Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti–β2-GPI Abs into Rag-1−/− mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.

During an ischemic event, the lack of blood flow to an organ induces tissue damage. However, return of blood flow during reperfusion enhances pathology significantly. The inflammatory response to ischemia/reperfusion (IR)-induced organ damage may subsequently lead to a systemic inflammatory response with multiple organ failure. Intestinal IR results in severe inflammatory-induced mucosal damage, barrier dysfunction, and subsequent bacterial translocation leading to sepsis (1) and frequently results in liver and lung damage (2).

Mesenteric IR-induced tissue injury is mediated by at least two components of the innate immune response, neutrophil infiltration, and complement activation (35). Initial studies demonstrated that neutrophil depletion attenuated intestinal IR-induced injury (4, 5). However, the presence of neutrophils was not sufficient for tissue damage when complement activation was inhibited (6). Complement activation increased adhesion molecule expression after IR and released a cascade of inflammatory mediators including leukotriene B4 (LTB4) and PGE2, which also contributed to tissue damage (79). In addition, depletion or inhibition of complement activation products prevented both local and remote organ injury in response to intestinal IR (1013).

Cells subjected to hypoxic conditions express cryptic Ags on the plasma membrane (14, 15). Cryptic Ags expressed on apoptotic cells are recognized by natural Abs, which frequently exhibit low-affinity binding (16). Previous studies indicated that administering naturally occurring mAbs reconstituted IR-induced intestinal damage in Ab-deficient Rag-1−/− mice (15, 1719). Multiple natural Abs, which recognized intracellular Ags, DNA, nonmuscle myosin and ribonucleoprotein, and cardiolipin-induced damage in the IR-resistant Rag-1−/− mouse, suggesting that the Abs and Ags are critical to IR-induced damage (1821). In conjunction with anti-phospholipid mAb, Abs to the serum protein, β2-glycoprotein I (β2-GPI) also restored tissue damage in Rag-1−/−, IR-resistant mice (19). These data suggest that ischemia induces a cellular response resulting in expression of multiple cryptic Ags targeted by low-affinity, naturally occurring Abs also found in autoimmune diseases.

The serum protein β2-GPI, also known as apolipoprotein H, is a member of the complement control protein family (22, 23) but has no known complement regulating function (24). However, β2-GPI is a cofactor for plasminogen activation (25) and an opsonin for the clearance of apoptotic cells by phagocytes (26). By binding to anionic phospholipids, DNA, or other negatively charged molecules (22), β2-GPI is the major antigenic target for anti-phospholipid Abs found in the serum of anti-phospholipid Ab syndrome (APLS) patients (27). Increased anti–β2-GPI Ab titer also correlated with increased risk of ischemic stroke or heart disease in APLS or systemic lupus erythematosus patients, respectively (28, 29). Taken together, these data suggest anti–β2-GPI Abs are involved in ischemic events.

Based on these data, we hypothesized that during reperfusion, serum protein β2-GPI binds ischemic cell membranes and is recognized by naturally occurring Abs, which leads to complement activation and inflammation. Using an in vitro model, our findings demonstrate that anti–β2-GPI Abs recognized β2-GPI bound to the surface of hypoxic endothelial cells. In a mouse model of intestinal ischemia, β2-GPI binding to damaged ischemic intestinal tissue correlated with tissue injury, and reduction of anti–β2-GPI Abs mitigated intestinal damage and inflammation. As reduction of Abs in vivo is difficult, we injected β2-GPI peptides to compete with β2-GPI binding to the tissue. Importantly, injection of peptides specific for the lipid-binding domain of β2-GPI blocked intestinal injury as well as eicosanoid and cytokine production. Administration of peptides containing the phospholipid-binding, lysine-rich region, and adjacent regions were most effective. Taken together, these data suggest that β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.

C57BL/6 and Rag-1−/− (backcrossed to C57BL/6 for 10 generations) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred and maintained under 12-h light/dark cycles at Division of Biology, Kansas State University (Manhattan, KS). All mice were allowed access to food and water ad libitum and maintained under specific pathogen-free conditions. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and was approved by the Institutional Animal Care and Use Committee.

Animals were subjected to IR similar to previously described studies (30). Briefly, ketamine (16 mg/kg)- and xylazine (80 mg/kg)-anesthetized mice were administered buprenorphine (0.06 mg/kg) for pain. After laparotomy and a 30-min equilibration period, a small vascular clamp (Roboz Surgical Instruments, Gaithersburg, MD) was applied to the isolated superior mesenteric artery. After 30 min of ischemia, the clamp was removed, and the intestines were reperfused for up to 2 h. Sham animals sustained the same surgical intervention without superior mesenteric artery occlusion. Mice treated with the various β2-GPI peptides underwent the same procedure with i.v. administration of the peptides (40 μM) 5 min prior to artery occlusion. Peptides 296, 305, and 296Cys to Ser were soluble in normal saline and injected i.v. in 100-μl volumes. Peptides 100 and 181 were dissolved in DMSO prior to diluting 1/100 in normal saline. Additional mice received peptides prior to sham treatment.

In some experiments, 200 μl C57BL/6 sera with or without the reduction of anti–β2-GPI Ab was administered i.v. to Rag-1−/− mice 20 min prior to clamp application. After euthanization, midjejunum 10–20 cm distal to the gastroduodenal junction was removed for analysis. Survival was not significantly different between treatment groups.

To decrease β2-GPI binding to the cells and tissue, peptides from mouse β2-GPI were designed. As domain V is proposed to contain the lipid binding site (31), we designed three overlapping 24–25 aa peptides from domain V (peptides 296, 305, and 322) using the National Center for Biotechnology Information sequence AAB30789 (32). Peptide 296 contains the lysine-rich region previously identified as the critical lipid-binding region (31, 33). The overlapping peptide 305 contains the final three residues of the lysine-rich region and continues into the tail region that is proposed to insert into the lipid membrane. Peptide 322 is contained within the tail region. Additional control peptides 100 and 181 are contained within domains II and III, respectively. Most peptides used in this study were purchased from Invitrogen (Carlsbad, CA), and the manufacturer determined purity (>90%) and sequence. Production of β2-GPI peptide 296Cys to Ser was generated by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, as described in detail previously (34). The peptides were purified by reversed-phase HPLC and characterized by matrix-assisted laser desorption time-of-flight mass spectroscopy. All lyophilized peptides were stored at −20°C until time of use.

Immediately after euthanasia, a 2-cm midjejunum tissue section was immediately fixed in 10% buffered formalin and embedded in paraffin, and 8-μm sections were cut transversely and H&E stained. Mucosal injury was graded on a six-tiered scale adapted from Chiu et al. (35) as described previously (36). Briefly, the average damage score of the intestinal section (75–150 villi) was determined after grading each villus from 0 to 6. Normal villi were assigned a score of 0; villi with tip distortion were assigned a score of 1; a score of 2 was assigned when Guggenheims’ spaces were present; villi with patchy disruption of the epithelial cells were assigned a score of 3; a score of 4 was assigned to villi with exposed but intact lamina propria with epithelial sloughing; a score of 5 was assigned when the lamina propria was exuding; and villi that displayed hemorrhage or were denuded were assigned a score of 6. Photomicrographs were obtained from H&E-stained slides using a ×20, 0.5 Plan Fluor objective on Nikon 80i microscope (Nikon, Melville, NY), and images were acquired at room temperature using a Nikon DS-5M camera with DS-L2 software.

Ex vivo generation of eicosanoids by midjejunal tissue was determined as described previously (30). Immediately after collection, a 2-cm intestinal section was minced, washed, resuspended in 37°C oxygenated Tyrode’s buffer (Sigma-Aldrich, St. Louis, MO), and incubated at 37°C for 20 min, and the supernatants were collected. PGE2 and LTB4 concentrations were determined using enzyme immunoassay kits (Cayman Chemical, Ann Arbor, MI). IL-6 and IL-12 concentrations were determined using a Milliplex MAP immunoassay kit (Millipore, Bedford, MA) and read on a Milliplex Analyzer (Millipore). All eicosanoid and cytokine concentrations were standardized to the total tissue protein content.

After euthanasia, a 2-cm intestinal section was immediately snap frozen in OCT freezing medium, and 8-μm sections were placed on slides for immunohistochemistry. The C3 deposition and F4/80 expression on the tissue sections was detected by staining with a purified rat-anti–mouse C3 (Hycult Biotechnology, Uden, The Netherlands) or F4/80 (eBioscience, San Diego, CA) Ab, followed by a Texas Red-conjugated donkey-anti–rat IgG secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). CD31 (PECAM-1) and CD106 (VCAM-1) were detected by FITC-conjugated rat anti-mouse CD31 or CD106 (BioLegend, San Diego, CA) Abs. Each experiment contained serial sections stained with the appropriate isotype control Abs. All slides were mounted with ProLong Gold (Invitrogen). Images were obtained at room temperature using a Nikon eclipse 80i microscope equipped with a CoolSnap CF camera (Photometrics, Tucson, AZ) and analyzed using Metavue software (Molecular Devices, Sunnyvale, CA). The fluorescence was semiquantitated using ImageJ software (National Institutes of Health, Bethesda, MD) using the fluorescent area fraction after setting threshold for each experiment. The average of the isotype control was subtracted from each photo. The average of 6–10 photos/tissue from three to five animals per treatment group is reported.

Midjejunum (25–30 mm) was longitudinally opened, adhered to a 6-well plate, and incubated at 4°C for 2 h in freshly oxygenated Tyrode’s buffer containing 15 μg/ml FC1 mAb (mouse IgG1, anti–β2-GPI) (37). The cross-linker 3,3′-dithiobis[sulfosuccinimidylpropionate] (Pierce, Rockford, IL) was added to the Ab solution at a final concentration of 1.5 mM and incubated at 4°C for an additional 2 h. The reaction was stopped with Tris (pH 7.5), and the washed mucosa was lysed in 1 ml MES/Brij58 (145 mM NaCl, 0.2 mM EDTA, 0.5% w/v Brij58 [Sigma-Aldrich], and 25 mM MES [Sigma-Aldrich] [pH 6.5]). The lysate was incubated for 30 min on ice with periodic vortexing and clarified by centrifuging at 5000 × g for 10 min at 4°C. Ab was immunoprecipitated overnight at 4°C with protein G beads (Pierce), and the samples were boiled in nonreducing Laemmli sample buffer prior to SDS-PAGE (10%) and Western blot analysis. Human β2-GPI (Fitzgerald) was used as a positive control. The blots were probed with anti–β2-GPI Ab MAB1066 (Chemicon International, Temecula, CA), followed by goat anti-mouse IgG HRP conjugate (Pierce). Protein was visualized using SuperSignal Detection Kit (Pierce) according to the manufacturer’s protocol.

Hypoxia was conducted similar to previous studies with the following modifications (38). Hypoxic MS-1 endothelial cells (American Type Culture Collection, Manassas, VA) received degassed, serum-free DMEM and were placed in a hypoxia chamber containing 94% nitrogen and 5% CO2. Normoxic cells received DMEM supplemented with 10% heat-inactivated sera from Rag-1−/− mice in 8% CO2. After 4 h at 37°C, all cells received fresh medium containing 10% heat-inactivated Rag-1−/− sera and were incubated in normoxic conditions for 1 h at 37°C. Additional peptide studies were performed by addition of peptides (40 μM final concentration) during the hypoxic period. Cells were methanol fixed and stained with the anti–β2-GPI mAb (Millipore), followed by an anti-mouse IgG Ab to determine β2-GPI binding. Anti–β2-GPI binding was determined by allowing anti–β2-GPI mAb (Millipore) to bind during the 1-h normoxic period. The cells were then stained with anti-mouse IgG Abs (Jackson ImmunoResearch Laboratories) as described previously (19). The fluorescence was determined in a blinded manner using a Nikon 80i fluorescent microscope with a ×40 Plan Fluor objective, and images were acquired using a CoolSnap Cf camera (Photometrics) and MetaVue Imaging software (Molecular Devices).

Anti–β2-GPI concentrations were determined based on optimal conditions described previously (39, 40). The specific isotypes of anti–β2-GPI Abs were determined after binding serum in duplicate to coated and blocked wells and incubating for 1 h. After washing, the appropriate biotinylated anti-mouse Ig isotype Abs were added to each well for 1 h at room temperature while gently shaking. After incubation with avidin peroxidase (Sigma-Aldrich), the plate was developed using tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

An ELISA plate was coated for 2 h at room temperature with 2 μg β2-GPI (Fitzgerald, Concord, MA) in PBS. After blocking for 2 h with 100 μl 3% BSA in PBS, 50 μl heat-inactivated C57BL/6 sera was added to half of the coated wells for 2 h at room temperature. The sera were then transferred to the remaining coated set of wells and incubated for an additional 2 h at room temperature. The reduced serum was removed, pooled, and then administered as described above. The reduction procedure removed ∼50% of the anti–β2-GPI Abs as determined by ELISA.

Data are presented as mean ± SEM and significance (p < 0.05) determined by one-way ANOVA with Newman-Keuls post hoc analysis (GraphPad/Instat Software, San Diego, CA).

C57BL/6 mice were subjected to ischemic injury, followed by 5, 10, or 15 min of reperfusion. Compared with pooled sham-treated animals, significant midjejunal mucosal injury was observed after 15 min of reperfusion and increased up to 2 h postreperfusion (Fig. 1A). In contrast, Rag-1−/− mice did not sustain intestinal damage at any time point analyzed (Fig. 1A). When analyzed for β2-GPI, sera from both C57BL/6 and Rag-1−/− mice contained similar concentrations of β2-GPI (data not shown). As previously shown, anti–β2-GPI binds ischemic-damaged tissue within 2 h following reperfusion (19); however, we were interested in determining the early kinetics of β2-GPI binding to tissue following ischemia. To examine the kinetics, tissue harvested after 5, 10, or 15 min of reperfusion was probed with the anti–β2-GPI mAb FC1. The Ab/Ag complexes were cross-linked to the surface of the villi prior to immunoprecipitation and Western blotting. Immunoprecipitation indicated the presence of β2-GPI bound to the cell surface at 15 min postreperfusion but not at the earlier time points (Fig. 1B). The apparent molecular mass difference between human and mouse is likely due to differential glycosylation and different isoelectric points (41). In addition, the presence of detectable levels of tissue-bound β2-GPI correlates positively with the earliest time point when significant damage was observed (Fig. 1A).

FIGURE 1.

The presence of β2-GPI and anti–β2-GPI correlates with IR-induced intestinal damage. A, Midjejunal sections collected from C57BL/6 or Rag-1−/− mice at 5, 10, 15, and 120 min after reperfusion or from sham-treated mice were scored for intestinal injury (75–150 villi/animal with 3–10 animals/treatment and each treatment was performed on at least two separate days). B, β2-GPI was immunoprecipitated with FC1 from tissue sections collected at 5, 10, and 15 min postreperfusion or from sham-treated mice and subjected to Western blot analysis. Human β2-GPI (50 kDa) was run as a control for mouse β2-GPI (54 kDa). The blot is representative of four experiments. C, Serum concentrations of anti–β2-GPI Abs in C57BL/6, CR2−/−, or Rag-1−/− mice as determined by ELISA. Isotypes of the specific Abs bound to β2-GPI were determined using specific rat anti-mouse isotyping Abs. Each bar represents the mean ± SEM of three independent experiments. *p ≤ 0.05 compared with sham.

FIGURE 1.

The presence of β2-GPI and anti–β2-GPI correlates with IR-induced intestinal damage. A, Midjejunal sections collected from C57BL/6 or Rag-1−/− mice at 5, 10, 15, and 120 min after reperfusion or from sham-treated mice were scored for intestinal injury (75–150 villi/animal with 3–10 animals/treatment and each treatment was performed on at least two separate days). B, β2-GPI was immunoprecipitated with FC1 from tissue sections collected at 5, 10, and 15 min postreperfusion or from sham-treated mice and subjected to Western blot analysis. Human β2-GPI (50 kDa) was run as a control for mouse β2-GPI (54 kDa). The blot is representative of four experiments. C, Serum concentrations of anti–β2-GPI Abs in C57BL/6, CR2−/−, or Rag-1−/− mice as determined by ELISA. Isotypes of the specific Abs bound to β2-GPI were determined using specific rat anti-mouse isotyping Abs. Each bar represents the mean ± SEM of three independent experiments. *p ≤ 0.05 compared with sham.

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MS-1 endothelial cells were subjected to hypoxia or normoxia to validate β2-GPI binding in vitro. Addition of sera from Rag-1−/− mice during the subsequent normoxia (reperfusion) stage provided the β2-GPI. After hypoxic but not normoxic treatment, cells were positive for β2-GPI (Fig. 2A–C). The addition of anti–β2-GPI mAb to the cells during reperfusion, again, showed that only hypoxic- but not normoxic-treated cells stained positively for anti–β2-GPI Abs (Fig. 2D–F). Similar to the in vivo results, in vitro studies showed that hypoxia-induced cellular changes facilitated the binding of both β2-GPI and anti–β2-GPI Abs to the surface of ischemic cells.

FIGURE 2.

β2-GPI and anti–β2-GPI Abs bind to MS-1 cells following hypoxia. Cells were subjected to 4 h of normoxia in media containing 10% heat-inactivated Rag-1−/− sera (A, D) or hypoxia under serum-free conditions (B, C, E, F), followed by 1 h of normoxia in media containing 10% Rag-1−/− serum in the absence (A–C) or presence (D, E) of anti–β2-GPI or isotype control (F) Ab. The cells were fixed with methanol, probed with a primary anti–β2-GPI Ab (A, B) or isotype control Ab (C), and then stained with an anti-mouse secondary or stained with secondary Ab only (red; D–F). Slides were mounted with DAPI (blue) to identify the nuclei. Each photomicrograph is representative of three experiments with four to six photomicrographs per treatment group in each experiment. Scale bar, 40 μm. Original magnification ×400.

FIGURE 2.

β2-GPI and anti–β2-GPI Abs bind to MS-1 cells following hypoxia. Cells were subjected to 4 h of normoxia in media containing 10% heat-inactivated Rag-1−/− sera (A, D) or hypoxia under serum-free conditions (B, C, E, F), followed by 1 h of normoxia in media containing 10% Rag-1−/− serum in the absence (A–C) or presence (D, E) of anti–β2-GPI or isotype control (F) Ab. The cells were fixed with methanol, probed with a primary anti–β2-GPI Ab (A, B) or isotype control Ab (C), and then stained with an anti-mouse secondary or stained with secondary Ab only (red; D–F). Slides were mounted with DAPI (blue) to identify the nuclei. Each photomicrograph is representative of three experiments with four to six photomicrographs per treatment group in each experiment. Scale bar, 40 μm. Original magnification ×400.

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To further understand the role of anti–β2-GPI Abs, we examined the presence of these Abs in wild-type and Rag-1−/− mice. As shown in Fig. 1C, we determined that ∼60 ng/ml anti–β2-GPI Ab (total Ig) is present in C57BL/6 serum, but as expected, Rag-1−/− serum contained no detectable Abs (Fig. 1C). Interestingly, serum from IR-resistant, Cr2−/− mice contains significantly less anti–β2-GPI Ab (Fig. 1C). These results indicate that naturally occurring Abs against β2-GPI exist in wild-type mice. The anti–β2-GPI Ab concentration in wild-type sera was determined to be primarily of the IgM and IgG2b isotypes with minor amounts of IgG3 and IgG1 isotypes (Fig. 1C). The presence of IgG2b, IgG3, and IgM isotypes is consistent with complement activation. Therefore, β2-GPI represents a significant target for forming Ab/Ag complexes capable of facilitating complement-mediated tissue damage.

The effects of anti–β2-GPI Ab reduction on IR-mediated damage were assessed by subjecting Rag-1−/− mice to IR after reconstitution with wild-type serum after two rounds of adsorption to bound β2-GPI. When Rag-1−/− mice were reconstituted with nonadsorbed C57BL/6 serum, significant damage was observed after 2 h reperfusion (Fig. 3A) similar to previous results for C57BL/6 mice (Fig. 1A). However, when mice were administered anti–β2-GPI-reduced serum, no damage was observed similar to that seen in Rag-1−/− IR control mice (Fig. 3A, 3D–F). Moreover, the effects of anti–β2-GPI reduction extended to dramatically decreasing the intestinal inflammatory response. The IR-induced increase in PGE2 and LTB4 production was abrogated with the Ab-reduced serum to concentrations similar to Rag-1−/− IR controls (Fig. 3B, 3C). These data suggest that inhibition of anti–β2-GPI Abs may provide a therapeutic target for IR-induced tissue damage.

FIGURE 3.

Reduction of anti–β2-GPI Ab attenuates tissue injury and inflammation. A, Midjejunal sections were scored (75–150 villi/animal) from Rag-1−/− mice with or without injection of C57BL/6 sera or anti–β2-GPI Ab-reduced C57BL/6 serum prior to sham or IR treatment. PGE2 (B) or LTB4 (C) production was measured in Rag-1−/− mice injected with C57BL/6 sera or anti–β2-GPI Ab-reduced C57BL/6 serum prior to sham or IR treatment. Values are represented as picograms per milligram of intestinal protein. *p ≤ 0.05 compared with sham; ϕp ≤ 0.05 compared with animals receiving nonreduced sera. Each animal is represented by an individual point with the bar representing the average. Each treatment was performed on at least two separate days. D–I, Representative intestinal sections H&E stained (D–F) or stained for C3 deposition (G–I) from IR-treated Rag-1−/− mice (D, G), IR-treated Rag-1−/− mice receiving C57BL/6 serum (E, H), or IR-treated Rag-1−/− mice receiving anti–β2-GPI Ab-reduced C57BL/6 serum are shown (F, I). Microphotographs are representative of three to four animals stained in at least three independent experiments. H&E scale bar, 50 μm; immunohistochemistry scale bar, 40 μm. Original magnification ×200.

FIGURE 3.

Reduction of anti–β2-GPI Ab attenuates tissue injury and inflammation. A, Midjejunal sections were scored (75–150 villi/animal) from Rag-1−/− mice with or without injection of C57BL/6 sera or anti–β2-GPI Ab-reduced C57BL/6 serum prior to sham or IR treatment. PGE2 (B) or LTB4 (C) production was measured in Rag-1−/− mice injected with C57BL/6 sera or anti–β2-GPI Ab-reduced C57BL/6 serum prior to sham or IR treatment. Values are represented as picograms per milligram of intestinal protein. *p ≤ 0.05 compared with sham; ϕp ≤ 0.05 compared with animals receiving nonreduced sera. Each animal is represented by an individual point with the bar representing the average. Each treatment was performed on at least two separate days. D–I, Representative intestinal sections H&E stained (D–F) or stained for C3 deposition (G–I) from IR-treated Rag-1−/− mice (D, G), IR-treated Rag-1−/− mice receiving C57BL/6 serum (E, H), or IR-treated Rag-1−/− mice receiving anti–β2-GPI Ab-reduced C57BL/6 serum are shown (F, I). Microphotographs are representative of three to four animals stained in at least three independent experiments. H&E scale bar, 50 μm; immunohistochemistry scale bar, 40 μm. Original magnification ×200.

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We hypothesized that if inhibition of Ab binding to β2-GPI on the tissue attenuated injury, then peptides that block the lipid-binding domain of β2-GPI may inhibit β2-GPI binding and attenuate IR-induced intestinal damage and inflammation as well. Peptides were designed to match sequences from multiple domains of mouse β2-GPI, including domains II and III and lipid-binding domain V as indicated in Fig. 4 and Table I. Within domain V, three overlapping peptides were created, 296, 305, and 322 (Fig. 4, Table I), to cover the lysine-rich domain (296) and the tail, which is inserted into the lipid membrane (322) with peptide 305 spanning the intervening region. Additional peptides from domains II and III were used as controls. Initial in vitro studies tested the ability of the peptides to block β2-GPI binding to hypoxic endothelial cells. As indicated in Fig. 5, after 4 h hypoxia, anti–β2-GPI mAb bound to untreated MS-1 cells significantly more than isotype control mAb. β2-GPI peptides 100 or 322 did not inhibit Ab binding to the hypoxic endothelial cell line. In contrast, anti–β2-GPI mAb did not bind to hypoxic MS-1 cells, which were pretreated with peptides 296 or 305. Taken together, these data indicated that the overlapping peptides 296 and 305 were capable of preventing β2-GPI from binding to hypoxic endothelial cells. As these two peptides contain three cysteines and may bind nonspecifically, the cysteines of peptide 296 were changed to serine and used in the in vitro hypoxia assay. Similar to peptide 296, the cysteine-serine substituted peptide also attenuated β2-GPI binding to the hypoxic cells.

FIGURE 4.

Location of overlapping β2-GPI peptides. A, Ribbon diagram of human β2-GPI with peptide locations identified by color, peptide 100 (gold), peptide 181 (green), peptide 296 (red), peptide 322 (dark blue), and overlapping peptide 305 (light blue). Inset is magnification of domain V. B, Cartoon of β2-GPI binding to lipid membrane with peptide locations indicated. C, Sequence identification of overlapping regions of peptides 296, 305, and 322. Red indicates regions of overlap. Peptides were designed based on the published sequences (32) to mimic the lipid binding domain and tail inserted into the lipid bilayer.

FIGURE 4.

Location of overlapping β2-GPI peptides. A, Ribbon diagram of human β2-GPI with peptide locations identified by color, peptide 100 (gold), peptide 181 (green), peptide 296 (red), peptide 322 (dark blue), and overlapping peptide 305 (light blue). Inset is magnification of domain V. B, Cartoon of β2-GPI binding to lipid membrane with peptide locations indicated. C, Sequence identification of overlapping regions of peptides 296, 305, and 322. Red indicates regions of overlap. Peptides were designed based on the published sequences (32) to mimic the lipid binding domain and tail inserted into the lipid bilayer.

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Table I.
β2-GPI peptide sequences
Peptide NameSequenceaResidue NumbersMolecular Mass (Da)
100 H-KNISFACNPGFFLNG-NH2 105–118 1627 
181 H-GNDTVMCTEQQN-NH2 182–193 1338 
296 H-IHFYCKNKEKKCSYTVEAHCRDGTI-OH 296–320 2974 
296 Cys-Ser H-IHFYSKNKEKKSSYTVEAHSRDGTI-OH 296–320 2925 
305 H-KKCSYTVEAHCRDGTIEIPSCFKEHS-OH 305–330 2969 
322 H-IPSCFKEHSSLAFWKTDASELTPC-NH2 322–345 2629 
Peptide NameSequenceaResidue NumbersMolecular Mass (Da)
100 H-KNISFACNPGFFLNG-NH2 105–118 1627 
181 H-GNDTVMCTEQQN-NH2 182–193 1338 
296 H-IHFYCKNKEKKCSYTVEAHCRDGTI-OH 296–320 2974 
296 Cys-Ser H-IHFYSKNKEKKSSYTVEAHSRDGTI-OH 296–320 2925 
305 H-KKCSYTVEAHCRDGTIEIPSCFKEHS-OH 305–330 2969 
322 H-IPSCFKEHSSLAFWKTDASELTPC-NH2 322–345 2629 
a

Amino acid sequence based on National Center for Biotechnology Information sequence AAB30789 as described in 1Materials and Methods.

FIGURE 5.

β2-GPI peptides inhibit anti–β2-GPI staining of hypoxic MS-1 cells. Cells were subjected to 4 h of hypoxia under serum-free conditions without (A) or with (B–G) β2-GPI peptides prior to 1 h normoxia in media containing 10% heat-inactivated Rag-1−/− sera. The cells were fixed with methanol, probed with a primary anti–β2-GPI Ab (A–F) or isotype control Ab (G), followed by a Texas Red-labeled, anti-mouse secondary Ab. Slides were mounted with DAPI (blue) to identify the nuclei. Each photomicrograph is representative of three experiments with four to six photomicrographs per treatment in each experiment. Scale bar, 40μm. Original magnification ×400.

FIGURE 5.

β2-GPI peptides inhibit anti–β2-GPI staining of hypoxic MS-1 cells. Cells were subjected to 4 h of hypoxia under serum-free conditions without (A) or with (B–G) β2-GPI peptides prior to 1 h normoxia in media containing 10% heat-inactivated Rag-1−/− sera. The cells were fixed with methanol, probed with a primary anti–β2-GPI Ab (A–F) or isotype control Ab (G), followed by a Texas Red-labeled, anti-mouse secondary Ab. Slides were mounted with DAPI (blue) to identify the nuclei. Each photomicrograph is representative of three experiments with four to six photomicrographs per treatment in each experiment. Scale bar, 40μm. Original magnification ×400.

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The in vitro hypoxia studies suggested that peptides 296 and 305 may attenuate IR-induced tissue damage. To test this hypothesis, peptides were infused into C57BL/6 mice 5 min prior to intestinal IR, and mucosal damage and inflammation were evaluated. Similar to in vitro results, mice that received peptides 296, 305, or 296 Cys to Ser sustained attenuated mucosal damage in response to IR (Fig. 6). In contrast, peptides 100, 181, and 322 sustained IR-induced intestinal damage similar to untreated mice (Fig. 6). Thus, peptide inhibition of β2-GPI attenuates IR-induced intestinal damage.

FIGURE 6.

β2-GPI peptides attenuate IR-induced mucosal damage in wild-type mice. A, Midjejunal sections were scored (75–150 villi/animal) from C57BL/6 mice with or without injection of β2-GPI peptides prior to sham or IR treatment. B–J, Representative intestinal sections H&E stained from C57BL/6 sham-treated mice without (B) or with peptide (C), IR-treated C57BL/6 mice in the absence of peptide (D) or receiving β2-GPI peptide 100 (E), peptide 181 (F) peptide 296 (G), peptide 206Cys to Ser (H), peptide 305 (I), and peptide 322 (J). Microphotographs are representative of three to four animals stained in at least three independent experiments. Scale bar, 50 μm. Original magnification ×200. Each bar is representative of three to four animals, and each treatment was performed on at least two separate days. *p ≤ 0.05 compared with sham + peptide; Φp ≤ 0.05 compared with IR treatment animals not receiving peptides.

FIGURE 6.

β2-GPI peptides attenuate IR-induced mucosal damage in wild-type mice. A, Midjejunal sections were scored (75–150 villi/animal) from C57BL/6 mice with or without injection of β2-GPI peptides prior to sham or IR treatment. B–J, Representative intestinal sections H&E stained from C57BL/6 sham-treated mice without (B) or with peptide (C), IR-treated C57BL/6 mice in the absence of peptide (D) or receiving β2-GPI peptide 100 (E), peptide 181 (F) peptide 296 (G), peptide 206Cys to Ser (H), peptide 305 (I), and peptide 322 (J). Microphotographs are representative of three to four animals stained in at least three independent experiments. Scale bar, 50 μm. Original magnification ×200. Each bar is representative of three to four animals, and each treatment was performed on at least two separate days. *p ≤ 0.05 compared with sham + peptide; Φp ≤ 0.05 compared with IR treatment animals not receiving peptides.

Close modal

To examine the multiple pathways of inflammation involved in IR-induced damage, intestinal tissues from the peptide treated mice were examined for complement deposition, adhesion molecule expression, and the macrophage marker F4/80. As expected, IR induced C3 deposition on the intestines of C57BL/6 mice in response to IR but not sham treatment (Fig. 7A). Similar to injury results, peptides 100 and 322 did not significantly inhibit C3 deposition (Fig. 7A, 7D). In addition, infusion of peptides 296 and 296Cys to Ser prior to IR significantly decreased C3 deposition (Fig. 7A, 7D). Interestingly, peptide 305 was not significantly different from either sham or IR treatment (Fig. 7D). Similarly, the expression of adhesion molecules CD31and VCAM was inhibited after treatment with peptides 296, 305, and 296Cys to Ser but not after treatment with peptide 322 (Fig. 7B, 7D) (data not shown). However, peptide 100 was significantly different from both sham- and IR-treated mice. Expression of the mature macrophage marker increased in response to IR with or without peptide 322 (Fig. 7C, 7D). Treatment with peptides 100, 296, 305, and 296 Cys to Ser reduced macrophage infiltration to sham levels after IR treatment (Fig. 7C, 7D).

FIGURE 7.

β2-GPI peptides attenuate IR-induced complement deposition, adhesion molecule expression, and macrophage infiltration. Representative intestinal sections stained for C3 (A), CD31 (B), or F4/80 (C) from sham-treated C57BL/6 mice, IR-treated C57BL/6 in the absence or presence of β2-GPI peptides as indicated. Microphotographs are representative of three to four animals stained in at least three independent experiments. Scale bar, 40 μm; original magnification ×200 (A, B). Scale bar, 20 μm; original magnification ×400 (C). D, Fluorescence was semiquantitated using ImageJ software (National Institutes of Health) and is reported as fluorescent fraction of specific Abs after subtraction of the fluorescent fraction of isotype control Abs. Each bar is representative of three to five animals with 6–10 photos analyzed/animal. *p ≤ 0.05 compared with sham + peptide; Φp ≤ 0.05 compared with IR treatment animals not receiving peptides.

FIGURE 7.

β2-GPI peptides attenuate IR-induced complement deposition, adhesion molecule expression, and macrophage infiltration. Representative intestinal sections stained for C3 (A), CD31 (B), or F4/80 (C) from sham-treated C57BL/6 mice, IR-treated C57BL/6 in the absence or presence of β2-GPI peptides as indicated. Microphotographs are representative of three to four animals stained in at least three independent experiments. Scale bar, 40 μm; original magnification ×200 (A, B). Scale bar, 20 μm; original magnification ×400 (C). D, Fluorescence was semiquantitated using ImageJ software (National Institutes of Health) and is reported as fluorescent fraction of specific Abs after subtraction of the fluorescent fraction of isotype control Abs. Each bar is representative of three to five animals with 6–10 photos analyzed/animal. *p ≤ 0.05 compared with sham + peptide; Φp ≤ 0.05 compared with IR treatment animals not receiving peptides.

Close modal

The proinflammatory cytokines IL-12 and IL-6 and eicosanoids LTB4 and PGE2 increase rapidly in response IR (42). Therefore, we examined the ability of peptides 296, 305, and 296 Cys to Ser to attenuate production of these inflammatory molecules. Similar to previous results, IR induced IL-12 and IL-6 production, which was attenuated by protective peptides 296, 305, and 296Cys to Ser (Fig. 8A, 8B). Interestingly, peptide 100 also attenuated IL-6 production (Fig. 8B). However, peptide 322 did not inhibit IR-induced cytokine production (Fig. 8A, 8B). Thus, β2-GPI binding occurs prior to IR-induced, proinflammatory cytokine production.

FIGURE 8.

β2-GPI peptides attenuate IR-induced proinflammatory cytokine and eicosanoid production. IL-12 (A), IL-6 (B), PGE2 (C), or LTB4 (D) production was measured in C57BL/6 mice with or without injection of β2-GPI peptides prior to sham or IR treatment. Values are represented as pg/mg of intestinal protein. Each bar is representative of three to four animals, and each treatment was performed on at least two separate days. *p ≤ 0.05 compared with sham; Φp ≤ 0.05 compared with animals not receiving peptide.

FIGURE 8.

β2-GPI peptides attenuate IR-induced proinflammatory cytokine and eicosanoid production. IL-12 (A), IL-6 (B), PGE2 (C), or LTB4 (D) production was measured in C57BL/6 mice with or without injection of β2-GPI peptides prior to sham or IR treatment. Values are represented as pg/mg of intestinal protein. Each bar is representative of three to four animals, and each treatment was performed on at least two separate days. *p ≤ 0.05 compared with sham; Φp ≤ 0.05 compared with animals not receiving peptide.

Close modal

Previous studies demonstrated that IR also induces eicosanoid production within 2 h postischemia (36). To determine whether β2-GPI initiation of intestinal damage contributes to eicosanoid production, intestinal LTB4 and PGE2 production within the intestine was examined in mice subjected to sham or IR in the presence or absence of the various β2-GPI peptides. As demonstrated in injury, peptides 296 and 296Cys to Ser attenuated IR-induced production of both eicosanoids, whereas mice treated with peptides 100 and 322 sustained inflammation similar to untreated mice (Fig. 8C, 8D). Despite the ability to attenuate IR-induced intestinal damage, peptide 305 did not attenuate intestinal eicosanoid production (Fig. 8C, 8D). Taken together, these data indicate that β2-GPI has a role in IR-induced tissue damage and initiation of inflammation (Table II). Further studies are required to determine whether administration of β2-GPI peptides at later time points attenuate injury and as such may provide clinically relevant therapeutics for a condition with a high mortality rate.

Table II.
Summary of IR-induced injury and inflammation C57BL/6 (B6) mice with or without peptide treatment
B6 IRaB6 + β2-100B6 + β2-296B6 + β2-296c-sB6 + β2-305B6 + β2-322
Injuryb − − − 
C3 deposition − − − 
CD31 deposition − − 
F4/80 deposition − − − 
IL-12p40 induction +/− − − − 
IL-6 induction +/− − − − 
PGE2 production − − +/− 
LTB4 production − − 
B6 IRaB6 + β2-100B6 + β2-296B6 + β2-296c-sB6 + β2-305B6 + β2-322
Injuryb − − − 
C3 deposition − − − 
CD31 deposition − − 
F4/80 deposition − − − 
IL-12p40 induction +/− − − − 
IL-6 induction +/− − − − 
PGE2 production − − +/− 
LTB4 production − − 
a

B6 mice subjected to IR with or without peptide treatment.

b

Measure of injury or inflammation.

+, Significant difference from sham-treated mice; −, not significantly different from B6 sham-treated mice; +/−, no significant difference from B6 mice subjected to either sham or IR.

Ab-dependent complement activation is required for initiation of IR-induced tissue damage (1, 10). Although Abs against multiple intracellular Ags have been implicated in initiating damage in response to IR, the identification of an extracellular Ag remained unclear (14, 20). We hypothesized that a serum protein, β2-GPI, binding to ischemic tissues is likely responsible for initiating the complement cascade. Both peptide inhibition of β2-GPI activity in wild-type mice (Table II) and infusion of wild-type serum containing reduced levels of anti–β2-GPI Abs into Rag-1−/− mice prevented IR-induced intestinal damage and inflammation. Thus, our results demonstrate that natural Abs targeting β2-GPI play a critical role in initiating Ab/Ag complexes required for subsequent complement activation in response to IR. In addition, these data suggest that binding of β2-GPI to ischemic cells is critical for IR-induced damage and inflammation. Although many studies have associated anti-phospholipid Abs with autoimmunity and acute graft rejection (reviewed in Ref. 43), β2-GPI and anti–β2-GPI Abs also mediate reperfusion-induced organ damage.

We showed that β2-GPI binding to the tissue within 15 min of reperfusion correlates with initiation of tissue damage. The binding to injured tissue also correlates with previously determined IR-induced lipid changes (44). Domain V of β2-GPI is responsible for binding negatively charged substrates such as membranes containing phosphatidylserine and/or cardiolipin (4547), which are significantly increased in response to IR (44). Thus, peptides 100 or 181 from domains II and III, respectively, would not be expected to reduce damage in the IR model of tissue damage. In the analysis of domain V, mutagenesis studies suggested that the Lys286 in the CKNKEKKC sequence is critical for in vitro binding of β2-GPI to cardiolipin (48). In addition, mutation of Lys284, Lys286, and Lys287 abolished anti–β2-GPI binding as detected by ELISA (48). Peptide 296 contains this lysine-rich sequence with three cysteine residues. Correlating with previous findings that the Cys residues were not as critical as lysine residues, peptide 296Cys to Ser also inhibited IR-induced damage and inflammation. A recent in vivo study found that a related lysine-rich sequence from CMV (KEKRKKK) inhibited Ab-induced thrombosis by competing with β2-GPI (49). It is likely that a similar mechanism may be occurring in the peptide-treated mice. However, protective peptide 305 contains only the last three amino acids in the CKNKEKKC sequence, suggesting that additional residues are capable of binding to cells as well. Interestingly, although peptide 305 prevented injury and macrophage infiltration, the peptide did not prevent eicosanoid production. These data suggest that distinct residues may be critical for the inflammatory response and intestinal damage or that a critical threshold must be reached for complete injury.

Reperfusion is accompanied by the production of inflammatory mediators and immune cell infiltration (50), which are believed to be responsible for the subsequent systemic pathologies (1). IR-induced lipid changes result in increased arachidonic acid and subsequent production of the inflammatory mediators LTB4 and PGE2 (44), which may contribute to cellular infiltration. Interestingly, anti–β2-GPI Abs binding to β2-GPI induced cellular infiltration and eicosanoid generation. Importantly, all these inflammatory mediators and the IR-induced proinflammatory cytokines were blocked by peptides 296 and 296Cys to Ser, whereas peptide 305 inhibited IL-12 and IL-6 production but not eicosanoid production (summarized in Table II). Taken together, these data suggest that the inflammatory response is controlled by a larger sequence than the CKNKEKKC sequence of the lipid-binding domain. Activation of complement also initiates immune cell infiltration and activation as treatment with C5a receptor antagonists attenuated neutrophil infiltration (4, 6, 7, 50). Despite containing four complement regulatory domains, β2-GPI exhibits no known complement regulating function (24). However, Ab reduction or treatment with peptides 296, 305, and 296Cys to Ser attenuated complement activation, suggesting that Ab recognition of the serum protein β2-GPI initiates complement activation. As the inflammatory responses are also induced by LPS, TLR pathways involvement is also possible.

The mechanism of β2-GPI binding to cells is not fully understood. Previous studies demonstrated that mice lacking specific immune regulatory proteins such as TLR4 also render mice resistant to IR-induced damage (30). Despite having the proper Ab repertoire, TLR4lpsd mice remain resistant to intestinal IR-induced damage (30). One possible explanation is that anti–β2-GPI Abs recognize β2-GPI in conjunction with TLR4 (51, 52), resulting in signaling through TLR4. This hypothesis is supported by the fact that anti–β2-GPI Abs induce phosphorylation, NF-κB activation, and TNF production by monocytes (53). Another possibility is presented by recent evidence illustrating that β2-GPI binds TLR2 on endothelial cells (54). Correlating with these data, we demonstrated that peptides 296 and 296Cys to Ser inhibit IR-induced IL-12 and IL-6 as well as upregulation of adhesion molecules and subsequent increases in cellular infiltration. Thus, the lack of TLR expression may prevent intestinal damage by interfering with Ab recognition of β2-GPI. In contrast, other studies indicate that β2-GPI-Ab complexes interact with other proteins including Ro60 on apoptotic cells (55) or annexin II (56). Binding to either of these proteins would suggest the Ag/Ab complexes resulted in monocyte stimulation, because limited evidence exists for a transmembrane domain in either Ro60 or annexin II. Although the nature of the interaction remains unclear, binding of the serum protein β2-GPI appears to initiate the subsequent inflammatory response during IR.

Similar to the TLR4-deficient mice, Cr2−/− mice are also resistant to IR-induced tissue damage. Despite having normal serum levels of Abs, Cr2−/− mice do not produce the necessary Ab repertoire required for IR-mediated tissue damage (19, 36, 57). The lack of anti–β2-GPI Abs in the Cr2−/− mice correlates with initial studies indicating that infusion of an anti–β2-GPI mAb was sufficient to restore injury (19). Although the exact role that CR2 plays in generating β2-GPI–reactive Abs is unclear, CR2 is associated with the B cell IgR and therefore may influence the selection of β2-GPI–reactive B cells (58). Thus, the interactions of TLR4 and CR2 with β2-GPI and/or anti–β2-GPI Abs remain unclear and require additional investigation.

It has been proposed that binding of β2-GPI to the cell membrane exposes cryptic epitope(s) recognized by natural Abs (59). Natural Ab recognition of β2-GPI is a characteristic of APLS and results in tissue damage and fetal loss (6062). When compared with IR in normal patients, APLS patients have significantly higher anti–β2-GPI Ab titers, and the Abs exhibit greater affinity for the target Ag, which is suggested to result in damage (63). By recognizing stressed or damaged tissue, β2-GPI recognition by anti–β2-GPI Abs appears to lead to IR-induced pathology. Although CR2 may play a role in generating Abs against β2-GPI, it is unclear why, under normal immunological functioning, β2-GPI elicits autoantibodies. The generation of anti–β2-GPI Abs may be for removal of apoptotic cells by phagocytes. This hypothesis suggests that β2-GPI binds to ischemic tissue because the membrane changes are similar to early apoptotic cells and that the process will facilitate clearance of the damaged cells (26, 64). When significantly lower concentrations of anti–β2-GPI Abs exist, such as in Cr2−/− mice or in the reduced serum, the hypoxic cells are not targeted, and complement activation is significantly reduced. Similarly, transfer of Cr2−/− serum or Abs to Rag-1−/− mice did not restore IR-induced intestinal damage (36). Although the exact nature of the alterations occurring in IR or apoptotic tissues is not fully characterized, ischemia exposes changes in the lipid and/or protein composition of the membrane allowing β2-GPI binding and subsequent natural Ab recognition during reperfusion.

Our previous studies indicated that IR-induced damage in Rag-1−/− mice required a combination of two IgG mAbs recognizing β2-GPI and negatively charged phospholipids (19). Importantly, neither mAb alone was sufficient to induce damage. These data suggest that IR-induced damage requires a complex of Abs recognizing multiple Ags, including β2-GPI bound to phospholipids. Based on these results, prevention of either the phospholipid changes or β2-GPI binding would attenuate injury. Recently, we demonstrated that IR-induced lipid changes occur in both Rag-1−/− and C57BL/6 wild-type mice within 15 min postreperfusion (44). As lipid mobility is critical to cellular signaling, blocking the lipid changes may produce significant side effects. In contrast, peptide inhibition of either β2-GPI binding to the lipids or Ab binding to β2-GPI would prevent binding by one mAb and subsequently prevent intestinal damage. In addition, as a natural serum protein, the expected side effects may be significantly lower than lipid blockade.

Previous studies indicated that IR-induced damage is due to natural Abs with reactivity to nonmuscle myosin, glycogen phosphorylase, or annexin IV. However, attenuated damage following peptide inhibition of β2-GPI binding suggests that these additional target Ags may be exposed after β2-GPI binding. It is possible that β2-GPI binding induces a signal, which leads to either apoptosis with annexin IV expression or necrosis and nonmuscle myosin exposure. As specific β2-GPI peptides reduced IR-induced tissue damage to sham levels, β2-GPI appears to be a critical therapeutic target for mesenteric IR. In addition, reperfusion-induced tissue damage in response to myocardial infarction, stroke, and transplantation appears to use similar mechanisms (42, 65). Thus, understanding the exact role of β2-GPI itself or the natural Abs recognizing β2-GPI in mediating tissue damage may lead to effective strategies of preventing reperfusion injury in multiple organs.

We thank Andrew Fritze for excellent technical assistance and Dr. Maurizio Tomasi for insightful discussions.

Disclosures The authors have no financial conflicts of interest.

This work was supported by National Institutes of Health Grants AI061691, P20 RR017686, and RR016475 from the Institutional Development Award Program of the National Center for Research Resources and Kansas State University.

Abbreviations used in this paper:

APLS

anti-phospholipid Ab syndrome

β2-GPI

β2-glycoprotein I

IR

ischemia/reperfusion

LTB4

leukotriene B4.

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