The Journal of Experimental Medicine

Berlin, Richard D.; Wood, W. Barry

doi: 10.1084/jem.119.5.697pmid: 14159044

The metabolic reactions responsible for the release of endogenous pyrogen from rabbit granulocytes incubated in 0.15 M NaCl are specifically inhibited by the presence of K + (and by related alkali metal ions, Rb + and Cs + ) in the medium. The inhibitory action of K + apparently involves penetration of the cell membrane and is directly antagonized by the cardiac glycoside, ouabain. It is concluded, therefore, that the inhibition of pyrogen release by extracellular K + is due to transport of K + into the cell. Although the precise molecular mechanisms which are responsible for the release of pyrogen from granulocytes incubated in K-free saline have not been elucidated, further study of the process has revealed: ( a ) that it is preceded by the accumulation of pyrogen within the cell, ( b ) that it depends upon the catalytic action of one or more sulfhydryl-containing enzymes, ( c ) that it does not require energy, either from glycolysis or from reactions depending on molecular oxygen, and ( d ) that its inhibition by K + and by arsenite is qualitatively similar to the depression caused by these same reagents on the release of other leucocytic proteins; i.e ., lysozyme and aldolase. Footnotes Submitted: 1 January 1964

Berlin, Richard D.; Wood, W. Barry

doi: 10.1084/jem.119.5.715pmid: 14157026

1. Phagocytosis promotes the release of endogenous pyrogen from polymorphonuclear leucocytes. 2. The release of pyrogen, though initiated by the phagocytic event, is not synchronous with it. 3. The postphagocytic release mechanism is not inhibited by sodium fluoride and, therefore, appears not to require continued production of energy by the cell. 4. The release process, on the other hand, is inhibited by arsenite, suggesting the participation of one or more sulfhydryl-dependent enzymes in the over-all reaction. 5. Particle for particle, the ingestion of heat-killed rough pneumococci causes the release of approximately 100 times as much pyrogen as the ingestion of polystyrene beads of the same size. 6. The pyrogen release mechanism of polymorphonuclear leucocytes separated directly from blood, unlike that of granulocytes in acute inflammatory exudates, is not readily activated by incubation of the cells in K-free saline. Despite this difference, both blood and exudate leucocytes following phagocytosis release large amounts of pyrogen, even in the presence of K + . The fact that the postphagocytic reaction is uninhibited by the concentrations of K + which are present in plasma and extracellular fluids, suggests that this mechanism of pyrogen release may well operate in vivo . 7. As might be expected from the foregoing observations, the intravenous injection of a sufficiently large number of heat-killed pneumococci causes fever in the intact host. Intravenously injected polystyrene beads, on the other hand, are significantly less pyrogenic. Evidence is presented to support the conclusion that the fever in both instances is caused by pyrogen released from the circulating leucocytes which have phagocyted the injected particles. 8. The possible relationships of these findings to the pathogenesis of fevers caused by acute bacterial infections are discussed. Footnotes Submitted: 1 January 1964

Kirkpatrick, Charles H.; Wilson, W. E. C.; Talmage, David W.

doi: 10.1084/jem.119.5.727pmid: 14157027

Twenty-eight patients with chronic renal diseases and uremia were investigated with respect to their cutaneous responsiveness to a panel of antigens expected to elicit immediate and delayed hypersensitivity reactions. Compared to a control group, there was a marked decrease in the incidence of responses of both types. Eighteen patients received renal allografts from members of the control group and were available for restudy in the postoperative period prior to the institution of adrenal steroid therapy. Each recipient acquired delayed responsiveness with specificity identical with that of the kidney donor. The donor group was reactive to 49 antigens to which the recipients were non-reactive preoperatively. Postoperatively, 40 of these reactivities were observed in the recipients. This successful demonstration of the transfer of immunologically competent tissue in association with renal transplantation indicates that the cause of depressed cutaneous hypersensitivity in uremia is not an inability of the skin per se to react. Footnotes Submitted: 9 December 1963

Sainte-Marie, Guy; Coons, Albert H.

doi: 10.1084/jem.119.5.743pmid: 14157028

Cells from lymph nodes of rabbits injected repeatedly with bovine serum albumin were transferred subcutaneously to previously irradiated rabbits, and the recipients were immediately injected with bovine serum albumin. A good antibody response resulted. In a series of such animals killed on successive days, skin samples at sites of cell deposition were removed and examined by immunofluorescence and by light microscopy. In these tissues abundant plasmocytes were found to have multiplied and differentiated in a regular progression from immature, to medium, to mature plasmocytes. During the 6 days of the experiment the small plasmocytes accumulated until they reached 85 per cent of the total plasmocytic population. The mitotic index of the large and medium plasmocytes averaged 11 per cent, implying a generation time of 6.3 hours on the basis of a 1 hour mitotic time. This rate of growth is sufficiently rapid to account for all the plasmocytes on the 6th day as deriving from less than 1 per cent of the population initially transferred. This rate and the orderly progression in the evolution of the plasmocytic population, make it highly improbable that plasmocytes arise from transformation of lymphocytes, but rather indicate that they spring from specific precursors already present among the transferred cells. Footnotes Submitted: 19 January 1964

Zabriskie, John B.

doi: 10.1084/jem.119.5.761pmid: 14157029

Non-lysogenic, non-toxinogenic Group A streptococci when infected by temperate bacteriophages isolated from known scarlatinal toxin-producing strains acquire the capacity to form erythrogenic toxin. This toxin causes a characteristic erythematous reaction in the skin of rabbits and is readily neutralizable by standard scarlatinal antitoxins. The production of toxin appears to be related to the synthesis of mature phage particles since ultraviolet enhancement of phage production results in a concomitant increase in toxin titer. In contrast, there is no increase in the production of another extracellular product, deoxyribonuclease, by these lysogenized streptococci. Furthermore, cellular disruption studies indicate that the toxin probably does not exist in a preformed state within the cell. Double diffusion reactions in agar indicate that a newly formed protein appears in the lysogenic culture filtrate and is absent in the non-lysogenic filtrates. Footnotes Submitted: 12 January 1964

Lanman, Jonathan T.; Herod, Llewellyn; Fikrig, Senih

doi: 10.1084/jem.119.5.781pmid: 14157030

The effect of homograft sensitization of a mother against her fetuses has been studied. A female rabbit was repeatedly grafted from both members of a breeding pair. Their offspring should and do become objects of homograft sensitization induced in the female by grafts from the parents. After sensitization was established, fertilized ova were transplanted from the breeding pair to the sensitized female. At the same operation, an equal number of eggs was also transplanted to the same recipient from another breeding pair against which no sensitization had been induced. In 16 such families, the survival rates for offspring born of each type of egg were found to be almost identical, indicating that homograft sensitization in the mother directed against her fetuses did not demonstrably harm them. Footnotes Submitted: 23 January 1964

Olins, D. E.; Edelman, G. M.

doi: 10.1084/jem.119.5.789pmid: 14157031

Admixture of separated L and H polypeptide chains of 7S γ-globulins under appropriate conditions has been found to result in the reconstitution of 7S molecules. The chains were mixed in 0.5 N propionic acid and when dialyzed into neutral aqueous buffers interacted to form reconstituted material in greater than 30 per cent yield. This material had sedimentation coefficients of 6S to 7S, a weight average molecular weight of 160,000, and its antigenic structure and electrophoretic properties were the same as those of 7S γ-globulin. By the use of I 131 and I 125 labels on the different types of chains, combined with ultracentrifugation of chain mixtures in sucrose density gradients, the 7S product was found to contain both isotopes in ratios consistent with the presence of two L and two H chains. After hydrolysis with papain, the reconstituted material yielded products resembling S and F fragments. All of the isotope corresponding to L chains was found in the counterpart of the S fragment; the isotope corresponding to the H chain fraction was present in both fragments. The activity reconstituted from chains of a purified guinea pig antibody to f1 phage was found to be associated mainly with the 7S material. Hybrid molecules containing rabbit L chains and human H chains and of human L chains and rabbit H chains were formed by the same techniques of reconstitution. It was found that the interchain disulfide bonds of native 7S γ-globulins could be broken and reoxidized, as could those of reconstituted 7S material. Reduced L and H chains mixed in propionic acid, dialyzed against neutral buffers containing mercaptan, then against neutral buffers in the absence of mercaptan, formed stable 7S molecules of molecular weight 160,000, which were dissociable only after reduction. Footnotes Submitted: 12 January 1964

Gally, J. A.; Edelman, G. M.

doi: 10.1084/jem.119.5.817pmid: 14157032

The L polypeptide chains of certain Bence-Jones proteins of group I have been found in three forms: monomers of molecular weight of about 20,000, dimers which monomerize in dissociating solvents, and dimers which are stable in such solvents. The L polypeptide chains of some Bence-Jones proteins of group II were found to occur naturally only as stable dimers. The L chains of normal human γ-globulin have been obtained in a reduced unalkylated form, and a fraction of these chains was found to form stable dimers under oxidizing conditions. It is suggested that a single disulfide bond is involved in stabilization of the dimer. In experiments on the reconstitution of 7S γ-globulin, it was found that stable dimers of L polypeptide chains did not associate appreciably with H γ chains to form a soluble product. L chains in the monomeric form, both of a reduced alkylated Bence-Jones protein and of reduced unalkylated γ-globulin, combined with H γ chains to form a 7S product. After hydrolysis with papain, the 7S material containing the Bence-Jones L chains yielded fragments comparable to the fragments of papain-treated myeloma proteins. As indicated by spectrofluorometric measurements, dissociable dimers and stable dimers of the L chains of a Bence-Jones protein both underwent identical thermally induced transitions in the temperature range 48–58°C. When L polypeptide chains were present in reduced alkylated γ-globulin or reduced alkylated S fragments, no transition occurred until 65°C, the coagulation temperature of γ-globulin and S fragments. Above this temperature, L chains were released into solution. These experiments suggested that free L chains and L chains bound to H γ chains have different conformational stabilities. Footnotes Submitted: 12 January 1964

Cross, A. Marjorie; Leuchars, Elizabeth; Miller, J. F. A. P.

doi: 10.1084/jem.119.5.837pmid: 14157033

Experiments performed on CBA mice thymectomized in adult life, exposed to lethal doses of irradiation and given tissue therapy are described. Marrow, foetal liver, or spleen cells from syngeneic donors could protect the mice against the lethal effects of irradiation. Between 30 and 70 days' postirradiation, however, marrow-treated, thymectomized irradiated mice showed evidence of trophic disturbances, such as failure to gain weight, in contrast to sham-operated, irradiated, marrow-treated controls. The immune responses of experimental and control mice were tested up to 150 days' postirradiation by challenging with sheep erythrocytes and allogeneic skin grafts. Sham-operated irradiated controls, whether protected with marrow, foetal liver, or spleen cells, produced normal immune responses when challenged at 28, 60, or 150 days after irradiation. Neither foetal liver cells nor marrow cells, in doses of up to 40 million cells per mouse, enabled thymectomized irradiated mice to recover normal immune functions. Spleen cells, from normal donors but not from neonatally thymectomized donors, restored immunological capacity in such mice. It is concluded that immunologically competent cells are present in the spleen of normal adult donors and can function in the absence of the thymus. Bone marrow, on the other hand, does not contain an adequate population of such cells but has lymphoid precursor cells, the descendants of which can become immunologically competent only in the presence of a functioning thymus mechanism. Footnotes Submitted: 19 January 1964

Salvin, S. B.; Smith, R. F.

doi: 10.1084/jem.119.5.851pmid: 14157034

Adult guinea pigs were made unresponsive to a heterologous protein ( e.g . bovine gamma globulin, or BGG) or a hapten-protein conjugate ( e.g. p -aminobenzoic acid-bovine gamma globulin, or PABAγmiddot;BGG) by intraperitoneal injection of 80 mg cyclophosphamide and the specific antigen. This immunologic unresponsiveness developed to the specific antigen administered simultaneously with the cyclophosphamide, and not to any variants. Thus, animals unresponsive to PABAγmiddot;BGG remained unresponsive to the original antigen on challenge with a variant, but formed delayed hypersensitivity and circulating antibody to the variant. The specificity of immunologic unresponsiveness, therefore, seems more closely related to the whole antigen molecule than does delayed hypersensitivity. Footnotes Submitted: 29 January 1964