Showing papers in "Journal of Experimental Medicine in 1956"

Action of x-rays on mammalian cells.

Abstract: The effects of x-irradiation have been quantitatively studied on single cells of a human cervical carcinoma (HeLa) under conditions such that 100 per cent of the unirradiated cells reproduce in isolation to form macroscopic colonies. This technique eliminates complexities due to interactions of members of large cell populations. Survival of single cells (defined as the ability to form a macroscopic colony within 15 days) yields a typical 2 hit curve when plotted against x-ray dose. The initial shoulder extends to about 75 r, after which a linear logarithmic survival rate is obtained, in which the dose needed to reduce survivors to 37 per cent is 96 r. This radiation sensitivity is tens to hundreds of times greater than that of any microorganism for which the equivalent function bas been studied. Evidence, though not proof, is presented that the lethal effect is due to a radiation-induced genetic defect which, however, cannot be a simple single gene inactivation. The locus of the action could be chromosomal. Beginning at doses of 100 r, or possibly earlier, growth-delaying effects of radiation are visible. Cells in which the ability to reproduce has been destroyed by doses below 800 r, can still multiply several times. At higher doses even a single cell division is precluded. A large proportion of the cells killed by radiation at any dose gives rise to one or more giant cells. These metabolize actively, grow to huge proportions but never reproduce under the experimental conditions employed. Methods of preparing large populations of giant cells are described. These giants are particularly susceptible to virus action. Some of the irradiated cells disappear from the plate, presumably by disintegration. This action of radiation is by far the least efficient, since even after 10,000 r, 5 to 10 per cent of the original cell inoculum is recoverable as giants.

Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer.

Abstract: Two methods for simple and rapid plating of single HeLa cells, human, carcinomatous cells, are described. These result in growth and formation of colonies from each single cell. One of these procedures uses irradiated, non-multiplying "feeder" cells to condition the medium. The second requires more gentle handling of the cells, but otherwise is virtually the same as that used in plating bacteria on semisolid, nutrient media. By extension of these methods, it is possible to isolate single mutant colonies and grow pure clonal stocks of animal cells. These genetically uniform strains are much more homogeneous in their behavior than the parental HeLa cell population. Growth curves obtained from developing colonies are highly reproducible. The most active mutant stocks so far isolated display a generation time of 18 to 20 hours. In pooled human serum HeLa cells assume a highly stretched, ameboid form, with marked motility; whereas growth of the same cells in a variety of non-human sera results in tightly packed, columnar, epithelial-like morphology. The two cell types possess volumes, nuclear cross-sections, plating efficiencies, and generation times which are identical within experimental error, but display widely different cross-sectional areas, suggesting that the basic change occurs in the cell surface. It is conceivable that this change may be related to that which enables the cells of a compact tumor to become invasive. Animal cells subjected to the standard trypsinization procedures which involve mechanical trauma and repeated washings in incomplete media leak large amounts of P and suffer impaired ability to reproduce as isolated cells. Application of the methods described in this paper as a tool for quantitative study of normal mammalian cell growth, physiology, genetics, and biochemistry, and the response of cells to drugs, viruses, high energy radiation, and other agents have been indicated.

The preparation and function of the hypertensin-converting enzyme.

Abstract: It has been shown by use of isolated, perfused rat kidneys that hypertensin II is a potent vasoconstrictor substance while hypertensin I is not. Hence it would appear that in intact animals the pressor activity of hypertensin I results from its rapid conversion to hypertensin II. An enzyme which effects this conversion has been procured from horse plasma in a semipurified form by means of ammonium sulfate fractionation and isoelectric precipitation. A method is described for estimating the activity of the enzyme. An example of the use of the preparation in converting purified hypertensin I to hypertensin II has been described.

Genetics of somatic mammalian cells : i. demonstration of the existence of mutants with different growth requirements in a human cancer cell strain (hela).

Abstract: The parental HeLa cell population, a morphologically uniform, human cancer cell strain, grown for several years in tissue culture by procedures always involving massive inocula, has been shown to contain different mutant cell types. Two clonal lines have been isolated and established as reliable stock cultures. Both strains exhibit 100 per cent plating efficiency in high or low serum concentrations in the presence of a feeder system. In the absence of a feeder system and in low serum concentrations, the two strains are quantitatively differentiable: S3 still exhibits 100 per cent plating efficiency, while that of S1 lies in the neighborhood of zero. These differences have remained stable throughout 100 successive generations of growth of each strain including 2 single cell isolations. Application of these techniques to studies in the genetics of mammalian somatic cells and to specific cell-cell interactions has been indicated.

The properdin system and immunity iii. the zymosan assay of properdin

Abstract: A detailed method for the zymosan assay of properdin is described, together with procedures for the preparation and standardization of reagents employed in the test. The reliability and reproducibility of the assay, as well as its limitations, are discussed.

Studies on the o antigen of salmonella typhosa v. enhancement of antibody response to protein antigens by the purified lipopolysaccharide

Abstract: Quantitative studies have demonstrated that a purified lipopolysaccharide (endotoxin) derived from the O-901 strain of Salmonella typhosa markedly enhanced the antibody response of rabbits when given separately or in conjunction with protein antigens. The augmentation of antibody levels varied from 2- to 40-fold with the number of injections, the dosage of antigen and endotoxin, and the route of administration. This antibody-enhancing property was found to be common to a broad group of endotoxins from Gram-negative bacilli and was not restricted to the lipopolysaccharide derived from S. typhosa. Factors affecting this enhancement were investigated, and data are presented which indicate that host susceptibility to endotoxin is a prerequisite for elevation of antibody levels; the intact lipopolysaccharide, as isolated, might not be essential for this activity; and the rate of clearance of antigen from the circulation of rabbits was accelerated when endotoxin was given in conjunction with protein. The data obtained are discussed in relation to postulated mechanisms on the antibody-enhancing action of endotoxin.

The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug.

Abstract: Populations of tubercle bacilli of human origin exposed in vivo to pyrazinamide and a companion drug, vanished from the tissues of the mouse in so far as could be determined by microscopy, culture, or guinea pig subinoculation. The vanishing did not represent a complete elimination of the tubercle bacilli from all the animals. 90 days after the completion of treatment, tubercle bacilli could be cultured from approximately one-third of the animals examined at that time. This complete disappearance of the tubercle bacilli thus meets the definition of a truly latent infection in that the infection is present but is hidden beyond the limits of diagnostic reach. All but one of the strains of tubercle bacilli which survived in the animals and were detectable in the posttreatment period, were susceptible to pyrazinamide when tested under appropriate conditions in vitro. Only two factors could be identified which were essential for the uniform occurrence of the disappearance of tubercle bacilli: the administration of the pyrazinamide in a high daily dosage for at least eight of a total of 12 weeks of antimicrobial therapy; and the concurrent or prior exposure of the microbial populations to isoniazid or in some cases to other antituberculous drugs. The observations suggest that the ability of pyrazinamide-containing chemotherapies to bring about the disappearance of a tubercle bacillus is closely related to the occurrence of some alteration in the bacillus, essential for maximal pyrazinamide action, in response to environmental influences, including other antituberculous drugs present in the environment.

Reversible collapse of rabbit ears after intravenous papain, and prevention of recovery by cortisone

Abstract: A substance has been demonstrated in solutions of crude papain, which, when injected intravenously into 1 kilo rabbits, in amounts less than 5 mg., results in complete collapse of both ears. The phenomenon becomes visible 4 hours after injection, and is complete within 24 hours. 3 or 4 days after papain, the ears gradually reassume their normal form. Ear collapse is associated with depletion of the ear cartilage matrix, and the disappearance of basophilia from the matrix. Similar changes occur in all other cartilage tissues, including bones, joints, larynx, trachea, and bronchi. At the time when the ears are restored to normal shape, the basophilic matrix reappears in cartilage. Repeated injections of papain, over a period of 2 or 3 weeks, bring about immunity to the phenomenon of ear collapse. When the arterial circulation to one ear is occluded for 15 minutes at the time of injection of papain, this ear is protected against collapse. The effect of crude papain could not be reproduced by crystalline papain protease or crystalline papain lysozyme, which together comprise a considerable portion of the dry weight of papain. The nature of the responsible factor has not been determined, and the possibility that chymopapain may be implicated is currently under study. Cortisone prevents the return of papain-collapsed ears to their normal shape and rigidity. Possibly this reflects a capacity of cortisone to impede the synthesis or deposition of sulfated mucopolysaccharides in tissues.

5-Hydroxytryptamine and histamine as mediators of the vascular injury produced by agents which damage mast cells in rats.

Abstract: Each of the four agents, ovomucoid, dextran, 48/80, and testis extract, when injected beneath the skin of the dorsa of the paws of rats produces a local vascular injury characterized by a protein-rich edema. Each agent also produces damage to mast cells. Either 5-hydroxytryptamine or histamine produces a response similar in the gross to that elicited by the agents which damage mast cells; however, neither of these two agents produces mast cell damage. On a weight basis 5-hydroxytryptamine is a much more potent edema-producing agent than histamine. The edema-producing action of 5-hydroxytryptamine can be differentiated from the similar action of histamine by the use of specific antagonists; dibenamine is a 5-hydroxytryptamine antagonist and pyrilamine a histamine antagonist. The edema produced by the mast cell-damaging agents is partially inhibited by dibenamine but is not diminished by pyrilamine. It is completely inhibited by treatment of rats with both drugs. The drugs which inhibit edema do not prevent mast cell damage by ovomucoid, dextran, 48/80, or testis extract. The observations are consistent with the hypothesis that agents which damage mast cells, "release" both 5-hydroxytryptamine and histamine and that in the rat the edema associated with mast cell damage is mediated largely by 5-hydroxytryptamine.

Phagocytin: a bactericidal substance from polymorphonuclear leucocytes.

Abstract: 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°C. and lower for several hours, solutions of it gradually lose bactericidal activity on standing for prolonged periods at 4°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.

Fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. I. The persistence of drug-susceptible tubercle bacilli in the tissues despite prolonged antimicrobial therapy.

Abstract: Observations are presented on the behavior of populations of tubercle bacilli in the tissues of mice during the administration of antimicrobial drugs. The behavior of the populations during therapy with any particular drug was different depending upon whether the tubercle bacilli were subsisting in the lung or in the spleen. Moreover, the pattern of microbial behavior was distinctive and predictable for each drug studied. Changes in the size of the populations of tubercle bacilli in the tissues appeared to be a more sensitive reflection of drug influence than microscopic study of the number and character of the tuberculous lesions. Nevertheless, in untreated animals, pulmonary lesions evolved and progressed steadily to a fatal outcome despite the fact that the populations of tubercle bacilli had stabilized at a relatively high census early in the course of therapy. The uniform persistence of tubercle bacilli in the spleen throughout prolonged drug administration was demonstrated with every drug or multiple drug regimens except for pyrazinamide when accompanied by isoniazid. Cultures of the bacilli which survived in the tissues despite antimicrobial therapy were highly susceptible to the drugs employed when tested in vitro. Thus the survival of the tubercle bacilli in the tissues represented microbial persistence rather than drug resistance. When pyrazinamide and isoniazid were administered together, it was not possible to detect the microorganisms in the spleen or lungs of treated animals. A detailed investigation of this apparent abolition of microbial persistence forms the subject of an accompanying report.

Clonal growth in vitro of epithelial cells from normal human tissues.

Abstract: Tissue culture strains of cells from four different normal human tissues—liver, conjunctiva, kidney, and appendix—have been grown by the plating procedure previously developed for the HeLa strain of cervical carcinoma cells. This technique results in colony formation from isolated single cells, in a manner completely analogous to the plating of bacteria in semisolid nutrient media. Clonal cell strains have been isolated from each cell type. All behaved exactly alike in all properties studied except that some differences in plating efficiency were displayed in some of the growth media employed. The cells from normal human tissues resembled the HeLa S3 carcinomatous cell in the following properties:— (a) Single cells displayed a plating efficiency close to 100 per cent in an appropriate medium. (b) They all grew as an epithelial sheet on glass, the cells being closely packed and polygonal in shape. (c) They had mean generation times of 20 to 23 hours in the nutrient media employed, (d) The mitotic frequency was constant, and therefore the duration of mitosis was the same for all the strains studied, (e) The incidence of multinuclearity and giant formation was very low and similar in both types of cells. (f) Both classes of cells had the same total volume, and the same nuclear cross-sectional area. (g) Both also showed a tendency to spread more in the presence of human serum (concentration of 20 per cent or more) than in porcine serum. However, this differential morphological response was much more marked in the HeLa cell than in those from normal tissues. The only difference noted in the behavior of these two groups of cells lay in the tendency of the cells from normal tissues always to exhibit a greater cross-sectional area when spread on glass than the HeLa cell in the same medium. The frequency of occurrence of different types of multinuclearity in the HeLa cell and cells from normal tissues has been measured. The data suggest that multinuclearity depends on two factors: a necessary, predisposing state in the cell, and a random, independent event causing the appearance of an additional nucleus in such a prepared cell.

Experimental Enteric Shigella and Vibrio Infections in Mice and Guinea Pigs.

Abstract: A method has been devised for inhibiting the normal enteric flora, permitting long term asymptomatic enteric infections of mice and guinea pigs with streptomycin-resistant strains of Shigella flexneri or Vibrio cholerae. Introduction of a streptomycin-resistant strain of E. coli into the intestinal tract of experimental animals resulted in a rapid elimination of the enteric pathogens studied. No in vitro production of antibiotic substances by this coli strain could be demonstrated. Active and oral passive immunization did not noticeably influence the number of Shigella or Vibrio organisms recoverable from the feces of infected animals.

The amino acid sequence of hypertensin. II.

Abstract: The amino acid sequence of horse hypertensin II has been determined by the use of chymotrypsin, the fluorodinitrobenzene method, and stepwise phenylisothiocyanate degradation. The results indicate that the amino acids of hypertensin II are arranged in the following order: asp-arg-val-tyr-iso-hist-pro-phe.

Association of a new type of cytopathogenic myxovirus with infantile croup.

Abstract: Viruses producing an unusual "sponge-like" cytopathogenic change in monkey kidney tissue culture were isolated from the pharyngeal swabs of 2 of 12 infants with croup. The infants from whom the viruses were isolated and 3 additional patients developed significant increases in neutralizing or hemagglutination-inhibition and complement-fixing or all 3 varieties of antibody during convalescence. The isolated agents appeared to be similar antigenically. Fluid from infected monkey kidney tissue culture agglutinated chick erythrocytes and in lower titer human "O" red cells. Hemagglutination occurred best at 4°C. and pH 8.0. Agglutination was reversed at 37°C. but resuspension and sedimentation of red cells at 4°C. resulted in a restitution of positive patterns. In addition, the virus was capable of partially removing receptors from the erythrocyte surface, but only when large quantities of virus were incubated with red cells for 24 hours at 37°C. and small doses of hemagglutinin used to test the treated cells. RDE removed both the erythrocyte receptors and the inhibitor for hemagglutinin present in certain normal sera. Low level multiplication occurred at a slow rate in the amniotic cavity of the fertile hen' egg. Gradocol membrane filtration yielded a size of 90 to 135 mµ. The virus was stable at –70°C. but infectivity was lost after treatment with 20 per cent ether for 15 hours. The properties of the isolated viruses were consistent with those required for classification in the myxovirus group. No antigenic relationship with influenza A, A', B, and C, Newcastle or Sendai viruses was found. The viruses were distinct from mumps virus but the existence of a common antigen was suggested. The high incidence of infection with this new virus in one group of croup patients suggests that it may be at least one of the etiologic agents of this clinical syndrome, but more extensive control studies will be necessary to establish a specific etiologic association. For the present the group will be referred to as CA viruses; i.e., croup-associated viruses.

The properdin system and immunity. V. The bactericidal activity of the properdin system.

Abstract: Methods for the preparation and standardization of reagents suitable for studies on the bactericidal action of the properdin system are described. The preparation and properties of serum free of properdin (RP(b)) are presented in detail because of the necessity for a suitable RP(b) in these studies. The properdin system is responsible for the bactericidal action of normal human serum against a variety of microorganisms. The present work shows that the removal of properdin from serum also removes bactericidal activity. Addition of properdin to properdin-deficient serum restores bactericidal activity. A quantitative relationship exists between the final properdin concentration and bactericidal activity against sensitive organisms. The possibilities of a bactericidal assay for properdin are discussed. It is demonstrated that, in addition to properdin, the four components of complement (present in RP(b)) are necessary for the destruction of properdinsensitive bacteria. If any component is missing, bactericidal activity is lost; when the component is replaced, bactericidal activity is restored. Magnesium is also necessary for the bactericidal activity of the properdin system. Maximal bactericidal activity is obtained with magnesium concentrations similar to that of normal human serum (10(-3) to 10(-4)M). The bactericidal activity of the properdin system occurs only at temperatures above 15 degrees . Resistant strains have been encountered in species of bacteria sensitive to the properdin system. Resistance or sensitivity is a characteristic of the individual strain and not of the species. The widespread occurrence of the properdin system in normal mammalian serum and the variety of bacteria destroyed by it suggest that the properdin system is a factor in natural resistance.

Experimental production of gross atherosclerosis in the rat.

Abstract: Gross atherosclerosis was produced in the rat by feeding purified diets containing cholesterol, sodium cholate, and thiouracil for periods up to 363 days In a few weeks a marked increase of the serum cholesterol and beta lipoproteins as well as of the liver lipides was observed Lesions visible in the gross were found on the intimal surfaces of the vessels of all 46 animals examined These were most prominent in the heart valves and aortic arch The earliest lesions, which were seen at 31 days, required Sudan staining for gross demonstration Older lesions were visible without staining Microscopic coronary artery lesions were present and in one instance were accompanied by massive myocardial infarction Vascular lesions were characterized by medial and intimal lipide infiltration and cellular intimal plaque formation In a part of this study the protein level of the diet was altered at the expense of sucrose The hypercholesteremic response among the rats varied according to the dietary protein level The lowest response was observed among those animals receiving the highest level of dietary protein A difference, however, in the severity and extent of the arterial lesions among these relatively small groups of rats could not be established under these experimental conditions In all these experiments a close correlation existed between the serum cholesterol levels and beta lipoproteins of the S(f)20-100 range

Increased resistance to infection and accompanying alteration in properdin levels following administration of bacterial lipopolysaccharides

Abstract: It has been shown that injection of lipopolysaccharides, derived from a variety of Gram-negative bacterial species, evokes in mice a rapidly developing rise in resistance to infection with Gram-negative pathogens. This is accompanied by an elevation in properdin titer, at times to levels 2 to 3 times the normal. The rate, magnitude, and duration of these responses are dependent on many factors, the most important of which are the quantity and timing of the lipopolysaccharide administered. The increased resistance to infection evoked in mice by lipopolysaccharides was effective against infections produced by endotoxin-bearing organisms-bacterial species highly susceptible in vitro to the bactericidal action of the properdin system. Properdin titers of mice prior to infection provide an incomplete picture of the subsequent reaction of the host to the infective agent. Following infection with Gram-negative organisms, properdin levels accurately reflect the bacteriologic course and outcome of the infection. Thus, in control animals, properdin titers progressively declined and the animals died, while in mice appropriately treated with lipopolysaccharide, properdin levels were either maintained in the normal range or increased, depending on the dose and time of administration of lipopolysaccharide; this was always accompanied by successful management of the infection. The complex nature of the alterations produced in the host by lipopolysaccharides is stressed. It is pointed out that the increase in the ability of the host to cope with Gram-negative infections may be the result of stimulation of other defense mechanisms, in addition to the properdin system.

Variation in the Group-Specific Carbohydrate of Group A Streptococci. II. Studies on the Chemical Basis for Serological Specificity of the Carbohydrates.

Abstract: Soil organisms have been isolated which elaborate induced enzymes capable of attacking group A and variant (V) streptococcal carbohydrates. The V enzyme hydrolyzes V carbohydrate extensively to dialyzable split products with resultant total loss of precipitating activity with homologous antisera. The split products inhibit the reaction between intact V carbohydrate and its antiserum: evidence is presented which indicates that rhamnose oligosaccharides are responsible for the inhibitory effect. The serological specificity of the V carbohydrate thus appears to be primarily dependent on a rhamnose-rhamnose linkage. The effect of the A enzyme on A carbohydrate is characterized by the removal of 50 to 70 per cent of the total glucosamine in the form of free N-acetyl-glucosamine. As a result of this treatment, the residual carbohydrate loses its reactivity with specific group A antisera and at the same time develops markedly increased cross-reactivity with V antisera. This cross-reactivity is in turn eliminated by treatment with V enzyme. The evidence suggests that the specificity of group A carbohydrate is determined to a large extent by side chains of N-acetyl-glucosamine which also serve to mask underlying rhamnose-rhamnose linkages with V specificity.

Reversible changes in the susceptibility of mice to bacterial infections. I. Changes brought about by injection of pertussis vaccine or of bacterial endotoxins.

Abstract: A study has been made of the time variations in the susceptibility of albino mice to experimental infections with Klebsiella pneumoniae, Staphylococcus aureus , and Mycobacterium tuberculosis . Susceptibility to infection was determined by two criteria: ( a ) mortality rates following intravenous injection of a known infective dose; ( b ) numbers of bacterial colonies that could be recovered from the tissues of the infected animals at various times after infection. When measured in terms of either one of these two criteria, susceptibility was consistently modified by temporary deprivation of food, and by various changes in the composition of the diet. Increase in susceptibility to infection could be detected within a few hours to a few days depending upon the nature and intensity of the nutritional disturbance imposed upon the animal. Under the proper conditions, return to a normal state of resistance could also occur within a very short time. There was commonly observed an explosive bacterial multiplication in mice receiving the infective dose shortly after being subjected to nutritional disturbances. In the case of infection with Klebsiella pneumoniae , however, this phase of increased susceptibility was often followed a few hours later by one during which the numbers of living bacteria that could be recovered from the various organs fell to a very low level. Many of the animals which had exhibited severe but transient bacteriemia recovered rapidly and survived the infection. Mice rendered more susceptible to infection by being fed deficient diets progressively recovered their normal resistance while being kept on the same inadequate regimens. This adaptation occurred even though the weight of the animals on these regimens remained much lower than the weight of the animals fed a complete diet ad lib . The weight of the animal at the time of infection appeared to have little bearing on susceptibility. However, susceptibility consistently increased during the periods while the animals were losing weight—whatever the cause of the weight loss. The facts disclosed in the present study, as well as findings reported by other investigators, make clear that profound changes in susceptibility to infection can be brought about by varied non-specific procedures. These changes can occur within very short periods of time and are often rapidly reversible.

Immunohistochemical analysis of amyloid by the fluorescence technique

Abstract: The immunohistochemical composition of amyloid deposits in secondary human amyloidosis and experimental amyloidosis in rabbits was studied by means of the "fluorescent antibody" technique of Coons et al. Quantitative studies of the relative amounts of gamma globulin present in the amyloid deposits by the use of radioiodinated fluorescent antibody are reported. It is concluded that amyloid deposits in several organs from cases of secondary human amyloidosis and experimental amyloidosis in rabbits contain considerable concentrations of gamma globulin. The presence of gamma globulin in amyloid might be interpreted as either a metabolic deposition of circulating globulin present in high concentrations in the plasma or as a result of an immunologic reaction involving antigen and antibody.

The amino acid composition of hypertensin ii and its biochemical relationship to hypertensin i

Abstract: Preparations of hypertensin II, obtained from the treatment of hypertensin I by the action of the hypertensin converting enzyme of plasma and purified by countercurrent distribution, were quantitatively analyzed for their amino acid content. Chromatography on ion exchange columns showed the presence of equimolar amounts of aspartic acid, proline, valine, isoleucine, tyrosine, phenylalanine, histidine, and arginine. Hypertensin I was found to contain one mole of leucine and one mole of histidine in addition to the amino acids of hypertensin II. These two amino acids were isolated from the conversion products of hypertensin I and identified as the peptide histidylleucine. Carboxypeptidase digestion of hypertensin I showed the carboxyl terminal sequence of amino acids to be residue-phenylalanyl-histidylleucine. Similar studies of hypertensin II demonstrated residue-phenylalanine. It was concluded that the conversion of hypertensin I by the plasma hypertensin converting enzyme involved hydrolysis of the phenylalanyl-histidine bond to form hypertensin II and histidylleucine. The further removal by carboxypeptidase of phenylalanine from hypertensin II destroyed all of the vasoconstrictor activity.

The role of epinephrine in the reactions produced by the endotoxins of gram-negative bacteria i. hemorrhagic necrosis produced by epinephrine in the skin of endotoxin-treated rabbits

Abstract: Extensive lesions of dermal hemorrhagic necrosis occurred in rabbits when epinephrine (or norepinephrine) was injected into the skin within 4 hours after an intravenous injection of endotoxin. As little as 5 µg. of intradermal epinephrine, and 1 µg. of intravenous endotoxin, were sufficient to produce lesions. Similar lesions, but smaller in size and surrounded by a zone of acute inflammation, were produced by intradermal injection of a mixture of comparable amounts of endotoxin and epinephrine. No lesions were produced by combinations of endotoxin with serotonin, pitressin, or ephedrine. Both types of epinephrine-endotoxin lesion were prevented by pretreatment with cortisone, dibenzyline, and chlorpromazine. They were not prevented by heparin or nitrogen mustard. The lesions produced by intradermal mixtures of epinephrine and endotoxin were greatly enhanced in size and severity in animals treated with nitrogen mustard. Both types of lesion were prevented in rabbits rendered "tolerant" by repeated injections of sublethal amounts of endotoxin. It is concluded that endotoxin has the property of altering the reactivity of blood vessels to epinephrine in such a way that this hormone becomes a potent necrotizing agent. The possibility that this effect may represent a basic mechanism in the various intoxicating actions of endotoxin, and certain implications of this hypothesis, are discussed.

Structure and development of viruses observed in the electron microscope. III. Influenza virus.

Abstract: Rods and spheres believed to represent viral particles were observed at the free surface of entodermal cells of the chorioallantoic membrane 6 to 44 hours after infection. Although occasional short rods revealed poorly defined internal bodies, the majority, as well as all the longer rods (filaments), exhibited no visible internal structure. The spheres presumed to lie central to the plane of section contained an inner body 20 to 22 mmicro in diameter. Both forms possessed a dense, sharply defined limiting membrane 30 A thick and a diffuse external coat of lesser density. Where superimposition within the section was minimal, the viral particles were separated by a relatively constant distance. Measured to include this spacing, on the assumption that it reflected the presence of a component of the outer coat, the diameters of a majority of the rods were 50 to 60 mmicro, whereas the spheres averaged 60 to 70 mmicro. The rods appeared to form by a process of extrusion from the cell wall and became detached either singly or in bundles of variable length. The spheres seemed to differentiate at the cell surface and to acquire the inner body, limiting membrane, and outer coat as they migrated through the membrane of the host cell. No characteristic changes were seen in the nuclei or adjacent cytoplasm, and recognizable viral particles were never encountered in these areas of the cell. No support was obtained for the assumption that the spheres developed primarily by segmentation of the rods. It is suggested that the spherical form of the virus is the elemental infectious unit and that the filamentous form is largely or completely non-infective.

THE CARBOHYDRATE OF γ-GLOBULIN AND MYELOMA PROTEINS

Abstract: Various preparations of gamma-globulin homogeneous in the ultracentrifuge showed a similar content of hexose, hexosamine, fucose, and sialic acid. Subfractionation of Fr. II gamma-globulin by zone electrophoresis revealed multiple components of different mean mobilities but containing similar amounts of carbohydrate. Gamma globulin isolated directly from normal serum by zone electrophoresis showed a heavy component in addition to the usual 7 S material. The heavy component (s(20, w) = 19 S) concentrated by preparative ultracentrifugation was found to be considerably richer in carbohydrate than the rest of the gamma-globulin and accounted for small differences in carbohydrate content between different preparations of gamma-globulin. Pathological sera with marked elevation in gamma-globulin showed a carbohydrate-protein ratio for the gamma-globulin similar to that found for the corresponding 7 S fraction in normal serum. This was only partially true of the myeloma proteins with a mobility in the gamma-globulin region. Certain of these proteins showed slight but significant differences. The myeloma proteins of faster mobility (beta-myelomas) contained considerably more carbohydrate. The possible role of these carbohydrates in accounting for some of the mobility and immunological differences in the myeloma proteins is discussed. The pathological proteins found in two cases of macroglobulinemia showed a high carbohydrate content similar to but slightly lower than the normal 19 S component of gamma-globulin.

A serologic recapitulation of past experiences with influenza a; antibody response to monovalent vaccine

Abstract: The results of the present study provide a striking demonstration of antibody orientation produced by the dominant antigens of the strains of influenza virus encountered at childhood. The homologous and heterologous antibody response to monovalent vaccines containing swine, PR8, FM1, or Cuppett viruses show that the antibody-forming mechanisms of children born after 1943 are oriented to strains of influenza A-prime; of recruits to strains of influenza A; and of persons over 30 years of age to a strain of swine influenza. It was shown that antibody response to heterotypic viruses appears to provide a serologic index of the amount of experience each of the segments of the general population studied have had with antigenic variants of influenza virus. Finally it was demonstrated that the antibody response of humans to strains of influenza A or influenza A-prime is remarkably uniform for isolates of each antigenic type.

Transfer of delayed hypersensitivity to diphtheria toxin in man.

Abstract: Simultaneous transfer of delayed hypersensitivity to diphtheria toxin and to tuberculin has been accomplished in eight consecutive instances in man using extracts from washed leucocytes taken from the peripheral blood of tuberculin-positive, Schick-negative donors who were highly sensitive ( i.e ., pseudoreactors) to purified diphtheria toxin and toxoid. The leucocyte extracts used for transfer contained no detectable antitoxin. The recipient subjects were Schick-positive (<0.001 unit antitoxin per ml. serum) and tuberculin-negative at the time of transfer. All the recipients remained Schick-positive for at least 2 weeks following transfer and in every case their serum contained less than 0.001 units antitoxin at the time when they exhibited maximal skin reactivity to toxoid. Evidence is presented which indicates that the transfer factor may be released from leucocyte suspensions under mild conditions in which most of the cells appear to remain morphologically intact. Four adult Schick-positive subjects have been sensitized to diphtheria toxoid by intradermal injection of a few micrograms of purified toxoid in the form of a washed toxoid-antitoxin precipitate. Two of these sensitized individuals showed severe delayed skin reactions specifically directed against diphtheria toxin (or toxoid) at a time when their serum antitoxin level was less than 0.001 units/ml.

Experimental pyelonephritis. I. Effect of ureteral ligation on the course of bacterial infection in the kidney of the rat.

Abstract: A study has been made of the effect of ureteral ligation on the susceptibility of the kidney to pyogenic infection. In most experiments a strain of E. coli was employed as the test organism, being injected intravenously in varying quantity either before or after ureteral ligation. A few experiments were also carried out with S. marcescens. Preliminary observations were made on the distribution and persistence of E. coli following its inoculation into the blood stream of normal rats. Rapid reduction in number of bacteria in the circulation occurred during the first 30 minutes, but bacteriemia persisted at a comparatively low level for at least 48 hours. Large proportions of the inoculated bacteria were arrested and apparently destroyed in the liver, spleen, and lungs. Comparatively small numbers were deposited in the kidneys; nevertheless, these continued to be demonstrable during the 1st week, without notable tendency to increase or decrease, then disappeared during the 2nd week. There was no acceleration in rate of disposal of the bacteria in the kidney when a second injection was made 1 week after the first. In rats with one ureter ligated the number of bacteria lodging in the kidneys after intravenous inoculation did not differ from that found in normal animals. It appears, therefore, that the increased susceptibility of the obstructed kidney to infection via the blood stream is not attributable to an increased trapping of circulating bacteria. 4 to 6 hours after the intravenous injection, however, an increased number of bacteria could be demonstrated in the obstructed kidney, apparently due to local multiplication, and by the end of 24 hours purulent infection was usually obvious. A comparatively large number of bacteria was required to cause infection, even in the kidney with obstruction. This appeared to be related to the small proportion of the intravenous inoculum which lodged in the kidney initially. Although bacteria could be demonstrated in the normal kidney for a week or more following intravenous injection it was not possible to induce active infection with equal regularity by ligating the ureter throughout this time. During the first 3 days the majority of obstructed kidneys developed infection, but after 5 or more days this occurred in only a small proportion of animals so treated. The reason for the difference, in relation to interval between intravenous injection and time of ligation, is not apparent. When the ureter was ligated but no intravenous injection of bacteria was given, staphylococcal infection developed in the obstructed kidney within 2 weeks in about one-third of the animals. Reasons are given for the belief that this was blood-borne infection, and not the result of contamination at the time of operation. Staphylococci were not recovered from the normal rat kidney. These "spontaneous" staphylococcal infections seldom developed when E. coli was injected intravenously at the time of ureteral ligation.

The nutritional requirements for the propagation of poliomyelitis virus by the HeLa cell.

Abstract: Only minimal amounts of poliomyelitis virus were formed by HeLa cells placed in a medium free from glucose and glutamine, even if the medium contained an otherwise full complement of essential and non-essential amino acids, purines, pyrimidines, NB(4) (+), and serum protein. Conversely, within one log of the optimal yield of virus was formed by HeLa cells in a medium containing only glucose, glutamine, and salts, even if the cells had been starved in this medium for 12 hours prior to their inoculation. The presence of glucose alone caused an average 170-fold increase in viral output beyond the amounts formed by the glucose- and glutamine-depleted cells. The addition of glutamine alone caused an average 2000-fold increase; and the addition of both increased the viral formation 40,000-fold. Qualitatively similar results were obtained with unstarved cells, not previously depleted of glucose and glutamine. It follows that only a small proportion of HeLa cells are capable of forming virus unless either glucose or glutamine, or both, are present in the medium. The elaboration of virus was significantly delayed in media containing glucose but no glutamine. The absence of glucose and glutamine did not prevent the fixation of poliomyelitis virus by the cell. When these compounds were added to previously depleted cells even 6 hours after inoculation, and after the excess free virus had been removed by washing and by the addition of specific antiserum, normal amounts of virus were formed despite the degenerative changes caused by the previous glucose and glutamine deprivation. Possible functions of glucose and glutamine in the elaboration of virus are discussed in the text. Such factors other than glucose, glutamine, or salts (e.g. amino acids, purines, pyrimidines, vitamins, protein, or NH(4) (+)) as may be needed by HeLa cells for the propagation of poliomyelitis virus, need not be present in the medium and cannot be easily washed out of the cell. Even 12 hours' total deprivation of the cells in salt solution prior to inoculation only slightly decreased their virus-synthesizing capacity in a similarly deficient medium, provided only that adequate amounts of glucose and glutamine were retained.

Studies on the interaction between phagocytes and tubercle bacilli : i. observations on the metabolism of guinea pig leucocytes and the influence of phagocytosis

Abstract: As a basis for studies on the interaction of phagocytes and tubercle bacilli experiments were carried out to obtain information on some biochemical characteristics of exudate leucocytes from guinea pigs. It was found that total cellular phosphorus was the most suitable measure of protoplasm. Cell counts were less reliable because of unavoidable clumping in the suspension, and dry weight measurements were less specific when contamination with erythrocytes occurred. The utility of phosphorus measurements in this connection depends upon the fact that an erythrocyte contains only 4 per cent of the phosphorus present in a leucocyte. From measurements on mixed suspensions consisting of varying known proportions of polymorphonuclear and mononuclear leucocytes as determined by differential counting, it was possible to compute true values for these two cell types with respect to oxygen consumption and lactic acid production. Thus it was found that monocytes consumed considerably more oxygen and produced more lactate than polymorphonuclear leucocytes. From the data obtained it is suggested that differences in metabolic activity found when comparing cell suspensions obtained by the use of different irritants are due to different proportions of the two cell types. In particular, the effects of oxygen tension and pH on the activities of the cells were studied. It was found that decreasing the proportion of oxygen in the atmosphere from that of air to 1 per cent reduced oxygen consumption by about 80 per cent, whereas lactic acid production was increased by about 45 per cent. It was also found that decreasing the pH of the medium below pH 7.5 caused a considerable reduction in the respiration, lactate production, and viability of polymorphonuclear leucocytes. The monocytes proved less sensitive to similar changes in pH, especially with regard to lactic acid production and viability. Observations were made of oxygen consumption and lactate production during phagocytosis. During the hour following the addition of heat-killed tubercle bacilli to the phagocytes, the oxygen consumption of suspensions rich in polymorphonuclear leucocytes rose by 60 per cent, and that of suspensions rich in monocytes rose by nearly 100 per cent. Lactic acid production was unchanged during phagocytosis.