Bioconjugate Chem. 2006, 17, 1288−1293
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A Method To Optimize PEG-Coating of Red Blood Cells Sameereh Hashemi-Najafabadi,† Ebrahim Vasheghani-Farahani,*,† Seyed Abbas Shojaosadati,† Mohammad Javad Rasaee,‡ Jonathan K. Armstrong,§ Mostafa Moin,| and Zahra Pourpak| Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, Tehran, P. O. Box 14115-143, I. R. Iran, Medical Biotechnology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, P. O. Box 14115-331, I. R. Iran, Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, and Immunology, Asthma and Allergy Research Institute, Children Medical Center, Tehran University of Medical Sciences, Tehran, P. O. Box 14185-863, I. R. Iran. Received March 7, 2006; Revised Manuscript Received June 26, 2006
Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. A proposed method to overcome donor-recipient blood group incompatibility is to mask the blood group antigens by the covalent attachment of poly(ethylene glycol) (PEG) to the red blood cell (RBC) membrane. Despite much work in the development of PEG-coating of RBCs, there is a paucity of data on the optimization of the PEG-coating technique; it is the aim of this study to determine the optimum conditions for PEG coating using a cyanuric chloride reactive derivative of methoxy-PEG as a model polymer. Activated PEG of molecular mass 5 kDa was covalently attached to human RBCs under various reaction conditions. Inhibition of binding of a blood-type specific antiserum (anti-D) was employed to evaluate the effect of the PEG-coating, quantified by hemocytometry and flow-cytometry. RBC morphology was examined by light and scanning electron microscopy. Statistical analysis of experimental design together with microscopy results showed that the optimum PEGylation conditions are pH ) 8.7, temperature ) 14 °C, and reaction time ) 30 min. An optimum concentration of reactive PEG could not be determined. At high polymer concentrations (>25 mg/mL) a predominance of type III echinocytes was observed, and as a result, a concentration of 15 mg/mL is the highest recommended concentration for a linear PEG of molecular mass 5 kDa.
INTRODUCTION The immunological response to transplanted allogeneic tissue and cells has been a significant barrier in both organ transplantation and blood transfusion. The most common example of allogeneic cell exposure is the use of red blood cells (RBCs) in transfusion. The cell surface serves as an important biological interface and biochemical barrier. The extreme complexity of proteins, carbohydrates, and lipids comprising the cell surface serves as the primary loci for tissue rejection. For blood banking, following Landsteiner’s discovery of the ABO blood group antigens on RBCs a century ago (1), the major cause of this immune reaction had been identified. Blood samples from donor and recipient were each tested and classified into one of the four phenotypes, O, A, B, or AB, which allowed the donor and recipient phenotypes to be matched. Since this initial discovery, there are currently over 200 different known antigens (2). In most transfusions, ABO and D (Rhesus) blood typing is sufficient to identify appropriate donors. More often, problems are encountered in individuals who receive multiple transfusions, such as patients with sickle cell anemia and thalassemia. In such patients, alloimmunization against minor RBC antigens can make it nearly impossible to identify appropriate blood donors (3-5). * To whom correspondence should be addressed. Tel.: +98-21-8 8005040; Fax: +98-21-88005040. E-mail:
[email protected]. † Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University. ‡ Medical Biotechnology Department, Faculty of Medical Sciences, Tarbiat Modares University. § University of Southern California. | Tehran University of Medical Sciences.
More than 20 years ago, Abuchowski et al. developed a technique to modify proteins by the covalent attachment of poly(ethylene glycol) (PEG). They showed that PEG-modified bovine albumin was less immunogenic than its unmodified counterpart (6, 7). The covalent attachment of PEG is now commonly used to modify a variety of proteins, enzymes, drugs, and artificial surfaces (8-12). Despite these applications, RBCs have only recently been considered for use as substrate for PEG modification. An overview of PEG-coating of RBCs by Fisher (13) summarizes the development of the field, and Lublin (14) reviewed the potential for PEG-coated RBCs as a component for the future creation of a safe, “universal” blood. The PEGcoating of RBCs provides a protective shell around the RBC that potentially excludes large molecules, such as antibodies, but does not appear to inhibit the passage of small molecules, such as glucose and oxygen (13). Several reactive derivatives of methoxy-PEG (mPEG) have been used to covalently attach mPEG to the surface of RBCs (15-18). All of these reactive derivatives preferentially target epsilon amino acids (almost exclusively lysine residues) on the extracellular surface of the plasma membrane (13, 19). All cells can be readily modified using cyanuric chloride activated PEG with only slight variations in pH, temperature, and time. Cyanuric chloride’s strengths as a coupling agent include the malleability of its chemistry, the efficacy in membrane protein modification, and the chemical stability of the modified proteins (20). Despite the development of this technology to PEG-coat RBCs, optimization of the reaction conditions has not been thoroughly investigated. In this study, RBCs were coated with a reactive cyanuric chloride mPEG derivative of 5 kDa
10.1021/bc060057w CCC: $33.50 © 2006 American Chemical Society Published on Web 08/05/2006
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molecular mass. The effects of process variables (polymer concentration, temperature, time, and pH) were investigated. Full factorial design and Taguchi analysis were employed to identify the optimum reaction conditions for PEGylation of RBCs. To assess the effectiveness of the PEG-coating, inhibition of agglutination by blood-type specific antisera was measured, using a hemocytometer to count the remaining single RBCs. In addition, the hemocytometery data were confirmed by flow cytometric analysis of fluorescein-labeled anti-D binding to PEG-coated RBCs. Finally, the morphology of PEG-RBCs was studied by light microscopy and scanning electron microscopy (SEM).
EXPERIMENTAL PROCEDURES Reagents. Methoxy-poly(ethylene glycol) of 5 kDa molecular mass, triethanolamine (TEA), and fluorescein isothiocyanate (FITC) were purchased from Sigma (St. Louis, MO). Cyanuric chloride (2,4,6-trichloro-1,3,5-triazine), benzene, anhydrous sodium carbonate, D-glucose, sodium chloride, and potassium chloride were obtained from Merck-Schuchardt (Darmstadt, Germany). Cyclohexane was purchased from Roth (Karlsruhe, Germany). Packed Rh-positive RBCs were obtained from Iranian Blood Transfusion Organization. Anti-D was purchased from CinnaGen Inc. (Tehran, Iran). Polymer Derivatization. Derivatization of 5 kDa mPEG with cyanuric chloride was performed using a modification of the method described by Abuchowski et al. (6). Briefly, 5 g of vacuum-dried mPEG (overnight at 80 °C) was dissolved in 40 mL of hot anhydrous benzene, cooled to 15 °C, and then slowly added to a solution of cyanuric chloride in benzene (5-fold molar excess). One gram of anhydrous sodium carbonate was then added. The mixture was stirred for 48 h at 15 °C under an atmosphere of dry nitrogen, and then the sodium carbonate was removed by vacuum filtration. The PEG derivative was precipitated with anhydrous cyclohexane, collected by vacuum filtration, and then resuspended in benzene. This process was repeated five times to remove any unreacted cyanuric chloride (21). The product was then dried under vacuum, using a freeze dryer system (Zirbus Technology, Vaco 5), and stored at -70 °C, under vacuum, until use (22). Coating of Red Blood Cells with mPEG. Packed Rhpositive RBCs were resuspended to a 10% hematocrit in TEA buffer (30 mM TEA, 110 mM NaCl, 4 mM KCl, 5 mM D-glucose). A fresh cold stock solution of the derivatized polymer was prepared in 0.9% NaCl, containing 1 mM HCl, and appropriate volumes were immediately added to the RBC suspensions to yield a final polymer concentration of 5-45 mg/ mL; an equivalent volume of buffer was added to the control samples. This acidic solution retards hydrolysis of the reactive PEG derivative prior to exposure to the RBCs; since only small volumes of the stock solution were added, the final pH of the suspensions remained unaltered. The samples were incubated with gentle mixing under various conditions (pH, temperature, time, and polymer concentration, defined in subsequent sections). After two washes with isotonic phosphate buffer solution (PBS, pH ) 7.4) at 200 g for 10 min, packed RBCs were prepared for evaluation of the polymer coating (23). RBC Agglutination by Anti-D. Inhibition of anti-D mediated agglutination was employed to assess the effectiveness of the polymer coating. Four hundred microliters of a control or PEGylated RBC (Rh-positive) suspension (6% hematocrit in isotonic saline) (24) was mixed with a solution of anti-D in PBS with a known concentration (ratio of anti-D: PBS ) 1: 3) and were incubated with a gentle mixing at room temperature for 30 min. The RBC s were then centrifuged at 200g for 1 min. One microliter of the pellet was re-suspended in 1 mL of PBS (0.1% hct), and then using a dye exclusion test with trypan
Table 1. Selected Experimental Variables with Corresponding Values at Two Levels for Full Factorial Design variable A: B: C: D:
temperature of reaction (°C) time of reaction (min) mPEG concentration (mg/mL) pH of TEA buffer
low level (1)
high level (2)
4 30 5 8
25 60 45 9.5
blue (viable cells remained uncolored, dead cells showed blue color) and light microscopic system (Nikon, E200), nonagglutinated viable free cells were counted using a hemocytometer (Improved Neubauer Ruling). The Improved Neubauer Ruling is a 3 by 3 mm (9 mm2) grid, subdivided into nine secondary squares, each 1 by 1 mm (1 mm2). The smallest squares in the center of the grid have an area of 1/400 mm2 and are arranged in groups of 16. Single free erythrocytes in five of the 25 sections of 16 small squares of the hemocytometer (four corner sections and the center square) were counted, which is equal to the number of free erythrocytes in 0.02 µL of suspension. For determination of free cells per 1 mL of RBCs at 100% hct, the number of counted free cells per 0.02 µL at 0.1% hct was multiplied by 5 × 107. The higher the number of free cells, the greater the effectiveness of RBC PEGylation. Flow Cytometry. Labeling of anti-D with FITC was achieved by following the procedure described by Coligan et al. (25). Fluorescein-labeled anti-D was added to a dilute suspension of RBCs in PBS. The ratio of fluorescein-labeled anti-D to RBCs, obtained by titration, was high to prevent agglutination (60 µL of 0.1% hematocrit in PBS, added to 200 µL of fluoresceinlabeled anti-D solution with a protein concentration of 0.16 mg/ mL). The samples were incubated for 30 min at room temperature in the dark with gentle mixing, centrifuged, and washed twice with PBS at 500g for 2 min. The labeled cells were then resuspended in 1 mL of PBS. The fluorescence intensity of FITC-anti-D labeled PEG-cells was measured using a FACSTAR PLUS flow cytometer (Becton Dickinson, San Jose, CA). Ten thousand cells were counted for each sample (23). The fluorescence intensity of FITC-anti-D labeled cells was recorded for each sample and expressed as a ratio to the intensity of FITCanti-D labeled control (uncoated) cells. The lower this ratio, the greater the effectiveness of RBC PEGylation. Microscopy. The morphology of PEG-RBCs and control (uncoated) RBCs at physiological pH was studied by SEM (XL 30, Philips, Netherlands). The procedure described by Kayden and Bessis (26) to prepare RBCs for SEM was followed. Additionally, RBCs were suspended in compatible plasma and the RBC morphology examined by light microscopy at 40 × magnification. Design of Experiments. Statistically designed experiments are highly efficient in that they give a fixed amount of information with much less effort than the classical one-variableat-a-time approach, and many of them give additional information about interactions between variables. Factorial and fractionalfactorial experiments are the most powerful (statistical) techniques in research, and well chosen fractional-factorials are particularly economical in assessing multivariable systems (27). Full Factorial at Two Levels. The first step (using the Yates Table) was to identify which variables have the largest effects on the PEGylation reaction. Selection of these factors was based on previous studies in literature for RBC PEGylation. The evaluated variables were temperature and time of PEGylation reaction, polymer concentration, and pH of TEA buffer (Table 1). Taguchi Design. We have employed Taguchi statistical technique to study the impact of multiple variables on single outputs (prevention of agglutination and antibody binding). In this technique, the results of the experiments are analyzed to achieve one or more of the following three objectives: (1) To
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Table 2. Selected Experimental Variables with Corresponding Values at Three Levels for Taguchi Design variable
first level (1)
second level (2)
third level (3)
A: temperature of reaction (°C) B: mPEG concentration (mg/mL) C: pH of TEA buffer
4 5 8
14 25 8.7
25 45 9.5
Table 3. Yates Table Analysis of a 24 Full Factorial Design (Four Variables in Two Levels) trial
TCa
temp (A) (°C)
time (B) (min)
concn (C) (mg/mL)
pH (D)
responseb × 10-7
F-valuec
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
l a b ab c ac bc abc d ad bd abd cd acd bcd abcd
4 25 4 25 4 25 4 25 4 25 4 25 4 25 4 25
30 30 60 60 30 30 60 60 30 30 60 60 30 30 60 60
5 5 5 5 45 45 45 45 5 5 5 5 45 45 45 45
8 8 8 8 8 8 8 8 9.5 9.5 9.5 9.5 9.5 9.5 9.5 9.5
55 100 60 100 70 125 65 135 95 75 50 100 125 125 90 175
160.33 0.01 39.47 115.12 12.79 1.58 1.13 22.43 15.70 0.23 30.35 17.27 0.23 1.58 0.01
a Treatment combination: The low level of any variable is denoted by l, and the high level of any variable is denoted by its lower-case letter. b Each value is the average number of free cells, per 1 mL suspension, for each test with two replicates. c SSx ) (f2x)/N, which fx is the amount in the final column of Yates analysis corresponding to x (not presented here) and N is the total number of treatment combinations with their replications (32), MSx ) SSx/øx, which øx is the degree of freedom corresponding to x, TSS ) Σy2i - (Σyi)2/N ) 36311.75 × 1014, yi is the result, RSS ) TSS - ΣSSx ) 1338 × 1014, error degree of freedom (ø) ) 16, residual error variance ) RSS/ø ) 83.60 × 1014, F-value ) MSx/(residual error variance), F-critical (ø1 ) 1, ø2 ) 16) ) 8.575 (obtained from standard table).
establish the best or the optimum condition for a product or a process. (2) To estimate the contribution of individual factors. (3) To estimate the response under the optimum conditions. The optimum condition is identified by studying the main effects of each of the factors. The process involves minor arithmetic manipulation of the numerical results. The main effects indicate the general trend of the influence of the factors. If a systematic, sequential approach to experimental design is adopted, it is usually sufficient to restrict the design to twolevel experiments. Sometimes, however, it is advantageous to work at three levels or more. The usual reason for employing either of these techniques is that a maximum or minimum is being approached. This design estimates the nonlinear (quadratic) effects (28). To obtain the optimum condition for PEGylation of RBCs, an L27 array of Taguchi (28) for three variables at three levels was designed. The variables and their corresponding values are presented in Table 2. This design was also employed to compare the hemocytometry results with the flow cytometry data. All experiments with RBCs were performed in duplicate unless stated otherwise.
RESULTS The results and statistical analysis for full factorial design (using Yates Table) at two levels are presented in Table 3. All the responses are presented as the number of viable free cells per 1 mL of packed cells. An increase in the number of free cells for PEGylated versus control (uncoated) RBCs shows that the PEG-RBCs were protected against agglutination. By comparison the obtained F-values with a critical F(1,16) value of 8.575 (obtained from the F-Table) (27), it is apparent that three
Table 4. An L27 Taguchi Array for RBC PEGylation trial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
temp (°C)
concn (mg/mL)
pH
responsea × 107
responseb
4 4 4 4 4 4 4 4 4 14 14 14 14 14 14 14 14 14 25 25 25 25 25 25 25 25 25
5 5 5 25 25 25 45 45 45 5 5 5 25 25 25 45 45 45 5 5 5 25 25 25 45 45 45
8 8.7 9.5 8 8.7 9.5 8 8.7 9.5 8 8.7 9.5 8 8.7 9.5 8 8.7 9.5 8 8.7 9.5 8 8.7 9.5 8 8.7 9.5
65 90 90 130 135 150 125 120 120 140 120 195 195 200 205 170 210 165 150 135 160 180 180 130 175 155 110
0.82 0.83 0.84 0.80 0.71 0.77 0.79 0.71 0.76 0.64 0.55 0.64 0.51 0.47 0.51 0.49 0.38 0.62 0.52 0.70 0.53 0.59 0.52 0.45 0.45 0.53 0.45
a Each value is the average number of free cells, per 1 mL suspension for each test with two replicates. b Each value is the average mean fluorescence intensity ratio of FITC labeled anti-D cells of PEG-RBCs versus control (uncoated) RBCs, for each test with two replicates.
variables (temperature, polymer concentration, and pH) and their interactions have F-values greater than critical F. Hence they were selected as important factors for PEGylation of RBCs. The effect of reaction time was less important. In fact after 30 min, the reaction was complete and any further increase in incubation time did not have a significant effect on the responses. As a result, the reaction time was fixed at 30 min to study the effect of other variables on the extent of PEGylation. The obtained results, using an L27 array of Taguchi, with both data from cell counting and flow cytometry methods are presented in Table 4. The main effects of the experimental variables on the number of free single RBCs and also the mean fluorescence intensity ratio of FITC-anti-D labeled cells of PEGRBCs versus control (uncoated) RBCs, as two indicators of the extent of PEGylation, are shown in Figures 1 and 2, respectively. These figures show the effect of one variable, when the others vary. Each point in each curve presents an average of the obtained responses from 9 duplicate experiments (18 experiments). These figures show that the optimum temperature and pH of reaction medium are 14 °C and pH ) 8.7, respectively. But the optimum polymer concentration, obtained by cell counting and flow cytometry methods, was 25 and 45 mg/mL, respectively. The morphology of control (uncoated) and PEG-coated RBCs (5, 10, 15, 25, and 45 mg/mL of activated mPEG) that were prepared under optimum conditions (30 min, 14 °C and pH ) 8.7) and then returned to a physiological pH with PBS (pH ) 7.4) are presented in Figure 3 (images from SEM). RBC morphology was classified according to Bessis (29). At 5 mg/ mL, type I echinocytes were observed with a few cells of echinocyte type II or III. At 10 mg/mL, type II echinocytes predominate with some cells of type I still observable. At 15 mg/mL, an equal amount of type II and III echinocytes were observed, and at 25 and 45 mg/mL only type III echinocytes existed. Light microscopic examination of RBCs suspended in compatible plasma confirmed the SEM observations.
Optimization of RBC PEGylation
Figure 1. The effect of different factors on free cell (nonagglutinated cell) number. Shown are the effect of (A) temperature of reaction, (B) polymer concentration, and (C) pH of TEA buffer.
DISCUSSION The selection of the levels of factors for optimization were based on the range given in the literature. Jackson et al. (30) showed that pH ) 9.2 is effective for coupling a cyanuric chloride activated mPEG to a protein. Other researchers used an elevated pH (pH ) 8-9.2) for coupling an activated PEG with RBCs (23, 31, 32). The range of 30-60 min and 4-25 °C for the time and temperature of RBC PEGylation, respectively, have also been used. Fisher (13) in his review noted that at polymer concentrations below 1 mM (5 mg/mL), the 5 kDa-activated mPEG is not very effective for masking antigens. Higher concentrations up to 50 mg/mL (10 mM) have also been used (16, 32), but some abnormality in the morphology of RBCs was reported. Light microscopic examination of linear mPEG 5 kDa-coated RBCs in autologous plasma showed marked echinocytosis above a mPEG concentration of 2 mM (33) and poor in ViVo survival of mPEG 5 kDa-coated RBCs has been observed by others in the mouse model at a concentration of 0.6mM and above (31). More effective antigen masking and maintenance of discocytic morphology was achieved with reactive derivatives of branched PEGs (33). SEM and light microscopy results from our work presented here show that a polymer concentration of 15 mg/mL is the highest recommended level for PEGylation using a linear PEG
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Figure 2. The effect of different factors on the mean fluorescence intensity ratio of PEG-RBCs versus control (uncoated) RBCs incubated with FITC labeled anti-D. Shown are the effect of (A) temperature of reaction, (B) polymer concentration, and (C) pH of TEA buffer.
of molecular mass 5 kDa. Above this concentration only type III echinocytes existed. RBCs of echinocyte type I and II may circulate in the host body, but cells of echinocyte type III would not circulate, because such cells are not deformable and hence would tend to get trapped in the microcirculation (34). Therefore the optimum concentration, 25 mg/mL, obtained by the cell counting method, and 45 mg/mL, obtained by the flow cytometry method, cannot be selected as optimum concentrations due to the loss of discocytic morphology. The observation of increasing echinocytosis with increasing reactive PEG concentration is solely related to the presence of PEG on the cell surface, and not exposure to high pH, as all control cells showed normal discocytic morphology after returning to a physiological pH. For the purposes of the work undertaken here, it was reasonable to select a linear PEG of molecular mass 5 kDa, as this is the most commonly used PEG derivative. It is possible that PEGs of larger molecular mass or of different geometry may sustain discocytic RBC morphology, while being effective antigen masking agents (33), but investigation into these variables was beyond the scope of the current study. During the incubation period at elevated pH, virtually no lysis (30 mg/mL) resulted in significant lysis after 24 h of incubation at 37 °C (but not at lower temperatures and times) (32).
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Figure 3. The morphology of mPEG-derivatized RBCs (3000×). The reactive mPEG 5kDa incubation concentrations are (A) 0 mg/mL control (uncoated), (B) 5 mg/mL, (C) 10 mg/mL, (D) 15 mg/mL (E) 25 mg/mL, and (F) 45 mg/mL.
The optimum temperature and pH of reaction medium, obtained by Taguchi design, are 14 °C and pH ) 8.7. At 4 °C the rate of reaction is low, and more time is required for completion of the reaction. At temperatures higher than 20 °C, the activated polymer may show a less active form (30) that suggests a moderate temperature is more favorable. As mentioned earlier, a pH >7.0 is required for reaction of the PEG derivative with primary amine residues. It can be seen that the optimum conditions at which the number of free cells was maximum, and the fluorescence intensity ratio of FITC-anti-D bound cells of the PEG-RBCs versus control (uncoated) RBCs was at a minimum, as shown in Table 4, are very similar to those deduced from Figures 1 and 2. It shows that agglutination between RBCs decreased and the corresponding number of free cells increased with increasing PEG concentration. Also as a result of RBC coating by mPEG, attachment of FITC labeled-anti-D to RBCs decreased and the corresponding ratio of mean fluorescence intensity of the PEGRBCs versus control (uncoated) RBCs was decreased. It is worth mentioning that inhibition of agglutination does not necessarily indicate that antibody binding was inhibited relative to uncoated cells and may merely reflect the physical prevention of Igs bridging between cells. However, inhibition of agglutination does facilitate a way to quantify the PEG-coating of RBCs and is sufficient to determine the optimum conditions for PEGylating. Direct measurement of inhibition of anti-D binding to PEG-RBCs by flow cytometric analysis, using a FITC-labeled anti-D, demonstrates that the polymer coating does prevent antibody binding. Cyanuric chloride-activated mPEG-5 kDa was covalently attached to human RBCs, and the optimum (best) conditions
for this reaction were determined by different methods of experimental design. The optimum pH of TEA buffer, temperature, and reaction time were pH ) 8.7, 14 °C, and 30 min, respectively. An optimum polymer concentration could not be determined by the methods employed here; however, a maximum concentration of 15 mg/mL, as the best condition, is recommended for a cyanuric chloride derivative of mPEG 5 kDa due to the observation of increasing echinocytosis with increasing concentration. Finally, concerning the good agreement between the results from hemocytometry and flow cytometry analyses, it can be concluded that the cell counting method is a simple and suitable technique to assess RBC PEGylation.
ACKNOWLEDGMENT This work was supported by grants from the Ministry of Science, Research and Technology, and Tehran University of Medical Sciences. The authors also wish to thank Iranian Blood Transfusion Organization for providing the packed Rh-positive RBCs.
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