Abstract
Objective
Quantify the concentration of α2-macroglobulin (A2M), immunomodulatory cytokines, and TGF-β1 factors in 4 commercially available autologous blood-based products including conditioned A2M (CA2M; Alpha2EQ; Astaria Global), autologous protein solution (APS; Pro-Stride; Zoetis), platelet-rich plasma (PRP; Restigen; Zoetis), and autologous conditioned plasma (ACP; Arthrex ACP). We hypothesized that CA2M would have higher concentrations of A2M and lower concentrations of cytokines and growth factors compared to APS, PRP, and ACP.
Methods
Blood was obtained from 6 healthy, adult horses and processed into CA2M, APS, PRP, and ACP. The concentration of immunomodulatory cytokines, including IL-1β, IL-4, IL-6, IL-10, IL-17a, and TNF-α, and the concentration of the growth factor TGF-β1 were quantified using immunoassays. The concentration of the IL-1 receptor antagonist was quantified using ELISA. The concentration of A2M was quantified using mass spectrometry.
Results
No differences in the concentrations of IL-1β, IL-4, IL-6, IL-10, and IL-17a were found. Median TGF-β1 was significantly higher in APS (10,801 pg/mL; P < .05), PRP (6,219 pg/mL; P < .05), and ACP (5,263 pg/mL; P < .05) compared to CA2M (2,090 pg/mL). The IL-1 receptor antagonist was significantly higher in APS (58.78 ng/mL) and PRP (40.45 ng/mL). Median A2M concentration was significantly higher in APS (4.08 mg/mL; P < .001) compared to CA2M (1.99 mg/mL).
Conclusions
Autologous blood-based products have notably different immunomodulatory and growth factor profiles. These differences likely reflect variable concentrations of platelets and WBCs, as well as processing methods.
Clinical Relevance
Equine veterinarians should be aware of the constituents of the different orthobiologics available before use.
The use of orthobiologics, including autologous blood-based products, to treat musculoskeletal injuries in both human and equine athletes has increased in recent years. Due to the limited healing capabilities of tendons, ligaments, and cartilage, chronic lameness, reinjury, and reduced performance are common. Therefore, there is a strong interest in developing new therapies that can bolster healing and prevent recurrence. Autologous blood-based products concentrate immunomodulatory cytokines and growth factors produced by leukocytes and platelets and have been used as intralesional and intraarticular therapies with documented therapeutic benefits.1–3 Several products are commercially available including platelet-rich plasma (PRP; Restigen; Zoetis), autologous protein solution (APS; Pro-Stride; Zoetis), and autologous conditioned plasma (ACP; Arthrex ACP). More recently, a new product that is reported to contain highly concentrated α2-macroglobulin (A2M) referred to as conditioned A2M (CA2M; Alpha2EQ; Astaria Global) from hereonin has been introduced to the equine market. Orthobiologics are being used in horses with increasing frequency due to increasing reports of efficacy in addition to concerns with the routine administration of intra-articular corticosteroids including chondrotoxicity and laminitis, especially in horses with underlying endocrinopathies.4–7
Alpha2EQ, Pro-Stride APS, Restigen PRP, and Arthrex ACP are all produced through different processing steps following the sterile collection of blood. The efficacy of these autologous blood-based products is thought to be due to increased concentrations of immunomodulatory cytokines released from leukocytes and degranulation of platelet α-granules releasing growth factors.5 Concentrations of anti-inflammatory and immunomodulatory cytokines such as IL-1 receptor antagonist (IL-1Ra), IL-10, and IL-6 tend to be more increased in products that concentrate leukocytes.8,9 Increased concentrations of growth factors, such as TGF-β1, PDGF, and VEGF, are a key feature of platelet-rich products with benefits derived from the promotion of extracellular matrix production, angiogenesis, and tissue repair.10–12
The protein A2M has been proposed as a potential therapeutic protein in the treatment of joint disease and tendon/ligament injury due to its potential for inhibition of proteases, including matrix metalloproteinases and aggrecanases, involved in the degradation of the extracellular matrix of these tissues.13 It is an endogenous plasma protease inhibitor composed of 2 covalently bound dimers that have a bait region in the molecule’s hollow center that binds proteases thereby preventing downstream degradative effects.14–16 Following cleavage, the A2M molecule rapidly experiences a conformational change that ensnares the protease and inhibits proteolytic activity, and eventually, the A2M-protease complex is removed by the liver.13,16 While there are no data on the therapeutic potential of A2M in the horse, A2M has been investigated using different in vivo small animal models and has been shown to significantly decrease cartilage degradation after anterior cruciate ligament transection in rats17 and improve healing in a rotator cuff injury model in rats.18
Although autologous blood-based products are used commonly in equine medicine, there continues to be limited information about their constituents. Therefore, the objective of this study was to quantify and compare the concentration of A2M and select immunomodulatory cytokines and growth factors in 4 commonly used, commercially available autologous blood-based products including PRP, APS, ACP, and CA2M. We hypothesized that CA2M would have higher concentrations of A2M and lower concentrations of cytokines and growth factors compared to PRP, APS, and ACP.
Methods
Animals
Six healthy, adult horses owned by the University of Pennsylvania were enrolled in the study, with ages ranging from 1 to 10 years (median, 5.67 years). There were 4 Thoroughbreds, 1 Appaloosa, and 1 Quarter Horse including 3 geldings and 3 mares. The study was approved by the University of Pennsylvania’s IACUC (No. 806715), and the IACUC’s guidelines were followed.
Blood collection and autologous blood-product preparation
Blood was drawn from the jugular vein of all horses using an 18-gauge needle following aseptic preparation. For the preparation of CA2M, 35 mL blood was collected into two 60-mL syringes containing 5 mL of the anticoagulant acid citrate dextrose solution-A (ACD-A; Jorgensen Laboratories), transferred to vacutainer tubes, and processed according to Astaria Global’s guidelines. Then, APS and PRP were prepared in accordance with the manufacturer’s protocol. Briefly, 55 mL blood was gently drawn into a 60-mL syringe containing 5 mL ACD-A before transfer to the single-use separator device. The device was then centrifuged at 1,745 X g for 15 minutes. For PRP, the platelet-poor plasma was aspirated and discarded, and then the PRP fraction was aspirated, aliquoted, and frozen at −20 °C until further evaluation. For APS, the PRP fraction was aspirated from the device, transferred to the APS concentrator containing polyacrylamide beads, and centrifuged at 682 X g for 2 minutes. The final APS product was aspirated from the device, aliquoted, and frozen at −20 °C until further evaluation. The Arthrex ACP Double-Syringe System containing 1.5 mL ACD-A was used to collect blood up to the 15-mL fill line. The syringe was sealed and centrifuged at 1,500 X g for 5 minutes. The final product was collected using the inner syringe, aliquoted into microcentrifuge tubes, and stored at −20 °C until further analysis. All analyses were performed following a single thaw within 6 months of collection.
Cytokine and growth factor quantification
An equine-specific cytokine/chemokine fluorescent magnetic bead-based multiplex immunoassay (Milliplex; Millipore; EQCYTMAG-93K) was used to quantify concentrations of IL-1β, IL-4, IL-6, IL-10, IL-17a, and TNF-α on the Luminex 200 instrument following the manufacturer’s instructions and as previously reported.19 Transforming growth factor-β1 was measured using a fluorescent magnetic bead-based singleplex immunoassay (Milliplex; Millipore; TGFBMAG-64K-01). Briefly, 25 μL of standard, control, or sample was added to the appropriate well after which 25 μL of serum matrix or assay buffer was added to standard and sample wells, respectively. Finally, 25 μL of premixed antibody-immobilized beads was added, and the plate was incubated on a plate shaker overnight at 4 °C. The plate was washed 3 times, and 25 μL of detection antibody was then added to each well followed by a 1-hour incubation on a plate shaker at room temperature (RT; approx 20 °C). Next, 25 μL of streptavidin-phycoerythrin was added to each well, and the plates were incubated for 30 minutes on a shaker at RT. The plate was rewashed 3 times, and 150 μL of sheath fluid was added before analysis. The plate was run on the Luminex 200 instrument and the xPONENT software parameters of 100-μL sample size and 50 events per bead.
The concentration of IL-1Ra was measured using an equine-specific sandwich ELISA (R&D Systems; DY2466). Following the manufacturer’s protocol, a clear 96-well microplate was coated with 100 μL diluted capture antibody and incubated overnight at RT. The plate was washed with 400 μL of wash buffer 3 times and blocked by adding 300 μL reagent diluent to each well. The plate was incubated at RT for 1 hour and then washed 3 times. Following this, 100 μL of standard or sample was added to appropriate wells and incubated at RT for 2 hours. The plate was then washed 3 times, and 100 μL of detection antibody was added to each well and incubated at RT for 2 hours. Finally, the plate was rewashed 3 times, 100 μL of streptavidin-horseradish peroxidase was added to each well, and the plate was incubated at RT for 20 minutes. Stop solution (50 μL) was added to each well, and optical density was determined immediately using a microplate reader at 450 nm with wavelength correction set at 570 nm.
The concentration of A2M was determined using selected reaction monitoring (SRM) mass spectrometry as previously reported.20 Briefly, the SRM assay was used to measure a specific A2M peptide as a surrogate of the whole A2M protein (Table 1). The tryptic peptide LLVYTILPDGEVVGDSAK was measured in all samples in a targeted, SRM quantitative assay after trypsin digestion. Peptide analytes included in the assay were standard, target, and internal control. Standard and target peptides were identical but derived from synthetic and endogenous sources, respectively. The standard peptide was used to make a series of peptide concentrations to generate a standard curve. The target peptide was enzymatically liberated from the A2M protein in the test samples. The synthetic internal control peptide was identical to the corresponding target and standard peptide amino acid sequence except that the C-terminal lysine residue was substituted with a stable isotope-labeled form of lysine. The same amount of internal control peptide was spiked into every sample (including standard peptides) to account for variations due to matrix effects on ionization and instrument performance.
Peptide sequences within α2-macroglobulin used for selected reaction monitoring mass spectrometry.
| Amino acid sequence | Peptide type | Molecular mass (Da) | Source |
|---|---|---|---|
| LLVYTILPDGEVVGDSAK | Standard | 1,889 | Syntheticb |
| LLVYTILPDGEVVGDSAK | Target | 1,889 | Equine plasma |
| LLVYTILPDGEVVGDSAKa | Internal control | 1,897 | Syntheticb |
Trypsin digestion was performed by diluting samples 1:100 with Tris (20 µL of sample into 1,980 µL of 50 mM Tris, pH 8.5). There were 2 analysis volumes, 20 µL and 40 µL, analyzed in duplicate in a 96-well plate. Each sample well was spiked with 50 mM Tris pH 8.5 to a total volume of 120 µL. Standard peptide solutions were added to the plate at 120 µL/well. Proteins in all wells were reduced for 60 minutes at 56 °C in the presence of 5 mM tris(2-carboxyethyl)phosphine. This was followed by alkylation for 30 minutes at RT in the presence of 6.3 mM iodoacetamide. Proteins were then trypsin digested for 17 hours at 37 °C. The trypsin solution also contained internal control peptide so that each well received consistent amounts of trypsin (3 µg) and internal control peptide (9 ng). Digestion was halted with the addition of formic acid (0.5% vol/vol).
Selected reaction monitoring was performed using a Waters TQS-Micro triple quadrupole mass spectrometer to measure A2M peptide concentration (picomoles of A2M peptide per milliliter). Digested peptides were analyzed using reversed-phase liquid chromatography (Waters CORTECS C18; 2.7-µm column, 2.1 X 50 mm) that eluted into the mass spectrometer. The first quadrupole of the mass spectrometer was used as a mass filter. Only analytes within 1 mass unit of the peptide masses in Table 1 were passed through to the second stage of the mass spectrometer. The second quadrupole of the mass spectrometer was programmed to fragment the peptides into a sequence-specific product ion by collision-induced dissociation. The third quadrupole increased the specificity by mass filtering the fragment ion pool to only allow the A2M sequence-specific fragment ion to pass through to the ion detector for quantitation. The mass spectrometer recorded peak area ion counts of the target peptides and the internal control peptide. The ratio of the target peptide ion count to the internal control peptide ion count was the response. A standard curve of the responses (y-axis) from known concentrations of standard peptide (x-axis) was plotted, and a linear 1/X-weighted fit was performed. The response from each unknown was interpolated from the standard curve to determine the A2M peptide concentration (picomoles of A2M peptide per milliliter). Two input volumes from a sample were analyzed in duplicate, and the average was reported as the final A2M peptide concentration. To determine A2M protein concentration (milligrams per milliliter), the concentration of picomoles of peptide per milliliter concentration was multiplied by 0.00016 as 1 pmol A2M protein/mL (based on an intact A2M protein molecular weight of 160,000 Da) = 0.16 mg A2M protein/mL.
Statistical analysis
Power analysis was calculated using a paired t test using previous studies21 evaluating concentrations of TGF-β1 in PRP and assuming means of 15% and 10% (assumed 5% decrease in the means) and an SD of 2.5. We assumed an α of 5%, power of 80%, and expected change δ of 5%. Based on the above-outlined assumptions, the sample size of animals in the study sample was n = 6. Histograms of data were visually inspected for a Gaussian distribution, and a Shapiro-Wilk test was performed to determine normal distribution before statistical analysis. Data were not normally distributed and were analyzed using the Kruskal-Wallis test with multiple comparisons made between groups using the Steel-Dwass method. All samples that were below the limit of detection (LOD) on the immunoassays were assigned a value that was 50% of the lowest detectable amount as stated by the assay manufacturer. Statistical analysis was performed with JMP Pro 17 software (JMP), with the level of significance set at P < .05.
Results
Cytokine and growth factor concentrations
The concentrations of IL-4, IL-6, IL-10, IL-17a, TNF-α, and TGF-β1 in the 4 autologous blood-based products are shown in Figure 1. There were no differences in concentrations of IL-4, IL-6, IL-10, IL-17a, and TNF-α between the different products. Platelet-rich plasma (median, 6,219 pg/mL; range, 3,245 to 7,915 pg/mL; P < .05), APS (median, 10,801 pg/mL; range, 4,384 to 19,176 pg/mL; P < .05), and ACP (median, 5,623 pg/mL; range, 3,632 to 7,287 pg/mL; P < .05) had higher concentrations of TGF-β1 compared to CA2M (median, 2,090 pg/mL; range, 1,685 to 3,023 pg/mL).
The concentration (pg/mL) of cytokines IL-4 (A), IL-6 (B), IL-10 (C), IL-17a (D), tumor necrosis factor-α (TNF)-α (E), and TGF-β1 (F) in 4 autologous blood-based products including conditioned α2-macroglobulin (CA2M), autologous conditioned plasma (ACP), autologous protein solution (APS), and platelet-rich plasma (PRP). Box plots show median (line), upper and lower quartiles (box), and minimum and maximum points (whiskers). Individual animals are depicted by different symbols. Each animal is represented by the same symbol in each product. *P < .05; n = 6.
Citation: American Journal of Veterinary Research 86, 4; 10.2460/ajvr.24.11.0363
Interleukin-1β and IL-1Ra concentrations
The concentrations of IL-1β and IL-1Ra and the ratio of the 2 are shown in Figure 2. There were no differences in the concentration of IL-1β between the 4 products. Both APS (median, 58.78; range, 30.22 to 81.99; P < .05) and PRP (median, 40.45 ng/mL; range, 31.43 to 104.89 ng/mL; P < .05) contained significantly higher concentrations of IL-1Ra compared to CA2M and ACP, which were both below the LOD. The ratio of IL-1Ra:IL-1β in APS (median, 1,682.23; range, 12.50 to 3,567.45; P < .01) was significantly higher than both CA2M and ACP. The ratio of IL-1Ra:IL-1β in PRP (919.39; range, 17.64 to 2,194.29; P < .01) was also significantly higher than both CA2M and ACP, which were both below the LOD of the IL-1Ra assay.
The concentration of IL-1β (A; pg/mL), IL-1 receptor antagonist (IL-1Ra; B; ng/mL), and the ratio of IL-1Ra:IL-1β (C) in 4 autologous blood-based products including CA2M, ACP, APS, and PRP. Box plots show median (line), upper and lower quartiles (box), and minimum and maximum points (whiskers). Individual animals are depicted by different symbols. Each animal is represented by the same symbol in each product. *P < .05; n = 6.
Citation: American Journal of Veterinary Research 86, 4; 10.2460/ajvr.24.11.0363
Concentration of A2M
The concentration of A2M (mg/mL) in the 4 autologous blood-based products is shown in Figure 3. There was significantly more A2M in APS (median, 4.08 mg/mL; range, 3.54 to 4.77 mg/mL; P < .001) than CA2M (median, 1.99 mg/mL; range, 1.76 to 2.66 mg/mL). No other differences were noted between groups.
The concentration of A2M (mg/mL) in 4 autologous blood-based products including CA2M, ACP, APS, and PRP is shown. Box plots show median (line), upper and lower quartiles (box), and minimum and maximum points (whiskers). Individual animals are depicted by different symbols. Each animal is represented by the same symbol in each product. ***P < .001; n = 6.
Citation: American Journal of Veterinary Research 86, 4; 10.2460/ajvr.24.11.0363
Discussion
Autologous blood-based products offer a stall-side, orthobiologic approach to treating various musculoskeletal injuries in the horse. Immunomodulatory cytokines and growth factors that modulate the inflammatory cascade and improve healing are reported to be increased in autologous blood-based products; however, little data are available that quantify the amount of these mediators in some new products such as Alpha2EQ. Additionally, few studies have quantified the amount of A2M in autologous blood-based products.20 In this comparative study, we found that there were no differences in the concentrations of several immunomodulatory cytokines, including IL-1β, IL-4, IL-6, IL-10, and IL-17a, among the 4 different products evaluated. However, we did find significantly higher quantities of the growth factor TGF-β1 in APS, PRP, and ACP compared to CA2M. In addition, IL-1Ra was below the LOD in ACP and CA2M. In APS and PRP, the IL-1Ra:IL-1β ratio was > 100, above the previously stated ratio needed to effectively downregulate IL-1β.22 Interestingly, A2M concentration was highest in APS, and this was significantly greater than CA2M.
Interleukin-1β is considered to be a master proinflammatory cytokine in osteoarthritis causing upregulation of the inflammatory cascade via NF-κB activation.17 Because of this, significant efforts have been directed toward developing therapies that contain high concentrations of the endogenous protein IL-1Ra that competitively inhibits IL-1β when it binds to the IL-1 receptor.12 Originally, incubation of whole blood for 24 hours at 37 °C was used to increase IL-1Ra production by monocytes that were activated by borosilicate beads.9 More recent studies8,18,23 have demonstrated that large quantities of IL-1Ra can be produced at the patient’s side without incubation. In this study, we found that high concentrations of IL-1Ra were present in both APS and PRP. Although we did not quantify IL-1Ra in unprocessed serum, previous studies8,24 have reported IL-1Ra concentrations of approx 0.2 to 4.0 ng/mL in equine serum, whereas we found approx 60 ng/mL in APS and approx 40 ng/mL in PRP. While high concentrations of IL-1Ra are expected in APS due to the concentration of leukocytes during processing, the high concentration of IL-1Ra in PRP was somewhat unexpected. It is possible that leukocyte-rich PRPs such as PRP lead to final products with high levels of IL-1Ra. Previous studies22 have shown that 10- to 100-fold higher amounts of IL-1Ra are required to effectively inhibit IL-1β. In this study, we found that both APS and PRP had more than 100-fold more IL-1Ra than IL-1β.
In terms of the immunomodulatory cytokines that we quantified, including IL-1β, IL-4, IL-6, IL-10, IL-17a, and TNF-α, no differences were noted between products. While this may seem like an intriguing finding when evaluating products that concentrate leukocytes, such as APS, similar findings have been previously reported.19 Although we did not quantify the concentration of these immunomodulatory cytokines in whole blood or unprocessed serum, previous studies8,25,26 have reported concentrations of IL-1β (approx 50 pg/mL), IL-6 (approx 10 pg/mL), IL-10 (approx 100 pg/mL), and TNF-α (approx 25 pg/mL) in equine serum to be similar to the amounts in the final processed products evaluated in this study. This suggests that processing does not affect the concentration of these cytokines, although a direct comparison to equine serum obtained from the same group of horses would be of interest.
Another important consideration when quantifying proteins in autologous blood-based products is that in acellular (serum-based) or hypocellular (leukocyte-poor) products, no further change in the product’s final protein profile would be expected as there are no cells to continue production and release of proteins. In products containing leukocytes, continued production of various proteins would be expected in vivo when the product is used fresh. There is conflicting evidence regarding the effect of leukocyte concentration on the therapeutic benefits of treatment with some studies27,28 showing that leukocyte-rich PRP may be more proinflammatory due to concentration of catabolic cytokines. However, a recent study29 demonstrated no difference in treatment outcomes of humans with knee osteoarthritis treated with leukocyte-poor or leukocyte-rich PRP. Interestingly, a recent study30 examining leukocyte phenotype pre- and postprocessing of human APS showed that processing led to differential regulation of genes in leukocytes, particularly monocytes and macrophages, where enrichment of M2 macrophages was noted in the final product. It is possible that leukocyte phenotype in the final product is more important than total leukocyte concentration. Finally, it should be noted that while one of the stated benefits of CA2M is that the product is a purer concentrate of a single protein, A2M, without the presence of other cytokines, our study suggests that CA2M does contain similar amounts of this select group of cytokines when compared to other autologous blood-based products.
Transforming growth factor-β1 is released from platelets upon α-granule degranulation, and thus, the higher quantities of TGF-β1 in ACP, APS, and PRP can be explained by the method of preparation of these products in which platelets are concentrated during one of the processing steps.11 Transforming growth factor-β1 is involved in the development and maintenance of cartilage, and while some studies have demonstrated upregulation of hyaluronic synthesis in TGF-β1–treated equine chondrocytes31 and enhanced chondrogenic differentiation of bone marrow–derived mesenchymal stem cells treated with TGF-β1,32 other studies33 have suggested that TGF-β1 is a key growth factor involved in the development of osteophytes. At this time, the ideal amount of TGF-β1 in both healthy and diseased equine joints remains unknown, and further work is needed.
There is strong interest in investigating A2M as an orthobiologic in joint disease due to its protease-inhibiting properties. Both aggrecanases and matrix metalloproteinases are considered to be important degradative enzymes involved in the degeneration of the extracellular matrix of articular cartilage.13 In the study presented here, we found that the concentration of A2M was highest in APS, and this was significantly greater than the concentration of A2M in CA2M. This finding is similar to previously published results demonstrating high concentrations of A2M in APS compared to CA2M.20 Further research is needed to determine the effects of A2M in equine joints and the ideal concentrations required to yield an optimized therapeutic effect.
This study has certain limitations that need to be considered. First, we compared 4 autologous blood-based products to each other and did not compare these to unprocessed serum. Instead, we made comparisons to previously published data that quantified the concentration of cytokines and growth factors in equine blood. In addition, we only quantified a select group of cytokines and growth factors. Examination of other mediators may provide further insight into the potential mechanism of action of autologous blood-based products. All quantification of cytokines, growth factors, and A2M was performed after a single thaw within 6 months of collection. At this time, the effect of storage time at −20 °C on the specific products investigated in this study is unknown and could alter concentrations to some degree. Finally, this study is only quantifying the constituents of these products and not their functionality; therefore, further research into the in vivo effects in the treatment of clinical disease is needed.
In conclusion, while APS, PRP, ACP, and CA2M contain similar amounts of several immunomodulatory cytokines, including IL-1β, IL-4, IL-6, IL-10, IL-17a, and TNFα, APS and PRP were found to have significantly higher quantities of IL-1Ra, an important anti-inflammatory mediator. In addition, APS contained the highest concentration of A2M. While all 4 products can be used as a part of a multimodal approach to treat musculoskeletal injuries, some products such as APS and PRP may be better suited for concentrating desirable anti-inflammatory and antidegradative mediators.
Acknowledgments
The authors thank Dr. Cade Torcivia for assistance with horse handling and care. The authors thank Lindsay Baltrusch for help with the preparation of CA2M. We also thank the Univerisity of Pennsylvania Institute for Infectious and Zoonotic Diseases and the Institute for Medical Translation New Bolton Center for support in purchasing key equipment used in this research.
Disclosures
Zoetis performed the SRM mass spectrometry for quantification of A2M. The authors have no financial interests in Zoetis and have no conflicts of interest to declare.
No AI-assisted technologies were used in the generation of this manuscript.
Funding
This study was funded by the Raymond Firestone Trust and Raker Tullener’s Fund, New Bolton Center, University of Pennsylvania. Pro-Stride and Restigen kits were provided as in kind support by Zoetis Animal Health. Stipend support for Dr. Barot was provided by the MARS Equestrian Veterinary Research Scholar program.
ORCID
K. Ortved https://orcid.org/0000-0003-0398-2444
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