Footpad Test Research Articles

Response of hypersensitive mice to the footpad injection of living homologous or heterologous mycobacteria: preliminary report.

Mice sensitized by the injection of viable mycobacteria into one of the hind footpads responded to a second injection of mycobacteria (3 to 4 weeks later), introduced into the contralateral foot, with a degree of footpad swelling that was both accelerated and exaggerated beyond that observed after the first inoculation. The degree of specificity of this reaction (i.e., response to homologous versus heterologous mycobacteria) was comparable to that previously reported for dermal reactions of hypersensitive guinea pigs to tuberculin or tuberculin-like antigens from mycobacteria. In preliminary studies it was impossible to achieve this state of specific sensitization by vaccinating mice subcutaneously with water-in-oil emulsions of heat-killed mycobacteria; reasons for the failure are discussed. It is suggested that this tool could prove useful in both taxonomic and immunological investigations. Advantages and disadvantages of the mouse footpad test in relation to the dermal skin test in guinea pigs are discussed.

Migration Inhibitory Factor and Interferon in the Circulation of Mice with Delayed Hypersensitivity

When mice infected with Mycobacterium tuberculosis strain BCG were inoculated intravenously with old tuberculin (OT) or living BCG cells, both migration inhibitory factor (MIF) and interferon appeared in the circulation within a few hours. In such animals, which showed delayed hypersensitivity by footpad tests, as little as 1.5 mg of OT or as few as 1.7 x 10(6) bacteria per mouse were capable of eliciting circulating MIF and interferon. Uninfected animals inoculated with large doses of OT or living BCG cells did not produce MIF or interferon. When nonspecific stimuli such as bacterial lipopolysaccharide (LPS; from Salmonella typhimurium strain LT-2), heat-killed Brucella abortus, Newcastle disease virus (NDV), and polyinosinic acid:polycytidilic acid (poly I:C) were inoculated intravenously into BCG-infected mice, MIF was produced in the circulation of animals challenged with LPS or Brucella but not in those challenged with NDV or poly I:C, although all the stimuli were capable of eliciting an interferon response. The interferon elicited in BCG-infected mice by specific antigen differed in at least one important property from the viral inhibitor produced by the nonspecific stimuli. The interferon which appeared after injection of OT or living BCG cells was destroyed by treatment at pH 2 for 24 hr at 4C, whereas the interferons produced after injection of the nonspecific stimuli were stable under the same conditions. The MIF activity in plasma from sensitized mice inoculated with specific antigen was also destroyed by treatment at pH 2. When mouse plasma containing both MIF and interferon activity was filtered through Sephadex G-100, both mediators were excluded in the same peak fractions. Sensitization of mice with complete Freund adjuvant instead of infection with BCG cells produces a different pattern of response. Although hypersensitive to specific antigen by footpad swelling tests, mice sensitized with complete Freund adjuvant failed to produce MIF or interferon when they were inoculated intravenously with OT or living BCG cells.

Response of Hypersensitive Mice to the Footpad Injection of Living Homologous or Heterologous Mycobacteria: Preliminary Report

Mice sensitized by the injection of viable mycobacteria into one of the hind footpads responded to a second injection of mycobacteria (3 to 4 weeks later), introduced into the contralateral foot, with a degree of footpad swelling that was both accelerated and exaggerated beyond that observed after the first inoculation. The degree of specificity of this reaction (i.e., response to homologous versus heterologous mycobacteria) was comparable to that previously reported for dermal reactions of hypersensitive guinea pigs to tuberculin or tuberculin-like antigens from mycobacteria. In preliminary studies it was impossible to achieve this state of specific sensitization by vaccinating mice subcutaneously with water-in-oil emulsions of heat-killed mycobacteria; reasons for the failure are discussed. It is suggested that this tool could prove useful in both taxonomic and immunological investigations. Advantages and disadvantages of the mouse footpad test in relation to the dermal skin test in guinea pigs are discussed.

Effect of dilantin in mice

Four hundred and thirty two mice of strains C3H/f, C57Bl, and SJL/J were divided into 9 groups and fed normal mouse pellets, a liquid chocolate flavored diet (Metrecal), and the liquid diet with dipenyl hydantoin, respectively, over a period of 16 months. An additional 60 C67Bl mice were sensitized with bovine serum albumin (BSA) and complete Freund's adjuvant while on dipenyl hydantoin and studied for their humoral and cellular immune reactivity by passive hemagglutination microassays and foot-pad tests. Mice of all 3 strains showed lesions in their lymphoreticular tissues from the second month of the experiment on, varying from partial activation to atrophy. Primarily affected was the paracortical zone of lymph nodes. C3H/f and C57Bl mice developed thymic lymphosarcomas with a frequency of 3 of 24 mice each. SJL/J mice also showed similar tumors early during the experiment, i.e. 6 of 42 mice during the 4th to the 8th month. After the 8th month SJL/J mice developed up to 90% reticulum cell sarcomas type B, a lesion known to occur in these mice in a high frequency; however, its occurrence was about 2–3 months earlier than usual. Immunologic studies in dipenyl hydantoin fed mice revealed variable but not significantly decreased serum anti-BSA titers while the foot-pad tests were constantly negative for a delayed hypersensitivity reaction. It is concluded from these results that diphenyl hydantoin accelerates the spontaneous development of lymphoma in susceptible animals and may also induce such tumors at low frequencies. The mode of action probably is through interference with the normal immune response by simultaneous immunostimulation and immunosuppression.

Macrophage Inhibition Produced by Guinea Pigs after Sensitization with a Larval Antigen of Trichinella spiralis

Experimental guinea pigs were sensitized both with a protein fraction of T. spiralis larvae (Melcher's antigen) and M. tuberculosis H37Ra in Freund's complete adjuvant. Controls were injected with the adjuvant alone. The experimental animals showed typical delayed skin reactions of the tuberculin type in separate sites tested with Melcher's antigen and Purified Protein Derivative (PPD), whereas the controls reacted only to PPD. Studies were made with peritoneal exudate cells to determine the effect of Melcher's antigen and PPD on macrophage inhibition in capillary tubes. The macrophages from all of the experimental animals were inhibited strikingly by both Melcher's antigen and PPD, but those from the control animals were inhibited only by PPD. These results suggest that in mice Melcher's antigen might increase the reproducibility of the inhibition test results shown previously with a crude saline extract of T. spiralis larvae. There is considerable evidence to support the suggestion that immunity to the intestinal stages of T. spiralis in the mouse host is due to delayed (cellular) hypersensitivity (Larsh, 1967). Recently, a crude saline extract of T. spiralis larvae has been used as a means for artificially sensitizing donors so that this immunity could be studied under more controlled conditions than are possible when using infected mice for this purpose. Donors sensitized with this crude antigen produced immunity, and after transfer of their spleen cells, immunity was conferred upon recipients (Larsh et al., 1969, 1970a, b). Although the results of these cell transfer studies tend to support the view that the transferred cells produced delayed sensitivity in the recipients, more information is needed to prove this hypothesis. In the first place, it must be shown that the sensitized donors have, in fact, developed delayed sensitivity as result of these antigen injections. Various in vivo tests are available for this purpose, such as skin and footpad tests. However, in view of its great potential value in such studies, and its acceptance as an in vitro correlate of delayed hypersensitivity, we chose first to make tests in capillary tubes for the inhibition of migration of macrophages. In 60% of the tests, the crude saReceived for publication 30 July 1970. * This investigation was supported in part by the Postdoctoral Fellowship (1F02GM44719-01, National Institute of General Medical Sciences) of the senior author. line extract antigen in the presence of splenic lymphocytes from artificially sensitized mice inhibited the migration of macrophages (Cypess and Larsh, 1970). The failure to obtain inhibition in a greater percentage of tests was attributed tentatively to the use of a crude antigen. Therefore, it was decided to select a more refined antigen for a similar study. The only reports to date on delayed skin responses to T. spiralis antigens in experimental hosts have dealt with studies in guinea pigs (Kim, 1966; Kim et al., 1967a, b). In these studies, a saline extract antigen similar to the one mentioned above was used for the most part, but some work was done by Kim (1966) with a more refined substance (Melcher's antigen), which is an acid-soluble protein fraction. Delayed skin reactions of the tuberculin type were demonstrated with both antigen preparations. In view of the available information on Melcher's antigen in guinea pigs, it was decided to use this system for the present study of macrophage inhibition. It seemed advisable to include an organism (Mycobacterium tuberculosis) known to produce consistently both delayed skin reactions and macrophage inhibition in tests with sensitized guinea pigs. This provided a means for checking the effectiveness of our sensitizing procedures, and the specificity of the responses produced as tested by skin reactions and inhibition of migration of

The effect of heterologous Anti-Lymphocyte serum on Delayed-Type hypersensitivity in mice

<h2>Summary</h2> Rabbit anti-mouse lymphocyte serum (ALS) was capable of depressing delayed (24- and 48-hour) footpad reactions to sheep erythrocytes in mice previously sensitized with sheep erythrocytes in adjuvant. The degree of depression depended on the mode of preparation of the ALS, the dose, and the time of injection relative to the footpad test. After intraperitoneal injection of ALS there occurred; lymphopenia in blood and peritoneal fluid, the appearance of blast cells in peritoneal fluid, and a temporary loss of macrophages followed by the appearance of larger and ultrastructurally changed macrophages in the peritoneal fluid; temporary loss of macrophages also followed the injection of normal rabbit serum. The ability of different batches of ALS to depress delayed footpad reactions could not be correlated exactly with any of the other changes or with the titre of antibodies to lymph node or peritoneal lymphocytes as measured by immune adherence. Delayed footpad reactions to allogeneic lymphocytes in mice sensitized by skin allografts were depressed by ALS given just before footpad testing. In mice which had combined early (Arthus) and delayed-type hypersensitivity to sheep erythrocytes, ALS treatment also depressed the early component of the footpad reactions.

Relationship of footpad sensitivity to purified protein derivatives and resistance to airborne infection with Mycobacterium tuberculosis of mice vaccinated with mycobacterial cell walls.

Footpads of mice sensitized by oil-treated Bacillus Calmette-Guérin (BCG) cell walls given either intravenously, subcutaneously, intradermally, intraperitoneally, or intramuscularly became swollen and reddened after injection of purified protein derivative (PPD). This reaction, greatest after intradermal and subcutaneous sensitization, generally reached a maximum about 24 hr after challenge and was still marked at 48 hr. The histological response was characterized by infiltration with both polymorphonuclear and mononuclear cells. The proportion of mononuclear cells increased with time and they predominated at 48 hr. The footpad reaction could be detected as early as 1 week after sensitization and persisted for at least 37 weeks. Footpad sensitivity to PPD and acquired resistance to airborne infection with Mycobacterium tuberculosis H37Rv were correlated in that (i) both reached a peak approximately 1 month after sensitization of the mouse, and (ii) cell walls treated with NaOH or given without oil neither protected mice against challenge infection nor sensitized them to PPD. Although, as we reported previously, mice vaccinated subcutaneously or intradermally exhibited little or no enhanced resistance to experimental infection, mice given oil-treated cell walls by these routes were highly sensitive to footpad inoculation of PPD. Therefore, the footpad test cannot be used to determine immunity of the mouse to pulmonary infection with tubercle bacilli.

Induction of Delayed Hypersensitivity to Influenza and Mumps Viruses in Mice

Abstract Delayed hypersensitivity was produced in mice to mumps and influenza virus antigens as a preliminary step to studies of the role of delayed hypersensitivity in resistance to viral infections. Since the footpad test was not found to be sufficiently quantitative or reliable, the in vitro technique of macrophage-migration-inhibition of George and Vaughan was adapted to the mouse model. Cells from sensitized mice were significantly inhibited from migrating when challenged with the homologous antigen. There was some inhibition of migration of sensitized cells with heterologous antigens, but this did not differ significantly from inhibition of non-sensitized cells so challenged, and could be largely circumvented by appropriate dilution of the challenge antigen. The migration of sensitized cells not challenged with either antigen was in the same range as that of the control, non-sensitized, unchallenged cells. It is concluded that macrophage-migration-inhibition provides an assay system for delayed hypersensitivity that is both sensitive and specific for the particular myxoviruses tested.

STUDIES ON CYTOPHILIC ANTIBODIES.

SummaryNon‐inbred Swiss mice were immunized by intraperitoneal or sub‐cutaneous injections of sheep erythrocytes in saline. Freund's incomplete adjuvant or Freund's complete adjuvant. Titrations were made of macrophage cytophilic antibodies. complement‐fixing (immune adherence) antibodies and direct haemagglutinating antibodies in sera obtained at weekly intervals up to 4 weeks. Early and delayed hypersensitivities were estimated by footpad tests with sheep erythrocytes. When the intraperitoneal route of immunization was used, cytophilic antibodies and delayed‐type hypersensitivity developed consistently only after the erythrocytes had been injected in Freund's complete adjuvant. When the subcutaneous route was used, cytophilic antibodies and delayed‐type hypersensitivity developed after the erythrocytes had been injected in complete or incomplete adjuvant but not after they had been injected in saline. There was a correlation between the intensity of delayed (48 hr.) footpad reactions and the levels of cytophilic antibodies in serum as well as between the intensity of early (4 hr.) reactions and the levels of complement‐fixing antibodies. A second injection of antigen in saline (footpad test) was usually followed by a rise in titre or by the reappearance, or even by the first appearance, of cytophilic antibodies in the serum. Cytophilic antibodies could appear in the absence of detectable complement‐fixing antibodies and vice versa, and there was overall no correlation between the levels of cytophilic and complement‐fixing antibodies in the sera.