The Journal of Experimental Medicine

Eisen, Herman N.; Tabachnick, Milton

doi: 10.1084/jem.108.6.773pmid: 13598812

When one or two drops of a dilute, non-irritating solution of 2,4-dinitrochlorobenzene (DNCB) is applied to a small area of skin of the intact guinea pig, about 20 per cent of the applied material, or some derivative of it, is soon excreted in urine. In normal, as well as in specifically sensitized guinea pigs, DNCB at the site of local application becomes rapidly bound to skin protein through primary chemical bonds. Twenty-four hours after application roughly half of the material present at the local skin site is still extractable with organic solvents. Of the non-extractable dinitrophenyl groups, about 99 per cent are in epidermis, and about 85 per cent are substituted in ϵ-NH 2 groups of lysine residues. Only traces of bound dinitrophenyl groups were observed in the corium. It is uncertain whether these are formed in situ , or are experimental contaminants, or are migratory epidermally formed conjugates. Even when DNCB is injected intradermally it combines predominantly with overlying epidermis and with epidermal components of hair follicles, but only slightly with corium. The 2,4-dinitrophenyl conjugates which are localized in the deeper, viable half of the epidermis, close to the epidermal-dermal junction, are inferred to be the agents responsible for specifically evoking the allergic response in sensitized animals. Conjugates which are situated in the outer, cornified half of the epidermis are shown to be incapable of eliciting the allergic response. The results furnish a basis for interpreting a common pattern of lesions in allergic contact dermatitis as it occurs spontaneously in man. Footnotes Submitted: 24 July 1958

Shope, Richard E.; Mangold, Robert; MacNamara, Lester G.; Dumbell, Keith R.

doi: 10.1084/jem.108.6.797pmid: 13598813

A naturally occurring cutaneous fibroma of deer has proven to be experimentally transmissible in deer. The causative agent is a virus that is readily alterable through Berkefeld N candles and that survives in fibroma tissue for at least as long as 27 months in glycerol-saline at –20°C. The experimentally produced deer fibroma has an incubation period of about 7 weeks, a very slow rate of growth, and a high regression rate. Footnotes Submitted: 21 July 1958

Krause, Richard M.

doi: 10.1084/jem.108.6.803pmid: 13598814

The lysis of cell walls of hemolytic streptococci by a phage-associated lysin has been described. A method is presented for preparing the lysin from Group C streptococcal phage lysates. Following lysis almost all of the cell wall carbohydrate is recovered in solution. This material has the serological reactivity, physical-chemical properties, and values for nitrogen, rhamnose, and glucosamine similar to those of the carbohydrate isolated from the cell walls by the Streptomyces albus enzyme. Group C carbohydrate isolated by either enzyme inactivates Group C bacteriophage. The protein liberated by the lysin from Group A Type 6 cell walls gives a type-specific precipitin reaction with homologous rabbit antiserum. Preliminary data are presented on the ammonium sulfate fractionation and the electrophoretic separation of a protein fraction with the serological reactivity of M protein. Footnotes Submitted: 25 July 1958

Raffel, Sidney; Newel, J. Michael

doi: 10.1084/jem.108.6.823pmid: 13598815

The "delayed hypersensitive" reactivity induced by antigen-antibody complexes has been studied from the standpoints of the role of such complexes in establishing this state, and the relationship of this state to classical delayed hypersensitivity. It has been shown that the reactivity established by antigen-antibody complexes appears early after injection, disappears within a few days, and is characterized by several properties which make it appear similar to true delayed hypersensitivity, including its appearance, its relative persistence for 48 hours, and its occurrence in the absence of antibodies. By the same tokens, it may be distinguished from hypersensitive reactions of the immediate type. It is referred to here as reactivity of the Jones-Mote type. Antigen alone stimulates exactly the same kind of early reactive state, but with larger doses of antigen this is later replaced by other immunologic responses including circulating antibodies and Arthus reactivity. If sufficiently small doses of antigen are employed, however, the "monophasic" reaction which follows antigen-antibody complexes consisting of the Jones-Mote type of skin responsiveness may be seen. The dermal reactivity under discussion is unlike classical delayed hypersensitivity chiefly in its evanescent character; it is present only during a few days early after antigen administration. It is suggested that this kind of reactivity, which may perhaps require a category of its own, may be related to the "tissue immunity" to tumor transplants which has been observed in mice. Footnotes Submitted: 9 July 1958

Weller, Thomas H.; Witton, Helen M.; Bell, E. John

doi: 10.1084/jem.108.6.843pmid: 13598816

Fourteen strains of virus derived from the cutaneous lesions of cases of varicella and eight from patients with herpes zoster were propagated serially in primary explant cultures of human preputial or embryonic skin-muscle tissue. Infectious material could not be demonstrated in the fluid phase of infected cultures and inocula for passage therefore consisted of suspensions of infected tissue. Such tissue suspensions when stored in the frozen state did not regularly retain infectivity. The cytopathic process was focal and appeared to develop as the result of transfer of infectious material from cell to contiguous cell. Optimum development of the focal lesions in vitro related directly to conditions favoring optimum tissue growth and was further influenced by the spatial relationship of the tissue outgrowth. A variety of types of cells of human origin and several of monkey origin were susceptible to infection and responded with the formation of intranuclear inclusion bodies. The cellular response otherwise was variable, ranging from simple rounding with little change in size to the formation of large multinucleated cytoplasmic syncytia. Strains of virus recovered from patients with varicella and from patients with herpes zoster could not be distinguished on the basis of their cultural attributes. Footnotes Submitted: 30 July 1958

Weller, Thomas H.; Witton, Helen M.

doi: 10.1084/jem.108.6.869pmid: 13598817

The preparation of antigenic materials capable of specific fixation of complement in the presence of convalescent phase sera from patients with varicella and herpes zoster is described. Satisfactory antigens were obtained by the repetitive harvest and subsequent concentration of the pooled nutrient fluids from bottle cultures of human embryonic skin-muscle tissue or of monkey kidney tissue infected with varicella or herpes zoster viruses. Specific fixation of complement was also demonstrated with antigens prepared from extracts of the infected cell sheets harvested from bottle cultures. In individuals with varicella, complement-fixing antibody usually appeared in the serum 4 or 5 days after the development of the exanthem and further significant increases in titer were characteristically observed during the 2nd week of illness. The complement-fixing antibody response in herpes zoster tended to follow the same pattern as in varicella, with the exception that in sera from some individuals relatively high titers were present in the acute phase specimen. Complement-fixing antigens prepared from varicella strains or from a zoster strain reacted to essentially the same degree with convalescent sera from the homologous and the heterologous clinical entities. The varicella-zoster antigens did not fix complement in the presence of paired sera obtained from a limited number of individuals with primary infections due to herpes simplex virus or from individuals with generalized vaccinia infection. Specific inhibition in vitro of the focal cytopathic process produced by the varicella-zoster viruses was demonstrated. This was accomplished by the incorporation of the sera under test as constituents of nutrient media of the cultures, either prior to or at the time of their inoculation with virus. The neutralization of focal cytopathogenicity thus obtained was relative in degree and never absolute; it was therefore assayed by repetitive counts of the number of focal lesions per culture in the various test groups. Inhibition of varicella-zoster viral cytopathogenicity occurred in the presence of convalescent serum from either clinical entity. The results of the immunologic studies with the viruses of herpes zoster and varicella as propagated in vitro are considered as providing further evidence in support of the concept of the close relationship and probable identity of the two agents. Footnotes Submitted: 30 July 1958

Uhr, Jonathan W.; Pappenheimer, A. M.

doi: 10.1084/jem.108.6.891pmid: 13598818

Guinea pigs rendered hypersensitive (delayed-type) to protein antigen can be completely and specifically desensitized by a single injection containing a sufficient amount of the corresponding antigen. Although 1 to 2 mg. of specific antigen are required for complete desensitization, as little as 20 µg. suffices to decrease the size of specific skin reactions in sensitized animals. The duration of non-reactivity lengthens as the amount of antigen in the desensitizing injection is increased, but skin reactivity eventually returns and is accompanied by the appearance of excess circulating antibody. Desensitization can be accomplished with the antigen-antibody complex as well as by "free" antigen. The appearance of delayed skin reactions can be prevented in fully sensitized animals by intravenous desensitization 2 or more hours after intradermal challenge or by simply skin testing with a desensitizing dose of specific antigen. Injection of a desensitizing dose of antigen into specifically sensitized animals also results in a transient anergic state, the implications of which are discussed. Footnotes Submitted: 26 June 1958

Uhr, Jonathan W.; Brandriss, M. W.

doi: 10.1084/jem.108.6.905pmid: 13598819

Guinea pigs with delayed hypersensitivity to protein antigens show a specific febrile response accompanied by a lymphopenia following injection of a desensitizing dose of specific antigen. No signs of shock are observed in highly sensitive animals following this injection. The response is not prevented in sensitive guinea pigs by inducing endotoxin tolerance or by pretreating with cortisone before specific challenge. Using a suitable antigen in sufficiently sensitive animals as little as 100 µg. can elicit a pronounced febrile response. Injection of a desensitizing dose of antigen specifically abolishes systemic as well as skin reactivity for several days. Normal or hypersensitive (delayed-type) animals passively sensitized with sufficient amounts of serum antibody show hypothermia after specific challenge and may show a delayed type of fatal shock. Differences were noted between their systemic reactivities, however, and the reactivity seen in specifically challenged tuberculous animals. Footnotes Submitted: 26 June 1958

Hirsch, James G.

doi: 10.1084/jem.108.6.925pmid: 13598820

The arginine-rich fraction of calf thymus histone (histone B) exerts bactericidal activity on various coliform bacilli and micrococci under certain conditions in vitro . Final concentrations of less than 1 µg. histone per ml. kill susceptible microbes without detectable morphological alteration or lysis. Among the microorganisms highly susceptible to histone are Escherichia, Salmonella, Shigella, Pseudomonas, Klebsiella , and Micrococcus pyogenes var. albus . Less susceptible or completely resistant are Proteus, Serratia, Micrococcus pyogenes var. aureus , and various types of hemolytic streptococci. Coliforms grown on solid media are much more resistant to the lethal effect of histone than are those cultured in liquid media. This difference is apparently related to the physiological state of the bacteria; agar grown microorganisms washed with water remain resistant to histone, whereas incubation in broth rapidly renders them more susceptible. Histone is adsorbed onto heat-killed E. coli K-12 under conditions suitable for lethal action on this organism. The bactericidal activity of histone is but little affected by pH of the test system, but ionic strength of the medium exerts a marked influence, the lethal action being reduced or blocked as the salt concentration reaches levels higher than that of 0.15–0.2 M NaCl. Relatively high concentrations of rabbit serum or of bovine plasma albumin reduce the bactericidal activity of histone in a medium at pH 7; these serum preparations are, however, essentially without effect in the test system at pH 5.6. The bactericidal effect of histone is antagonized by addition to the medium of small amounts of certain basic substances (protamine, spermine), or of various acid polysaccharides (heparin, nucleic acid, bacterial lipopolysaccharides). The rate of killing of E. coli K-12 by histone increases as the temperature and the concentration of histone are raised. Within the limits studied, this rate also appears to be directly proportional to the concentration of bacteria in the system. Footnotes Submitted: 29 July 1958

Puck, Theodore T.; Cieciura, Steven J.; Robinson, Arthur

doi: 10.1084/jem.108.6.945pmid: 13598821

A methodology designed to eliminate mitotic inhibitor action and involving use of pretested fetal calf serum and careful pH and temperature control has been described by which cells from normal human and animal tissue can be maintained in active growth for long periods in vitro without development of aneuploidy. By means of this procedure, it is possible reliably to establish cell cultures from minute skin biopsies which can be taken from any individual. Clones of mammalian cells with chromosomal markers have been isolated by this means from x-irradiated non-irradiated cell cultures. Application of these techniques to chromosome delineation in large numbers of human subjects; determination of chromosomal sex in patients; spontaneuos and induced genetic changes in somatic mammalian cells in vivo and in vitro ; comparison of metabolic differences between normal and cancerous cells and other problems have been indicated. Footnotes Submitted: 24 July 1958