* Department of Microbiology and Immunology, College of Veterinary Medicine, and Institute for Comparative and Environmental Toxicology, Cornell University, Ithaca, New York 148536401; and
Wadsworth Center, New York State Department of Health, Albany, New York 122010509
Received December 12, 2000; accepted August 15, 2001
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ABSTRACT |
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Key Words: lead acetate; developmental immunotoxicity; gender; stage of development.
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INTRODUCTION |
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Pb is a heavy metal that has been shown to be a developmental immunotoxicant defined as a xenobiotic, which through human exposure results in immune function changes and has the potential to adversely affect human health. When Pb is administered throughout full gestation, persistent effects occur in the immune system of the offspring (Bunn et al., 2001a, Chen et al., 1999
, Faith et al., 1979
, Luster et al., 1978
, Miller et al., 1998
). An indicator of T helper type-1 immunity, such as the delayed-type hypersensitivity response, is depressed and production of interferon-gamma (IFN-
) is reduced. Concurrently, production of interleukin-4 (IL-4) and total serum IgE are increased (indicators of T helper type-2 responses). This is consistent with in vivo adult exposure to Pb, which has also been associated with an increase in IgE and IL-4 concomitant with decreased IFN-
production (Heo et al., 1996
, 1997
). Little is known about the effects of Pb or other immunotoxins when administered during different stages of embryonic development. In the chicken, Pb administered at 12 days of embryonic age depresses the DTH response in females. In contrast, when Pb is given as late as 9 days of embryonic age, the juvenile DTH response is not affected (Lee et al., 2001
).
Differential gender effects have been observed after in utero exposure to Pb throughout full gestation (Bunn et al., 2001a; Ronis et al., 1998
). Juvenile and adult male rats do not exhibit decreased DTH responses, in contrast to sexually immature and mature female rats whose DTH responsiveness is significantly depressed. When potential gender differences were examined after a single low-level in ovo exposure to Pb (Bunn et al., 2000
), gender-based immune response capabilities differed as well. In the chicken embryo with Pb exposure at 5 days of age, males exhibited increased antibody production in response to both foreign- and self-antigenic challenge, whereas female antibody production was unchanged.
A recent workshop considered approaches to identify critical windows of exposure during development relative to immunotoxicological risk (Dietert et al., 2000). With this in mind, and based on our previous observations in the chicken, we hypothesized that exposure to Pb in mammals at different stages during the ontogeny of the immune system (either before migration of the stem cells or during colonization of the thymus and bone marrow) may lead to differential immunotoxicity in the adult offspring. Both males and females were examined for gender differences in immune function after developmental-timed Pb exposure. The results show that, indeed, persistent immune changes are evident after a pulsed in utero exposure to Pb either early or late in gestation. However, the effects of the exposure differ depending upon the timing of the exposure and the gender of the offspring.
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MATERIALS AND METHODS |
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Experimental design.
The studies were conducted in the CD rat, using 20 dams per treatment group. Animals were randomly assigned to either a control or a 500-ppm (mg/l) Pb (as Pb acetate) treatment group. Control rats received sodium acetate in their drinking water at equivalent acetate levels to the Pb treatment group. Acetic acid (0.00125%) was added to both control and treated water to aid in dissolution. The treated water was administered ad libitum either early in gestation (days 39) or late in gestation (days 1521). The Pb dose delivered to the dam was calculated by determining the amount of Pb-treated water consumed/rat/day multiplied by 500 mg Pb/1000 ml water. Before and after exposure, animals received normal drinking water. After parturition, the offspring were culled at 7 days of age to 4 males and 4 females per litter. Offspring were weaned at 21 days of age and housed individually. Twenty animals (each male or female derived from a different dam) from each treatment group were used for experimental analyses. Males and females received a primary and secondary sensitization with Keyhole limpet hemocyanin (KLH) in the caudal tail fold in a 200-µl volume of sterile water (5 mg/ml) at 10 and 11 weeks of age (Exon et al., 1990). At 12 weeks, all groups received a challenge injection of heat-aggregated (80°C for 1 h) 20 mg/ml KLH in 0.1 ml of saline in the footpad. Control animals received an injection of saline alone in the footpad. The delayed-type hypersensitivity response (DTH) was measured after 24 h, using spring-loaded calipers (Dyer, Model 304). Animals were sacrificed at this time point (12 weeks old) with blood samples drawn, tissues excised, and body weights recorded. Offspring were assessed for immunotoxicity endpoints as adults, as opposed to juveniles, since prior evidence from our laboratory (Bunn et al., 2001a
) indicated that following full gestational Pb exposure, a greater spectrum of immunotoxic effects was observed when offspring were assessed as adults vs. juveniles.
Reagents.
Pb acetate, sodium acetate, o-phenylenediamine dihydrochloride (OPD), LPS, and ConA were purchased from Sigma-Aldrich (St. Louis, MO). Peroxidase-conjugated goat anti-rat IgG was obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Calbiochem (San Diego, CA) supplied the KLH. ELISA kits for IFN-, IL-2, IL-4, IL-6, IL-10, IL-12, MCP-1, and TNF-
were purchased from Biosource International (Camarillo, CA). Cayman Chemical (Ann Arbor, MI) provided the ELISA kit for PGE2 analysis. Bioactive TGF-ß was measured by an ELISA assay obtained from Promega (Madison, WI).
Blood collection and Pb analysis.
For blood Pb and anti-KLH IgG antibody determinations, peripheral blood was obtained by cardiac puncture of offspring at the time of sacrifice. Heparin was used as an anticoagulant for whole blood Pb determinations, while serum was obtained for analysis of KLH antibody, total IgE, and MCP-1. Blood Pb concentrations were analyzed by atomic absorption (Parsons and Slavin, 1993). The minimum reportable blood lead concentration was 2 µg/dl. Front and hind limb bones were collected from 1-day-old pups and analyzed for Pb (Zong et al., 1996
) at Wadsworth Center, New York State Department of Health, Albany, NY. Total leukocyte counts were performed on whole blood diluted 1:20 with erythrocyte lysing buffer. Leukocytes were enumerated on a hemocytometer. Differential cell counts were performed on blood smears stained with a Diff-Quick Staining Set (Dade Behring AG, Dudingen, Switzerland).
Splenocyte preparation.
Spleens were removed aseptically at the time of sacrifice and single-cell suspensions were prepared by forcing the spleens through 400-µm sterile nylon mesh. Splenocytes were washed, then erythrocytes were lysed with a buffered solution (pH 7.2) of 0.15 M NH4Cl, 1.0 mM KHCO3 and 0.1 mM EDTA. After washing with Hank's balanced salt solution (HBSS), cells were plated at a concentration of 3 x 106 cells/well for unseparated preparations and at 6 x 106 for adherent cell preparations. For the latter, protein content was analyzed in wells using the bicinchoninic acid (BCA) method (Pierce Biochemical, Rockford, IL). Adherent cells were stimulated with 0, 0.1, or 1.0 µg/ml LPS. Unseparated splenocytes were stimulated with either 0 or 5 µg/ml ConA.
ELISA for cytokine analysis.
IL-2, IL-4, IL-6, IL-10, IL-12 (p40), IFN- and TGF-ß were measured in 24-h unseparated splenocyte supernatants stimulated with 5 µg/ml ConA in RPMI 1640 supplemented with 2% fetal calf serum (< 0.06 EU/ml). TNF-
and PGE2 were measured in 24-h supernatants generated from adherent splenocytes exposed to 1.0 µg/ml LPS. MCP-1 was measured in serum.
Antigen-specific antibody ELISA.
IgG antibody against KLH antigen was measured by ELISA as previously described (Miller et al., 1998), similar to Exon and Talcott (1995). An initial screening assay was performed to determine the optimal dilution of the sera. Briefly, KLH antigen was bound to a 96-well microtiter plate and incubated overnight at 4°C. The following day serum samples were diluted 1/500, added to the plates, and incubated for 1 h at 37°C. After washing, peroxidase-labeled mouse anti-rat IgG was added and incubated for 1 h. OPD was used to develop the plates, and the absorbance was read at 450 nm. Relative absorbances were compared with positive and negative pooled control samples.
Total IgE ELISA.
Total serum IgE was measured by a sandwich ELISA using the method supplied by PharMingen. Briefly, plates were coated with purified anti-rat IgE (PharMingen, San Diego, CA), washed and blocked, and sera added. Biotinylated anti-rat IgE (PharMingen, San Diego, CA) was used as the detection antibody. Purified rat IgE kappa (Serotec, Raleigh, NC) was used as the standard. After incubation with substrate (OPD), plates were read using an ELISA reader (Biotek EL312).
Nitric oxide (NO).
Adherent splenocytes were incubated for 24 h in RPMI 1640 medium supplemented with 2% fetal calf serum (< 0.06 EU/ml) and stimulated with 0, 0.1, or 1.0 µg LPS. NO production was measured by the Griess reaction (Green et al., 1982), which assesses accumulation of nitrite.
Histopathologic analysis of thymuses.
Thymuses were removed from all animals, trimmed of fat, and weighed before storage in 10% buffered formalin. After fixation, thymuses were embedded in paraffin and sectioned at 6 µm. Staining of tissues was done with Harris's hematoxylin and eosin. Image Pro software (Media Cybernetics, Silver Spring, MD) was utilized for analysis of thymus sections. The ratio of cortical to medullar areas of the thymic section was determined and calculated by the formula:
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Organ collection.
Thymuses and spleens were collected, trimmed of adherent fat, and weighed at the time of sacrifice.
Statistical analysis.
Data were analyzed by a General Linear Model analysis of variance (ANOVA), followed by Fisher's least-significant-difference test (Minitab Statistical Software, Minitab Inc., State College, PA), a multiple comparison test carried out if significance was indicated by ANOVA. Results were considered significant with a probability level of p < 0.05. For statistical analysis, the minimum reportable blood-lead level was divided by 2, so as not to bias the data toward the null hypothesis.
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RESULTS |
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Hematological parameters in mature offspring.
Total leukocyte counts, along with relative and absolute leukocyte values, were assessed following exposure to Pb either early or late in gestation. There were no significant differences in total leukocyte counts between treatment groups with Pb exposure at either stage, although male offspring had higher total leukocyte counts than females (Table 4). When relative and absolute leukocyte values were determined, females, but not males, exposed to Pb late in gestation had significantly elevated relative numbers of monocytes when compared to the late gestation, gender-matched controls (Table 5
). Other leukocyte subsets were not altered by Pb treatment at different developmental stages of exposure.
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DISCUSSION |
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The present results indicate that differences in Pb-induced immunotoxicity do exist depending upon the timing of in utero exposure. Additionally, gender differences were evident in functional measures as well as cytokine and metabolite production, following early and late gestational exposures. The finding supports the tenet that differential windows of vulnerability exist during development for the immunotoxic effects of Pb. Additionally, gender of the offspring can influence immunotoxic risk.
Pb exposure levels of the dams in this study ranged from 4762 mg lead acetate/day. While the Pb exposure of the dams is relatively high for a short period of time, the levels are relevant to human exposure. Lewin et al. (1999) reported that Pb levels near four Superfund sites had mean dust Pb levels of 1108 mg/kg and soil Pb levels of 505 mg/kg and that increased soil Pb levels were associated with blood Pb levels in children. In addition to Pb exposure levels, blood-lead levels measured for 2 days at the end of exposure in the dams coincided with blood-lead levels still observed worldwide (Rojas et al., 2000, Fischbein et al., 1993
, Sata et al., 1998
). Therefore, human exposures to Pb at these levels still exist.
Male and female offspring exposed to Pb late in gestation had elevated blood Pb levels at one day of age when compared to the blood Pb levels of dams sampled at the same time point. The offspring levels were increased approximately 4-fold compared to those in dam blood (51 µg/dl vs. 12 µg/dl). When bone Pb burdens were assessed, the late-gestation exposed animals had bone Pb levels of
14 µg/g in male offspring and 12 µg/g in females. While male offspring stored slightly more Pb in their bones than females, both genders had equivalent blood Pb levels. Several prior studies have found equivalent or lower cord blood Pb levels compared with maternal blood Pb levels (Campagna et al., 1999
, Nashashibi et al., 1999
, Raghunath et al., 2000
, Wan et al., 1996
), but increased cord blood Pb levels have also been reported (Rothenberg et al., 1996
). The high concentration of Pb in the late-gestation Pb-exposed offspring suggests that differences in intake and distribution of Pb in the dam during early vs. late gestation and possible differential absorption into soft tissues of the offspring compared to the dam could be factors.
Consumption data indicate no difference among dams for Pb intake among the early- vs. late-pregnancy exposures. Danielsson et al. (1983) reported that early exposure to Pb in mice was restricted to embryonic blood, because yolk sac placenta erythrocytes incorporated Pb into the embryonic hemoglobin. When Pb was administered after day 14, much of the Pb was accumulated in the bone. In this study, bone Pb levels of the late-exposed animals were significantly greater than those of those exposed early. Danielsson et al. (1983) reported that fetal Pb concentrations (µg Pb per whole fetus) were also higher at day 18 than at day 12. Based on that study and our results, it is likely that placental transfer of lead is more efficient when the fetus is exposed in late, rather than early gestation. It might be possible that more lead is transferred to the fetus by virtue of increased calcium mobilization in the dam from the gut and its own bone stores. It is also possible that the Pb was more readily absorbed into the fetus from the dam late in gestation at the Pb dosage employed. No other Pb dosages were examined in this study due to experimental size constraints. Since differential immunotoxicity was observed between the sexes, despite equivalent blood Pb levels, and although with slightly higher bone Pb levels in the males, it seems unlikely that gender differences in immunotoxicity are related to embryonic exposure differences.
Despite similar Pb intake, dams in the present study had higher blood Pb levels when exposed late in gestation than when exposed earlier in gestation (compared 2 days postexposure). This is consistent with the finding of Danielsson et al. (1983) who reported dam Pb levels were higher on gestation day 18 (4-h post i.v. Pb injection) than on day 12. Hackett et al. (1982) found no difference in distribution of Pb to the major organs (liver, kidney, spleen, and lung) in the rat dam with Pb exposure at 9 or 15 days of gestation. Therefore, the dam blood Pb levels might be elevated later in pregnancy while Pb distribution to the major organs might not be affected by the stage of pregnancy.
Differential lactational exposure could have contributed to the differential immunotoxicity observed with Pb exposure during different developmental stages. Snyder et al. (2000) examined maternal transfer of Pb during gestation only, lactation only, and gestation with lactation exposures; they found that lactational exposure alone could cause immunotoxicity at certain doses in offspring; but also they reported that gestational exposure alone resulted in equivalent immunotoxic effects. In this study, the blood Pb levels of the dams exposed late in gestation to Pb were 12 µg/dl. With a blood Pb level this low only 2 days after the end of exposure, it is doubtful that lactational Pb exposure played a significant role in the differential immunotoxicity with differential gestational exposure to Pb. In addition, Pb exposure during discrete stages of chicken embryonic development produced differential immunotoxic effects in the offspring that were dependent on the stage of developmental exposure. The DTH response was depressed in females with Pb exposure at 12 days of embryonic age, whereas exposure to Pb at 5 days of embryonic age had no effect on the female DTH response (Lee et al., 2001). The chicken embryo develops in the absence of a maternal system and the same depressed DTH response was observed with later Pb exposure.
Differential gender effects have been observed previously with full gestational exposure to Pb in the rat (Bunn et al., 2001a). Gender-based immunotoxicity has also been observed after a one-time, low-level exposure to Pb at 5 days of incubation in the chicken (Bunn et al., 2000
). Our present data indicate that gender may influence the outcome of Pb exposure on the developing fetus. At the Pb dosage used, adult female offspring exposed to Pb late in gestation exhibited more profound immunotoxic effects. Depression of the DTH response, along with elevation of IL-10 production, increased thymic weights, and an increased relative number of monocytes were evident. On the other hand, males exhibited increased IL-12 production and decreased IL-10 production with late-gestational exposure to Pb. The gender differences occurred despite similar blood and bone Pb burdens. These findings suggest that the macrophage may be an important target cell in both males and females with late gestational Pb exposure.
It is unclear how gender in utero can affect Pb-induced immunotoxicity. Hormonal differences might influence either bioavailability of Pb to immune compartments or the relative susceptibility of developing immune target cells to Pb. Since blood and bone Pb concentrations tended to be similar between genders, either the distribution differences occurred only among soft tissues or the target cell susceptibility hypothesis would be favored. Chao et al. (1994) reported a differential gender-based sensitivity for nitrite production by the macrophage with varying concentrations of estrogen and progesterone, and suggested that female sex hormones may alter reactive oxygen and nitrite intermediate release in vivo. Early in gestation, adherent splenocyte nitrite production was significantly depressed in Pb-treated males and the same trend, although not significant, was observed in females. Late-gestation control and Pb-treated animals had decreased nitrite levels compared to the early gestation controls. The depression of nitrite levels late in gestation may reflect an acetate effect on adherent splenocyte function when administered late in gestation.
In addition, sex steroid hormones modulated by developmental exposure to Pb may affect the developing thymocyte since thymocytes and thymic epithelial cells bear receptors for estrogens and androgens. Pb has been reported to decrease steroid sex hormone levels in juveniles after in utero exposure to Pb acetate (Ronis et al., 1996, Wiebe et al., 1982
). Leposavic et al. (1996) found differential effects of gonadectomy on the developing thymocyte in weanling and adult rats. In immature rats, ovariectomy resulted in an increased percentage of CD4 + CD8-thymocytes while gonadectomy in the weanling male resulted in increased CD4 + CD8-, TCR
ß+ thymocytes and decreased double-positive and double-negative thymocytes.
IL-12 is produced primarily by macrophages and is a heterodimeric (p70) cytokine comprised of p40 and p35 subunits. The cytokine is involved in cell-mediated immunity. The IL-12 ELISA kit utilized for this set of experiments measured free p40 as well as the heterodimer (p70). Kishikawa et al. (1997) reported enhanced IL-12 p40 monomers with Pb exposure in mice and suggested that excess p40 protein could interfere with IL-12 function. It is known that the p40 chain of IL-12 inhibits the effects of the IL-12 heterodimer. Therefore, it is not surprising that elevated IL-12 production in late Pb-exposed males did not correspond to an increased DTH response. However, the combination of depressed IL-12 production and elevated IL-10 production in late Pb-exposed females is a potential factor in the observed suppressed DTH response from the rats.
The observed effect on the DTH response in females may be attributed to Pb's effect on IL-10 in both genders. An increase in IL-10 has been associated with a decrease in DTH (Bickerstaff et al., 2000,D'Orazio et al., 1998, Manickan et al., 1998
). Bickerstaff et al. (2000) determined that antibodies to IL-10 or TGF ß recovered the DTH response in cardiac allograft-acceptor mice. D'Orazio et al. (1998) also investigated the role of IL-10 in the induction of the DTH response. The authors showed that TGF-ß induced the macrophage to secrete IL-10, which, in turn, suppressed the DTH response. APC incubated with anti-IL-10 and TGF-ß were not able to depress Th1 immune responses, and APC produced robust DTH responses and increased production of IL-12 in the absence of TGF-ß. IL-10 knockout mice were used to confirm the suppressive effect on the DTH immune response. Altered cytokine production by offspring exposed to Pb late in gestation could be influenced by altered concentrations of sex steroid hormones modulated by Pb. Wilcoxen et al. (2000) reported that female SJL mice preferentially secreted high levels of IL-12 and low-levels of IL-10 and that IL-10 regulated the secretion of IL-12 by the APC. When males (which normally secrete high IL-10 and low IL-12) were castrated or treated with anti-IL-10, IL-12 and IFN-
levels increased and DTH responses were enhanced. The authors concluded that gonadal hormones could alter APC cytokine secretion. Since Pb has been associated with the modulation of sex steroid hormone concentrations as well as steroid hormone receptors (Ronis et al 1996
, Wiebe and Barr, 1988
, Wiebe et al., 1982
), it is possible that Pb could exert an effect on the developing macrophage through modulation of sex steroid hormones at a critical time point during embryogenesis.
Pb exposure is associated with decreased IFN- production (a T helper type-1 associated activity) while T helper type-2 activities (increased IL-4 and total IgE) are increased in children (Lutz et al., 1999
), adult rodents (Heo et al., 1996
,1997
), and female rodent offspring exposed to Pb throughout full gestation (Chen et al., 1999
, Miller et al., 1998
). A decrease in the DTH response has been the reported hallmark outcome as well in both adults and in female offspring with embryonic Pb exposure (Bunn et al., 2001a
, Chen et al., 1999
, Lee et al., 2001
, McCabe et al., 1999
, Miller et al., 1998
). In the present study, and in contrast with complete gestational exposure, pulsed exposure to Pb (either early or late in gestation) had no persistent effects on the classical T-helper type cytokines, with the exception of IL-10. Since IL-10 is also produced by macrophages, the macrophage is probably an important link to the prototypic suppression of the DTH response.
An increase in relative thymic weights was observed in females with late gestational exposure to Pb. However, histological analysis of the thymuses as suggested by Kuper et al. (2000) revealed no differences between treatment groups or genders. Since this approach does not provide a complete picture of thymic cellularity at different developmental time points, it would be useful to examine additional aspects of the Pb-thymocyte interaction.
The ontological development of the immune system in the embryo involves the rapid differentiation of progenitor cells. Around day 9 of gestation in the rat, migration of stem cells to the fetal liver and spleen occur with colonization of the bone marrow and thymus occurring around day 13 until birth (Dietert et al., 2000, Holladay and Smialowicz, 2000
). Thymocytes express CD4 and CD8 by day 14 and monocytes are observed in the circulation at day 12 or later (Holladay and Siamowicz, 2000). The time of gestational development of these cells corresponds primarily to the first half of gestation in humans. With exposure to Pb at these critical time points during fetal development, persistent immunotoxicity is observed and offspring gender influences the eventual immune outcome after Pb exposure. These findings suggest that the risk to the fetus of Pb exposure during human pregnancy might not be comparable at all stages of pregnancy. Given the relative dearth of developmental immunotoxicity data compared with adult exposure information, the issue of differential health risk during ontogeny warrants further examination.
Previous work done by our laboratory suggested that immunotoxicity can be detected in juvenile, as well as adult rats, with full gestational exposure to Pb but that the full spectrum of immunotoxic effects may not be observed unless the offspring are assessed as adults (Bunn et al., 2001b, in press). In this study, a persistent increase in IL-10 production was observed in late Pb-treated females concomitant with a decrease in the DTH response, an in vivo measure of cell-mediated immunity. In addition, relative numbers of monocytes and thymic weights were increased. These changes indicate that cell-mediated immunity to viruses, bacteria, and tumors may be altered following Pb exposure late in gestation, and that females may be more susceptible to the immunotoxic effects of Pb than males. A suppressed DTH response has been reported for patients with acute infective illnesses (Bennett et al., 1998
). An increase in IL-10 has been associated with increased susceptibility of systemic lupus erythematosus (van der Linden et al., 2000
). Females are already more susceptible to SLE than males; therefore, potential exposure to Pb late in gestation may compromise health by increasing susceptibility to autoimmune diseases as well. The results of this study indicate that exposure to Pb during a specific stage of embryonic development has a differential outcome on the offspring's resultant immune capabilities, depending on stage of exposure and/or gender. Ironically, the offspring were more profoundly affected with late-gestational Pb exposure of the dam compared to early- gestational Pb exposure. Given the nature of the immune changes induced by early exposure to Pb, it is feasible that host resistance to disease, including hypersensitive and certain autoimmune diseases, would be affected.
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ACKNOWLEDGMENTS |
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NOTES |
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