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Neuronal Nitric Oxide Synthase Is Necessary for Elimination of Giardia lamblia Infections in Mice1 Erqiu Li, Ping Zhou, and Steven M. Singer2 NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-L-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-L-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection. The Journal of Immunology, 2006, 176: 516 –521.
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nfections with Giardia lamblia are one of the most common intestinal maladies in the world (1). These infections can lead to acute diarrhea, cramps, and nausea, although asymptomatic infections are the most common. Although most infections are controlled by an effective immune response, some individuals develop chronic disease. It is unclear what immune mechanisms are responsible for effective control of infections (2, 3). Although IgA has been shown to have a role in controlling infections (4), it is clear that IgA-independent mechanisms are also able to eliminate Giardia and that IL-6 appears to be pivotal for these pathways (5–7). In addition to Abs, a number of different mechanisms have been proposed that might control Giardia infections. These include antimicrobial peptides, NO, and mast cell products (8 –12). We have recently found that mice that are unable to produce a mast cell response are deficient in the elimination of Giardia infections (13). In contrast, Eckmann (3) reported that mice lacking functional ␣-defensins due to deletion of the gene for matrilysin control G. muris infections as well as wild-type mice. NO has been recognized as playing a pivotal role in the control of infections with numerous microbes, including Leishmania spp., Toxoplasma gondii, Salmonella typhimurium, and Mycobacterium tuberculosis (14, 15). Because NO can also inhibit G. lamblia replication and differentiation in vitro, we decided to examine the role Department of Biology, Georgetown University, Washington, D.C. 20057 Received for publication December 29, 2004. Accepted for publication October 19, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
This work was supported by National Institutes of Health (NIH) Grant AI49565 (to S.M.S.). These studies were conducted using the animal core facility supported by National Cancer Institute-Cancer Center support grant and the histopathology and tissue shared resource facility constructed with support from Research Facilities Improvement Grant C06RR14567 from the National Center for Research Resources, NIH. 2 Address correspondence and reprint requests to Dr. Steven M. Singer, Department of Biology, Reiss Science Building, Room 406, Georgetown University, Washington, D.C. 20057. E-mail address:
[email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
of NO during infections of mice with G. lamblia. Using both genetargeted mice and inhibitors of NO synthase (NOS)3 we show that it is not the inducible isoform of NOS (NOS2), but, instead, the neuronal isoform (NOS1) that is responsible for contributing to elimination of this infection.
Materials and Methods Mice, parasites, and infections C57BL/6J, B6.129S2-Il6tm1Kopf/J, B6.129P2-Nos2tm1Lau/J, B6;129S4Nos1tm1Plh/J, B6129SF2/J, and B6.CB17-Prkdcscid/SzJ mice were all obtained from The Jackson Laboratory. Females, between 5 and 8 wk of age, were used for all experiments. The GS/H7 clone of G. lamblia was propagated in vitro and used for infections as previously described (5). Briefly, mice were gavaged with 106 trophozoites in PBS on day 0 and killed on different days after infection. Parasite numbers in the distal duodenum and proximal jejunum were counted by mincing tissue in ice-cold PBS, chilling for 15 min, and counting with a hemocytometer. All animal experiments were approved by the Georgetown University animal care and use committee.
Enzyme inhibitors All inhibitors were obtained from Sigma-Aldrich. The nonselective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), was administered in drinking water at a concentration of 0.5 mg/ml (16). Water was replaced every other day. The NOS1-selective inhibitor, 7-nitroindazole (7-NI), was dissolved in DMSO and injected i.p. at a dose of 25 mg/kg (16). The NOS2-selective inhibitor, N-iminoethyl-L-lysine (L-NIL), was dissolved in PBS and injected i.p. at a dose of 10 mg/kg (16). The peristalsis inhibitor, loperamide, was dissolved in water and administered orally at a dose of 25 mg/kg (17). Inhibitors were given starting on day 2 after infection and every other day thereafter.
RT-PCR Total RNA was isolated from the jejunum, immediately distal to the segment used for quantifying parasite numbers, using RNA-STAT-60 (TelTest). cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen Life Technologies), and cDNA was amplified with specific primers. Primer and probe sequences for TaqMan real-time PCR of GAPDH, NOS2, TNF-␣, IL-4, and IFN-␥ (18) and of NOS1 (19) have 3
Abbreviations used in this paper: NOS, NO synthase; L-NAME, NG-nitro-L-arginine methyl ester; L-NIL, N-iminoethyl-L-lysine; 7-NI, 7-nitroindazole. 0022-1767/05/$02.00
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FIGURE 1. Expression of NOS isoforms and TNF-␣ after Giardia infection. Intestinal RNA from wild-type (f) and IL-6-deficient (䡺) C57BL/6 mice was collected from uninfected (day 0) or infected mice on the indicated days after infection. RNA was reverse transcribed, and NOS2 (A), NOS1 (B), and TNF-␣ (C) cDNA levels were determined using real-time PCR. Expression levels relative to uninfected C57BL/6 mice were determined by normalizing values to GAPDH. n ⫽ 3/group. Data are representative of duplicate experiments. ⴱ, p ⬍ 0.05 compared with wild-type mice.
been previously described. Reactions were performed using TaqMan reagents and an ABI 7000 (PerkinElmer). Normalization with GAPDH was performed using the ⌬⌬Ct method described by PerkinElmer.
Immunohistology Segments of distal jejunum were snap-frozen and sectioned using OCT (Miles). Eight-microliter sections were thawed on glass slides, fixed with cold acetone for 5 min, and stained with rabbit anti NOS1 or rabbit antiNOS2 antisera (Santa Cruz Biotechnology). Staining was revealed with goat anti-rabbit FITC (Santa Cruz Biotechnology) and visualized with a Zeiss Axiophot epifluorescence microscope equipped with a CoolSnap FX CCD camera (Roper Scientific) and connected to a Macintosh G3 computer running OpenLab 3.0 (Improvision). Imagers were further processed using Photoshop (Adobe Systems).
Gastrointestinal motility Mice were fasted overnight before measuring motility. Water was freely available during this period. Mice were gavaged with 0.2 ml of 10% charcoal in 5% gum acacia (both from Sigma-Aldrich). Thirty minutes later, mice were killed, and the intestinal tracts were carefully removed. The total length from the pylorus to the cecum was measured, as was the distance from the pylorus to the leading edge of the charcoal. Motility is expressed as the percentage of the total intestinal length traversed by the charcoal.
Statistics The numbers of parasites, PCR results, and gastrointestinal motility were compared between groups using Students t test statistics (PRISM 3.0; GraphPad). The nonparametric Mann-Whitney U test was used to compare parasite number data that included animals with undetectable parasites.
not shown). No significant differences in IFN-␥ mRNA levels were observed (data not shown). Similar to NOS2, TNF-␣ mRNA levels were decreased in the Giardia-infected, IL-6-deficient mice compared with wild-type mice (Fig. 1C). However, these differences were only 3-fold on day 5 and 2-fold on day 12. Differences in TNF-␣ and IL-4 may account for the reduced NOS2 expression in IL-6-deficient mice after Giardia infection. To test whether NOS2 production of NO was necessary for control of infections, we infected NOS2-deficient mice (22) with G. lamblia. We also treated wild-type mice with the isoform-nonspecific NOS inhibitor, L-NAME (16). Fig. 2 shows that 5 days after infection, L-NAME-treated mice had three to four times more parasites in the small intestine than untreated mice and almost as many parasites as IL-6-deficient mice. In contrast, NOS2-deficient mice had 8 –10 times fewer parasites than wild-type controls 5 days after infection. By 10 days after infection, the untreated wildtype mice and NOS2-deficient mice no longer had detectable parasites, whereas all the L-NAME-treated mice still had measurable parasite loads. This difference between NOS2-deficient mice and L-NAME-treated mice could represent compensatory mechanisms at work in the genetically deficient mice or the inhibition of additional isoforms of NOS, e.g., the neuronal isoform NOS1 or the endothelial isoform NOS3, by L-NAME. To determine whether NOS-deficient or L-NAME-treated mice had markedly altered immune responses, we analyzed the production of anti-parasite IgA
Results Because IL-6-deficient mice have a defect in the control of Giardia infections and because NO can inhibit parasite replication and differentiation in vitro, we began by investigating whether inducible NOS (NOS2) was up-regulated during infections of wild-type mice, but not IL-6-deficient mice. Fig. 1A shows that NOS2 mRNA levels were ⬃3-fold higher in wild-type mice 5 days after infection. In this model, parasite numbers typically remain high until between 5 and 7 days after infection. By day 12, when most parasites have typically been eliminated, a 17-fold increase in NOS2 mRNA was seen. Uninfected IL-6-deficient mice had ⬃70% less NOS2 mRNA than wild-type mice. Consistent with their inability to eliminate the infection, NOS2 mRNA only increased 3-fold by day 12 after infection and remained lower than in uninfected wild-type mice. TNF-␣ is a key inducer of NOS2 expression (20), whereas IL-4 has been shown to inhibit NOS2 induction (21). We therefore examined TNF-␣ and IL-4 mRNA levels in the small intestines of these mice. Similar to the results previously reported in mesenteric lymph nodes by Bienz et al. (7), IL-4 mRNA levels were increased in IL-6-deficient mice infected for 12 days compared with 12-day infected, wild-type mice or uninfected, IL-6-deficient mice (data
FIGURE 2. Inhibition of NOS activity, but not deletion of the NOS2 gene, delays parasite elimination. Mice were infected with Giardia on day 0 and parasite numbers in the small intestine were determined on either day 5 (f, Œ, , and ⽧) or day 11 (‚, 䡺, ƒ, and 〫) after infection. Wild-type C57BL/6 mice received no drug (f and 䡺) or L-NAME (‚ and Œ). NOS2deficient mice ( and ƒ) and IL-6-deficient mice (⽧ and 〫) received no drugs. Each symbol represents an individual mouse. Data are representative of three independent experiments. ⴱ, p ⬍ 0.05.
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FIGURE 5. Localization of NOS1 in the small intestines of Giardiainfected mice. Frozen sections from wild-type mice not infected (A) or infected for 10 days with Giardia (B) were stained with Abs to NOS1 as described in Materials and Methods. The absence of staining for NOS1 in Giardia-infected NOS1-deficient mice (C) or wild-type mice stained with normal rabbit IgG (D) confirmed the specificity of primary and secondary Abs.
FIGURE 3. NOS1, rather than NOS2, activity is necessary for the elimination of Giardia. A, Groups of four mice were infected with Giardia, and parasite numbers were determined on day 10 after infection. Wild-type C57BL/6 mice were treated with the nonspecific NOS inhibitor L-NAME or the NOS1-specific inhibitor 7-NI; NOS2-deficient mice were treated only with L-NAME. Data from L-NAME-treated and NOS2-deficient mice are representative of three independent experiments. 7-NI data are representative of two independent experiments. B, Wild-type (f) and NOS1deficient (䡺) B6 ⫻ 129 F2 mice were infected with Giardia, and the number of intestinal trophozoites was determined 5 and 10 days after infection. Each point represents an individual mouse, and bars indicate the means. Data are representative of two independent experiments. ⴱ, p ⬍ 0.05.
and cytokine responses in infected mice. No differences were seen in either IgA or cytokine responses (data not shown), suggesting that manipulation of NOS2 did not have widespread effects in this
FIGURE 4. Localization of NOS2 in the small intestines of Giardia-infected mice. Frozen sections of uninfected (A) and 10-day Giardia-infected (C and D) wild-type mice were stained with Abs to NOS2 as described in Materials and Methods. The absence of staining for NOS2 in Giardia-infected NOS2-deficient mice (B) confirmed the specificity of primary and secondary Abs. D, Larger view of an image taken at the same original magnification as in C.
system. Nevertheless, we can conclude that NOS2 is not absolutely required to control infections with G. lamblia in mice. Indeed, the ability of both IFN-␥- and IL-4-deficient mice to control infections (5) suggested that there are probably redundant mechanisms able to control this infection. To determine whether inhibition of NOS1 or NOS3 by L-NAME was effecting elimination of Giardia, we infected NOS2-deficient mice and treated them with L-NAME. If the observed effect of L-NAME was due to inhibition of NOS2, we reasoned that treatment of the NOS2 mutant mice should have no effect in these mice. Fig. 3A shows that L-NAME had a similar effect when given to NOS2-deficient mice as when given to wild-type mice, however. Thus, L-NAME inhibition of either NOS1 or NOS3 must be involved in the elimination of Giardia. Wild-type mice were therefore also treated with 7-NI, an NOS inhibitor that shows selective activity against NOS1 (16). Treatment with 7-NI resulted in extended infections similar to those produced by treatment with LNAME (Fig. 3A), supporting the idea that NOS1 is indeed important for the elimination of Giardia. To further confirm a role for NOS1, but not NOS2, we treated wild-type mice with L-NIL (data not shown), an inhibitor that has a preference for NOS2 over NOS1 (16). As expected from results with NOS2-deficient mice, C57BL/6 mice treated with L-NIL had no detectable parasites 10 days after infection, similar to untreated mice (n ⫽ 4/group). We also infected NOS1-deficient mice to confirm the results found with 7-NI. Although both wild-type B6 ⫻ 129 F2 mice and NOS1-deficient mice had readily detectable infections 5 days after inoculation with parasites, the number of parasites per mouse was significantly higher in NOS1-deficient mice at this time (Fig. 3B). Moreover, by 12 days after infection the wild-type mice had eliminated all detectable parasites, whereas the NOS1-deficient mice were still heavily infected. These results confirmed that NOS1 plays an essential role in the elimination of Giardia infections. NOS1-positive neurons have been described in the intestinal tract and are known to play a role in regulating intestinal motility (23, 24). Fig. 4 shows that NOS2 in the small intestine of both uninfected and infected mice is predominantly expressed just under the epithelial cell layer of the villi (Fig. 4, C and D). Consistent
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FIGURE 6. Inhibition of gastrointestinal motility delays elimination of Giardia infections. A, Wild-type C57BL/6 mice were infected with Giardia and not treated (f) or treated every other day, beginning on day 2 after infection, with either L-NAME (Œ) or loperamide (). Parasite numbers were determined on day 10 after infection. Each symbol represents an individual mouse. B, Gastrointestinal motility was measured in uninfected C57BL/6 mice (⽧), untreated mice infected with Giardia (f), and infected mice treated on days 2–7 after infection with L-NAME (). C, Gastrointestinal motility was measured in uninfected C57BL/6 mice (⽧), untreated mice infected with Giardia (f), and infected mice treated with loperamide on days 2, 4, and 6 after infection (). D, Gastrointestinal motility was measured in C57BL/6 (f and ⽧) and C57BL/6 SCID (䡺 and 〫) mice either before (⽧) or 7 days after (f) infection with Giardia. For B, C, and D, motility was measured on day 7 after infection. Data are presented as the percentage of the small intestine traveled in 20 (B and C) or 30 (D) min. Each point represents an individual mouse. Data are representative of two independent experiments. ⴱ, p ⬍ 0.05.
with the results from real-time PCR (Fig. 1), more NOS2 appears in infected mice (Fig. 4C) than in uninfected mice (Fig. 4A). Importantly, no NOS2 staining was seen using NOS2-deficient mice (Fig. 4B), confirming the specificity of the reagents used for these studies. NOS1 expression was seen in long processes within the intestinal muscle layers and in patches of cells in the submucosa (Fig. 5, A and B). Thus, it is unlikely that NO produced by NOS1 could directly affect Giardia within the lumen of the small intestine. NOS1-expressing neurons and interstitial cells of Cajal have been described previously in the myenteric and submucosal plexuses of the enteric nervous system (25–27). Some neuronal NOS immunoreactivity was also seen within the villi, possibly reflecting neuronal processes into this tissue. Little change was observed in the level of NOS1 staining between uninfected and infected mice. This is in agreement with the real-time PCR data presented in Fig. 1B, which shows that the level of NOS1 mRNA is essentially identical among uninfected and infected, wild-type and IL-6-deficient C57BL/6 mice. Because NOS1-expressing neurons in the enteric nervous system are involved in regulating gastrointestinal motility and because Giardia-infected gerbils have increased intestinal transit rates, we asked whether changes in motility were involved in the elimination of Giardia. We measured gastrointestinal motility by feeding mice charcoal meals and determining the distance traveled by the charcoal in a fixed amount of time (17). This method measures both gastric emptying and intestinal transit together. We treated infected mice with either L-NAME or loperamide as a positive control for inhibiting motility. Loperamide is a -opiate receptor agonist commonly used to inhibit gastrointestinal motility and to treat diarrhea (28, 29). It can also inhibit Ca2⫹ channel function in neurons (28). Treatment with loperamide extended Giardia infections in wild-type mice similar to treatment with LNAME (Fig. 6A). As expected from results on intestinal transit in Giardia-infected gerbils (30), infection significantly increased the rate of motility in infected C57BL/6 mice (Fig. 6, B–D). Importantly, blocking NOS activity with L-NAME partially reversed this increase in transit (Fig. 6B), as did treatment with loperamide (Fig. 6C). Finally, no increase in motility was seen after infecting SCID mice with Giardia (Fig. 6D) despite the presence of high parasite loads in these mice (5). Thus, because both loperamide and LNAME block the increase in motility and prolong Giardia infec-
tions, we conclude that the increase in intestinal motility caused by Giardia infection is important for the elimination of the parasites from the intestinal tract. Furthermore, this increase in motility depends on adaptive immune responses, because no changes were observed in SCID mice.
Discussion Control of intestinal infections requires an effective immune response that must be balanced against destructive pathology. In mice, as in most humans, infected with G. lamblia, the parasites are effectively eliminated by the immune system without causing severe inflammation or diarrhea. Multiple mechanisms have been shown to be able to inhibit or kill Giardia in vitro. However, to date none has been found to be essential for controlling this infection in vivo. For example, although IgA against the parasite is clearly toxic in vitro, B cell- and IgA-deficient mice eliminate 90% or more of the parasites between 1 and 2 wk after infection (4, 5). This contrasts with SCID mice and c-kitw/wv mice that maintain infections for at least several months and IL-6-deficient mice that eliminate infections between 4 and 8 wk after infection (5, 6, 13). This probably reflects the fact that elimination of Giardia occurs through several redundant mechanisms, and it is only in mouse mutants deficient in multiple or nonredundant mechanisms that a clear phenotype is observed. We have shown that IL-6-deficient mice infected with G. lamblia have decreased levels of TNF-␣ and NOS2 mRNA in the small intestine compared with wild-type mice. Although production of NOS2 by intestinal epithelial cells can prevent parasite replication in vitro (9), treatment of wild-type mice with L-NIL, which specifically inhibits this isoform of NOS and infections in mice lacking the NOS2 gene, clearly shows that this pathway is not necessary for the elimination of infections in vivo. However, these data do not rule out a nonredundant role for NOS2 in parasite elimination. In contrast, the isoform-nonspecific inhibitor, L-NAME, and the NOS1-specific inhibitor, 7-NI, both resulted in inhibition of parasite elimination, indicating that the neuronal enzyme NOS1 is necessary for control of Giardia infection. Enhanced infections in NOS1-deficient mice confirmed the assignment of NOS1 as the essential NOS isoform for eliminating this infection. In a previous analysis of Giardia infections in IL-6-deficient mice, Bienz et al. (7) found elevated IL-4 mRNA in the mesenteric
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lymph nodes, but no NOS2 expression was detected at this site. They also reported that neither wild-type nor IL-6-deficient mice had detectable NOS2 mRNA in the mesenteric lymph nodes after infection. However, because Giardia replicates exclusively in the lumen of the small intestine, one would expect that NO production by intestinal epithelial cells would be more relevant for eliminating the infection, as suggested by Eckmann et al. (9). Our immunolocalization of NOS2 showed that NOS2 is found in cells underlying the intestinal epithelium. This is consistent both with an absence of NOS2 expression in the mesenteric lymph nodes and with the lack of effect of inhibiting this enzyme on the outcome of infection. Normal intestinal motility is a consequence of the coordinated contraction and relaxation of intestinal smooth muscles. This coordination is achieved by the enteric nervous system. Smooth muscle contraction is the result of cholinergic stimulation of smooth muscle, whereas relaxation is mediated in part through inhibitory signaling via NO. We propose that immune responses during infection increase motility, and that inhibition of NOS activity with L-NAME resulted in reduced gastrointestinal motility by interfering with muscle relaxation. Our measurements of gastrointestinal motility reflect both gastric emptying as well as intestinal transit, and it is therefore possible that gastric emptying and/or intestinal motility are important in the elimination of this infection. Indeed, NOS1-deficient mice have been shown to have reduced gastric emptying (31). In contrast, the major action of loperamide is to reduce intestinal transit, rather than gastric emptying (32, 33). Nevertheless, although loperamide is primarily an agonist of -opioid receptors, it can also block Ca2⫹ channels and inhibit calmodulin-mediated effects in neurons (28). Because NOS1 activity is regulated by calmodulin (34), we suggest that the role of loperamide in prolonging Giardia infection may be mediated through reducing intestinal motility by -opioid receptors and perhaps also via inhibition of NOS1 activity. Additional studies will be needed to determine whether altered gastric emptying has a role in the elimination of Giardia infection. The effects of intestinal infection on motility have been investigated previously in several models. Gerbils infected with G. lamblia were shown to have increased intestinal transit rates, but roles for transit and NO in eliminating the infection were not investigated (30). One of the best-studied models of infection-induced changes in motility is infections with the nematode Trichinella spiralis, where infection leads to hypermotility and mastocytosis similar to Giardia infections (35). Interestingly, treatment with L-NAME prevented hypermotility in Trichinella-infected rats, but led to increased expulsion of worms (36), in contrast to the effect of L-NAME on parasite numbers in Giardia-infected mice. This suggests that although hypermotility is not needed for the elimination of Trichinella, it is needed for the elimination of Giardia. Human infection with G. lamblia often results in severe abdominal cramps and malabsorptive diarrhea. We have now shown that Giardia infections in mice lead to increased rates of intestinal transit, and that this transit is required for elimination of the infection. It is likely that similar changes in intestinal transit in humans contribute to the symptoms associated with this infection. Additional investigation into the pathophysiology of Giardia infection is needed, because no pathogenic mechanisms have yet been identified. Furthermore, these data indicate that signals mediated by the enzyme NOS1 play a key role in the elimination of this infection, but NO production by NOS2 is clearly not required. Although NOS2 has been implicated in the elimination of numerous infections, this is the first example, of which we are aware, in which NOS1 activity is required.
Acknowledgments We gratefully acknowledge Dr. Heidi Elmendorf for suggesting the loperamide experiments and Dr. Terez Shea-Donohue for helpful discussions.
Disclosures The authors have no financial conflict of interest.
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