The Journal of Experimental Medicine

Rifkind, Richard A.; Godman, Gabriel C.; Howe, Calderon; Morgan, Councilman; Rose, Harry M.

doi: 10.1084/jem.114.1.1pmid: 13741584

Sequential stages in the development and release of ECHO 9 virus have been illustrated and described. It is suggested that viral particles differentiate and become oriented in columns upon a fine filamentous lattice at cytoplasmic template sites which are distinct from the endoplasmic reticulum. Subsequently, virus is dispersed in the peripheral cytoplasm and gains egress from the cell through rents in the plasma membrane. Complete cellular disruption with viral release may supervene. The virus consists of a 13 to 15 mµ dense core and a poorly defined outer membrane, 22 to 24 mµ in diameter. Incomplete forms, lacking the core, are observed in the cytoplasm but have not been seen in the extracellular space. Footnotes Submitted: 12 March 1961

Watson, Barbara K.

doi: 10.1084/jem.114.1.13pmid: 13783417

Variation in the amount and quality of influenza virus injected into the amniotic sac of the chick embryo led to differences not only in the yield of virus but also in the immunofluorescent cytology of the infection. The production of infectious virus was associated with a predominance of cells in which the virus antigens were first detected along the cell surface in contact with the amniotic fluid, later deeper in the cytoplasm, and finally, though to a lesser extent, in the nucleus. When the virus yield was primarily non-infectious hemagglutinin the virus antigens appeared in reverse sequence; i.e ., nucleus first, then adjacent cytoplasm, and ultimately throughout the entire cell. Under conditions of "autointerference," the immunofluorescence seen in some of the cells failed to progress beyond the nuclear or early cytoplasmic stage, while many other cells remained unreactive throughout the experimental period. With the largest dose used a pale intranuclear reaction was localized 1 hour after injection but the embryo died shortly thereafter. Footnotes Submitted: 21 March 1961

Austen, K. F.; Brocklehurst, W. E.

doi: 10.1084/jem.114.1.29pmid: 13685196

The anaphylactic release of histamine from perfused, chopped guinea pig lung is very sensitive to changes in the NaCl concentration of the containing medium, and it is ionic strength rather than particle concentration which is critical. Consequently, in studies with inhibitors care must be taken to avoid inadvertently increasing ionic strength and thereby misinterpreting the cause of the inhibition. Since immune hemolysis exhibits a similar sensitivity to changes in the NaCl concentration of the suspending medium, salicylaldoxime and phlorizin, which prevent the participation of the third component of complement in immune hemolysis, were investigated for their effect on the anaphylactic reaction. Salicylaldoxime is a potent inhibitor of in vitro anaphylaxis in guinea pig lung, but phlorizin is only a weak inhibitor. Potassium cyanide, 1 m M , inhibits the anaphylactic release of histamine most effectively if the duration of contact between the tissue and the cyanide prior to antigen addition is minimal; preincubation of the tissue with cyanide prior to antigen addition results in progressive diminution of inhibition even when there is only minimal loss of cyanide from the containing medium. The anaphylactic release of histamine from perfused whole lungs or suspensions of blood-free chopped lung is not prevented by the cytochrome oxidase inhibitor, carbon monoxide. In addition, 2-heptyl 4 hydroxyquinoline N oxide and malonic acid, which inhibit aerobic metabolism at different sites, do not prevent the reaction. These studies and those with cyanide indicate that the anaphylactic release of histamine in guinea pig lung is not dependent on cytochrome-mediated aerobic metabolism. Footnotes Submitted: 20 February 1961

Webb, Tom; Lapresle, Claude

doi: 10.1084/jem.114.1.43pmid: 13783579

An insoluble specific adsorbent for anti-human serum albumin antibodies was prepared by coupling human serum albumin (H.S.A.) to polystyrene by azo bonds. Rabbit anti-H.S.A. immune serum was passed through a column of the adsorbent. It was shown that different volumes of the immune serum were required for the saturation of the different determinant groups of H.S.A. by their corresponding antibodies. The elution of the anti-H.S.A. antibodies adsorbed on the column was achieved by passing successively through the column an acetate buffer pH 3.0 and a solution of 0.1 N HCl in 0.15 M NaCl. The antibodies were eluted in three different fractions, each of which was composed of γ-globulins only. These three fractions contained different proportions of antibodies of different specificities. Footnotes Submitted: 12 March 1961

Garvin, James E.

doi: 10.1084/jem.114.1.51pmid: 13703800

Some factors affecting the retention of human polymorphonuclear neutrophils (PMN), lymphocytes, and platelets on a siliconized glass bead column were explored. PMN were more effectively retained when the flow rates were slow and the columns long. They were found largely on the upper portions of the columns except with rapid flow rates when they spread down the columns. PMN retention on the columns was greatest in the range 30°–43°C. Both magnesium and calcium ions were required for full adhesiveness; calcium ions alone were unable to restore adhesiveness to PMN from blood which had been treated with a chelating resin to remove divalent cations. The adhesiveness of the PMN was independent of cyanide and dinitrophenol, but was almost completely eliminated by iodoacetamide. Under all the conditions mentioned above in which adhesiveness was lost there was a concurrent loss of the usual ability of the PMN to migrate, but at least in the presence of EDTA, a capacity to change shape by pseudopod formation remained. Lymphocytes were retained on the columns to a much lesser extent than the PMN under all conditions and, within limits, this retention was not related to either flow rate or column length. Maximum lymphocyte retention occurred in the range 30°–43°C. No dependence of lymphocyte adhesiveness was shown for divalent cations, cyanide, dinitrophenol, or iodoacetamide, but such dependence is not excluded by the data obtained. Platelets were largely retained by the glass bead columns under most conditions and this was unrelated to temperature in the range 0°–50°C. Their adhesiveness was found to require either magnesium or calcium ions and to be blocked by iodoacetamide. Footnotes Submitted: 9 March 1961

Fong, Jacob; Chin, Dennis; Elberg, Sanford S.

doi: 10.1084/jem.114.1.75pmid: 13700570

Passage of the virulent H37Rv strain of tubercle bacillus in normal or immune systems (normal or immune monocytes suspended in the corresponding serum) resulted in decreased virulence of the bacilli; this was evidenced by the very low mortality rates in mice inoculated intravenously with passaged bacilli. Passaged bacilli when cultivated directly in tween-albumin medium or when grown on glycerol-blood agar plates after recovery from infected mouse tissues proved as virulent as unpassaged bacilli. The decreased virulence of passaged H37Rv was accompanied by loss of ability to bind neutral red. Passaged H37Rv was more sensitive than unpassaged bacilli to inactivation by sodium oleate and by normal monocyte lysate; however, passaged H37Rv was more resistant than unpassaged bacilli to inhibition by streptomycin. Footnotes Submitted: 21 March 1961

Goldstein, Gerald; Slizys, Irene S.; Chase, Merrill W.

doi: 10.1084/jem.114.1.89pmid: 13706641

1 . A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2 . In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3 . Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: ( a ) conjugating only gamma globulins with fluorescein isothiocyanate; ( b ) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; ( c ) use of crystalline preparations of fluorescein isothiocyanate. 4 . Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5 . The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6 . A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules. Footnotes Submitted: 9 March 1961

Weigle, William O.

doi: 10.1084/jem.114.1.111pmid: 13783813

Immunological tolerance produced in rabbits by neonatal injections of BSA can be terminated by a series of injections of certain heterologous serum albumins which cross-react with BSA. Injections of albumins distantly related to BSA were more effective in terminating the tolerant state than injections of albumins closely related to BSA. It was concluded from results obtained with several heterologous albumins that immunological tolerance to BSA is directed to both the over-all antigenic or physical-chemical composition of the protein and the individual determinant groups present on the protein. Several possible mechanisms were given to explain the ability of cross-reacting albumins to terminate the tolerant state of BSA-tolerant rabbits. A possible relationship between the termination of tolerance in BSA-tolerant rabbits injected with cross-reacting albumins and autoimmunily was discussed. It was also suggested that the relative ease with which tolerance could be established to heterologous serum proteins in comparison to bacterial antigens is the result of the close serological and physical-chemical relationship of the heterologous serum proteins to the serum proteins of the rabbit. Rabbits injected with 500 mg of BSA during the first 5 days of life failed to form antibody capable of either eliciting an immune elimination of an injection of I* BSA given 3 to 4 months later or complexing with the circulating I* BSA. Footnotes Submitted: 8 March 1961

Krause, Richard M.; McCarty, Maclyn

doi: 10.1084/jem.114.1.127pmid: 13754097

Lysis of trypsinized Group A streptococcal cell walls with phage-associated lysin releases into solution dialyzable and non-dialyzable mucopeptide fractions composed of N -acetylglucosamine, N -acetylmuramic acid and alanine, glutamic acid, lysine, and glycine in addition to the characteristic group-specific carbohydrate. The latter substance contains appreciable amounts of N -acetylmuramic acid and the amino acids as well as N -acetylglucosamine and rhamnose. Hot formamide extraction of the cell walls results in a soluble fraction of group-specific carbohydrate and an insoluble residue. The Group A carbohydrate in this instance is composed of rhamnose and N -acetylglucosamine. The composition of the insoluble residue is similar to that of the mucopeptide fractions released from the cell wall by phage-associated lysin. This residue was shown by electron microscopy to be composed of discrete discs which appear similar in structure to the intact cell wall. The specific carbohydrate obtained by hot formamide extraction of Group A-variant cell walls was composed almost exclusively of rhamnose. The residue fraction was similar to that of Group A. The residue of cell walls extracted with hot formamide is extensively solubilized not only by phage-associated lysin and S. albus enzyme, but also by lysozyme, which has no measurable effect on the intact streptococcal cell wall. Footnotes Submitted: 9 April 1961

Schneider, H.; Formal, Samuel B.; Baron, L. S.

doi: 10.1084/jem.114.1.141pmid: 13748213

Antibiotic-pretreated mice were fed orally an Hfr culture of streptomycin-resistant E. coli and 1 day later, a streptomycin-resistant F - S. typhimurium culture. Hybrids were recovered in relatively small numbers from the feces of these mice within 24 hours demonstrating that genetic recombination can occur within the intestinal tract of a mammalian host under experimental conditions. These hybrids multiplied rapidly and persisted throughout the course of the experiment. In addition, hybrids were recovered which had not been observed in single matings performed in vitro . Footnotes Submitted: 9 March 1961