The Journal of Experimental Medicine

Dvorak, Harold F.; Waksman, Byron H.

doi: 10.1084/jem.116.1.1pmid: 13888780

Normal Dutch rabbit lymph node and spleen minces, lymph node cell suspensions, and residues from lymph node cell suspensions were cultured in Millipore chambers with slices of autologous or homologous (New Zealand) ear skin. for varying time intervals. Lymphoid cells exposed to New Zealand ear skin for more than 4 days were found capable of producing typical "transfer reactions" in the specific New Zealand ear skin donor, similar in every way to reactions produced by cells from lymph nodes sensitized in the intact Dutch animal. Heat-killed cells and cells exposed to New Zealand ear skin for less than 4 days (in chambers or in the intact animal) or to Dutch ear skin for any period of time were incapable of eliciting such reactions. It is concluded that normal lymphoid tissues undergo primary sensitization when exposed to homografts in Millipore chambers for suitable periods of time. Footnotes Submitted: 6 February 1962

Schwab, John H.

doi: 10.1084/jem.116.1.17pmid: 13909623

Specific antibody and a glucosaminidase enzyme react with the serologically active sites on C polysaccharide, and local injection of these reagents will neutralize the toxic effect of C polysaccharide complexes even after an interval of 24 to 72 hours. The cell wall lysins in S. albus filtrate and from phage-lysed Group C streptococci, break down the cell wall structure of Group A streptococci but leave the serological reactive sites of the C polysaccharide intact. These reagents lose much of their ability to neutralize the C polysaccharide toxin when superinjected after an interval of 4 hours following toxin injections. Toxic C polysaccharide cannot be recovered from an injection site by intercellular perfusion of the excised skin area after an interval of 1 to 4 hours following toxin injection. It is concluded that toxic particles of C polysaccharide complexes combine firmly with the dermal tissue of rabbits within 1 to 4 hours following intracutaneous injection. The cell wall lysins neutralize the toxin by reducing the particle size of C polysaccharide complexes. This minimum particle size is required for the initial reaction with tissue and when this has occurred these reagents are no longer able to influence the development of the lesion. Footnotes Submitted: 4 March 1962

Eagle, Harry; Piez, Karl

doi: 10.1084/jem.116.1.29pmid: 13888938

At least seven compounds synthesized by cultured cells in amounts which should suffice for sustained growth have nevertheless proved under certain conditions necessary for survival (asparagine, cystine, glutamine, homocystine inositol, pyruvate, serine). In every instance so far examined, that requirement has been population-dependent, disappearing at cell densities sufficiently large to bring the concentration in the medium and in the cellular pool to metabolically effective levels before the cells died of the specific deficiency. At population densities of less than 100 cells per ml, serine was required by all cultured cells so far studied. With more exigent strains, such as the RT6 strain of rabbit fibroblast and the P388 mouse leukemia, the serine requirement disappeared only at populations in excess of 50,000 and 150,000 cells per ml, respectively. The requirement for pyruvate by the latter cell as an alternative to serine also disappeared at that population density. In a cystine-free medium there were population-dependent requirements for cystine, homocystine, or serine, depending on the experimental conditions. With methionine and glucose as cystine precursors, the critical population density permitting cellular survival and growth was in excess of 200,000 cells/ml. The provision of homocystine as an intermediate reduced the critical population density to 10,000 to 60,000 cells/ml; with the further provision of serine, the critical cell concentration permitting growth was reduced to 50 to 500 cells/ml. Cells adapted to glutamic acid, and capable of utilizing it as a substitute for glutamine, nevertheless required exogeneous glutamine at cellular densities of less than 50,000 cells per ml. In some experiments, the provision of asparagine reduced the critical population density to 10,000 cells/ml, presumably because of its glutamine-sparing action. Inositol is required by most cell lines, despite their demonstrated capacity to synthesize it from glucose. With at least one cell line (HeLa), sustained growth was occasionally achieved in an inositol-free medium if the population density was maintained in excess of 240,000 cells/ml. The possible implications of these findings with respect to the loss of specific organ functions in dispersed cell culture are discussed. Footnotes Submitted: 19 March 1962

Smiley, J. Donald; Yeager, Henry; Ziff, Morris

doi: 10.1084/jem.116.1.45pmid: 13914038

Chick embryos were sacrificed at intervals after simultaneous injection of BAPN and proline-C 14 , the collagen separated into neutral salt-extractable and residue fractions, and total hydroxyproline and hydroxyproline specific radioactivity determined in each fraction. Extractable collagen, measured as hydroxyproline, increased markedly and had a higher specific activity in BAPN-treated embryos than in corresponding controls. Hydroxyproline of the residue collagen in the treated animals, however, had a lower specific activity. When proline-C 14 was injected 24 hours prior to BAPN, the specific radioactivity of the soluble collagen of treated embryos was similar to that of controls, in spite of the fact that the specific activity of the residue fraction was higher than that of the soluble fraction at the time of BAPN administration. These results suggest that the increased amount of soluble collagen in lathyrism induced by administration of BAPN does not arise from the collagen insoluble prior to administration of the drug, but rather that BAPN acts by blocking the formation of mature collagen fibers, perhaps by preventing the formation of cross-linkages between α-collagen chains. Footnotes Submitted: 8 April 1962

Smuckler, Edward A.; Iseri, Oscar A.; Benditt, Earl P.

doi: 10.1084/jem.116.1.55pmid: 13914520

The morphological and certain metabolic effects of carbon tetrachloride intoxication were studied in the rat with emphasis on liver alterations. Morphological changes were investigated by histological and electron microscopical means. Functional changes were investigated using histochemical and amino acid incorporation, techniques. The liver constituents were examined chemically. Plasma volume alterations were measured using dye and homologous protein dilution techniques. The histological appearance of the liver of treated animals included cellular swelling, dispersal of the cytoplasmic basophilia, and necrosis. Electron micrographs showed an early (3 hours following carbon tetrachloride administration) and widespread dislocation of the ribonucleoprotein particles from the membranes of the rough endoplasmic reticulum, but no apparent alteration in the mitochondrial structure. Histochemical examination of two mitochondrial enzyme systems, α-ketoglutarate dehydrogenase and succinic dehydrogenase, revealed no alterations in activities until a later time (6 to 12 hours following carbon tetrachloride administration). ATPase showed a gross quantitative decrease in activity at 6 and 12 hours, but not earlier. There was a decreased amino acid incorporation into two liver-produced proteins, viz ., albumin and fibrinogen. This decrease is not explicable on the basis of the inability of the liver to take up the amino acid, an altered dilution volume into which the amino acid or formed protein is placed, or an impaired capacity of the liver to excrete protein once formed. It is concluded that the decreased amino acid incorporation rate reflects depressed synthesis of protein by the liver. Other pathological changes in the liver, including necrosis, fatty change, and mitochondrial alterations may be dependent upon severe impairment of protein synthesis. Footnotes Submitted: 22 February 1962

Rüde, Erwin; Goebel, Walther F.

doi: 10.1084/jem.116.1.73pmid: 14495108

The somatic antigen of the non-colicinogenic bacillus E. coli K235 L - OC - has been isolated, and its chemical and serological properties have been compared with those of colicine K. The antigen of the non-colicinogenic bacillus has a protein content significantly lower than that of the C + antigen, a difference which might be related to the antibacterial activity of the latter. The lipocarbohydrate components of the two antigens are chemically very similar; both contain the same proportions of galactose, glucose, heptose, rhamnose, glucosamine, and mannosamine. When tested by agar diffusion, the two antigens are indistinguishable, as are their lipocarbohydrate components. Our studies indicate that the bactericidal activity of colicine K does not reside in its lipocarbohydrate but in its protein component. Footnotes Submitted: 27 March 1962

Michael, J. Gabriel; Massell, Benedict F.

doi: 10.1084/jem.116.1.101pmid: 14473315

Endotoxins derived from several species of Gram-negative bacteria, while inducing non-specific resistance to typhoid bacilli in mice, failed to increase the resistance of these animals to infection with virulent strains of Group A streptococci. However, if administration of endotoxin was followed by injection of minute amounts of type-specific antiserum, a substantial degree of protection against the streptococcal pathogen was obtained. The same amount of type-specific antiserum given to the animals by itself did not have any effect on the outcome of the streptococcal infection. Fresh rabbit blood, obtained from animals pretreated with endotoxin, together with anti-M protein immune serum, was found strongly bactericidal for streptococci. These observations suggest that, at least with regard to streptococcal infection, both humoral and cellular factors are required for induction of non-specific resistance. Footnotes Submitted: 9 April 1962

Bollet, Alfred Jay; Davis, John S.; Hurt, John O.

doi: 10.1084/jem.116.1.109pmid: 13870814

Antibodies to crystalline bovine hepatic L -glutamic dehydrogenase were induced in rabbits. These antibodies inhibited the bovine glutamic dehydrogenase used as antigen, and also inhibited glutamic dehydrogenases from rat, rabbit, human, pigeon, and frog livers, as well as frog renal and muscle glutamic dehydrogenase. The antibody did not inhibit yeast glutamic dehydrogenase which differs from the animal enzymes in cofactor requirement. The kinetic characteristics of the inhibition of the bovine glutamic dehydrogenase indicate mixed competitive and non-competitive inhibition, suggesting reaction with multiple antigenic sites; the data indicate competitive inhibition of the enzyme from other species, suggesting reaction with the catalytic site. Footnotes Submitted: 4 April 1962