Article Figures & Data
Figures
- FIG. 1.
Percent worm burdens in mice primed with L3 E/S products, L3-L4 cuticles, L3 larvae, and RPMI. Three sets of BALB/cByJ mice (eight mice per group) were primed with various worm fractions on the same day. The first group of mice received 25 live L3 B. pahangi larvae per mouse. The second group received 25 cuticles obtained from the L3-L4 molt during in vitro culture per mouse. The third group received the supernatants collected from the culture of 25 worms per mouse (L3-L4 E/S products). A group of four mice injected with 0.5 μl RPMI each served as controls. After a month, these four sets of mice were challenged with 50 live L3 larvae. The mice were sacrificed 10 days after the challenge, and the number of worms surviving in the peritoneal cavity were counted. The bars represent the average percent worm burdens (depicted on the y axis) in these groups of mice. The results marked with an asterisk are significantly different (P < 0.05) from those for the RPMI group. This experiment is representative of four similar experiments.
- FIG. 2.
Percent worm burdens in secIgM−/− mice reconstituted with serum from L3-primed, naïve, and E/S-primed BALB/cByJ mice. A group of BALB/cByJ mice were primed with E/S products from 25 L3 larvae. Two other groups of BALB/cByJ mice were injected with either L3 larvae or RPMI. Each group contained three to five mice. Sera were obtained from each of these groups 2 weeks after infection and pooled groupwise. Three groups of secIgM−/− mice (six to eight mice per group) were infected with 50 B. pahangi L3 larvae. Twelve days later, these mice were reconstituted with serum from one of the three groups described above. Six days after the transfer, the mice were sacrificed and their percent worm burdens were calculated. The bars represent the average percent worm burdens (depicted on the y axis) in these groups of mice. The results marked with an asterisk are significantly different (P < 0.05) from those for the group receiving serum from naïve mice. This experiment is representative of three similar experiments.
- FIG. 3.
Percent worm burdens in mice primed with RPMI, L3 larvae, L3 E/S products, L4 E/S products, microfilarial E/S products, and adult E/S products. L4 larvae, adults, or microfilariae were cultured in serum-free culture medium for 7 days. At the end of the 7 days, the culture supernatants were collected, and groups (five mice per group) of BALB/cByJ mice were primed with these E/S products. A month later, these mice were challenged with 50 L3 larvae, and 10 days later, the mice were sacrificed and the number of surviving worms was counted. As controls, a group of five mice were injected with 0.5 ml RPMI and another group were injected with 25 L3 larvae. The bars represent the average percent worm burdens (depicted on the y axis) in these groups of mice. The results marked with an asterisk are significantly different (P < 0.05) from those for the group primed with RPMI. This experiment is representative of three similar experiments.
- FIG. 4.
Identification of 1E9. Hybridomas were prepared as described in the text. Cell-free supernatants of different clones were collected. Worms were plated at five worms per well. Twenty microliters of supernatant or serum from primed wild-type (WT) mice or naïve WT mice were added to each well. PECs were harvested from secIgM−/− mice infected with B. pahangi L3 larvae 14 days prior. A total of 106 PECs were added per well, and the volume was made up to 200 μl per well. The plate was incubated at 37°C in 5% CO2 for 1 h. After an hour the plates were removed and adhesion was scored by a blinded observer. Mean adhesion scores (± SEM) for each of the supernatants are shown. The experiment was repeated with a selected cohort of supernatants for reproducibility. Five clones with adhesion scores greater than or equal to that with primed serum were selected. Of these, 1E9 was selected for reproducibility of results and growth characteristics.
- FIG. 5.
Percent worm burdens in secIgM−/− mice reconstituted with serum from naïve mice, L3-primed mice, and purified 1E9 antibodies. Three sets of five secIgM−/− mice were challenged with 50 L3 larvae. On day 12, these mice were reconstituted with primed serum, naïve serum, or purified 1E9 antibodies. On day 18, the mice were sacrificed and the worms in the peritoneal cavity were quantitated. The bars represent the average percent worm burdens (depicted on the y axis) in these groups of mice. Statistically significant (P < 0.05) changes compared to results for naïve serum-reconstituted mice are designated with an asterisk. This experiment is representative of three similar experiments.
- FIG. 6.
Dot blot testing the reactivities of 1E9 to ALT-2, TCTP, E4 (a leishmanial protein), a tick salivary protein, whole E/S, OVA, and BSA. Various proteins were blotted onto a strip of nitrocellulose membrane. The strip was blocked using 5% milk in PBS (blocking buffer). The strip was then transferred to a 1:100 dilution of 1E9 or isotype control Sp6 in blocking buffer. It was then washed with PBS-0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO). It was then transferred to a solution containing 3 μg/10 ml horseradish peroxidase (HRP)-conjugated anti-IgM antibody. This experiment is representative of four similar experiments.
- FIG. 7.
Percent worm burdens in mice primed with E4, BmTCTP, or BmALT-2. Four groups of mice of five mice each were injected with E4, TCTP, or ALT-2 mixed with 1E9 conjugated beads and were challenged with 50 L3 larvae 3 weeks after the priming injections. The mice were sacrificed on day 10 of the challenge, and the worm burdens were quantitated. An E/S product-primed group served as positive control, while mice primed with E4 were regarded as negative control. The bars represent the average percent worm burdens (depicted on the y axis) in these groups of mice. Statistically significant differences (P < 0.05) compared to results for E4 are indicated with an asterisk. This experiment is representative of three similar experiments.
- FIG. 8.
Surface staining of live L3 larvae with 1E9 antibody. Larvae on which nonspecific binding was mitigated by prolonged culture in serum-containing medium were incubated with either 1E9 (a to d) or Sp6 (e and f) antibodies. (a, b, e, and f) Larvae were incubated with goat anti-mouse IgM-FITC. (c and d) Larvae were incubated with the isotype control goat anti-mouse IgM-FITC. This is a representative of two similar experiments. Each experiment included approximately five larvae per group. All larvae stained uniformly.
- FIG. 9.
L3 larvae in granulomas stained with 1E9 antibody. B. pahangi L3 larvae were injected into wild-type mice, and the granulomas that were produced were harvested. These granulomas were fixed with 2% glutaraldehyde and embedded in plastic. One-micrometer sections were cut. The sections were blocked with FBS and incubated with 1E9 (a) or isotype matched control antibody Sp6 (b) followed by colloidal gold-conjugated anti-mouse IgM. The gold particles were silver enhanced (arrows) and counterstained with toluidine blue. This is representative of two similar experiments.
