1Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo; Departments of 2Tropical Medicine and Parasitology and 4Internal Medicine, School of Medicine, Keio University, Tokyo; and 3Yakult Central Institute for Microbiological Research, Kunitachi City, Tokyo, Japan
Submitted 28 June 2004 ; accepted in final form 15 November 2004
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ABSTRACT |
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interleukin-7; colitis; memory T cells; lamina propria lymphocytes; proliferation; high-level interleukin-7 receptor
We have also shown a potential role for IL-7/IL-7R-mediated immune responses in intestinal inflammation (30, 31). First, IL-7 transgenic (Tg) mice developed chronic colitis that mimicked histopathological characteristics of human ulcerative colitis (UC). As chronic colitis developed, IL-7 Tg mice showed decreased expression of IL-7 protein in epithelial cells and significant infiltration of CD4+IL-7R+ T cells in the lamina propria. Second, we clarified that mucosal CD4+ T cells expressing high levels of IL-7R (IL-7Rhigh) are the pathogenic T cells that induce chronic colitis and are thus potential targets for treatment of murine chronic colitis (32). We demonstrated that transfer of IL-7Rhigh CD4+ LPLs isolated from colitic mice in immunodeficient mice induces chronic severe colitis. Importantly, the selective elimination of IL-7Rhigh LPLs by administering small amounts of toxin-conjugated anti-IL-7R antibody completely ameliorated ongoing colitis (32). Third, dysregulation of the IL-7/IL-7R system is also observed in colonic mucosa of patients with UC at the active stage (unpublished data).
The mechanism of expansion and survival of IL-7Rhigh CD4+ LPLs in intestinal mucosa is, however, poorly understood. IL-7 is believed to act synergistically with IL-15 to regulate the potential for survival and homeostatic proliferation of memory CD8+ T cells but not memory CD4+ T cells (5, 24, 27), but controversy remains about whether IL-7 itself has a role in supporting the turnover of memory CD4+ T cells. Recent reports indicated that memory CD4+ T cells expressed high levels of IL-7R and responded to IL-7 by prolonged survival in vitro (10, 13, 25). This result indicated that IL-7 has previously unrecognized roles in the regulation of the survival and maintenance in at least a certain subset of memory CD4+ T cells. Especially, it remains unknown whether IL-7 is involved in the dysregulated homeostasis of memory-type pathogenic T cells in autoimmune disorders. Because we have shown that CD4+ LPLs in colitic mice express high levels of IL-7R, we sought to address whether IL-7 contributes to the expansion of those cells under pathogenic autoimmune conditions.
Here we provide evidence that, among LPLs generally exhibiting memory cell phenotypes, only those in mice with chronic colitis express high levels of IL-7R. Stimulation with exogenous recombinant IL-7 (rIL-7) alone in vitro induced significant proliferation of IL-7Rhigh memory CD4+ LPLs isolated from colitic mice but had no effect on that of memory CD4+ LPLs from wild-type (WT) mice. Other epithelial cell-derived cytokines, including IL-15 and thymic stromal lymphopoietin (TSLP), did not induce the proliferation of IL-7Rhigh CD4+ LPLs. Of note, in vivo administration of rIL-7 caused colitis more quickly and in greater severity in mice transferred with CD4+ LPLs from colitic mice. Moreover, inhibition of the IL-7/IL-7R pathway by administration of anti-IL-7R monoclonal antibody (MAb) significantly ameliorated colitis. These data indicate that IL-7 plays a crucial role in expansion of IL-7Rhigh mucosal T cells in memory CD4+ cells and development of chronic colitis. They also suggest that therapeutic approaches targeting this pathway may be feasible in the treatment of human inflammatory bowel disease.
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METHODS |
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Purification of T cell subsets.
Spleens and mesenteric lymph nodes (MLNs) were mechanically dissociated, and the red blood cells were lysed by treatment with 0.84% NH4Cl. Cells were filtered through a 100-µm-pore-size nylon mesh and suspended in RPMI (Sigma-Aldrich, St. Louis, MO) containing 10% FCS. Splenic CD4+ T cells were isolated from the cell suspension using the anti-CD4 MACS beads system (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. For isolation of colonic LPLs, entire colons were opened longitudinally, washed with PBS, and cut into small (3 mm) pieces. The dissected specimens were incubated twice with Ca2+- and Mg2+-free Hanks' balanced salt solution containing 1 mM dithiothreitol (GIBCO-BRL, Gaithersburg, MD) for 30 min at 37°C with gentle stirring. The residual tissue fragments were washed and incubated with collagenase A (Roche, Mannheim, Germany) for 2 h at 37°C. The supernatants were collected and washed, and the lymphocyte fraction was isolated on discontinuous Percoll gradients of 75 and 40%. Enriched CD4+ LP T cells were obtained by positive selection using the anti-CD4 MACS magnetic separation system as described above. The resultant cells were shown to contain >96% CD4+ cells by analysis using the FACS Calibur (Becton-Dickinson, Sunnyvale, CA).
Induction of colitis.
In this study, the well-characterized CD4+CD45RBhigh-induced RAG-2/ colitis (23) and spontaneous TCR-/ colitis (15) models were used. The LP CD4+ T cells isolated from colitic TCR-
/ (20-wk-old) mice were transferred into RAG-2/ mice as previously described (32). All animals were weighed, and diarrhea was evaluated as an indicator of colitis.
Flow cytometry.
The profiles of spleen, MLN and LP T cells were analyzed by flow cytometry. Cells were preincubated with a FcR-blocking MAb (CD16/32; 2.4G2; BD PharMingen, San Diego, CA) for 20 min and then incubated with FITC-, phycoerythrin (PE)-, or biotin-labeled specific antibodies for 30 min on ice. Antibodies used were anti-CD4 MAb (RM45; BD PharMingen), anti-TCR-
MAb (H57597; BD PharMingen), anti-CD25 MAb (HT-2; BD PharMingen), anti-CD44 MAb (IM7; BD PharMingen), anti-CD62L MAb (MEL-14; BD PharMingen), anti-CD69 MAb (H1.2F3; BD PharMingen), and anti-IL-7R MAb (A7R34; kindly provided by Dr. Tetsuo Sudo, Toray Industries, Tokyo, Japan). Biotinylated antibodies were detected with PE-streptavidin (BD PharMingen). Standard two-color flow cytometric analysis was performed using a FACS Caliber (Becton-Dickinson) with the CellQuest software. Staining with isotype-matched control MAbs was performed to assess background fluorescence intensity. Dead cells were eliminated from analysis by 7AAD (BD PharMingen) staining.
Proliferation assay. Splenic (2 x 105 cells/well), MLN (1 x 105 cells/well) and LP (1 x 105 cells/well) CD4+ T cells were seeded on 96-well round microtiter plates and cultured in 10% FCS-containing RPMI, supplemented with 2-mercaptoethanol (GIBCO), penicillin-streptomycin (GIBCO), L-glutamine (GIBCO), and HEPES (GIBCO). After 72 h of incubation, cells were pulsed for 12 h with [3H]thymidine (1 µCi/well), and the [3H]thymidine incorporation was measured with a scintillation counter (Filtermate Harvester; Perkin-Elmer, Boston, MA). Recombinant murine (rm) IL-7 (Peprotech EC, London, UK), rmIL-15 (R&D Systems, Minneapolis, MN), and rmTSLP (R&D Systems) were used for cellular stimulation at concentrations of 0.550 ng/ml.
Detection of apoptosis. Isolated colonic CD4+ LPLs resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin were cultured in 96-well round microtiter plates at 1 x 105 cells/well in the absence or presence of rmIL-7 (50 ng/ml). After 10 days of culture, cells were washed with PBS and resuspended in a mixture of 100 µl diluted binding buffer and 5 µl of annexin V-FITC (BD PharMingen) solution, and then 2 µl of 7-amino-actinomysin D were added. After 15 min of incubation at room temperature in the dark, flow cytometry analysis was performed using a FACS Caliber (Becton-Dickinson). A portion of cells was stained with PE-anti-CD4 MAb in parallel, and the percentage of surviving CD4+ cells was determined by evaluating annexin V-negative cells. To analyze Bcl-2 expression in CD4+ LPLs, these cells were cultured in medium supplemented with rIL-7 (50 ng/ml). After 16 h of incubation, cells were fixed and permeabilized with BD Cytofix/Cytoperm solution before intracellular cytokine staining with FITC-conjugated anti-Bcl-2 MAb (3F11; BD PharMingen).
Cytokine assay.
To measure cytokine production, isolated CD4+ LPLs were cultured in medium supplemented with 1 µg/ml soluble anti-CD28 MAb (37.51; BD PharMingen) in 96-well plates precoated with 10 µg/ml anti-CD3 MAb (1452C11; BD PharMingen) in PBS. Culture supernatants were collected, and the cytokine concentrations of IFN-, TNF-
, IL-2, IL-4, and IL-5 were determined by flow cytometry with a cytometric bead array set (BD PharMingen) according to the manufacturer's instructions.
Administration of IL-7 in colitic memory IL-7Rhigh CD4+ LPL-transferred RAG-2/ mice.
CD4+ LP T cells were isolated from colitic TCR-/ mice (20 wk of age) by use of anti-CD4 MACS beads. The purified CD4+ LPLs (1.5 x 105) were intraperitoneally transferred to RAG-2/ mice. Either rmIL-7 (5 µg/day ip on days 07) or PBS alone was administered to these mice intraperitoneally, and the effects of IL-7 on the development of colitis were evaluated.
Administration of anti-IL-7R MAb for diseased TCR-/ mice.
TCR-
/ mice (8-wk-old) were treated with rat anti-murine IL-7R
MAb (A7R34) by intraperitoneal injection of a 1-mg dose one time per week for 8 wk. Control mice were treated with the same amounts of purified rat IgG (Sigma-Aldrich). All mice were killed on the day after the last treatment, and colitic lesions were evaluated.
Clinical and histological analysis. Mice were weighed and monitored for appearance and signs of soft stool or diarrhea three times per week. After death, clinical scores were assessed as the sum of the following four parameters: hunching and wasting, 0 or 1; colon thickening, 03 (0, no colon thickening; 1, mild thickening; 2, moderate thickening; 3, extensive thickening); and stool consistency, 03 (0, normal beaded stool; 1, soft stool; 2, diarrhea; 3, gross bloody stool; see Ref. 9). For histological scoring, the area most affected was selected and graded by the number and severity of lesions. The mean degree of inflammation in the colon was calculated using a modification of a previously described scoring system (2).
Statistical analysis. The results are expressed as means ± SD. For statistical analysis, we used the program Statview for Macintosh and MS Office (Excel) and analyzed the data by Student's t-test.
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RESULTS |
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Stimulation with rIL-7 alone in vitro induced expansion and survival of IL-7Rhigh memory CD4+ LPLs isolated from colitic mice.
To clarify the mechanism by which CD4+IL-7Rhigh LPLs infiltrated and expanded in the LP, we performed in vitro cell proliferation studies. To determine the effect of IL-7 on CD4+ LPLs from diseased or nondiseased TCR-/ mice, diseased CD4+CD45RBhigh T cell-transferred mice and WT mice were stimulated in vitro with exogenously added rIL-7. Surprisingly, stimulation with rIL-7 alone induced significant increase in DNA synthesis of IL-7Rhigh memory CD4+ LPLs from both types of colitis mice in a concentration-dependent manner (Fig. 2). In sharp contrast, rIL-7 alone did not enhance the proliferation of CD4+ LPLs from WT mice. Interestingly, CD4+ LPLs from diseased TCR-
/ mice showed higher proliferative responses to rIL-7 than with those from diseased CD4+CD45RBhigh-transferred mice, reflecting the higher levels of IL-7R expression, as shown in Fig. 1A. Consistent with the lower expression of IL-7R on LPLs from nondiseased TCR-
/ mice (Fig. 1A) than that from the paired diseased TCR-
/ mice, the proliferative responses of LPLs by rIL-7 from nondiseased mice were significantly lower than those from the paired diseased mice (Fig. 2). Furthermore, rIL-7 alone induced a marginal effect on the proliferation of spleen CD4+ cells and MLN CD4+ cells from diseased and nondiseased TCR-
/ mice or diseased CD4+CD45RBhigh-transferred mice and WT mice.
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In vivo administration of IL-7 exacerbates colitis in RAG-2/ mice transferred with IL-7RhighCD4+ TCR-/ LPLs.
To assess whether the enhancing effect of IL-7 on the expansion of IL-7RhighCD4+ LPLs in vitro is likewise observed in vivo, we tested whether in vivo rIL-7 administration might influence the colonic inflammation-induced RAG-2/ mice transferred with IL-7RhighCD4+ LPLs isolated from colitic TCR-
/ mice (Fig. 5A). Our previous studies showed that recipient mice transferred with 1.5 x 105 IL-7Rhigh CD4+ LPLs from colitic TCR-
/ mice developed colitis within 6 wk after the transfer. rIL-7 was intraperitoneally injected in those recipient mice daily on days 07 after the cell transfer. Intriguingly, IL-7-treated recipient mice, but not PBS-treated mice, rapidly showed severe weight loss and wasting disease at 23 wk after the transfer, and we killed all recipient mice at 3 wk after the transfer.
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Cytokine production of isolated CD4+ LPLs in RAG-2/ mice after stimulation with anti-CD3 MAb and anti-CD28 MAb were quite different. Isolated CD4+ LPLs from mice treated with rIL-7 produced significantly higher amounts of IFN- (P < 0.001) and TNF-
(P < 0.001), but not IL-2, IL-4, and IL-5 (P < 0.05), compared with those from PBS-treated mice (Fig. 5G).
Interestingly, flow cytometric analysis revealed that the degree of IL-7R expression in isolated CD4+ LPLs of recipient mice treated with rIL-7 or PBS was significantly decreased compared with that in freshly isolated CD4+ LPLs from colitic TCR-/ mice but still higher than that on normal CD4+ LPLs (Fig. 5H). In CD4+ LPLs of recipient mice treated with rIL-7, TCR-
dim cells were significantly increased and IL-7R expression on CD4+ LPLs was higher than that in spleen CD4+ cells (Fig. 5I). In contrast, the expression of IL-7R on LPLs from WT mice was much lower than that in mice treated with rIL-7. These infiltrated CD4+IL-7Rhigh LPLs from rIL-7-treated recipient mice with severe colitis mainly consisted of memory cells with CD44high, CD62Llow, and CD45RBlow (Fig. 5J). Collectively, these data indicate that in vivo administration of rIL-7 worsens chronic colitis, probably through the expansion of memory CD4+ LPLs in RAG-2/ mice transferred with LPLs from colitic mice.
Blockade of the IL-7/IL-7R pathway ameliorated colitis with decrease of memory CD4+ LPLs.
To prove the role of IL-7 in the exacerbation of chronic colitis with expansion of IL-7Rhigh LPLs, we finally attempted to control chronic colitis by inhibiting the IL-7/IL-7R pathway using a blocking antibody-based strategy. We found that 50 µg/ml of anti-IL-7R MAb used in this assay efficiently inhibited in vitro proliferation of CD4+ LPLs from chronically inflamed mucosa of TCR-/ mice (data not shown). In this setting, we used established TCR-
/ mice rather than the above-mentioned RAG-2/ mice reconstituted with CD4+IL-7Rhigh memory T cells, with the scope of clinical application to human inflammatory bowel disease. Because TCR-
/ mice were shown to develop colitis at 1216 wk of age in our breeding laboratory, we started the treatment at 8 wk of age. We diagnosed these mice by observing weight loss, ruffle appearance, signs of soft stool and diarrhea. We treated chronic colitis in TCR-
/ mice by intraperitoneal injection of 1 mg anti-IL-7R MAb weekly for 8 wk (Fig. 6A). Blockade of the IL-7/IL-7R pathway by anti-IL-7R MAb significantly ameliorated the development of chronic colitis in TCR-
/ mice. In clear contrast, TCR-
/ mice treated in parallel with an isotype-matched control antibody developed severe colitis, as shown by diarrhea, bloody stool, and weight loss. Gross inspection and histological analysis of the colon in anti-IL-7R MAb-treated TCR-
/ mice revealed marked reduction in the inflammatory activity (Fig. 6B). This difference was confirmed by histological scoring of multiple colon sections, which gave scores of 1.33 ± 0.21 in anti-IL-7R MAb-treated mice vs. 2.67 ± 0.21 in control rat IgG-treated mice (P < 0.01; Fig. 6C). In addition, the number of CD4+ LPLs was significantly decreased in mice treated with anti-IL-7R MAb (Fig. 6D). These results indicate that blockade of the IL-7/IL-7R pathway leads to inhibition of chronic colitis with decreased memory CD4+ LPLs.
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DISCUSSION |
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We have previously demonstrated the potential roles of the IL-7-IL-7R pathway in intestinal inflammation in a series of studies. First, we showed that IL-7 Tg mice developed chronic colitis that mimicked histopathological characteristics of UC in humans (30). In the colonic mucosa of colitic IL-7 Tg mice, marked infiltration of IL-7Rhigh LPLs was demonstrable. Second, we demonstrated that IL-7RhighCD4+ LPLs in colitic TCR-/ mice were pathogenic, because immunodeficient mice in which these cells were transferred developed similar colitis. Third, we showed that these IL-7Rhigh LPLs are potential targets for the treatment of chronic colitis (32). In RAG-2/ mice transferred with colitic TCR-
/ LPLs, the expansion of transferred IL-7RhighCD4+ LPLs reached 100 times the colitic mucosa of recipient mice, and their selective elimination by small amounts of toxin-conjugated anti-IL-7R
antibody completely ameliorated ongoing colitis (32). The reason for the substantial proliferative response of the IL-7RhighCD4+ LPLs remains unclear, because IL-7 production in the epithelial cells was conversely decreased according to goblet depletion (unpublished observations). Recent studies have indicated that IL-7 is implicated in the survival and homeostatic proliferation of naive CD4+ T cells (12, 24, 27, 28). It was also demonstrated that IL-7 could have previously unrecognized roles in the survival and generation of memory CD4+ cells (10, 13, 25). For instance, low doses of IL-7 serve as a survival factor for effector memory CD4+ cells, and high doses of IL-7 induce proliferation of memory cells (13). IL-7 directly regulates both survival and expansion of memory CD4+ cells (10, 13, 25). Kondrack et al. (10) demonstrated that memory CD4+ cells expressing comparable levels of IL-7R to naive T cells show sustained cellular survival in response to IL-7 in vivo. Thus IL-7 would be a good candidate for proliferation and survival of colitogenic IL-7RhighCD4+ memory LPLs. In the current study, we demonstrated that in vitro stimulation with IL-7, but not IL-15 and TSLP, induced a proliferative response in IL-7RhighCD4+ LPLs from colitic TCR-
/ mice or colitic CD4+CD45RBhigh T cell-transferred mice. In sharp contrast, memory CD4+ LPLs from nondiseased TCR-
/ mice or WT mice did not respond to IL-7. Interestingly, CD4+ LPLs from colitic TCR-
/ mice showed more potent ability to proliferate than those from colitic CD4+CD45RBhigh-transferred mice, suggesting that the proliferation response on IL-7 stimulation depends on the expression levels of IL-7R. However, the possibility cannot be excluded that reactivities to IL-7 are dependent on the Th2-dominant (TCR-
/ mice) or Th1-dominant (CD4+CD45RBhigh-transferred mice) immune model. Upon continuous in vitro stimulation with IL-7, colitic CD4+ LPLs survived and expanded for 10 days in this study (Fig. 4D), and actually survived for several weeks in the following study (unpublished observation). These data indicate that IL-7 promotes both expansion and survival in vitro of IL-7Rhigh memory CD4+ LPLs that infiltrated the colonic mucosa with chronic colitis.
In the present study, we also demonstrated that in vivo administration of IL-7 worsened chronic colitis with expansion of IL-7Rhigh memory CD4+ LPLs in RAG-2/ mice transferred with those LPLs from colitic TCR-/ mice. Moreover, blockade of IL-7R by an antibody-based strategy in vivo led to inhibition of colitis with decreased memory CD4+ LPLs. Therefore, we conclude that IL-7 itself plays crucial roles in the expansion of IL-7Rhigh memory CD4+ mucosal T cells and exacerbation of chronic colitis, although it is also possible that yet-unknown cytokines are involved in this mechanism.
We have recently demonstrated that intestinal epithelial cells also express IL-7R and that bone marrow-derived epithelial cells can differentiate into the epithelium of the gastrointestinal tract in humans (18, 19). Epithelial cells of male donor origin were distributed throughout the entire gastrointestinal tract of female bone marrow transplant recipients. Donor-derived epithelial cells remarkably repopulated the gastrointestinal tract during epithelial regeneration. Therefore, we suggest that the IL-7-IL-7R pathway may have previously unrecognized roles in the mucosal immune system.
Surprisingly, however, we found that IL-7R expression on IL-7-stimulated IL-7RhighCD4+ LPLs was significantly downmodulated compared with that on freshly isolated IL-7RhighCD4+ LPLs in vitro. Similarly, Park and colleagues (21) very recently demonstrated a novel regulatory mechanism that specifically suppressed IL-7R expression on normal murine T cells in response to IL-7. Furthermore, we have previously demonstrated that IL-7 expression in colonic epithelial cells is decreased in colonic mucosa with murine chronic colitis, such as IL-7 Tg mice and TCR-
/ mice (30). Therefore, we assessed the effect of other cytokines, such as IL-15 and TSLP, secreted by colonic epithelial cells on the expansion of IL-7Rhigh memory CD4+ LPLs in chronic colitic lesions. Our results revealed that neither IL-15 nor TSLP alone induced the proliferation of IL-7Rhigh memory CD4+ LPLs from colitic mice or IL-7Rlow memory CD4+ LPLs from WT mice. Collectively, these results indicate that IL-7 from a source outside the intestine or yet-unknown cytokines may be responsible for the proliferation of IL-7RhighCD4+ LPLs on colitic mucosa. Consistent with this hypothesis, a recent report has shown that serum concentration of IL-7 is strongly upregulated, and IL-7 is produced by dendritic-like cells within peripheral lymphoid tissues in human immunodeficiency virus-infected patients (17). Furthermore, it is also possible that colitogenic IL-7 highly sensitive IL-7Rhigh CD4+ LPLs can sufficiently respond to decreased IL-7 in the inflamed mucosa, although the mechanism of the sustained IL-7Rhigh expression on pathogenic CD4+ LPLs remains largely unclear. Further study will be needed to address this issue.
We have some evidence that the IL-7-IL-7R pathway in the colonic mucosa is disturbed in human UC (unpublished observation). Human inflammatory bowel disease is thought to result from inappropriate activation of the mucosal immune system driven by luminal antigens. The activation of important cell populations is eventually accompanied by the production of a wide variety of nonspecific mediators of inflammation. including many other inflammatory and proinflammatory cytokines, chemokines, and growth factors. We suggest that IL-7Rhigh memory CD4+ LPLs are one such important cell population. Therefore, this study provides a basis for practical application of therapy targeting the IL-7-IL-7R pathway for the treatment of chronic intestinal inflammation in human inflammatory bowel disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* E. Okada and M. Yamazaki contributed equally to this work.
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REFERENCES |
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