Use of drug-specific antibodies to identify ethidium adducts produced in Trypanosoma brucei by photoaffinity labeling.

A photoreactive azido analog of the trypanocide ethidium bromide, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, attached covalently to calf thymus DNA (CT DNA) by photoaffinity labeling, was used to generate antibodies for the drug analog. The specificity of the antiserum was tested using enzyme-linked immunoadsorbant assays (ELISA) against immobilized antigen (photoaffinity labeled DNA) and by both the avidin-biotin peroxidase reaction and indirect immunofluorescence performed on smears of drug treated trypanosomes. The reaction of the antiserum with the covalently bound drug adduct was diminished effectively by prior incubation with an excess of ethidium monoazide, ethidium diazide, and ethidium bromide, and to a lesser extent by the DNA-ethidium complex, the diazide-DNA or RNA adduct, and the monoazide-RNA adduct. DNA which had been photoaffinity labeled with either the propidium or the acridine moiety did not react. The antiserum recognition of DNA photoaffinity labeled with ethidium monoazide was based on the substituted phenanthridinium ring system of the parent ethidium, as evidenced by competition binding studies involving the free monoazido analog (EA1), the diazido analog (EA2), and the parent compound, ethidium bromide (EB). This approach and the sensitivity it provides should prove useful for identifying the distribution and fate of covalently bound drugs resulting from antiparasitic drug treatment, and for studying their roles in antiparasitic action.

Pulmonary granuloma formation in murine toxocariasis: transfer of granulomatous hypersensitivity using bronchoalveolar lavage cells.

A major drawback to studying granuloma formation in murine toxocariasis is the ability of the second-stage larva of Toxocara canis to escape from a developing granuloma, migrate elsewhere, and initiate granuloma formation anew. In an attempt to circumvent this difficulty, 2 different T. canis-derived antigenic preparations were covalently attached to Sepharose 4B beads and embolized into the microvasculature of the lungs of CBA/J mice that had been infected 10 days previously with 25 T. canis ova. Both T. canis egg extract (TEE) and T. canis exoantigens (TEX) were able to elicit antigen-specific granulomas 6 days postembolization as determined by both histologic and morphologic criteria. Histologically, the eosinophil-rich granulomas forming around antigen-coated beads embolized into infected mice resembled the developing granuloma previously described forming around the second-stage larva. Attempts to transfer granulomatous reactivity in this model using either immune spleen cells or immune serum were unsuccessful. Successful transfer of granulomatous hypersensitivity was achieved using cells obtained by bronchoalveolar lavage of previously infected mice. The results suggest the feasibility of using this embolic model of granuloma formation in murine toxocariasis.

Use of bronchoalveolar lavage to compare local pulmonary immunity with the systemic immune response of Toxocara canis-infected mice.

Following infection of mice with larvae of the canine roundworm Toxocara canis, there is a persistent pneumonitis. Heretofore, nothing was known about the immunologic potential of the cells that constitute this inflammatory exudate. By performing bronchoalveolar lavage (BAL), enough inflammatory cells were obtained to compare the local pulmonary immune response to T. canis infection with the systemic immune response as reflected in the peripheral blood and spleen cells of the same mice. Groups of C57BL/6J female mice were given 100 infective ova and BAL, peripheral blood, and spleen cells collected on days 8, 11, 14, and 17 postinfection. The percentage of eosinophils in the BAL averaged about 80% and was four to five times as great as that in the peripheral blood at all times assayed. Use of concanavalin A (ConA)-elicited lymphocyte blastogenesis to evaluate T-lymphocyte activity revealed that BAL T-cell activity was low on day 8 and peaked on day 11. When the B-cell mitogen lipopolysaccharide was used in the assay, there appeared to be far less BAL cell reactivity compared with BAL T-cell activity. Both B- and T-cell responses of the BAL cells were only a fraction of the responses seen concurrently in spleen cells. Use of Toxocara exoantigens in the blastogenesis assay revealed that Toxocara exoantigens could elicit between 20 and 95% of the ConA response in BAL cells, while in spleen cells Toxocara exoantigens could only elicit 1 to 5% of the ConA response. These results suggest that BAL is a useful method for recovering local inflammatory cells that possess detectable immunologic activity. In the case of pulmonary toxocariasis, eosinophils account for the majority of the cells that are present, with most of the remaining cells being T. canis antigen-specific T lymphocytes.

Immune responses of CBA/J mice to graded infections with Toxocara canis.

The immunological responsiveness of CBA/J mice infected with various numbers of the canine ascarid, Toxocara canis, was characterized after a single infection to ascertain the smallest infection capable of perturbing the immune system of the host. Mice receiving the lowest inoculations (5 eggs per mouse or 0.25 larvae per g of body weight) had detectable alterations in the number of circulating peripheral blood eosinophils and spleen weight-to-body weight ratios. Mice infected with 25 eggs each (1.25 larvae per g of body weight) showed augmented concanavalin A-elicited splenic lymphocyte transformation and a positive lymphocyte transformation in response to a toxocaral antigen preparation in addition to even higher eosinophil counts and heavier spleens. Spleen cells from mice receiving the two largest inocula (125 eggs and 250 eggs per mouse or 6.25 and 12.5 larvae per g of body weight, respectively) had in addition to the above responses a sixfold increase in spontaneous DNA synthesis. An enzyme-linked immunosorbent assay for mouse antibody responses to T. canis indicated that the time of onset as well as the magnitude of the antitoxocaral humoral response is directly proportional to the size of the inoculation used to initiate the infection. Finally, we showed that allowing the infection to become protracted results in some responses increasing somewhat in magnitude, but regardless of length of infection, the magnitude of any of the responses examined is proportional to the size of the infection. The results indicate that different host responses have different thresholds of sensitization and suggest that larvacidal reactions which require intricate interactions among several components of the immune system may not occur in very small infections.