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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Biology, Hai Nam Normal University, Hai Kou, China (Y.H.Y.); and Department of Pharmacology (Y.H.Y., W.K.K.W., E.K.K.T., H.P.S.W., E.K.Y.L., W.H.L.S., V.Y.S., C.H.C.) and Research Center of Infection and Immunology (C.H.C.), Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Received February 6, 2006; accepted April 28, 2006.
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Abstract |
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(TGF), which is a ligand of EGFR, by small interfering RNA completely nullified the mitogenic signals evoked by rCRAMP in RGM-1 cells. These findings suggest that rCRAMP exhibits prohealing activity in stomachs through TGF-dependent transactivation of EGFR and its related signaling pathway to induce proliferation of gastric epithelial cells.
). Many endogenous molecules that regulate these cellular responses have been identified (Tarnawski et al., 2001; Taupin and Podolsky, 2003). Little studied in this panoply of factors are the host defense peptides, which were originally characterized as small cationic peptides that could kill microbes. This group of peptides is now recognized to contain multifunctional molecules that can modulate many host cellular responses (Li et al., 2000; Selsted and Ouellette, 2005).
Cathelicidins constitute a class of host defense peptides in mammals (Zaiou and Gallo, 2002). They are synthesized as preproprotein, which is characterized by an N-terminal signal sequence, a well conserved cathelin-like domain, and a C-terminal peptide domain that is proteolytically cleaved to release a small molecular weight host defense peptide. The mature peptides vary markedly among different species. Many mammals such as pigs and cattle have multiple cathelicidin genes, whereas humans, mice, and rats have only one, designated as LL-37/hCAP-18, mCRAMP, and rCRAMP, respectively. The peptide is present in phagocytic granulocytes, the first type of cell to be recruited from the blood to sites of infection and injury, and on surfaces in contact with the outside environment like skin epithelium (Termen et al., 2003). In the gastrointestinal tract, LL-37, the mature peptide of human cathelicidin, is produced constitutively by differentiated surface and upper crypt epithelial cells in the colon and by Brunner's glands in the duodenum (Hase et al., 2002). LL-37 is also produced in the stomach by surface epithelial cells, as well as chief and parietal cells, and is found in the gastric juice. Recent findings also reveal that LL-37 is up-regulated in the gastric secretion and epithelium inflamed by Helicobacter pylori infection (Hase et al., 2003).
Wound repair and inflammation are crucial adaptations to tissue damage and bacterial infection. Therefore, it comes as no surprise that soluble peptide factors have evolved to orchestrate all these processes, including killing bacteria together with regulation of inflammation and wound healing. The human cathelicidin LL-37, in this respect, not only possesses microbicidal activity against a broad spectrum of microorganisms but also increases chemotaxis of neutrophils, monocytes, T cells, and mast cells (Yang et al., 2000; Niyonsaba et al., 2002). LL-37 also induces secretion of interleukin (IL)-8 and other chemokines from monocytes, macrophages, dendritic cells, and epithelial cells and elicits maturation and release of IL-1
in lipopolysaccharide (LPS)-primed monocytes (Scott et al., 2002; Tjabringa et al., 2003; Bowdish et al., 2004; Davidson et al., 2004; Elssner et al., 2004). On the other hand, LL-37 inhibits the expression of specific proinflammatory genes up-regulated by LPS, such as nuclear factor 
B1 (p105/p50) and tumor necrosis factor -induced protein 2, accompanied by reduced nuclear translocation of nuclear factor B subunits p50 and p65 (Mookherjee et al., 2006). LL-37 also reduces nitric oxide production induced by IL-1 and LPS (Ciornei et al., 2003).
In the context of tissue repair, cathelicidins LL-37 and mCRAMP are strongly expressed in skin epithelium during wound healing in humans and mice, respectively (Dorschner et al., 2001). In addition, the expression of LL-37 is low or absent in chronic skin ulcers, and antibodies to this peptide inhibit post-wounding re-epithelialization (Heilborn et al., 2003). The ability of LL-37 to induce angiogenesis further highlights its potential role in wound repair (Koczulla et al., 2003). It has been proposed that the prohealing effects of cathelicidins may be mediated through modification of growth factor/receptor interactions and angiogenesis (Gallo et al., 1994; Li et al., 2000; Chon et al., 2001). However, definitive proof of the involvement of these mechanisms in wound healing has not yet been obtained. Although H. pylori-associated inflammation in the human gastric mucosa is known to induce LL-37 expression, little is known about its role in mucosal repair. In this connection, rCRAMP, the rat cathelicidin, has a similar expression pattern and biological activity to human cathelicidin (Travis et al., 2000; Termen et al., 2003). rCRAMP and LL-37 also share a similar secondary structure, which is characterized by an amphipathic -helix. Therefore, it has been proposed that the rat model is an appropriate experimental system to study the role of cathelicidin in human diseases (Termen et al., 2003). Nevertheless, it is also worthwhile to note that rCRAMP is cytotoxic at high concentration and shares stronger homology to mCRAMP than to LL-37 (Termen et al., 2003). In the present study, we aim to investigate whether this peptide promotes gastric ulcer healing in rats through a defined mitogenic mechanism in gastric epithelial cells.
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Materials and Methods |
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Induction of Experimental Ulcer and Sample Collection. The study was approved by the Committee on the Use of Live Animals for Teaching and Research at the University of Hong Kong. Male Sprague-Dawley rats (180-200 g) were reared on a standard laboratory diet (Ralston Purina, Chicago, IL) and given tap water. Rats were deprived of food but had free access to tap water 24 h before ulcer induction. Gastric kissing ulcers were produced by luminal application of acetic acid. Animals were sacrificed on days 1, 4, 7, and 10 after ulcer induction, and the ulcer area was measured as described previously (Ma et al., 2000) by a person who was unaware of the type of treatment. After measuring the ulcerated area, a longitudinal section of stomach along the greater curvature, including the ulcer base and both sides of the ulcer margin, was fixed in 4% buffered formalin for 24 h at 4°C for histologic study. The remaining glandular mucosa around the ulcer (including the ulcer margin and adjacent normal mucosa) was scraped with a glass slide on an ice-cold dish and immediately frozen in liquid nitrogen. The mucosal samples were stored at -70°C for isolation of RNA and protein.
Gene Therapy with rCRAMP Plasmid. Full-length rCRAMP complementary DNA was cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) under a cytomegalovirus promoter. The same plasmid without the rCRAMP insert was used as control. All the plasmids were amplified in DH5 Escherichia coli-competent cells (Invitrogen) and purified with an endo-free plasmid mega-prep kit (Qiagen, Valencia, CA). Twenty-four male Sprague-Dawley rats were fasted for 24 h before experiments. Gastric ulcers were induced as described earlier. Immediately after ulcer induction, each of 12 rats was injected with 100 µg of the plasmid DNA with rCRAMP insert, and the remaining rats were injected with the same amount of control plasmid DNA. The plasmid DNA was injected from four sides of the ulcer induction site into the submucosa as described by Jones et al. (2001).
Histology and Immunohistochemistry. Sections were stained for proliferative cells and microvessels by immunohistochemistry as described previously (Shin et al., 2004). To assess cell proliferation, sections were digested with trypsin for 15 min at room temperature and incubated with a blocking agent (LSAB kit; DAKO, Copenhagen, Denmark) for 1 h. They were then incubated with a monoclonal primary antibody against mouse proliferating cell nuclear antigen (PCNA) (1:200) overnight at 4°C. Sections were incubated with Link reagent (LSAB kit) for 1 h, followed by streptavidin for another hour. Finally, they were incubated with hydrogen peroxidase-diaminobenzidine to visualize PCNA-positive cells. After washing with tap water, sections were counterstained with Mayer's hematoxylin, dehydrated and mounted. The number of stained cells was counted under a microscope at 400x magnification. Microvessel density was measured with a procedure similar to PCNA staining, except that rabbit anti-human von Willebrand factor (1:200) (DAKO) was used to identify microvessels, and Mayer's hematoxylin counterstaining was omitted. Microvessel density was expressed as the number of microvessels per square millimeters in five to eight randomly selected fields (x200).
Cell Culture and Viability Assay. Rat gastric mucosal epithelial cell line RGM-1 (RCB-0876; Riken Cell Bank, Tsukuba, Japan) was grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium (GIBCO, Grand Island, NY) supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin, and 20% fetal bovine serum (GIBCO, Gaithersburg, MD) in an incubator at 37°C, 95% humidity, and 5% CO2. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction method as described previously (Shin et al., 2004).
[3H]Thymidine Incorporation Assay. Cell proliferation was assessed as DNA synthesis by the [3H]thymidine incorporation assay with modifications. RGM-1 cells were seeded in 24-well culture plates at 
5 x 104 cells/ml and were allowed to grow in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 20% fetal bovine serum for 24 h. Afterward, cells were growth-arrested in serum-free medium overnight, and then various concentrations of rCRAMP (5-20 µg/ml) were incubated with the cells for 24 h to study the mitogenic effect of rCRAMP in the presence or absence of AG1478 (1 µM), U0126 (25 µM), or GM6001 (25 µM), which were administered 30 min before rCRAMP treatment. In the next step, 0.5 µCi of [3H]thymidine was added to each well, and the cells were incubated for a further 4 h. The final incorporation of [3H]thymidine into cells was measured with a liquid scintillation counter (LS-6500; Beckman Instruments, Inc., Fullerton, CA).
Immunoprecipitation and Western Blot Analysis. RGM-1 cells were harvested in radioimmunoprecipitation assay buffer containing proteinase and phosphatase inhibitors as described previously (Shin et al., 2004). Protein was quantified with a protein assay kit (Bio-Rad Laboratories, Hercules, CA). To immunoprecipitate EGFR, lysates (500 µg) were incubated overnight at 4°C with 1 µg of anti-EGFR antibody and 20 µl of 50% protein A-Sepharose slurry. Beads were washed with radioimmunoprecipitation assay buffer three times. They were then boiled in 2x Laemmli sample buffer and run on SDS-polyacrylamide gels. For the Western blot analysis, equal amounts of protein (40 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Hybond C nitrocellulose membranes (Amersham Biosciences, Arlington Heights, IL). For mature rCRAMP, SDS-PAGE was performed using 16.5% tricine gels. Membranes were probed with anti-epidermal growth factor (EGF), anti-vascular endothelial growth factor, anti-ERK1/2, anti-phospho-ERK1/2, anti-EGFR, and antiphosphotyrosine antibodies overnight at 4°C and incubated for 1 h with secondary peroxidase-conjugated antibodies. The proteins were visualized with an enhanced chemiluminescence system (Amersham Biosciences).
Reverse Transcription-Polymerase Chain Reaction for rCRAMP, Transforming Growth Factor , and -Actin. The total RNA was isolated from rat gastric tissues and RGM-1 cells with TRIzol reagent (Invitrogen). Two micrograms of total RNA was reverse-transcribed using the Thermoscript reverse transcription-polymerase chain reaction (RT-PCR) system (Invitrogen). PCR was performed for rCRAMP, transforming growth factor (TGF) , and -actin using the following primer pairs: rCRAMP, sense primer 5'-TCTGAGCCCCAAGGGGATGAGGA-3' and antisense primer 5'-CCAAGGCAGGCCTACTGCTCTAT-3' [product size 356 base pair (bp)]; TGF, sense primer 5'-CTGGGTATCCTGGTAGCTGTGT-3' and antisense primer 5'-GACCACTGTCTCAGAGTGGC-3' (product size 322 bp); and -actin, sense primer 5'-GTGGGGCGCCCCAGGCACCA-3' and antisense primer 5'-CTCCTTAATGTCACGCACGATTTC-3' (product size 540 bp). The PCR conditions were as follows: the template cDNA was first denatured at 94°C for 5 min. During 40 cycles of amplification, the denaturation step was at 94°C for 1 min, the annealing step at 55°C for 1 min, and the extension step at 72°C for 1 min. The final extension step was at 72°C for 7 min. The PCR products were electrophoresed on 1.5% (w/v) agarose gels containing 0.5 µg/ml ethidium bromide. Gel photographs were then analyzed semiquantitatively in a multianalyzer (BioRad, Hercules, CA).
Knockdown of TGF by Small Interfering RNA. Small interfering RNA (siRNA) targeting rat TGF (TGF-siRNA) was designed online and synthesized by Invitrogen. The sequence of TGF-siRNA, which corresponded to the coding regions 139 to 164 relative to the first nucleotide of the start codon, were as follows: 5'-CAGAUUCCCACACUCAGUAUUGUUU-3' and 5'-AAACAAUACUGAGUGUGGGAAUCUG-3'. Transfection was performed using Oligofectamine reagent (Invitrogen) according to the manufacturer's instructions. The efficacy of TGF knockdown was assessed by RT-PCR. Assays were performed 2 days after transfection.
Statistical Analysis. Results were expressed as the mean ± S.E.M. Statistical analysis was performed with analysis of variance, followed by the Tukey t test. P < 0.05 was considered statistically significant.
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; Ma et al., 2000), we determined the protein levels of these factors in the gastric mucosa on day 4 after ulcer induction and found that their expression was not affected by the rCRAMP gene therapy (data not shown).
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Synthetic rCRAMP Stimulated Proliferation of Cultured Gastric Epithelial Cells. Given the marked increase in the number of proliferative cells at the ulcer margin induced by rCRAMP plasmid treatment, we examined further whether rCRAMP directly affected the growth of cultured rat gastric epithelial cells (RGM-1). We found that rCRAMP treatment significantly induced cell proliferation in a dose-dependent manner. At 5 and 10 µg/ml, the peptide increased cell proliferation by 20.5 and 36.1%, respectively. EGF was used as a positive control, which stimulated cell proliferation by 29.4% (Fig. 4). At all the concentrations used, rCRAMP exerted no cytotoxic effect on the gastric epithelial cells.
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), the effect of rCRAMP on ERK1/2 activation in RGM-1 cells was studied. Results showed that rCRAMP induced a concentration-dependent increase in ERK1/2 phosphorylation (Fig. 5A) that was significant over the concentration range of 5 to 20 µg/ml and maximal at 10 µg/ml. In cells treated with 10 µg/ml rCRAMP, ERK1/2 phosphorylation was maximal at 15 min after incubation and returned to the basal level at 60 min (Fig. 5B). In addition, activation of ERK1/2 by rCRAMP was fully inhibited by pretreatment with MEK inhibitor U0126 (Fig. 5C). Treatment of RGM-1 cells with rCRAMP also caused an increase in tyrosine phosphorylation of EGFR, which was completely abolished by AG1478 (an EGFR kinase inhibitor) (Fig. 6A). Moreover, treatment of RGM-1 cells with AG1478 dramatically decreased the phosphorylation of ERK1/2 induced by rCRAMP without altering total protein levels of ERK1/2 (Fig. 6B).
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Inhibitors of MMP, EGFR Tyrosine Kinase, and MEK Abolished the Mitogenic Effect of rCRAMP. To determine the involvement of MMP, EGFR, and MEK in rCRAMP-induced cell proliferation, cell proliferation was assayed in the absence or presence of respective inhibitors. Results revealed that inhibition of MMP, EGFR tyrosine kinase, and MEK with GM6001, AG1478, and U0126, respectively, abolished rCRAMP-induced cell proliferation (Fig. 8), indicating that rCRAMP-induced cell proliferation was dependent on these factors.
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Knockdown Nullified the Effect of rCRAMP on EGFR Phosphorylation and Cell Proliferation. Although the results presented so far clearly indicated that rCRAMP induced cell proliferation through EGFR transactivation, whether this phenomenon is ligand-dependent had not yet been determined. It has been reported that gastric mucosa and gastric cancer cell lines produce heparin-binding EGF-like growth factor (HB-EGF), TGF, and amphiregulin, all of which can be mobilized by MMP and thereby activate EGFR (Naef et al., 1996; Tanida et al., 2004). In RGM-1 cells, the mRNA of TGF and amphiregulin, but not HB-EGF, were detected by RT-PCR. The expression level of TGF was higher than that of amphiregulin (data not shown). Furthermore, down-regulation of TGF by siRNA significantly suppressed rCRAMP-induced EGFR phosphorylation (Fig. 9, A and B) and cell proliferation (Fig. 9C). These findings are in accordance with the speculation that rCRAMP-induced EGFR transactivation is dependent on the release of TGF. Furthermore, knockdown of TGF decreased basal DNA synthesis by approximately 15% (P < 0.05), indicating that TGF is required for the maintenance of basal levels of proliferation in RGM-1 cells.
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-defensins and cathelicidins, serve important innate immune functions by acting as "natural antibiotics" to provide first-line defense against infection (Schutte and McCray, 2002; Elsbach, 2003; Selsted et al., 2005). These peptides are expressed on epithelial surfaces and in neutrophils and are up-regulated on bacterial infection. In the present study, we showed for the first time that the rat cathelicidin rCRAMP was involved in tissue repair in the stomach. To this end, ulceration up-regulated the expression of rCRAMP in the gastric mucosa. Further induction of rCRAMP expression by plasmid-based gene therapy accelerated ulcer healing by promoting angiogenesis and cell proliferation in the gastric mucosa. In accord with these findings, human cathelicidin LL-37 has been reported to promote proliferation and migration of human airway epithelial cells, as well as cutaneous wound repair. In addition, LL-37 is known to directly induce proliferation and formation of vessel-like structures in cultivated endothelial cells. Mice deficient in mCRAMP also exhibit significantly decreased vascular structures and delayed wound closure (Heilborn et al., 2003; Koczulla et al., 2003; Shaykhiev et al., 2005). All these findings suggest that cathelicidin is an important endogenous mitogenic and proangiogenic factor in gastric ulcer healing. However, the mechanism by which ulceration up-regulates rCRAMP expression in the gastric mucosa is presently unknown. Moreover, whether rCRAMP, a known modulator of inflammation (Yang et al., 2000; Niyonsaba et al., 2002; Mookherjee et al., 2006), can possibly alter the cytokine environment and thereby facilitate the subsequent healing process remains unexplored. In this regard, our results show that the up-regulation of rCRAMP could be detected early after ulcer induction (on day 1 and day 4), implicating that rCRAMP might be involved in the modulation of acute inflammatory response during gastric ulceration.
Previous studies showed that EGFR and its related signaling pathway are involved in the proliferation of gastric epithelial cells (Tarnawski et al., 1992; Pai et al., 1998). Here we report that rCRAMP increased RGM-1 cell proliferation and EGFR and ERK1/2 phosphorylation, which was abolished by inhibition of MMP or knockdown of TGF, indicating that the mitogenic action of rCRAMP is signaling through MMP-mediated TGF-dependent transactivation of EGFR. In addition, the MMP inhibitor had no effects on the EGF-induced phosphorylation of EGFR and ERK1/2, suggesting that activation of the EGFR and ERK1/2 pathway by rCRAMP may involve cleavage of membrane-anchored EGFR ligands by MMP. In this regard, it is known that EGFR ligands such as TGF and HB-EGF are synthesized as transmembrane precursor molecules, which can be cleaved by cell surface proteinases known as ADAM (a disintegrin and metalloproteinase) to release the soluble form, a process known as ectodomain shedding (Xiao and Majumdar, 2001; Sahin et al., 2004). Therefore, it is believed that the mitogenic signal evoked by rCRAMP in gastric epithelial cells is effected by ADAM-mediated ectodomain shedding of TGF, which can be blocked by MMP inhibitors (Fig. 10). In this context, ADAM17 has been shown to be the major convertase responsible for the shedding of TGF (Sahin et al., 2004). Indeed, it has been shown that LL-37 can transactivate EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR ligands to induce IL-8 release (Tjabringa et al., 2003). Prostaglandin E2 also signals through a similar pathway to transactivate EGFR and phosphorylate ERK1/2 by stimulating MMP-dependent cleavage of TGF in colon cancer and gastrointestinal hypertrophy (Pai et al., 2002). However, the molecular mechanisms underlying cathelicidin-induced activation of MMP are presently unknown but may involve signals mediated by a G protein-coupled receptor (Shaykhiev et al., 2005).
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Recently, antimicrobial peptides have generated intense interest because of their therapeutic potential against antibiotic-resistant pathogens. There is also growing concern over H. pylori resistance to antibiotics. For example, H. pylori resistance to macrolide, metronidazole, amoxicillin, and tetracycline is increasingly reported and may limit the efficacy of current treatment (Megraud, 2004). Therefore, second-line therapy for H. pylori eradication is promptly needed. To this end, cathelicidin has been shown to be bactericidal for several strains of H. pylori, including SD4, SD14, and SS1 (Hase et al., 2003). In the current study, we also show that overexpression of cathelicidin can promote ulcer healing in rats. Therefore, cathelicidin may be a potential natural antibiotic for the eradication of H. pylori in gastric ulcer patients while at the same time promoting ulcer repair in the gastric mucosa. Although direct administration of synthesized peptide seems unlikely because of its poor chemical stability in the stomach, endoscopic injection of cathelicidin-encoding plasmids or inoculation of bioengineered bacteria that actively produce cathelicidin (e.g., Lactobacillus transformed with cathelicidin-encoding plasmids) could be a clinically effective method for delivery of cathelicidin at the ulcer site (Steidler et al., 2000; Jones et al., 2001).
To conclude, our experimental findings not only establish a novel role for cathelicidin in ulcer healing but also define the mechanistic pathways of its mitogenic action on gastric epithelial cells. This unique and naturally produced protein that combines antimicrobial and ulcer healing actions may provide a new therapeutic approach in the treatment of infectious gastric ulcer.
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Footnotes |
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Y.H.Y. and W.K.K.W. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; GM6001, N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide; EGFR, epidermal growth factor receptor; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; PCNA, proliferating cell nuclear antigen; PAGE, polyacrylamide gel electrophoresis; EGF, epidermal growth factor; RT-PCR, reverse transcription-polymerase chain reaction; TGF, transforming growth factor; siRNA, small interfering RNA; HB-EGF, heparin-binding epidermal growth factor-like factor; ADAM, a disintegrin and metalloproteinase.
Address correspondence to: Dr. Chi H. Cho, Department of Pharmacology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong, China. E-mail: chcho{at}hkusua.hku.hk
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