Footpad Test Research Articles

Experimental paracoccidioidomycosis in the mouse. III. Histopathological and immunological findings after intravenous infection in the presence or absence of previous immunization

Fifty male white Swiss mice aged 4 weeks were inoculated with 5 x 10(5) viable yeast forms of Paracoccidioides brasiliensis (strain 18). Ten of these animals had been previously immunized with particulate P. brasiliensis antigenfor 4 weeks by intradermal injection. The controls consisted of 10 animals that were only immunized and 10 animals submitted to no treatment. The animals were sacrificed 2, 4, 7,11 and 16 weeks later. We studied: 1) the anti-P. brasiliensis delayed hypersensitivity response measured by the footpad test 24 hours prior to sacrifice; 2) the specific antibody production measured by double immunodiffusion in agar gel; 3) the histopathology of lungs, liver, spleen, adrenals and kidneys. We observed that: a) the immunized animals developed more intense cell-immune responses than the infected ones; b) infection reduced the cell- immune response of the immunized animals; c) intravenous infection of mice with P. brasiliensis was characterized by a systemic and progressive granulomatous inflammation. The animals infected after previous immunization showed less extensive lung inflammation, with smaller granulomas and fewer fungi. The results indicate that the present murine model mimics some findings of the human subacute form of paracoccidioidomycosis (systemic disease with depressed cellular immunity) and that the extrapulmonary immunization scheme was able to induce a certain degree of protection of the lung from infection with P. brasiliensis

Complete regression of Lewis lung carcinoma by cyclophosphamide in combination with immunomodulators.

Several types of combination therapy, including OK432, cyclophosphamide (CY), and/or lentinan plus bacterial lipopolysaccharide (LPS), reported to have strong antitumor activity against some tumors, were only slightly effective on Lewis lung carcinoma (LC) in C57BL/6 mice. Combination therapy by intralesional injection of OK432 followed by intraperitoneal administration of CY, lentinan and LPS caused almost complete regression of intradermal solid-type LC. Maximal antitumor activity was observed when lentinan and LPS were administered later than CY. Mice in which LC had regressed due to this combination therapy showed an antitumor delayed hypersensitivity reaction (measured by the footpad test), but they were not resistant to rechallenge with LC. These results provide a new model for combination therapy against weakly immunogenic tumors.

Rifampin in the treatment of leprosy.

The minimal inhibitory concentration of rifampin for Mycobacterium leprae is less than 1 microgram/ml. Therapy with rifampin has proved efficacious both in mice experimentally infected with M. leprae and in humans with leprosy. Rifampin kills M. leprae more rapidly than do other antileprosy drugs currently available. Consequently, M. leprae bacilli from patients with lepromatous disease are rendered noninfectious within three weeks after the institution of rifampin therapy, as determined in the mouse footpad test system. Administration of this antibiotic substantially reduces the quantities of M. leprae discharged in the nasal secretions of lepromatous patients within three weeks, thus rapidly decreasing the potential infectivity of these individuals. Intermittent rifampin therapy for leprosy has been successful, with a low incidence of adverse reactions to the drug. Worldwide, the prevalence of primary and secondary resistance of M. leprae to dapsone has increased markedly. Therefore, the World Health Organization recommends a multidrug regimen that includes intermittent administration of rifampin for the treatment of leprosy.

Combination therapy of murine tumors with lentinan, bacterial lipopolysaccharide and a Streptococcus preparation, OK432.

Combination therapy by intralesional injection of OK432 followed by intraperitoneal administration of lentinan and bacterial lipopolysaccharide caused almost complete regression of solid-type tumor MH134. All three components were needed for maximal antitumor activity. Mice in which MH134-tumor had regressed due to this combination therapy showed an augmented antitumor delayed hypersensitivity reaction (measured by the footpad test) and resistance to rechallenge with MH134, but they had no cytolytic antibodies in their serum detectable by the complement cytotoxicity test. The possible importance of local inflammation induced by OK432 in this combination therapy is discussed.

Immune reaction of tumor-bearing mice to Propionibacterium acnes and the antitumor effect of the bacteria.

The relationship between immunological reaction to Propionibacterium acnes (PA) and the antitumor effect of the injected bacterium was investigated. The aim was to determine whether the strength of the immune reaction to the bacterium can be used to predict its antitumor effectiveness. C3Hf/Sed mice received SC injections (right thigh) of viable cells of a methylcholanthrene-induced fibrosarcoma. When the tumor grew to 5 mm, the hosts received 350 micrograms PA IV as the antitumor treatment. Cellular immunity (footpad test) to PA was assayed in one group of these mice 14 days later, and in the other anti-PA agglutinins were determined 28 days later. The PA injection cured 22 of 58 mice in the first, and 20 of 46 mice in the second group. Footpad reaction and agglutinin titers to PA in cured mice were not statistically different from those in mice eventually killed by the tumor. Therefore, the strength of the immune reaction to PA in tumor-bearing mice could not be used to predict the antitumor effectiveness of the bacterium.

Experimental pulmonary paracoccidioidomycosis in mice: morphology and correlation of lesions with humoral and cellular immune response.

The present paper describes a murine model for pulmonary paracoccidioidomycosis injecting 6 X 10(5) yeast forms of Paracoccidioides brasiliensis (Pb) by the direct intratracheal route. The sequential histopathology of lung and dissemination lesions together with humoral (immunodiffusion test) and cellular immune response (footpad test and macrophage inhibition factor assay - MIF assay) were investigated since the 1st to the 360th day after infection. All infected animal showed pulmonary Pbmycosis up to Day 30; onwards the lesions subsided being found only in one mouse at Day 360. Dissemination lesions were observed in paratracheal and cervical lymph nodes in 9 out of 68 infected animals. Histologically early lesions were rich in polymorphonuclear cells and evolved to a macrophage desquamative pneumonitis at Day 15 and to typical epithelioid granulomata from Day 30 up to Day 360. Specific precipitating antibodies were first detected 15 days after infection, peaked from Day 30 to 60 and were not observed at Day 360. Significant cell-mediated immunity to Pb was noted at Day 15 with the peak reaction at Day 60 and 90. The intratracheal route represents a highly effective way of infecting mouse with Pb. This experimental pulmonary Pbmycosis is a granulomatous inflammation which courses with specific humoral and cellular immune response. It may be a good tool for further investigation in the pathogenesis and natural history of the disease.

Immunological relatedness among Candida albicans and other pathogenic Candida species

Membrane-mitochondrial (butanol-hot phosphate-buffered saline) and cytosol (soluble cytoplasmic substances) extracts from seven pathogenic species of Candida were used in in vivo and in vitro immunological assays to study antigenic similarities among the strains with respect to C. albicans. Mice were sensitized with C. albicans serotype A for footpad testing or to provide cells for lymphocyte stimulation assays, and guinea pigs were immunized with whole cells or butanol-hot phosphate-buffered saline extracts of C. albicans to obtain antisera for immunodiffusion assays. When extracts from each of the seven species were used in the assays, they consistently segregated, as determined by statistical or subjective analyses, into three groups. Extracts of C. albicans serotype A or B and C. stellatoidea were the most immunologically reactive in all assays, indicating close similarities between those two species, whereas extracts of C. tropicalis and C. parapsilosis elicited only moderate responses. Extracts from C. krusei, C. guilliermondii, and C. pseudotropicalis were hypo- or nonreactive in the assays, indicating a low level of antigenic relatedness to C. albicans.

Enhancement of delayed-type hypersensitivity to Trypanosoma cruzi in mice treated with Mycobacterium bovis BCG and cyclophosphamide.

C57BL/10J mice treated with Mycobacterium bovis BCG and cyclophosphamide were immunized with disrupted epimastigotes or with living blood trypomastigotes from Trypanosoma cruzi and assayed for delayed hypersensitivity by footpad testing with epimastigote antigens. Enhanced and lasting reactions were observed in mice pretreated with BCG or cyclophosphamide or both and immunized with epimastigotes. Whereas BCG pretreatment clearly reduced the mortality rates of mice immunized with living blood forms, no enhancement of the delayed hypersensitivity responses was observed in animals treated with BCG or cyclophosphamide or both before infection. The production of high levels of delayed hypersensitivity in the absence of infection and its adoptive transfer with cells could help to evaluate the participation of cell-mediated immunity in the protection against T. cruzi.

In vitro phagocytosis of Candida albicans by peritoneal mouse macrophages.

The ability of sensitized mouse peritoneal macrophages to phagocytose and inhibit Candida albicans was studied in an in vitro system. Mice were sensitized to C. albicans by intraperitoneal infection with viable organisms or by intracutaneous injection of heat-inactivated cells in Freund complete adjuvant. Development of delayed hypersensitivity to C. albicans was evaluated by footpad tests with cytoplasmic and cell wall antigens as well as by macrophage migration inhibition by these antigens and by whole heat-inactivated cells. Inhibition of macrophage migration by heat-inactivated cells was significantly greater when the mice were sensitized by viable organisms. The macrophages from these mice were also larger and showed a greaer ability to inhibit germ tube production by phagocytosed yeasts. This suggests that macrophages may play a protective role in infection by C. albicans.

In vivo detection of specific cell-mediated immunity in street rabies virus infection in mice.

In street rabies-infected mice, in vivo expression of delayed type hypersensitivity (DTH) measured by the footpad test was revealed by challenge with inactivated fixed rabies virus (RV). The use of BCG as an adjuvant cell-mediated immunity (CMI) was necessary for the production of significant DTH levels. Typical DTH kinetics were obtained, with a maximum at 24 h after the challenge. DTH was also found to be at highest levels 4 days after infection with street rabies virus. DTH could also be revealed with street rabies virus in RV immunized mice. Adoptive transfer of lymphoid cells from a street rabies infected donor to normal recipient mice was performed and DTH was tested with RV. Susceptibility of DTH to immunosuppression by cyclophosphamide treatment was also assayed in street rabies virus-infected mice and in adoptively-sensitized recipient mice. These results and the relationship between DTH and CMI in rabies infection and immunization are discussed.

Transfer factor from BCG-sensitized mice.

A mouse model was established for the study of transfer factor (TF). TF was extracted from the spleens of sensitized mice and examined for activity by the footpad test, which showed that mouse and human TF possessed similar properties. Parenteral administration of TF imparted to unsensitized mice immunologically specific, delayed type hypersensitivity within 24 hr. The magnitude of the response the recipients was proportional to the dose of TF. TF activity was relatively heat-stable, detectable in a fraction of molecular weight of ca 1,000 and apparently contained nucleic acids. Nude mice responded negatively to TF but following administration of viable naive spleen cells a positive response was observed, which suggests that the thymus plays an important role in the expression of TF phenomenon and that the target of TF is the T cells.

Kinetics of different specific immunological parameters after rabies vaccination in mice.

A significant protection to an intracerebral challenge of 70 mean lethal doses of a standard live rabies virus strain was obtained in BCG-pretreated mice or in normal mice which had been immunized with a single subcutaneous injection of a beta-propiolactone-inactivated rabies vaccine. Concomitantly, levels of delayed-type hypersensitivity (measured in vivo by the footpad test) and serum-neutralizing activity were evaluated at various times after immunization. All immune criteria were significantly augmented in the BCG-pretreated, rabies-immune mice as compared to normal, rabies-immune mice. However, peak levels of protection, delayed-type hypersensitivity, and serum-neutralizing activity did not occur at the same times. For instance, in the BCG-pretreated, rabies-immune mice, delayed-type hypersensitivity peaked on day 7, protection peaked on day 21, and serum-neutralizing activity peaked on day 60. In BCG-pretreated mice, which did not receive the rabies vaccine, positive delayed-type hypersensitivity, some protection, and serum neutralizing activity were observed 4 to 5 weeks after BCG pretreatment. The possible relationships between specific and nonspecific immunity provoked by rabies virus antigens, tissue culture cell-associated antigens (derived from the bovine fetal kidney cells in which the rabies virus was grown, and BCG are discussed.

Suppression of tuberculin hypersensitivity during influenza infection in mice

Tuberculin hypersensitivity was examined during acute influenza infection in mice. Employing the footpad test as a measure of delayed-type hypersensitivity, it was noted that tuberculin hypersensitivity was suppressed temporarily beginning on day 3 and continuing through days 10 to 16 following intranasal infection with influenza A/PR8. These changes occurred at a time when influenza virus was replicating in lung tissue. Suppression of footpad swelling was not detected when mice were administered live virus intravenously, were given Formalin-inactivated virus intranasally, or were immunized against influenza before intranasal infection. Transient reduction of total circulating lymphocytes also occurred during influenza infection but did not correlate with the duration of footpad suppression. Because this model system reproduces many of the alterations in immunological function reported to occur during influenza infection in humans, it should provide a useful tool for investigating mechanisms of influenza-induced immunosuppression.

Delayed-type hypersensitivity responses in infected mice elicited by cytoplasmic fractions of Cryptococcus neoformans

Four subcellular fractions of Cryptococcus neoformans prepared by differential centrifugation of disrupted whole yeast and a 3-day culture filtrate were examined for their ability to elicit delayed-type hypersensitivity in sensitized animals. The methods used to detect sensitization were (i) the footpad swelling test and inhibition of peritoneal macrophage migration in mice and (ii) skin testing in guinea pigs. Two entities, the post-mitochondrial supernatant and the culture filtrate, showed considerable activity in the footpad test, with 26- and 30-microliter 24-h swellings, respectively, at 6 weeks after infection. With the latter there was interference from a strong antibody-mediated 4-h skin reaction. The post-mitochondrial supernatant produced strong delayed-type hypersensitivity in guinea pigs at a dose of 69 microgram, and there was no demonstrable cross-reactivity in animals sensitized with heterologous fungi. The footpad swelling in mice correlated well with the macrophage migration inhibition test, with 71% inhibition in mice infected subcutaneously with C. neoformans at 6 weeks. However, mice infected intravenously developed poorer cell-mediated immunity than the subcutaneously infected mice. The post-mitochondrial supernatant was found to contain detectable amounts of cryptococcal capsular polysaccharide.

Delayed-type hypersensitivity to rabies virus in mice: assay of active or passive sensitization by the footpad test

With a purified beta-propiolactone-inactivated rabies virus, a significant increase in footpad swelling was elicited in normal or in BCG-pretreated mice after immunization with varying doses of rabies vaccine. These footpad reactions were shown to peak at 24 h and to be associated with an infiltration of newly formed blood monocytes demonstrated by histology and [125I]deoxyuridine labeling. A relationship between the lymphoproliferation and the degree of sensitization is described, and the susceptibility to cyclophosphamide treatment is also examined. Adoptive transfer of specific reactivity to normal recipient mice with immune lymphoid cells, but not with immune serum, was demonstrated, and the results represent another argument for a cell-mediated immunological mechanism.

Correlation of immune response with clinical stage in Lewis lung tumor-bearing mice.

The effect of Lewis lung tumor growth in mice on the induction of primary immune response to SRBC, was investigated by PFC assay for measuring antibody activity and by footpad test as a correlate for delayed type hypersensitivity reactions. With the appearance of micrometastases in the lungs there was a decline in the humoral and cellular immune response to the SRBC. An increase of number and size of metastases in the lungs led to a further depression of the immune reactivity. Since the reduction of general immune response in mice bearing this tumor is not due to a direct influence of tumor cells, it might be assumed that suppressor cells or factors, are actively abrogating the general and also the tumor directed immune reactions.

The Macrophage Disappearance Reaction (MDR) as an in Vivo Test of Delayed Hypersensitivity in Mice

The macrophage disappearance reaction (MDR), as an invivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 h after immunized mice received an intraperitoneal injection of 200 microng of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.

Interferon and delayed-type hypersensitivity to a viral antigen.

Delayed hypersensitivity to Newcastle disease virus was measured with use of a footpad test in mice. When tissue culture interferon was administered 24 hr before sensitization, footpad swelling upon subsequent challenge with the virus was significantly decreased. In animals already sensitized to the virus, footpad swelling was similarly decreased if interferon was administered a few hours before footpad challenge. This result indicates that interferon can affect both the afferent and the efferent pathways of delayed hypersensitivity to Newcastle disease virus and is in agreement with previous results obtained with nonviral antigens.

Delayed Hypersensitivity in the Mouse

Publisher Summary This chapter discusses the delayed-type hypersensitivity (DTH), the prototype model of cellular immune phenomena as it occurs in mice. Most of the recent explosive development in the field of cellular immunology has emphasized the in vitro demonstrations of cellular responses to immunologic challenge and the various products of such stimulated cells, which possibly have in vivo phlogogenic or cytotoxic potential. However, before the full significance of cellular immune responses in in vivo situations can be determined, it will be necessary to transfer the in vitro observations to in vivo delayed hypersensitivity reactions. For this purpose, the mouse appears to be the most likely subject. Footpad tests, flank skin tests, and epicutaneous tests all have become well established and routinely used indicators of DTH in mice. The excellence of mice for studying the pathogenetic roles of DTH in infectious disease is well illustrated by a series of recent experiments on the formation of infectious granulomas around schistosome eggs, deposited in the lungs of mice by intravenous injection.

Skin testing of guinea pigs and footpad testing of mice with a new antigen for detecting delayed hypersensitivity to Cryptococcus neoformans.

This study was undertaken to evaluate the potential of a cryptococcal culture filtrate antigen, cryptococcin C184, for detecting delayed hypersensitivity in Cryptococcus neoformans-injected animals. The antigen was tested on guinea pigs which had received saline or C. neoformans and on animals sensitized to Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans, or Sporothrix schenckii. A delayed-type hypersensitivity response was elicited by cryptococcin C184 in C. neoformans-injected guinea pigs, whereas no indurations or erythemas were seen at 48 h after skin testing of saline controls or heterologously sensitized guinea pigs. Besides being specific for Cryptococcus, the antigen showed a high degree of sensitivity and was reproducible. Footpad tests were conducted with the antigen on mice which had previously received either 10(5) viable C. neoformans cells or saline. Delayed hypersensitivity was indicated in the C. neoformans-injected mice by the increase in thickness of antigen-injected footpads when compared with the saline-injected footpads. In control mice, antigen- and saline-injected footpads were comparable in thickness 24 h after injection. Mice sensitized to B. dermatitidis were footpad tested with C184, and no cross-reactivity was demonstrated.