasm. During in vitro cultivation there are marked changes in cell ultrastructure. Most prominent is the formation of large electron-opaque granules, some of which have a complex matrix containing both electron-opaque and lucent vesicles. In addition, there is an increase in size of the Golgi apparatus with the appearance of new lamellae and tiny, smooth surfaced vesicles. With continued cultivation, large lipid droplets are found in apposition to the rough endoplasmic reticulum. The formation and size of electron-opaque granules as well as the enlargement of the Golgi region is stimulated by high concentrations of serum in the medium. Cells obtained from the peritoneal cavity of lipopolysaccharide stimulated animals demonstrated changes in ultrastructure similar to those seen in cells cultured in vitro.

Rabbit alveolar macrophages were incubated in vitro with radioactive protein precursors. Plasma membranes were isolated from these cells, dissolved in phenol-urea-acetic acid, and separated by acrylamide gel electrophoresis. (3)H-leucine was rapidly incorporated into membrane protein. The rate of labeling with (3)H-leucine was markedly different from one protein band to another, indicating heterogeneous or multistep synthesis and assembly of proteins in the alveolar macrophage plasma membrane. Cells incubated with (3)H-choline incorporated this compound into membrane lecithin. On gel electrophoresis the label derived from choline was located in the two bands migrating most rapidly towards the cathode. Studies on cells incubated with (3)H-glucosamine revealed incorporation of label into two protein bands, one located near the origin and the other migrating rapidly towards the cathode. The in vitro techniques were also employed for pulse-chase studies to gain information on rate of turnover of macrophage plasma membrane proteins. This turnover rate was rapid, with a half-life of approximately 8 hr. The radioactivity disappeared from the several protein bands at the same rate, suggesting bulk removal of membrane rather than catabolism of the individual proteins in situ. Endocytosis seems a likely mechanism to account for a major part of the plasma membrane removal. Studies on the protein components of phagolysosomal membranes from cells which had been labeled with (3)H-leucine revealed the presence of all of the major labeled protein bands characteristic of the plasma membrane except one, thus confirming the bulk interiorization of large segments or units of plasma membrane by endocytic processes.

The intracellular flow of tritiated lysine as revealed by electron microscope radioautography was studied in heterophilic myelocytes of rabbit marrow. Label over the Golgi complex rose to a maximum of 37% of total cytoplasmic grains 30 min after initial exposure to the tracer and fell to 11% after 3 to 4 hr of incubation. Coincident with decrease in label over the Golgi complex, grain counts over granules rose to 32% after 3 to 4 hr. The time sequence of incorporation and flow of tritiated lysine and the per cent distribution of label was similar in bone marrow myelocytes under in vivo and in vitro conditions. The results demonstrate a function of the Golgi complex in incorporating or packaging certain basic amino acids or proteins into cytoplasmic granules of heterophilic myelocytes.

The bactericidal activity of phagocytin on Gram-negative enteric bacilli is influenced by the reaction of the medium; the more acid the environment, the more marked is the activity. Phagocytin exerts approximately the same action whether citrate, acetate, or phosphate salts are used as buffer, and the addition of glucose, casein hydrolysate, or cation binding agents does not produce notable change. Although proteins in general have but little effect, the inclusion in the medium of a high concentration of bovine plasma albumin neutralizes the lethal action of phagocytin on enteric bacilli. Very high concentrations of magnesium or calcium ions antagonize but do not completely block the bactericidal effect. The bactericidal activity of phagocytin is essentially independent of the numbers of bacteria exposed; a 100-fold increase m the numbers of enteric bacilli results in approximately a 2-fold increase in the concentration of phagocytin required to kill 90 per cent of them. When susceptible microorganisms are exposed to phagocytin at 0 degrees C., practically no killing takes place. At higher temperatures the bactericidal action is rapid, being well advanced in 5 minutes and complete within 30 minutes.

A crystalline substance capable of suppressing the growth of a variety of mycobacteria in vitro has been isolated from extracts of tissue in acidified dilute ethanol. This inhibitory material was found to be equally active against virulent, attenuated, and avirulent variants of human and bovine tubercle bacilli, but had little or no effect on saprophytic mycobacteria and on several non-acid-fast microorganisms under the conditions of the test. Its inhibitory activity on the growth of tubercle bacilli was essentially independent of the size of the inoculum within the limits studied. The crystalline material appeared to exert a bactericidal action on the susceptible organisms. Tubercle bacilli maintained in the presence of the agent for 4 days failed to grow when transferred to inhibitor-free media. The findings were not appreciably altered by minor variations in the composition of the medium or by shift in its reaction. When certain preparations of whole serum were used in the medium in place of albumin, no antimycobacterial activity was observed; however, this activity was restored by adding bovine albumin (fraction V) to the media containing whole serum. By chemical purification and analysis, the inhibitory material was identified as spermine, an organic base widely distributed in animal tissues.

A combined morphological, autoradiographic, and cytochemical study at the electron microscope level has been directed towards the formation of electron-opaque granules of cultured macrophages. Labeling of the membrane-bound vesicular structures of pinocytic origin was accomplished with colloidal gold. The initial uptake of gold occurred within micropinocytic vesicles. These electron-lucent vesicles subsequently fused with and discharged their contents into larger pinocytic vacuoles. Colloidal gold was homogeneously distributed in the large pinosomes. In contrast, gold was initially deposited in the periphery of preformed dense granules indicating that these structures were also in constant interaction with the external environment. Colloidal gold was not observed within the cisternae of the endoplasmic reticulum nor within the saccules or vesicles of the Golgi apparatus. There were, however, many small, gold-free vesicles, indistinguishable from Golgi vesicles, which were preferentially aligned about and appeared to fuse with the large pinosomes. The intracellular flow of leucine-H(3)-labeled protein was followed by electron microscopic autoradiography. After a 15 min pulse of labeled amino acid there was initial labeling of the rough endoplasmic reticulum. Subsequently, much of the label appeared in the Golgi complex. At still later time periods the cytoplasmic dense granules contained the majority of the isotope. Acid phosphatase activity was localized to the dense granules and in the majority of cells to the Golgi apparatus. It is suggested that hydrolytic enzymes are initially synthesized in the endoplasmic reticulum and are then transferred to the Golgi apparatus. Here they are packaged into small Golgi vesicles which represent the primary lysosome of macrophages. The Golgi vesicles subsequently fuse with pinosomes, thereby discharging their hydrolases and forming digestive granules or secondary lysosomes.

Control and phagocyting populations of rabbit polymorphonuclear leucocytes have been compared in terms of the content and distribution of phagocytin and selected hydrolytic enzymes. Following incubation at 37 degrees C., the cells were disrupted by homogenization and separated into an 8,200 g pellet and supernatant fluid. The high speed pellet from control leucocytes contained all the larger particulate elements of the cell including intact cytoplasmic granules. The ingestion of large numbers of heat-killed bacteria was accompanied by a fourfold reduction in the total phagocytin content of the leucocyte, whereas phagocytosis did not influence the recovery of histone-like bactericidin from nuclei. Engulfment of microorganisms led to a progressive decrease in the activity of phosphatases, beta glucuronidase, and cathepsin extractable from the 8,200 g pellet, with a concomitant increased activity of these enzymes in the supernatant fraction. No significant difference in the total enzyme content of control and phagocyting cells was noted. These findings are consistent with the lysis of granules following phagocytosis and the liberation of granule constituents into the cytoplasm of the leucocyte.

PHAGOCYTIN AND HISTONE DIFFER SIGNIFICANTLY IN THE FOLLOWING REGARDS: (a) the bactericidal action of histone is rapidly lost on peptic digestion, while that of phagocytin is but little affected; (b) the lethal effect of phagocytin on coliform bacteria is much more resistant than that of histone to antagonism by spermine or by increasing ionic strength of the medium; (c) phagocytin can be extracted from disrupted granulocytes with dilute citric acid whereas effective extraction of histone requires stronger mineral acid or strong salt solution; (d) phagocytin is limited in distribution to polymorphonuclear leucocytes while histone is demonstrable in many tissues. A new technique has been devised which permits extraction of phagocytin essentially free of lysozyme and histones. Phagocytin thus prepared kills certain Gram-positive bacteria as well as Gram-negative bacilli under appropriate in vitro test conditions. Among susceptible Gram-positive microbes are Group A streptococci and staphylococci. Phagocytin is demonstrable in citric acid extracts of granulocytes obtained from rabbit, man, horse, and guinea pig, the only species thus far investigated. Circulating blood leucocytes as well as exudate cells contain this bactericidal substance. The lethal effects of phagocytin on bacteria may be influenced, depending on the particular microorganism, by either pH or ionic strength of the medium. The bactericidal action of phagocytin is only slightly reduced following digestion with trypsin, chymotrypsin or papain. The active ingredient is, however, non-dialyzable and apparently precipitated by trichloracetic acid. Data available at present are insufficient to define the chemical nature of phagocytin.

A marked reduction in numbers of cytoplasmic granules in rabbit and human polymorphonuclear leucocytes takes place following ingestion of various microorganisms or of a yeast cell wall preparation. The degranulation occurs within 30 minutes of phagocytosis, and is directly related to the quantity of material engulfed. White cells completely degranulated following phagocytosis of large numbers of microorganisms remain viable for at least 1 hour. The granules of polymorphonuclear leucocytes contain the antimicrobial agent, phagocytin, and various digestive enzymes. These substances thus are released into the cytoplasm or into vacuoles following ingestion of foreign material. The granule system and granule lysis mechanism may well play a central role in the primary function of these specialized cells; namely, that of destroying invading microorganisms.

Antisera to rabbit polymorph granules and to rabbit erythrocytes have been prepared in guinea pigs. Both antigranule and antierythrocyte sera are hemolytic and both exhibit striking cytotoxicity on leucocytes. The sequence of toxic events, as observed by phase contrast cinemicrophotography and electron microscopy, consists of explosive granule lysis, cell swelling, cytoplasmic liquifaction, and nuclear fusion. Other rabbit cells are also susceptible to these cytotoxic effects, but cells, including polymorphs, of other mammals are not. Cytotoxic action of the antisera requires, in addition to the antibody, heat-labile serum factors and divalent cations, suggesting that the action is a combined one of antibody and complement. The morphologic observations have been supported by biochemical studies demonstrating release into the medium of granule-bound hydrolases following exposure of polymorphs or of isolated granules to the antigranule or antierythrocyte sera. Granulolytic activity of the antisera can be reduced or removed by absorption with either rabbit leucocyte granules or with erythrocytes, indicating that leucocyte granules and erythrocytes have an identical or similar membrane constituent. The observations lend support to the notion that lysosomal hydrolases may exert autolytic effects in some situations.

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

Metallic copper in the uterine or abdominal cavities of rats or monkeys stimulates an impressive local exudation of polymorphonuclear leukocytes. This cellular response to copper persists for at least 7 months, without significant local tissue damage or detectable systemic effects on the test animal. This finding provides a possible explanation for the capacity of copper to increase impressively the antifertility effect of polyethylene intrauterine contraceptive devices.

Phagocytosis of yeast cell walls and of Bacillus megaterium by human, rabbit, and chicken polymorphonuclear leucocytes has been observed by phase contrast microscopy and recorded on motion picture film. In suitably thin preparations intracellular events could be visualized well. Lysis of cytoplasmic granules began early in the course of the ingestion process, rupture occurring only in granules adjacent to the microorganism being engulfed. Formation of a visible vacuole about the ingested particle frequently followed degranulation. Chicken polymorphonuclear leucocytes, with their large phase-dense granules, were particularly suitable subjects for observations on detailed morphologic aspects of granule lysis. Rupture took place rapidly (0.1 second or less); in place of the granule there appeared a clear zone, often with a small phase-dense round structure in its center. Also accompanying granule lysis was an increase in phase density of the adjacent surface of the microorganism. Over the course of the following few seconds the darkening on the organism faded, the dense small body disappeared from view, and the clear zone contracted towards the engulfed particle. The observations are discussed in relation to the hypothesis that fusion takes place between the granule membrane and the invaginated cell membrane overlying the ingested particle, with discharge of granule contents directly into the phagocytic vacuole.

A chronic infiltration of polymorphonuclear leukocytes was invariably found in the infertile regions of uteri containing foreign bodies in conventional rats, germfree rats, mice, and rabbits. Polymorphonuclear leukocytes were never found in the fertile regions of these uteri. A foreign body in the uterus of the rat, and probably also the mouse, was associated with a bacterial infection which spread the inflammatory response throughout the horn containing the foreign body, and in the mouse occasionally into the control horn as well. No bacteria could be cultured from the rabbit uterine horn containing a foreign body. In the germfree rat, both the infiltration of polymorphonuclear leukocytes into the uterus and fertility were significantly different from that observed in the conventional rat. Whereas in the conventional rat the inflammation and infertility extended along the entire length of the uterine horn containing a small foreign body, in the germfree rat the inflammation and infertility were closely correlated to the position of the foreign body. As judged by measurements of lysozyme in the uterine lumens of rats and rabbits, polymorphonuclear leukocytes released their contents into solution in the uterine lumen. It is concluded that some substance derived from polymorphonuclear leukocytes may exert toxic effects on fertilized ova or on spermatozoa and thus be responsible for the infertility of uteri containing foreign bodies.

Spermine inhibits the multiplication of tubercle bacilli in vitro only if another tissue substance is present in the culture medium. This activating substance occurs as a chemical contaminant of the albumin in commercial bovine plasma fraction V ordinarily added to culture media; it is also found in whole bovine and sheep serum and in aqueous extracts of the guinea pig kidney. No detectable spermine activator is contained in whole human, guinea pig, or rabbit serum. The serum constituent which renders spermine inhibitory for the growth of acid-fast bacteria has the properties of a protein of the alpha globulin classification. This protein is an enzyme which acts on spermine. Presumably, some product of the enzymatic reaction exerts a toxic effect on mammalian tubercle bacilli.