The Journal of Experimental Medicine

Minden, Percy; Farr, Richard S.

doi: 10.1084/jem.130.5.931pmid: 4981514

Studies were undertaken to find a substance or substances for use in primary binding types of tests to detect humoral antibodies in rabbits and monkeys exposed to the tubercle bacillus that would distinguish between strains of mycobacteria. The antigen employed was a component of the 5159 strain of Mycobacterium tuberculosis that was obtained following sonification, ultracentrifugation, electrophoresis, and elution from a preparative polyacrylamide column. When the antigen was labeled with 131 I, specific binding was observed in sera from immunized rabbits and BCG-protected rhesus monkeys by the radio-gel electrophoresis and ammonium sulfate tests. This component was partially characterized, and its major antigenic determinants were associated with anionic mucopolysaccharides. Electrophoretically at pH 8.3 it migrated anodally to albumin, its molecular weight was between 9,000 and 12,000, and it was soluble in 50% saturated ammonium sulfate. Binding to antibody was destroyed after treatment with Pronase, but not after DNase or RNase. Inhibition of the reaction, as measured by the ammonium sulfate test, between the 131 I-labeled component and antisera from rabbits that had been immunized with sonicated 5159 organisms, was studied. These experiments demonstrated a capacity to define subtle similarities and differences among different mycobacteria and mycobacterial components. Some microorganisms not clearly related to mycobacteria also partially inhibited this reaction, suggesting that they shared antigenic groups with the component derived from 5159 organisms. The studies described suggested the advisability of using direct primary tests and purified components of mycobacteria to differentiate further the antigenic groups between individual pathogenic mycobacteria and between pathogenic and nonpathogenic organisms. Footnotes Submitted: 16 June 1969

Lee, Chi-Jen; Dubos, Rene

doi: 10.1084/jem.130.5.955pmid: 4310504

Physicochemical and immunological techniques have been used in an attempt to characterize a filterable agent, separated from the intestines of mice raised under ordinary conditions of husbandry, which produces a lasting depression of weight in specific pathogen-free (SPF) mice when administered to them orally shortly after birth. Although this agent has not yet been identified, it will be tentatively designated here as enterovirus. The mouse enterovirus can be readily sedimented by ultracentrifugation and by precipitation at pH 4.3; it does not pass through cellophane membranes. Its infective power is completely destroyed by ultraviolet radiation, but is resistant to heating at 56°C, exposure to ether, treatment with trypsin, ribonuclease, and deoxyribonuclease. Dialysis and treatment with ether and nucleases greatly increase the infective activity of the intestinal filtrates containing the enterovirus, a finding which suggests that these procedures eliminate or destroy some inhibitory substance(s). The mouse enterovirus causes hemagglutination of mouse red blood cells. When injected into rabbits, it elicits in them an immune response that renders their serum capable of neutralizing its weight-depressing activity. As measured by inhibition of hemagglutination or complement fixation, the sera of infected mice do not exhibit any significant activity against usual mouse viruses. Centrifugation of the mouse enterovirus in 50%–20% sucrose gradient gave almost complete recovery of the infectivity and of hemagglutinating activity in the same fraction. In contrast, the protein content of the material was distributed through the various fractions. Consequently, this procedure resulted in a marked increase of specific activity. Footnotes Submitted: 11 July 1969

Dubos, Rene; Lee, Chi-Jen; Costello, Richard

doi: 10.1084/jem.130.5.963pmid: 5347698

The effects of neonatal influences on the growth and longevity of mice were studied by using animals derived from a highly inbred germfree colony that had been reassociated with a microbial flora free of known pathogens. The size of the animals at weaning time could be conditioned predictably by manipulating the diet of their mothers during gestation and lactation or by shortening or lengthening the period of lactation. A deficient diet during gestation or during lactation decreased the metabolic efficiency of the adult animal, even if it was fed an optimum diet after weaning. The effect was greatest when malnutrition occurred during both pregnancy and lactation. In contrast, an optimum diet during gestation and lactation rendered the animal less susceptible to the depressing effects of nutritional deficiency during adult life. A marked and lasting growth depression could be reproducibly achieved by contaminating newborn mice orally with an unidentified enterovirus. But neonatal infection with enterobacteria or mycobacteria even though severe, did not significantly alter the growth rate. Regardless of its initial cause, the depression of the growth rate during the preweaning period persisted throughout the whole life span of the animals, even when they were placed under optimum sanitary and nutritional conditions after weaning. Agencies (nutritional or infectious) which brought about a depression of whole body weight also affected the absolute and relative sizes of the various organs, especially of the brain . By manipulating neonatal influences, it was possible to produce at will in a given colony of highly inbred mice a family of strikingly different growth curves. This could be done without causing the death of any animal or affecting longevity. Footnotes Submitted: 11 July 1969

Aoki, Tadao; Hämmerling, Ulrich; de Harven, Etienne; Boyse, Edward A.; Old, Lloyd J.

doi: 10.1084/jem.130.5.979pmid: 5347699

The representation of mouse alloantigens belonging to three systems, H-2, θ and TL, on the surface of cells from thymus, spleen, lymph nodes, and peritoneal cavity, was studied by electron microscopy with ferritin-labeled antibody. As expected from earlier serological data, TL was confined to thymocytes, θ was found on thymocytes and lymphocytes, and H-2 occurred to some extent on all cell types observed. On reticular cells, lymphocytes, plasma cells, and eosinophils, the majority of the cell surface was occupied by H-2; thymocytes had considerably less H-2, and erythrocytes and peritoneal macrophages least of all. In every instance the representation of antigen was discontinuous, the fraction of the cell surface covered being characteristic both of the antigen and of the type of cell. H-2 and θ provide a striking example of this; H-2 is present in far higher amounts on lymphocytes than on thymocytes, whereas the converse is true of θ. Within areas positive for H-2 or θ, protuberances of the surface membrane were often antigen-negative. A better definition of cell surface structure, gained from studies such as this, is necessary for further inquiry into how the cell surface is assembled, and into selective gene action in relation to cellular differentiation. Footnotes Submitted: 20 June 1969

Ambrose, Charles T.

doi: 10.1084/jem.130.5.1003pmid: 5347691

Two opposite effects of actinomycin D on antibody synthesis have been studied in organ cultures of rabbit lymph node fragments. These cultures were prepared from previously primed rabbits and stimulated with antigen(s) on day 0 to yield a secondary response, whose inductive phase extended to about day 9 and whose productive phase may last for several months in the serum-free medium described here. Concentrations of actinomycin D above 0.01 µ M (0.012 µg/ml) produce inhibition of antibody synthesis during both phases of the response. However, antibody synthesis is about 10 times more sensitive to inhibition by this drug when it is added during the inductive phase than during the productive phase. During the latter phase, synthesis is more rapidly terminated as the drug level approaches 10 /µ M (12.5 µg/ml). At this level the 50% synthesis time is about 2.8 hr, which is identical with that found when 5–10 µ M puromycin is added to the medium of parallel cultures. Transient enhancement of antibody synthesis is frequently produced by a brief exposure to low levels of actinomycin D (generally less than 0.01 µ M ). Enhancement appears in precise temporal association with actinomycin pulses added for 2 days or less only between days 6 and 16. This apparent enhancement of antibody synthesis resembles the increased enzyme synthesis described by Garren et al. (6) and led to a search for an antibody-inhibitory material (AIM) whose synthesis might be stopped preferentially by low levels of the drug. Stimulated lymph node cultures produce between days 6 and 15 a nondialyzable material which inhibits antibody synthesis during the productive phase of heterologous antigen-antibody culture systems. Just as enhancement with low levels of actinomycin D appears within 2 hr after the drug has been added to cultures, so inhibition occurs within 4 hr of adding AIM to cultures during their productive phase. These observations suggest that AIM is analogous to the translational "repressor" postulated by Garren et al. (6). AIM has relevance in two areas of immunology: ( a ) it may be the explanation for many examples of antigenic competition, and ( b ) it may represent a normal control mechanism for the productive phase. Footnotes Submitted: 16 June 1969

Schlossman, Stuart F.; Herman, Judith; Yaron, Arieh

doi: 10.1084/jem.130.5.1031pmid: 5347692

Studies of the immunochemical specificity of antigen-induced thymidine-2- 14 C incorporation in lymph node cells obtained from animals immunized to a series of closely related α-DNP-oligolysines, ϵ-DNP-oligolysines, and oligolysines have shown that the sensitized cell exhibits an extraordinary degree of specificity for antigen. The sensitized cell is maximally stimulated by the homologous immunizing antigen and can discriminate among compounds which differ from one another only in the position of a dinitrophenyl group or D -lysine residue on an identical oligolysine backbone. These studies support the view that the immunogen is not degraded prior to the induction of the immune response, and that the majority of cells produced as a consequence of immunization have stereospecific antigen receptors for the DNP-oligolysine used to induce the response; a smaller and more variably sized population of cells is produced with receptors specific for the oligolysine portion of the immunizing antigen. When specifically sensitized lymph node cell cultures are stimulated in vitro by heterologous DNP-oligolysines, the oligolysine- and not the DNP-oligolysine-sensitive population of cells appears to play a crucial role in the specificity of such cross-reactions. It is concluded from these studies that the antigen receptor on the sensitized lymph node cell differs in both kind and degree from conventional antibody. The chemical nature of the receptor and the means by which this receptor reacts with antigen to initiate the biosynthetic or proliferative cellular immune response still remain undefined. Footnotes Submitted: 19 May 1969

Daugharty, Harry; Hopper, John E.; MacDonald, A. Bruce; Nisonoff, Alfred

doi: 10.1084/jem.130.5.1047pmid: 5347693

Specifically purified anti- p -azobenzoate antibodies of the IgG class from individual rabbits were used to elicit anti-idiotypic antibodies in recipient rabbits. Allotypes of each donor and recipient were matched. When polymerized antibodies were used for immunization, more than 80% of the recipients responded with the formation of antibodies that precipitated the monomeric donor antibody. Percentages of precipitable molecules in the donor antibody population (D) varied from 4 to 56. As little as 4% was readily detectable by the Ouchterlony method or precipitin test. Specificity of the reaction was tested by double diffusion in agar gel against a panel of purified antibenzoate antibodies from 14 heterologous rabbits and, quantitatively, in three systems by measurement of the extent of coprecipitation of heterologous, radiolabeled antibenzoate antibodies. No cross-reactions were observed. Reactions were shown to be attributable to antibenzoate antibodies in the donor serum, and contributions of allotypic reactions were excluded. In three systems investigated quantitatively, and in one studied qualitatively, two recipients of the same donor antibody produced anti-antibody that reacted with essentially the same subfraction of the donor antibody population. The findings that only a portion of the D population is immunogenic, and that the same subfraction is frequently immunogenic in different recipients, suggest that the immunogenic population comprises a limited number of homogeneous groups of antibody molecules. This is supported by the small number of bands usually observed by the Ouchterlony technique. Quantitative methods of analysis should provide an approach to the study of cell populations producing antibodies of a particular idiotype. Footnotes Submitted: 23 June 1969

Swanson, John; Hsu, Konrad C.; Gotschlich, Emil C.

doi: 10.1084/jem.130.5.1063pmid: 5347694

The presence of M antigens on group A streptococci is associated with hairlike fimbriae that cover the surface of the streptococcal cell wall and are demonstrable by electron microscopy. These fimbriae also may be associated with R antigen. Like M protein, the surface fimbriae are destroyed by trypsin treatment and reappear when "trypsinized" streptococci are reincubated in fresh, trypsin-free broth. Ferritin-conjugated, type-specific antibodies localize on homologous M+ cells in a pattern suggestive of several M antigenic sites along the length of individual surface fimbria. The M-associated fimbriae remain on the residual cell wall after removal of the bulk of group-specific polysaccharide through nitrous acid extraction. This suggests attachment of the fimbriae to the mucopeptide and minor polysaccharide components remaining in the nitrous acid-extracted wall. The pattern of localization of ferritin-conjugated antibodies on homologous streptococci before and after trypsin exposure and upon reincubation of the trypsinized cells in fresh medium suggests the following hypothesis: M antigen is secreted by the cell, is partially excreted through the otherwise intact cell wall, and is bound by the wall so that M protein occupies a peripheral, exposed position on the surfaces of the streptococcal cell wall. Footnotes Submitted: 3 July 1969

Cerottini, Jean-Charles; Lambert, Paul-Henri; Dixon, Frank J.

doi: 10.1084/jem.130.5.1093pmid: 4186796

The immune responsiveness of (NZB x NZW) F 1 hybrid mice (NZB/W) has been compared with that of three other strains of mice, A/J, BALB/c, and CBA/J. The antigens used included sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and human γ-globulin (HGG). It was found that important strain differences existed in the amount of antibody produced, but the relative immune responsiveness depended very much upon the nature of antigen. By comparison with the other strains tested, NZB/W mice had a higher antibody production to some antigens (SRBC and BSA) but were low responders to others (KLH). Induction of unresponsiveness to HGG by treatment with ultracentrifuged HGG was studied in the strains cited above. NZB/W mice became tolerant after injection of HGG ultracentrifuged at 100,000 g for 2 hr. Similar experiments carried out with another preparation of HGG (centrifuged at 20,000 g for 30 min) failed to reveal any abnormal behavior of NZB/W mice as compared to BALB/c or A/J mice. These results do not support the concept that NZB/W mice possess a general immune hyperreactivity or a relative inability to be made tolerant to protein antigens. However, they do not rule out the possibility that these mice have a genetically determined hyperresponsiveness to some antigens, in particular to nuclear antigens. Footnotes Submitted: 7 July 1969

Foerster, John; Green, Ira; Lamelin, Jean-Pierre; Benacerraf, Baruj

doi: 10.1084/jem.130.5.1107pmid: 4899853

Hartley guinea pigs genetically unresponsive to hapten-PLL (poly- L -lysine) conjugates were lethally irradiated and given allogeneic bone marrow from Hartley responder animals. Many of the animals died of graft versus host disease before their response to 2,4-dinitrophenyl-PLL (DNP-PLL) could be measured. The immune response of the surviving recipient animals was evaluated by anti-DNP antibody production, development of delayed hypersensitivity to DNP-poly- L -lysine, as well as by lymph node cell stimulation in vitro by this antigen. 12 of 14 recipient animals thus treated made an immune response as measured by 2 of the 3 parameters. Strain 13 guinea pigs, genetically unable to respond immunologically to DNP-PLL and to DNP-GL (2,4-dinitrophenyl- L -glutamic acid L -lysine copolymer) were lethally irradiated and given bone marrow from (2 x 13) F 1 responder animals or strain 13 bone marrow and (2 x 13) F 1 lymph node and spleen cells. A high proportion of the animals survived this procedure; no evidence of graft versus host disease was observed. Three of three strain 13 animals irradiated and, given strain 13 bone marrow and (2 x 13) F 1 lymph node and spleen, and then immunized with DNP-PL, made a specific immune response. 7 of 10 irradiated strain 13 animals given strain 13 bone marrow and (2 x 13) F 1 lymph node and spleen made an immune response to DNP-GL. However, only one of six irradiated strain 13 animals made a vigorous immune response to DNP-GL after reconstitution with (2 x 13) F 1 bone marrow alone. The ability to transfer the immune response to PLL antigens from responder to nonresponder animals demonstrates unequivocally that the defect in the non-responder animals is immunological rather than due to some other type of non-immunological mechanism. The bone marrow contains all the immunological cells necessary for the expression of the PLL gene. However, the finding that (2 x 13) F 1 lymph node and spleen cells were more effective than (2 x 13)F 1 bone marrow cell populations (known to be a rich source of monocyte precursors) suggests that the cells in which the PLL gene function is expressed may be lymphocytes rather than monocytes and macrophages. Footnotes Submitted: 14 July 1969