CBA/J mice were obtained from The Jackson Laboratory and housed in the American Association for Accreditation of Laboratory Animal Care– approved animal care facilities of the Centers for Disease Control and Prevention. Newborn (<24 h old) male mice from uninfected parents received transthoracic, intraperitoneal injections of 50 μg of immunoaffinity-purified anti-SEA Abs, commercial normal mouse IgG (NoMoIgG; Sigma), human anti-SEA mAb E5 (12), or commercial normal human IgG (NoHuIgG; Sigma) in PBS. To prepare affinity-purified, polyclonal anti-SEA Abs, pooled sera from mice infected for either 8 or 20 wk (the latter segregated into MSS and HSS) were passed over a column of SEA coupled to cyanogen-bromide–activated Sepharose 4B (Sigma). Bound anti-SEA Abs (Id) were eluted using 0.1 M glycine-HCl (pH 2.8) and collected into 0.025 M borax. The eluates were concentrated and dialyzed against saline, and their protein concentrations were determined. All Id preparations (i.e., those from different serum sources) used in a given experiment were prepared over the same column. Mice that had been neonatally manipulated were infected at 8 wk of age by subcutaneous injection of 45 cercariae of a Puerto Rican strain of S. mansoni that had been maintained in Biomphalaria glabrata snails. The number of live animals was recorded weekly to give survival proportions.

Levels of adult worm circulating cathodic antigen (CCA) were quantified in the sera from infected mice using the 5H11 monoclonal sandwich assay as described previously (13). Absorbance levels from serum samples were compared with those obtained on an adult schistosome worm antigenic preparation (SWAP) standard curve run in parallel to obtain a SWAP equivalent that could be used to compare results from samples run on different days.

For enumeration of eggs in the liver, a portion (∼0.5 g) of weighed liver from infected mice was removed and frozen until digestion. For digestion, 5 ml of 5% KOH (14) was added to each tube and incubated at 37°C until the tissue was completely digested (2–4 h). Duplicate 25-μl aliquots of the digest were placed on a glass slide, and eggs were counted under microscope. Data are expressed as eggs per gram of liver tissue.

Ab isotype analyses were performed by specific ELISAs to detect anti-SEA activity and to evaluate what effects the cytokine environment may have on Ab class switching in these animals (15, 16). SEA (0.25 μg/ well in 0.1 M NaHCO₃, pH 9.6) was adsorbed onto flat-bottomed microtiter plates (Immunolon II; Dynatech Laboratories, Inc.) overnight, at 4°C. After a 1-h blocking step of PBS with 0.3% Tween 20 (Sigma), plates were incubated with optimized dilutions of infected mouse sera. Anti-SEA Abs bound to the SEA-coated plate were detected using peroxidase-conjugated IgG fractions of isotype-specific goat anti–mouse IgG1, anti– mouse IgG2a, or anti–mouse IgG2b (Southern Biotechnology Associates, Inc.). Assays were developed by the addition of TMB peroxidase substrate solution (Kirkegaard & Perry Laboratories), the reaction was halted by addition of 1 M H₂SO₄, and the color reaction was read at 450 nm on a microplate reader (Molecular Devices Corp.).

Spleen cells were cultured in 48-well plates (Costar Corp.) at 2.5 × 10⁶ cells/well in 1 ml RPMI 1640 supplemented with 2% fetal bovine serum and 2% penicillin/streptomycin (GIBCO BRL). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂ and stimulated with medium alone, SEA (4 μg/ml), or anti-SEA Id preparations (40 μg/ml). Supernatant fluids were harvested after 48 h and stored at −70°C. Cytokine levels were measured by capture ELISA as described previously (11).

Upon killing of the experimental animals, livers were removed and a portion of each liver was preserved in 10% buffered formalin. For histologic analysis, tissues were imbedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin. Granuloma areas were measured with the aid of computerized morphometrics (ImagePro Plus; Media Cybernetics). Only those granulomas with a single, central egg were measured. Granulomas containing more than one egg or that were contiguous with other granulomas were ignored. A minimum of 25 granulomas was measured per mouse to calculate the average granuloma size for individual mice. The means for all mice in each group were then averaged for statistical comparison of the groups. For evaluation of fibrosis, some liver sections were stained with Gomori's trichrome stain. Computerized morphometrics were used to calculate the percent fibrotic area of 25 contiguous fields, and the average fibrotic tissue area was calculated for each mouse. As with the granuloma measurements, the means for each mouse in a group were averaged for statistical comparison.

Statistical analyses were performed using Instat and Prism software (GraphPad Software for Science). Group means were compared by two-tailed t test for comparison of two groups; two-tailed analysis of variance and multiple comparison tests were used for comparison of three or more groups. Statistical analyses of survival proportions were performed using two-tailed log–rank tests and two-sided Fisher's exact test. P values <0.05 were considered to be statistically significant.

To determine the long-term effects of neonatal exposure to Id, we monitored the survival of chronically infected mice for 20 wk of infection. Mice were injected with 50 μg of different Id preparations within the first 24 h of life and then infected with 45 cercariae when they became adults. There were no deaths in any group before 8 wk of infection. Mice that received 8WkId lived significantly longer (P < 0.05) than mice that received NoMoIgG (Fig. 1, top and middle). A similar, significant prolongation of survival was also observed after neonatal injection of MSS Id compared with NoMoIgG or HSS Id (Fig. 1, middle and bottom). All mice that had received 8WkId or MSS Id displayed the less severe characteristics of MSS at 20 wk of infection. The percent survival at 20 wk for each of these experiments is shown in Table I. Interestingly, the mice that received HSS Id showed decreased survival proportions compared not only with 8WkId- and MSS Id–injected animals (P < 0.0001) but also with animals that received NoMoIgG (P < 0.002).

Although 8WkId, MSS Id, and HSS Id were all prepared on the same SEA-Sepharose column, we also tested survival of S. mansoni–infected mice injected neonatally with an mAb (E5) to counter the possibility that the effects observed in the survival studies were a consequence of SEA that had leached off the affinity column into the Id preparations. E5 was produced from a human heterohybridoma that shares CRIs with 8WkId and MSS Id (9, 12, 17). Because this Ab was not passed over the SEA affinity column, there is no chance that the preparation was contaminated with SEA. As with the polyclonal 8WkId or MSS Id preparations, neonatal exposure to E5 resulted in longer survival times than for mice that received NoHuIgG (Fig. 2).

We next analyzed the histopathological features of mice that were neonatally exposed to NoMoIgG, 8WkId, MSS Id, or HSS Id preparations and subsequently infected with S. mansoni. Mice that received 8WkId or MSS Id as newborns had a significantly smaller acute (8 wk) infection mean granuloma size (Fig. 3 A) and a significantly decreased percentage of fibrotic liver tissue at 8 wk after infection (Fig. 3 B) than mice that received NoMoIgG or HSS Id at birth. The histologic appearance of granulomas and fibrotic areas representative of these means is presented in Fig. 4. We also observed that mice receiving 8WkId or MSS Id at birth had decreased 8 wk–infection spleen to body weight ratios than animals neonatally injected with NoMoIgG or HSS Id (Table II). To determine whether these differences in pathology were simply an effect of differences in worm burdens, we measured CCA levels in these animals but found no significant difference between mice in any of the neonatal injection groups (Table II). However, in spite of the comparable worm burdens, mice that received HSS Id as newborns were found to have a significantly higher mean number of eggs per gram of liver than mice that had been injected with MSS Id (Table II), resulting in the fibrosis per egg between these two groups not being significantly different (data not shown). The numbers of eggs per gram of liver for mice neonatally injected with NoMoIgG or 8WkId were not significantly different from each other or from either the MSS Id or HSS Id injection groups (Table II).

To study whether neonatal exposure to Id has an effect on cytokine profiles expressed in response to SEA or Id (11) during subsequent S. mansoni infections, spleen cells were harvested from 8 wk–infected mice that had been exposed to the different Id preparations 16 wk previously, as newborns. The spleen cells were stimulated with SEA or Id preparations, and the supernatants were assayed for cytokines. Spleen cells from mice infected for 8 wk that had received 8WkId or MSS Id as newborns produced higher levels of IFN-γ in response to SEA or stimulatory Id preparations (8WkId or MSS Id) than did spleen cells from mice neonatally exposed to NoMoIgG or HSS Id (Fig. 5). Consistent with previous results (11), and similar to anti-SEA Id preparations from hepatosplenic patients (2), HSS Id was not stimulatory for any of the measured responses for cells from any treatment group.

Because isotype switching to IgG2a in mice is at least in part effected by IFN-γ (15, 16), we investigated the anti-SEA IgG subclasses present in sera of the neonatally manipulated mice (Fig. 6). At 8 wk of infection, SEA- and SWAP-specific IgG2a and IgG2b levels in sera of mice neonatally injected with 8WkId or MSS Id were higher than those of mice neonatally injected with NoMoIgG or HSS Id (P < 0.0001). SEA- and SWAP-specific IgG1 levels were similar among the various neonatal injection groups, indicating that the differences in granuloma and fibrotic regulation were not related to a failure to make schistosome-specific Abs in mice neonatally treated with NoMoIgG or HSS Id.