The Journal of Experimental Medicine

Chedid, Louis; Skarnes, Robert C.; Parant, Monique

doi: 10.1084/jem.117.4.561pmid: 14020462

The incubation of endotoxin with Na 2 Cr 51 O 4 yielded a product which was well labeled. That the label was fixed on the endotoxin itself was shown by autoradiography on specific lines of precipitation formed in agar. Ultracentrifugation at 40,000 RPM sedimented 80 per cent of the total weight of the starting Boivin preparation. Agar diffusion patterns with subsequent autoradiographs demonstrated that the chromium tag was associated only with the heavy fractions of the pellet. The supernatant contained precipitable, but unlabeled endotoxin. Toxicity measurements showed that more than 99 per cent of the total toxicity resided in the pellet fractions. The chromate-tagged endotoxin was specifically identified in plasma samples taken up to 6 hours after intravenous administration of LD 50 or sublethal doses. The endotoxin was not totally detoxified in vivo since plasma collected 6 hours after the injection of even the sublethal dose was toxic when assayed in adrenalectomized mice. The endotoxin was specifically identified in urine specimens but it was no longer toxic or radioactive. Agar diffusion experiments indicated that only degraded material was present. Footnotes Submitted: 7 November 1962

Meyer, Franz; Putnam, Frank W.

doi: 10.1084/jem.117.4.573pmid: 13935269

Bence Jones protein injected into rabbits was excreted rapidly into the urine. The excreted protein retained the physicochemical and immunochemical properties of the injected Bence Jones protein. In spite of the rapid excretion, a significant portion of the injected Bence Jones protein was found to be metabolized. After reinjection of radioactive Bence Jones protein into the donor patient, only a little radioactivity was recovered in the urinary protein. The implications of these results are discussed. Footnotes Submitted: 5 December 1962

Hahn, Jerome J.; Cole, Roger M.

doi: 10.1084/jem.117.4.583pmid: 13951944

The formation and destruction of long chains by growth of Group A streptococci in the presence of type-specific antibody have been studied with the fluorescent antibody technique. Long chain formation has been shown to depend on the presence of free antibody during the growth of the bacteria. Destruction of long chains has been shown to depend on the continued growth and division of the bacteria in the absence of free antibody. Univalent antibody fragments formed by proteolytic digestion of antibody globulin have been shown to have the combining properties of untreated antibody but do not result in the production of long chains. A model involving end-to-end agglutination during growth of Group A streptococci has been presented to explain the mechanism of production of long chains by growth of Group A streptococci in the presence of type-specific antibody. Footnotes Submitted: 4 December 1962

Fishman, M.; Adler, F. L.

doi: 10.1084/jem.117.4.595pmid: 13945311

The diffusion chamber technique permitted the demonstration of specific antibody formation in x-irradiated recipients of such chambers filled with normal lymph node cells and a cell-free homogenate of macrophages which had been incubated in intro with T2 bacteriophage. The activity of the cell-free homogenate was retained in its RNA fraction isolated by means of the phenol method. No antibody formation occurred if such RNA was treated with RNAase. On sucrose gradients (5 to 20 per cent), the active RNA was found to be present in the top third layer. The question of the possible presence of antigen complexed to the RNA is discussed. Footnotes Submitted: 19 December 1962

Fireman, Philip; Vannier, Wilton E.; Goodman, Howard C.

doi: 10.1084/jem.117.4.603pmid: 13945231

1. The removal of the ß 2 A-globulins from three sera from treated ragweed-sensitive individuals by immune absorption was associated with the loss of all detectable skin-sensitizing antibody activity as demonstrated by Prausnitz-Küstner testing. 2. Gel filtration studies, with sephadex G-200, indicated that the fractions containing only macroglobulins were devoid of all detectable skin-sensitizing antibody activity. 3. The immune absorption of the γ-globulins from a serum fraction containing ß 2 A-globulins, γ-globulins, and a trace of ß 2 M-globulins had no detectable influence on the skin-sensitizing antibody activity. 4. The removal of a portion of the albumin from the allergic sera by immune absorption, with retention of the skin-sensitizing activity, indicated that the loss of skin-sensitizing antibody was not due to non-specific absorption on an antigen-antibody precipitate. 5. No inhibition of the Prausnitz-Küstner reaction was observed when sheep serum, normal human serum, or normal human γ-globulins were tested in concentrations described. 6. We conclude that the skin-sensitizing antibody activities in the three sera from treated ragweed-sensitive individuals studied were associated with the ß 2 A-globulins. Footnotes Submitted: 6 December 1962

Dietrich, Felix M.; Weigle, William O.

doi: 10.1084/jem.117.4.621pmid: 14027809

C57BL/6 mice were rendered tolerant to one or another of 13 soluble protein antigens. Tolerance was induced by a single injection of 20 mg protein within 24 hours after birth. The duration of the unresponsive state was measured and compared with the rates of catabolism of the antigens as determined in adult and new born mice. The data presented fail to show a correlation between the persistence of labeled protein antigen and the duration of tolerance. In several occasions, even an inverse relationship between duration of the unresponsive state and persistence was demonstrated. The results, therefore, strongly indicate that the duration of tolerance is not dependent on the rates of catabolism of the antigens. Several of the commercial protein preparations used in this study contained minor impurities to which the animals were generally not rendered tolerant. By means of diffusion in agar techniques, it was demonstrated that mice injected at birth with a tolerance-inducing dose of antigen would generally not reveal precipitating antibodies to this antigen after the tolerant state had been abolished. A speculative explanation was given in terms of quantitative or qualitative differences of antibodies found in such animals as compared to the immunized control mice. After the 3rd or 4th day of life, newborn mice catabolized I 131 -labeled heterologous proteins at the same rates as adult mice. The apparent slow elimination during the first days of life was, at least in part, the result of retention of nonprotein-bound I 131 . Footnotes Submitted: 18 December 1962

Flick, John A.; Pincus, William B.

doi: 10.1084/jem.117.4.633pmid: 13945543

In order to gain insight into the pathogenesis of the vesicular lesion of local primary vaccinia infection, newborn rabbits were injected with 0.5 mg of purified inactivated vaccinia virus in an attempt to render them immunologically tolerant. Within a few days these, and control normal rabbits of the same age, were infected on the skin with active vaccinia virus. Most of the tolerant-prepared rabbits failed to develop a local lesion of vaccinia but some developed a very atypical lesion. Successful virus isolation from some, and the presence of inclusions in the tissues of others, indicated successful infection with the virus. Skin allergy to the active virus failed to develop in the test animals but did in the controls. Thus, there was a high degree of correlation between inability to produce delayed hypersensitivity to the viral antigens and failure to develop a vaccinial skin lesion, indicating the probable allergic nature of the primary lesion. There was also a high mortality rate in the group of tolerant-prepared, infected animals. It was associated with a spreading of the virus from the site of infection to the organs, suggesting that generalized vaccinial infection was the cause of death. The observations were compatible with the hypothesis that death was due to viral toxicity. The observations also suggest that, in the animal possessing normal immunological function, active immunity develops rapidly, perhaps at the level of the draining lymph node, to prevent appreciable virus from leaving the site of infection. The absence of detectable immunological activity toward vaccinia virus early in the tolerant-prepared animals and even after 1 month in some of the survivors, indicates that a high degree of immunological tolerance was produced against these microbial antigens. Footnotes Submitted: 9 December 1962

Evans, Robert S.; Bingham, Margaret; Weiser, Russell S.

doi: 10.1084/jem.117.4.647pmid: 19867227

A disease characterized by frequent association of enteritis and polyagglutinable cells often develops in weanling rabbits. The red cell lesion renders the cells susceptible to agglutination and hemolysis in normal rabbit sera. The degree of red cell abnormality varies among different animals and disappears when the animals recover. The abnormality of the red cells responsible for their polyagglutinability and susceptibility to hemolysis was resistant to the action of trypsin or papain and persisted in heated stroma preparations derived from polyagglutinable cells. The factors necessary for agglutination and hemolysis of the polyagglutinable cells are present in normal rabbit sera but are lacking in the sera of affected rabbits. These factors returned to normal levels as the polyagglutinable cell lesion disappeared. The sera of rabbits with polyagglutinable cells contained normal levels of complement and properdin. Whereas the agglutinating factor in normal sera is heat-stable at 56°C for 30 minutes, the hemolytic factor is heat labile. The hemolytic factor is apparently distinct from complement and properdin since it was adsorbed from normal rabbit serum by zymosan or by polyagglutinable cells at 0°C. However, complement was fixed when normal rabbit serum was reacted with stroma from polyagglutinable cells. Hemolysis of polyagglutinable cells by normal rabbit serum at 25°C was inhibited by preliminary incubation of the mixture at 0°C prior to incubation at 25°C. Evidence was obtained which indicated that this inhibition was due to progression of a reaction involving Ca ++ independent of a reaction involving Mg ++ . Footnotes Submitted: 6 November 1962

Greisman, Sheldon E.; Carozza, Frank A.; Hills, J. Dixon

doi: 10.1084/jem.117.4.663pmid: 13950296

Pyrogenic tolerance following 7 daily intravenous injections of 2.0 µg/kg E. coli endotoxin in albino rabbits was associated with significant increases in RES phagocytic activity as measured with colloidal carbon. Nevertheless, 4 hours after RES blockade with thorotrast (3 ml/kg), the tolerant rabbits exhibited significantly lower fever indices following intravenous endotoxin challenge than did non-tolerant control animals despite comparably depressed capacities to clear carbon from the blood. Moreover, plasma from rabbits tolerant to endotoxin induced significant tolerance in normal rabbits prepared by thorotrast blockade without enhancing the depressed carbon clearance. This passive protection extended to heterologous endotoxins. Analysis of the data indicates that RES blockade does not abolish tolerance; rather blockade resets the reactivity to endotoxin in the normal and tolerant animal, rendering both exquisitely reactive, but permitting retention of the major portion of tolerance. Apparently the tolerant animal possesses a dual endotoxin defense system. One system is abolished by thorotrast; the other is in part humoral, accounts for the greater portion of tolerance, and is thorotrast-resistant. The nature of the humoral component is not defined but is consistent with that of an opsonin with high endotoxin specificity. Footnotes Submitted: 30 December 1962

Terasaki, Paul I.; McClelland, John D.

doi: 10.1084/jem.117.4.675pmid: 13980634

Antigenic differences between certain inbred strains of mice which could not be revealed by hemagglutination techniques were readily disclosed by lymphocyte cytotoxicity. With an improved cytotoxicity test lymphotoxic titers were as high as 1:512 with non-hemagglutinating A anti CBA antisera. In other mouse strain combinations, a close parallel of both types of antibody activity was obtained. Though both activities were absorbed from antisera proportionally by erythrocytes and lymph node cells, 100 to 1000 times as many erythrocytes as lymphocytes were necessary to produce an equivalent reduction in antibody activity. These findings suggest that erythrocytes may possess only subthreshold quantities of certain antigens which are present in readily detectable levels on lymphocytes. Lymphocyte cytotoxicity therefore may assay a wider range of allogenic antigens than hemagglutination. Footnotes Submitted: 6 January 1963